Factores de transcrição e epigenética no cancro da mama : novos achados sobre a regulação do gene CDH3/P-Caderina

Tese de doutoramento em Ciências da Saúde – Ciências da SaúdeBreast cancer is the leading cause of cancer amongst women in the westernized world. It is a heterogeneous disease ranging from premalignant hyperproliferation to invasive and metastatic carcinomas. Disease progression is poorly understood but is likely due to the accumulation of genetic alterations leading to widespread changes in gene expression, ultimately affecting cell biology and often increasing growth capacity and survival advantage. Consistent with this, recent studies have shown that different breast tumour subclasses display distinct gene expression profiles. In addition to genetic alterations, there is increasing evidence for gross epigenetic alterations in tumour cells, both at the levels of DNA methylation and histone marks. Epigenetic alterations target and modulate several important genes in breast cancer, contributing, not only for its initiation, but also for its development and response to therapies. One of the key genes in breast biology and cancer, which expression and molecular function is strongly regulated by epigenetic modifications, is the ligand-activated transcription factor oestrogen-receptor α (ERα), the primary mediator of the ovarianproduced steroid hormone oestrogen action in breast mammary cells. Breast cancer development and progression is, in fact, closely associated with the presence or absence of ERα being an important prognostic and predictor indicator in this disease. Consequently, the leading drugs used for endocrine therapy of breast cancer, namely anti-oestrogens such as tamoxifen or fulvestrant, block ERα activity. Despite the efficacy of these target agents during short/medium time regimens, the use of endocrine therapy is limited by the onset of drug resistance, normally mediated by epigenetic alterations, in which most patients, who initially respond favourable to endocrine therapy, eventually recur. In the present thesis we present an immunohistochemical approach involving two ERα- signalling pathways-related transcription factors, FOXA1 and GATA-3, in order to assess whether their expression could be useful as prognostic markers in breast cancer patients. We demonstrated that patients harbouring FOXA1-positive tumours show a better diseasefree survival in a 5 years follow-up time and that FOXA1 expression associates with good prognosis clinicopathological features. More importantly, and for the first time, we established that this forkhead-box transcription factor has a power for recurrence risk stratification among the poor prognostic ERα-negative breast cancer patients, demonstrating the clinical importance of this biomarker in breast cancer prognosis. We still demonstrated that GATA-3 does not constitute a strong predictor for breast cancer diseasefree survival nor a good prognostic marker, but was shown to be a robust luminal differentiation marker. Taken together, the expression assessment of FOXA1 and GATA-3 can provide important clinical information, not only regarding the favourable prognostic outcome, but can also constitute an important tool to define and assess the luminal A subtype in breast cancer and, eventually, response to endocrine therapy. ERα-negative breast cancers are resistant to endocrine therapies and have a worse prognosis than ERα-positive breast cancers. DNA methylation and chromatin remodelling are two epigenetic mechanisms that have been linked with a generation of an aggressive ERα-negative phenotype in breast cancer. Endocrine therapies have been reported to interfere with the expression of ERα-repressed genes, ultimately leading to the ERα- negative phenotype of breast cancer, but most importantly, with the induction of breast cancer cell aggressiveness and invasiveness in specific cell contexts. We have been focussed on studying CDH3/P-cadherin gene expression, as an ERα-repressed gene in breast cancer; enlightening a mechanism for its regulation in a breast cancer cell model. The mechanism by which ERα-signalling inhibition led to P-cadherin overexpression was completely unknown until now. Herein, we described a chromatin remodelling (H3K4me2) at CDH3 gene regulatory region which, being induced by ICI 182,780 treatment, can modulate CDH3 promoter activity and the expression of a pro-invasive protein in breast cancer, as Pcadherin. Moreover, this study allowed the identification of a new transcription factor, C/EBPα that is able to regulate CDH3 activity in breast cancer cell lines and which expression associates with P-cadherin in breast cancer patient samples. Overall, the work summarized in this thesis discusses molecular characteristics that may influence the risk for ERα-negative breast cancer patients to recur, also suggesting two new markers which, in addition to ERα assessment, might be useful for predict breast cancer outcome in patients non-responsive to endocrine therapies. Moreover, we demonstrated a chromatin remodelling event provoked by the pure anti-oestrogen ICI 182,780, which is able to increase CDH3 promoter activity and therefore, the expression of the pro-invasive protein P-cadherin in breast cancer cells, contributing to the elucidation of how breast cancer cells may acquire aggressive properties after loss of oestrogen signalling.O cancro da mama constitui a neoplasia mais frequente entre as mulheres dos países ocidentalizados. A elevada heterogeneidade da doença permite variações fenotípicas que vão desde a hiper-proliferação pré-maligna ao carcinoma invasivo