Dissertations / Theses on the topic 'Metastatic cancer'

The complex PI3K/mTOR pathway regulates tumor progression via effects on cellular proliferation, apoptosis, autophagy, and motility. New drugs that inhibit the catalytic site of both PI3K and mTOR have shown promise in clinical trials. Here, we report the first use of a novel, dual PI3K/mTOR catalytic site inhibitor (PF-04691502, PF1502) in a xenograft model of breast cancer metastasis to bone. Metastatic MDA-MB-1833 cells showed PI3K/mTOR activation relative to parental MDA-MB-231. Low-dose PF1502 significantly impaired tumor cell motility and invasion in vitro without causing cell cycle arrest, apoptosis, or reduced proliferation. Pre-treatment of tumor cells at this dose reduced bone metastatic outgrowth in vivo. The atypical tumor suppressor, p27KIP1, is phosphorylated in its C-terminal region by multiple AGC kinases downstream of PI3K/mTOR. These phosphorylation events promote cytoplasmic mislocalzation of p27 which, in turn, facilitates inhibition of the RhoA cytoskeletal regulatory protein. The resulting turnover of the actin cytoskeleton is thought to underlie the increased cellular motility attributed to cytoplasmic p27. In MDA-MB-1833 cells, PI3K/mTOR inhibition reduced p27 C-terminal phosphorylation at T157 and T198 and reduced cytoplasmic p27 levels. Overexpression of a p27T157D/T198D phospho-mimetic mutant conferred resistance to the anti-motility effects of PF1502 in vitro. MDA-MB-1833 cells demonstrate p27-dependent inhibition of RhoA-ROCK signaling, as well as p27-dependent motility and invasion in vitro, however, RhoA knockdown did not confer resistance to the anti-motility effects of PF1502. p27shRNA dramatically impaired the bone metastatic outgrowth of MDA-MB-1833 in vivo. In an effort to explore potentially novel RhoA-independent mechanisms whereby cytoplasmic p27 might drive tumor cell motility and metastasis, we turned to the process known as epithelial-to-mesenchymal transition (EMT). The EMT program has been implicated as a critical driver of tumor metastasis in a variety of cancer models. PI3K/mTOR inhibition and shRNA p27 treatment both reversed expression of EMT markers in MDA-MB-1833. Thus, PI3K/mTOR appears to drive p27-dependent motility and metastasis at least in part by induction of an EMT-like phenotype, a novel mechanism through which p27 might act to promote tumor progression. These results provide an important new clinical rationale supporting the use of PI3K/mTOR inhibitors as anticancer agents via their inhibition of tumor invasion and metastasis.

In most randomised controlled trials (RCTs), a large number of patients need to be followed over many years, for the clinical benefit of the drug to be accurately quantified (1). Using an early proxy, or a surrogate endpoint, in place of the direct endpoint of overall survival (OS) could theoretically shorten the duration of RCTs and minimise the exposure of patients to ineffective or toxic treatments (2, 3). This thesis examined the relationship between surrogate endpoints and OS in metastatic colorectal cancer (CRC), advanced non-small cell lung cancer (NSCLC) and metastatic breast cancer (MBC). A review of the literature identified 144 RCTs in metastatic CRC, 189 in advanced NSCLC and 133 in MBC. The publications were generally of poor quality with incomplete reporting on many key variables, making comparisons between studies difficult. The introduction of the CONSORT statement was associated with improvements in the quality of reporting. For CRC (337 arms), NSCLC (429 arms) and MBC (290 arms) there were strong relationships between OS and progression free survival (PFS), time to progression (TTP), disease control rate (DCR), response rate (RR) and partial response (PR). Correlation was also demonstrated between OS and complete response (CR) in CRC and duration of response (DOR) in MBC. However, while strong relationships were found, the proportion of variance explained by the models was small. Prediction bands constructed to determine the surrogate threshold effect size indicated that large improvements in the surrogate endpoints were needed to predict overall survival gains. PFS and TTP showed the most promise as surrogates. The gain in PFS and TTP required to predict a significant gain in overall survival was between 1.2 and 7.0 months and 1.8 and 7.7 months respectively, depending on trial size and tumour type. DCR was a better potential predictor of OS than RR. The results of this study could be used to design future clinical trials with particular reference to the selection of surrogate endpoint and trial size.

Metoder for å måle utfall av behandling hos pasienter med prostatakreft uten spredning. Prostatakreft uten påvist spredning kan behandles med operasjon, strålebehandling med eller uten hormonbehandling, eller hormonbehandling alene. Svært mange menn med nylig påvist prostatakreft vil imidlertid kunne leve i mange år uten plager av sykdommen også uten behandling, og en stor andel dør til slutt av helt andre årsaker enn prostatkreft. Man mangler imidlertid helt sikre metoder for å velge ut pasienter som ikke trenger behandling, men i noen tilfeller hvor sykdommen er i tidlig fase er et regelmessig kontrollopplegg hvor kurativ behandling iverksettes først hvis sykdomen viser tegn til utvikling, forsvarlig. Imidlertid kan prostatakreft også være en agressiv sykdom med stor risiko for spredning og forkortet levetid. Pasienter med nylig påvist prostatakreft uten spredning og intermediær til høy risiko for progresjon regnes derfor å være behandlingstrengende. Det kan ta mange år før man merker symptomer på et tibakefall etter en behandling som ikke har gitt ønsket kurasjon. Da vil sykdommen ofte ha kommet for langt til å kunne helbredes. Påvisning av manglende behandlingseffekt før et tilbakefall av sykdommen gir symptomer er imidlertid kun viktig hvis det da finnes effektiv tileggsbehandling. Siden overlevelse vanligvis ansees som det viktigste effektmålet ved kurativ kreftbehandling, blir det også viktig å avklare om det å påvise et tidlig tilbakefall eller manglende behandlingseffekt virkelig kan forutsi om pasienten på sikt vil utvikle plager av kreftsykdommen og i verste fall dø av den. I tillegg er det også svært viktig at bivirkningene ved utredning og behandling er så lite plagsomme som mulig. Denne avhandlingen omhandler metoder for å måle effekt/utfall av behandling hos pasienter med prostatakreft uten spredning. Avhandlingen inbefatter 4 studier hvor hovedvekten er lagt på resultater av vevsundersøkelser av prostatkjertelen etter åpen radikal prostatectomi (operasjon) samt resultater i form av overlevelse og bivirkninger når kombinert strålebehandling og hormonbehandling sammenlignes med hormonbehandling alene. Videre undersøkes hvor hyppig det påvises kreftceller i systematiske vevsprøver fra prostata 3-4 år etter at de to sistnevne behandlingene ble iverksatt. Endelig undersøkes bivirkninger av vevsprøvetakning etter kombinert strålebehandling og hormonbehandlig sammenlignet med hormonbehandling alene. Resultatene av studiene viser at andel pasienter som fikk prostatakreften fullstendig fjernet ved operasjon bedømt ut fra kirurgiske marginer gradvis økte de første årener etter at operasjonsmetoden ble innført. Det gir en indikasjon på at operasjonsteknikken bedret seg vesentlig i samme periode. Strålebehandling kombinert med hormonbehandling ga vesentlig bedre totaloverlevelse enn hormonbehandling alene. Kombinasjonsbehandlingen ga en lett øktning av bivirkninger, men bivirkningene var akseptable. De pasientene som fikk denne kombinasjonsbehandlingen hadde vesentlig skjeldnere gjenværende kreftceller i vevsprøvene sammenlignet med de som bare fikk hormonbehandling. Videre hadde pasienter med gjennværende kreftceller i prostatakjertelen vesentlig høyere risiko for tilbakefall påvist med blodprøve (stigende PSA) . Bivirkningene ved å ta systematiske vevsprøver fra prostata etter hormonbehandling med eller uten stråleterapi var små og gikk stort sett over i løpet av en uke.

Gastric cancer is one of the main cancers in the world, and is responsible for a large number of deaths each year. In Europe, the main issue is that it is only detected at latter stages, when metastases have developed. Although metastasis is the main cause of death from such tumours, the process is a very complex one and still not fully understood. In order to unravel this mechanism, microarrays have been used to study the expression pattern of a large number of genes in primary tumours and metastatic samples. These studies aim at indentifying genes that may playa role in metastasis. A number of microarray studies have already been carried out on gastric cancer to pursue knowledge. However, they have been mainly carried out in Asian populations, which are thought to present different gastric tumours than Europeans, possibly due to genetic and environmental parameters. The present thesis therefore aimed to carry out the first microarray study on gastric tumours from a European population, to identify genes that might playa role in gastric cancer metastasis, and assess whether there were any differences between Asian and European samples. In order to achieve this aim, the printing of in-house microarrays and methods to amplify and hybridise the samples first needed to be developed. This included the development of a new amplification method, the SMARTff7 protocol. Results showed that this method allowed more genes to be amplified than with an .established protocol. In addition, a microarray study published in 2003 identified a metastasis specific gene expression signature that could differentiate between metastatic and nonmetastatic primary tumours from different sites. However, this study did not include samples from gastric cancer. It was thus decided to test whether this signature could be applied to primary gastric tumours, using the new MetriGenix® platform. Although the analysis seemed to indicate that the signature did not apply to gastric tumours, technical issues meant that the results were inconclusive. On the other hand, a larger microarray analysis using the techniques developed during this project allowed the indentification of a number of genes of interest which may playa role in the metastatic spread of gastric cancer.

