Nair Lopes

Advanced prostate cancers frequently develop resistance to androgen-deprivation therapy with serious implications for patient survival. Considering their importance in this type of neoplasia, epigenetic modifications have drawn attention as alternative treatment strategies. The aim of this study was to assess the antitumoral effects of the combination of hydralazine, a DNA methylation inhibitor, with enzalutamide, an antagonist of the androgen receptor, in prostate cancer cell lines. Several biological parameters, such as cell viability, proliferation, DNA damage, and apoptosis, as well as clonogenic and invasive potential, were evaluated. The individual treatments with hydralazine and enzalutamide exerted growth-inhibitory effects in prostate cancer cells and their combined treatment displayed synergistic effects. The combination of these two drugs was very effective in decreasing malignant features of prostate cancer and may become an alternative therapeutic option for prostate cancer patient management.

Among the well-established alterations contributing to prostate cancer (PCa) pathogenesis, epigenetics is an important player in its development and aggressive disease state. Moreover, since no curative therapies are available for advanced stage disease, there is an urgent need for novel therapeutic strategies targeting this subset of patients. Thus, we aimed to evaluate the combined antineoplastic effects of DNA methylation inhibitor hydralazine and histone deacetylase inhibitors panobinostat and valproic acid in several prostate cell lines. The effect of these drugs was assessed in four PCa (LNCaP, 22Rv1, DU145 and PC-3) cell lines, as well as in non-malignant epithelial (RWPE-1) and stromal (WPMY-1) cell lines, using several assays to evaluate cell viability, apoptosis, proliferation, DNA damage and clonogenic potential. We found that exposure to each epidrug separately reduced viability of all PCa cells in a dose-dependent manner and that combined treatments led to synergic growth inhibitory effects, impacting also on colony formation, invasion, apoptotic and proliferation rates. Interestingly, antitumoral effects of combined treatment were particularly expressive in DU145 cells. We concluded that hydralazine and panobinostat attenuate malignant properties of PCa cells, constituting a potential therapeutic tool to counteract PCa progression.

Oesophageal cancer is a life-threatening disease, accounting for high mortality rates. The poor prognosis of this malignancy is mostly due to late diagnosis and lack of effective therapies for advanced disease. Epigenetic alterations may constitute novel and attractive therapeutic targets, owing to their ubiquity in cancer and their reversible nature. Herein, we offer an overview of the most important studies which compared differences in expression of enzymes that mediate epigenetic alterations between oesophageal cancer and normal mucosa, as well as in vitro data addressing the role of these genes/proteins in oesophageal cancer. Furthermore, The Cancer Genome Atlas database was interrogated for the correlation between expression of these epigenetic markers and standard clinicopathological features. We concluded that most epigenetic players studied thus far are overexpressed in tumours compared to normal tissue. Furthermore, functional assays suggest an oncogenic role for most of those enzymes, supporting their potential as therapeutic targets in oesophageal cancer.

Background Gastric cancer is the fifth most common cancer and the third cause of global cancer mortality. CDX2 is an intestinal differentiation marker with prognostic value in gastric cancer and transcriptionally regulates the expression of glycoprotein A33 (GPA33) and liver intestine cadherin (LI-cadherin). Methods This study evaluated the clinical significance of the combined expression of CDX2 and its targets GPA33 and LI-cadherin in gastric cancer by fluorescence-based multiplex immunohistochemistry together with digital image analysis and chromogenic immunohistochemistry in 329 gastric cancer samples arranged in tissue microarrays. Additionally, publicly available RNA-seq expression data from 354 gastric cancer samples from the TCGA database were used to validate the immunohistochemistry results. Results Expression of the three markers (CDX2, GPA33, and LI-cadherin) was strongly correlated, defining an intestinal differentiation panel. Low or negative protein expression of the intestinal differentiation panel identified patients with particularly poor overall survival, irrespective of the methodology used, and was validated in the independent series at the RNA-seq level. Conclusions Expression of the intestinal differentiation panel (CDX2, GPA33, and LI-cadherin) defines a set of biomarkers with a strong biological rationale and favourable impact for prognostication of gastric cancer patients.

