The prospects of DNA damage repair variants guiding platinum compounds in the treatment of triple negative breast cancer / 中华肿瘤杂志

Triple negative breast cancer (TNBC) is prone to recurrence and metastasis, which is the subtype of poorest prognosis. Chemotherapy is the main treatment, although there is lack of effective adjuvant chemotherapy regimens. The unsatisfactory efficacy of chemotherapy has been a bottleneck in improving the outcome of TNBC. Platinum compounds act directly on DNA to kill tumor cells, and they have a stronger killing effect on tumor cells carrying DNA damage repair (DDR) defects, which is an important entry point to improve the efficacy of TNBC. Biomarkers for predicting the efficacy of platinum drugs in TNBC treatment have always been a hot topic. The DDR pathway contains a large number of related genes, and recent studies have shown that deficiencies in the DDR pathway may be associated with the efficacy of platinum drugs, which is expected to be a biomarker for predicting the efficacy of platinum drugs in breast cancer treatment.

Progress in systemic therapy for triple-negative breast cancer / 医学前沿

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with a heterogeneous genetic profile. Chemotherapy exhibits substantial activity in a small subset of these patients. Drug resistance is inevitable. Major progress has been made in the genetic analysis of TNBC to identify novel targets and increase the precision of therapeutic intervention. Such progress has translated into major advances in treatment strategies, including modified chemotherapy approaches, immune checkpoint inhibitors, and targeted therapeutic drugs. All of these strategies have been evaluated in clinical trials. Nevertheless, patient selection remains a considerable challenge in clinical practice.

Tumor suppressive function of Matrin 3 in the basal-like breast cancer

BACKGROUND: Basal-like breast cancer (BLBC) or triple-negative breast cancer (TNBC) is an aggressive and highly metastatic subtype of human breast cancer. The present study aimed to elucidate the potential tumor-suppressive function of MATR3, an abundant nuclear protein, in BLBC/TNBC, whose cancer-relevance has not been characterized. METHODS: We analyzed in vitro tumorigenecity by cell proliferation and soft agar colony formation assays, apoptotic cell death by flow cytometry and Poly (ADP-ribose) polymerase (PARP) cleavage, epithelial-mesenchymal transition (EMT) by checking specific EMT markers with real-time quantitative PCR and in vitro migration and invasion by Boyden Chamber assays. To elucidate the underlying mechanism by which MATR3 functions as a tumor suppressor, we performed Tandem affinity purification followed by mass spectrometry (TAP-MS) and pathway analysis. We also scrutinized MATR3 expression levels in the different subtypes of human breast cancer and the correlation between MATR3 expression and patient survival by bioinformatic analyses of publicly available transcriptome datasets. RESULTS: MATR3 suppressed in vitro tumorigenecity, promoted apoptotic cell death and inhibited EMT, migration, and invasion in BLBC/TNBC cells. Various proteins regulating apoptosis were identified as MATR3-binding proteins, and YAP/TAZ pathway was suppressed by MATR3. MATR3 expression was inversely correlated with the aggressive and metastatic nature of breast cancer. Moreover, high expression levels of MATR3 were associated with a good prognosis of breast cancer patients. CONCLUSIONS: Our data demonstrate that MATR3 functions as a putative tumor suppressor in BLBC/TNBC cells. Also, MATR3 potentially plays a role as a biomarker in predicting chemotherapy-sensitivity and patient survival in breast cancer patients.

miR-29b-3p promotes progression of MDA-mB-231 triple-negative breast cancer cells through downregulating TRAF3

BACKGROUND: Breast cancer is the second common malignant cancer among females worldwide. Accumulating studies have indicated that deregulation of miRNA expression in breast cancer will contribute to tumorigenesis and form different cancer subtypes. However, the reported studies on miR-29b-3p-regulated breast cancer are limited so far. Herein, we investigated the role and mechanism of miR-29b-3p in the triple negative breast cancer cell line MDA-MB-231. METHODS: The relative miR-29b-3p expression in different breast cancer cell lines were determined by qRT-PCR. CCK8 and colony formation assay were used to determine the influence of miR-29b-3p on cell proliferation. Migration assay and invasion assay were performed for cell migration and invasion respectively. To study the cell integrity immunofluorescence was performed. TUNEL assay, flow cytometry assay, hoechst staining and western blot were conducted to determine the influence of miR-29b-3p inhibitor on cell apoptosis. TRAF3 was found to be the target gene of miR-29b-3p using bioinformatics predictions. Dual-luciferase assay was performed to determine the relative luciferase activity in NC, miR-29b-3p mimic, miR-29b-3p inhibitor with TRAF3 3'-UTR wt or TRAF3 3'-UTR mt reporter plasmids. The proteins expression of NF-κB signaling pathway in MDA-MB-231 after transfection with NC, miR-29b-3p mimic, miR-29b-3p inhibitor were determined by western blot. RESULTS: The miR-29b-3p expression was significantly increased in MDA-MB-231 compare with MCF-10A. miR-29b-3p inhibitor reduced the cell viability of MDA-MB-231 and inhibited cell migration and invasion. Cell cytoskeleton integrity destroyed after miR-29b-3p inhibitor treatment. Furthermore, we identified the mechanism and found miR-29b-3p targets the TRAF3 and activates NF-κB signaling pathway. CONCLUSIONS: From the above studies, our results indicated that miR-29b-3p acts as a promoter for the development of MDA-MB-231.