Les cancers du sein, premiers cancers féminins et causes aujourd’hui encore de nombreux décès, sont classés en 3 sous-types moléculaires : luminaux –les plus répandus, HER2 et triple-négatifs (TN) –les plus agressifs. Lors du diagnostic, l’envahissement des ganglions lymphatiques axillaires par les cellules tumorales est établi. Il s’agit, en plus de la classification moléculaire, d’un marqueur pronostique utilisé en clinique pour stratifier les patientes, car il informe sur le risque de développement ultérieur de métastases, cause majeure des décès à l’heure actuelle. Les tumeurs solides, et notamment les cancers du sein, sont des écosystèmes complexes comprenant de nombreux types cellulaires qui interagissent avec les cellules cancéreuses. Parmi eux, les fibroblastes associés au cancer (CAF) sont les plus abondants et participent activement à de nombreux aspects de la tumorigenèse dont la croissance, l’invasion, l’angiogenèse, l’immunosuppression. Cependant, ils constituent une population hétérogène et à ce jour, très peu d’études ont analysé cette hétérogénéité tout en la liant aux diverses fonctions décrites des CAF. Dans ce projet, nous nous sommes intéressés au rôle de cette hétérogénéité fibroblastique dans la dissémination métastatique des cancers du sein. En combinant l’étude de plusieurs marqueurs de CAF, nous avons montré que les ganglions lymphatiques envahis par les cellules tumorales sont constitués de 4 sous-populations de CAF (CAF-S1, S2, S3 et S4), similaires à celles identifiées dans les tumeurs primaires appariées. De façon intéressante, ce sont les deux sous-types de CAF myofibroblastiques (αSMA+), CAF-S1 et particulièrement CAF-S4, qui s’accumulent préférentiellement dans les ganglions métastatiques. Ils présentent les mêmes signatures transcriptomiques entre les deux localisations tissulaires (ganglions envahis et tumeurs primaires correspondantes). Or, ces deux populations CAF-S1 et CAF-S4 augmentent le phénotype invasif des cellules tumorales, en régulant des fonctions complémentaires. D’un côté, les CAF-S1 sont hautement motiles, et stimulent la prolifération, la migration, l’invasion et l’initiation d’une transition épithélio-mésenchymateuse des cellules de cancer du sein. De l’autre, les CAF-S4 sont très contractiles, capables de remodeler la matrice extracellulaire et promeuvent ainsi l’invasion et la motilité des cellules tumorales dans des systèmes en 3 dimensions. Des expériences fonctionnelles suggèrent que l’action des CAF-S1 implique CXCL12 et TGFβ tandis que celle des CAF-S4 dépend de la voie NOTCH. En accord avec ces résultats, l’accumulation des CAF et leur identité dans les ganglions envahis constituent deux nouveaux facteurs pronostics dans les cancers du sein, indépendants du sous-type de cancers du sein et de l’envahissement ganglionnaire. En effet, un fort contenu en CAF-S4 y est associé avec un développement ultérieur de métastases à longue distance. Ainsi, analyser le contenu fibroblastique des ganglions axillaires au diagnostic pourrait constituer une information nouvelle et utile à la prise en charge des patientes
Breast cancers are the most common cancers in women and despite great improvements in treatments, they are still responsible for many deaths worldwide. They are classified into 3 main molecular subtypes: Luminal cancers are the most frequent ones, while HER2 and TN are the most aggressive. At diagnostic, lymph node involvement is also assessed as it constitutes, in addition to molecular classification, a strong prognostic marker. Indeed, it informs on the risk to develop further distant metastases, which is the main cause of death by cancer. Solid tumors, including breast cancers, are complex ecologies comprising numerous different cell types that interact with cancer cells. Among them, cancer-associated-fibroblasts (CAF) are the most abundant and actively participate in many tumor hallmarks such as tumor growth, invasion, immunosuppression and angiogenesis. However, they do not constitute a homogeneous population but so far, only few studies have characterized this heterogeneity and linked it to CAF previously described functions. In this project, we focused on the potential involvement of CAF heterogeneity in breast cancer metastatic spread. Combining the analysis of several CAF markers, we showed that invaded LN comprise 4 CAF subsets (CAF-S1, S2, S3 and S4), similar to those found in primary tumors. Interestingly, the two myofibroblastic subsets (αSMA+) CAF-S1 and especially CAF-S4 preferentially accumulate in metastatic LN and present the same transcriptomic profiles in both tumors and LN. Importantly, both CAF-S1 and CAF-S4 display pro-invasive properties, by acting at different levels on tumor cells. On the one hand, highly motile CAF-S1 stimulate breast cancer cell proliferation, migration and EMT initiation. On the other hand, CAF-S4 exhibit an important contractility and by remodeling the matrix they are able to promote tumor cell invasion in 3D. Functional studies highlight a CXCL12/TGFβ involvement in CAF-S1 functions while CAF-S4 pro-invasive phenotype appears to be Notch-dependent. In agreement with these data, we found that CAF accumulation and subset enrichment in involved LN were two new prognostic factors, independent of breast cancer molecular subtypes and LN status at diagnosis. Indeed, stromal rich LN with a predominance of CAF-S4 are associated with long distance metastases development and poor overall survival. Thus, we propose that analyzing LN fibroblastic content at diagnosis could constitute new and useful information to breast cancer patients’ care

Metastatic prostate cancer is currently incurable. Metastasis is thought to result from changes in the expression of specific metastasis-driving genes, leading to a cascade of activated downstream genes setting the metastatic process in motion. As such, metastasis-driving genes could provide effective therapeutic targets and prognostic biomarkers for improved disease management. In search of potential metastasis-driving genes, genes with elevated expression in patient-derived metastatic LTL-313H prostate cancer tissues, as distinct from non-metastatic LTL-313B tissues, were identified. Among these genes, TIMELESS and DLX1 were promising. Unfortunately, their silencing and overexpression in prostate cancer cells did not lead to inhibition of metastatic properties, indicating that they were not metastasis-driving genes. A different, novel approach was used based on the notion that metastasis-driving genes can activate genes in an amplification cascade fashion. Accordingly, I used the IPA’s Upstream Regulator Analysis tool to analyze the differential gene expression profile of the metastatic and non-metastatic tissues to predict the upstream master regulatory (metastasis-driving) genes accountable for the differential expression. Six candidate genes were identified, including GATA2, a pioneer factor-encoding gene. Elevated GATA2 expression in clinical metastatic prostate cancer specimens correlated with poor patient prognosis. Furthermore, GATA2 gene silencing in human prostate cancer LNCaP cells led to marked reduction in cell proliferation, cell migration, tissue invasion, focal adhesion disassembly and a dramatic change in transcriptional activity, indicating that GATA2 plays a critical role in prostate cancer metastasis. As such, GATA2 could represent a metastasis-driving gene and a potential therapeutic target for inhibiting the growth and metastasis development in prostate cancer. Further analysis of GATA2-regulated genes led to the development of a GATA2-based metastatic gene signature. Its prognostic value was confirmed using two prostate cancer patient cohorts. In addition, it was shown to be a prognostic factor for risk assessment of metastasis development, independent of the widely used D’Amico prognostic classification system. However, a thorough validation is critical and, if successful, the GATA2-based gene signature could lead to a paradigm shift in the management of early prostate cancer. In conclusion, the findings of this study appear to be potentially useful for improved management of metastatic prostate cancer.
Medicine, Faculty of
Graduate

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers with an overall 5-year survival rate < 5%, a rate that has not improved for a long time. The dismal prognosis for PDAC is part due to late detection, often at advanced stages of the disease where patients have developed distant metastases, but also due to chemotherapeutical resistance. Patients eligible for surgical resection of the pancreatic tumour has the best prognosis, but even when resection is successful, patients often relapse with distant metastases within 2 years after surgery. Macrophages promote tumourigenesis and enhance metastasis in many cancer types; however, the role of macrophages in PDAC metastasis is poorly understood. Using an experimental mouse model of liver metastasis, we find that PDAC liver metastasis critically depends on the early recruitment of granulin-secreting metastasis-associated macrophages (MAMs) to the liver that promote metastatic colonisation. Mechanistically, we demonstrate that granulin secretion by MAMs activates resident hepatic stellate cells (HSTCs) into myofibroblasts that secrete extracellular matrix components, including periostin, resulting in a fibrotic microenvironment that sustains metastatic tumour growth. Disruption of MAM recruitment in PI(3)Kγ-deficient mice, chemical depletion of MAMs from established lesions or genetic ablation of granulin reduces HSTC activation and liver metastasis. Adjuvant CTX is standard for patients after surgical resection to eliminate any residual cancer cells, and it improves survival for patients after resection. We find that 45% of mice with metastatic lesions respond to gemcitabine treatment with a reduction in metastatic burden. The metastatic lesions in these mice are characterised by a reduction in αSMA+ myofibroblasts. HSTCs do not seem to promote cancer cell survival in the presence of chemotherapy, but rather constitutes a protective niche that promotes relapse by promoting the growth of pancreatic cancer cells after gemcitabine treatment.

Over the last 10-15 years the medical management of metastatic breast cancer has improved survival, so women are living longer with progressive disease. Little is understood about women’s problems and needs and how they live their everyday lives. This study aimed to explore the experience of women with metastatic breast cancer by applying three phases: a cross-sectional survey exploring quality of life, experience of care and where they turned for support; exploration of the narratives of 30 women considering the social consequences of living with progressive breast cancer on identity; and finally triangulating medical and nursing documentation, a measure of physical functioning and ten women’s narratives to define the illness trajectory of metastatic breast cancer. Phase 1: Quality of life was found to be poor with women experiencing a significant symptom burden. Experience of care was poor with unmet information and support needs. There was little evidence of General Practitioner or palliative care involvement in care. Phase 2: In weathering the oscillations of progressive disease, women faced threats to their social identity. Women sought ways to maintain their social roles and social order to avoid social isolation. To do this they adopted contingent identities: stoicism or absolved responsibility. Women used these contingent identities to mediate any discontinuity between the self, the body and social order. These self-representations were used by women to maintain their social roles and social order and in doing so avoiding being discredited and socially isolated. Phase 3: Mapping women’s illness trajectories identified three typical trajectories. The illness trajectories demonstrated the complexity of living over time with progressive disease, through phases which give definition to a previously ill-defined pathway. Living with metastatic breast cancer challenges the personal resources of the individual and poses interesting questions about how healthcare professionals provide information, effective symptom control, and emotional and practical support to women. Current models of care are not meeting women’s needs and new approaches to care provision need to be considered.

Tumor biologist have long appreciated that both cell to cell and cell to extracellular matrix (ECM) interactions are involved in the invasive and metastatic events that are characteristic of malignancy. Cancer cell attachment to and invasion of an ECM has been associated with metastatic potential of cell lines of the Dunning rat prostate model. It was postulated that differences observed in the metastatic potential of four Dunning cell lines may correlate with cell-matrix interactions. Four cell lines, highly metastatic ML, MLL, AT-3 and non-metastatic AT-1 were studied. The adhesive, invasive and chemoinvasive capability of each cell line was compared. Cell adhesion was examined by plating the cells on plastic dishes coated with various components of the ECM (fibronectin, laminin and collagen) as well as EHS Natrix (a natural ECM) . Invasion was determined by examining cells ability to traverse a matrigel barrier. Correlations were found between the cells' adhesive and invasive abilities in response to the ECM. These observations suggest that ECM components are highly involved in prostate cancer cell activities and loss may contribute to tumor progression and metastasis.

