Tesis sobre el tema "ER-positive"

Identification of positive regulators of T-cell immunity induced during autoimmune diseases is critical for developing novel therapies. The endoplasmic reticulum resident ubiquitin ligase Hrd1 has recently emerged as a critical regulator of dendritic cell antigen presentation, but its role in T-cell immunity is unknown. Here we show that genetic deletion of Hrd1 in mice inhibits T-cell proliferation, production of IL-2, and differentiation of Th1 and Th17 cells, and consequently protects mice from experimental autoimmune encephalomyelitis. Hrd1 facilitates T-cell proliferation by the destruction of cyclin-dependent kinase inhibitor p27(kip1), and deletion of p27(kip1) in Hrd1-null T-cells rescues proliferative capacity but not the production of cytokines, including IL-2, IFN-γ and IL-17. T-cell expression of Hrd1 is higher in patients with multiple sclerosis than in healthy individuals, and knockdown of Hrd1 in human CD4(+) T cells inhibits activation and differentiation to Th1 and Th17 cells. Our study identifies Hrd1 as a previously unappreciated positive regulator of T cells and implies that Hrd1 is a potential therapeutic target for autoimmune diseases.

Breast cancer is the most frequently diagnosed cancer and remains the second leading cause of death among women in Europe and United States. In this malignancy, metastasis remains to be an incurable condition, and therefore the major cause of death. Metastatic lesions can appear within a wide time ranging from months to years or decades after primary tumor resection. In particular, in the estrogen receptor (ER) positive breast cancer subgroup metastatic latency continues to be a major challenge for the researchers, clinicians and patients. This thesis reports the identification and functional analysis of molecular mechanisms involved in the latency of ER positive breast cancer. For that purpose we based our research on a comprehensive approach that relies on genetically engineered human breast cancer cells, experimental mouse models, unbiased whole- genome screen and clinical data. The first part of the thesis describes a novel mouse model of breast cancer dormancy. We showed that metastatic cells home the bone and enter the latency phase as micrometastatic lesions where tumor growth is restricted mainly due to the equilibrated ratios of cell proliferation and cell death. This experimental mouse model was used to identify genes relevant for long- latent relapse. To this end, we performed in vivo loss-of- function shRNA screening. In the screening we challenged a whole-genome library of shRNA to uncover genes whose depletion negatively regulates dormancy. Among the candidate genes revealed by the screen we focused on MSK1 as a long-latent metastasis regulator. The in vivo and in vitro validation results indicate that MSK1 plays a role in homing and differentiation of metastatic cells. We showed that MSK1 promotes the expression luminal transcription factors - FOXA1 and GATA-3. Therefore, MSK1 depletion is beneficial for metastatic cells leading to a partial phenotype shift towards a more aggressive and poorly differentiated basal population. Furthermore, our data suggest that MSK1 may be involved in metastatic cell plasticity by remodeling the chromatin. Importantly, low MSK1 gene expression levels associate with early metastasis in ER positive breast cancer.
El cáncer de mama es el tipo de cáncer más frecuentemente diagnosticado, siendo la segunda causa de muerte entre las mujeres de Europa y Estados Unidos. En esta enfermedad, la metástasis sigue siendo incurable, y por ello es la principal causa de muerte. Las lesiones metastásicas pueden aparecer dentro de un amplio periodo de tiempo que va desde meses hasta años o incluso décadas después de la extirpación del tumor primario. Concretamente, en el subgrupo de cáncer de mama RE positivo, este largo periodo de latencia es el principal desafío para investigadores, médicos y pacientes. En esta tesis se muestra la identificación y el análisis funcional de mecanismos moleculares implicados en la latencia del cáncer de mama RE positivo. Para este propósito, nuestros estudios se han llevado a cabo mediante una estrategia experimental basada en líneas celulares de cáncer de mama genéticamente modificadas, modelos experimentales de ratón, análisis global del genoma y datos clínicos. La primera parte de la tesis describe un novedoso modelo de ratón de dormancia de cáncer de mama. Observamos que, en nuestro modelo, las células metastásicas llegan al hueso y entran en una fase de latencia en forma de lesiones micrometastásicas en la que el crecimiento del tumor se ve impedido, principalmente debido a que la tasa de proliferación celular se iguala a la tasa de muerte celular. Este modelo experimental de ratón se usó para identificar genes relevantes en el proceso de latencia y por tanto en la recurrencia a largo plazo. Para ello, llevamos a cabo un análisis in vivo de pérdida de función con shRNA. En este análisis utilizamos una amplia librería de shRNA para descubrir genes cuya eliminación regula la dormancia de manera negativa. Entre los genes candidatos identificados en este análisis nos focalizamos en MSK1 como un regulador de la metástasis latente. La validación in vitro e in vivo indica que MSK1 juega un papel en el anidamiento y la diferenciación de las células metastásicas.

