Análisis del papel de beta2-quimerina en cáncer de mama

Resumen

The Rho/Rac GTPases are a family of small G-proteins, which play an important role in cytoskeleton rearrangements. These proteins regulate numerous cellular processes including cellular adhesion, migration, proliferation, survival, differentiation and malignant transformation. Therefore, the precise control of the spatial and temporal activation of Rho GTPases has critical importance to cells. The Rho/Rac GTPases function as molecular switches that cycle between an inactive, GDP-bound form and an active, GTP-bound form. The fluctuation between the active and inactive state of GTPases is highly regulated by three types of proteins: GEFs (Guanine nucleotide Exchange Factors) which activate the GTPases by increasing the release rate of bound nucleotides, GAPs (GTPase Activating Proteins) that stimulate the endogenous activity of GTPase and lead G-protein inactivation, and the third class GDIs (GDP Dissociation Inhibitors) which prevent the replacement of GDP by GTP . The most studied members of this family in humans are: Rho (RhoA, RhoB and RhoC), Rac (Rac1, Rac2, Rac3 and Rac1b) and Cdc42 (Cdc42). These subfamilies are well known for their role in each of the multiple steps in tumorigenesis: upregulation of proliferation, cytoskeletal organization, de-differentiation, invasion, migration and metastasis. Hyperactivation of these GTPases are frequently found in tumors. However, there is no evidence for activating mutations of Rho GTPases in human tumors and the increased GTP loading (activation) is mainly mediated by alterations in their regulatory proteins. As an example, many Rho-GEFs have been identified as oncogenes such as Vav and Tiam1. As proteins with opposite function to GEFs, GAPs might be expected to function as tumor suppressors. This function has been demonstrated for DLC, GRAF, P190 and IQGAP, and emerging evidences suggest also this role for ß2-chimaerin, GAP protein object of our study. ß2-chimaerin is one of the members of the chimaerin family of GAP proteins. Structurally, ß2-chimaerin is composed of an N-terminal SH2 domain involved in heteromolecular interactions, a C1 domain that binds diacylglycerol (DAG) and a catalytic GAP domain with specificity for the Rac GTPase. Several studies show the downregulation of ß2-chimaerin in malignant gliomas, in breast cancer cells and duodenal adenocarcinomas, suggesting a role for ß2-chimaerin as a tumour suppressor in these types of cancer. Inactivation of ß2-chimaerin would results in increased Rac activity and supporting this hypothesis, hyperactive Rac1 has been associated with increased, proliferation, and invasion of breast cancer. Rac1 regulates the rearrangement of the actin cytoskeleton required for the formation of lamellipodia that are specific for forward migration during invasion. The control of cytoskeleton reorganization via Rac involves at least two different mechanisms. One involves the activation of Arp2/3, which has a prominent role in actin polymerization through WAVE/Scar indirect activation. A second mechanism is mediated by Pak (p21-activated kinase). PAK activated by Rac/Cdc42 induces formation of lamellipodia, filopodia, the membrane ruffles, stress fibers and remodeling of focal adhesion complexes. Proteins involved in the cytoskeletal reorganization include the main components of the actomyosin cytoskeleton, intermediate filaments, microtubules, integrins, and a variety of proteins associated with the above. Rac1 also has essential roles in the regulation of cytoskeletal dynamics controlling cell-cell and cell-matrix adhesion. The deregulation of these processes favors migration and invasiveness and is a hallmark of tumor progression. All the data summarized here suggest that ß2-chimaerin may play an important role in the control of actin cytoskeleton through the modulation of Rac activity and that inactivation of ß2-chimaerin in tumors may contribute to the invasive properties of cancer cells due to Rac hyperactivation. In the present study we analyze the role of ß2-chimaerin as a tumor suppressor in breast cancer with two different experimental approaches. One is the analysis of breast tumor development in ß2-chimaerin knock-out mice. A second approach is the overexpression of ß2-chimaerin in human breast cancer cell lines. The mouse model was generated by crossing the ß2-chimaerin knock-out mice (KO) with the MMTV-Neu mice (chn2-/-/Neu), a well known genetic model for breast carcinogenesis. The MMTV-Neu mice have an amplification of the gene encoding ErbB2, whose hyperactivation is associated with a high number of breast tumors. To study the effect of ß2-chimaerin downregulation in the development of breast tumors several experiments were performed including monitoring breast tumor (incidence, latency and tumor multiplicity) and mammary gland (preneoplastic lesions). Our results from the