Le cancer de la prostate est le cancer le plus fréquent et constitue la troisième cause de décès par cancer chez l'homme dans les pays industrialisés. Le traitement systémique de première intention des formes métastatiques consiste en une thérapie de déprivation androgénique par castration chirurgicale ou médicamenteuse. Le cancer de la prostate métastatique résistant à la castration (mCRPC) est considéré comme incurable. Son traitement repose sur de la chimiothérapie ou des inhibiteurs de la voie du récepteur des androgènes (ARPIs) tels que l'enzalutamide ou l'abiratérone. Même si les ARPIs sont efficaces initialement chez la majorité des patients, environ 20% des patients n'en tirent pas bénéfice (résistance primaire) et ceux chez qui le traitement est efficace finissent par développer une maladie résistante aux ARPIs (résistance secondaire ou acquise). Les mécanismes de résistance sont multiples et extrêmement complexes. Plusieurs études se sont concentrées sur les mécanismes de résistance primaire, mais il existe très peu d'études axées sur les mécanismes de résistance secondaire. Des études antérieures, dont une majorité utilisant des modèles précliniques, ont suggéré que les altérations du récepteur aux androgènes lui-même telles que les variants d'épissage (en particulier AR-V7) sont associés à la résistance primaire et secondaire. Cependant, ce biomarqueur n'est pas utilisé en pratique courante. L'activation de voies de signalisation alternatives peut être également responsable de la résistance. D'autres mécanismes de résistance, encore peu connus et étudiés, incluent des modifications épigénétiques et l'activation d'autres facteurs de transcription.L'objectif de cette thèse était d'étudier les mécanismes de résistance aux ARPIs chez les patients atteints de mCRPC.Pour cela, nous avons développé une étude prospective (MATCH-R) permettant de réaliser des biopsies tumorales de patients traités par des ARPIs au stade métastatique. Un séquençage de l'exome et du transcriptome ont été réalisés. Les relations entre les anomalies génomiques et la survenue de résistance aux ARPIs ont été analysées.Les résultats suggèrent que la résistance primaire aux ARPIs résulte de l'activation de plusieurs phénomènes biologiques incluant (1) une réduction de l'activité de la voie du RA (2) une activation de la transition-épithélio-mésenchymateuse et de programmes de cellules souches, et (3) l'activation de la voie de signalisation Hedgehog (Hh), suggérant un bénéfice potentiel de l'inhibition de la voie Hh chez les patients présentant une activation Hh. Aucune anomalie liée à l'ADN est associée à la résistance primaire. La collection de plusieurs échantillons au moment de la résistance secondaire a permis de caractériser l'évolution clonale de ces tumeurs qui suit essentiellement un modèle d'évolution « en branche » avec acquisition séquentielle de nouvelles altérations de gènes importantes (AR, PTEN, RB1). L'analyse du transcriptome montre l'activation de voie de signalisation impliquée dans la prolifération cellulaire et de voies conduisant à une différenciation neuro-endocrine.Les résultats de cette étude doivent être confirmés par des études indépendantes au stade mCRPC ou à des stades moins avancés compte-tenu de l'utilisation plus précoces des ARPIs en particulier dans la situation de cancer de la prostate hormono-sensible.Des travaux sont en cours au laboratoire pour valider fonctionnellement l'intérêt potentiel des inhibiteurs de Hh qui sont déjà utilisés dans le traitement de plusieurs cancers et développer des options thérapeutiques ciblées
Prostate cancer is the most common cancer in men worldwide and the third most common cause of cancer deaths in industrialized countries. First line systemic therapy for metastatic prostate cancer usually includes androgen-deprivation therapy using surgical or pharmacological castration. Metastatic castration-resistant prostate cancer (mCRPC) is considered incurable. Its treatment is usually based on the chemotherapy or androgen receptor pathway inhibitors (ARPIs) such as enzalutamide or abiraterone. Although ARPIs are initially effective in the majority of patients, up to 20% of patients do not benefit from them (primary resistance) and those in whom the treatment is effective eventually develop ARPI-resistant disease (secondary or acquired resistance). The mechanisms of resistance are multiple and extremely complex. Several studies have focused on primary resistance mechanisms, but there are very few studies focused on mechanisms of secondary resistance. Previous studies, a majority of which used preclinical models, suggested that alterations in the androgen receptor itself such as splice variants (particularly AR-V7) are associated with primary and secondary resistance. However, this biomarker is not used in routine practice. Activation of alternative signaling pathways may also be responsible for resistance. Additional mechanisms of resistance, although still poorly known and studied, include epigenetic modifications and activation of other transcription factors.The aim of this thesis was to study the mechanisms of resistance to the new ARPIs in patients with mCRPC. To do so, we developed a prospective study (MATCH-R) to collect tumor biopsies from patients treated with ARPIs. Exome and transcriptome sequencing were performed. The relationships between genomic alterations and the occurrence of resistance to ARPIs were analyzed. The results suggest that primary resistance to ARPIs results from the activation of several biological pathways including those related to (1) a reduction in AR pathway activity (2) activation of the epithelial-mesenchymal transition and stem cell program, and (3) activation of the Hedgehog (Hh) signaling pathway protein, suggesting a potential benefit of Hh pathway inhibition among patients with Hh activation. DNA abnormalities that may cause primary resistance have not yet been identified. Collection of multiple samples at the time of secondary resistance allowed characterization of the clonal evolution of these tumors, which essentially follows a "branching" evolutionary pattern with sequential acquisition of important new gene alterations (AR, PTEN, RB1). Transcriptome analysis shows the activation of signaling pathways involved in cell proliferation and pathways leading to neuroendocrine differentiation. Further research is needed to validate these results in independent studies at the mCRPC stage or at earlier stages given the earlier use of ARPIs, particularly in the situation of hormone-sensitive prostate cancer.Ongoing work is underway in the laboratory with preclinical experimental models to functionally validate the potential value of Hh inhibitors that are already used in the treatment of other cancer in order to develop targeted therapeutic options

L’homéostasie sodique joue un rôle prépondérant lors de la carcinogénèse prostatique. Cependant, le rôle du canal de fuite sodique NALCN lors de la carcinogénèse prostatique était totalement inconnu. L’objectif principal de cette étude était d’étudier le rôle de NALCN dans la dérégulation de l’homéostasie sodique lors de la tumorigenèse prostatique. Tout d’abord, nous avons montré sur des coupes de tissus prostatiques que l’expression de NALCN est augmentée dans les cancers. De plus, NALCN est surexprimé dans les lignées cancéreuses prostatiques les plus agressives. Nous avons vérifié la fonctionnalité du canal NALCN et de ses protéines associées via des expériences d’imagerie sodique dans ces lignées. Grâce à notre étude, nous montrons que NALCN n’est pas impliqué dans le cycle cellulaire, la viabilité cellulaire, l’apoptose ni la prolifération. En revanche, nous avons démontré que ce canal affecte grandement la motilité, la migration et l’invasion de nos lignées cellulaires cancéreuses prostatiques. Nous avons montré que NALCN et le proto-oncogène Src sont co-localisées dans le cancer de la prostate, notamment au niveau de structures appelées invadopodes. Enfin, nous avons prouvé par des études in vivo que la croissance tumorale et la formation de métastases sont inhibées lorsque l’expression du canal NALCN est diminuée. En conclusion, nos données mettent en évidence que le canal NALCN est un acteur important dans l’augmentation du potentiel métastatique des cellules cancéreuses de prostate à la fois in vitro et in vivo. NALCN peut donc être considéré comme une nouvelle cible thérapeutique permettant de diminuer l’agressivité des cancers de la prostate
Importantly, altered Na+ homeostasis was implemented in prostate carcinogenesis. However, until now nothing was known about a newly discovered Na+ leak channel, NALCN, and its role in prostate malignancy. Therefore, the main objective of this study was to investigate the involvement of NALCN as a potential candidate of the deregulated Na+-dependent signalling mechanisms in prostate cancer. Interestingly, NALCN represented distinctly different localization patterns and levels of expression between human healthy and cancer prostate tissues. Indeed, NALCN was expressed preferentially in highly aggressive prostate cancer cell lines. Na+ imaging results verified on functionality of NALCN channelosome in these cells. Our study also revealed that NALCN was not involved in cell cycle, viability, apoptosis and proliferation, but significantly affected motility, migration and invasiveness of the prostate cancer cells. Interestingly, it was already reported that protooncogene Src family kinase is recruited to the NALCN complex. In this study, we confirmed that NALCN and Src kinase are co-localized in human prostate cancer cells, particularly in the structures that represent invadopodia formation sites. Furthermore, in vivo studies confirmed that NALCN downregulation inhibits tumour growth and metastasis formation. Overall, these data provide evidence on NALCN contribution to the increased metastatic potential of human prostate cancer cells in vitro and in vivo. Therefore, NALCN could provide new perspective molecular target for the disease suppression, in particular at its advanced stages

Colorectal cancer remains the third largest cause of mortality from cancer related death, with approximately 90% of these deaths attributed to metastatic spread of the disease. There is a need to improve chemotherapy, with the dietary polyphenol curcumin, derived from turmeric, representing a potential candidate as it possess very few side effects and it has shown efficacy in mouse models. Recent advances in our understanding of tumour development have highlighted the existence of tumour initiating cells (TIC), which possess clonogenic potential, are essential for tumour growth, and represent an important therapeutic target. This study sought to determine whether curcumin in combination with oxaliplatin+5-Fluorouracil (OX+5-FU) represented a better combination for targeting TICs, using a variety of models consisting of cells derived directly from colorectal liver metastasis (CRLM). ALDHHigh activity, CD133 and CD26 were all found to mark a spheroid forming population, a method that tests for clonogenicity and selects for TICs. Curcumin significantly reduced the number of spheroids compared to DMSO, and enhanced efficacy of OX+5-FU. The only marker to decrease after treatment was ALDHHigh activity, which was also positively associated with spheroid growth. CD26 expressing cells were identified as a possible chemo-resistant population, however, this population remained unaffected by curcumin. Ex vivo analysis using explant cultures demonstrated that curcumin could significantly decrease ki67 and increase cleaved capase-3 expression, notably enhancing the effects of oxaliplatin in a proportion of patients. A pilot study was undertaken using non-obese diabetic/severe combined immunodeficient (NOD-SCID) mice to reflect a clinical regimen, and assess in vivo whether curcumin could enhance efficacy of OX+5-FU, but the results advocate the use of higher doses as little effect was seen. Overall this body of work contributes to knowledge on propagating CRLM TICs, their expression of known TIC markers, and response to curcumin.

