Nellore A, etal., J Clin Endocrinol Metab. 2009 Mar;94(3):1026-32. doi: 10.1210/jc.2008-1042. Epub 2008 Dec 9.
CONTEXT: Rap1 GTPase-activating protein (GAP) regulates the activity of Rap1, a putative oncogene. We previously reported Rap1GAP was highly expressed in normal human thyroid cells and decreased in five papillary thyroid carcinomas (PTCs). OBJECTIVES: To confirm the significance of these findings,
we analyzed Rap1GAP expression in a larger set of benign tumors (adenomas and hyperplastic nodules) and PTCs. We determined whether the presence of the BRAF(V600E) mutation or allelic loss of Rap1GAP related to changes in Rap1GAP protein expression. To determine the consequences of Rap1GAP loss, we targeted Rap1GAP in culture using small interfering RNA. DESIGN, PATIENTS, AND METHODS: A highly specific Rap1GAP antibody was applied to sections of 55 human thyroid tissues. Genomic DNA was analyzed for the presence of the BRAF(V600E) mutation, and loss of Rap1GAP. Rap1GAP expression in rat thyroid cells was abolished using small interfering RNA. RESULTS: We observed that down-regulation of Rap1GAP in benign lesions and PTCs was common. Rap1GAP expression was more severely decreased in PTCs. Loss of Rap1GAP expression was observed in multiple histological variants of PTCs. Approximately 20% of PTCs and adenomas exhibited allelic loss of Rap1GAP. Loss of Rap1GAP was not associated with the presence of the BRAF(V600E) mutation. In vitro, loss of Rap1GAP was sufficient to increase Rap1 activity in thyroid cells. CONCLUSIONS: These data indicate that loss of Rap1GAP is a frequent event in PTC. The more frequent and greater down-regulation of Rap1GAP in PTCs compared with adenomas suggests a role for Rap1GAP depletion in the progression of human thyroid tumors, possibly through unrestrained Rap activity.
Although abundant in well-differentiated rat thyroid cells, Rap1GAP expression was extinguished in a subset of human thyroid tumor-derived cell lines. Intriguingly, Rap1GAP was downregulated selectively in tumor cell lines that had acquired a mesenchymal morphology. Restoring Rap1GAP expression to
these cells inhibited cell migration and invasion, effects that were correlated with the inhibition of Rap1 and Rac1 activity. The reexpression of Rap1GAP also inhibited DNA synthesis and anchorage-independent proliferation. Conversely, eliminating Rap1GAP expression in rat thyroid cells induced a transient increase in cell number. Strikingly, Rap1GAP expression was abolished by Ras transformation. The downregulation of Rap1GAP by Ras required the activation of the Raf/MEK/extracellular signal-regulated kinase cascade and was correlated with the induction of mesenchymal morphology and migratory behavior. Remarkably, the acute expression of oncogenic Ras was sufficient to downregulate Rap1GAP expression in rat thyroid cells, identifying Rap1GAP as a novel target of oncogenic Ras. Collectively, these data implicate Rap1GAP as a putative tumor/invasion suppressor in the thyroid. In support of that notion, Rap1GAP was highly expressed in normal human thyroid cells and downregulated in primary thyroid tumors.
Zhang Z, etal., Am J Pathol. 2006 Feb;168(2):585-96.
Rap1, a growth regulatory protein that is strongly expressed in human squamous cell carcinoma (SCC), is inactivated by rap1GAP. Recent evidence in normal rat cells suggests that rap1GAP regulates proliferation. The objective of the current study was to investigate whether rap1GAP functions as a tumo
r suppressor in SCC. Using a pull-down assay, active GTP-bound rap1 was up-regulated in SCC compared to normal or immortalized keratinocytes. Because both rap1A and rap1B isoforms of rap1 are expressed in SCC, the rap1GAP inactivation of both rap1 isoforms was verified using cells transfected with EGFP-rap1A or EGFP-rap1B or co-transfected with FLAG-tagged rap1GAP. The results demonstrate that expression of rap1GAP in oropharyngeal SCC down-regulated active rap1, ERK activation, and proliferation. Incubation of stably transfected SCC cells with nocodazole, an inhibitor of mitosis, caused a slower accumulation of rap1GAP-transfected cells in the G2 phase, in comparison to the vector control, indicating that rap1GAP-transfected cells have slower progression through the cell cycle. This was supported by down-regulation of cyclin D1, cdk4, and cdk6 in rap1GAP-transfected SCC cells. Furthermore, SCC cells transfected with rap1GAP produced significantly smaller tumors in nude mice as compared to controls (P < 0.01). These novel findings suggest that rap1GAP acts as a tumor suppressor protein in SCC.
