RESUMO
Triple negative breast cancers (TNBC) present a poor prognosis primarily due to their resistance to chemotherapy. This resistance is known to be associated with elevated expression of certain anti-apoptotic members within the proteins of the BCL-2 family (namely BCL-xL, MCL-1 and BCL-2). These regulate cell death by inhibiting pro-apoptotic protein activation through binding and sequestration and they can be selectively antagonized by BH3 mimetics. Yet the individual influences of BCL-xL, MCL-1, and BCL-2 on the sensitivity of TNBC cells to chemotherapy, and their regulation by cancer-associated fibroblasts (CAFs), major components of the tumor stroma and key contributors to therapy resistance remain to be delineated. Using gene editing or BH3 mimetics to inhibit anti-apoptotic BCL-2 family proteins in TNBC line MDA-MB-231, we show that BCL-xL and MCL-1 promote cancer cell survival through compensatory mechanisms. This cell line shows limited sensitivity to chemotherapy, in line with the clinical resistance observed in TNBC patients. We elucidate that BCL-xL plays a pivotal role in therapy response, as its depletion or pharmacological inhibition heightened chemotherapy effectiveness. Moreover, BCL-xL expression is associated with chemotherapy resistance in patient-derived tumoroids where its pharmacological inhibition enhances ex vivo response to chemotherapy. In a co-culture model of cancer cells and CAFs, we observe that even in a context where BCL-xL reduced expression renders cancer cells more susceptible to chemotherapy, those in contact with CAFs display reduced sensitivity to chemotherapy. Thus CAFs exert a profound pro-survival effect in breast cancer cells, even in a setting highly favoring cell death through combined chemotherapy and absence of the main actor of chemoresistance, BCL-xL.
Assuntos
Fibroblastos Associados a Câncer , Resistencia a Medicamentos Antineoplásicos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Neoplasias de Mama Triplo Negativas , Proteína bcl-X , Humanos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Proteína bcl-X/metabolismo , Proteína bcl-X/genética , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Resistencia a Medicamentos Antineoplásicos/genética , Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidoresRESUMO
Controversial reports have suggested that SARS-CoV E and 3a proteins are plasma membrane viroporins. Here, we aimed at better characterizing the cellular responses induced by these proteins. First, we show that expression of SARS-CoV-2 E or 3a protein in CHO cells gives rise to cells with newly acquired round shapes that detach from the Petri dish. This suggests that cell death is induced upon expression of E or 3a protein. We confirmed this by using flow cytometry. In adhering cells expressing E or 3a protein, the whole-cell currents were not different from those of the control, suggesting that E and 3a proteins are not plasma membrane viroporins. In contrast, recording the currents on detached cells uncovered outwardly rectifying currents much larger than those observed in the control. We illustrate for the first time that carbenoxolone and probenecid block these outwardly rectifying currents; thus, these currents are most probably conducted by pannexin channels that are activated by cell morphology changes and also potentially by cell death. The truncation of C-terminal PDZ binding motifs reduces the proportion of dying cells but does not prevent these outwardly rectifying currents. This suggests distinct pathways for the induction of these cellular events by the two proteins. We conclude that SARS-CoV-2 E and 3a proteins are not viroporins expressed at the plasma membrane.
Assuntos
COVID-19 , SARS-CoV-2 , Cricetinae , Animais , Cricetulus , Membrana Celular , Células CHORESUMO
Cancer-associated fibroblasts (CAF) are a major cellular component of epithelial tumors. In breast cancers in particular these stromal cells have numerous tumorigenic effects in part due to their acquisition of a myofibroblastic phenotype. Breast CAFs (bCAFs) typically express MCL-1. We show here that pharmacological inhibition or knock down of this regulator of mitochondrial integrity in primary bCAFs directly derived from human samples mitigates myofibroblastic features. This decreases expression of genes involved in actomyosin organization and contractility (associated with a cytoplasmic retention of the transcriptional regulator, yes-associated protein-YAP) and decreases bCAFs ability to promote cancer cells invasion in 3D coculture assays. Our findings underscore the usefulness of targeting MCL-1 in breast cancer ecosystems, not only to favor death of cancer cells but also to counteract the tumorigenic activation of fibroblasts with which they co-evolve. Mechanistically, pharmacological inhibition of MCL-1 with a specific BH3 mimetic promotes mitochondrial fragmentation in bCAFs. Inhibition of the mitochondrial fission activity of DRP-1, which interacts with MCL-1 upon BH3 mimetic treatment, allows the maintenance of the myofibroblastic phenotype of bCAFs.