Each year approximately 26,000 British women develop breast cancer and 16,000 die from their disease. Breast cancer is the most common cancer in women in the United Kingdom. Previously, most studies have focused on the needs of women following surgery for the treatment of primary breast cancer. However, few systematic studies have monitored the needs of women with metastatic disease. The median survival of women with metastatic breast cancer is 19 months and therefore it would seem appropriate to monitor the rehabilitation needs of these women. This study examines the physical, psychological, and social rehabilitation needs of a consecutive series of 80 patients following definitive diagnosis of metastatic breast cancer. These patients were interviewed every eight weeks at home on eight separate occasions and were asked to complete the following standardised questionnaires: The Cancer Rehabilitation Evaluation System (CARES); The Hospital Anxiety and Depression Scale (HAD); and The Rotterdam Symptom Checklist (RSCL). In addition, the researcher completed an interview schedule to detail demographic details, current treatment, and which members of the medical team the patient had seen in the previsou month. The researcher also completed the Edinburgh Rehabilitation Status Scale (ERSS) which gives a total score of disability. The results of the descriptive component demonstrated that patients had a range of different rehabilitation needs throughout the course of their illness as defined by the CARES and the ERSS. These needs do not change throughout the course of the metastatic phase of the disease but detection of these problems is extremely low and, as a result, referral to appropriate services does not usually occur. Demographic factors such as age, marital status, social class, and number and age of children were not found to be associated with rehabilitation status. A significant problem in this group of patients was found to be that of mood disturbance and a complex inter-relationship was found to exist between rehabilitation status, age, physical symptomatology and mood using multiple stepwise regression analyses and factor analysis.

Metastatic breast cancer is a leading cause of cancer-related mortality worldwide. While existing interventions are effective at treating localized tumors, disseminated malignancies remain incurable. Vaccine-induced anti-tumor immunity is a promising approach for treating disseminated tumors, as immune responses are systemic, have antigen-restricted cytotoxicity, and generate protective immune “memory” populations. Our group has developed a novel heterologous prime/boost vaccine protocol that treats established 4T1 murine mammary tumors. Briefly, this approach entails a vaccine prime consisting of tumor lysate antigens encapsulated within poly(lactic-co-glycolic) acid (PLGA) microparticles (MPs). The vaccine prime was followed by a vaccine boost consisting of tumor lysates plus adjuvants. Spontaneous 4T1 lung metastasis was evaluated at a pre-determined endpoint in vaccinated versus untreated mice. Vaccinated mice demonstrated significant, but incomplete, reductions in metastatic tumor burdens relative to untreated control mice. Encouraged by these results, we evaluated additional vaccine variations with the goal of improving therapeutic responses. The addition of immunomodulatory chemotherapy or checkpoint blockade immunotherapy failed to significantly improve the initial vaccine’s efficacy. Conjugation of streptavidin/biotin complexes to the PLGA MP significantly improved vaccine efficacy, with vaccinated mice demonstrating 88% less metastatic tumor burdens than their untreated counterparts. These findings illustrate that vaccines based upon PLGA MP-mediated delivery of tumor lysates can form the basis of an effective treatment for metastatic breast cancer and suggest that similar approaches may be both efficacious and well-tolerated in the clinic.

Cancer cell metastasis is induced by actin-dependent cell migration and is affected by cytoskeletal remodelling proteins. FMNL2 is one such protein which promotes colorectal cancer (CRC) cell metastasis and amoeboid style invasion of melanoma cells. FMNL2 mRNA is subject to alternative splicing and studies suggest that the resulting encoded proteins are likely to differ in their regulation, subcellular localization and activity. We identified four FMNL2 isoforms (ITM, YHY, PMR and TQS) expressed in non-invasive (SW480) and invasive (SW620) CRC cells, as well as in highly invasive A375 amoeboid melanoma cells. qPCR data suggests that an “invasive” isoform (TQS) may be preferentially expressed in highly invasive and amoeboid cell lines. Boyden chamber invasion assay results show that FMNL2 knockdown inhibits amoeboid style invasion in two melanoma cell lines and that TQS is the most efficient isoform at rescuing the invasive phenotype. This study provides a further understanding of FMNL2’s role in invasion and metastasis and identifies specific targets for the development of future antimetastatic therapies.

Le cancer de l’ovaire est le cancer gynécologique avec la plus grande mortalité due à un diagnostique tardif au stade de maladie extensive péritonéale. Malgré les progrès de la chirurgie radicale et de la chimiothérapie les récurrences abdominales demeurent la cause la plus fréquente de mortalité. Il existe peu d’études de la maladie métastatique péritonéale. Notre hypothèse de travail est que les différences entre la maladie métastatique et la tumeur primaire sont primordiales dans la survenue d’une maladie résiduelle ou récurrente. Nous avons utilisé une approche exhaustive comprenant des études du transcriptome, des variations du nombre de copie (VNC) et des sequençages des exomes pour caractériser les différences entre lésions primaires, métastases péritonéales et métastases lymphatiques.Résultats: Notre étude démontre que les VNC varient de façon significative entre la tumeur primaire et la métastase peritonéale. Les différences d’expressions géniques bien que mineures permettent de retrouver les voies de signalisation primordiales pour le développement des métastases. Le séquençage des exomes montre très peu de différences en terme de polymorphisme. Par ailleurs la majorité des polymorphismes présents dans les métastases se retrouvent à une faible fréquence dans la tumeur primaire de façon concordante avec la théorie clonale. Conclusion: L’ensemble des résultats montre la possibilité d’une origine clonale de la maladie métastatique des cancers de l’ovaire comportant la majorité des anomalies au niveau des variations du nombre de copie. L’intégration de ces données permettrait d’optimiser les thérapeutiques ciblées
Ovarian cancer is the most deadly gynecological cancer. The high rate of mortality is due to the large tumor burden with extensive metastatic lesion of the abdominal cavity. There are few studies on genetic alterations and their consequences in peritoneal metastatic tumors when compared to their matched ovarian primary tumors. Our hypothesis is that differences between the metastatic and primary lesions might be the cause of residual disease and, most importantly may have a role in post-chemotherapeutic recurrences. Methods: We conducted integrated genomics analysis on matched primary and metastatic tumors from 9 patients. In the papers presented here we analyze genome-wide Copy Number Variations (CNVs) using SNP Arrays targeting peritoneal metastasis differences, Gene expression differences using Microarrays also targeting peritoneal metastasis differences, and for some patients, Single Nucleotide Polymorphisms (SNPs) in genes through Exome sequencing.Results: Here we show that CNVs vary significantly between primary and metastatic tumors and include genes that have been considered potential chemotherapeutic targets based on primary tumor only data. Gene expression differences, while minor, showed highly statistically significant enrichment of genes in ovarian cancer critical pathways. In agreement with findings in other cancers, exome sequencing data revealed very few SNP differences of which most metastasis enriched SNPs were present at very low levels in the primary tumor. The results presented here should allow better design of therapies to target residual ovarian cancer disease

Skeletal metastasis is a lethal component of many advanced cancers including prostate, the second most common cancer among men. Patients whose prostate cancer is localized and detected early benefit from multiple treatment options ranging from active surveillance to radiation and surgery, resulting in a 5-year survival rate of nearly 100%. Unfortunately, the prognosis and survival for patients with advanced metastatic disease is much worse due to the highly aggressive nature of the disease and a paucity of treatment options. Understanding the mechanisms and interactions that occur between metastatic cancer cells and the bone will enable the future treatment landscape for bone metastatic prostate cancer to expand, thereby improving patient outcomes. Our current knowledge of how metastatic prostate cancer cells interact with the bone is summarized in a model known as the “vicious cycle.” Numerous fundamental vicious cycle factors have been identified, including parathyroid hormone-related protein (PTHrP), while additional elements, such as matrix metalloproteinases (MMPs), are progressively being discovered and added to the model. PTHrP is a critical regulator of bone resorption and augments osteolysis in skeletal malignancies. In Chapter 2, we report that the mature PTHrP1-36 hormone is processed by MMPs to yield a stable product, PTHrP1-17. PTHrP1-17 retains the ability to signal through PTH1R to induce calcium flux and ERK phosphorylation but not cyclic AMP production or CREB phosphorylation. Notably, PTHrP1-17 promotes osteoblast migration and mineralization in vitro, and systemic administration of PTHrP1-17 augments ectopic bone formation in vivo. Further, in contrast to PTHrP1-36, PTHrP1-17 does not affect osteoclast formation/function in vitro or in vivo. Finally, immunoprecipitation-mass spectrometry analyses using PTHrP1-17-specific antibodies establish that PTHrP1-17 is indeed generated by cancer cells. Thus, MMP-directed processing of PTHrP disables the osteolytic functions of the mature hormone to promote osteogenesis, indicating important roles for this mechanism in bone remodeling in normal and disease contexts. MMPs have traditionally been associated with cancer progression based on their extracellular matrix degrading activities. However, it has become evident that their regulation of non-extracellular matrix substrates can exert both contributive and protective effects during tumorigenesis. Previous studies of matrix metalloproteinase-3 (MMP-3) have demonstrated tissue dependent pro- and anti-tumorigenic effects, but despite elevated expression, its roles have not been explored in bone metastatic prostate cancer. In Chapter 3, we show that tumor-derived MMP-3 contributes to prostate tumor growth in bone. In vitro, we observe that silencing MMP-3 reduces prostate cancer cell proliferation. Further, we found increased levels of IGFBP3, a known MMP-3 substrate, and decreased IGF-1R, ERK, and AKT phosphorylation in the MMP-3 silenced cells. Notably, we also observe reduced tumor growth and proliferation in in vivo intratibial models when tumor-derived MMP-3 expression is silenced. These data suggest that increased MMP-3 expression by prostate cancer cells contributes to their proliferation in bone by regulating the activity of the IGF/IGF-1R signaling axis. Taken together, our studies indicate that MMPs possess important functional roles in bone metastatic prostate cancer. We believe that elucidation of these mechanisms and their contributions to the vicious cycle of bone metastasis will offer novel opportunities to design effective therapeutic treatment options.