In clinical practice, patients with advanced ER+/HER2+ BC are treated with a combination of anti-HER2 and chemotherapy as first choice but it is clear that a proportion of these derive prolonged benefit from the combination of hormonal therapy (HT) and anti-HER2 therapy (AH2T). Preclinical evidence from cellular models of BC indicates that ER and HER2 pathways are strictly interdependent and that targeting both pathways in ER+/HER2+ BC might be an effective therapeutic strategy; few trials investigated this combination showing a significant, albeit modest, clinical improvement. CDK4/6 pathway acts downstream of both the ER and HER2 pathways. CDK4/6 inhibitors (PD)have shown synergistic activity with HT or AH2T but data on the combination of PD with both HT and AH2T in ER+/HER2+ BC are lacking. PD activity is dependent on an intact RB pathway; therefore we analyzed whether Cyclin D/Rb/E2F pathway might help discriminating the subgroup of ER+/HER2+ BC resistant to PD therapy. We developed a gene expression signature of Rb-loss-of-function, the RBsig, that included a final set of 87 genes that is predictive of resistance to PD in BC cell lines. An in-silico analisys on a meta-dataset of neoadjuvant trials has also shown a good performance of RBsig in prediction of a better response to neoadjuvant chemotherapy. These data suggest RBsig as a potential marker for identifying patients with differential benefit from chemotherapy or HT+PD, both in combination with AH2T.

Over 1 million women a year are diagnosed with Breast Cancer. The majority, approximately 70% express the oestrogen receptor (ER). ER positive breast cancer has historically been perceived as a ‘good cancer’, although many woman with ER+ breast cancer still succumb to their disease and globally breast cancer is the leading cause of female cancer deaths. The advent of gene expression profiling and the definition of the molecular instrinsic subtypes has defined at least two subtypes of ER positive breast cancers (luminal A and luminal B) that differ markedly in terms of biological behaviour, response to adjuvant therapies and most importantly patient outcome. The focus of this research is ER+ breast cancer and targeting patient therapy in this heterogeneous group. This work attempts to translate our understanding of the biology of the ER and cell signalling interactions to aid the correct identification of patients for both current therapy and more novel therapeutic approaches. Following a hypothesis generating pilot study examining whether the level of ER influences response to endocrine therapy, 557 formalin fixed paraffin embedded (FFPE) breast cancer specimens retrieved at time of definitive surgery from early breast cancer patients with available accurate 15 years follow up data were analysed to measure ER, Progesterone receptor (PgR), HER2 and Ki67 expression using immunohistochemistry. Tumour expression of ER, PgR and the combined endocrine receptor (CER), which considers the expression level of both hormone receptors and hypothesised to more accurately quantify endocrine responsiveness by acting as a surrogate marker of a functioning ER signalling pathway, were analysed. The results suggest that in this cohort of ER+ endocrine treated patients CER is a better predictor of endocrine response than either the ER of PgR independently. The CER was thereafter utilised as a surrogate marker of oestrogen receptor signalling pathway to develop a scoring system which included HER2 IHC expression and tumour histological grade, as surrogate markers of the 3 key pathways (ER signalling, HER2 signalling and proliferation). These were chosen as previous studies comparing various gene prognostic profiles indicate commonality in sampling groups of the genes representing their activation. The scoring system, named the Clinical Outcome Score (COS) was developed to represent a pragmatic equivalent of gene prognostic profiles utilising currently routinely measured tumour markers. We hypothesised that COS as an indicator of tumour biology may aid identification of risk in the very challenging group, ER+/HER2 negative patients with intermediate grade and low disease burden, and may help guide adjuvant therapy decisions particularly the indication for chemotherapy . In this exploratory analysis, the distribution of COS scores (2-10) followed a linear response with a notable separation between low scores (2-4) and high scores (5-10). Importantly, when analysed in combination with tumour burden, low COS may help identify patients with nearly 100% long term survival, however in all analysis high COS was associated with a highly significant poorer outcome in terms of early recurrence, late recurrence and 15 year breast cancer specific survival. This group of high risk ER+ breast cancer patients represent a real challenge (and concern) in the treatment of early breast cancer, as there is increasing evidence that ER+ tumours are relatively chemo-in senstive and the response to chemotherapy agents is limited. As a secondary analysis, within our cohort of ER+/ HER2- endocrine treated patients we retrospectively analysed the benefit of chemotherapy in patients with low and high COS scores and the results indicate lack of benefit in the cohort of patients diagnosed 1995-1998. Investigating novel therapeutic targets focusing on the subtypes of breast cancer, and tumour biology involved in endocrine resistance is now beginning to take precedence in breast cancer research. Two potential new therapeutic targets in ER+ breast cancer were studied. The first is the sodium iodide symporter, NIS, a transmembrane glycoprotein which has been exploited for the safe delivery of radio-iodide in the treatment of thyroid cancers for many years. NIS is expressed in many breast cancers, however most breast cancers expressing NIS lack functional uptake as demonstrated by scintography studies and in vivo animal work. In vitro results suggest that the ER is important in NIS regulation and function. In addition MAPK and PI3K-Akt signalling pathways may have a role in NIS regulation- both these pathways are often activated in ER+ breast cancer and known to have extensive crosstalk with the ER. Utilising ER+ and ER negative breast cancer cell lines we examined NIS function following gene delivery with a human NIS (hNIS) transfected plasmid and assessed function and expression of NIS following ER knockdown by siRNA. Our results suggest that the ER phenotype is important but not necessarily the ER per say. We examined NIS expression in a mixed ER+ and ER- cohort (n=50) of patient tumour samples using real time RT-PCR, and report high levels of NIS mRNA expression was limited to ER+ breast tumours. Prompting analysis of NIS expression, cellular location and correlations with cell signalling proteins in 300 ER+ breast cancers using IHC . Significant correlations were identified with key members of the PI3K-Akt and MAPK supporting their role in NIS regulation in vivo. Importantly, in both patient cohorts NIS was found to be significantly associated with poor outcome, and we hypothesis that this is an effect of enhanced growth factor signalling and activation of pathways in biologically more aggressive ER+ cancer (ER+/PgR-) may also regulate NIS and suggest future directions of research. Lastly, as a pilot study expression of Src kinase, a non receptor tyrosine kinase implicated in tamoxifen resistance and breast cancer virulence, was analysed by IHC in the ER+ breast cancer patient cohort. Interestingly nuclear Src kinase was found to be associated with improved outcome and hypothesise that Src Kinase expression in breast cancer may have varying roles in the different subtypes of breast cancer, an important consideration as Src Kinase inhibitors are currently in clinical trials. This pilot study formed a hypothesis that was subsequently examined in another student’s PhD thesis.

