Rozprawy doktorskie na temat „PI3K TARGET”

Deregulated activation of phosphatidylinositol 3-kinase (PI3K) pathway is central to many human malignancies. The functions of this pathway are critical for normal cell metabolism, proliferation, and survival. In lung cancers, the PI3K pathway activity is often aberrantly driven by multiple mutations, including EGFR, KRAS, and PIK3CA. Molecules targeting the PI3K pathway are intensely investigated as potential anti-cancer agents. Although inhibitors of the pathway are currently in clinical trials, rational and targeted use of these compounds, alone or in combination, requires an understanding of isoform-specific activity in context. We sought to identify class IA PI3K enzyme (p110a/PIK3CA, p110b/PIK3CB, p110d/PIK3CD) activities using isoform-specific inhibitors in a lung cancer model system. Treatment of non-small cell lung cancer (NSCLC) cell lines with PIK3CA, PIK3CB, PIK3CD or PIK3CB/D inhibitors resulted in pharmacokinetic and pharmacodynamic responses that frequently tracked with a specific mutation status. Activation of PIK3CA dictated response to the PIK3CA-specific inhibitor while deletion of PTEN phosphatase indicated response to the PIK3CB inhibitor. The PIK3CD isoform-specific inhibitors lacked efficacy in all NSCLC cell lines tested, however treatment at increased concentrations likely provide concurrent inhibition of both PIK3CB/D isoforms improving activity of either agent alone but did not track with a single biomarker. The observed pharmacodynamic and proliferation responses to isoform-specific inhibitors suggested that PI3K isoforms may functionally compensate for loss of another in certain genetic backgrounds. These studies demonstrate unanticipated cellular responses to PI3K isoform inhibition in NSCLC, suggesting that patient populations with specific mutations can benefit from certain isoform-selective inhibitors, or combinations, allowing for rational and targeted clinical use of these agents.

Acute Lymphoblastic Leukemia (ALL) is a malignant disorder characterized by the abnormal clonal proliferation of B-cell progenitors (B-ALL) or immature stage thymocytes (T-ALL). Constitutive activation of the PI3K/Akt/mTOR network is a common feature of B- and T-ALL, influencing cell growth and survival. The PI3K/Akt/mTOR inhibitors are currently being developed for clinical use either as single agents or in combination with conventional chemotherapy for T-ALL patient treatment. In this study it has been investigated the effects of a panel of PI3K/Akt/mTOR inhibitors on healthy human CD4+ T-cells when compared with T-ALL cell lines. Then, it has been verified whether a multi-inhibition treatment against Akt protein could enhance the efficacy of individual drug administration and overcome drug resistance as well as to obtain a decrease in single drug concentration, by testing the effects of combined treatments with three Akt inhibitors with different mode of action, GSK690693, MK-2206 and Perifosine on T-ALL cell lines. Combined administration of the drugs displayed a significant synergistic cytotoxic effect and affected PI3K/Akt/mTOR pathway at much lower concentration than single drug use. Highest synergistic effect for full inhibition of Akt was also related to the timing of every drug administration. The results obtained suggested that targeting Akt as a key protein of PI3K/Akt/mTOR pathway with multiple drugs might represent a new and promising pharmacological strategy for treatment of T-ALL patients. It has also been investigated the role of microRNAs (miRNAs), a class of small noncoding RNAs, which play a role in various biological processes, including proliferation, apoptosis and tumorigenesis. The dysregulation of miRNAs is implicated in invasion in several human cancer types and leukemia is not an exception. By using in vitro models, it has been done an analysis of the effect of PI3K signaling inhibitors on expression of miRNA level involved in ALL disease and PI3K activation. The results obtained have shown that these drugs could modulate miRNA expression. Therefore, the regulation of miRNA expression profiling in ALL by using PI3K signaling inhibitors could be used as a new therapeutic approach in the near future. In addition, it has been analyzed the efficacy of PI3K signaling pathway inhibitors in B- and T-ALL cell lines harboring the Abl1 tyrosine kinase gene fusion that lead to an aberrant cell proliferation. It has been studied the effects of anti Bcr-Abl1 drugs such as Imatinib, Nilotinib and GZD824 associated with PI3K signaling inhibitors. Drugs against PI3K/Akt/mTOR cascade administered in combination with Imatinib, Nilotinib and GZD824 decreased cell viability, induced apoptosis and autophagy in a marked synergistic manner. These findings suggested that selected PI3K/Akt/mTOR inhibitors used in combination with anti Bcr-Abl1 drugs may be an attractive novel therapeutic intervention in Ph+ B- and T-ALL.
La Leucemia Linfoblastica Acuta (LLA) è un tumore maligno ematologico caratterizzato da una proliferazione clonale incontrollata di progenitori della linea cellulare di tipo B (LLA-B) o timociti allo stadio immaturo (LLA-T). L’attivazione della via di trasduzione del segnale di PI3K/Akt/mTOR è una caratteristica comune della LLA-B e T ed influisce sulla crescita e sopravvivenza cellulare. Gli inibitori della via di PI3K/Akt/mTOR sono attualmente in fase di studio per uso clinico, sia come singoli agenti che in combinazione con la convenzionale chemioterapia utilizzata nel trattamento dei pazienti affetti da LLA-T. In questo studio sono stati analizzati gli effetti di un pannello di inibitori della via di PI3K/Akt/mTOR su linfociti T-CD4+ di individui sani e confrontati con linee cellulari tumorali umane di LLA-T. Successivamente è stato verificato se il trattamento di inibizione multipla della proteina Akt potesse aumentare l’efficacia dei farmaci somministrati singolarmente e superare la resistenza al farmaco ottenendo la riduzione della concentrazione del singolo agente. Pertanto, sono stati studiati e testati gli effetti di tre inibitori su linee cellulari umane di LLA-T diretti contro Akt ma con differenti modi di azione: GSK690693, MK-2206 e Perifosina. Questa combinata somministrazione di farmaci ha mostrato un significativo effetto sinergico ed ha influito sulla via di PI3K/Akt/mTOR ad una concentrazione molto più bassa rispetto a quella del singolo farmaco. Il più elevato effetto sinergico per una totale inibizione di Akt è stato associato alla tempistica adottata per ciascuna somministrazione. I risultati ottenuti hanno suggerito che, mirare Akt come bersaglio chiave nella via del segnale di PI3K/Akt/mTOR con la somministrazione multipla di farmaci, potrebbe rappresentare una nuova e promettente strategia per il trattamento dei pazienti affetti da LLA-T. E’ stato inoltre studiata l’azione dei microRNA (miRNA), una classe di piccoli RNA non codificanti che giocano un ruolo in vari processi biologici, quali la proliferazione, la morte cellulare e la genesi del cancro. La regolazione incontrollata dei miRNA è implicata nell’invasione di diversi tumori umani e la leucemia non è esclusa. Usando modelli in vitro è stata eseguita un’analisi degli effetti degli inibitori della via del segnale di PI3K sui livelli di espressione dei miRNA coinvolti nella LLA e nell’attivazione di PI3K. I risultati emersi hanno mostrato che questi farmaci potrebbero modulare l’espressione dei miRNA, pertanto, la regolazione dei loro profili di espressione nella LLA, utilizzando gli inibitori diretti contro la via di PI3K, potrebbe costituire un nuovo terapeutico approccio per il prossimo futuro. Infine, è stata valutata l’efficacia degli inibitori della via del segnale di PI3K nelle linee cellulari di LLA-B e T caratterizzate dalla proteina di fusione Abl1 che causa una proliferazione cellulare incontrollata. Sono stati studiati gli effetti di farmaci contro il gene Bcr-Abl1 come Imatinib, Nilotinib e GZD824 utilizzati in combinazione con i farmaci diretti contro la via di PI3K. La combinazione di questi farmaci ha mostrato una ridotta vitalità cellulare, innescando il processo di morte e autofagia cellulare in maniera sinergica. Questi dati hanno suggerito che la selezione di inibitori diretti contro la via di PI3K/Akt/mTOR somministrati in combinazione con farmaci contro il gene di fusione Bcr-Abl1, potrebbe rappresentare un allettante nuovo intervento terapeutico da prendere in considerazione nel trattamento della LLA-B e T portatrice del cromosoma Philadelphia (Ph+).

In Europa, la sopravvivenza a 5 anni dei pazienti affetti da leucemia mieloide acuta (LMA) è solo del 20%. La duplicazione interna in tandem del gene FLT3 (FLT3-ITD), che codifica per il recettore della tirosina chinasi FLT3, è la mutazione più frequente (~ 25%) nella LMA con cariotipo normale, dove porta all'attivazione costitutiva della chinasi FLT3. Nonostante risultati iniziali molto promettenti con inibitori di FLT3 (FLT3i) nei pazienti con questa mutazione, pochi pazienti hanno remissioni prolungate, evidenziando la necessità di nuove e più efficaci terapie. La persistenza delle cellule staminali leucemiche guida la leucemogenesi della LMA ed è responsabile della resistenza ai farmaci e della ricaduta dopo chemioterapia convenzionale. L'attivazione costitutiva di FLT3 porta all’attivazione del signaling a valle, e in particolare della via PI3K/AKT/mTOR, una cascata di segnale fortemente associata alla sopravvivenza delle cellule staminali leucemiche e al crosstalk tra le cellule staminali leucemiche e le cellule stromali associate al microambiente tumorale midollare. La nicchia midollare fornisce protezione alle cellule leucemiche FLT3-ITD nei confronti degli inibitori FLT3. Pertanto, la via PI3K/AKT/mTOR può rappresentare un bersaglio terapeutico nella AML FLT3-ITD. Questo studio mira a verificare l'ipotesi che l'inibizione di PI3K/AKT/mTOR sensibilizzi le cellule AML FLT3-ITD alla terapia mirata con RTKi utilizzando linee cellulari AML umane e blasti di pazienti primari. In particolare, ho definito il profilo fenotipico delle linee cellulari FLT3-ITD rispetto a quelle FLT3 wildtype dopo trattamento con un pannello di FLT3i o PI3K/AKT/mTORi che non hanno dimostrato sufficiente efficacia clinica se utilizzato come monoterapia. Successivamente, ho valutato l’effetto del farmaco sulla crescita cellulare e sul ciclo cellulare e l'apoptosi. I risultati ottenuti dimostrano che BAY-806946 (pan PI3Ki) e PF-04691502 (inibitore duale PI3K/mTORi) sono in grado di inibire la crescita poiché causano arresto del ciclo cellular in fase G1 e apoptosi, un effetto che appare indipendente dallo stato mutazionale di FLT3. Dimostrano inoltre che l’arresto della crescita cellulare indotto dall’inibitore di FLT3 (FLT3i) quizartinib è causato principalmente dall’induzione di apoptosi. Tuttavia l’efficacia rimane inferiore rispetto al trattamento con chemioterpia convenzionale (AraC). Inoltre, la proof of concept per l’utilizzo della combinazione del quizartinib con BAY-806946 è stata ottenuta in linee cellulari AML FLT3-ITD e blasti primari da paziente. Nel valutare i blasti primari da paziente, è stato considerato il ruolo protettivo delle cellule stromali mesenchimali in co-coltura, e dei fattori di crescita per riprodurre le condizioni del microambiente midollare. Pertanto, blasti primari da paziente LAM sono stati mantenuti in co-coltura con cellule stromali MS5 in presenza di concentrazioni fisiologiche di fattori di crescita quali IL-3, TPO e GM-CSF. Come atteso, il BAY-806946 potenzia l’effetto citostatico e citotossico del quizartinib nelle cellule MOLM-13 e nei blasti primari da paziente con mutazione FLT3-ITD in condizione di co-coltura. E’ importante sottolineare l’incremento di apoptosi osservato anche nella sottopopolazione staminale leucemica CD34+CD38-. Infine, ho valutato il profilo delle citochine e delle fosfoproteine persistenti come bersagli putativi dopo il trattamento di combinazione. Complessivamente, questo studio dimostra il potenziale di PI3Ki per migliorare l'efficacia di RTKi quizartinib nel trattamento della LMA FLT3-ITD.
Acute myeloid leukemia (AML) has a very poor 5-year survival of ~20% in Europe. The internal tandem duplication (ITD) mutation of the Fms-like receptor tyrosine kinase 3 (FLT3) (FLT3-ITD) is the most frequent mutation (~25%) in normal karyotype AML. In recent clinical studies, few patients display prolonged remissions with receptor tyrosine kinase (RTK) inhibitors, such as FLT3 inhibitors (FLT3i) therapy, highlighting a substantial unmet need for novel effective treatment. Persistence of leukemia stem cells (LSC) drive AML leukemogenesis, responsible for drug resistance and disease relapse following conventional chemotherapy. Growing evidence recognizes that FLT3-ITD mutation leads to the constitutive activation of FLT3 kinase and its downstream pathways, including PI3K/AKT/mTOR signaling, strongly associated with LSC survival and crosstalk between LSC and stromal cells associated bone marrow (BM) tumor environment (TME). The TME provides protection of FLT3-ITD AML cells against FLT3 inhibitors. Thus, the PI3K/AKT/mTOR pathway may represent as a putative target for FLT3-ITD AML. This study aims to test the hypothesis that PI3K/AKT/mTOR inhibition could sensitize FLT3-ITD AML cells to RTKi-lead targeted therapy using human AML cell lines and primary patient blasts. First, I uncover the phenotypic profile of FLT3-ITD versus FLT3 wildtype cell lines following treatment with selected FLT3i or PI3K/AKT/mTORi that have failed treatment of AML as monotherapy in clinical studies. More specifically, I determine the drug efficacy by means of cell growth measurement and assessment of cell cycle status and apoptosis. I was able to demonstrate that BAY-806946 (pan PI3Ki) and PF-04691502 (dual PI3K/mTORi) exerted growth inhibitory activity caused by G1 cell cycle arrest and apoptosis, and this effect was irrespective of FLT3 status. Quizartinib (FLT3i) selectively inhibited cell growth in FLT3-ITD AML and this effect was mainly caused by apoptosis. The observed drug-induced apoptotic effect was however not as efficient as chemotherapy. Next, I provide proof-of-concept for the combination of quizartinib and BAY-806946 using both FLT3-ITD AML cell lines and primary patient blasts. When evaluating on primary patient blasts, I take into consideration the protective role of mesenchymal stromal cells and physiological growth factors to mimic the BM microenvironment. Hereby, I co-cultured FLT3-ITD AML blasts with stromal cell line MS-5 and added growth factors essential for AML survival and differentiation such as IL-3, TPO and G-CSF at physiological concentration. As expected, treatment with BAY-806946 enhanced both cytostatic and cytotoxic effect of quizartinib in FLT3-ITD AML cell line MOLM-13 as well as primary patient blasts in co-culture. More importantly, enhanced apoptosis was measured in the stem cell like CD34+CD38- population. Lastly, I elucidate the cytokine profile and persistent phosphoproteins as putative targets following combination treatment. Ultimately, this study demonstrates the potential of PI3K/AKT/mTORi to enhance the efficacy of RTKi quizartinib for the treatment of FLT3-ITD AML.