Assuntos
Neoplasias da Mama , Fibroblastos Associados a Câncer , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Ecossistema , Feminino , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , FenótipoRESUMO
Resistance of solid cancer cells to chemotherapies and targeted therapies is not only due to the mutational status of cancer cells but also to the concurring of stromal cells of the tumor ecosystem, such as immune cells, vasculature and cancer-associated fibroblasts (CAFs). The reciprocal education of cancer cells and CAFs favors tumor growth, survival and invasion. Mitochondrial function control, including the regulation of mitochondrial metabolism, oxidative stress and apoptotic stress are crucial for these different tumor progression steps. In this review, we focus on how CAFs participate in cancer progression by modulating cancer cells metabolic functions and mitochondrial apoptosis. We emphasize that mitochondria from CAFs influence their activation status and pro-tumoral effects. We thus advocate that understanding mitochondria-mediated tumor-stroma interactions provides the possibility to consider cancer therapies that improve current treatments by targeting these interactions or mitochondria directly in tumor and/or stromal cells.
RESUMO
Emerging evidence supports neurotensin as a trophic and antiapoptotic factor, mediating its control via the high-affinity neurotensin receptor (NT1 receptor) in several human solid tumors. In a series of 51 patients with invasive ductal breast cancers, 34% of all tumors were positive for neurotensin and 91% positive for NT1 receptor. We found a coexpression of neurotensin and NT1 receptor in a large proportion (30%) of ductal breast tumors, suggesting a contribution of the neurotensinergic signaling cascade within breast cancer progression. Functionally expressed NT1 receptor, in the highly malignant MDA-MB-231 human breast cancer cell line, coordinated a series of transforming functions, including cellular migration, invasion, induction of the matrix metalloproteinase (MMP)-9 transcripts, and MMP-9 gelatinase activity. Disruption of NT1 receptor signaling by silencing RNA or use of a specific NT1 receptor antagonist, SR48692, caused the reversion of these transforming functions and tumor growth of MDA-MB-231 cells xenografted in nude mice. Our findings support the contribution of neurotensin in human breast cancer progression and point out the utility to develop therapeutic molecules targeting neurotensin or NT1 receptor signaling cascade. These strategies would increase the range of therapeutic approaches and be beneficial for specific patients.
Assuntos
Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neurotensina/biossíntese , Receptores de Neurotensina/biossíntese , Animais , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patologia , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Progressão da Doença , Ativação Enzimática , Feminino , Humanos , Imuno-Histoquímica , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Pessoa de Meia-Idade , Invasividade Neoplásica , Transplante HeterólogoRESUMO
We investigated the expression of the neurotensin high-affinity receptor 1 (NTS1) during inflammatory bowel disease (IBD)-related colorectal oncogenesis, in colonic samples from 30 patients with IBD-related adenocarcinomas, dysplasias, and inflammatory mucosa (IM). The percentage of NTS1-positive epithelial cells progressively increased from the inflammatory condition to adenocarcinoma and was significantly higher in adenocarcinomas than in IM (p=0.0169). In parallel, the percentage of neurotensin (NT)-positive epithelial cells increased during the IBD-related oncogenesis. Finally, as NTS1 is a ss-catenin inducible gene, we found that a number of preneoplastic lesions and adenocarcinomas co-expressed NTS1 and beta-catenin without NT expression. Therefore, this study suggests two pathways of NTS1 overexpression during IBD-related oncogenesis: one triggered by NT overexpression, and a second associated with an activation of the APC/beta-catenin pathway, these two pathways being not mutually exclusive.
Assuntos
Transformação Celular Neoplásica , Neoplasias do Colo/complicações , Doenças Inflamatórias Intestinais/metabolismo , Neurotensina/metabolismo , Receptores de Neurotensina/metabolismo , beta Catenina/metabolismo , Células Cultivadas , Humanos , Imuno-Histoquímica , Doenças Inflamatórias Intestinais/complicações , Ligantes , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In tumours, accumulation of chemoresistant cells that express high levels of anti-apoptotic proteins such as BCL-XL is thought to result from the counter selection of sensitive, low expresser clones during progression and/or initial treatment. We herein show that BCL-XL expression is selectively advantageous to cancer cell populations even in the absence of pro-apoptotic pressure. In transformed human mammary epithelial cells BCL-XL favours full activation of signalling downstream of constitutively active RAS with which it interacts in a BH4-dependent manner. Comparative proteomic analysis and functional assays indicate that this is critical for RAS-induced expression of stemness regulators and maintenance of a cancer initiating cell (CIC) phenotype. Resistant cancer cells thus arise from a positive selection driven by BCL-XL modulation of RAS-induced self-renewal, and during which apoptotic resistance is not necessarily the directly selected trait.