Glial cell line-derived neurotrophic factor (GDNF) was originally recognized for its ability to promote survival of midbrain dopaminergic neurons, but it has since been demonstrated to be crucial for the survival and differentiation of many neuronal subpopulations, including motor neurons, sympathe
tic neurons, sensory neurons and enteric neurons. To identify possible effectors or regulators of GDNF signaling, we performed a yeast two-hybrid screen using the intracellular domain of RET, the common signaling receptor of the GDNF family, as bait. Using this approach, we identified Rap1GAP, a GTPase-activating protein (GAP) for Rap1, as a novel RET-binding protein. Endogenous Rap1GAP co-immunoprecipitated with RET in neural tissues, and RET and Rap1GAP were co-expressed in dopaminergic neurons of the mesencephalon. In addition, overexpression of Rap1GAP attenuated GDNF-induced neurite outgrowth, whereas suppressing the expression of endogenous Rap1GAP by RNAi enhanced neurite outgrowth. Furthermore, using co-immunoprecipitation analyses, we found that the interaction between RET and Rap1GAP was enhanced following GDNF treatment. Mutagenesis analysis revealed that Tyr981 in the intracellular domain of RET was crucial for the interaction with Rap1GAP. Moreover, we found that Rap1GAP negatively regulated GNDF-induced ERK activation and neurite outgrowth. Taken together, our results suggest the involvement of a novel interaction of RET with Rap1GAP in the regulation of GDNF-mediated neurite outgrowth.
To find the underlying causes of primary myelodysplastic syndrome (MDS), the gene expression profiling of both CD34+ cells and bone marrow mononuclear cells from MDS patients was performed using oligonucleotide microarray and cDNA microarrays, respectively. Several candidate genes which were differe
ntially expressed in MDS patients versus normal controls were selected and confirmed in expanding samples by quantitative real-time reverse transcription-polymerase chain reaction after clustering and bioinformatics analysis. one of these genes, RAP1GAP, was found to be expressed at a significantly higher level in patients with MDS in comparison with those suffering from other hematopoietic diseases including leukemia (P < 0.01). We propose that over-expression of RAP1GAP gene may play a role in the pathogenesis of MDS.
Zhang L, etal., Cancer Res. 2006 Jan 15;66(2):898-906.
Human chromosome 1p35-p36 has long been suspected to harbor a tumor suppressor gene in pancreatic cancer and other tumors. We found that expression of rap1GAP, a gene located in this chromosomal region, is significantly down-regulated in pancreatic cancer. Only a small percentage of preneoplastic p
ancreatic intraductal neoplasia lesions lost rap1GAP expression, whereas loss of rap1GAP expression occurred in 60% of invasive pancreatic cancers, suggesting that rap1GAP contributes to pancreatic cancer progression. In vitro and in vivo studies showed that loss of rap1GAP promotes pancreatic cancer growth, survival, and invasion, and may function through modulation of integrin activity. Furthermore, we showed a high frequency of loss of heterozygosity of rap1GAP in pancreatic cancer. Collectively, our data identify rap1GAP as a putative tumor suppressor gene in pancreatic cancer.