Class I phosphatidylinositol 3-kinases (PI3Ks) are heterodimeric lipid kinases consisting of a regulatory subunit and one of four catalytic subunits (p110α, p110β, p110γ or p110δ). p110γ/p110δ PI3Ks are highly enriched in leukocytes. In general, PI3Ks regulate a variety of cellular processes including cell proliferation, survival and metabolism, by generating the second messenger phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). Their activity is tightly regulated by the phosphatase and tensin homolog (PTEN) lipid phosphatase. PI3Ks are widely implicated in human cancers, and in particular are upregulated in T-cell acute lymphoblastic leukemia (T-ALL), mainly due to loss of PTEN function. These observations lend compelling weight to the application of PI3K inhibitors in the therapy of T-ALL. At present different compounds which target single or multiple PI3K isoforms have entered clinical trials. In the present research, it has been analyzed the therapeutic potential of the pan-PI3K inhibitor BKM120, an orally bioavailable 2,6-dimorpholino pyrimidine derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. BKM120 treatment resulted in cell cycle arrest and apoptosis, being cytotoxic to a panel of T-ALL cell lines and patient T-lymphoblasts. Remarkably, BKM120 synergized with chemotherapeutic agents currently used for treating T-ALL patients. BKM120 efficacy was confirmed in in vivo studies to a subcutaneous xenotransplant model of human T-ALL. Because it is still unclear which agents among isoform-specific or pan inhibitors can achieve the greater efficacy, further analyses have been conducted to investigate the effects of PI3K inhibition, in order to elucidate the mechanisms responsible for the proliferative impairment of T-ALL. Overall, these results indicated that BKM120 may be an efficient treatment for T-ALLs that have aberrant up-regulation of the PI3K signaling pathway and strongly support clinical application of pan-class I PI3K rather than single-isoform inhibitors in T-ALL treatment.

Class I phosphatidylinositol 3-kinases (PI3Ks) are heterodimeric lipid kinases consisting of a regulatory subunit and one of four catalytic subunits (p110α, p110β, p110γ or p110δ). p110γ/p110δ PI3Ks are highly enriched in leukocytes. In general, PI3Ks regulate a variety of cellular processes including cell proliferation, survival and metabolism, by generating the second messenger phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). Their activity is tightly regulated by the phosphatase and tensin homolog (PTEN) lipid phosphatase. PI3Ks are widely implicated in human cancers, and in particular are upregulated in T-cell acute lymphoblastic leukemia (T-ALL), mainly due to loss of PTEN function. These observations lend compelling weight to the application of PI3K inhibitors in the therapy of T-ALL. At present different compounds which target single or multiple PI3K isoforms have entered clinical trials. In the present research, it has been analyzed the therapeutic potential of the pan-PI3K inhibitor BKM120, an orally bioavailable 2,6-dimorpholino pyrimidine derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. BKM120 treatment resulted in cell cycle arrest and apoptosis, being cytotoxic to a panel of T-ALL cell lines and patient T-lymphoblasts. Remarkably, BKM120 synergized with chemotherapeutic agents currently used for treating T-ALL patients. BKM120 efficacy was confirmed in in vivo studies to a subcutaneous xenotransplant model of human T-ALL. Because it is still unclear which agents among isoform-specific or pan inhibitors can achieve the greater efficacy, further analyses have been conducted to investigate the effects of PI3K inhibition, in order to elucidate the mechanisms responsible for the proliferative impairment of T-ALL. Overall, these results indicated that BKM120 may be an efficient treatment for T-ALLs that have aberrant up-regulation of the PI3K signaling pathway and strongly support clinical application of pan-class I PI3K rather than single-isoform inhibitors in T-ALL treatment.

In this study, we investigated differential activation and the role of two mTOR complexes in cell migration of prostate cancer cells. Specific knock-down of endogenous RAPTOR and RICTOR by siRNA resulted in decreased cell migration in LNCaP, DU145, and PC3 cells indicating that both mTORC1 and mTORC2 are required for cell migration. EGF treatment induced the activation of both mTORC1 and mTORC2 as determined by complex-specific phosphorylation of mTOR protein. Specific knock-down or inhibition of Rac1 activity in PC3 cells blocked EGF-induced activation of mTORC2, but had no effect on mTORC1 activation. Furthermore, the over-expression of constitutively active Rac1 (Rac1Q61L) resulted in significant increase in cell migration and activation of mTORC2 in PC3 cells, but had no effect on mTORC1 activation. Constitutively active Rac1 (Rac1Q61L) in PC3 cells was localized in the plasma membrane and was found to be in a protein complex which contained mTOR and RICTOR proteins, but not RAPTOR. In conclusion, we suggested that EGF-induced activation of Rac1 causes the phosphorylation/activation of mTORC2 via RICTOR, specific regulator of mTORC2 activation in numerous cancer cells. The major role played by mTOR in a wide array of cancers has in the recent decades led to the development of numerous mTOR inhibitors. One of the drawback of these first generation mTOR inhibitors are that m TORC1 activity is inhibited but effect on mTORC2 activity require high dosages and prolonged exposure in different cancer cell types including HeLa, PC3, LNCaP, and A549. High dosage of rapamycin and its associated rapalogs required for mTORC2 inhibition is clinically unsuitable. Studies have shown that the dual mTORC1/C2 inhibitors trigger feedback loops causing metastasis and affect the cell viability of normal tissues in vitro and in vivo. There is a need for specific mTORC1 and mTORC2 inhibitor, which overcome the disadvantages of the previously developed mTOR inhibitors. The Rac1-RICTOR axis suggested in this study could be used as a potential target for the development of mTORC2 inhibitor and lead to a potential therapeutic treatment for aggressive prostate cancer.

Endometrial cancer (EC) is the fourth most common cancer in women in developed countries, such as North America, Europe and Australia. Patients with low-grade, early-stage disease usually have a favourable survival rate. However, patients that present at an advanced stage of disease have an average survival of only 12 months. Current treatments for these patients are radiation and chemotherapy, which offer limited clinical benefit. There is no efficient treatment for advanced EC. Improved therapeutic approaches are needed for the treatment of recurrent and metastatic endometrial cancer. Recent advances in cancer biology have resulted in the development of molecular targeted therapies. The Fibroblast Growth Factors Receptor (FGFR) family and their ligands (fibroblast growth factors, FGFs) regulate a broad spectrum of physiological processes as well as tissue patterning and organogenesis during embryogenesis. Abnormally activated FGFRs have been identified in various cancers and are emerging as potential therapeutic targets. The Pollock laboratory and other groups have demonstrated that 10-20% of endometrioid ECs carry FGFR2 mutations that may be a novel therapeutic target in endometrial carcinoma. Preclinical studies show that inhibition of FGFR can inhibit EC cell growth in vitro. However, FGFR inhibitors are not as efficient at inhibiting tumour growth in vivo. We aim to find a way to improve the efficacy of FGFR inhibition in cancer treatment. About 90% of EC patients harbour genetic aberrations in the components of the PI3K/AKT pathway which indicates this signalling pathway plays an important role in the development of EC. Work from our lab demonstrates that inhibition of FGFR results in abrogation of MAPK activation in sensitive EC cells, however, the PI3K/AKT signalling pathway remains unaffected. PI3K/AKT signalling plays a vital role in cancer cell proliferation and survival, furthermore crosstalk between the MAPK and PI3K/AKT signalling pathways is associated with resistance to targeted therapies. Thus, the first aim of this study was to examine whether combination of the FGFR inhibitor (BGJ398) with various different PI3K inhibitors was synergistic in FGFRi sensitive EC cells. We present data that the combination of the pan-FGFR inhibitor (BGJ398) with pan-PI3K inhibitors (GDC-0941, BKM120) or a p110α-selective PI3K inhibitor (BYL719) was synergistic in inhibiting cell growth. Significantly more cell death and inhibition of long-term cell survival was observed in the combination treatments compared to each of the single drug treatments. Importantly, these effects could also be observed at lower concentrations. This study is the first to indicate that partial inhibition of the PI3K signalling pathway could significantly increase cell death when combined with the FGFR inhibitor BGJ398 in FGFR2 mutant EC cells. These data provide evidence that sub-therapeutic doses of PI3K inhibitors could enhance the efficacy of anti-FGFR therapies and a combination therapy may represent a superior therapeutic treatment in FGFR2 mutant EC patients. The in vivo work (conducted by Dr Vanessa Bonazzi) shows that the combination of BGJ398 and GDC-0941 and BYL719 resulted in tumour regression, while single drug treatment only slowed tumour growth. Interestingly, BYL719 alone resulted in increased tumour growth in tumour xenografts of AN3CA but not JHUEM2. In the first results chapter we further investigated the mechanism of enhanced cell death from the combination of BGJ398 and PI3K inhibitors. The activation of ERK and AKT has been inhibited by the combination of BGJ398 and PI3K inhibitors. However, the combination of the MEK inhibitor trametinib and the PI3K inhibitors induced less cell death than inhibition of the FGFR and PI3K signalling pathways. BGJ398 but not trametinib or GDC-0941 inhibited the activity of PLCγ1. We have also found trametinib up-regulated PLCγ1 activity, which is a novel finding in the field. We next employed several pharmacological inhibitors to investigate whether PLCγ1 is involved in the cell death observed following the combination of BGJ398 and GDC-0941 treatment. As there is no PLCγ1 inhibitor available currently, we used two different pan-PLC inhibitors, manoalide and U73122. Co-inhibition of the MAPK, PI3K/AKT and PLC signalling recapitulated cell growth inhibition seen with the combination of FGFR and PI3K inhibitor in both cell lines. Cell death induced by the combination of PLC inhibitors with trametinib and GDC0941 was similar as the combination BGJ398 and GDC0941 in AN3CA, but significantly less than the combination BGJ398 and GDC0941 in JHUEM2. Unfortunately, Western blotting was unable to show inhibition of PLCγ1 bringing into question whether these PLC inhibitors inhibited PLC function sufficiently, and whether the phenotypic effects of manoalide and U73122 when added to the trametinib and GDC0941 combination are due to inhibition of PLCγ1. The second results chapter reports efforts to identify the mechanism of intrinsic resistance to FGFR inhibition in EC cell lines carrying FGFR2 activating mutations but showing intrinsic resistance to FGFR inhibition (EI, EN1078D, and MFE319) with comparisons to the two sensitive EC cell lines (JHUEM2 and AN3CA). We have observed sustained activation of ERK in the resistant cells after treatment with an FGFR inhibitor, while ERK was inhibited in the sensitive cells. Inhibition of the MAPK signalling pathway could not sensitise the resistant cells to FGFR inhibition. Although several other receptor tyrosine kinases (RTKs) were hyperactivated in these cells, pharmacological inhibition did not show they were reliant on these RTKs. Co-inhibition of these kinases did not sensitise these cells to BGJ398. Knockdown of FGFR2 by shRNA in the sensitive cells induced moderate cell death, but limited cell death in the resistant cells. Interestingly, co-inhibition of the MAPK, PI3K/AKT and PLC signalling pathways has induced markedly less cell growth inhibition in the resistant cells compared to the sensitive cells, suggesting the resistant cells are less dependent on these central signalling pathways than the sensitive cells. Western blotting results showed that FGFR2 expression was considerably lower in the resistant cells than in the sensitive cells. Based on these results we have concluded that FGFR2 mutation status is not the only factor that determines sensitivity to FGFR inhibition, high expression of mutant FGFR2 is also important. This is a novel finding in the field and one which could guide patient select criteria in future clinical trials. Lastly, we show that FGFR2 knockdown in medium containing 10% FBS has little impact on downstream ERK phosphorylation whereas pan FGFR inhibition with BGJ398 could totally abrogate ERK phosphorylation. In cells grown overnight in serum starved conditions, FGFR2 knockdown did reduce downstream ERK phosphorylation but not to the same extent as pan FGFR inhibition in full growth medium. These data suggest that inhibition of FGFR2 alone is insufficient and that inhibition of multiple FGFRs will be more effective as a cancer treatment.

Background: Radiotherapy is used in the treatment of over 50% of cancer patients and bar surgery, is the most effective cancer intervention. However, in the clinic secondary malignancies have been observed following radiotherapy and in vitro increased cell migration and invasion have been seen following radiation. The Src/FAK signalling pathway is known to play an important role in the metastatic phenotype through its involvement in cell adhesion, migration and invasion and we have previously demonstrated that radiotherapy can activate this pathway along with the phosphoinositide 3-kinase (PI3K) pathway, also associated with tumour metastases and an aggressive phenotype. Using pharmacological inhibitors, we have investigated combination approaches to evaluate whether Src and PI3K targeting is beneficial in a radiotherapy context, especially focusing on metastatic phenotype. We wished to relate pathway activation to cellular phenotype and increase understanding of the metastatic cascade, the processes involved and the signalling pathways taking the lead. Method: Using thyroid carcinoma cell lines FTC133 and 8505c the effects of Src inhibition using AZD0530, FAK inhibition using FAKi and PI3K inhibition using GDC-0941 were studied. The effects of radiotherapy alone, and in combination with the above inhibitors, were also studied. In vitro MTT, apoptosis and clonogenic assays were used to assess cell proliferation and cell survival and scratch assays, cell adhesion and cell spreading assays were used to assess the effects of the drugs on metastatic characteristics. In vivo tumour growth, survival and ex vivo clonogenics were used to measure the effects of AZD0530 and GDC-0941. Western blotting, immunofluorescence and immunohistochemistry was used to observe the effects on pathway activation and protein localisation. Results: Src and FAK inhibition reduced metastatic characteristics of thyroid carcinoma cell lines in vitro such as cell spreading and migration. FAK inhibition showed a greater effect on cell survival by MTT, clonogenic and apoptosis. In the thyroid carcinoma cell lines radiotherapy enhanced the metastatic phenotype. This was seen by enhanced activation of the Src and PI3K pathways, increased migration and invasion in vitro and enhanced tumour metastasis in vivo. By combining Src inhibition with radiation a reduction in metastatic characteristics was observed and by combining PI3K inhibition with radiotherapy radiosensitivity could be improved. With the triple combination of Src and PI3K inhibition with radiotherapy a significant reduction in cell survival was demonstrated in vitro compared to radiation alone and either inhibitor combined with radiation, with a corresponding significant reduction in tumour growth being observed in vivo. With the combination of Src and PI3K inhibition significant reductions in metastatic characteristics were also observed both in vitro and in vivo seen by a reduction in cell migration and tumour metastasis. Finally combined inhibition of the Src and PI3K pathway reduced the radiation enhanced activation of several pathways in vivo including Src and PI3K.Conclusions: Together these results suggest that the Src and PI3K pathways play a role in radiation enhanced metastatic characteristics in thyroid carcinoma and through combined inhibition of the pathway the negative effects of radiation, enhanced migration and invasion, can be inhibited and the cells can be made more radiosensitive. Full characterisation of the pathways involved in radiation induced motility and radioresistance will provide further rationale for combination therapies and provide potential for application of these therapies in the clinic.