Gastric cancer is one of the most common types of cancer and the third cause of cancer-related death worldwide. The aggressiveness of the disease is related to, among other factors, tumour differentiation. CDX2 is a known intestinal differentiation marker with prognostic value in gastric cancer and two of its targets are expressed at the cell surface: GPA33 (glycoprotein A33) and LI-cadherin (liver intestine cadherin). Whether these membrane proteins are good biomarkers in gastric cancer and could refine the significance of CDX2 expression remains unknown. In order to answer these questions, we evaluated the expression of CDX2, GPA33 and LI-cadherin using immunohistochemistry in 350 gastric cancer samples arranged in TMAs (tissue microarrays) and evaluated the co-localization of the biomarkers using fluorescent multiplex immunohistochemistry. CDX2 was expressed in 36% of the cases, while GPA33 and LI-cadherin were positive in 55% and 66%, respectively. All markers were significantly correlated with each other and are more often expressed in early stage (I and II) cancers (p < 0.05). Overall survival analysis showed that both GPA33 and LI-cadherin predict better outcome. When stratifying the series in early (I and II) and late (III and IV) stages, both proteins significantly associate with better outcome for late stages of disease progression. Overall, these data indicate that the presence of an intestinal differentiation program in gastric cancer is a marker of good prognosis. The concordance rate between CDX2 and GPA33 and CDX2 and LI-cadherin expression was 68% and 64%, respectively. Since CDX2 displays a heterogeneous pattern of expression and TMA sampling can, by chance, select a negative area in a positive tumour, we also compared CDX2 expression between whole-tissue sections and TMAs. In our series we obtained 68 discrepant cases (positive for CDX2 in whole-tissue sections and negative in the TMAs). For the majority of these cases (50, corresponding to 74%) the combined expression of CDX2 targets, GPA33 and LI-cadherin, can rescue the underrepresentation of CDX2 expression in the TMAs and identifies CDX2-dependent intestinal differentiation. We conclude that CDX2, GPA33 and LI-cadherin are significantly associated with each other and are commonly expressed in early stages of gastric cancer. The expression of GPA33 and LI-cadherin is associated with better overall survival, particularly in late-stage disease patients. Finally, GPA33 and LI-cadherin are good cell surface surrogate markers for CDX2 expression, not only because of the good concordance of expression, but also because the combined expression of GPA33 and LI-cadherin rescues false negative CDX2 cases in TMAs versus whole-tissue samples. Citation Format: Nair Lopes, Christian Bergsland, Merete Bjornslett, Jarle Bruun, Ragnhild Lothe, Raquel Almeida, Leonor David. CDX2 targets, GPA33 and LI-cadherin, are novel biomarkers with prognostic value in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 565.

Introduction Gastric cancer is one of the most frequent tumours and the third leading cause of cancer-related death worldwide. The investigation of new biomarkers that can predict patient outcome more accurately and allow better treatment and follow-up decisions is of crucial importance. The transcription protein SRY-box 9 (SOX9) is a member of the high-mobility-group box class DNA-binding proteins. SOX9 is an important regulator of cell-fate decisions in embryogenesis and adulthood, playing critical roles in differentiation and proliferation, also in the gastrointestinal tract. SOX9 has been correlated to tumour behaviour in different tissues, including in gastric cancer, nevertheless with contradictory results. In this work we sought to ascertain the relevance of SOX9 transcription factor as a prognostic marker in gastric cancer. Material and methods SOX9 expression was analysed by immunohistochemistry in a series of 333 cases of gastric adenocarcinoma, and its association with clinico-pathological and follow-up data was evaluated. A second gastric cancer validation cohort consisted of 354 cases from the cancer genome atlas (TCGA), showing high versus low SOX9 expression. Results and discussions SOX9 expression was present in 83% of gastric cancer cases. Loss of SOX9 expression was significantly associated with relapse, however SOX9 expression was more frequent in stage IV cases. SOX9 loss of expression predicted worse disease-free survival but not overall survival, in this series. The prognostic value of SOX9 was independent of Laurén classification but it was more pronounced in tumours with expansive versus infiltrative growth (p=0.008). In patients that presented with disease in stage I to III, loss of SOX9 expression was significantly associated with venous invasion and lymph node metastases. In an independent series of gastric cancer, where SOX9 expression was assessed at the mRNA level (TCGA), low SOX9 expression levels were also associated with poor patient outcome. Functional studies, after down- and up-regulation of SOX9, are now being undertaken in order to better understand the clinico-pathological observations and the relevance of SOX9 as a potential biomarker in gastric cancer. Conclusion We have identified SOX9 as a marker of relapse in gastric cancer. Further experiments are needed to elucidate its biological relevance at the cellular level and to test its potential role in invasion.