Cornichon (CNI) proteins are a conserved family of proteins among eukaryotes, from Erv14 in the yeast Saccharomyces cerevisiae to CNI homologs (CNIHs) in mammals and plants. Erv14 functions as a cargo receptor of coat protein complex II (COPII) for protein trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus, en route to their final destinations. By interacting with specific cargo proteins, CNI proteins regulate key steps of embryo polarity in Drosophila, budding in yeast, and synaptic transmission in the mammalian brain. However, we have very limited understanding of plant CNIHs. Positive-strand RNA viruses assemble their viral replication complexes (VRCs) at specific host organelle membranes. With a better understanding of host factors involved in targeting viral replication proteins to the preferred organelles, we expect to block trafficking of viral replication proteins and thus, viral infection, by manipulating the required host proteins. Brome mosaic virus (BMV) is a model of positive-strand RNA viruses and its replication can be recapitulated in yeast. Importantly, BMV replication protein 1a is the only required viral protein to form VRCs at the perinuclear ER membrane in yeast. I demonstrate that Erv14 and COPII coat proteins are required for targeting BMV 1a to the perinuclear ER in yeast, suggesting a novel function of COPII vesicles in protein trafficking to the perinuclear ER membrane and in the BMV VRC formation. As for cellular functions, I show that plant CNIHs complement the defective distribution of BMV 1a in yeast mutant lacking Erv14. Taking advantage of Arabidopsis thaliana knockout mutants and knockdown of gene expression in Nicotiana benthamina, I also discover that CNIHs unexpectedly play crucial roles in pollen development, infection of a bacterial pathogen, and maintenance of ER tubules. I further confirm that CNI proteins are also required for maintaining ER tubules in yeast, suggesting a novel and conserved role in shaping ER morphology. Therefore, these findings indicate the functional diversity and redundancy of CNI proteins in key cellular processes and suggest a novel strategy to control plant pathogenic viruses and bacteria by manipulating plant CNIHs.
Ph. D.

Positive-strand RNAviruses invariably assemble their viral replication complexes (VRCs) by remodeling host intracellular membranes. How viral replication proteins are targeted to specific organelle membranes to initiate VRC assembly remains elusive. Brome mosaic virus (BMV), whose replication can be recapitulated in Saccharomyces cerevisiae, assembles its VRCs by invaginating the outer perinuclear endoplasmic reticulum (ER) membrane. Remarkably, BMV replication protein 1a (BMV 1a) is the only viral protein required for such membrane remodeling. We show that ER-vesicle protein of 14 kD (Erv14), a cargo receptor of coat protein complex II (COPII), interacts with BMV 1a. Moreover, the perinuclear ER localization of BMV 1a is disrupted in cells lacking ERV14 or expressing dysfunctional COPII coat components (Sec13, Sec24 or Sec31). The requirement of Erv14 for the localization of BMV 1a is bypassed by addition of a Sec24-recognizable sorting signal to BMV 1a or by overexpressing Sec24, suggesting a coordinated effort by both Erv14 and Sec24 for the proper localization of BMV 1a. The COPII pathway is well known for being involved in protein secretion; our data suggest that a subset of COPII coat proteins have an unrecognized role in targeting proteins to the perinuclear ER membrane.