e metastático. Apesar das vias de progressão da doença ainda não se encontrarem totalmente caracterizadas, a causa mais comum e provável será a acumulação de alterações genéticas, as quais, produzem alterações na expressão de vários genes e afectam a biologia da célula, conferindo capacidades de crescimento e de sobrevivência. De facto, tem-se verificado, em estudos recentes, que diferentes sub-grupos de cancro da mama exibem perfis distintos de expressão genética. Associadas a estas alterações genéticas, as modificações epigenéticas ocorridas nas células tumorais, essencialmente ao nível do padrão de metilação do ADN e da acetilação/metilação da cromatina, têm sido alvo de crescente interesse na compreensão do processo da carcinogénese. As alterações epigenéticas controlam uma série de genes importantes no cancro da mama, contribuindo não só para a sua iniciação, mas também para o seu desenvolvimento e resposta terapêutica. Um dos genes centrais na biologia da glândula mamária e na sua carcinogénese é o receptor de estrogénio (REα), um factor de transcrição que constitui a principal ferramenta que permite a acção da hormona esteróide – estrogénio, sobre as células mamárias. O desenvolvimento e progressão do cancro da mama estão por isso fortemente associados à presença ou ausência de expressão do REα, constituindo este, um importante factor de prognóstico e preditivo da resposta terapêutica endócrina nesta neoplasia. Consequentemente, as principais drogas usadas para terapia endócrina de cancro da mama, nomeadamente as anti-estrogénicas, como o Tamoxifeno e o Fulvestrant, visam bloquear a actividade do REα. Não obstante a eficácia da utilização destes fármacos, o uso da terapia endócrina tem vindo a demonstrar algumas limitações, uma vez que a maioria dos doentes que inicialmente respondem a esta terapia, vêem tardiamente a desenvolver resistência endócrina, eventualmente com recidiva, provocada normalmente por alterações epigenéticas não específicas. Num contexto de genes associados à via de sinalização mediada pelo REα, estudou-se por imunohistoquímica a expressão de dois factores de transcrição, FOXA1 e GATA-3, no sentido de avaliar a sua importância como factores de prognóstico em pacientes com cancro da mama. Demonstrou-se que pacientes com tumores positivos para FOXA1 apresentam uma melhor tempo-livre de doença num período de estudo de 5 anos e que a expressão de FOXA1 está associada a perfis clínico-patológicos de bom prognóstico. Demonstrou-se ainda que a expressão de FOXA1, num sub-grupo de tumores REα-negativos, permite a estratificação do risco de recorrência durante o mesmo período de estudo, realçando assim a importância clínica deste factor de transcrição no prognóstico do cancro da mama. Relativamente à expressão da proteína GATA-3, observou-se que este factor de transcrição não constitui nem um factor de prognóstico robusto, nem um indicador de melhor tempolivre de doença, revelando, no entanto, ser um importante e consistente marcador de diferenciação luminal. Assim, a avaliação da expressão de FOXA1 e GATA-3 poderá ser útil, não só sob o ponto de vista da prática clínico-patológica, no sentido de fornecer uma importante informação relativamente ao prognóstico da doente, mas também como uma ferramenta essencial para definir o subtipo luminal A em cancro da mama. Os tumores mamários REα-negativos são resistentes a terapias endócrinas e apresentam pior prognóstico relativamente a tumores REα-positivos. A metilação do ADN e a remodelação da cromatina são dois mecanismos epigenéticos que têm sido relacionados com a aquisição de um fenótipo mais agressivo de cancro da mama. Tem sido descrito que as terapias endócrinas podem modelar a expressão de genes reprimidos pelo REα, conduzindo o tumor à aquisição progressiva de um fenótipo REα-negativo, com consequente indução de invasão celular em ensaios in vitro. O gene CDH3/P-caderina é, no modelo de cancro da mama, um gene reprimido pelo REα, embora o mecanismo pelo qual a inibição da sinalização mediada pelo REα e resultante α sobre-expressão de P-caderina, fosse ainda desconhecido. Demonstrou-se assim que o tratamento de linhas celulares de cancro da mama REα-positivas com o antagonista de REα, ICI 182,780, leva a uma remodelação da cromatina ao nível do promotor do gene CDH3, através de uma proeminente marca activadora H3K4me2, a qual se sugere ser causal da indução da expressão da proteína próinvasiva P-caderina. Este estudo permitiu ainda identificar um novo factor de transcrição com capacidade de regular a actividade do promotor CDH3 em linhas celulares, e cuja expressão se associa com a P-caderina em tumores de pacientes com cancro da mama. Em conclusão, o trabalho constante desta tese explora características moleculares que podem influenciar o risco de recidiva de doentes com cancro da mama hormono-negativos e onde a avaliação de dois novos marcadores, conjuntamente com a determinação da expressão de REα, pode ser importante na avaliação do prognóstico de doentes com neoplasia mamária não-responsiva às terapias endócrinas. Demonstrou-se ainda que determinadas alterações da cromatina, induzidas pelo ICI 182,780, são responsáveis pela activação do promotor CDH3 e consequente expressão de P-caderina em células de cancro da mama. Este estudo permite assim elucidar um mecanismo pelo qual células de cancro da mama adquirem propriedades agressivas após perda de sinalização mediada pelo REα.Fundação para a Ciência e a Tecnologia (FCT), com a referência SFRH/BD/15316/2005.Programa Operacional Ciência e Inovação (POCI), POCI/BIA – BCM/59252/2004Fundo Social Europeu (FSE)

