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Mutant K-RAS Promotes Invasion and Metastasis in Pancreatic Cancer Through GTPase Signaling Pathways.

ABSTRACT: Pancreatic ductal adenocarcinoma is one of the most aggressive malignancies, characterized by the local invasion into surrounding tissues and early metastasis to distant organs. Oncogenic mutations of the K-RAS gene occur in more than 90% of human pancreatic cancers. The goal of this study was to investigate the functional significance and downstream effectors of mutant K-RAS oncogene in the pancreatic cancer invasion and metastasis. We applied the homologous recombination technique to stably disrupt K-RAS oncogene in the human pancreatic cell line MiaPaCa-2, which carries the mutant K-RAS (G12C) oncogene in both alleles. Using in vitro assays, we found that clones with disrupted mutant K-RAS gene exhibited low RAS activity, reduced growth rates, increased sensitivity to the apoptosis inducing agents, and suppressed motility and invasiveness. In vivo assays showed that clones with decreased RAS activity had reduced tumor formation ability in mouse xenograft model and increased survival rates in the mouse orthotopic pancreatic cancer model. We further examined molecular pathways downstream of mutant K-RAS and identified RhoA GTP activating protein 5, caveolin-1, and RAS-like small GTPase A (RalA) as key effector molecules, which control mutant K-RAS-dependent migration and invasion in MiaPaCa-2 cells. Our study provides rational for targeting RhoA and RalA GTPase signaling pathways for inhibition of pancreatic cancer metastasis.

SUBMITTER: Padavano J

PROVIDER: S-EPMC4612127 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Mutant K-RAS Promotes Invasion and Metastasis in Pancreatic Cancer Through GTPase Signaling Pathways.

Padavano Julianna J Henkhaus Rebecca S RS Chen Hwudaurw H Skovan Bethany A BA Cui Haiyan H Ignatenko Natalia A NA

Cancer growth and metastasis 20151019 Suppl 1

Pancreatic ductal adenocarcinoma is one of the most aggressive malignancies, characterized by the local invasion into surrounding tissues and early metastasis to distant organs. Oncogenic mutations of the K-RAS gene occur in more than 90% of human pancreatic cancers. The goal of this study was to investigate the functional significance and downstream effectors of mutant K-RAS oncogene in the pancreatic cancer invasion and metastasis. We applied the homologous recombination technique to stably di ...[more]

PMID: 26512205

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Project description:BACKGROUND:Signaling pathways critical for embryonic development re-emerge in adult pancreas during tumorigenesis. Aspartate ?-hydroxylase (ASPH) drives embryonic cell motility/invasion in pancreatic development/differentiation. We explored if dysregulated ASPH is critically involved in pancreatic cancer pathogenesis. METHODS:To demonstrate if/how ASPH mediates malignant phenotypes, proliferation, migration, 2-D/3-D invasion, pancreatosphere formation, immunofluorescence, Western blot, co-immunoprecipitation, invadopodia formation/maturation/function, qRT-PCR, immunohistochemistry (IHC), and self-developed in vitro metastasis assays were performed. Patient-derived xenograft (PDX) models of human pancreatic ductal adenocarcinoma (PDAC) were established to illustrate in vivo antitumor effects of the third-generation small molecule inhibitor specifically against ASPH's ?-hydroxylase activity. Prognostic values of ASPH network components were evaluated with Kaplan-Meier plots, log-rank tests, and Cox proportional hazards regression models. RESULTS:ASPH renders pancreatic cancer cells more aggressive phenotypes characterized by epithelial-mesenchymal transition (EMT), 2-D/3-D invasion, invadopodia formation/function as demonstrated by extracellular matrix (ECM) degradation, stemness (cancer stem cell marker upregulation and pancreatosphere formation), transendothelial migration (mimicking intravasation/extravasation), and sphere formation (mimicking metastatic colonization/outgrowth at distant sites). Mechanistically, ASPH activates SRC cascade through direct physical interaction with ADAM12/ADAM15 independent of FAK. The ASPH-SRC axis enables invadopodia construction and initiates MMP-mediated ECM degradation/remodeling as executors for invasiveness. Pharmacologic inhibition of invadopodia attenuates in vitro metastasis. ASPH fosters primary tumor development and pulmonary metastasis in PDX models of PDAC, which is blocked by a leading compound specifically against ASPH enzymatic activity. ASPH is silenced in normal pancreas, progressively upregulated from pre-malignant lesions to invasive/advanced stages of PDAC. Expression profiling of ASPH-SRC network components independently/jointly predicts clinical outcome of PDAC patients. Compared to a negative-low level, a moderate-very high level of ASPH, ADAM12, activated SRC, and MMPs correlated with curtailed overall survival (OS) of pancreatic cancer patients (log-rank test, ps?<?0.001). The more unfavorable molecules patients carry, the more deleterious prognosis is destinated. Patients with 0-2 (n?=?4), 3-5 (n?=?8), 6-8 (n?=?24), and 9-12 (n?=?73) unfavorable expression scores of the 5 molecules had median survival time of 55.4, 15.9, 9.7, and 5.0?months, respectively (p?<?0.001). CONCLUSION:Targeting the ASPH-SRC axis, which is essential for propagating multi-step PDAC metastasis, may specifically/substantially retard development/progression and thus improve prognosis of PDAC.

Dataset Information

Mutant K-RAS Promotes Invasion and Metastasis in Pancreatic Cancer Through GTPase Signaling Pathways.

Publications

Mutant K-RAS Promotes Invasion and Metastasis in Pancreatic Cancer Through GTPase Signaling Pathways.

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