Cytochrome P450 enzymes (P450s) are involved in cancer development and treatment due to their roles in the oxidative metabolism of various endogenous (e.g. oestrogen) and exogenous (e.g. tamoxifen) compounds. It is well-known that intermediate P450 metabolites derived from oestrogen metabolism are associated with breast carcinogenesis. The main aim of this project was to profile the cytochrome P450 and P450-regulatory nuclear receptor mRNAs in a series of breast cancer cell lines (BCCs) and compare this profile with normal breast cells. This study used the qualitative reverse transcriptasepolymerase chain reaction (RT-PCR) to detect mRNA expression of target genes. Results showed CYP1B1, CYP2D6, CYP2J2, CYP2R1, CYP2U1 and CYP4X1 mRNA to be present in all cell lines. CYP2A6, CYP2C8, CYP2C18, CYP2F1 and CYP4Z1 mRNA were expressed in oestrogen receptor (ER)-positiveCaucasian and ER-negative Afro- Caribbean BCCs. Although no differences in P450 mRNA were observed between the different ethnic groups, these preliminary findings suggest potential similarities in the ERpositive Caucasian and ER-negative Afro-Caribbean BCCs which warrant further investigation The CYP4Z1 PCR product was identified as two distinct bands. Specific primer sets were used to demonstrate potential intron retention in CYP4Z1. Using established in vitro models for the study of regulatory mechanisms of CYP4Z1, T47D and ZR-75-1 breast cancer cell lines were used to determine the appropriate nuclear receptors (i.e. progesterone receptor, glucocorticoid receptor or peroxisome proliferator-activated receptor alpha ). These findings suggest that there may be an alternative receptor mechanism involved in CYP4Z1 mRNA induction in these cells. In conjunction, pre-treatment of these two cell lines with the RNA synthesis inhibitor actinomycin D followed by the agonists showed a significant reduction (p < 0.05) of CYP4Z1 mRNA levels and inhibited CYP4Z1 induction by either progesterone, dexamethasone or pirinixic acid, indicating that these agonists have effects on CYP4Z1 mRNA transcription or stability. In contrast, cycloheximide differentially affected the level of CYP4Z1 mRNA induction by these agonists. Taken together, these results suggest that CYP4Z1 mRNA induction in T47D and ZR-75-1 is mediated through differential cell type specific regulatory mechanisms and there is evidence for differential regulation of the splice variants.

La chimiothérapie néoadjuvante (CNA) est utilisée dans les cancers du sein agressifs ou localement avancés (CS). Au delà des bénéfices cliniques, elle représente une opportunité pour monitorer in vivo la sensibilité d’une tumeur à un traitement.A partir de l’analyse de sets de données de patients traités par CNA, nous souhaitons identifier des mécanismes associes à la résistance ou sensibilité au traitement. Dans la première partie, nous avons évalué des paramètres, cliniques, anatomopathologiques et transcriptomiques. Nous avons démontré que des éléments non explorés comme la présence d’embols après CNA revêtaient une information pronostique importante. Dans une 2ème partie, nous avons analysé l’impact de l’infiltrat immunitaire dans le cancer du sein, et avons décrit les changements observés entre des échantillons avant et après CNA. Nous avons montré que l’impact pronostique des TILs était différent avant et après CNA, et était opposé dans les CS triple négatif ou HER2-positif. Finalement, nous avons analysé l’impact des comédications pendant la CNA. Nous avons trouvé des effets positifs – via l’augmentation de l’infiltrat immunitaire et la réponse au traitement – et des effets négatifs avec des effets délétères dans certains sous groupes de patients. En conclusion, la situation néoadjuvante représente une plateforme pour générer et potentiellement valider des hypothèses de recherche. La mise à disposition de jeux de données de patients traités par chimiothérapie néoadjuvante constituerait une ressource majeure pour accélérer la recherche contre le cancer du sein
Neoadjuvant chemotherapy (NAC i.e. chemotherapy before surgery) is increasingly being used for aggressive or locally advanced breast cancer (BCs). Beyond clinical benefits, it represents an opportunity to monitor in vivo sensitivity to treatment. Based on the analysis of datasets of BCs patients treated with NAC, we aimed at identifying mechanisms associated with resistance or sensitivity to treatment.In the first part, we evaluated biological, clinical, pathological and transcriptomic patterns. We demonstrated that unexplored pathological features such as post-NAC lymphovascular invasion may carried an important prognostic information.In a second part, we analyzed impact of imune infiltration in BC and we described extensively the changes of tumor infiltrating lymphocytes (TILs) between pre and post-NAC samples. We showed that the prognostic impact of TILs was different before and after NAC, and was opposite in TNBC and HER2-positive BCs. Finally, we investigated the impact of comedications use during NAC. We found both positive effects - while enhancing immune infiltration and response to treatment - and negative effects with deleterisous oncologic outcomes in specific patients subgroups. In conclusion, the neoadjuvant setting represents a platform to both generate and potentially validate research hypotheses aiming at increasing the efficacy of treatment. The public release of real-life datasets of BC patients treated with NAC would represent a major resource to accelerate BC research

碩士
國立中興大學
生物化學研究所
104
Past studies have pointed out that when estrogen binds with its receptor estrogen receptor, ER, the event induces gene expression sufficient for cell division and breast cancer progression. Therefore, understanding how to reduce the expression of ER is the most importance for treatment. Luteolin, a flavone found in some vegetables, has been reported to exhibit antioxidant, antiinflammatory, and anticancer activities. Luteolin also can inhibit angiogenesis and induce apoptosis of cancer cells but the molecular mechanisms of it is not clear. In this study, we used luteolin to treat breast cancer cell line MCF7 and T47D which overexpress the estrogen receptor. To address the hypothesis that luteolin could inhibit ER-α expression, cell survival and cancers metastasis via the inhibition of ILK signailing pathway in ER-α-positive breast cancer cells as well as discuss the molecular mechanisms of luteolin. Our data indicates that luteolin could inhibit ER-α-positive breast cancer cell survival through the inhibition of ER-α expression of protein and mRNA by using Western blot and qPCR. From previous studies we know that ER-α is regulated by YB-1, which is regulated by Twist, and they were be regulated by ILK related signailing pathway. Thence, this study uses luteolin treatment ILK signal pathway-related protein, and then observed performance. Finally study confirmed luteolin can inhibit the expression of ER-α through inhibition of ILK related pathways, and further inhibiting the survival of ER- positive breast cancer cells. According to previous studies, twist and metastasis of cancer are closely related. So we used wound healing assays and invasion assays to confirm that luteolin can inhibit the metastasis and invasion of ER- positive breast cancer cell lines. So luteolin is considered to have cancer chemopreventive and chemotherapeutic potential for ER- positive breast cancer cells.