The shallow-water Polyplacophora of the Azores and some comments on the biogeographical relationships of the Azorean malacofauna.

The Mollusc's reference collection of the Department of Biology of the University of the Azores (DBUA) was checked for Polyplacophora. A taxonomic list is given and new data is provided regarding the geographic distribution and bactymetric range of the Azorean Polyphacophora. Some comments are made about the biogeographical relationships of Gastropoda, Bivalvia, Cephalopoda and Polyplacophora of the Azores

P-cadherin overexpression Is an indicator of clinical outcome in invasive breast carcinomas and is associated with CDH3 promoter hypomethylation

Purpose: P-cadherin overexpressionhas been reported in breast carcinomas,where itwas associated with proliferative high-grade histological tumors. This study aimed to analyze P-cadherin expression in invasive breast cancer and to correlate it with tumor markers, pathologic features, and patient survival. Another purpose was to evaluate the P-cadherin promoter methylation pattern as the molecularmechanismunderlying this gene regulation. Experimental Design: Using a series of invasive breast carcinomas, P-cadherin expressionwas evaluated and correlated with histologic grade, estrogen receptor, MIB-1, and p53 and c-erbB-2 expression. In order to assess whether P-cadherin expression was associated with changes in CDH3 promoter methylation, we studied the methylation status of a gene 5V-flanking region in these same carcinomas.This analysis was also done for normal tissue and for a breast cancer cell line treatedwith a demethylating agent. Results: P-cadherinexpression showeda strong correlationwithhighhistologic grade, increased proliferation, c-erbB-2 and p53 expression, lack of estrogen receptor, and poor patient survival. This overexpressioncanbe regulatedby gene promotermethylationbecause the 5-Aza-2V-deoxycytidine treatment ofMCF-7/AZ cells increased P-cadherinmRNA and proteinlevels. Additionally, we found that 71% of P- adherin-negative cases showed promoter methylation,whereas 65% of positive ones were unmethylated (P = 0.005). The normal P-cadherin-negative breast epithelial cells showed consistent CDH3 promotermethylation. Conclusions: P-cadherin expressionwas strongly associatedwith tumor aggressiveness, being a good indicator of clinical outcome. Moreover, the aberrant expression of P-cadherin in breast cancermight be regulated by gene promoter hypomethylation

P-cadherin expression in glandular lesions of the uterine cervix detected by liquid-based cytology

Objective: To study P-cadherin aberrant expression as a possible marker for cervical adenocarcinomas in cytological samples. Methods: We studied P-cadherin immunoexpression in liquid-based cervical cytology samples of biopsy-proven cervical lesions. Results: We found a statistically significant correlation between P-cadherin expression and a cytological diagnosis of malignancy, either glandular or squamous (P < 0.0001). Twenty-two of 33 malignant cases showed P-cadherin membrane staining. None of the 30 benign cases tested showed membrane staining, but three of them displayed an aberrant nuclear P-cadherin expression. Conclusions: We concluded that P-cadherin can be used to discriminate between malignant and benign cervical cytological specimens, but not to discriminate glandular from squamous lesions

The transcription factor FOXO3a is a crucial cellular target of gefitinib (Iressa) in breast cancer cells