Breast cancer is the most common cancer among Canadian women and 14-20% will develop lethal metastases within 5 years. A potential novel therapeutic target is Focal Adhesion Kinase (FAK), a cytoplasmic tyrosine kinase. FAK’s expression is inversely correlated with survival and is known to regulate cell migration, proliferation and invasion. While tyrosine kinase inhibitors are historically ineffective as single agents, they are commonly used as part of combination therapies. Therefore, given its central role in tumor cell biology and cell signaling, we hypothesized that inhibiting FAK in combination with pharmacological agents commonly used to treat metastatic breast cancer patients will result in enhanced anti-tumor activity. We combined a commercial FAK inhibitor (PF-562271) with a range of chemotherapeutic agents commonly used to treat metastatic breast cancer and searched for synergistic partners. Only DNA topoisomerase inhibitors showed potential to synergistically reduce cell viability when paired with low doses of the FAK inhibitor. However, the combination does not induce an increase in cell death or apoptosis. It was then discovered that both agents in isolation and in combination produce increased levels of ROS, a toxic metabolite. This, along with other more preliminary data, provides clues for a novel proposed mechanism of action for this interaction.

CD24 is a small (81 amino acids) GPI anchored protein which is involved in promoting cell motility and stemness and may be a part of the metastatic process. It is a heavily glycosylated molecule and contains numerous O-glycosylation sites together with two N-glycosylation sites. N-glycosylation is thought to be important in protein function, and therefore, the aim of this study is to (a) investigate the importance of N-glycosylation in the function of CD24, (b) identify other potentially functional sites in CD24 by deletion mapping, (c) define downstream targets of CD24, and (d) identify the extrinsic signals of which activate CD24. (a) Through site-directed mutagenesis, we changed the glycosylated residues N32 (ACC to CAA) and Q52 (AAT to CAG) in CD24. Mutating each of these sites individually, when compared to pCCD24WT (wild-type CD24), caused a partial reduction in ability to induce cell motility and cell invasion (cell motility p=0.0001 cell invasion p=0.0001) and, unexpectedly, resulted in significantly enhanced cell proliferation (p=0.0001). Mutation of both sites resulted in a near loss of motility induction and retained cell proliferation. (b) We mapped the functional sites of CD24 by deleting seven amino acid segments of the whole of the mature peptide. Apart from the N-glycosylation sites, no other functional domains were identified which altered cell motility or proliferation. (c) Previously, in our lab it has been shown that Cten is downstream motility-inducing target of CD24. We hypothesised that CD24 may signal through the Notch pathway since Notch1 has an important role in maintaining CSCs. Results showed that forced expression of CD24 upregulates Notch1 and Cten whilst knockdown of CD24 causes loss of Notch1 and Cten expression. However, forced expression of CD24 with simultaneous knockdown of Notch1 resulted in failure to induce Cten. (d) CD24 is reported to act as a ligand of P-selectin. We found that stimulating CD24 expressing cell lines induced with P-selectin induced cell motility (p=0.0011) and caused an increased in the protein expression of downstream targets of CD24. Stimulating cell lines expressing CD24 with mutant glycosylation sites resulted in a failure to induce motility or CD24 targets. We conclude, the removal of the N-glycosylation sites in CD24 resulted in a loss of cell migration and invasion, thereby suggesting the importance of these sites in mediating the migration and invasion functions of CD24. Unexpectedly, these mutations also appeared to stimulate cell proliferation, suggesting that wild type CD24 can functionally inhibit cell proliferation. Deletion mapping did not reveal any other functional sites on the mature CD24 suggesting that O-glycosylation is relatively affecting the glycosylation in the biology of CD24. Notch1 was to be an important downstream target of CD24 and a regulator of Cten. The binding of P-selectin with CD24 resulted in increased motility of CD24 which is also dependent on N-glycosylation.

Metastatic, castrate-resistant prostate cancer (mCRPC) is one of the most prevalent cancers and is the third leading cause of cancer death among men. Several treatment options have been developed to combat mCRPC, however none have produced any tangible benefits to patients' overall survivability. As part of a crowd-sourced algorithm development competition, participants were asked to develop new prognostic models for mCRPC patients treated with docetaxel. Such results could potentially assist in clinical decision making for future mCRPC patients.

In this thesis, we present a new ensemble prognostic model to perform risk prediction for mCRPC patients treated with docetaxel. We rely on traditional survival analysis model like the Cox Proportional Hazard model, as well as more recently developed boosting model that incorporates smooth approximation of the concordance index for direct optimization. Our model performs better than the the current state-of-the-art mCRPC prognostic models for the concordance index performance measure and is competitive with these models on the integrated time-dependent area under the receiver operating characteristic curve.

"Colorectal cancer is a global health concern. The high incidence of colorectal metastasis, mainly in the liver, triggers an increase of the mortality rate and greatly reduces the effective cure chances. For this reason, the investigation in the area is now focused on efficient detection and elimination of metastasis. Nanotechnology has become a fundamental research field since it provides promising perspectives regarding specific, oriented and sustained delivery of loaded nanoparticles for nanoteragnostic approaches. The current project aims to develop a nanocarrier, strategically constructed for specific administration, recognition and therapy of colorectal liver metastasis. Concerning this goal, two different approaches were concurrently developed: a green-therapeutic technology and a novel nanoparticulate system, by nanoprecipitation and inverse microemulsion, respectively. (…)"
N/A

The skeleton is the most common site of prostate cancer bone metastasis, and at present, there are no curable treatments for these patients. To further understand what stimulates tumor cell growth in the bone microenvironment and to find suitable therapies, reliable model systems are needed. For this purpose, we have developed an in vitro co-culture system that can be used to study interactions between tumor cells and murine calvarial bones. To validate the model, we measured the release of collagen fragments and monitored changes in expression levels of genes normally expressed during active bone remodeling. One of the major reasons why prostate cancer cells colonize bone is the abundance of tumor-stimulating factors, such as insulin-like growth factors (IGFs), present in this milieu. We found that the IGF-1 receptor (IGF-1R) was one of the most highly activated receptor tyrosine kinases in tumor cell lines stimulated with bone conditioned media. Since IGF-1 is known to be a strong survival factor for tumor cells, we hypothesized, that concurrent inhibition of IGF-1R signaling can enhance the effects of apoptosis-inducing therapies, such as castration. We used our co-culture model to target human prostate cancer cell lines, PC-3 and 22Rv1, with simvastatin (an inhibitor of the mevalonate pathway and an inducer of apoptosis), in combination with anti-IGF-1R therapy. Tumor cell viability declined with either one of the therapies used alone, and the effect was even more pronounced with the combined treatment. The hypothesis was also tested in rats that had been inoculated with rat prostate cancer cells, Dunning R3327-G, into the tibial bone, and treated with either anti-IGF-1R therapy, castration, or a combination of both therapies. Immunohistochemistry was used to evaluate therapeutic effects on tumor cell proliferation and apoptosis, as well as tumor cell effects on bone remodeling. The tumor cells were found to induce an osteoblastic response, both in vivo in rats, and in vitro using the co-culture model. Interestingly, the therapeutic response differed depending on whether tumor cells were located within the bone marrow cavity or if they had leaked out into the knee joint cavity, highlighting the role of the microenvironment on metastatic growth and therapeutic response. Therapies targeting the IGF-1R have been tested in clinical trials, unfortunately with disappointing results. By immunohistochemical evaluation of bone metastases from patients with castration-resistant prostate cancer, we found a large variance in IGF-1R staining within this group of patients. Hence, we postulate that the effects of anti-IGF-1R therapies could be more beneficial in patients with high tumoral IGF-1R-activity than in IGF-1R negative cases. We also believe that side effects, such as hyperglycemia, associated with anti-IGF-1R therapy, could be reduced if this treatment is administered only to selected patients and for shorter time periods. In a separate study, using whole-genome expression data from bone metastases obtained from prostate cancer patients, we present evidence that a high activity of osteoblasts is coupled to a high activity of osteoclast. Moreover, we found that high bone remodeling activity is inversely related to tumor cell androgen receptor (AR) activity. The results from this study may be of importance when selecting therapy for patients with bone metastatic cancer, especially when bone-targeting therapies are considered, and could aid in the search for novel therapeutic targets. In summary, we present an in vitro model for studies of the bidirectional interplay between prostate cancer cells and the bone microenvironment. We also demonstrate the importance of IGF-1 in prostate cancer bone metastases and suggest that inhibition of IGF-1R signaling can be used to treat prostate cancer as well as to enhance effects of other treatments such as androgen deprivation therapy. Furthermore, we emphasize the possibility of molecular tumor characterization when designing treatment plans for individual patients, thereby maximizing the therapeutic effects.

Melanoma incidence is increasing faster than any other cancer worldwide.1 Early detection is often curative, but metastatic melanoma is lethal (5-year survival <20%) due to the development of resistance to all approved drugs.1 However, emerging evidence suggests that differences in melanoma metabolism relative to non-malignant cells may provide a target to improve treatment.2-14 Specifically, melanoma cells have increased mitochondrial electron transport chain (ETC) activity, elevated levels of reactive oxygen species, and a simultaneous hyperpolarized mitochondrial membrane potential relative to non-malignant cells.4, 8, 11, 15-17 Furthermore, melanoma cells have upregulated glucose consumption and concurrent increased levels of glucose transporters (GLUTs) relative to non-malignant cells; the products of glycolysis (pyruvate and NADPH) aid in the detoxification reactive oxygen species (ROS), while the intermediates are utilized in energy production via increased oxidative metabolism.15, 18 Collectively, melanoma cells exhibit alterations in metabolic, mitochondrial, and cell-surface targets that can be potentially exploited for therapeutic strategies for selective cancer cell killing relative to non-malignant cells. The research presented here demonstrates the therapeutic potential for a new class of mitochondrial-targeted fluorescent lipophilic-cations: pyridinium derivatives (UIRF 17023.186PV1 U.S. Provisional Patent Application No. 62/268,980 Patent Pending). Importantly, the pyridinium derivatives presented in this study have not been previously investigated as a mitochondrial-targeted therapy.19-21 Furthermore, the research presented outlines the feasibility of improving melanoma cellular accumulation of these pyridinium derivatives by including a GLUT targeting moiety in the form of a hexosamine. The addition of a hexosamine molecule to pyridinium derivatives has the potential to increase melanoma cell accumulation by targeting upregulation of GLUT expression in melanoma cells relative to normal cells. Thus, the results of this study identified: (1) a triphenylvinylpyridine (TPVP) lipophilic cation derivative that increased melanoma oxidative metabolism and decreased melanoma cell viability; and (2) the targeting potential for GLUT-mediated melanoma cell specific delivery of glucosamine-modified TPVP derivatives. These findings support the hypothesis that TPVP-based therapies can be developed to exploit fundamental differences in glucose and mitochondrial metabolism to selectively kill melanoma cells relative to non-malignant cells.