Interleukin-13 (IL-13) has been implicated as a critical inducer of a number of features of allergy and asthma including the induction of nonspecific airway hyperresponsiveness (AHR), eosinophilic inflammatory response, eotaxin production, excess mucus formation, and fibrosis. Determining the mechanism(s) of AHR, a hallmark of asthma, is crucial to our understanding of both the pathogenesis and successful treatment of asthma. After carrying out initial experiments to determine the effect of IL-13-induced AHR on murine and rat tracheal rings, mice tissues were chosen for subsequent experiments due to their consistent results and the fact that the mouse genetic map was completed in 1996, which will enable subsequent gene therapy work. Human and mouse share a high percentage of their genes with an average of 85% homology. Numerous IL-13 signalling studies have concentrated on the JAK/STAT6 pathway. IL-13 also activates phosphoinositide 3-kinase (PI3K) and downstream effector molecules. In experiments presented in this thesis pharmacological and genetic approaches implicate the involvement of PI3K and its individual isoform PI3Kδ in IL-13 induced AHR in vitro and this involvement was confirmed using a small interference RNA (siRNA) technology approach. However, IL-13 induced an early activation of PI3K, whereas increased responsiveness was not observed until overnight incubation. Arginase I induction was demonstrated to be another PI3K-dependent potential mechanism of IL-13-induced hyperresponsiveness. The epithelium is also implicated in IL-13-induced hyperresponsiveness, however, the induction of arginase I was demonstrated in both intact and denuded epithelium tracheal rings. The siRNA approach was also employed in 9HTEo-, A549 and BEAS-2B cell lines using different transfecting agents. From these findings, it is concluded that class IA p110δ could be a useful target for the treatment of asthma by preventing IL-13-induced airway smooth muscle hyperresponsiveness and also that arginase I may be involved in IL-13-induced hyperresponsiveness through PI3K- and epithelial-dependent pathways.

Introduction: Over the past decades, considerable advances in understanding of cell biology at genetic, epigenetic and proteomic levels led to development of new strategies for better outcome of cancer therapy. One of these new strategies is targeting the class I PI3K/Akt/mTOR signaling pathway, in that this pathway plays a key role in regulation of many cellular functions, including proliferation, survival, metabolism, autophagy and motility. Dysregulation of the class I PI3K/Akt/mTOR pathway has been documented in a variety of human tumours and inhibition of this pathway has been observed to hamper tumour proliferation in vitro and prevent tumour progression in vivo and in clinic. More recently, emerging evidence suggests that the class I PI3K/Akt/mTOR pathway is associated with Cancer Stem Cell (CSC) biology, in light of maintenance, viability and conventional therapy resistance of CSCs. The CSC theory conceptualizes that a subset of tumour cells with Stem Cell-like properties, including self-renewal, multipotency, differentiation, and resistance to chemotherapy and radiotherapy, can recapitulate new tumours and resistance to cancer therapy. Materials and Methods: To explore class I PI3K/Akt/mTOR signaling pathway and CSCs as therapeutic targets in canine oncology, in one series of experiments, smallmolecular inhibitors Wortmannin, ZSTK474, KP372-1 and Rapamycin, which selectively target pan-class I PI3K, pan-class I PI3K, Akt and mTOR, respectively, were utilized to treat canine cancer cell lines using inhibitors alone or in combination with conventional therapeutic drugs. The human acute lymphoblastic leukaemia of T-cell origin cell line (Jurkat T cell line) was used as a comparative control. In another, a stem cell culture system was performed to isolate CSCs from canine glioma J3T cell line. Subsequently, microarray analysis of transcriptional expression profiles of J3T spheres (the putative CSCs) versus J3T parental cells was performed. Results: In this study, small molecules ZSTK474 and KP372-1 were found to significantly decrease cell viability at lower micromolar and nanomolar ranges, respectively. Rapamycin decreased cell viability at lower micromolar concentrations. However, the efficacy of Wortmannin varied from one cell line to another. Dissection of the mechanism of these inhibitors using Western Blot analysis and annexin V staining showed that all inhibitors functioned by decreasing phosphorylation of class I PI3K pathway members. Notably, the efficacy of Wortmannin for this pathway inhibition is confined to certain cell lines. In addition, Wortmannin had shorter drug duration than the other three inhibitors. Annexin V staining showed that KP372-1 was a potent inducer of apoptosis, with decreasing potency in hierarchy order, Rapamycin, Wortmannin and ZSTK474. The data obtained from the combination of pan-class I PI3K inhibitor (Wortmannin or ZSTK474) and mTOR inhibitor (Rapamycin) suggested that additive/synergistic effects were, in part, due to inactivation of Akt. The class I PI3K pathway inhibitors enhanced the efficacy of Doxorubicin in SB cells but not in canine REM, 3132 and J3T cells. The CSC colonies of canine glioma J3T cells were successfully isolated and expanded in the neurosphere formation assay. By microarray analysis, several class I PI3K signaling network-associated genes, particularly IGFBP2 (27-fold), FYN (9.3- fold), and DDIT4 (8.5-fold), were found to be highly up-regulated in the J3T CSCs. However, the genes encoding components, such as Akt1 and eIF4E, of class I PI3K/Akt/mTOR axis signaling were either unchanged or down-regulated in the CSCs. The majority of the genes encoding translation initiation factors were also downregulated in the CSCs. Conclusions: This study demonstrates that class I PI3K/Akt/mTOR signaling pathway is critical for proliferation and survival of cell lines derived from human acute lymphoblastic leukemia of T cell origin (Jurkat T cell line) and a variety of canine tumours. However, it appears that this pathway is dispensible for maintainence and viability of the CSCs isolated from canine gloma J3T cell line. This study suggests that the strategy of dual inhibition of class I PI3K and mTOR kinases may have better outcomes than the combination inhibitors of this pathway (such as ZSTK474 and KP372-1) with Doxorubicin in canine oncology.

A subgroup of non-small cell lung cancer (NSCLC) cases harbour mutations in classical oncogenes, which can affect therapy response and prognosis. By therapeutically targeting the corresponding proteins with inhibitory drugs, the clinical outcome for these lung cancer patients may be improved. One of these oncogenes is the phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) which encodes the catalytic subunit of the phosphatidylinositol 3 kinase (PI3K). PIK3CA is a central regulator in the PI3K/Akt/mTOR pathway, which controls cell growth and apoptosis. Mutations in the PIK3CA gene are considered to up-regulate the kinase activity in tumour cells and through that dysregulate fundamental cellular processes. PI3K inhibitors are currently tested in clinical trials and present a promising therapy option in lung cancer patients. In this study, a pyrosequencing assay for detection of PIK3CA mutations in tumours from patients with NSCLC was established. The three "hot-spot" codons 542, 545 and 1047 of the PIK3CA gene were analysed. The sensitivity of this assay was determined to the presence of 5 % of mutant alleles. In agreement with previous reports, three of the 60 lung cancer cases revealed PIK3CA mutations (5 %). All mutations occurred in exon 9 codon 542 or 545. In line with previous reports, two of the three samples harboured concurrent mutation in the EGFR or KRAS gene. The established pyrosequencing analysis for PI3KCA mutations provides a reliable and cost-effective assay for clinical diagnostics. The determination of the PI3KCA mutation status may help to distinguish patients for treatment targeting the PI3K pathway.

Cancer remains the second leading cause of death in the United States and the world, despite years of research and the development of different treatments. One reason for this is cancer cells are able to survive through adaptation to their environment and aberrantly activated growth signaling. As such, developing new therapies that overcome these hurdles are necessary to combat cancer. Previous work in our laboratory using RNA interference screening identified genes that regulate the survival of glioblastoma (GBM) or autophagy in chronic myelogenous leukemia (CML) cancer cells. One screen identified Phosphatidylinositol-4,5-bisophosphate 3-kinase catalytic subunit beta (PIK3CB) in the family of Phosphatidylinositol 3-kinases (PI3K) as a survival kinase gene in GBM. Work contained in this dissertation set out to study PIK3CB mediated GBM cell survival. We report that only PIK3CB, in its family of other PI3K genes, is a biomarker for GBM recurrence and is selectively important for GBM cell survival. Another screen identified the long non-coding RNA, Linc00467, as a gene that regulates autophagy in CML. Autophagy is a dynamic survival process used by all cells, benign and cancerous, where cellular components are broken down and re-assimilated to sustain survival. Work contained in this dissertation set out to characterize the role that Linc00467 serves in regulating autophagy in a myriad of cancers. Collectively our data have showed Linc00467 to actively repress levels of autophagy in cancer cells. Further, our data revealed an important role for Linc00467 in regulating the stability of the autophagy regulating protein serine-threonine kinase 11 (STK11). Because of the unique role that Linc00467 serves in regulating autophagy we renamed it as, autophagy regulating long intergenic noncoding RNA or ARLINC. Taken together the work in this dissertation unveils the inner-workings of two important cancer cell survival pathways and shows their potential for development into therapeutic targets to treat cancer.
Ph. D.

Cancer remains the second leading cause of death in the United States and the world, despite years of research and the development of different treatments. One reason for this is cancer cells are able to survive through adaptation to their environment and aberrantly activated growth signaling. As such, developing new therapies that overcome these hurdles are necessary to combat cancer. Previous work in our laboratory using RNA interference screening identified genes that regulate the survival of glioblastoma (GBM) or autophagy in chronic myelogenous leukemia (CML) cancer cells. One screen identified Phosphatidylinositol-4,5-bisophosphate 3-kinase catalytic subunit beta (PIK3CB) in the family of Phosphatidylinositol 3-kinases (PI3K) as a survival kinase gene in GBM. Work contained in this dissertation set out to study PIK3CB mediated GBM cell survival. We report that only PIK3CB, in its family of other PI3K genes, is a biomarker for GBM recurrence and is selectively important for GBM cell survival. Another screen identified the long non-coding RNA, Linc00467, as a gene that regulates autophagy in CML. Autophagy is a dynamic survival process used by all cells, benign and cancerous, where cellular components are broken down and re-assimilated to sustain survival. Work contained in this dissertation set out to characterize the role that Linc00467 serves in regulating autophagy in a myriad of cancers. Collectively our data have showed Linc00467 to actively repress levels of autophagy in cancer cells. Further, our data revealed an important role for Linc00467 in regulating the stability of the autophagy regulating protein serine-threonine kinase 11 (STK11). Because of the unique role that Linc00467 serves in regulating autophagy we renamed it as, autophagy regulating long intergenic noncoding RNA or ARLINC. Taken together the work in this dissertation unveils the inner-workings of two important cancer cell survival pathways and shows their potential for development into therapeutic targets to treat cancer.
Ph. D.

Histiocytic sarcoma (HS) is an exceptionally rare malignant neoplasm derived from dendritic cells and histiocytes, with no available effective treatment options. Akt signaling and proteasome dysfunction have been implicated in the pathogenesis of the disease, both in humans and dogs. Our work aims to investigate the importance of the Akt signaling pathway and evaluate the potential of Akt-targeted therapy in a canine model of histiocytic sarcoma. We demonstrated Akt signaling to be active in 9 out of 10 canine HS tumor samples, regardless the presence of PTEN. Moreover, the Akt signaling pathway appears to be constitutively active in DH82 cells — a cell line model of canine HS, when compared to control canine dendritic cells. Pharmacologic Akt inhibition resulted in significant decrease in Akt S473 phosphorylation, GSK-3β S9 phosphorylation, Akt activity, cell viability, increased apoptosis, and resulted in sensitization to proteasome inhibition-depended cell death in a synergistic manner. Proteasome inhibition using carfilzomib, an irreversible proteasome inhibitor, induced dose-depended/caspase-3 independent cell death, at clinically relevant drug concentrations. The therapeutic effect of Akt inhibition was validated in vivo using a DH82 xenograft murine model. Akt inhibition lead to reduced tumor growth, prolonged overall survival, and ameliorated splenomegaly, but not affected the lung metastasis. Moreover, the therapeutic effect of Akt inhibition was potentiated in combination with carfilzomib. In conclusion, targeting Akt signaling may represent an attractive potential therapeutic target for the HS. Future studies are required to examine the clinical efficacy of Akt-targeted therapy in dogs with HS using novel selective Akt inhibitors.
Ph. D.

Ebolavirus (EboV) is a highly pathogenic enveloped virus that causes outbreaks of zoonotic infection in Africa. The clinical symptoms are manifestations of the massive production of pro-inflammatory cytokines in response to infection and in many outbreaks, case fatality rate exceeds 75%. The unpredictable onset, ease of transmission, rapid progression of disease, high mortality and lack of effective vaccine or therapy have created a high level of public concern about EboV. Here we report the properties of a benzylpiperazine adamantane diamide-derived compound identified in a screen for inhibitors of EboV infection. We found that the inhibitor is specific, reversible, and that the target(s) for inhibition are present in cells and not in virus particles. The compound is not an inhibitor of acid pH-dependent endosome protease activity, which is required for EboV infection. Treatment of cells with this compound causes accumulation of cholesterol in late endosomes and lysosomes (LE/LY), suggesting it inhibits one or more proteins involved in regulation of cholesterol uptake into cells. Using mutant cell lines and informative derivatives of the inhibitor, we found the inhibitor target is the endosomal membrane protein Niemann-Pick C1 (NPC1). NPC1 is a polytopic LE/LY membrane protein that mediates uptake of lipoprotein-derived cholesterol into cells. We find that NPC1 is essential for EboV infection, that NPC1 binds to the protease-cleaved GP1 subunit of the EboV glycoprotein, and that the anti-viral compound inhibits infection by targeting NPC1 and interfering with binding to GP1. Furthermore, analysis of viral variants resistant to the anti-viral compound revealed that the residues which confer resistance are located on the surface of the receptor binding domain of GP1. Combined with the results of previous studies of GP structure and function, our findings support a model of EboV infection in which cleavage of the GP1 subunit by endosomal cathepsin proteases removes heavily glycosylated domains to expose the N-terminal domain, which is a ligand for NPC1 and regulates membrane fusion by the GP2 subunit. Thus, NPC1 is essential for EboV entry and a target for anti-viral therapy.