Gefitinib is a specific inhibitor of the epidermal growth factor receptor (EGFR) that causes growth delay in cancer cell lines and human tumor xenografts expressing high levels of EGFR. An understanding of the downstream cellular targets of gefitinib will allow the discovery of biomarkers for predicting outcomes and monitoring anti-EGFR therapies and provide information for key targets for therapeutic intervention. In this study, we investigated the role of FOXO3a in gefitinib action and resistance. Using two gefitinib-sensitive (i.e., BT474 and SKBR3) as well as three other resistant breast carcinoma cell lines (i.e., MCF-7, MDA-MB-231, and MDA-MB-453), we showed that gefitinib targets the transcription factor FOXO3a to mediate cell cycle arrest and cell death in sensitive breast cancer cells. In the sensitive cells, gefitinib treatment causes cell cycle arrest predominantly at the G(0)-G(1) phase and apoptosis, which is associated with FOXO3a dephosphorylation at Akt sites and nuclear translocation, whereas in the resistant cells, FOXO3a stays phosphorylated and remains in the cytoplasm. The nuclear accumulation of FOXO3a in response to gefitinib was confirmed in tumor tissue sections from breast cancer patients presurgically treated with gefitinib as monotherapy. We also showed that knockdown of FOXO3a expression using small interfering RNA (siRNA) can rescue sensitive BT474 cells from gefitinib-induced cell-proliferative arrest, whereas reintroduction of active FOXO3a in resistant MDA-MB-231 cells can at least partially restore cell-proliferative arrest and sensitivity to gefitinib. These results suggest that the FOXO3a dephosphorylation and nuclear localization have a direct role in mediating the gefitinib-induced proliferative arrest and in determining sensitivity to gefitinib.Supported by the German Cancer Aide Foundation (J. Krol)and the Association of International Cancer Research (R. Francis).Andrew Sunters and Andreas Polychronic were fellows funded by CancerResearch UK. Andre Albergaria is a recipient of a grant from Fundação para a Ciência e a Tecnologia, Portugal. This work was sponsored by theBreast Cancer Research Trust and Cancer Research U

Evaluation of tissue and circulating mir-21 as potential biomarker of response to chemoradiotherapy in rectal cancer

This research received funding from European Structural & Investment Funds through the COMPETE Programme—Programa Operacional Regional de Lisboa—Programme Grant LISBOA-01-0145-FEDER-016405,and from National Funds through FCT—Fundação para a Ciência e a Tecnologia—Programme Grant SAICTPAC/0019/2015.Response to chemoradiotherapy (CRT) in patients with locally advanced rectal cancer (RC) is quite variable and it is urgent to find predictive biomarkers of response. We investigated miR-21 as tissue and plasma biomarker of response to CRT in a prospective cohort of RC patients; The expression of miR-21 was analyzed in pre-and post-CRT rectal tissue and plasma in 37 patients with RC. Two groups were defined: Pathological responders (TRG 0, 1 and 2) and non-responders (TRG 3). The association between miR-21, clinical and oncological outcomes was assessed; miR-21 was upregulated in tumor tissue and we found increased odds of overexpression in pre-CRT tumor tissue (OR: 1.63; 95% CI: 0.40–6.63, p = 0.498) and pre-CRT plasma (OR: 1.79; 95% CI: 0.45–7.19, p = 0.414) of non-responders. The overall recurrence risk increased with miR-21 overexpression in pre-CRT tumor tissue (HR: 2.175, p = 0.37); Significantly higher miR-21 expression is observed in tumor tissue comparing with non-neoplastic. Increased odds of non-response is reported in patients expressing higher miR-21, although without statistical significance. This is one of the first studies on circulating miR-21 as a potential biomarker of response to CRT in RC patients.publishersversionpublishe

Evaluation of Tissue and Circulating miR-21 as Potential Biomarker of Response to Chemoradiotherapy in Rectal Cancer

Response to chemoradiotherapy (CRT) in patients with locally advanced rectal cancer (RC) is quite variable and it is urgent to find predictive biomarkers of response. We investigated miR-21 as tissue and plasma biomarker of response to CRT in a prospective cohort of RC patients; The expression of miR-21 was analyzed in pre- and post-CRT rectal tissue and plasma in 37 patients with RC. Two groups were defined: Pathological responders (TRG 0, 1 and 2) and non-responders (TRG 3). The association between miR-21, clinical and oncological outcomes was assessed; miR-21 was upregulated in tumor tissue and we found increased odds of overexpression in pre-CRT tumor tissue (OR: 1.63; 95% CI: 0.40–6.63, p = 0.498) and pre-CRT plasma (OR: 1.79; 95% CI: 0.45–7.19, p = 0.414) of non-responders. The overall recurrence risk increased with miR-21 overexpression in pre-CRT tumor tissue (HR: 2.175, p = 0.37); Significantly higher miR-21 expression is observed in tumor tissue comparing with non-neoplastic. Increased odds of non-response is reported in patients expressing higher miR-21, although without statistical significance. This is one of the first studies on circulating miR-21 as a potential biomarker of response to CRT in RC patients.info:eu-repo/semantics/publishedVersio