Aspectos moleculares envolvidos no surgimento do Tumor Triplo-Negativo de Mama em pacientes portadoras ou não de mutação germinativa em BRCA1 / Molecular aspects involved in development of triple-negative breast cancer in patients with or without germline mutation in BRCA1

O Câncer de Mama Triplo-Negativo (CMTN), caracterizado pela perda de expressão dos receptores de estrógeno (RE), de progesterona (RP) e pela não super-expressão/amplificação do receptor do fator de crescimento epidermal humano do tipo 2 (HER-2), é considerado um subtipo bastante agressivo e heterogêneo molecularmente. Clinicamente, o CMTN apresenta prognóstico ruim, altas taxas de recorrência e menor sobrevida global em relação aos outros subtipos de câncer de mama. Ele corresponde a aproximadamente 15% dos casos de câncer de mama e não apresenta nenhuma terapia alvo efetiva. Mutação patogênica germinativa nos genes BRCA1 e BRCA2 leva a um aumento de risco para o desenvolvimento de Câncer de Mama e Ovário, sendo que mutação germinativa em BRCA1 está associada ao desenvolvimento de CMTN, especialmente em pacientes diagnosticadas antes dos 50 anos. A deficiência de BRCA1 leva ao mecanismo ineficiente do reparo do DNA e ao desenvolvimento do tumor. Recentemente, nosso grupo classificou as pacientes diagnosticadas com CMTN em grupo Hereditário (com mutação patogênica germinativa em BRCA1) e grupo Esporádico (sem mutação germinativa em BRCA1). Estes foram ainda classificados em tumores BRCA1-deficiente (com hipermetilação no promotor de BRCA1) e em tumores BRCA1-proficiente (sem hipermetilação no promotor de BRCA1 e sem mutação germinativa em BRCA1). As diferenças moleculares entre esses grupos de CMTN são de grande interesse clínico e biológico, embora ainda não sejam conhecidas. Dentro desse contexto, nosso objetivo foi investigar o perfil transcricional de CMTN sob diferentes aspectos de deficiência de BRCA1. Para isso, foi avaliado o sequenciamento do RNA (RNA-Seq) de 37 casos de CMTN em idade jovem (≤ 50 anos), sendo 9 casos Hereditário (com mutação patogênica germinativa em BRCA1) e 28 casos Esporádico. Dos casos Esporádicos, 9 foram BRCA1-deficiente (com o promotor de BRCA1 hipermetilado) e 19 BRCA1-proficiente (com o promotor de BRCA1 não hipermetilado). Utilizamos o RNA total a partir das 37 amostras tumorais e 25 amostras normais pareadas adjacentes ao tumor. As bibliotecas de cDNA foram construídas a partir do RNA total através do método de depleção do rRNA e sequenciadas na plataforma NextSeq (Illumina). Para a normalização dos dados de expressão de cada gene, foi aplicada a medida FPKM. Os critérios para a obtenção dos genes diferencialmente expressos (GDEs), nas amostras tumorais em relação às normais, foram fold change ≥ 4,0 e ≤ - 4,0 e p valor ajustado ≤ 0,05). Para classificação molecular dos tumores TN, foi utilizada a ferramenta online TNBCtype. As curvas de sobrevida global de acordo com essa classificação molecular foram calculadas através do método de Kaplan-Meier. Para verificar os processos biológicos envolvidos com a tumorigênese do CMTN, os GDEs foram submetidos a uma análise funcional in silico através do programa Ingenuity Pathway Analysis (IPA). Em média, 48.627.204 milhões de sequências foram geradas por amostra, das quais 79,5% foram mapeadas no genoma humano referência, revelando, em média, 15.071 genes expressos com pelo menos 10 sequências única mapeada por amostra. O perfil transcricional das amostras CMTN permitiu a classificação nos 7 subtipos moleculares de CMTN, sendo a maioria das amostras classificadas no subtipo imunomodulador (IM) e mesenquimal (M). No grupo das amostras BRCA1-deficiente, observamos um predomínio do subtipo IM enquanto que, nas amostras BRCA1-proficiente, houve um maior número de amostras classificadas em M. Não observamos diferença nas curvas de sobrevida global entre os dois grupos de amostras. Porém, os resultados mostraram que o subtipo IM parece ter uma tendência de melhor sobrevida global comparado com os outros subtipos, independente do status de BRCA1. A expressão diferencial das amostras tumorais em relação às amostras normais no grupo de CMTN Hereditário revelou 1965 GDEs, sendo 589 mais expressos e 1376 menos expressos no tumor; e, no grupo de CMTN Esporádico, a análise revelou 1837 GDEs, sendo 645 mais expressos e 1192 menos expressos no tumor. A partir dos GDEs de cada grupo, a análise do IPA mostrou que os dois grupos apresentaram vias canônicas enriquecidas comuns significantemente ativadas e envolvidas, de forma geral, com a Regulação do ciclo celular. Entretanto, as vias canônicas significantemente inibidas mostraram-se exclusivas em cada grupo: a via de Sinalização do receptor do glutamato foi a mais significativa (z-score = -2,12) nas amostras de CMTN Hereditário; e a via de Ativação de LXR/RXR foi a mais significativa (z-score = -2,98) nas amostras CMTN Esporádico. Além disso, observamos reguladores transcricionais significantemente ativados e inibidos exclusivamente em cada grupo. Os genes TNF e E2F1 foram os mais significantemente ativados apenas nas amostras de CMTN Hereditário (z-score = 4,06) e de CMTN Esporádico (z-score = 2,22), respectivamente. Por outro lado, o microRNA mir-21 e o gene PPARG foram os reguladores inibidos exclusivamente nas amostras CMTN Hereditário (-score = -4,68) e nas amostras CMTN Esporádico (z-score = -4,24), respectivamente. Esse estudo, de forma geral, revelou potenciais vias e genes reguladores de cascatas de sinalização envolvidos na tumorigênese do CMTN no contexto da deficiência de BRCA1, contribuindo na elucidação da complexidade funcional de tumores TN