L’adenocarcinoma pancreatico e’ una delle neoplasie con piu’ alta mortalita’ nei paesi occidentali, con una sopravvivenza media di 6 mesi e una percentuale estremamente bassa di sopravvivenza a lungo termine. L’evento principale nello sviluppo dell’adenocarcinoma pancreatico e’ la mutazione del gene KRAS, che tuttavia e’ particolarmente difficile da colpire a livello molecolare. Strategie terapeutiche piu’ efficaci per l’adenocarcinoma pancreatico possono derivare dall’impiego di terapie molecolari. L'obiettivo di questo lavoro e’ quello di identificare nuovi meccanismi e molecolari e strategie terapeutiche per l’adenocarcinoma pancreatico attraverso l’uso modelli cellulari e murini KRAS mutati. Metodi Abbiamo analizzato i risultatiti dello screening di 50 molecole su piu’ di 500 linee cellulari tumorali (incluso 46 di adenocarcinoma pancreatico). Abbiamo disegnato due strategie differenti incluso: 1) un inibitore di JAK2 che inibisce funzionalmente STAT3 e 2) un inibitore di MEK1/2 (AZD-6244), in monoterapia o in combinazione con un inibitore di PI3K (BKM-120 e GDC-0941), in un modello murino KRAS mutato che ricapitola la progressione istopatologica dell’adenocarcinoma pancreatico. Risultati 1) JAK2: lo screening su larga scala di linee cellulari con un inibitore di JAK2 ha mostrato che l’adenocarcinoma pancreatico mostra una sensitivita’ aumentata ( fino a >30 volte) per questa molecola. La sensitivita’ correla con il livello di espressione di pSTAT3 e gp130, un recettore a monte della cascata di STAT3. Abbiamo inoltre osservato che il sottogruppo di adenocarcinoma pancreatico con alti livelli di STAT3 mostra una attivazione costante del pathway IL6/LIF-gp130. Per definire la funzione di STAT3 nell’adenocarcinoma pancreatico, abbiamo quindi sviluppato un modello murino condizionale per STAT3. STAT3 e’ necessario precocemente per lo sviluppo the di lesioni pre-neoplastiche come PanIN (Neoplasia pancretica intraepiteliale) e ADM (Metaplasia acino-duttale). In aggiunta, l’inattivazione di STAT3 blocca lo sviluppo dell’adenocarcinoma pancreatico in un modello ortotopico. I nostri risultati dimostrano che STAT3 ha un ruolo critico nella inizio e progressione dell’adenocarcinoma pancreatico, indicando che la sua inibizione possa essere una strategia terapeutica efficace. Inoltre dimostra che l’espressione di gp130 e pSTAT3 possa essere un biomarker efficace per predire la risposta a un inbitore di JAK2. 2) MEK/PI3K: un altro dato emerso dal nostro screening cellulare e’ che l’adenocarcionoma pancreatico e’ relativamente resistente a monoterapie. Il profilo dell’inibitore di MEK1/2. AZD-6244, ha mostrato una alta efficacia per l’adenocarcinoma pancreatico. Abbiamo tuttavia notato che per indurre apoptosi era necessario associare un inibitore di PI3K (BKM-120 o GDC-0941). Abbiamo testato questa combinazione in modelli murini di adenocarcinoma pacreatico e comparato con trattamento in monoterapia, agenti tradizionali e placebo. La combinazione di MEK e PI3K ha mostrato di ritardare l’insorgenza dei tumori e di aumentare la soopravvivenza in tumori avanzati, sebbene i tumori tendessero a ricorrere nel lungo termine. Conclusioni Questo lavoro dimostra che 1)JAK2 e’ un target molecolare per il trattamento dell’adenocarcinoma pancreatico, in particolare in tumori con alti livelli di GP130; 2) MEK e PI3K sono essenziali nella progressione dell’adenocarcinoma pancreatico e la loro inibizione combinata potrebbe rappresentare una efficace strategia terapeutica.
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers in western countries, with a median survival of 6 months and an extremely low percentage of long-term surviving patients. KRAS mutations are known to be a driver event of PDAC, but targeting mutant KRAS has proved challenging. As new targeted agents are becoming available for clinical trial we aimed to design improved therapeutic approaches for the treatment of pancreatic ductal adenocarcinoma by means of in vitro and in vivo models of pancreatic adenocarcinoma. Methods We analyzed the results of a high-throughput screening of >500 human cancer cell lines (including 46 PDAC lines), for sensitivity to 50 clinically-relevant compounds. We designed two different strategies including 1) a JAK2 inhibitor that blocks STAT3 function and 2) a MEK1/2 inhibitor, AZD-6244, for efficacy alone or in combination with the PI3K inhibitors, BKM-120 or GDC-0941, in a KRASG12D-driven GEMM that recapitulates the multi-step pathogenesis of human PDAC. Results 1) JAK2 inhibitor: Large-scale screening of cancer cell lines with a JAK2 inhibitor that blocks STAT3 function revealed a >30-fold range in sensitivity in PDAC, and showed a close correlation of sensitivity with levels of tyrosine-phosphorylated STAT3 and of the gp130 receptor, an upstream signaling component. Correspondingly, upregulation of the IL6/LIF-gp130 pathway accounted for the strong STAT3 activation in PDAC subsets. To define functions of STAT3 in vivo, we developed mouse models that test the impact of conditional inactivation of STAT3 in KRAS-driven PDAC. We showed that STAT3 is required for the development of the earliest pre-malignant pancreatic lesions, acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN). Moreover, acute STAT3 inactivation blocked PDAC initiation in a second in vivo model. Our results demonstrate that STAT3 has critical roles throughout the course of PDAC pathogenesis, supporting the development of therapeutic approaches targeting this pathway. Moreover, our work suggests that gp130 and phospho-STAT3 expression may be effective biomarkers for predicting response to JAK2 inhibitors. 2) MEK1/2/PI3K inhibitors: In vitro screens revealed that PDAC cell lines are relatively resistant to single-agent therapies. The response profile to the MEK1/2 inhibitor, AZD-6244, was an outlier, showing the highest selective efficacy in PDAC. While MEK inhibition alone was mainly cytostatic, apoptosis was induced when combined with PI3K inhibitors (BKM-120 or GDC-0941). When tested in a PDAC GEMM and compared to the single agents or vehicle controls, the combination delayed tumor formation in the setting of prevention and extended survival when used to treat advanced tumors, although no durable responses were observed. Conclusions: Our studies point to 1)JAK2 as a therapeutic target in GP130 high pancreatic cancers and 2) important contributions of MEK and PI3K signaling to PDAC pathogenesis suggesting that dual targeting of these pathways may provide benefit in some PDAC patients.

Thesis (Ph. D.)--Ohio State University, 2005.
Title from first page of PDF file. Document formatted into pages; contains xix, 94 p.; also includes graphics (some col.). Includes bibliographical references (p. 87-94). Available online via OhioLINK's ETD Center

La teràpia endocrina és un dels majors avenços en el tractament de càncer de mama (CM) dels últims 30 anys. Tot i això, les pacients acaben recaient degut a mecanismes de resistència, com ara les alteracions de la via de PI3K/mTOR i de FGFR. La via de PI3K està freqüentment alterada en CM, especialment en el conjunt luminal on el 30-40% de les pacients tenen mutacions al gen PIK3CA. S’han desenvolupat inhibidors específics d’mTOR i de PI3K (PI3Kinh), i alguns han sigut aprovats per la FDA per a CM ER+ metastàtic. Tanmateix, mecanismes de resistència han estat limitant la resposta. En aquest sentit, alteracions posteriors o en paral·lel a PI3K, com la amplificació de la regió 11q13, són recurrents en CM metastàtic i és important esbrinar si aquestes confereixen resistència a PI3Kinh. L’amplificació de FGFR1 es troba en 10% dels casos de CM metastàtic. Inhibidors específics contra FGFR1/2/3 (FGFRinh) han mostrat una eficàcia limitada en el conjunt d’amplificats de FGFR1. D’altra banda, els inhibidors multi-tirosina quinasa (MTKI, específics per a les famílies de FGFR, VEGFR i PDFGR) han mostrat una millor eficàcia però amb més toxicitat. En aquest estudi, hem investigat mecanismes de resistència a PI3Kinh i biomarcadors de resposta a FGFRinh. En la primera part, hem determinat l’activitat antitumoral d’alpelisib, un PI3Kinh específic per a la isoforma α, en una cohort de 24 xenògrafts derivats de tumor de pacient (PDX). Hem detectat una agregació d’alteracions posteriors a PI3K i amplificacions en l’11q13 entre els models resistents i les hem validat en models in vitro, incloent la sobreexpressió de ciclina D1 i FGFs. A més, 2 de 3 models PDX amb alteracions possiblement activadores d’mTORC1 han mostrat una eficàcia superior amb l’inhibidor d’mTORC1/2 (mTORC1/2inh) en comparació amb l’PI3Kinh. En la segona part d’aquesta tesi hem analitzat l’activitat antitumoral d’un FGFRinh, rogaratinib, en una cohort de 17 PDXs que contenen amplificacions en FGFR1/4 i/o FGF3/4/19 (l’amplicó 11q13). D’entre els potencials biomarcadors de resposta analitzats (nombre de còpies de gen de FGFR1-4, expressió gènica de FGFR1-4, i co-amplificació i expressió de lligands FGF), hem identificat que alts nivells de mRNA de FGFR1-4 prediuen la resposta a FGFRinh. Hem comparat l’eficàcia de rogaratinib amb un MTKI, lucitanib, on hem observat una millor eficàcia. A més, hem observat un elevat bloqueig de proliferació i vascularització amb lucitanib. Testant teràpia antiangiogènica en dos models resistents a rogaratinib, hem observat nivells similars d’eficàcia al lucitanib, suggerint que la resposta a lucitanib és deguda al bloqueig vascular en ambdós models. En conclusió, hem determinat que models que contenen alteracions activadores d’mTORC1 (e.g. TSC1del) són resistents a PI3Kinh però sensibilitzen a mTORC1/2inh i que la sobreexpressió de ciclina D1, FGF i ambdues alteracions generen resistència a PI3Kinh. Tanmateix, hem determinat que nivells alts d’mRNA de FGFR1-4 prediuen la resposta a FGFRinh i que els MTKI presenten millor eficàcia degut a un elevat bloqueig de la proliferació i la vasculatura. Creiem que aquests resultats ajudaran a millorar la selecció de pacients en futurs assajos clínics de teràpia dirigida a CM ER+.
Endocrine therapy has been one of the major advances in the treatment of breast cancer (BC) for the past 30 years. However, patients eventually relapse due to mechanisms of resistance. These involve several pathways related to proliferation and growth, including the PI3K/mTOR and FGFR pathways. The PI3K pathway is frequently altered in BC, especially in the luminal setting where 30-40% of patients harbor PIK3CA mutations. Specific inhibitors targeting key nodes in the PI3K pathway have been developed, such as mTOR and PI3K inhibitors (PI3Kinh). Clinical trials combining these inhibitors with endocrine therapy showed improved responses, leading to FDA approval of some of these inhibitors for metastatic ER+ BC. However, mechanisms of resistance are a major concern. In this sense, alterations downstream PI3K or in parallel pathways are recurrent in the metastatic disease, and it is important to understand if they confer resistance to PI3K inhibitors. Another common alteration in luminal BC is the amplification of the 11q13 amplicon, containing important genes for proliferation such as CCND1, FGF3/4/19, PAK1 and RPS6KB2, whose impact on PI3K sensitivity is unknown. FGFR1 amplification is found in 10% of metastatic luminal BC. Specific inhibitors against FGFR1/2/3 (FGFRinh) have shown limited efficacy in the FGFR1amp BC. This is in contrast with the clinical efficacy observed previously with Multi-targeted Tyrosine Kinase Inhibitors (MTKI, targeting FGFR, VEGFR and PDGFR families), albeit these agents showed severe adverse effects. In this study, we investigated mechanisms of resistance to PI3Kinh and biomarkers of response to FGFRinh. In the first part, we determined the antitumoral activity of alpelisib, an α-specific PI3Kinh, in a cohort of 24 genetically annotated Patient-Derived Xenografts (PDX). We observed an aggregation of genetic alterations downstream PI3K and 11q13 amplifications among the resistant models and validated these with in vitro models as mechanisms of resistance to alpelisib, including overexpression of cyclin D1 and high FGF-signaling. In addition, in 2 out of 3 PDX models with putative mTORC1 activating alterations, we observed higher efficacy of an mTORC1/2 inhibitor (mTORC1/2inh) compared with a PI3Kinh. In the second part of this thesis we analyzed the antitumor activity of an FGFRinh, rogaratinib, in a cohort of 17 PDX harboring amplifications in FGFR1/4 and/or FGF3/4/19 (11q13 amplicon). Amongst the potential biomarkers of response tested (FGFR1-4 gene copy number, FGFR1-4 gene expression, and FGF ligand co-amplification and expression), we identified that high mRNA of FGFR1-4 could predict for FGFRinh response. We compared rogaratinib efficacy with a MTKI, lucitanib, and we observed higher efficacy of lucitanib across 7 models. We further analyzed the mode of action of rogaratinib vs. lucitanib and we observed an increased blockade of proliferation and vascularization under lucitanib treatment. However, when testing an antiangiogenic therapy in two rogaratinib-resistant models, similar levels of efficacy as with lucitanib monotherapy were achieved, suggesting that lucitanib efficacy was due to the blockade of the vascularization in both models. In conclusion, we established that PDX models harboring genetic alterations consistent with mTORC1 activation (e.g. TSC1del) are resistant to PI3Kinh but sensitive to an mTORC1/2inh and that cyclin D1 overexpression, high FGF and both alterations together result in resistance to PI3Kinh. We further determined that high mRNA of FGFR1-4 predicts for FGFRinh response and that a MTKI exhibits higher efficacy than specific FGFRinh due to a higher blockade of proliferation and vascularization. These results will help for a better patient selection in future clinical trials for ER+ BC targeted therapy.

Acute lymphoblastic leukemia (ALL) is a hematologic malignancy characterized by the uncontrolled proliferation of lymphoblasts that accumulate in the blood, bone marrow and other organs. It represents 20% of adult acute leukemia and is the most common leukemia in children. The signal transduction pathway mediated by phosphatidylinositol 3-kinase (PI3K)/ Akt/mammalian target of rapamycin (mTOR) plays a key role in the regulation of important events for cells such as proliferation, differentiation and apoptosis, but also for the development of cancer and resistance to chemotherapy. In fact, the genes involved in the PI3K/Akt/mTOR pathway are often mutated and the activation signal mediated by these proteins is frequently altered in many cancer types, including ALL. Therefore, the components of this pathway are potential new targets for the development of innovative targeted therapies which use molecules that inhibit the key components of those signal transduction pathways with high specificity, and which have a central role in the oncogenesis process. The aim of this study is to evaluate the effects of small-molecule inhibitors (SMIs) on the PI3K/Akt/mTOR pathway using a panel of human leukemic cell lines and primary patient samples. Drugs directed against the Akt and mTOR proteins were used, administered either alone or in combination, to assess their synergistic effects on cells. In particular, MK-2206, GSK690693 and Perifosine are specific inhibitors of Akt, while RAD001 and CCI-779 are directed against mTORC1 and Torin-2 is directed against mTORC1/2. MK-2206, RAD001 and Torin-2 showed a specific cytotoxicity, inducing apoptosis and also caused cell cycle arrest in G0/G1 phase and autophagy both in cell lines and patient samples. They also down-regulated Akt and mTOR, as well as their downstream substrates. Moreover, potential synergies between drugs that hit at different levels of the PI3K/Akt/mTOR signal transduction pathway have been studied: a dual action on two targets, has been analyzed, using either MK-2206 or GSK690693 and RAD001 or CCI-779, that showed a synergistic effect, not observable with Torin-2. Also the efficacy of a triple hit on the same target, i.e. Akt, has been analyzed using MK-2206, GSK690693 and Perifosine: the three drugs displayed a synergistic effect that allowed to minimize drug concentrations. These results emphasize the increasing interest in studying pharmacological and personalized strategies for the development of new potential therapeutic protocols for cancer patients’ treatment, in order to overcome resistances and to improve clinical outcome.