碩士
國立中興大學
生物化學研究所
106
Estrogen receptor-α is a ligand-activated transcription factor implicated in breast cell and plays an important role in cancer cell proliferation and metastasis. The binding of estrogen and the estrogen receptor-α in the cytoplasm causes two estrogen receptors to dimerize spontaneously, and translocated into the nucleus. Citrus fruits and its related genera contain about 300 limonoids have been known nowadays. In the present study, we found that limonoids inhibit breast cancer cell proliferation. Interestingly, the effect of limonoids inhibited the growth of ER-α positive breast cancer cells was better than the ER-α negative breast cancer cells. Therefore, we aimed at the relationship between limonoids and ER-α signaling pathway. Our data indicated that among the abundant of the limonoids, the cytotoxic effect of Nomilin was stronger than Limonin in breast cancer cells. Moreover, we demonstrated that Nomilin reduced the expression of ER-α protein levels in ER-α positive breast cancer cells. Nomilin repressed cell proliferation through the inhibition of AKT/mTOR signaling pathway in MCF7 cells. We also found that Nomilin inhibited ER-α positive breast cancer cell metastasis through the regulation of E-cadherin. According to previous studies, Twist was transcription factor that can induced transcription of Y-box binding protein-1. We found that Nomilin reduced the expression of Y-box binding protein-1 through the inhibition of ER-α. This work sheds light on the pathways by which Nomilin inhibits ER-α positive breast cancer cells proliferation and metastasis.

The estrogen receptor (ER) is a ligand dependent transcription factor that regulates the expression of a number of gene products involved in the proliferation and progression of breast neoplasms. Given the critical functions of the ER in the breast was well as the prognostic relationships between ER expression and breast cancer, it is necessary to elucidate the mechanism(s) responsible for the failed expression of the ER gene in nearly 40% of all breast tumors. Based upon information from other eukaryotic genes, it is known that the maintenance of the integrity of cis-elements located in the upstream regulatory region is crucial for the proper regulation of gene expression. Thus, we have hypothesized that sequence alteration in the upstream regulatory region of the ER gene may account for the failure of the ER gene to be transcribed in a number of breast tumors Sequence analyses of the upstream regulatory region of the ER gene from both the ER-positive MCF-7 and the ER-negative MDA-MB-231 breast cancer cell lines revealed a number of alterations/variations, These sequence alterations/variations included base pair additions, deletions, and substitutions. When the functional importance of these alterations/variations was tested utilizing chloramphenicol acetyl transferase (CAT) assays, two of the alterations located in the upstream region of the ER-negative MDA-MB-231 cells, a guanine insertion at -1976 and a cytosine to thymine transition at -1752, were determined to be associated with a significant 50% decrease in transcriptional activity upon comparison to control levels. From subsequent studies, this decrease in transcriptional activity was determined to be dependent upon the concurrent presence of both alterations as well as upon the exposure to estrogen and progesterone. Gel mobility analyses of DNA segments containing either of these alterations revealed no variations in binding patterns, thereby suggesting that these alterations do not independently affect the binding of trans-acting factors In addition, a number of alterations were identified between these two breast cancer cell lines and the sequence that has been previously published for leukocytes. The most prominent of these was a cluster of 17 base pair insertions located approximately 300 base pairs upstream of the Exon 1' upstream open reading frame (ORF). The role of these base pair insertions in the regulation of the expression of Exon 1' sequences was tested utilizing Northern blot analysis, RNase protection analysis, RT-PCR, and subsequent sequencing analysis upon additional breast cancer cell lines. The results of these efforts indicate that the cluster of 17 base pair insertions is not a factor in determining the expression of the Exon 1' transcript. However, the expression of this alternative ER transcript does appear to be related to tissue culture conditions and cell passage number
acase@tulane.edu

Tese de mestrado, Ciências Biofarmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2018
