RESUMO
BACKGROUND: Rab proteins constitute a large family of monomeric GTP-binding proteins that regulate intracellular vesicle transport. Several Rab proteins, including rab31, have been shown to affect cancer progression and are related with prognosis in various types of cancer including breast cancer. Recently, the gene encoding rab31 was found to be overexpressed in estrogen receptor-positive breast cancer tissue. In a previous study we found a significant association of high rab31 mRNA expression with poor prognosis in node-negative breast cancer patients. In the present study, we aimed to investigate the impact of rab31 (over)-expression on important aspects of tumor progression in vitro and in vivo. METHODS: Breast cancer cells displaying low (MDA-MB-231) or no (CAMA-1) endogenous rab31 expression were stably transfected with a rab31 expression plasmid. Batch-transfected cells as well as selected cell clones, expressing different levels of rab31 protein, were analyzed with regard to proliferation, cell adhesion, the invasive capacity of tumor cells, and in vivo in a xenograft tumor model. Polyclonal antibodies directed to recombinantly expressed rab31 were generated and protein expression analyzed by immunohistochemistry, Western blot analysis, and a newly developed sensitive ELISA. RESULTS: Elevated rab31 protein levels were associated with enhanced proliferation of breast cancer cells. Interestingly, weak to moderate overexpression of rab31 in cell lines with no detectable endogenous rab31 expression was already sufficient to elicit distinct effects on cell proliferation. By contrast, increased expression of rab31 in breast cancer cells led to reduced adhesion towards several extracellular matrix proteins and decreased invasive capacity through Matrigel(TM). Again, the rab31-mediated effects on cell adhesion and invasion were dose-dependent. Finally, in a xenograft mouse model, we observed a significantly impaired metastatic dissemination of rab31 overexpressing MDA-MB-231 breast cancer cells to the lung. CONCLUSIONS: Overexpression of rab31 in breast cancer cells leads to a switch from an invasive to a proliferative phenotype as indicated by an increased cell proliferation, reduced adhesion and invasion in vitro, and a reduced capacity to form lung metastases in vivo.
Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Proteínas rab de Ligação ao GTP/genética , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/metabolismo , Feminino , Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Transplante Heterólogo , Proteínas rab de Ligação ao GTP/imunologia , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
mRNA levels of the urokinase receptor splice variant uPAR-del4/5 are associated with prognosis in breast cancer. Its overexpression in cancer cells affects tumor biologically relevant processes. In the present study, individual breast cancer cell clones displaying low vs. high uPAR-del4/5 expression were analyzed demonstrating that uPAR-del4/5 leads to reduced cell adhesion and invasion in a dose-dependent manner. Additionally,matrix metalloproteinase-9 (MMP-9) was found to be strongly upregulated in uPAR-del4/5 overexpressing compared to vector control cells. uPAR-del4/5 may thus play an important role in the regulation of the extracellular proteolytic network and, by this, influence the metastatic potential of breast cancer cells.
Assuntos
Neoplasias da Mama/genética , Mama/metabolismo , Splicing de RNA , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Mama/citologia , Mama/patologia , Neoplasias da Mama/patologia , Adesão Celular , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metaloproteinase 9 da Matriz/genética , Invasividade Neoplásica/genética , Regulação para CimaRESUMO
uPAR, the three-domain membrane receptor of the serine protease urokinase, plays a crucial role in tumor growth and metastasis. Several mRNA splice variants of this receptor have been reported. One of these, uPAR-del4/5, lacking exons 4 and 5, and thus encoding a uPAR form lacking domain DII, is specifically overexpressed in breast cancer and represents a statistically independent prognostic factor for distant metastasis-free survival in breast cancer patients. The aim of the present study was to examine the molecular and cellular properties of the encoded uPAR-del4/5 protein. To investigate the impact of the uPAR-del4/5 overexpression on in vitro and in vivo aspects of tumor progression (e.g., proliferation, adhesion, invasion, metastatic seeding, and/or metastatic growth), we combined the analysis of transfected cancer cell lines with a murine xenograft tumor model. Increased expression of uPAR-del4/5 in human cancer cells led to reduced adhesion to several extracellular matrix proteins and decreased invasion through Matrigel, while cell proliferation was not affected in vitro. Moreover, invasion of uPAR-del4/5 overexpressing cells was not altered by addition of urokinase, while that of uPAR-wild-type overexpressing cells was drastically increased. Accordingly, we observed that, in contrast to uPAR-wild-type, uPAR-del4/5 does not interact with urokinase. On the other hand, when overexpressed in human breast cancer cells, uPAR-del4/5 distinctly impaired metastatic dissemination and growth in vivo. We demonstrate that the uPAR-del4/5 mRNA splice variant mediates tumor-relevant biological processes in vitro and in vivo. Our results thus illustrate how tumor-specific alternative splicing can distinctly impact the biology of the tumor.