The nuclear structme of 17Ne and 13O has been studied by the 20Ne(3He,6He)17Ne and 16O(3He,6He)13O reactions at 70 MeV and 80 MeV, respectively. Fifteen levels were identified, and angular distributions have been measured for nine of these levels in 17Ne, while for 13O eighteen levels were identified, but angular distributions were obtained for only ten levels. The observed transferred angular momentum dependence of these angular distributions allowed spin-parity assignments. The T= 3/2 quartet analog states in mass A=17 have been completed for six levels. The results of the isobaric multiplet mass equation analysis show a slight linear dependence of the b and c coefficients on the excitation energy. It was found that the coefficients for the positive parity states do not follow the systematics of the negative parity states. The absolute values of the b and c coefficients are larger for the positive parity states. An analysis in terms of Coulomb energy displacement indicates a possible configuration mixing or core polarization effect in these states. The d coefficient also has a large deviation from zero, only for the positive parity states indicating a possible expansion of the radial wavefunction or some isospin symmetry breaking effects. Further detailed theoretical interpretation of these effects may bring valuable information about the configuration and structure of these states. The leveis in 13O were measured with good energy resolution, and thus, it was possible to identify the first excited state unambiguously. However, if one identifies this state as the analog of the known first excited state in the mirror nucleus 13B, this leads to one of the largest level shifts known in literature.
The nuclear structme of 17Ne and 13O has been studied by the 20Ne(3He,6He)17Ne and 16O(3He,6He)13O reactions at 70 MeV and 80 MeV, respectively. Fifteen levels were identified, and angular distributions have been measured for nine of these levels in 17Ne, while for 13O eighteen levels were identified, but angular distributions were obtained for only ten levels. The observed transferred angular momentum dependence of these angular distributions allowed spin-parity assignments. The T= 3/2 quartet analog states in mass A=17 have been completed for six levels. The results of the isobaric multiplet mass equation analysis show a slight linear dependence of the b and c coefficients on the excitation energy. It was found that the coefficients for the positive parity states do not follow the systematics of the negative parity states. The absolute values of the b and c coefficients are larger for the positive parity states. An analysis in terms of Coulomb energy displacement indicates a possible configuration mixing or core polarization effect in these states. The d coefficient also has a large deviation from zero, only for the positive parity states indicating a possible expansion of the radial wavefunction or some isospin symmetry breaking effects. Further detailed theoretical interpretation of these effects may bring valuable information about the configuration and structure of these states. The leveis in 13O were measured with good energy resolution, and thus, it was possible to identify the first excited state unambiguously. However, if one identifies this state as the analog of the known first excited state in the mirror nucleus 13B, this leads to one of the largest level shifts known in literature.

El càncer de bufeta avançat s'associa amb un mal pronòstic i amb opcions limitades de tractament. Tot i l’èxit recent amb l'ús d'inhibidors immunitaris, no tots els pacients responen a la teràpia i encara hi ha la necessitat d'opcions alternatives. La ruta de PI3K/AKT/mTOR està sobreactivada sovint en aquesta patologia i pot ser un objectiu terapèutic potencial per a la intervenció terapèutica. Es va estudiar l'eficàcia antitumoral del TAK-228, un inhibidor oral de mTORC1 / 2 en models preclínics de càncer de bufeta amb alteracions en la transducció de senyals d'aquesta via. Es va demostrar una forta inhibició de la proliferació cel·lular in vitro, i la reducció del creixement del tumor in vivo. Es van analitzar possibles biomarcadors de resposta. Es van observar forts efectes sinèrgics amb la combinació de TAK-228 i TAK-117 (inhibidor de PI3Kα). El TAK-228 va mostrar una millor eficàcia quan es va combinar amb el paclitaxel. Es necessitaran estudis addicionals amb el TAK-228 per veure si aquesta eficàcia terapèutica preclínica es tradueix en benefici clínic per a pacients amb càncer de bufeta.
Advanced bladder cancer is associated with a poor prognosis and with limited treatment options. Despite of the recent success with the use of immune check-point inhibitors, not all patients will respond to therapy and there is still need for alternative options. PI3K/AKT/mTOR pathway is frequently activated in this pathology and can be a potential therapeutic target for treatment intervention. We studied the antitumor efficacy of TAK-228, an oral mTORC1/2 inhibitor in preclinical models of bladder cancer with signal transduction alterations of this pathway. We demonstrated strong inhibition of cell proliferation in vitro, and reduction of the tumor growth in vivo. Potential biomarkers of response are analysed. Strong synergistic effects were observed with the combination of TAK-228 and TAK-117 (PI3Kα inhibitor). TAK-228 did show improved efficacy when combined with paclitaxel. Further studies with TAK-228 will be needed to see if this preclinical therapeutic efficacy is translated into clinical benefit for selected bladder cancer patients.

Carvacrol is a phenol monoterpene found in aromatic plants specially in Lamiaceae family, which has been evaluated in an experimental model of breast cancer. However, any proposed mechanism based on its antitumor effect has not been reported. In our previous study, carvacrol showed a protective effect on 7,12-dimethylbenz[α]anthracene- (DMBA-) induced breast cancer in female rats. The main objective in this research was to evaluate by using in silico study the carvacrol on HER2, PI3Kα, mTOR, hERα, PR, and EGFR receptors involved in breast cancer progression by docking analysis, molecular dynamic, and drug-likeness evaluation. A multilevel computational study to evaluate the antitumor potential of carvacrol focusing on the main targets involved in the breast cancer was carried out. The in silico study starts with protein-ligand docking of carvacrol followed by ligand pathway calculations, molecular dynamic simulations, and molecular mechanics energies combined with the Poisson–Boltzmann (MM/PBSA) calculation of the free energy of binding for carvacrol. As result, the in silico study led to the identification of carvacrol with strong binding affinity on mTOR receptor. Additionally, in silico drug-likeness index for carvacrol showed a good predicted therapeutic profile of druggability. Our findings suggest that mTOR signaling pathway could be responsible for its preventive effect in the breast cancer.
Revisión por pares

The development of a deuterium-deuterium/ tritium-deuterium (D-D/ D-T) pulsed neutron generator based on the principle of the Plasma Immersion Ion Implantation (PI3) technique is presented, in terms of investigating development of a compact system to generate an ultra short burst of mono-energetic neutrons (of order 1010 per second) during a short period of time (<
20&mu
s) at repetition rates up to 1 kHz. The system will facilitate neutron detection techniques, such as neutron back-scattering, neutron radiography and time-of-flight activation analysis.

Aspects addressed in developing the system includes (a) characterizing the neutron spectra generated as a function of the target configuration/ design to ensure a sustained intense neutron flux for long periods of time, (b) the system was also characterised as a function of power supply operating conditions such as voltage, current, gas pressure and plasma density.

Le cancer du sein triple négatif (CSTN) représente 10-15% des cancers du sein. Son pronostic est sombre en particulier face à la rareté des thérapies ciblées adaptées à ce sous type. Sa complexité de prise en charge est directement liée à sa grande hétérogénéité tant au niveau moléculaire que morphologique.Dans ce contexte, nous avons développés des modèles de Patient Derived Xenograft (PDX) issus de CSTN. Ce modèle, robuste, a la particularité de retenir les caractéristiques (histologiques, génotypiques mais aussi phénotypiques) des tumeurs observées chez les patients.Dans notre cohorte de 61 PDX de CSTN, nous avons confirmé l’hétérogénéité anatomopathologique et génomique de ce sous type. Les différentes anomalies moléculaires mises en évidence sont de faible fréquence (<10%) mais 88% de nos modèles ont une altération potentiellement ciblables et plus de la moitié ont au moins 2 altérations ciblables. Nous nous sommes particulièrement intéressés à 2 sous types de CSTN : (i) le sous -type LAR (Luminal Androgen Receptor) dont nous avons décrit les premiers modèles de PDX : ces modèles présentent des altérations fréquentes de la voie PI3K ainsi que des réponses majeures aux inhibiteurs de cette voie ; (ii) le sous type métaplasique, dont 4 de nos 9 modèles présentent une double altération genomique dans les voies PI3K et RTK-MAPK ainsi que des réponses complètes et durables à la combinaison d’inhibiteurs de PI3K et de MAPK.Dans les autres sous-types de CSTN, nous avons également mis en évidence des taux de réponse importants aux inhibiteurs de la voie PI3K et MAPK. Les biomarqueurs de réponse à ces différentes thérapies ciblées testées sont en cours d’étude en particulier par intégration des données génomique et protéique de nos modèles
Triple negative breast cancer (TNBC) accounts for 10-15% of breast cancers. Its prognosis is worse, particularly due to the rarity of targeted therapies adapted to this subtype. Its complexity of management is directly related to its high heterogeneity, both at the morphological and genomical levels.In this context, we developed Patient Derived Xenograft (PDX) models from TNBC. This robust model has the specificity of retaining the characteristics (histological, genotypic but also phenotypic) of the tumors observed in patients.In our cohort of 61 PDXs of TNBC, we confirmed the anatomopathological and genomical heterogeneity of this subtype. Majority of targeted alterations are of low frequency (<10%) but 88% of our models harbour a potential targetable alteration and more than half have at least 2 targetable alterations. We were particularly interested in 2 subtypes of TNBC: (i) the LAR subtype for which we have described the first PDX models: these models present frequent alterations of the PI3K pathway as well as major responses to PI3K inhibitors; (ii) the metaplastic subtype, of which 4 of our 9 models show double alterations in the PI3K and RTK-MAPK pathways and complete and durable responses to the combination of PI3K-MAPK inhibitors.In the other CSTN subtypes, we have also demonstrated significant response rates to PI3K and MAPK inhibitors. Biomarkers of response to these various targeted therapies tested are being studied, in particular by integrating the genomic and protein data from a higher number of PDX models

Contexte. Le cancer bronchopulmonaire (CBP) demeure la première cause de mortalité par cancer dans le monde. Malgré l'espoir suscité par le développement des thérapies ciblées, son pronostic demeure sombre, particulièrement dans les cas de CBP à petites cellules (CBP-PC) et de CBP non à petites cellules (CBP-NPC) présentant une activation de l'oncogène KRAS. Matériel et Méthodes. Nous avons mené 3 études successives, visant à (i) radiosensibiliser des modèles de CBP-PC par l'ajout d'un inhibiteur de BCL2, (ii) cibler des modèles de CBP-NPC mutés KRAS par l'association d'un inhibiteur de mTOR et d'un inhibiteur de RAF, et (iii) créer un modèle préclinique orthotopique murin de CBP reproduisant la progression tumorale observée en clinique. Résultats. Dans la première étude, l'inhibiteur de BCL2 oblimersen a présenté un effet radiosensibilisant sur des modèles de CBP-PC, in vitro et in vivo. Dans la seconde étude, l'association de l'inhibiteur de mTOR everolimus et de l'inhibiteur de RAF/VEGFR RAF265 a présenté un effet synergique sur des lignées cellulaires de cancers présentant la double mutation de KRAS et de PIK3CA, in vitro et in vivo. Dans la troisième étude, l'injection orthotopique d'une lignée bioluminescente de CBP-NPC chez des souris nude a permis d'établir des tumeurs intra pulmonaires évoluant vers une extension métastatique ganglionnaire et hématogène, et de détecter la présence de cellules tumorales circulantes. Conclusion. L'association d'un inhibiteur de BCL2 à la radiothérapie est une stratégie intéressante dans le CBP-PC, l'association d'un inhibiteur de mTOR et d'un inhibiteur de RAF/VEGFR est une stratégie intéressante dans le CBP-NPC présentant une double mutation KRAS-PIK3CA, mais ces données doivent être confirmées sur des modèles orthotopiques afin de gagner en pertinence avant d'envisager un transfert en clinique.

碩士
長庚大學
生物醫學研究所
100
Chronic hepatitis B virus (HBV) infection has been identified as a major risk factor in hepatocellular carcinoma (HCC), which is one of the most common cancers worldwide. Evidence suggests that the HBV X protein (HBx) plays a crucial role in HCC development. Genome-wide analysis identified earlier that the receptor for hyaluronan-mediated motility (RHAMM) represents a putative oncogene overexpressed in many human cancers, especially HCC. However, the role of RHAMM in establishment of tumorigenesis and chemoresistance in HCC is still unclear. This study presents our finding that HBx upregulates the expression of RHAMM through PI3K/Akt/C/EBPβ pathway, and its implication in cancer motogenicity and target therapy. First﹐upregulation of RHAMM expression by HBx was discovered in HCC cells through the PI3K/Akt pathway. Furthermore, departure of C/EBPβ from RHAMM gene promoter by HBx was critical for the RHAMM upregulation as revealed by RNAi and ChIP assays. By scratch assay, we also found that knock-down RHAMM partly impaired motogenicity and, surprisingly, partly reversed growth inhibition by mitotoxin drugs (Taxol, Vincristine) in HCC cells. Nevertheless, altered expression of RHAMM by HBx did affect these drugs-induced apoptosis. Using public dataset of HCC patients, we also found enhanced RHAMM mRNA in the patients of poor prognosis. Our findings uncover the molecular mechanism that underlies the upregulation of RHAMM in HBx-positive HCC cells. These results also provide an explanation for cell motogenicity and cancer metastasis of HBV-positive HCC, and the potential relevance of RHAMM in target therapy as well.

During my PhD in Clinical and Experimental Oncology at the University of Florence, I focused on the molecular underpinnings in Philadelphia-negative Myeloproliferative neoplasms (MPNs). MPNs, including Essential Thrombocythemia (ET), Polycythemia Vera (PV) and Primary Myelofibrosis (PMF), are characterized by clonal proliferation of hematopoietic progenitor cells and dysregulated production of blood cells of different lineages. MPNs are characterized by occurrence of driver mutations in JAK2V617F, CALR and MPL. The exciting discovery of new genetic abnormalities in patients with MPNs, occurred in the last decade, holds the promise for advancing our understanding of the pathogenesis of these disorders as well as improving the therapeutic management of patients. In my thesis, I will present the results of two research topics. In the first project I investigated the molecular basis of the functional crosstalk between the main signalling pathways activated by JAK2 mutation, that include the JAK/STAT and PI3K/mTOR pathways, as well how to exploit the added value of target drug combination aimed at improving efficacy, and ameliorating toxicity, of treatments in MPNs. As regard the second project, I focused on the most recently described MPN-associated driver mutations in CALR, whose function and downstream targets are still poorly known. To this end, I generated new cellular models of CALR mutations using target genomic editing and analysed the effects of mutations on cellular phenotypic traits.