Breast cancer is one of the most common forms of cancer worldwide and the second leading cause of cancer-related death. Oestrogen receptor positive (ER+) breast cancer makes up the majority of breast cancer cases, where oestrogens play a key role in promoting cancer cell growth and tumour progression. Besides the therapeutic success of the endocrine therapies and their clinical effectiveness in the treatment of this type of tumours, the side effects associated with these therapies, along with the development of endocrine resistance, emphasise the importance and the need to find new and improved therapies. In recent years, several studies on different cancer cell models, including breast cancer, have demonstrated and enhanced the anticancer properties of cannabinoids. Considering this, in this study, the in vitro effects of the phytocannabinoids, cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), as well as of the endocannabinoid anandamide (AEA), were investigated on an ER+ breast cancer cell line that overexpresses the enzyme aromatase (MCF-7aro) and on a resistant ER+ breast cancer cell line (LTEDaro), which mimics the late-stage of resistance to endocrine therapy. A non-tumour fibroblastic cell line (HFF-1) was also used to explore whether these compounds are toxic towards non-cancerous cells. Our results demonstrate that AEA, CBD and THC are non-toxic towards the non-cancerous cells, and have the ability to reduce MCF-7aro cell viability and inhibit and decrease the levels of aromatase, as well as ERα, in these cells. Moreover, in MCF-7aro cells, these compounds also caused cell cycle arrest and induced apoptotic cell death in, through the mitochondrial pathway. Curiously, AEA and CBD also caused an up-regulation of ERβ levels in these cells, which along with aromatase inhibition may be a therapeutic advantage for this type of tumour. Contrary to CBD, the effects induced by THC on these cells were dependent on cannabinoid receptors CB1 and CB2, while for AEA were only CB2-dependent. In addition, it was also shown that CBD induced autophagy in MCF-7aro cells as a promoter mechanism of apoptosis. Interestingly, the resistant LTEDaro cells were sensitive to cannabinoid treatment. In conclusion, these cannabinoids show promising anti-tumour properties regarding ER+ breast cancer treatment, and even in cases of late-stage resistance. Thus, the results from this study will provide relevant information for future research involving cannabinoids and cancer, which may lead to their potential use in the clinic for the treatment of this disease.
O cancro de mama é uma das formas mais comuns de cancro em todo o mundo e a segunda principal causa de morte relacionada com cancro. A maioria dos casos de cancro de mama são recetor de estrogénio positivo (ER+), onde os estrogénios desempenham um papel fundamental na promoção do crescimento e progressão do tumor. No entanto, apesar do sucesso terapêutico e da eficácia clínica das terapias endócrinas utilizadas neste tipo de tumores, os efeitos adversos associados a estas terapias, juntamente com o desenvolvimento de resistência endócrina, realçam a importância e a necessidade da procura de novas terapias mais eficazes. Nos últimos anos, vários estudos em diferentes modelos celulares, incluindo cancro de mama, demonstraram a possível relevância das propriedades anticancerígenas dos canabinóides. Tendo isto em consideração, neste trabalho foram estudados os efeitos in vitro dos fitocanabinóides, canabidiol (CBD) e Δ9-tetrahidrocanabinol (THC), assim como do endocanabinóide anandamida (AEA), numa linha celular de cancro de mama ER+ que sobreexpressa a enzima aromatase (MCF-7aro) e numa linha celular resistente de cancro de mama ER+ (LTEDaro), que mimetiza a fase tardia da resistência à terapia endócrina. Uma linha celular de fibroblastos não-tumoral (HFF-1) foi também utilizada, de forma a explorar se estes compostos são tóxicos para células não-cancerígenas. Os nossos resultados demonstram que AEA, CBD e THC não são tóxicos para as células não-cancerígenas, contudo têm a capacidade de reduzir a viabilidade das células MCF-7aro e inibir e diminuir os níveis da aromatase, bem como do ERα. Além disso, em células MCF-7aro, estes compostos causaram uma paragem do ciclo celular e induziram a morte celular por apoptose, através da via mitocondrial. Curiosamente, AEA e CBD também causaram um aumento dos níveis do ERβ nessas células, o que, juntamente com a inibição da aromatase, poderá ser uma vantagem terapêutica para esse tipo de tumores. Ao contrário do CBD, os efeitos induzidos pelo THC nestas células foram dependentes dos recetores canabinóides CB1 e CB2, enquanto que para a AEA foram apenas dependentes do CB2. Para além disso, foi demonstrado também que o CBD induziu autofagia nas células MCF-7aro como um mecanismo promotor da apoptose. Curiosamente, as células resistentes LTEDaro foram sensíveis ao tratamento com os canabinóides. Em conclusão, estes canabinóides apresentaram propriedades anti-tumorais promissoras para o tratamento do cancro de mama ER+, até mesmo em casos de uma resistência tardia. Assim, os resultados deste estudo poderão fornecer informações relevantes para pesquisas futuras envolvendo canabinóides e cancro, o que poderá conduzir ao seu potencial uso na clínica para o tratamento desta doença.