Potential of miR-21 to Predict Incomplete Response to Chemoradiotherapy in Rectal Adenocarcinoma

Funding: This work has received funding from European Structural and Investment Funds through the COMPETE Programme Grant LISBOA-01-0145-FEDER-016405, from National Funds through Fundação para a Ciência e Tecnologia Programme grant SAICTPAC/0019/2015 and by a cholar from the Portuguese Society of Coloproctology as Investigation in Coloproctology Research Prize 2016–2018.Background: Patients with locally advanced rectal adenocarcinoma (LARC) are treated with neoadjuvant chemoradiotherapy (CRT). However, biomarkers for patient selection are lacking, and the association between miRNA expression and treatment response and oncological outcomes is unclear. Objectives: To investigate miRNAs as predictors of response to neoadjuvant CRT and its association with oncological outcomes. Methods: This retrospective study analyzed miRNA expression (miR-16, miR-21, miR-135b, miR-145, and miR-335) in pre- and post-chemoradiation rectal adenocarcinoma tissue and non-neoplastic mucosa in 91 patients treated with neoadjuvant CRT (50.4 Gy) and proctectomy. Two groups were defined: a pathological complete responders group (tumor regression grade—TRG 0) and a pathological incomplete responders group (TRG 1, 2, and 3). Results: miR-21 and miR-135b were upregulated in tumor tissue of incomplete responders comparing with non-neoplastic tissue (p = 0.008 and p < 0.0001, respectively). Multivariate analysis showed significant association between miR-21 in pre-CRT tumor tissue and response, with a 3.67 odds ratio (OR) of incomplete response in patients with higher miR-21 levels (p = 0.04). Although with no significance, patients treated with 5-fluorouracil (5-FU) presented reduced odds of incomplete response compared with those treated with capecitabine (OR = 0.19; 95% confidence interval (CI) 0.03–1.12, p = 0.05). Moreover, significant differences were seen in overall survival (OS) in relation to clinical TNM stage (p = 0.0004), cT (p = 0.0001), presence of distant disease (p = 0.002), mesorectal tumor deposits (p = 0.003), and tumor regression grade (p = 0.04). Conclusion: miR-21 may predict response to CRT in rectal cancer (RC).publishersversionpublishe

Potential of miR-21 to Predict Incomplete Response to Chemoradiotherapy in Rectal Adenocarcinoma

Background: Patients with locally advanced rectal adenocarcinoma (LARC) are treated with neoadjuvant chemoradiotherapy (CRT). However, biomarkers for patient selection are lacking, and the association between miRNA expression and treatment response and oncological outcomes is unclear. Objectives: To investigate miRNAs as predictors of response to neoadjuvant CRT and its association with oncological outcomes. Methods: This retrospective study analyzed miRNA expression (miR-16, miR-21, miR-135b, miR-145, and miR-335) in pre- and post-chemoradiation rectal adenocarcinoma tissue and non-neoplastic mucosa in 91 patients treated with neoadjuvant CRT (50.4 Gy) and proctectomy. Two groups were defined: a pathological complete responders group (tumor regression grade—TRG 0) and a pathological incomplete responders group (TRG 1, 2, and 3). Results: miR-21 and miR-135b were upregulated in tumor tissue of incomplete responders comparing with non-neoplastic tissue (p = 0.008 and p < 0.0001, respectively). Multivariate analysis showed significant association between miR-21 in pre-CRT tumor tissue and response, with a 3.67 odds ratio (OR) of incomplete response in patients with higher miR-21 levels (p = 0.04). Although with no significance, patients treated with 5-fluorouracil (5-FU) presented reduced odds of incomplete response compared with those treated with capecitabine (OR = 0.19; 95% confidence interval (CI) 0.03–1.12, p = 0.05). Moreover, significant differences were seen in overall survival (OS) in relation to clinical TNM stage (p = 0.0004), cT (p = 0.0001), presence of distant disease (p = 0.002), mesorectal tumor deposits (p = 0.003), and tumor regression grade (p = 0.04). Conclusion: miR-21 may predict response to CRT in rectal cancer (RC).info:eu-repo/semantics/publishedVersio

Co-expression of E- and P-cadherin in breast cancer: role as an invasion suppressor or as an invasion promoter?

Sem resumo disponível.publishe