Introduction: One of the main obstacle to the successful development of therapeutic strategies remains the identification of biomarker underlying drug resistance. Recently, investigators have become more aware the role of the tumor microenvironment (TME) in cancer and the potential therapeutic opportunities that derive from suppressing potential resistance mechanisms arising microenvironmental interactions. The aim of this study was to set-up multicellular culture models to uncover the molecular mechanisms by which stromal/endothelial cells modulate response to signaling inhibitors and to identify potential therapeutic targets in PTEN-loss contexts. Methods and Materials: Isogenic CRC cell lines (X-MAN™ HCT116 and HCT116 PTEN-/-) were treated with MAPKi and PI3K/mTORi alone or in combination, in the presence or absence of stromal fibroblasts or fibroblast/endothelial cell conditioned medium (CM). Cytofluorimetric analysis and Crystal Violet assay were used to analyse functional response to targeted agents; pathways activation and cytokine/chemokine profile were analysed using Western blot and ELISA assay respectively. Results and Discussion: In co-culture CRC models, the response to MAPK and PI3K inhibitors is the result of interaction between tumor cells and their surrounding stroma. The response to PI3K/mTORi is mainly influenced by microenvironmental interactions: direct cell-to-cell tumor/stroma contact renders stromal cells resistant to PI3K/mTORi, while the presence of stromal cell-derived soluble factors sensitizes PTEN-competent CRC cells to PI3K/mTORi-mediated growth inhibition. This effect was confirmed using CM from different types of stromal cells (fibroblast/endothelial) that similarly affected the response of CRC cell lines to signalling inhibitors; this is probably due to similar profile of cytokine/chemokine production in stromal cell and is subjected to a “saturation” effect. The presence of stromal CM upregulates MAPK activation regardless of PTEN status, whereas mTOR pathway upregulation is observed mainly in PTEN-competent CRC cellsin PTEN-competent cells soluble factors released by stromal elements paradoxically impair PTEN function, leading to downstream mTORC1 complex formation and pathway activation. This paradoxical mTORC1 activation upon exposure to stroma-derived soluble factors results in functional hypersensitivity of PTEN-competent CRC cells to the growth inhibitory effects of double PI3K/mTOR inhibitors. . Conclusions: The presence of stromal cells (fibroblasts/endothelium) profoundly influences CRC response to PI3K/mTOR-targeting agents. Understanding the mechanisms underlying microenvironmental interactions (tumor, stroma, soluble factors) may be of fundamental importance to overcome therapeutic resistance and develop more effective therapies for patients affected by cancer.

The PI3K-PKB/Akt-PTEN signal transduction pathway orchestrates a variety of fundamental cell processes and its deregulation is implicated in several human diseases, including cancer. While the importance of this pathway to many cellular functions is well established, the mechanisms leading to context-specific physiological outcomes in response to a variety of stimuli remain largely unknown. Spatial restriction of signaling events is one of the means to coordinate specific cellular responses. To investigate the subcellular locus-specific roles of the major PI3K effector PKB/Akt in various cell processes, I have devised a novel experimental system employing cellular compartment-directed PKB/Akt pseudosubstrate inhibitors. The work herein describes the development and characterization of the localized PKB/Akt pseudosubstrate inhibitor system and its application to investigate potential locus-specific functions in established PKB/Akt-regulated cellular processes. Subcellular compartment-restricted PKB/Akt inhibition in the 3T3L1 adipocyte differentiation model revealed that nuclear and plasma membrane, but not cytoplasmic, PKB/Akt activity is required for terminal adipocyte differentiation. Nuclear and plasma membrane pools of PKB/Akt were found to contribute to distinct stages of adipocyte differentiation, revealing that PKB/Akt activity impacts multiple points of this program. The localized PKB/Akt pseudosubstrate inhibitor system was also utilized to investigate the importance of distinct subcellular pools of PKB/Akt in breast epithelial cells. MCF-10A human breast epithelial cells can be grown in three-dimensional culture to form acinar structures that recapitulate in vivo mammary glandular architecture. Expression of the plasma membrane PKB/Akt inhibitor during cell growth in three-dimensional culture severely impaired acinar formation. On the other hand, expression of the nuclear PKB/Akt inhibitor during acinar development resulted in the formation of large, misshapen, multi-acinar structures. Assessment of the migratory capacity of MCF-10A cells upon localized PKB/Akt inhibition revealed that nuclear PKB/Akt inhibition promoted, while plasma membrane PKB/Akt inhibition impaired, MCF-10A cell migration. The development of locus-specific PKB/Akt inhibitors represents the first attempt to prioritize the targets of this kinase based on their subcellular localization. This work and its immediate extensions will further our understanding of the biology of PKB/Akt, a multi-tasking kinase with profound roles in development, cellular and organismal homeostasis and disease.

The PI3-kinase/AKT/mTOR pathway plays a central role in cancer signaling. While p110α is the catalytic α-subunit of PI3-kinase and a major drug target, PTEN is the main negative regulator of the PI3-kinase/AKT/mTOR pathway. PTEN and p110α protein expression in tumors is commonly analyzed by immunohistochemistry, which suffers from poor multiplexing capacity, poor standardization, and antibody cross-reactivity, and which provides only semi-quantitative data. Here, we present an automated, and standardized immuno-matrix-assisted laser desorption/ionization mass spectrometry (iMALDI) assay that allows precise and multiplexed quantitation of PTEN and p110α concentrations, without the limitations of immunohistochemistry. IMALDI, which combines immuno-enrichment with analysis using a benchtop MALDI-Time-of-Flight (TOF) mass spectrometer, is an especially well-suited method for translating mass-spectrometry based assays into the clinical lab. We systematically optimized the iMALDI workflow regarding sensitivity, robustness, and throughput while developing highly flexible automation protocols using a Bravo 96LT liquid handling robot. We further developed custom R scripts to improve data visualization and analysis. One hour digestion using a protein to trypsin ratio of 1:2, followed by direct immuno-enrichment for 1 h yielded high and consistent peptide recoveries. We demonstrated that the PTEN and p110α iMALDI assays can be multiplexed using both simultaneous and sequential enrichment, reducing the amount of required sample material as well as simplifying the workflow. The PTEN+p110α iMALDI assay was validated and demonstrated high accuracy for both target proteins (90-112% recovery of known spiked-in concentrations) as well as high precision and 5-day reproducibility (overall CVs of 9%) across the linear range of the assay (0.6 to 20 fmol). Lower limits of quantitation below 1 fmol were achieved. Endogenous PTEN and p110α were quantified in cell lines as well as fresh-frozen tumor tissue samples. A novel two-point internal calibration strategy (2-PIC) was developed, based on spiking two peptide isotopologues into the sample as internal standards, avoiding the need for an external calibration. We quantified endogenous PTEN in a Colo-205 cell line using the PTEN iMALDI assay, as well an orthogonal PTEN immuno-multiple reaction monitoring (immuno-MRM) method to demonstrate this technique. Excellent agreement was shown between both calibration approaches (residual standard deviation between 2-PIC and external calibration of 1.6-5.8%), as well as high correlation between PTEN iMALDI and PTEN immuno-MRM (R²= 0.9966) and good agreement between quantified amounts (0.48±0.01 and 0.29±0.02 fmol/µg of total protein). Finally, we analysed a set of patient samples from a AKT inhibitor AZD5363 drug trial using a multi-site workflow combining the developed PTEN+p110α assay with established AKT1+AKT2 iMALDI assays and untargeted proteomics. We demonstrated how the combination of targeted and untargeted proteomics approaches may be used to gain novel insights into the tumor biology of patient tissue samples. Further, we showed that the PTEN iMALDI assay has good correlation with a comparable immunohistochemistry method (R²=0.86), and that our assays can be further multiplexed, reducing the required amount sample material. Thus, we showed that iMALDI is promising tool for biomarker quantitation.
Graduate
2022-08-12

Dissertação de Mestrado em Biotecnologia Farmacêutica apresentada à Faculdade de Farmácia
O cancro é, atualmente, a doença responsável pela segunda maior taxa de mortalidade a nível mundial. Entre todas as doenças oncológicas, o cancro do pulmão é o mais frequente e o mais fatal, por isso, representa uma necessidade médica relevante.Embora existam estratégias terapêuticas inovadoras para cancro do pulmão introduzidas recentemente, como os inibidores de tirosina cinase e os agentes anti-angiogénicos, a sua eficácia não é isenta de fenómenos de resistência. Para além disso, a quimioterapia convencional recorrendo a fármacos com elevado potencial citotóxico, quer individualmente ou em combinação, embora muitas vezes eficaz, também apresenta limitações: a toxicidade em tecidos saudáveis (responsável por efeitos secundários nefastos para o doente) ou a dificuldade em fazer chegar rácios farmacologicamente vantajosos ao tumor (devido às suas diferentes características farmacocinéticas). Neste contexto, torna-se relevante o desenvolvimento de novas estratégias terapêuticas que simultaneamente ultrapassem a resistência a fármacos inerente à heterogeneidade tumoral, atingindo diferentes populações celulares do tumor, e reduzam os efeitos secundários sobre tecidos saudáveis.O presente trabalho teve como objetivo avaliar o potencial citotóxico de uma nova estratégia terapêutica contra o cancro do pulmão de não-pequenas células (CPNPC), o subtipo mais frequente de cancro do pulmão. A estratégia consiste em lipossomas sensíveis ao pH, PEGuilados, contendo combinações de doxorrubicina (inibidor da topoisomerase II) e ceramida-C6 (inibidor pró-apoptótico da via PI3K/Akt) e direcionados, pelo peptídeo F3, para a nucleolina, uma proteína que se encontra sobreexpressa na membrana celular das células tumorais e endoteliais do microambiente tumoral. Neste estudo, demonstrou-se que, através do ajuste rigoroso das condições de pH, a produção de lipossomas sensíveis a pH, contendo ceramida-C6 na bicamada lipídica, é exequível em condições acídicas moderadas sem interferir com as suas propriedades físicas básicas. Além disso, foi demonstrado que lipossomas funcionalizados com o peptídeo F3 se associam, pelo menos, 51 vezes mais com as células de CPNPC em comparação com os não direcionados. Apesar desta observação, os lipossomas funcionalizados com peptídeo F3 encapsulando uma combinação de doxorrubicina e ceramida-C6 não permitiram um aumento da citotoxicidade contra as linhas celulares de CPNPC (A549 e H1975), ao contrário do que se observou anteriormente em cancro da mama triplo negativo. Esta observação correlacionou-se com um impacto reduzido na desregulação dos níveis de fosforilação de Akt e, consequentemente, na inibição da cascata de sinalização a esta associada. Por outro lado, as células de CPNPC exibiram uma maior ativação basal de Akt em comparação com as linhas celulares de cancro da mama, o que as favoreceu em termos de resistência à ação da ceramida-C6. De um modo geral, o conhecimento gerado no presente trabalho sugere que o potencial citotóxico da ceramida-C6, em combinação com doxorrubicina e direcionada para a nucleolina, pode depender da ativação constitutiva da via de sinalização PI3K/Akt em células de diferentes origens histológicas, independentemente dos níveis de nucleolina na superfície celular e consequente internalização das nanopartículas.
Globally, cancer is the second leading cause of mortality and, among all malignant neoplasms, lung cancer is the most common and fatal disease, thereby representing an urgent medical need.Despite the development of innovative therapeutic approaches such as tyrosine kinase inhibitors and anti-angiogenic therapies, their efficacy is restricted by the emergence of drug resistance phenomena in lung cancer. Moreover, although often effective, standard chemotherapy using potent cytotoxic drugs, as single or combined agents, presents limitations. The undesired toxicity to healthy tissues (causing serious side effects) and the difficulty to delivery synergistic ratios of drug combinations to the tumor (due to the differences in the pharmacokinetics properties of the drugs) are some of the major challenges associated with standard chemotherapy strategies. In this context, it is relevant to develop innovative therapeutic approaches capable of bypassing drug resistance mechanisms, by affecting diverse cellular populations in the tumor microenvironment, while reducing the side effects in normal tissues.The present work aimed at evaluating the cytotoxic potential of a new therapeutic strategy towards non-small cell lung cancer (NSCLC), the most frequent subtype of lung cancer. The strategy is based on PEGylated pH-sensitive liposomes, containing combinations of doxorubicin (topoisomerase II inhibitor) and C6-ceramide (pro-apoptotic inhibitor of the PI3K/Akt pathway), and targeted by the F3 peptide to nucleolin, a protein which is overexpressed in the cell membrane of cancer and endothelial cells of the tumor microenvironment. Herein, it was demonstrated that fine tuning of pH conditions enables the manufacturing of pH-sensitive liposomes containing C6-ceramide in the lipid bilayer in mildly acidic conditions, without impact on basic physical properties. Furthermore, it was demonstrated that F3 peptide-targeted liposomes associated, at least, 51-fold higher with NSCLC cells, compared to the non-targeted counterpart. Despite this observation, the F3 peptide-targeted liposomes containing a combination of doxorubicin and C6-ceramide did not enable an increase in cytotoxicity against A549 and H1975 NSCLC cell lines, in contrast with previous observation in triple negative breast cancer. This observation correlated with the limited impact on the deregulation of phosphorylated Akt (p-Akt) levels, and thus consequent downregulation of the associated signaling pathway. Alongside, data demonstrated NSCLC cell presented higher basal Akt activation compared with breast cancer cell lines, favoring the former in terms of resistance to C6-ceramide action. Overall, the data generated herein suggested that the cytotoxic potential of C6-ceramide, in combination with doxorubicin, and targeted to nucleolin may depend on the constitutive activation of PI3K/Akt signaling pathway across histological origins, and independently of the nucleolin levels at cell surface and consequent internalization of the nanoparticles.
Outro - This work was supported by the European Regional Development Fund (ERDF), through the COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation and Portuguese national funds via FCT – Fundação para a Ciência e a Tecnologia, I.P., under projects Cancel Stem (reference POCI-01-0145-FEDER-016390), CENTRO-01-0145-FEDER-000012 (HealthyAging2020), Euronanomed 2 (FCT reference ENMed/0005/2015)" and CNC.IBILI (FCT reference UID/NEU/04539/2019).

Squamous cell lung cancer (SQLC) is the second most prevalent histologic type of lung cancer and accounting for approximately 30% of newly diagnosed non-small cell lung cancer (NSCLC) cases. Systemic treatments for SQLC patients include cytotoxic chemotherapy and immune-oncology approaches. In contrast with lung adenocarcinoma, which is the other main subtype of NSCLC, no patient-tailored treatments are available so far for SQLC. Accumulating evidence suggests that the PI3K/mTOR axis is one of the most frequently altered pathways in SQLC. However, despite a plethora of clinical trials with numerous PI3K/mTOR targeted inhibitors, no significant increase in patients’ survival has been observed as compared to standard treatment options. A possible explanation for the outcome of those clinical trials might be the lack of reliable predictive biomarkers for better patients’ stratification. We and others have reported Rictor copy number gain (CNG) in a set of SQLC patients by performing targeted DNA sequencing on archival tissues. Another group has suggested the existence of Rictor focal amplification in subsets of lung cancers, including SQLC, and further suggested Rictor as a potential predictive biomarker of response to targeted therapy. However, no conclusive data were presented to show that Rictor amplification is driving activation of the PI3K/mTOR pathway in SQLC cells or representing a valid biomarker predictive of response to targeted inhibition of the pathway. Here, we used three different SQLC cell lines and 60 tissue specimens to show that CNG of Rictor is a recurrent event in SQLC, yet this is due to the polysomy of the short arm of chromosome 5 rather than to focal amplification. All three cell lines tested showed different Rictor CNG and different levels of its transcript and protein. In particular, the SQLC cell line harboring the higher CNG (H-1869) accordingly displayed higher level of Rictor protein. Therefore, we sought to test the possibility that the dosage of Rictor might affect the activation of PI3K/mTOR pathway and sensitivity towards its targeting agents. Unexpectedly, we found that Rictor levels did not parallel the biochemical activation of the pathway nor the sensitivity to dual mTORC1/C2 or PI3K/mTOR inhibitions. These observations were confirmed by genetic perturbation analysis, as reduction of Rictor levels through RNA interference did not lead neither to reduced cell viability nor to significant changes in drug sensitivity in the two cell lines tested. Overall, our findings suggest that Rictor does not represent a predictive biomarker of response towards PI3K/mTOR directed therapy.

Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer, with a worldwide incidence of 275,000 new cases annually (Warnakulasuriya, 2009). Globally, the head and neck carcinoma represents a major cause of morbidity and mortality and is the sixth most commonly occurring cancer (Warnakulasuriya, 2009). A majority (>90%) of the head and neck cancers are squamous in origin and thus are linguistically referred to as head and neck squamous cell carcinoma (HNSCC) (Warnakulasuriya, 2009). HNSCC includes cancers of the oral cavity, larynx and pharynx; oral cancer being the most common (Warnakulasuriya, 2009). Although, HNSCC is the sixth most common cancer globally (Warnakulasuriya, 2009), the Indian scenario is graver. According to GLOBOCAN 2008 (http://globocan.iarc.fr), the worldwide age standardized incidence rate (ASR) for HNSCC (and thus OSCC) is 5.3 and 2.5 per 100,000 males and females respectively (Ferlay et al., 2010). In India, the ASR is 9.8 and 5.2 per 100,000 males and females respectively, clearly demonstrating a remarkably high incidence rate of OSCC (Ferlay et al., 2010; http://globocan.iarc.fr). OSCC is a peculiar cancer which is largely preventable and rarely presents as a familial disorder. The most common etiological factors associated with OSCC include tobacco and alcohol consumption (Johnson, 2001). Additionally, high risk human papillomaviruses (HPV strains 16 and 18) as well as genetic predispositions have been implicated. The treatment of OSCC mainly relies on surgical resection of the tumor. The site, size, depth of infiltration and proximity to the bone of the tumor determine whether a combination of surgery with radiation therapy or chemotherapy would be advised (Scully and Bagan, 2009). The concomitant chemo-radiation therapy is the most commonly used strategy in locally advanced cancer. Taxanes (e.g., paclitaxel and docetaxel) and platinum-based induction chemotherapy (e.g., cisplatin) are the options in the treatment of locally advanced cancer. Epidermal growth factor receptor (EGFR) targeted with cetuximab in combination with radiotherapy has been successfully tested in a large randomized trial and thus is currently a new option (Scully and Bagan, 2009). The success of cetuximab has paved the path for the development and implementation of molecules targeting various signaling pathways. Despite extensive research on oral squamous cell carcinoma (OSCC), the five-year survival rate has not changed in several decades with the exception of the targeted treatment strategies involving cetuximab as discussed above. The current chemotherapeutic approaches lack selectivity and are flagitious. Thus, effective treatment of OSCC requires the identification of molecular targets to design appropriate therapeutic strategies. To this end, the present study took three distinct approaches in order to validate the use of existing targets and to reveal novel prognostic biomarkers and therapeutic targets. 1) Targeting the PI3K-AKT-MTOR pathway in OSCC and identification of determinants of its sensitivity. 2) Gene expression analysis of ectopically overexpressed TSC2 to identify new therapeutic targets and prognostic biomarkers as well as to elucidate the genes regulated by it. 3) Expression profiling of CYP1B1 in order to validate the use of CYP1B1 based prodrug therapy in OSCC. Investigations pertaining to the changes in gene and protein expression profiles in malignant as well as pre-malignant lesions have documented the deregulation of the PI3K-AKT-MTOR (phosphoinositide 3-kinase-AKT-mechanistic target of rapamycin) and EGFR (epidermal growth factor receptor) pathways in OSCC which are being widely targeted in many therapeutic strategies (Molinolo et al., 2007; Chakraborty et al., 2008; Matta and Ralhan, 2009; Molinolo et al., 2009; Stransky et al., 2011). The PI3K-AKT-MTOR pathway is a central hub for controlling cellular proliferation and growth in response to various intracellular as well as extracellular stimuli. Crucial signaling cascades including WNT, RAS, HIF-1α and AMPK cross-talk with the PI3K-AKT-MTOR pathway at a variety of molecular junctions. Thus, making this pathway sensitive to perceiving various growth modulatory conditions, ranging from the presence of growth factors to hypoxia and nutrient deprivation (Sengupta et al., 2010; Yang and Guan, 2007). The aberrant expression of the PI3K-AKT-MTOR pathway in OSCC advocated the targeting of this coveted pathway (Chakraborty et al., 2008). In various cancers, the monotherapeutic treatments with inhibitors like LY294002 (PI3K inhibitor) and rapamycin (MTOR inhibitor) demonstrated reduced efficacies. Such reduced efficacies were attributed to the drug toxicity and non-specific action of LY294002 (Davies et al., 2000; Sun et al., 2005; Ikezoe et al., 2007; Wang et al., 2008; Liu et al., 2009), or the ablation of a feedback inhibition loop leading to the reactivation of the PI3K-AKT-MTOR pathway by rapamycin (O'Reilly et al., 2006; Carracedo et al., 2008). Thus, rapamycin or its analogues demonstrated mediocre efficacy due to cytostatic effects in clinical trials, primarily due to the paradoxical activation of major survival kinases namely MAPK and AKT (O'Reilly et al., 2006; Carracedo et al., 2008). The present study aimed at increasing the efficacy of these drugs by incorporating a combinatorial approach. The MTT assay demonstrated that prolonged monotherapeutic treatments with rapamycin led to a modest growth inhibition in three OSCC (KB, SCC131 and SCC084) and HeLa cell lines. Western blot analysis of the phosphorylation status of AKT and RPS6KB1 revealed that monotherapeutic treatments with rapamycin for 96 hr led to the reactivation of the PI3K-AKT-MTOR pathway. Thus, the modest growth inhibitory effect of rapamycin was attributed to the reactivation of the PI3K-AKT-MTOR pathway. A combinatorial treatment approach was hence believed to circumvent this problem in order to increase the efficacy of targeting the PI3K-AKT-MTOR pathway. The PI3K inhibitor LY294002 was used combinatorially with rapamycin. This prolonged dual combinatorial treatment regime was distinctly more efficacious than either of the drugs alone and led to a reduction in cellular viability accompanied by increased sub-G1 population, indicating marked cell death that was characterized as caspase-3 dependent apoptosis. The differential sensitivity of the cell lines towards this combinatorial treatment revealed a novel determinant of the sensitivity, the transactivation of EGFR. The cell lines (SCC131 and SCC084) that were capable of transactivating EGFR were relatively resistant to the dual targeting of PI3K and MTOR in comparison to cell lines that did not transactivate EGFR (HeLa and KB). Further, targeting PI3K, MTOR and EGFR simultaneously was more efficacious in the presence of EGFR transactivation than dually targeting PI3K and MTOR. The results conclusively proved that the combinatorial therapeutic approach dually targeting PI3K and MTOR is a promising treatment strategy as compared to a monotherapeutic treatment and a major factor determining the sensitivity towards this treatment is the status of autophosphorylation of EGFR (Tyr1173) which governs the potential for EGFR transactivation by the combinatorial treatment. Thus, this study demonstrated that the status of EGFR autophosphorylation (Tyr1173) can be used as a biomarker to predict the sensitivity towards the combinatorial targeting of PI3K and MTOR in OSCC. The PI3K-AKT-MTOR pathway is negatively regulated by TSC2 (tuberous sclerosis complex 2; tuberin) (Tee et al., 2002). The importance of the TSC2 gene in the regulation of cell growth and proliferation is irrefutable. TSC2 facilitates the crosstalk between a variety of cellular signals, making it a crucial hub where many cellular networks integrate like AKT, MAPK and AMPK (Clements et al., 2007; Rosner et al., 2007; Rosner et al., 2008). It is a tumor suppressor gene and is downregulated in many cancers including OSCC (Chakraborty et al., 2008). In order to identify the genes regulated by TSC2 in OSCC, we stably overexpressed TSC2 in KB cells and the changes in the gene expression profiles caused by this ectopic overexpression were observed using a whole genome expression microarray. The results showed differential regulation of 268 genes (107 genes were upregulated and 161 genes were downregulated, p<0.05, fold change ≥ 1.5). A majority of these genes were functionally associated with transcription, cell growth and proliferation, apoptosis, cell cycle and neurogenesis. Functional annotation and network analysis was performed by using the DAVID v6.7 and IPA version 8.7 softwares. The microarray data revealed a novel aspect in the crosstalk between WNT signaling and TSC2, namely the transcriptional regulation of WNT signaling by TSC2. Further, in the context of therapeutic applications, the microarray analysis revealed multiple genes that were functionally categorized to be involved in response to radiation, UV and drugs (e.g., SERPINB13 and IL1B). Future studies on the regulation of such genes that are involved in responses to drugs and radiation may give insights into the role of TSC2 in resistance or sensitivity towards chemotherapy and radiation therapy. Moreover, EREG, a member of the epidermal growth factor family, was found to be the most downregulated gene in the microarray analysis. Previous reports have documented elevated levels of EREG in tuberous sclerosis lesions and its association with poor clinical prognosis in OSCC patients (Li et al., 2008; Shigeishi et al., 2008), making its regulatory aspects intriguing. Additionally, published data on the transcriptional functions of TSC2 instigated us to analyze the role of TSC2 in the regulation of EREG. TSC2 has been shown to modulate the transcription mediated by members of the steroid receptor superfamily of genes (Henry et al., 1998) and was shown to bind specifically to ERα and inhibit estrogen induced proliferation (Finlay et al., 2004). Also, TSC2 has been shown to possess C-terminal transcriptional activation domains (Tsuchiya et al., 1996). We have therefore attempted to investigate the transcription related functional aspects of TSC2 by exploiting the observed transcriptional repression of EREG. The physiological roles of TSC1 and TSC2 that are independent of the PI3K-AKT-MTOR pathway have been termed as ‘non-canonical’ (Neuman and Henske, 2011). The repression of EREG by TSC2 was observed to be insensitive to rapamycin, suggesting that it was independent of MTORC1 and thus a non-canonical function of TSC2. To determine whether the repression in EREG was at the level of the promoter, we performed a dual luciferase reporter assay. The results showed that the EREG promoter was repressed by stable as well as transient overexpression of TSC2. In order to elucidate the mechanism of transcriptional regulation by TSC2, we performed the ChIP analysis to observe the in vivo binding of TSC2 to the EREG promoter. In the ChIP analysis with the anti-TSC2 antibody, we observed that TSC2 did not bind to the EREG promoter between the regions -857 bp to -302 bp or -325 bp to +165 bp. Further, in silico analysis revealed an interesting trend among the transcription factors that were differentially regulated by TSC2 and had putative binding sites on the EREG promoter. A majority of these transcription factors (17/21) were downregulated by the overexpression of TSC2. This observation suggested that the repression of EREG could be an indirect effect due to repression of transcription factors caused by overexpression of TSC2. On the whole, this study revealed novel functions of TSC2 in OSCC with implications in determining novel biomarkers and therapeutic targets. As discussed previously, OSCC has a very flagitious treatment regime. A prodrug approach is thought to aid in targeting chemotherapy (Rooseboom et al., 2004). CYP1B1, a member of the cytochrome P450 family, has been implicated in chemical carcinogenesis (Bandiera et al., 2005; Sliwinski et al., 2010). There exists a general accordance that this protein is overexpressed in a variety of cancers (e.g., colon, lung, renal, bladder, prostate, breast, endometrial and esophageal cancers), making it an ideal candidate for a prodrug therapy (McFadyen et al., 1999; Murray et al., 2001; McFadyen et al., 2004; Sissung et al., 2006; Wen and Walle, 2007; Sliwinski et al., 2010). The activation of the prodrug facilitated by CYP1B1 would enable the targeting of chemotherapy to tumor tissues in which CYP1B1 is specifically overexpressed as a result reducing the non-specific side effects that the current chemotherapy elicits (Rooseboom et al., 2004). This study was aimed at validating the use of CYP1B1 as a target for the prodrug therapy in OSCC. The expression profile of CYP1B1 was analysed in a panel of 51 OSCC tumors, their corresponding normal tissues, an epithelial dysplasia lesion and its matched normal tissue by qRT-PCR, Western blotting and Immunohistochemistry. Counterintuitively, CYP1B1 was found to be downregulated in 77.78% (28/36) tumor tissues in comparison to their corresponding normal tissues as well as in the epithelial dysplasia lesion compared to its matched normal tissue at the transcriptional level, and in 92.86% (26/28) of tumor tissues at the protein level. This clearly demonstrated the downregulation of CYP1B1 at the transcriptional and translational levels in tumor tissues in comparison to their corresponding normal tissues. These observations indicate that caution should be observed as this therapy may not be applicable universally to all cancers. Since CYP1B1 has been shown to be involved in the activation of pro-carcinogens (Murray et al., 2001; Bandiera et al., 2005; Sissung et al., 2006), its inhibition could facilitate the development of a prophylactic therapy for oral cancer. Overall, this study has identified the transactivation of EGFR as a determinant of sensitivity towards combinatorial targeting of PI3K and MTOR in OSCC and has demonstrated that the autophosphorylation of EGFR (Tyr1173) can be used as a marker to judge the sensitivity towards this treatment. In the clinical perspective, the identification of such markers would aid in predicting the efficacy of targeted therapies. Such investigations would enable the strategic treatment of OSCC patients, thus decreasing the time lost in trial and errors for determining the appropriate treatment. Additionally, this study elucidated a novel role of TSC2 in the transcriptional repression of EREG, a prognostic biomarker for OSCC. Further, the study revealed potential prognostic biomarkers as well as therapeutic targets that are regulated by TSC2 by using a whole genome expression microarray. Moreover, the counterintuitive downregulation of CYP1B1 in OSCC tumors suggested the possibility of a prophylactic therapy for oral cancer but also advised a precautionary note for the application of prodrug treatments based on CYP1B1 overexpression in OSCC.

Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer, with a worldwide incidence of 275,000 new cases annually (Warnakulasuriya, 2009). Globally, the head and neck carcinoma represents a major cause of morbidity and mortality and is the sixth most commonly occurring cancer (Warnakulasuriya, 2009). A majority (>90%) of the head and neck cancers are squamous in origin and thus are linguistically referred to as head and neck squamous cell carcinoma (HNSCC) (Warnakulasuriya, 2009). HNSCC includes cancers of the oral cavity, larynx and pharynx; oral cancer being the most common (Warnakulasuriya, 2009). Although, HNSCC is the sixth most common cancer globally (Warnakulasuriya, 2009), the Indian scenario is graver. According to GLOBOCAN 2008 (http://globocan.iarc.fr), the worldwide age standardized incidence rate (ASR) for HNSCC (and thus OSCC) is 5.3 and 2.5 per 100,000 males and females respectively (Ferlay et al., 2010). In India, the ASR is 9.8 and 5.2 per 100,000 males and females respectively, clearly demonstrating a remarkably high incidence rate of OSCC (Ferlay et al., 2010; http://globocan.iarc.fr). OSCC is a peculiar cancer which is largely preventable and rarely presents as a familial disorder. The most common etiological factors associated with OSCC include tobacco and alcohol consumption (Johnson, 2001). Additionally, high risk human papillomaviruses (HPV strains 16 and 18) as well as genetic predispositions have been implicated. The treatment of OSCC mainly relies on surgical resection of the tumor. The site, size, depth of infiltration and proximity to the bone of the tumor determine whether a combination of surgery with radiation therapy or chemotherapy would be advised (Scully and Bagan, 2009). The concomitant chemo-radiation therapy is the most commonly used strategy in locally advanced cancer. Taxanes (e.g., paclitaxel and docetaxel) and platinum-based induction chemotherapy (e.g., cisplatin) are the options in the treatment of locally advanced cancer. Epidermal growth factor receptor (EGFR) targeted with cetuximab in combination with radiotherapy has been successfully tested in a large randomized trial and thus is currently a new option (Scully and Bagan, 2009). The success of cetuximab has paved the path for the development and implementation of molecules targeting various signaling pathways. Despite extensive research on oral squamous cell carcinoma (OSCC), the five-year survival rate has not changed in several decades with the exception of the targeted treatment strategies involving cetuximab as discussed above. The current chemotherapeutic approaches lack selectivity and are flagitious. Thus, effective treatment of OSCC requires the identification of molecular targets to design appropriate therapeutic strategies. To this end, the present study took three distinct approaches in order to validate the use of existing targets and to reveal novel prognostic biomarkers and therapeutic targets. 1) Targeting the PI3K-AKT-MTOR pathway in OSCC and identification of determinants of its sensitivity. 2) Gene expression analysis of ectopically overexpressed TSC2 to identify new therapeutic targets and prognostic biomarkers as well as to elucidate the genes regulated by it. 3) Expression profiling of CYP1B1 in order to validate the use of CYP1B1 based prodrug therapy in OSCC. Investigations pertaining to the changes in gene and protein expression profiles in malignant as well as pre-malignant lesions have documented the deregulation of the PI3K-AKT-MTOR (phosphoinositide 3-kinase-AKT-mechanistic target of rapamycin) and EGFR (epidermal growth factor receptor) pathways in OSCC which are being widely targeted in many therapeutic strategies (Molinolo et al., 2007; Chakraborty et al., 2008; Matta and Ralhan, 2009; Molinolo et al., 2009; Stransky et al., 2011). The PI3K-AKT-MTOR pathway is a central hub for controlling cellular proliferation and growth in response to various intracellular as well as extracellular stimuli. Crucial signaling cascades including WNT, RAS, HIF-1α and AMPK cross-talk with the PI3K-AKT-MTOR pathway at a variety of molecular junctions. Thus, making this pathway sensitive to perceiving various growth modulatory conditions, ranging from the presence of growth factors to hypoxia and nutrient deprivation (Sengupta et al., 2010; Yang and Guan, 2007). The aberrant expression of the PI3K-AKT-MTOR pathway in OSCC advocated the targeting of this coveted pathway (Chakraborty et al., 2008). In various cancers, the monotherapeutic treatments with inhibitors like LY294002 (PI3K inhibitor) and rapamycin (MTOR inhibitor) demonstrated reduced efficacies. Such reduced efficacies were attributed to the drug toxicity and non-specific action of LY294002 (Davies et al., 2000; Sun et al., 2005; Ikezoe et al., 2007; Wang et al., 2008; Liu et al., 2009), or the ablation of a feedback inhibition loop leading to the reactivation of the PI3K-AKT-MTOR pathway by rapamycin (O'Reilly et al., 2006; Carracedo et al., 2008). Thus, rapamycin or its analogues demonstrated mediocre efficacy due to cytostatic effects in clinical trials, primarily due to the paradoxical activation of major survival kinases namely MAPK and AKT (O'Reilly et al., 2006; Carracedo et al., 2008). The present study aimed at increasing the efficacy of these drugs by incorporating a combinatorial approach. The MTT assay demonstrated that prolonged monotherapeutic treatments with rapamycin led to a modest growth inhibition in three OSCC (KB, SCC131 and SCC084) and HeLa cell lines. Western blot analysis of the phosphorylation status of AKT and RPS6KB1 revealed that monotherapeutic treatments with rapamycin for 96 hr led to the reactivation of the PI3K-AKT-MTOR pathway. Thus, the modest growth inhibitory effect of rapamycin was attributed to the reactivation of the PI3K-AKT-MTOR pathway. A combinatorial treatment approach was hence believed to circumvent this problem in order to increase the efficacy of targeting the PI3K-AKT-MTOR pathway. The PI3K inhibitor LY294002 was used combinatorially with rapamycin. This prolonged dual combinatorial treatment regime was distinctly more efficacious than either of the drugs alone and led to a reduction in cellular viability accompanied by increased sub-G1 population, indicating marked cell death that was characterized as caspase-3 dependent apoptosis. The differential sensitivity of the cell lines towards this combinatorial treatment revealed a novel determinant of the sensitivity, the transactivation of EGFR. The cell lines (SCC131 and SCC084) that were capable of transactivating EGFR were relatively resistant to the dual targeting of PI3K and MTOR in comparison to cell lines that did not transactivate EGFR (HeLa and KB). Further, targeting PI3K, MTOR and EGFR simultaneously was more efficacious in the presence of EGFR transactivation than dually targeting PI3K and MTOR. The results conclusively proved that the combinatorial therapeutic approach dually targeting PI3K and MTOR is a promising treatment strategy as compared to a monotherapeutic treatment and a major factor determining the sensitivity towards this treatment is the status of autophosphorylation of EGFR (Tyr1173) which governs the potential for EGFR transactivation by the combinatorial treatment. Thus, this study demonstrated that the status of EGFR autophosphorylation (Tyr1173) can be used as a biomarker to predict the sensitivity towards the combinatorial targeting of PI3K and MTOR in OSCC. The PI3K-AKT-MTOR pathway is negatively regulated by TSC2 (tuberous sclerosis complex 2; tuberin) (Tee et al., 2002). The importance of the TSC2 gene in the regulation of cell growth and proliferation is irrefutable. TSC2 facilitates the crosstalk between a variety of cellular signals, making it a crucial hub where many cellular networks integrate like AKT, MAPK and AMPK (Clements et al., 2007; Rosner et al., 2007; Rosner et al., 2008). It is a tumor suppressor gene and is downregulated in many cancers including OSCC (Chakraborty et al., 2008). In order to identify the genes regulated by TSC2 in OSCC, we stably overexpressed TSC2 in KB cells and the changes in the gene expression profiles caused by this ectopic overexpression were observed using a whole genome expression microarray. The results showed differential regulation of 268 genes (107 genes were upregulated and 161 genes were downregulated, p<0.05, fold change ≥ 1.5). A majority of these genes were functionally associated with transcription, cell growth and proliferation, apoptosis, cell cycle and neurogenesis. Functional annotation and network analysis was performed by using the DAVID v6.7 and IPA version 8.7 softwares. The microarray data revealed a novel aspect in the crosstalk between WNT signaling and TSC2, namely the transcriptional regulation of WNT signaling by TSC2. Further, in the context of therapeutic applications, the microarray analysis revealed multiple genes that were functionally categorized to be involved in response to radiation, UV and drugs (e.g., SERPINB13 and IL1B). Future studies on the regulation of such genes that are involved in responses to drugs and radiation may give insights into the role of TSC2 in resistance or sensitivity towards chemotherapy and radiation therapy. Moreover, EREG, a member of the epidermal growth factor family, was found to be the most downregulated gene in the microarray analysis. Previous reports have documented elevated levels of EREG in tuberous sclerosis lesions and its association with poor clinical prognosis in OSCC patients (Li et al., 2008; Shigeishi et al., 2008), making its regulatory aspects intriguing. Additionally, published data on the transcriptional functions of TSC2 instigated us to analyze the role of TSC2 in the regulation of EREG. TSC2 has been shown to modulate the transcription mediated by members of the steroid receptor superfamily of genes (Henry et al., 1998) and was shown to bind specifically to ERα and inhibit estrogen induced proliferation (Finlay et al., 2004). Also, TSC2 has been shown to possess C-terminal transcriptional activation domains (Tsuchiya et al., 1996). We have therefore attempted to investigate the transcription related functional aspects of TSC2 by exploiting the observed transcriptional repression of EREG. The physiological roles of TSC1 and TSC2 that are independent of the PI3K-AKT-MTOR pathway have been termed as ‘non-canonical’ (Neuman and Henske, 2011). The repression of EREG by TSC2 was observed to be insensitive to rapamycin, suggesting that it was independent of MTORC1 and thus a non-canonical function of TSC2. To determine whether the repression in EREG was at the level of the promoter, we performed a dual luciferase reporter assay. The results showed that the EREG promoter was repressed by stable as well as transient overexpression of TSC2. In order to elucidate the mechanism of transcriptional regulation by TSC2, we performed the ChIP analysis to observe the in vivo binding of TSC2 to the EREG promoter. In the ChIP analysis with the anti-TSC2 antibody, we observed that TSC2 did not bind to the EREG promoter between the regions -857 bp to -302 bp or -325 bp to +165 bp. Further, in silico analysis revealed an interesting trend among the transcription factors that were differentially regulated by TSC2 and had putative binding sites on the EREG promoter. A majority of these transcription factors (17/21) were downregulated by the overexpression of TSC2. This observation suggested that the repression of EREG could be an indirect effect due to repression of transcription factors caused by overexpression of TSC2. On the whole, this study revealed novel functions of TSC2 in OSCC with implications in determining novel biomarkers and therapeutic targets. As discussed previously, OSCC has a very flagitious treatment regime. A prodrug approach is thought to aid in targeting chemotherapy (Rooseboom et al., 2004). CYP1B1, a member of the cytochrome P450 family, has been implicated in chemical carcinogenesis (Bandiera et al., 2005; Sliwinski et al., 2010). There exists a general accordance that this protein is overexpressed in a variety of cancers (e.g., colon, lung, renal, bladder, prostate, breast, endometrial and esophageal cancers), making it an ideal candidate for a prodrug therapy (McFadyen et al., 1999; Murray et al., 2001; McFadyen et al., 2004; Sissung et al., 2006; Wen and Walle, 2007; Sliwinski et al., 2010). The activation of the prodrug facilitated by CYP1B1 would enable the targeting of chemotherapy to tumor tissues in which CYP1B1 is specifically overexpressed as a result reducing the non-specific side effects that the current chemotherapy elicits (Rooseboom et al., 2004). This study was aimed at validating the use of CYP1B1 as a target for the prodrug therapy in OSCC. The expression profile of CYP1B1 was analysed in a panel of 51 OSCC tumors, their corresponding normal tissues, an epithelial dysplasia lesion and its matched normal tissue by qRT-PCR, Western blotting and Immunohistochemistry. Counterintuitively, CYP1B1 was found to be downregulated in 77.78% (28/36) tumor tissues in comparison to their corresponding normal tissues as well as in the epithelial dysplasia lesion compared to its matched normal tissue at the transcriptional level, and in 92.86% (26/28) of tumor tissues at the protein level. This clearly demonstrated the downregulation of CYP1B1 at the transcriptional and translational levels in tumor tissues in comparison to their corresponding normal tissues. These observations indicate that caution should be observed as this therapy may not be applicable universally to all cancers. Since CYP1B1 has been shown to be involved in the activation of pro-carcinogens (Murray et al., 2001; Bandiera et al., 2005; Sissung et al., 2006), its inhibition could facilitate the development of a prophylactic therapy for oral cancer. Overall, this study has identified the transactivation of EGFR as a determinant of sensitivity towards combinatorial targeting of PI3K and MTOR in OSCC and has demonstrated that the autophosphorylation of EGFR (Tyr1173) can be used as a marker to judge the sensitivity towards this treatment. In the clinical perspective, the identification of such markers would aid in predicting the efficacy of targeted therapies. Such investigations would enable the strategic treatment of OSCC patients, thus decreasing the time lost in trial and errors for determining the appropriate treatment. Additionally, this study elucidated a novel role of TSC2 in the transcriptional repression of EREG, a prognostic biomarker for OSCC. Further, the study revealed potential prognostic biomarkers as well as therapeutic targets that are regulated by TSC2 by using a whole genome expression microarray. Moreover, the counterintuitive downregulation of CYP1B1 in OSCC tumors suggested the possibility of a prophylactic therapy for oral cancer but also advised a precautionary note for the application of prodrug treatments based on CYP1B1 overexpression in OSCC.

O cancro oral é definido pela Classificação Internacional de Doenças pelo conjunto de tumores malignos que afetam qualquer localização da cavidade oral, dos lábios à garganta, incluindo as amígdalas e a faringe. É importante referir que o cancro oral é um dos tipos de doença oncológica mais comum a nível mundial e apresenta uma elevada taxa de mortalidade. De uma forma genérica, esta doença tem como principais abordagens terapêuticas a intervenção cirúrgica, a radioterapia e a quimioterapia, ou até mesmo a combinação destas terapias. Esta dissertação tem como base expor e compreender de forma mais aprofundada as novas terapias alvo para cancro oral, que atualmente são estudadas e aplicadas como tratamento desta patologia oncológica e transmitir a importância das mesmas. Pretende-se realçar que estas terapias são distintas das convencionais, analisando o mecanismo de ação, o tipo de doentes e em que fase são utilizadas, os seus benefícios, malefícios e resposta por parte da doença. As terapias inovadoras que estão a ser desenvolvidas apresentam uma influência positiva em relação à sobrevivência global, à sobrevivência livre de progressão da doença e à qualidade de vida dos pacientes. Este trabalho trata-se de uma revisão bibliográfica, com recurso à biblioteca da Faculdade de Ciências da Saúde da Universidade Fernando Pessoa e às bases de dados Pubmed, Scielo e B-on.
Oral cancer is defined by the International Classification of Diseases by the set of malignant tumors that affect any localization of the oral cavity, from the lips to the throat, including the tonsils and the pharynx. It is important to note that oral cancer is one of the most common types of cancer worldwide and has a high mortality rate. Generally, this disease has as main therapeutic approaches surgical intervention, radiotherapy and chemotherapy, or even the combination of these therapies. This dissertation is based on exposing and comprehending in more depth the new target therapies for oral cancer, which are currently studied and applied as treatment of this oncological pathology and transmit the importance of them. It is intended to emphasize that these therapies are distinct from the conventional ones, analyzing the mechanism of action, the type of patients and in what phase they are used, their benefits, harms and response by the disease. Innovative therapies being developed have a positive influence on overall survival, progression-free survival, and patients' quality of life. This work is a bibliographical review, using the library of the Faculty of Health Sciences of the Fernando Pessoa University and the databases Pubmed, Scielo and B-on.