This project had the financial support from Fundação para a Ciência e Tecnologia (FCT), through the attribution of the Post-Doc grant to Cristina Amaral (SFRH/BPD/98304/2013) and by the project FCT/MEC (UID/MULTI/04378/2013 – POCI/01/0145/FEDER/007728), co-financed by FEDER and by national funds, under the Partnership Agreement PT2020.

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2016
O cancro da mama é uma doença heterogénea determinada por várias características clínicas e patológicas, incluindo parâmetros histológicos e marcadores moleculares como o recetor de estrogénio (RE), recetor de progesterona (RP) e o recetor 2 do fator de crescimento epidérmico humano (HER2). A classificação de cancro da mama em três principais grupos com interesse clínico reflete essencialmente a classificação molecular: subtipo Luminal, Luminal A (RE+ e/ou RP+, HER2-, Ki67 baixo) e Luminal B (RE+ e/ou RP+, HER2+ ou Ki67 elevado); subtipo com sobre-expressão de HER2 (RE-, PR-, HER2+); e subtipo triplo negativo (RE-, RP- HER2-). Os tumores da mama positivos para o RE são os mais frequentes (cerca de 80%) e são os que apresentam melhor prognóstico. No entanto, cerca de 30% irão progredir com metastização à distância, resultando num aumento da taxa de mortalidade. O cancro da mama RE+ é dependente de estrogénios que ativam o RE, um factor de transcrição importante. Neste contexto, o gene YBX1, que codifica a proteína YB-1 (Y-box binding protein 1), foi recentemente identificado como parte de um preditor de mau prognóstico em cancro da mama dependente de RE. A proteína YB-1 parece estar envolvida em todos os hallmarks do cancro, sendo um indicador de mau prognóstico, recidiva, invasão e resistência a fármacos. Em cancro da mama, a proteína YB-1 encontra-se normalmente sobreexpressa, e tem sido associada com a progressão da doença e com a ausência de RE e RP. Devido à sua capacidade de ligação aos ácidos nucleicos, a proteína YB-1 é um regulador importante de transcrição e tradução de vários genes relacionados com o cancro, como ERBB2, cyclin A/B1, E2F, MDR1, MYC, PIK3CA, e também um componente principal das partículas de ribonucleoproteínas mensageiras, contribuindo assim para a sua estabilização e regulação de tradução. O papel funcional da proteína YB-1 parece ser dependente da sua localização celular, que é estreitamente regulada. Em condições fisiológicas normais a proteína é maioritariamente citoplasmática, embora possa também ser encontrada no núcleo. A translocação nuclear surge, geralmente, em resposta a um stress ou estímulo, e pode ocorrer por clivagem proteolítica ou fosforilação da proteína. A proteína YB-1 é fosforilada no resíduo de serina 102 localizada no cold shock domain, pela cinase p90 ribossomal S6 e pela cinase Akt serina/treonina. Recentemente, utilizando um modelo animal de xenotransplantes ortotópicos de cancro da mama RE+ vs. RE-, com modulação dos níveis de estradiol, o nosso grupo mostrou que o gene YBX1 se encontra sobrexpresso em tumores RE+ que cresceram na presença de 17β-Estradiol (E2), comparativamente a tumores RE+ que cresceram na ausência de E2. Assim, o objetivo deste projeto foi explorar a relevância biológica e clínica da regulação de YB-1 mediada pelo RE em cancro da mama. A nossa hipótese é que as terapias dirigidas ao RE possam afectar o gene YBX1, levando a uma diminuição da sua expressão e que a cessação da hormonoterapia possa desencadear um aumento da sua expressão e consequentemente da proliferação tumoral. Para testar a nossa hipótese usámos uma abordagem translacional, baseada em modelos celulares de cancro da mama e na análise de grupos clínicos relevantes de pacientes com cancro da mama. O estudo do efeito do E2 na expressão do gene YBX1 na linha celular de cancro da mama positiva para RE MCF-7, mostrou que a adição de E2 ao meio de cultura per se não é suficiente para alterar os níveis de expressão de YBX1. De facto, a análise de um painel de 124 genes regulados pelo RE revelou que a sua regulação in vitro difere substancialmente do que foi observado in vivo, o que indica que existirão outros fatores e/ou mecanismos envolvidos, quer sejam intrínsecos da célula ou derivados do hospedeiro, que poderão afetar a relação entre o RE e YBX1. O efeito do E2 nos níveis de YB-1 foi também avaliado ao nível da proteína por Western Blot e imunofluorescência, utilizando anticorpos específicos contra YB-1 total e a forma fosforilada no resíduo de serina 102 (p-YB-1). O estímulo com E2 induziu um aumento da p-YB-1 e da sua localização nuclear. Este efeito mostrou ser dependente do RE, uma vez que foi inibido pelo tamoxifeno e não foi observado na linha celular RE negativa MDA-MB-231. Assim, embora não tenha sido observado um efeito da ativação da via do RE pelo E2 na expressão do gene YBX1, foi detetado um aumento na fosforilação da proteína YB-1, o que sugere que no modelo in vitro, a expressão e atividade de YB-1 pode ser modulada pela via RE ao nível