Rap1GAP inhibits tumor growth in oropharyngeal squamous cell carcinoma.
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 tumor 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.
Zhang
Zhaocheng
Z
Department of Oral Medicine, Pathology, and Oncology, University of Michigan, School of Dentistry, Ann Arbor, MI 48109-1078, USA.
Mitra
Raj S
RS
Henson
Bradley S
BS
Datta
Nabanita S
NS
McCauley
Laurie K
LK
Kumar
Pawan
P
Lee
Julia S-J
JS
Carey
Thomas E
TE
D'Silva
Nisha J
NJ
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D002294:Carcinoma, Squamous Cell / Q000201:enzymology* / Q000517:prevention & control*; D049109:Cell Proliferation; D002478:Cells, Cultured; D019938:Cyclin D1 / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4 / Q000378:metabolism; D051361:Cyclin-Dependent Kinase 6 / Q000378:metabolism; D016195:G2 Phase / Q000187:drug effects; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000201:enzymology; D007668:Kidney / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008938:Mitosis / Q000187:drug effects; D015739:Nocodazole / Q000494:pharmacology; D009959:Oropharyngeal Neoplasms / Q000201:enzymology* / Q000517:prevention & control*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D014162:Transfection; D020668:rap GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D020827:rap1 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
D000970:Antineoplastic Agents; D020690:GTPase-Activating Proteins; C430803:RAP1A protein, human; C484658:RAP1GAP protein, human; C485184:enhanced green fluorescent protein; D019938:Cyclin D1; D049452:Green Fluorescent Proteins; D051057:Proto-Oncogene Proteins c-akt; C495911:CDK6 protein, human; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C116753:RAP1B protein, human; D020668:rap GTP-Binding Proteins; D020827:rap1 GTP-Binding Proteins; D015739:Nocodazole