Triple-negative breast cancer (TNBC), characterized by lack of expression of the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), results in aggressive biology, early peak of recurrence, and shorter overall survival than other subtypes. It comprises approximately 15% of breast cancer cases and yet there is no effective therapy. Mutations in the BRCA1 and BRCA2 genes are associated with increased risk of breast and ovarian cancers since germline mutation in BRCA1 is associated with the development of TNBC, especially in patients diagnosed before age 50. BRCA deficiency leads to impaired DNA repair and tumor development. Recently, our group classified TNBC patients into hereditary BRCA1-mutated and sporadic BRCA1-proficient. These were further classified into BRCA1-deficient tumors (with BRCA1 promotor hypermethylation) and BRCA1-proficient tumors (no BRCA1 promoter hypermethylation neither BRCA1 germline mutation). Molecular differences between hereditary and sporadic TNBC groups are clinically and biologically interesting although it remains unclear. In this context, we aimed to investigate the transcriptional profile of TNBC-associated or not with BRCA1 deficiency. For that, RNA sequencing (RNA-seq) from 37 early-onset TNBC (≤ 50 years old) was evaluated, comprising 9 Hereditary (with BRCA1 germline pathogenic mutation) and 28 Sporadic cases, of which 9 BRCA1-deficient and 19 BRCA1-proficient. Total RNA from 37 tumors samples and 25 adjacent normal samples of paired cases were used to constructed RNAseq libraries by depleting ribosomal RNA and sequenced on Illumina NextSeq 500 platform. Expression values were normalized by FPKM. Differentially expressed genes (DEGs) between tumor and normal samples were obtained using fold-change ≥ |4| and p value adjusted ≤ 0.05 as statistical criteria. The TNBC samples were subtyping using the web-based prediction tool TNBCType. Overall survival curves were calculated using the Kaplan.Meir method. IPA software was used to detect activated/inactivated canonical pathways and relevant upstream regulators, considering a z-score ≤-2.0 and ≥ 2.0, respectively. On average, 49 million reads were generated per sample, of which 79.5% were mapped to the human reference genome revealing 15,071 expressed genes with at least 10 reads per sample. From the transcriptional profile of TNBC samples we classified into seven TNBC subtypes being the majority of tumors classified as immunomodulatory (IM) and mesenchymal (M) subtype. We detected no difference in overall survival for both groups. However, trends towards better overall were observed for TNBC samples classified as IM compared with other subtypes, without associations with BRCA1 status. Differential gene expression analysis between tumor and normal samples in the hereditary group revealed 1,965 DEGs, being 589 upregulated and 1,376 downregulated; and in the sporadic group, the analysis revealed 1,837 DEGs, being 645 upregulated and 1,192 downregulated. Using the DEGs of each group, the IPA analysis revealed that Cell Cycle Regulation signaling was activated in both groups. Regarding inactivated pathways, we detected the Glutamate Receptor signaling (z-score = -2,12) in hereditary TNBC and the LXR/RXR activation in sporadic TNBC (z-score = -2,98). Also, the IPA analysis revealed relevant specific transcription regulators of each group. The TNF and E2F1 were the most significantly activated genes in hereditary- and sporadic-TNBC, respectively. On the other hand, the mir-21 and PPARG were the most significantly unique inhibited regulators in hereditary and sporadic, respectively. In general, this study unveiled potential pathways and regulatory genes for signaling cascades involved in TNBC tumorigenesis considering the deficiency of BRCA1, contributing to the elucidation of the functional complexity of the tumorigenic process of TNBC patients

