Downregulation of Rap1GAP in Human Tumor Cells Alters Cell/Matrix and Cell/Cell Adhesion

Abstract

Rap1GAP expression is decreased in human tumors. The significance of its downregulation is unknown. We show that Rap1GAP expression is decreased in primary colorectal carcinomas. To elucidate the advantages conferred on tumor cells by loss of Rap1GAP, Rap1GAP expression was silenced in human colon carcinoma cells. Suppressing Rap1GAP induced profound alterations in cell adhesion. Rap1GAP-depleted cells exhibited defects in cell/cell adhesion that included an aberrant distribution of adherens junction proteins. Depletion of Rap1GAP enhanced adhesion and spreading on collagen. Silencing of Rap expression normalized spreading and restored E-cadherin, ␤-catenin, and p120-catenin to cell/cell contacts, indicating that unrestrained Rap activity underlies the alterations in cell adhesion. The defects in adherens junction protein distribution required integrin signaling as E-cadherin and p120-catenin were restored at cell/cell contacts when cells were plated on poly-L-lysine. Unexpectedly, Src activity was increased in Rap1GAP-depleted cells. Inhibition of Src impaired spreading and restored E-cadherin at cell/cell contacts. These findings provide the first evidence that Rap1GAP contributes to cell/cell adhesion and highlight a role for Rap1GAP in regulating cell/matrix and cell/cell adhesion. The frequent downregulation of Rap1GAP in epithelial tumors where alterations in cell/cell and cell/matrix adhesion are early steps in tumor dissemination supports a role for Rap1GAP depletion in tumor progression.Mammalian Rap proteins Rap1a/b and Rap2a/b/c are members of the Ras superfamily of small GTPases. Rap proteins are active when bound to GTP and inactive when bound to GDP. Cellular Rap activity is regulated by the concerted action of guanine nucleotide exchange factors that activate Rap (RapGEFs) and Rap-specific GTPaseactivating proteins (RapGAPs) that inactivate Rap (reviewed in reference 10). The Rap1GAP family is composed of several members, including Rap1GAP, Rap1GAPII, Spa-1/SIPA1, and E6TP1/SIPA1L1. Several lines of evidence suggest that RapGAPs function as tumor and/or invasion suppressors. Downregulation of E6TP1 by human papillomavirus protein E6 contributes to cervical cancer (20, 21), and Spa-1 deficiency in mice induces a spectrum of myelodysplastic disorders similar to chronic myelogenous leukemia (26). The SPA1 gene was identified as a candidate for the metastasis efficiency modifier locus in mice (38). Although the relevance of this observation to humans is not yet clear, single-nucleotide polymorphisms in the SPA1 gene in human breast tumors have been associated with lymph node involvement and poor survival (15). Intriguingly, Spa-1 interacts with Brd4 (18) and Rrp-1b (13), the protein products of genes associated with patterns of extracellular matrix protein gene expression characteristic of metastatic tumors (14).The RAP1GAP gene maps to 1p35-36, a chromosomal region subject to copy number alterations in human tumors (36, 49). Rap1GAP protein levels are decreased in pancreatic adenocarcinomas (53), pap...

Prostaglandin E2 Regulates Renal Cell Carcinoma Invasion through the EP4 Receptor-Rap GTPase Signal Transduction Pathway

“…7A) and previous studies suggest Rap1GAP and its family members Rap1GAPII, Spa1/SIPA1, and E6TP1 possess tumor metastasis suppressor activities. Rap1GAP protein levels are decreased in several types of cancer, including pancreatic adenocarcinoma (55), papillary thyroid carcinoma (56,57), colorectal carcinoma (58), and melanoma (59). The implication of Rap1GAP as a tumor suppressor was largely deduced based upon experimental results using forced overexpression of full-length Rap1GAP (60,61).…”

Slp2-a controls renal epithelial cell size through regulation of Rap–ezrin signaling independently of Rab27

“…S1B), indicating that the activity of Rap1 and Rap2 is regulated in close proximity to the plasma membrane by Rap1 GAPs. Because activation of Rap signaling, Rac signaling and/or ezrin signaling, has been shown to impair cell-cell adhesion by destabilizing E-cadherin at the cell-cell junctions (Lozano et al, 2008;Pujuguet et al, 2003;Tsygankova et al, 2010) and because reduced accumulation of E-cadherin at the cell-cell junctions was observed in the Slp2-a-KD cells (supplementary material Fig. S4A), it is highly possible that activation of these signals facilitates cell spreading by disrupting cell-cell adhesion.…”

Rap1GAP impairs cell-matrix adhesion in the absence of effects on cell-cell adhesion

“…The expression of Rap1GAP is decreased in tumors of the pancreas, skin, thyroid and colon. [1][2][3][4][5][6] Rap1GAP expression is further decreased in invasive compared to benign lesions, suggesting that depletion of Rap1GAP enhances tumor progression. 1,2,4,5 The advantages conferred to tumor cells by the downregulation of Rap1GAP are unknown.…”

“…5,9,10 We previously reported that silencing Rap1GAP expression in human colon cancer cells impaired cell-cell adhesion and enhanced spreading on collagen. 6 The weakening of cell-cell contacts together with alterations in matrix adhesion are hallmarks of tumor progression. These data suggest that loss of Rap1GAP endows cells with the ability to disseminate and provide a potential rationale for the progressive downregulation of Rap1GAP in human tumors.…”

“…Silencing Rap1GAP induced sustained increases in Rap activity. 6 Activated Rap enhances cell-matrix adhesion by regulating integrin affinity and avidity. [11][12][13] However, Rap activity promotes cell-cell adhesion (reviewed in refs.…”