analysis of the mouse model showed a similar incidence for breast tumor development independent on genotypes but a strong reduction in breast tumor latency in absence of ß2-chimaerin. This result suggests that KO mice are more susceptible to tumor development. Despite the fact that there were no differences in the breast tumor multiplicity, we observed an increased presence of preneoplastic lesions in the mammary glands. These findings suggest that ß2-chimaerin is playing an important role in the initial stage of breast cancer development. Moreover, the metastatic phenotype of chn2-/-/Neu mice showed reduced incidence in the development of lung metastasis than control mice, however the multiplicity of metastatic foci was higher in absence of ß2-chimaerin. Mice breast tumors were subjected to histological evaluation and immunohistological analysis with common markers for proliferation (Ki67, and cyclin D1), apoptosis (cleaved caspasa-3) and cell-cell junctions (E-cadherin). This examination was completed with biochemical studies to assess Rac activity as AKT and ERK activation. The overall results showed a tumor phenotype with minor differences between both genotypes in processes such as angiogenesis or dedifferentiation. However, ß2-chimaerin deficient mice had a markedly reduced tumor growth due to a decreased proliferation and increased cell death by apoptosis. Although tumors arising from ß2-chimaerin KO mice had higher levels of active Rac, they showed no differences in cyclin D1 expression, or phosphorylation of either AKT or ERK. In order to further study the role of ß2-chimaerin in metastasis, we analyzed the effect of the expression of this protein in human breast cancer cell lines. With that aim, we generated cells stably expressing ß2-chimaerin in MCF7 (MCF7-ß2) and LM2 (LM2-ß2), two cells lines with ß2-chimaerin downregulation, different cellular phenotype and metastatic potential. We first confirmed that re-expression of ß2-chimaerin in these cells resulted in a decrease in the levels of active Rac. Next, we studied the impact of ß2-chimaerin expression in the control of actin cytoskeleton and the dynamic processes that are involved in the epithelial-mesenchymal transition (EMT). Loss of cell-cell adherence is one of the most important changes that must take place during EMT, and allow tumor cells to migrate into blood vessels or lymphatic tissue, and to invade distant organs. We performed multi-parameter time-lapse imaging of the cells by analyzing wound healing, adhesion dynamics in an extracellular matrix (collagen IV) and invasion assays with Matrigel and Boyden chambers. Data obtained from these experiments revealed that cell migration and invasion are impaired in presence of ß2-chimaerin, paralleled with altered actin cytoskeleton structures (lamelipodia, stress fibres and protrusion formations). In cells of mesenchymal phenotype (LM2), expression of ß2-chimaerin increased either the area or the velocity of cell adherence to substratum, especially in presence of collagen IV, which suggests a role for ß2-chimaerin on cell spreading. On the other hand, focal adhesions showed equal size and were distributed homogeneously at peripheral attachment points. These adhesions appeared equidistant from the nucleus, which indicates lack of polarisation independently of ß2-chimaerin expression. Finally, LM2-ß2 cells had reduced capacity to migrate in a wound healing assay or thought matrigel compared with control cells. In epithelial cells (MCF7), expression of ß2-chimaerin resulted in a marked reduction of E-cadherin staining at the cell contacts, as analyzed by confocal microscopy, which paralleled with reduced E-cadherin protein levels observed by western blot analysis. In addition, we demonstrated that ß2-chimaerin reduce the strength of intercellular contacts with a hanging drop assay. Finally, we carried out a calcium switch assay to investigate whether ß2-chimaerin was involved in the initial formation, or in the stabilization of cell junctions. We observed that control cells restored the junctions almost completely after 15 minutes following the calcium switch, as revealed by E-cadherin and actin staining. Nevertheless, only a small fraction of the junctions between ß2-chimaerin expressing cells demonstrated proper E-cadherin localization 45 minutes after the calcium switch. Taken together, these results suggest a dual role of ß2-chimaerin in breast cancer. While downregulation of this protein in animal models accelerates breast tumor onset, it reduces tumor malignancy and susceptibility to develop metastasis. We also describe a different role for ß2-chimaerin depending on the cell type. In transformed breast mesenchymal cells, ß2-chimaerin may have a protective role in metastasis by decreasing cell migration and invasion, whereas expression of ß2-chimaerin in epithelial breast cancer cells results in reduced cell-cell adhesion.