Avaliação da prevalência de mutação no gene BRCA1 emtumores triplo-negativos de mama: associação da mutação comcaracterísticas clínicas e de resposta terapêutica / Evaluation of BRCA1 gene mutation prevalence in triple negative breast cancer: Association of mutation with clinical features and treatment response

O câncer de mama é o tipo de câncer mais incidente em mulheres em todo o mundo, excluindo os casos de câncer de pele não-melanoma. A estimativa de incidência para o biênio de 2014-2015 no Brasil é de mais de 57 mil novos casos por ano. O câncer de mama é uma doença heterogênea, podendo ser dividida em subtipos de acordo com o perfil imunofenotípico e de expressão gênica desses tumores. Em relação ao perfil imunofenotípico, o tumor de mama triplo-negativo (TN) é caracterizado pela ausência dos receptores hormonais de estrogênio (ER) e de progesterona (PR) além de não apresentar super-expressão/amplificação do receptor 2 do fator de crescimento epidérmico humano (HER2). Essa condição é importante, pois as terapias hormonais e moleculares efetivas em outros subtipos, não têm efeito nesses tumores, sendo o tratamento feito com base em quimioterapia sistêmica. Somado a isso, o tumor TN demonstra maior agressividade e padrões metastáticos distintos dos outros tumores, o que resulta em pior prognóstico e sobrevida para as pacientes portadoras desses tumores. Vários trabalhos, incluindo do nosso grupo de pesquisa, têm relatado alta prevalência de mutações germinativas patogênicas no gene BRCA1 em mulheres jovens portadoras de tumores TN de mama...

Breast cancer is the most frequent type of cancer in women worldwide, with exception of non-melanoma skin cancer. The incidence estimate for 2014-2015 biennium in Brazil is more than 57 thousand new cases per year.Breast cancer is a heterogeneous disease that is divided according to immunophenotypic and gene expression profiles of the tumors. Regarding the immunophenotypic profile, the triple-negative breast cancer (TNBC) is characterized by the lack of hormonal receptors for estrogen and progesterone (ER and PR) and also the absence of superexpression/ amplification of the Human Epidermal Growth Factor Receptor 2 (HER2). This condition is important because hormonal and molecular therapies have no effect on these tumors. Therefore systemic chemotherapy is the mainstay treatment. Moreover, TNBC displays higher aggressiveness and distinct metastatic pattern compared to other breast tumors, resulting in worse prognosis and survival for TNBC patients. Several researchers, including our research group, have reported high prevalence of germline pathogenic mutations in the BRCA1 gene among young women diagnosed with TNBC. This gene is involved primarily in the mechanism of DNA repair by homologous recombination and acts as a tumor suppressor gene. Thus, germline mutation that leads to loss of function of its respective protein may favor cancer development, mostly in the breast and ovarian of carriers. However, it is not well stablished the proportion of pathogenic...

beta-TrCP1 degradation is a novel action mechanism of PI3K/mTOR inhibitors in triple-negative breast cancer cells

An F-box protein, beta-TrCP recognizes substrate proteins and destabilizes them through ubiquitin-dependent proteolysis. It regulates the stability of diverse proteins and functions as either a tumor suppressor or an oncogene. Although the regulation by beta-TrCP has been widely studied, the regulation of beta-TrCP itself is not well understood yet. In this study, we found that the level of beta-TrCP1 is downregulated by various protein kinase inhibitors in triple-negative breast cancer (TNBC) cells. A PI3K/mTOR inhibitor PI-103 reduced the level of beta-TrCP1 in a wide range of TNBC cells in a proteasome-dependent manner. Concomitantly, the levels of c-Myc and cyclin E were also downregulated by PI-103. PI-103 reduced the phosphorylation of beta-TrCP1 prior to its degradation. In addition, knockdown of beta-TrCP1 inhibited the proliferation of TNBC cells. We further identified that pharmacological inhibition of mTORC2 was sufficient to reduce the beta-TrCP1 and c-Myc levels. These results suggest that mTORC2 regulates the stability of beta-TrCP1 in TNBC cells and targeting beta-TrCP1 is a potential approach to treat human TNBC.