Pharmaceutical Sciences
Ph.D.
Triptolide (TPL), a diterpenoid triepoxide that is extracted from a traditional Chinese herb called Tripterygium Wilfordii (also known as ‘Thunder God Vine’) has recently drawn increasing interests from pharmaceutical and biomedical researchers, especially in the aspect of its potential efficacy on multiple cancer treatment. TPL has shown significant growth and proliferation inhibition activities in a broad range of cancer cell types. Moreover, it has shown the inhibition of osteoclastogenesis by breast cancer bone metastasis. However, due to its limitation in toxicity, solubility and non-specific biodistribution, it is challenging for the application of TPL in clinical study. Besides, TPL can rapidly distribute in most vital organs and no evidences shown tissue accumulation of drug. It is indispensable to overcome those barriers and optimize the properties and performance of the promising drug molecule. Lipid-based nanocarriers such as nanostructured lipid carriers (NLC) have been extensively studied for delivery of poorly-water soluble drug compounds. They also have the potential to optimize the physicochemical properties of the drug and may enhance a targeted delivery of the drug to specific therapeutic site. Alendronate (Fosamax®), an FDA approved bisphosphonate drug for osteoporosis, osteogenesis imperfecta and several other bone diseases, has been used as a bone targeting decoration agent. Breast cancer cell line MDA-MB-231 and other type of cancer cell lines have been used to study the in vitro cytotoxicity of TPL and the carriers while MC3T3-E1 cell line was used for toxicity assessment. Rats have also been used to study the in vivo performance of the drug. After modifying and optimizing the formulation of the particle, the formulation had the ability to remain structurally and functionally stable when being in the bio-simulated media at 37 °C and in water at room temperature with high encapsulation efficiency. In vitro study illustrated that both TPL free drug (stock solution 10mg/mL dissolved in DMSO) and TPL nanoparticle without alendronate (TPL-NP) had similar cytotoxicity on MDA-MB-231 and some other type of cancer cell lines. The ALE decoration on the particle (ALE-NP-TPL) has enhanced the anti-cancer effect especially with breast cancer cell line. The in vivo study shows that after 24 hours of the dose injection at local bone site, the formulation and TPL can remained at the location without random distribution to other organs. TPL-NP has not only successfully optimized the physicochemical properties of the drug, but also shows great enhancement of therapeutic effect both in vitro and in vivo study.
Temple University--Theses

Solid epithelial tumours are complex structures in which the associated stroma supports cancer cells (Quail and Joyce, 2013). During metastatic progression, cancer cells disseminate from their tissue of origin and recapitulate the tumour structure at distant organs, including the stromal compartment. Metastasis initiating cells (MICs) are functionally discriminated among the bulk of cancer cells for their high ability to establish metastasis (Malanchi et al., 2012, Baccelli et al., 2013). Additionally, efficient metastasis requires the expression of specific molecules within the local microenvironment (Oskarsson et al., 2014). Thus, a favourable microenvironment or niche is a crucial early step in metastatic progression. However what features of MICs mediate metastatic niche activation is poorly characterised. One strategy adopted by metastatic cells to disseminate from primary tumours is the activation of the developmental programme epithelial-to-mesenchymal transition (EMT). However, EMT is a reversible programme that needs to be inhibited at the target site for tumour cells to re-acquire epithelial characteristics compatible with metastatic outgrowth (Nieto, 2013). To successfully metastasise cancer cells need to retain self-renewal and growth properties through epithelial plasticity. This implies that during metastasis 'stemness' should not be strictly coupled to EMT as previously suggested (Mani et al., 2008). To date, both the potential advantage of disseminated cancer cells mesenchymal status and the source of their epithelial plasticity at the metastatic site remain unknown. In this thesis we use metastatic breast cancer models to elucidate the enhanced niche-induction ability of mesenchymal MICs, its relationship to EMT and the source of its epithelial modulation during metastatic colonisation. Importantly, we identify THBS2 as a novel effector linked to the EMT status of cancer cells that enhances stromal niche activation. Subsequently, the newly activated stroma triggers cancer cell BMP-dependent re-epithelialisation promoting metastatic outgrowth. Thereby, we describe a temporally controlled metastatic colonisation where the EMT status of cancer cells promotes its own inhibition via a cancer cell-stromal crosstalk that initially enhances metastatic niche formation, and ultimately favours a cancer cell proliferative state compatible with metastatic outgrowth.

Background: Bone metastases occur in most patients with advanced hormone-refractory prostate cancer causing pain, pathologic fractures, and spinal cord compression. Few studies specifically address surgical treatment of metastatic spinal cord compression (MSCC) in prostate cancer. Criteria for identifying patients who may benefit from surgery are poorly defined. Most of the current knowledge regarding tumor biology in prostate cancer is based on studies of primary tumors or soft tissue metastases. The mechanisms regulating growth of bone metastases are not fully established. Aims: a) to evaluate outcome after surgery for MSCC in prostate cancer and to identify prognostic factors for survival and functional recovery; b) to evaluate current practice for referral of prostate cancer patients with MSCC; c) to analyze expression of androgen receptor (AR), cell proliferation, apoptosis, and prostate-specific antigen (PSA) in bone metastases with regard to survival after surgery for complications of bone metastases. Patients and Methods: We retrospectively evaluated the hospital records of 68 consecutive patients operated for metastatic spinal cord compression. Tumor tissue from bone metastases was obtained on spinal surgery (54 patients), fracture surgery (4 patients) and biopsy (2 patients), and analyzed by immunohistochemistry. Results: Study I: Mortality and complication rate after surgery was high. Patients with hormone-naïve disease and those with hormone-refractory disease with good performance status and without visceral metastases had more favorable survival. The ability to walk after surgery was related to better survival. Study II: A new score for prognosis of survival after surgery for spinal cord compression includes: hormone status of prostate cancer, Karnofsky performance status, evidence of visceral metastasis, and preoperative serum PSA. The score is simple, tumor specific, and easy to apply in clinical practice. Study III: Our results suggest that delays in diagnosis and treatment may have negative impact on functional outcome. Pretreatment ability to walk, hormone status of prostate cancer, and time from loss of ambulation influenced neurological recovery after surgery for spinal cord compression. Study IV: High nuclear AR immunostaining in bone metastases and high preoperative serum PSA were associated with a poor outcome after metastasis surgery in patients with hormone-refractory prostate cancer. Short-term effect of castration therapy disclosed that nuclear AR immunostaining was decreased and apoptosis was increased, but cell proliferation remained largely unaffected. Conclusion:  Prostate cancer patients with metastatic spinal cord compression represent a heterogeneous group. We identified prognostic factors for survival and functional outcome, which may help clinicians in making decisions about treatment. Our results also implicate the need for development of local and regional guidelines for treatment of patients with spinal cord compression, as well as the importance of information to patients at risk.

Tumor stroma is known to affect tumor growth and metastasis. Inhibiting PDGF signaling, with the goal of depleting PDGFRβ+ stromal cells, is a putative therapeutic approach in this context. PDGFRβ is widely accepted as a pericyte marker and targeting PDGF signaling primarily affects pericytes. Pericyte-endothelial cell interactions modulate angiogenesis and vascular stability in developmental and pathological contexts. Owing to this, pericytes are speculated to be important regulators of tumor growth and metastasis, although their role is not clear.

Five DBVs were synthesized, expressed, purified, and complexed with plasmid DNA for analysis using a range of physicochemical and biological assays. Four of the vectors (RMHT, RM3GT, T-RMG, and T-RMR) formed cationic nanoparticles with plasmid DNA, and were capable of transfecting prostate cancer cells. Conversely RMGT formed highly cytotoxic anionic particles incapable of transfecting cells. RMHT was the most promising vector as it protected the cargo from serum and facilitated specific delivery into castrate resistant prostate cancer cells. RAT, a fusogenic peptide, is composed of an arginine rich cell penetrating peptide, a steric alpha-helicallinker, and a metastatic tumour targeting ligand named TMTP1. RAT formed non-toxic cationic nanoparticles with plasmid DNA that were capable of specifically transfecting castrate resistant prostate cancer cells through receptor-mediated endocytosis. Furthermore, RAT nanoparticies remained stable over a wide temperature range, and protected DNA from degradation by serum endonucleases. RAT was investigated in combination with iNOS plasmids driven by constitutive (CMV) and tumour type specific (hOC) promoters. RAT facilitated iNOS gene expression, and subsequent generation of μM concentrations of the NO free radical. This equated to a maximum of 59% cell kill and less than 74% clonogenicity in prostate cancer cells. Systemic delivery in vivo slightly delayed tumour growth, and therefore future considerations will include dosing strategies ,and circulation enhancement. These bio-inspired gene delivery platforms overcome a multitude of biological barriers and the results generated in this thesis warrant further investigation.

Both mouse double minute (MDM2) and prostate-specific membrane antigen (PSMA) are known to be associated with the progressive properties of cancer. Moreover, overexpression of both molecules has been implicated in an increase in the proliferation, migration and invasion of tumour cells. MDM2 is a negative regulator of tumour suppressor of p53 but also is known to play multiple p53-independent roles in many cancer types. PSMA was originally thought to be solely expressed in prostate tissues and overexpression prostatic cancers; however, recently its expression was reported in various other solid tumours, including those of the breast. Our work showed a possible link between these proteins following knockdown of each molecule in breast cancer cell lines, ZR-75.1 and MDA-MB-231, with targeted siRNA molecules. A decrease of MDM2 and PSMA led to a decrease in the proliferative, adhesive, migratory and invasive capacities of the cell lines. Additionally, knockdown of MDM2 and PSMA led to similar changes in secretion of matrix metalloproteinases (MMPs), with decreases in MMP2 and MMP8 being seen from both breast cell lines investigated. It was then seen that a link between the two protein could be mediated through the phosphorylation status of serine 473 on protein kinase B (AKT). PSMA knockdown in both breast cancer cell lines led to a decrease of AKT phosphorylation and thus a decrease in MDM2 serine 188. Additionally, it was found that MDM2 siRNA leads to an increase in c-JUN serine 63 phosphorylation, and that PSMA siRNA can lead to an increase at the same site, depending on the cell line. These results indicate that MDM2, AKT and PSMA may represent a new pathway which could be targeted for therapy for breast tumours and perhaps other types of cancer.

