RESUMO
Rhomboid pseudoproteases are catalytically inactive members of the rhomboid superfamily that modulate the traffic, turnover and activity of their target proteins. Rhomboid domain containing 2 (RHBDD2) is a rhomboid family member overexpressed during mammary gland development and advanced stages of breast cancer. Interactome profiling studies have identified RHBDD2 as a novel binding partner of WW domaincontaining oxidoreductase (WWOX) protein. The present study characterized the RHBDD2WWOX interaction in proliferating and differentiated stages of normal mammary and breast cancer cells by coimmunoprecipitation and confocal microscopy. Normal breast and proliferating cancer cells showed significantly increased RHBDD2 mRNA levels compared with their differentiated counterparts. WWOX mRNA was primarily expressed in differentiated cells. WWOX coprecipitated with RHBDD2, indicating that endogenous RHBDD2 and WWOX were physically associated in normal and breast cancer proliferating cells compared with the differentiated stage. Colocalization assays corroborated the coimmunoprecipitation results, demonstrating the RHBDD2WWOX protein interaction in normal and proliferating breast cancer cells. RHBDD2 harbors a conserved LPPY motif at the Cterminus region that directly interacted with the WW domains of WWOX. Since WWOX serves as an inhibitor of the TGFß/SMAD3 signaling pathway in breast cells, modulation of SMAD3 target genes was analyzed in proliferating and differentiated mammary cells and in RHBDD2 silencing assays. Increased expression levels of SMAD3regulated genes were detected in proliferating cells compared with their differentiated counterparts. Follistatin and angiopoietinlike 4 mRNA was significantly downregulated in RHBDD2 transiently silenced cells compared with scrambled control small interfering RNA. Based on these results, WWOX was suggested to be a novel RHBDD2 target protein involved in the modulation of breast epithelial cell proliferation and differentiation.
Assuntos
Neoplasias da Mama/metabolismo , Glândulas Mamárias Humanas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Oxidorredutase com Domínios WW/metabolismo , Animais , Neoplasias da Mama/genética , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Proteínas de Membrana/química , Camundongos , Ligação Proteica , Domínios Proteicos , Transdução de Sinais , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Regulação para Cima , Oxidorredutase com Domínios WW/química , Oxidorredutase com Domínios WW/genéticaRESUMO
RHBDD2 is an intramembrane pseudoprotease member of the Rhomboid superfamily. Our previous studies in breast and colorectal cancer indicate an association between RHBDD2 overexpression and advanced tumor stages. Two alternative transcriptional variants have been described for RHBDD2, which would be encoding for different RHBDD2 protein isoforms. The expression of these RHBDD2 variants/isoforms and its association with breast cancer was the focus of this study. First, expression of RHBDD2 splicing variants was evaluated in normal and breast tumor samples. RHBDD2 variant 2 overexpression was detected in tumors in respect to normal breast tissues at the mRNA and protein levels (P<0.05). Moreover, RHBDD2 variant 2 expression was associated with poor prognostic factors such as basallike intrinsic subtype (P<0.05), high proliferation (P<0.01) and longterm riskofrecurrence (P<0.01) scores. Second, the expression of both variants was evaluated under nutritionaldeprived conditions in breast cancer cell lines. Results demonstrated that RHBDD2 splicing was switched from mRNA variant 1 to variant 2 in association with a significant increment of protein isoform B in response to glucose starvation treatment. Therefore, we propose that the switch from the RHBDD2 variant 1, expressed in normal epithelial cells, to variant 2 occurs as an adaptive phenotype to bypass the stressful tumor microenvironment and promote tumor progression. Finally, the RHBDD2 subcellular localization was corroborated at the Golgi apparatus and their associated vSNARE transport vesicles, suggesting a putative new role for RHBDD2 in the protein trafficking of human breast cancer cells.
Assuntos
Processamento Alternativo/genética , Neoplasias da Mama/genética , Variação Genética/genética , Proteínas de Neoplasias/genética , Estresse Fisiológico/genética , Mama/patologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Progressão da Doença , Células Epiteliais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Complexo de Golgi/metabolismo , Humanos , Células MCF-7 , Proteínas de Membrana , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , RNA Mensageiro/genética , Microambiente Tumoral/genéticaRESUMO
During the past years, molecular studies through high-throughput technologies have led to the confirmation of critical alterations in colorectal cancer (CRC) and the discovery of some new ones, including mutations, DNA methylations, and structural chromosomal changes. These genomic alterations might act in concert to dysregulate specific signaling pathways that normally exert their functions on critical cell phenotypes, including the regulation of cellular metabolism, proliferation, differentiation, and survival. Targeted therapy against key components of altered signaling pathways has allowed an improvement in CRC treatment. However, a significant percentage of patients with CRC and metastatic CRC will not benefit from these targeted therapies and will be restricted to systemic chemotherapy. Mechanisms of resistance have been associated with specific gene alterations. To fully understand the nature and significance of the genetic and epigenetic defects in CRC that might favor a tumor evading a given therapy, much work remains. Therefore, a dynamic link between basic molecular research and preclinical studies, which ultimately constitute the prelude to standardized therapies, is very important to provide better and more effective treatments against CRC. We present an updated revision of the main molecular features of CRC and their associated therapies currently under study in clinical trials. Moreover, we performed an unsupervised classification of CRC clinical trials with the aim of obtaining an overview of the future perspectives of preclinical studies.