pós-transducional. No contexto clínico, o nosso principal objetivo consistiu em estabelecer uma possível correlação entre a expressão de YB-1 e o cancro da mama RE+. Deste modo, analisámos pela primeira vez a expressão de p-YB-1 em amostras cancro da mama, num conjunto de 60 amostras de tumor primário e 32 metástases emparelhadas. Observou-se uma associação entre níveis elevados de p-YB-1 e tumores RE e RP negativos (P=0,006 e P=0,037, respetivamente). Relativamente aos outcomes clínicos, a expressão de YB-1 e p-YB-1 correlacionou-se com uma diminuição da sobrevivência livre de recidiva (P=0.0442, HR 0.5514 95%CI 0.3088-0.9846 e P=0.0108, HR 0.058 95%CI 0.1230-0.7606 respetivamente), mas não com a sobrevivência global (P=0.2473, HR 0.6097 95%CI 0.3704-1.292 e P=0.0687, HR 0.4789 95%CI 0.2168-1.058). Desta forma, YB-1 e p-YB-1 são importantes biomarcadores de pior prognóstico, nomeadamente para risco de recidiva, especialmente em pacientes com tumores RE-, RP-. Em amostras de metástases, observou-se uma correlação positiva entre a expressão elevada de p-YB-1 e metástases RP negativas (P=0,030), sendo que a marcação de YB-1 não mostrou associação significativa com nenhum dos parâmeros clínicos. Verificámos ainda não existirem níveis diferentes de expressão de ambos os marcadores nos tumores primários e metástases, refletindo uma alteração durante a progressão tumoral (Teste McNemar: P=0.7728 e P=0.0771, respetivamente; Paired t-test: P=0.5754 e P=0.1883, respetivamente). Este estudo consiste n a primeira análise da expressão de YB-1 e p-YB-1 em tumores primários da mama e metástases emparelhadas. Visto que a deteção da proteína YB-1 em tecidos tumorais é um marcador de mau prognóstico, procurámos de seguida avaliar se a deteção de YB-1 no soro de doentes com cancro da mama poderia ser igualmente significante. A deteção de níveis séricos de proteínas é uma técnica minimamente invasiva e de fácil aplicação. Para esta análise foi utilizado um grupo de amostras de soro disponível de doentes com cancro da mama e metástases ósseas. Foi detetada a presença de YB-1 no soro de 22 doentes, correlacionada com a presença de metástases extra-ósseas (P=0.044). A análise multivariada mostrou que os doentes com YB-1 no soro apresentaram uma progressão da doença óssea mais rápida (HR 3.29, 95% CI 1.13 – 9.60, P=0.029), no entanto sem diferenças ao nível da sobrevivência global (HR 2.04, 95% CI 0.86 – 4.87, P=0.108). Este estudo corresponde à primeira análise dos níveis séricos de YB-1 em doentes com cancro da mama. Em suma, os resultados obtidos neste projeto mostram que não só a proteína YB-1, mas também p-YB-1 e YB-1 secretada têm valor de prognóstico em doentes com cancro da mama, reforçando a sua utilidade clínica como fator de prognóstico e possível alvo terapêutico. Este estudo mostrou ainda a regulação in vitro e in vivo de YBX1 não é exclusivamente dependente da presença de E2. No geral, este estudo gerou dados significativos e colocou questões importantes que serão abordadas em projetos futuros.
Estrogen receptor-positive (ER+) tumors are the most frequent breast cancers (BC), and have the better prognosis. Nevertheless, about 30% of patients with ER+ BC will develop distant metastases, with increased mortality rates. ER+ BC is dependent on estrogens that activate ER, an important transcription factor. In this context YBX1, which encodes for Y-box binding protein 1 (YB-1), was recently identified as part of an ER-dependent poor prognosis predictor for ER+ BC. YB-1 is an oncoprotein overexpressed in BC, where it has been associated with disease progression and ER/PR negativity. Recently, using an orthotopic mouse model of ER+ vs. ER- BC, our group showed that YBX1 is overexpressed in ER+ tumors growing under the presence of 17β-Estradiol (E2). Therefore, this project aimed to establish a biological and clinical link between ER and YB-1 expression in BC. Using BC cell lines, we showed that E2 per se did not altered YB-1 expression, at the mRNA or protein level, but induced its phosphorylation. These results need further investigation to address the mechanism behind YB-1/ER connection we observe in the in vivo model. Next, we explored the prognostic value of p-YB-1 expression and secreted YB-1 in the clinical setting. We demonstrated that p-YB-1 is a biomarker of decreased distant metastases-free survival and overall survival, and that secreted YB-1 correlates with faster bone disease progression in patients with BC and bone metastases. In conclusion, the results obtained in this project demonstrate that not only YB-1 but also p-YB-1 and secreted YB-1 have prognostic value in BC patients, reinforcing its clinical utility as prognostic factor and putative target. We also showed that the in vitro and in vivo regulation of YBX1 is not exclusively dependent on the presence of E2. Overall, this work generated significant data and raised important questions to be addressed in future projects.