Class of 2016 Abstract
Objectives: To determine the number of patients treated with eribulin who required an alternate dosing schedule other than “day 1/day 8” due to side effects. Methods: Chart reviews were conducted on all patients who met inclusion criteria. Data collected included patient demographics, history of surgery/radiation, number of past chemotherapy treatments, and lab values prior to each eribulin cycle. Results: A total of 37 patients met inclusion criteria for this study. Ten patients were initially started on the “day 1/day 8” schedule and 3 of those patients required a change to the extended “day 1/day 15” schedule. The remaining 27 patients were started on the extended schedule. Conclusions: The number of patients requiring a dosing schedule change due to side effects was not statistically significant. This finding was due to the fact that the majority of patients were started on an alternate dosing schedule in the beginning of treatment. More extensive studies would be required to determine if a majority of patients would require this alternate dosing schedule, and if this should be initiated in all patients starting on eribulin.

Multiple research studies have demonstrated racial, socioeconomic status (SES), and neighborhood disparities in first-line treatment of colorectal cancer patients, including those with metastatic colorectal cancer. However, disparities in adjunct monoclonal antibody treatment disparities have not been explored. The purpose of this study was to assess racial, SES, and neighborhood disparities in adjunct monoclonal antibody treatment of elderly metastatic colorectal cancer patients. The research was rooted in 3 theories: the fundamental cause theory, the diffusion of innovations theory, and theory of health disparities and medical technology. Data from the SEER-Medicare database and logistic regression were used to assess the relationship between the variables of interest and adjunct monoclonal antibody therapy. In this study, race (p = 0.070), SES (p = 0.881), and neighborhood characteristics (p = 0.309) did not significantly predict who would receive monoclonal antibody therapy. The results demonstrated a potential improvement in historically documented colorectal cancer treatment disparities. Specifically, historical treatment disparities may not be relevant to newer therapies prescribed to patients with severe disease. The difference could be related to improved access to care or a change in treatment paradigm due to the severity of metastatic colorectal cancer. Future studies aimed at understanding the causes of this social change (i.e., reduced treatment disparities) are warranted. Understanding the root cause of the reduced treatment disparities observed in this study could be used to reduce treatment disparities in other cancer populations.

Hypoxia is a naturally occurring event in solid tumours due to aberrant growth rates, unchecked proliferation and unregulated vascularity. Intratumoural hypoxia has been shown to contribute to malignant progression through the regulation of hypoxia-inducible factors (HIFs), transcription factors that in turn regulate a variety of stem-like and promigratory genes. Several recent meta-analyses of cancer patients administered Non-Steroidal Anti Inflammatory Drugs (NSAIDs) over the long term show a reduction in both pre and post-operative risk ratios (RR) demonstrating that NSAIDs have anti-cancer activity. The main function of NSAIDs is as a pain relieving medication by inhibiting cyclooxygenase (COX). This was originally believed to be the cause for their anti-cancer activity as COX enzymes are known to be upregulated in a variety of cancers. However, several groups have contested this theory with a variety of methods including; using NSAID derivatives that do not inhibit COX, using amounts that are either above or below that required to inhibit COX enzymes, and by studies with COX-2 null animals or cell lines. Given the above observations for the anticancer activity of NSAIDs, it was important to understand more precisely the mechanism(s) through which NSAIDs may act as a cancer treatment. Therefore, 5 NSAIDs and 1 non-COX-2 inhibitory NSAID derivative were examined for their mechanisms of action as anticancer drugs in vitro using murine melanoma and breast cancer cell lines as models.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
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Background: Uveal melanoma (UM) is an extremely aggressive disease with approximately 50% of patients developing incurable metastatic disease. Therefore, accurate prognosis of a patient is necessary for closer follow up and the earlier implementation of systemic adjuvant therapies in those most likely to develop metastatic disease. Fortunately, UM can be classified into two distinct molecular classes based on clinically validated gene expression profiling, chromosomal aberrations and specific driver mutations, which accurately predict the metastatic propensity of the primary tumour. However, genetic testing currently requires biopsy of the eye which can lead to serious complications including permanent blindness. Therefore, an alternative source of primary tumour genetic material is needed to avoid these complications. Aims: We proposed that circulating tumour cells (CTCs) are a viable source of tumour genetic material in which patient prognosis could be analysed. Firstly, we aimed to increase the sensitivity of an immunomagnetic enrichment protocol to capture CTCs. Secondly, we aimed to evaluate whole genome amplification methods for accurate single cells analysis to determine the genomic profile of UM cells. The combination of both aims would allow the use of UM CTCs for determining disease prognosis from an easily accessible blood sample. Methodologies: Aim 1 - To refine and evaluate methods for multi-marker immunomagnetic capture of UM CTCs. A tissue microarray (TMA) was created from 1mm cores taken from archived primary UM tissue. Normal tissue and cutaneous melanoma were added as controls. The TMA was stained by immunohistochemistry (IHC) for melanoma, melanocyte, and stem cell markers. Stained tissue was assessed to determine intensity and coverage of staining. In addition to primary UM tissue, five UM cell lines were assessed for the same markers using flow cytometry and immunocytochemistry. Given their high level of staining of UM, 5HT2B, ABCB5, surface gp100 (BETEB), MCAM, and MCSP were coated to immunomagnetic beads and used to determine the retrieval rate of UM cell lines cells spiked into peripheral blood mononuclear cells at a known quantity. CTCs could be detected by immunofluorescent staining of MART1, gp100, and S100β. Aim 2 - Aim 2: To develop methodologies for the detection of genetic markers of metastatic propensity using single UM cells. Single UM cell line cells plus respective bulk genomic DNA whole genome amplified and bulk genomic DNA were amplified using PicoPlex and Repli-G WGA kits to determine each kits’ respective viability of detecting CNVs using low-pass (0.01-0.1x) whole genome sequencing (WGS) on the IonPGM platform. Peripheral blood mononuclear cells (PBMCs) were used as negative controls. In addition, we tested if these methods allowed accurate CNV data after fixation, permeabilisation, and immunostaining. After ensuring cell processing had no significant effects on genomic profile of single cells, blood samples from patients were processed to isolate CTCs from PBMCs. Isolated CTCs were then whole genome amplified using PicoPlex and shallow sequenced using the IonPGM system. Results: We validated several melanoma, melanocyte, and stem cell markers which have been previously shown to be expressed in cancer, cutaneous melanoma, or UM. We found that 5HT2B, and ABCB5, surface gp100 (BETEB), MCAM, and MCSP were highly expressed in primary UM tissue or UM cell lines and were able to immunomagnetically capture UM cell line cells. Concurrently, we validated the use of shallow (0.01x-0.1x depth) whole genome sequencing of single UM cells amplified using the PicoPlex WGA Kit and found that PicoPlex offered a robust method of amplifying single cells that have undergone immunomagnetic isolation, fixation, staining, and capture whilst retaining the original genetic profile of the parent cell line. Upon testing this in a patient, we found a gain of chromosome 8 which is an early event in UM tumourigenesis; aneuploidy of chromosome 8 is a genetic feature that may, with the aid of future studies, delineate patient metastatic risk.

CD4+Foxp3+ regulatory T cells (Tregs) are the main regulators of peripheral tolerance and prevent the development of fatal autoimmune disease in humans and mice. Furthermore, Tregs have also been implicated in suppressing anti-tumour immune responses and are often enriched at sites of primary and metastatic tumours. While studies have shown the effect of Treg ablation on the control of primary tumours, few studies have examined their contribution to metastasis progression. In this thesis I hypothesised that the depletion of Tregs could promote control over metastasis. To address this, a highly metastatic murine mammary carcinoma cell line 4T1 was injected into transgenic mice expressing the diphtheria toxin receptor in Foxp3+ cells. Foxp3+ cells were depleted by administration of diphtheria toxin and the impact of this on growth of primary tumours and metastases was assessed and measured in vitro clonogenic assays. Results of these experiments indicated that Tregdepletion led to control of primary tumour growth and in some mice to control of metastases. Control of metastases was linked to control of primary tumour growth. In order to measure metastasis in vivo, a PET/CT imaging technique was optimized. Primary tumours and large metastatic nodules were successfully imaged in mice using F18 FDG as a radiotracer. However, the studies described herein revealed that micrometastases in mouse lungs were too small to be reliably identified using PET data parameters. CT imaging did however enable detection of increases in tissue density within the lungs, which was suggestive of micrometastases. Data obtained in this way also indicated that Treg-depletion promotes control of metastasis in some mice. Collectively, the findings described in this thesis indicate that Tregdepletion can contribute to control of metastatic disease and should therefore represent an important component of novel immunotherapies.

Metastasis is a complex, multistep process requiring alterations in many aspects of tumour cell behaviour, including changes in invasive potential, motility and adhesive interactions. Alterations in these parameters are achieved by changes in extracellular matrix degrading protease production, extracellular matrix modulation (i.e. 1 integrins, CD44) and cell adhesion molecule production, to name a few. In this series of studies, the effects of several cytokine/growth factors which are present in the complex microenvironment of a solid tumour are investigated in primary and secondary renal and ovarian cell lines. The effects which were investigated in in vitro models examined the invasion potential, motility, adhesion, matrix metalloproteinase production, 1 integrin expression, CD44 expression, fibronectin production and fibronectin splicing. Major findings were that EGF (40ng/ml) in the primary renal carcinoma cell line, A704, increased the tumour cells invasion potential, motility, altered its adhesion to several extracellular matrix components, increased the production of matrix metalloproteinase production, altered the expression of several 1 integrins and increased the expression of CD44 and its variants. In a similar manner, stimulation of a primary ovarian carcinoma cell line, CaOv3 with TGF1 (5ng/ml) resulted in changes which paralleled those of the primary renal carcinoma cell line when stimulated with EGF. Other findings in the study included demonstration of the effect of other cytokine/growth factors (PDGF and HGF) on the aforementioned parameters, however, these were not as comprehensive as EGF and TGF1 on the primary renal and ovarian carcinoma cell lines. Therefore, it is postulated that cytokine/growth factors have an important role in modulating parameters which contribute to the metastatic propensity of tumour cells. In particular EGF and TGF1 may have important respective roles in the dissemination of renal and ovarian tumours.