Assuntos
Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Células-Tronco Neoplásicas/citologia , Transdução de Sinais , Proteína bcl-X/metabolismo , Proteínas ras/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Proteína HMGA2/metabolismo , Humanos , Células MCF-7 , Espectrometria de Massas , Camundongos , Camundongos Nus , Recidiva Local de Neoplasia , Fenótipo , Plasmídeos/metabolismo , Proteômica , Proteínas Proto-Oncogênicas c-fos/metabolismoRESUMO
Neurotensin is a tridecapteptide acting mostly in the brain and gastrointestinal tract. NT binds two G protein coupled receptors (GPCR), NTS1 and NTS2, and a single transmembrane domain receptor, NTS3/gp95/sortilin receptor. NTS1 mediates the majority of NT action in neurons and the periphery. Like many other GPCRs, upon agonist stimulation, NTS1 is internalized, endocytosed, and the cells are desensitized. It is tacitly acknowledged that the intensity and the lasting of cellular responses to NT are dependent on free and functional NTS1 at the cell surface. Understanding how NTS1 expression is regulated at the membrane should provide a better comprehension towards its function. This review analyzes and discusses the current cellular and molecular mechanisms affecting the expression of NTS1 at the cellular membrane upon acute and chronic NT stimulation.
Assuntos
Receptores de Neurotensina/agonistas , Receptores de Neurotensina/metabolismo , Animais , Transporte Biológico Ativo , Endocitose , Regulação da Expressão Gênica , Humanos , Membranas/metabolismo , Modelos Neurológicos , Neurotensina/metabolismo , Receptores de Neurotensina/genética , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Hath1, a bHLH transcription factor negatively regulated by the γ-secretase-dependent Notch pathway, is required for intestinal secretory cell differentiation. Our aim was fourfold: 1) determine whether Hath1 is able to alter the phenotype of colon cancer cells that are committed to a differentiated phenotype, 2) determine whether the Hath1-dependent alteration of differentiation is coupled to a restriction of anchorage-dependent growth, 3) decipher the respective roles of three putative tumor suppressor genes Hath1, MUC2 and P27kip1 in this coupling and, 4) examine how our findings translate to primary tumors. Human colon carcinoma cell lines that differentiate along a mucin secreting (MUC2/MUC5AC) and/or enterocytic (DPPIV) lineages were maintained on inserts with or without a γ-secretase inhibitor (DBZ). Then the cells were detached and their ability to survive/proliferate in the absence of substratum was assessed. γ-secretase inhibition led to a Hath1-mediated preferential induction of MUC2 over MUC5AC, without DPPIV modification, in association with a decrease in anchorage-independent growth. While P27kip1 silencing relieved the cells from the Hath1-induced decrease of anchorage-independent growth, MUC2 silencing did not modify this parameter. Hath1 ectopic expression in the Hath1 negative enterocytic Caco2 cells led to a decreased anchorage-independent growth in a P27kip1-independent manner. In cultured primary human colon carcinomas, Hath1 was up-regulated in 7 out of 10 tumors upon DBZ treatment. Parallel MUC2 up-regulation occurred in 4 (4/7) and P27kip1 in only 2 (2/7) tumors. Interestingly, the response patterns of primary tumors to DBZ fitted with the hierarchical model of divergent signalling derived from our findings on cell lines.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinoma/genética , Neoplasias do Colo/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Mucina-2/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Neoplasias do Colo/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/metabolismo , Humanos , Modelos Biológicos , Mucina-2/metabolismo , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Pesquisa Translacional Biomédica , Células Tumorais CultivadasRESUMO
PURPOSE: This study aimed to investigate the role of the neurotensin/neurotensin receptor I (NTSR1) complex in non-small cell lung cancer (NSCLC) progression. EXPERIMENTAL DESIGN: The expression of neurotensin and NTSR1 was studied by transcriptome analysis and immunohistochemistry in two series of 74 and 139 consecutive patients with pathologic stage I NSCLC adenocarcinoma. The findings were correlated with clinic-pathologic features. Experimental tumors were generated from the malignant human lung carcinoma cell line A459, and a subclone of LNM35, LNM-R. The role of the neurotensin signaling system on tumor growth and metastasis was investigated by small hairpin RNA-mediated silencing of NTSR1 and neurotensin. RESULTS: Transcriptome analysis carried out in a series of 74 patients showed that the positive regulation of NTSR1 put it within the top 50 genes related with relapse-free