The significance of the widespread downregulation of Rap1GAP in human tumors is unknown. In previous studies we demonstrated that silencing Rap1GAP expression in human colon cancer cells resulted in sustained increases in Rap activity, enhanced spreading on collagen and the weakening of cell-cell co
ntacts. The latter finding was unexpected based on the role of Rap1 in strengthening cell-cell adhesion and reports that Rap1GAP impairs cell-cell adhesion. We now show that Rap1GAP is a more effective inhibitor of cell-matrix compared to cell-cell adhesion. Overexpression of Rap1GAP in human colon cancer cells impaired Rap2 activity and the ability of cells to spread and migrate on collagen IV. Under the same conditions, Rap1GAP had no effect on cell-cell adhesion. Overexpression of Rap1GAP did not enhance the dissociation of cell aggregates nor did it impair the accumulation of beta-catenin and E-cadherin at cell-cell contacts. To further explore the role of Rap1GAP in the regulation of cell-cell adhesion, Rap1GAP was overexpressed in non-transformed thyroid epithelial cells. Although the formation of cell-cell contacts required Rap1, overexpression of Rap1GAP did not impair cell-cell adhesion. These data indicate that transient, modest expression of Rap1GAP is compatible with cell-cell adhesion and that the role of Rap1GAP in the regulation of cell-cell adhesion may be more complex than is currently appreciated.
Zheng H, etal., Cancer Res. 2009 Jan 15;69(2):449-57. doi: 10.1158/0008-5472.CAN-08-2399.
Melanoma is the most serious, highly aggressive form of skin cancer with recent dramatic increases in incidence. Current therapies are relatively ineffective, highlighting the need for a better understanding of the molecular mechanisms contributing to the disease. We have previously shown that activ
ation of Rap1 promotes melanoma cell proliferation and migration through the mitogen-activated protein kinase pathway and integrin activation. In the present study, we show that expression of Rap1GAP, a specific negative regulator of Rap1, is decreased in human melanoma tumors and cell lines. Overexpression of Rap1GAP in melanoma cells blocks Rap1 activation and extracellular signal-regulated kinase (ERK) phosphorylation and inhibits melanoma cell proliferation and survival. In addition, overexpression of Rap1GAP also inhibits focal adhesion formation and decreases melanoma cell migration. Rap1GAP down-regulation is due to its promoter methylation, a mechanism of gene silencing in tumors. Furthermore, treatment of melanoma cells with the demethylating agent 5-aza-2'-deoxycytidine reinduces Rap1GAP expression, followed by decreased Rap1 activity, ERK phosphorylation, and cell proliferation and survival-changes that are significantly blunted in cells transfected by small interfering RNA-mediated Rap1GAP knockdown. Taken together, our findings indicate that down-regulation of Rap1GAP via promoter hypermethylation promotes melanoma cell proliferation, survival, and migration.
Potla U, etal., J Clin Invest. 2014 Apr;124(4):1757-69. doi: 10.1172/JCI67846. Epub 2014 Mar 18.
Injury to the specialized epithelial cells of the glomerulus (podocytes) underlies the pathogenesis of all forms of proteinuric kidney disease; however, the specific genetic changes that mediate podocyte dysfunction after injury are not fully understood. Here, we performed a large-scale insertional
mutagenic screen of injury-resistant podocytes isolated from mice and found that increased expression of the gene Rap1gap, encoding a RAP1 activation inhibitor, ameliorated podocyte injury resistance. Furthermore, injured podocytes in murine models of disease and kidney biopsies from glomerulosclerosis patients exhibited increased RAP1GAP, resulting in diminished glomerular RAP1 activation. In mouse models, podocyte-specific inactivation of Rap1a and Rap1b induced massive glomerulosclerosis and premature death. Podocyte-specific Rap1a and Rap1b haploinsufficiency also resulted in severe podocyte damage, including features of podocyte detachment. Over-expression of RAP1GAP in cultured podocytes induced loss of activated beta1 integrin, which was similarly observed in kidney biopsies from patients. Furthermore, preventing elevation of RAP1GAP levels in injured podocytes maintained beta1 integrin-mediated adhesion and prevented cellular detachment. Taken together, our findings suggest that increased podocyte expression of RAP1GAP contributes directly to podocyte dysfunction by a mechanism that involves loss of RAP1-mediated activation of beta1 integrin.