T lymphocytes stimulated through their antigen receptor (TCR) preferentially express mRNA isoforms with shorter 3´ untranslated regions (3´‐UTRs) derived from alternative pre‐mRNA cleavage and polyadenylation (APA). However, the physiological relevance of APA programs remains poorly understood. CD5 is a T‐cell surface glycoprotein that negatively regulates TCR signaling from the onset of T‐cell activation. CD5 plays a pivotal role in mediating outcomes of cell survival or apoptosis, and may prevent both autoimmunity and cancer. In human primary T lymphocytes and Jurkat cells we found three distinct mRNA isoforms encoding CD5, each derived from distinct poly(A) signals (PASs). Upon T‐cell activation, there is an overall increase in CD5 mRNAs with a specific increase in the relative expression of the shorter isoforms. 3´‐UTRs derived from these shorter isoforms confer higher reporter expression in activated T cells relative to the longer isoform. We further show that polypyrimidine tract binding protein (PTB/PTBP1) directly binds to the proximal PAS and PTB siRNA depletion causes a decrease in mRNA derived from this PAS, suggesting an effect on stability or poly(A) site selection to circumvent targeting of the longer CD5 mRNA isoform by miR‐204. These mechanisms fine‐tune CD5 expression levels and thus ultimately T‐cell responses.

Cancer stem cells are hypoxia-resistant and present a preponderant glycolytic metabolism. These characteristics are also found in basal-like breast carcinomas (BLBC), which show increased expression of cancer stem cell markers. Recently, we demonstrated that P-cadherin, a biomarker of BLBC and a poor prognostic factor in this disease, mediates stem-like properties and resistance to radiation therapy. Thus, the aim of the present study was to evaluate if P-cadherin expression was associated to breast cancer cell populations with an adapted phenotype to hypoxia. Immunohistochemistry was performed to address the expression of P-cadherin, hypoxic, glycolytic and acid-resistance biomarkers in primary human breast carcinomas. In vitro studies were performed using basal-like breast cancer cell lines. qRT-PCR, FACS analysis, western blotting and confocal microscopy were used to assess the expression of P-cadherin after HIF-1α stabilization, achieved by CoCl2 treatment. siRNA-mediated knockdown was used to silence the expression of several targets and qRT-PCR was employed to evaluate the effects of P-cadherin on HIF-1α signaling. P-cadherin high and low breast cancer cell populations were sorted by FACS and levels of GLUT1 and CAIX were assessed by FACS and western blotting. Mammosphere forming efficiency was used to determine the stem cell activity after specific siRNA-mediated knockdown, further confirmed by western blotting. We demonstrated that P-cadherin overexpression was significantly associated with the expression of HIF-1α, GLUT1, CAIX, MCT1 and CD147 in human breast carcinomas. In vitro, we showed that HIF-1α stabilization was accompanied by increased membrane expression of P-cadherin and that P-cadherin silencing led to a decrease of the mRNA levels of GLUT1 and CAIX. We also found that the cell fractions harboring high levels of P-cadherin were the same exhibiting more GLUT1 and CAIX expression. Finally, we showed that P-cadherin silencing significantly decreases the mammosphere forming efficiency in the same range as the silencing of HIF-1α, CAIX or GLUT1, validating that all these markers are being expressed by the same breast cancer stem cell population. Our results establish a link between aberrant P-cadherin expression and hypoxic, glycolytic and acid-resistant breast cancer cells, suggesting a possible role for this marker in cancer cell metabolism.