survival. Immunohistochemistry revealed neurotensin- and NTSR1-positive staining in 60.4% and 59.7% of lung adenocarcinomas, respectively. At univariate analysis, NTSR1 expression was strongly associated with worse 5-year overall survival rate (P = 0.0081) and relapse-free survival (P = 0.0024). Multivariate analysis showed that patients over 65 years of age (P = 0.0018) and NTSR1 expression (P = 0.0034) were independent negative prognostic factors. Experimental tumor xenografts generated by neurotensin- and NTSR1-silenced human lung cancer cells revealed that neurotensin enhanced primary tumor growth and production of massive nodal metastasis via autocrine and paracrine regulation loops. CONCLUSION: NTSR1 expression was identified as a potential new prognostic biomarker for surgically resected stage I lung adenocarcinomas, as NTSR1 activation was shown to participate in lung cancer progression.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Neurotensina/genética , Receptores de Neurotensina/genética , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Idoso , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Metástase Linfática , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Análise Multivariada , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neurotensina/metabolismo , Avaliação de Resultados em Cuidados de Saúde , Prognóstico , Interferência de RNA , Receptores de Neurotensina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante HeterólogoRESUMO
BACKGROUND: The neurotensin (NTS) and its specific high affinity G protein coupled receptor, the NT1 receptor (NTSR1), are considered to be a good candidate for one of the factors implicated in neoplastic progression. In breast cancer cells, functionally expressed NT1 receptor coordinates a series of transforming functions including cellular migration and invasion. METHODS AND RESULTS: we investigated the expression of NTS and NTSR1 in normal human breast tissue and in invasive ductal breast carcinomas (IDCs) by immunohistochemistry and RT-PCR. NTS is expressed and up-regulated by estrogen in normal epithelial breast cells. NTS is also found expressed in the ductal and invasive components of IDCs. The high expression of NTSR1 is associated with the SBR grade, the size of the tumor, and the number of metastatic lymph nodes. Furthermore, the NTSR1 high expression is an independent factor of prognosis associated with the death of patients. CONCLUSION: these data support the activation of neurotensinergic deleterious pathways in breast cancer progression.
Assuntos
Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Regulação Neoplásica da Expressão Gênica , Receptores de Neurotensina/metabolismo , Idoso , Biópsia , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Progressão da Doença , Estradiol/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Invasividade Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , PrognósticoRESUMO
Alterations in the Wnt/APC (adenomatous polyposis coli) signalling pathway, resulting in beta-catenin/T cell factor (Tcf)-dependent transcriptional gene activation, are frequently detected in familial and sporadic colon cancers. The neuropeptide neurotensin (NT) is widely distributed in the gastrointestinal tract. Its proliferative and survival effects are mediated by a G-protein coupled receptor, the NT1 receptor. NT1 receptor is not expressed in normal colon epithelial cells, but is over expressed in a number of cancer cells and tissues suggesting a link to the outgrowth of human colon cancer. Our results demonstrate that the upregulation of NT1 receptor occurring in colon cancer is the result of Wnt/APC signalling pathway activation. We first established the functionality of the Tcf response element within the NT1 receptor promoter. Consequently, we observed the activation of NT1 receptor gene by agents causing beta-catenin cytosolic accumulation, as well as a strong decline of endogenous receptor when wt-APC was restored. At the cellular level, the re-establishment of wt-APC phenotype resulted in the impaired functionality of NT1 receptor, like the breakdown in NT-induced intracellular calcium mobilization and the loss of NT pro-invasive effect. We corroborated the Wnt/APC signalling pathway on the NT1 receptor promoter activation with human colon carcinogenesis, and showed that NT1 receptor gene activation was perfectly correlated with nuclear or cytoplasmic beta-catenin localization while NT1 receptor was absent when beta-catenin was localized at the cell-cell junction in early adenomas of patients with familial adenomatous polyposis, hereditary non-polyposis colorectal cancer and loss of heterozygosity tumours. In this report we establish a novel link in vitro between the Tcf/beta-catenin pathway and NT1 receptor promoter activation.