El càncer colorectal (CCR) representa la tercera causa de mortalitat per càncer en països occidentals, essent les metàstasis la principal causa de mort. Tot i el progrés en les estratègies de prevenció que han disminuït la incidència i mortalitat del CCR, prop d’un quart dels pacients encara són diagnosticats en estadis metastàtics avançats, amb una taxa de supervivència a cinc anys de només el 15%. És per això que la inhibició del desenvolupament de metàstasis actuant sobre cèl·lules mare canceroses incrementarà significativament els beneficis de les teràpies actuals. En col·laboració amb el grup de recerca en Nanobiotecnologia de la UAB, hem desenvolupat nanopartícules proteiques autoensamblables dirigides al receptor CXCR4, la sobreexpressió del qual es correlaciona amb la disseminació tumoral, baixa supervivència, i recurrència en pacients de CCR. L’avaluació preclínica de l’eficàcia i toxicitat del nanoportador T22-GFP-H6 i els seus derivats terapèutics requereix l’ús de models in vivo de CCR disseminats. Amb aquest objectiu, hem desenvolupat models de CCR subcutanis i altament metastàtics que sobreexpressen CXCR4, derivats de la línia cel·lular de CCR SW1417 o de la mostra de pacient SP5. Per tal d’incrementar l’eficiència metastàtica dels models CXCR4+ anteriors, vam implantar ortotòpicament cèl·lules SW1417 amb expressió de luciferasa en el cec de ratolins amb immunodeficiència severa. Els ratolins NOD/SCID (deficients en cèl·lules T i B), presentaren taxes metastàtiques baixes. Per contra, la microinjecció ortotòpica en ratolins NSG (deficients en cèl·lules T, B i NK), replicà el patró de disseminació observat en pacients, provocant la mort dels ratolins i un nombre i mida més gran de metàstasis hepàtiques i pulmonars en comparació als ratolins NOD/SCID. En l’avaluació de la biodistribució de les nanopartícules, tal i com indica l’emissió de fluorescència (al voltant del 70% del total), la T22-GFP-H6 va assolir una alta acumulació selectiva en tumors del model subcutani CXCR4+, mentre que l’acumulació en òrgans no tumorals fou només transitòria. Vam demostrar que l’acumulació al tumor del nanoportador és CXCR4-dependent, perquè el pretractament amb AMD3100, un antagonista de CXCR4, va reduir l’acumulació tumoral. A més, aquesta va incrementar amb la funcionalització del nanoportador amb el pèptid fusogènic HA2, que afavoreix l’escapament endosomal. Per altra banda, vam observar que el nanoconjugat terapèutic T22-GFP-H6-Aur era capaç de mantenir la biodistribució del nanoportador, però el seu efecte antitumoral fou sorprenentment baix. El T22-GFP-H6-Aur només va inhibir metàstasis transcel·lòmiques en un model de CCR altament metastàtic i a més, va activar una resposta immunogènica letal en la seva administració repetida en ratolins poc immunodeprimits. Com a opció terapèutica alternativa, vam substituir la proteïna GFP de la nanopartícula per la toxina PE24 desimmunitzada, per tal de reduir la seva immunogenicitat afegint al mateix temps una potent activitat citotòxica intrínseca. L’administració de dosis baixes de la nanotoxina T22-PE24-H6, va prevenir el desenvolupament de metàstasis limfàtiques i hematògenes en el model de CCR altament metastàtic sense toxicitat. Vam demostrar que la nanotoxina T22-PE24-H6 indueix la mort de cèl·lules tumorals per la via no apoptòtica de la piroptosi. En resum, l’ús de la nanotoxina T22-PE24-H6 podria ser una estratègia prometedora per a l’eliminació selectiva de cèl·lules mare de CCR CXCR4+ en absència de toxicitat sistèmica, aplicable a CCR metastàtics i resistents a la quimioteràpia que s’associïn a la sobreexpressió de CXCR4 i a mecanismes antiapoptòtics.
El cáncer colorrectal (CCR) representa la tercera causa de mortalidad por cáncer en países occidentales, siendo las metástasis la principal causa de muerte. Aunque el progreso en las estrategias de prevención ha reducido la incidencia y mortalidad del CCR, cerca de un cuarto de los pacientes aún son diagnosticados en estadios metastáticos avanzados, con una tasa de supervivencia a cinco años de solamente el 15%. Es por eso, que la inhibición del desarrollo de metástasis actuando sobre células madre cancerosas incrementará significativamente los beneficios de las terapias actuales. En colaboración con el grupo de investigación en Nanobiotecnología de la UAB, hemos desarrollado nanopartículas proteicas autoensamblables dirigidas al receptor CXCR4, la sobreexpresión del cual correlaciona con la diseminación tumoral, baja supervivencia, y recurrencia en pacientes de CCR. La evaluación preclínica de la eficacia y toxicidad del nanoportador T22-GFP-H6 y sus derivados terapéuticos, requiere el uso de modelos in vivo de CCR diseminados. Con este objetivo, hemos desarrollado modelos de CCR subcutáneos y altamente metastáticos que sobreexpresan CXCR4, derivados de la línea celular de CCR SW1417 o de la muestra de paciente SP5. Con el objetivo de incrementar la eficiencia metastática de los modelos CXCR4+ anteriores, implantamos ortotópicamente células SW1417 con expresión de luciferasa en el ciego de ratones con inmunodeficiencia severa. Los ratones NOD/SCID (deficientes en células T y B), presentaron tasas metastáticas bajas. Por contra, la microinyección ortotópica en ratones NSG (deficientes en células T, B y NK), replicó el patrón de diseminación observado en pacientes, provocando la muerte de los ratones y un mayor número y tamaño de metástasis hepáticas y pulmonares en comparación con los ratones NOD/SCID. En la evaluación de la biodistribución de las nanopartículas, tal y como indica la emisión de fluorescencia (alrededor del 70% del total), T22-GFP-H6 alcanzó una alta acumulación selectiva en tumores del modelo subcutáneo CXCR4+, mientras la acumulación en órganos no tumorales fue solamente transitoria. Demostramos que la acumulación en tumor del nanoportador es CXCR4-dependiente, porque el pretratamiento con AMD3100, un antagonista de CXCR4, redujo la acumulación tumoral. Además, la esta incrementó con la funcionalización del nanoportador con el péptido fusogénico HA2, que favorece el escapamiento endosomal. Por otra parte, observamos que el nanoconjugado terapéutico T22-GFP-H6-Aur mantuvo la biodistribución del nanoportador, pero su efecto antitumoral fue sorprendentemente bajo. T22-GFP-H6-Aur solamente inhibió metástasis transcelómicas en un modelo de CCR altamente metastático y además activó una respuesta inmunogénica letal en su administración repetida en ratones poco inmunodeprimidos. Como opción terapéutica alternativa, sustituimos la proteína GFP de la nanopartícula por la toxina PE24 desinmunizada, a fin de reducir su inmunogenicidad añadiendo al mismo tiempo una potent actividad citotóxica intrínseca. La administración de dosis bajas de la nanotoxina T22-PE24-H6, previno el desarrollo de metástasis linfáticas y hematógenas en el modelo de CCR altamente metastático sin toxicidad. Demostramos que la nanotoxina T22-PE24-H6 induce la muerte de las células tumorales por la vía no apoptótica de la piroptosis. En conclusión, el uso de la nanotoxina T22-PE24-H6 podría ser una estrategia prometedora para la eliminación selectiva de células madre de CCR CXCR4+ en ausencia de toxicidad sistémica, aplicable a CCR metastáticos y resistentes a la quimioterapia que se asocien a la sobreexpresión de CXCR4 y a mecanismos antiapoptóticos.
Colorectal cancer (CRC) remains the third cause of cancer-related mortality in Western countries, being metastases the main cause of death. Despite progress in prevention strategies that decreased CRC incidence and mortality, still nearly a quarter of patients are diagnosed at an advanced metastatic stage, with only a 15% five-year survival rate. Thus, inhibition of metastasis development by targeting cancer stem cells, which are associated with cancer dissemination, will significantly increase the benefits of current cancer therapies. In collaboration with the Nanobiotechnology group from de UAB, we developed self-assembling protein-based nanoparticles targeting the CXCR4 receptor whose overexpression correlates with tumor dissemination, poor survival, and recurrence in CRC patients. Preclinical evaluation of the efficacy and toxicity of the T22-GFP-H6 nanocarrier and its therapeutic derivatives required the use of adequate in vivo disseminated CRC models. For that purpose, we generated subcutaneous and highly metastatic models of CRC that overexpress CXCR4, derived from the SW1417 CRC cell line or the SP5 patient sample. In order to increase the metastatic efficiency of previous CXCR4+ CRC models, we orthotopically implanted luciferase expressing SW1417 cells in the cecum of severe immunodeficient mice. NOD/SCID mice (deficient in T and B cells) presented a low metastatic rate. In contrast, orthotopic microinjection in NSG mice (deficient in T, B and NK cells) replicated the dissemination pattern observed in patients, causing mice death and resulting in a higher number and size of hepatic and pulmonary metastases as compared to NOD/SCID mice. In the assessment of nanoparticles’ biodistribution, T22-GFP-H6 achieved a highly selective tumor uptake in a CXCR4+ CRC subcutaneous model, as detected by fluorescent emission (around 70% of the total), while displaying only transient accumulation in non-tumor organs. We demonstrated that the nanocarrier tumor accumulation was CXCR4-dependent because pre-treatment with AMD3100, a CXCR4 antagonist, reduced tumor uptake. Furthermore, tumor accumulation was increased by the functionalization of the nanocarrier with the fusogenic HA2 peptide, which promotes endosomal escape. On the one hand, we observed that the therapeutic nanoconjugate T22-GFP-H6-Aur maintained the nanocarrier’s biodistribution but its antitumoral effect was surprisingly poor. T22-GFP-H6-Aur inhibited only tanscelomic metastasis in a highly metastatic CRC model, while activating a lethal immunogenic response when repeatedly administered in low immunosuppressed mice. As an alternative therapeutic option, we replaced the GFP protein in the nanoparticle by the de-immunized PE24 toxin, to reduce its immunogenicity while promoting a potent and intrinsic cytotoxic activity. The administration of low doses of the T22-PE24-H6 nanotoxin prevented the development of lymphatic and hematogenous metastasis in the highly metastatic CRC model without toxicity. We demonstrated that the T22-PE24-H6 nanotoxin induced cancer cell death through the non-apoptotic pathway, pyroptosis. In conclusion, the use of the T22-PE24-H6 nanotoxin could be a promising strategy to selectively eliminate CXCR4+ CRC stem cells in the absence of systemic toxicity, applicable to chemotherapy-resistant and disseminated CRC associated with the upregulation of CXCR4 and antiapoptotic mechanisms.