K-Ras Mutation Induced Feedback ERK Activation Contributes To The Everolimus Resistance In Pancreatic Cancer

Mammalian target of rapamycin (mTOR) of the phosphatidylinositol3-kinase(PI3K)-related protein kinase family has recently emerged as a cancer therapeutictarget.mTORC1controls protein translation through phosphorylation and activation of itssubstrates, p70S6ribosomal kinase (p70S6K) and eukaryotic translational initiation factor4E (eIF4E) binding protein (4E-BP1). This mTORC1pathway is activated in many cancersand drives cancer progression. Rapamycin is a macrolide antibiotic that can inhibitsmTORC1phosphorylation of its substrates. The rapamycin analogs everolimus (RAD001),temsirolimus (CCI-779) and ridaforolimus (AP23573) have therefore been developed ascancer therapeutic agents. In phase III trials, single-agent everolimus and temsirolimusprolonged overall survival of patients with advanced renal cell carcinoma, mantle celllymphoma and pancreatic neuroendocrine tumor. In phase II trials, however, the single-agenttherapies have had limited clinical activity against glioblastomas, advanced melanomas,breast cancers and pancreatic cancer. The molecular basis of everolimus resistance in thesecancers is currently under intensive investigation.mTORC1acts as a central integrator for upstream inputs from growth factors, nutrientsand stress. Growth factors such as insulin and insulin-like growth factor-1(IGF-1) canactivate mTORC1through their cognate receptor tyrosine kinase (RTK)-mediatedphosphorylation and activation of PI3K and Akt. The growth factors can also signalmTORC1through Ras-extracellular signal-regulated kinase (ERK) signaling pathway. TheRas-ERK pathway consists of Ras GTPase and the protein kinases Raf, mitogen-activatedprotein kinase (MAPK)/ERK kinase (MEK) and ERK. RTK-mediated activation of the RasGTPase leads to the activation of these kinases through phosphorylation loops: Raf activatesMEK, MEK activates ERK and ERK activates p90ribosomal S6kinase (RSK).Both ERKand RSK promote the activation of mTORC1. This growth factor-mTORC1pathway isregulated through two negative feedback loops:mTORC1-S6K-mediated phosphorylationand degradation of insulin receptor substrate (IRS) and mTORC1-mediated phosphorylationof growth factor receptor-bound protein10(Grb10). Through these negative feedback loops,rapalogs inhibit mTORC1and activate PI3K and ERK in cancers, resulting in cancerresistance to the treatment. Ras GTPases are coded by the human H-Ras, K-Ras and N-Ras genes. K-Ras, but notH-Ras or N-Ras, is frequently mutated in nearly95%human pancreatic cancer. Mutant (mt)K-Ras proteins exhibit constitutive GTPase activity and thus contribute to the developmentand progression of pancreatic cancer. Here, we show that K-Ras mutations play a criticalrole in the everolimus resistance in pancreatic cancer. K-Ras mutations contribute to theeverolimus-induced feedback activation of IGF-1-Ras-Raf-ERK pathway and inhibition ofthe mt K-Ras abolishes the feedback ERK signal, reduces the everolimus resistance and thusenhance inhibitory effect of everolimus on the growth of K-Ras mt pancreatic cancercells-derived mouse xenografts. The results indicate that targeting of K-Ras mutation maylead to the development of therapies that overcome everolimus resistance in pancreaticcancer.The main results are as follows:①Western-blot and immunohistochemistry revealed an increase of p-S6protein inpancreatic cancer tissues surgically removed from patients as compared to normal pancreatictissues. we also confirmed the most common K-Ras mutation occurs at codon12inpancreatic cancer tissues and this mutation in four of five pancreatic cancer tissues byPCR-RFLP analysis. The expression of p-S6protein was detected in both K-Ras wild type(wt) and mt pancreatic cancer tissues. These results demonstrate mTORC1activation inpancreatic cancer tissues without correlation with K-Ras mutation in the tissues.②We selected pancreatic cancer cell lines that carry wt.(BxPC-3, Hs776T) or MTK-Raps (Capan-2, PANC-1) by PCR-RFLP analysis, then cell viability and colony formationassay and flow cytometer showed that the K-Ras MT cell lines, Capan-2and PANC-1, wereresistant to the everolimus treatment as compared to the K-Ras wt BxPC-3and Hs776T celllines, and the everolimus treatment led to a significant increase in G1phase in the K-Ras wtbut not the K-Ras mt cell lines. Western-Blot and Co-IP assay confirmed that everolimustreatment inhibits mTORC1but activates Ras-Raf-ERK pathway in K-Ras mt pancreaticcancer cells.③Western-blot showed that in the combination with everolimus, sorafenib eliminatedeverolimus-induced ERK pathways in the K-Ras mt PANC-1cells. cell viability and colonyformation assay showed that combination of sorafenib and everolimus synergisticallyinhibits the growth of K-Ras mt pancreatic cancer cells through the simultaneous inhibitionof mTORC1pathway and everolimus-induced ERK pathway. ④We took a knockdown approach to construct K-Ras shRNA expressing wt BxPC-3and K-Ras mt PANC-1cell lines, by cell viability and Flow cytometry assay showed that theknockdown of wt K-Ras did not affect the response of the K-Ras wt BxPC-3cells toeverolimus treatment but significantly reduced the resistance of the K-Ras mt PANC-1cellsto everolimus. Re-expression of K-RasG12Din K-Ras shRNA expressing PANC-1cell linesindicated that K-Ras mutation contributes to the everolimus resistance is through itsactivation of ERK pathway in pancreatic cancer cells.⑤We grew K-Ras mt PANC-1cell line in serum-free culture conditions supplementedwith growth factors and found that everolimus-induced ERK feedback loop occurs throughIGF-1signaling pathway. Then Western-Blot and cell cycle were employed to confirm thattreatment of NVP-AEW541significantly reduced the IGF-1-mediated feedback activation ofERK and Akt. These results suggest that mt K-Ras is required for the everolimus-inducedfeedback loop of IGF-1-induced ERK pathway. Targeting of mt K-Ras eliminates thefeedback loop and thus enhances the inhibitory effect of everolimus on the growth of K-Rasmutant pancreatic cancer cells.⑥We generated mouse subcutaneous xenografts from the K-Ras shRNA and emptyvector expressing K-Ras mt PANC-1cells, the results showed that everolimus treatmentsignificantly reduced the volumes of the xenografts derived from the K-Ras shRNAexpressing PANC-1as compared with the empty vector-expressing PANC-1cells.Western-blot confirmed the K-Ras knockdown and the inhibition of the mTORC1pathwayin the everolimus treated xenografts of the K-Ras shRNA expressing PANC-1cells. ThenImmunohistochemistry confirmed the presence of p-ERK protein in the xenografts derivedfrom the empty vector but not shRNA expressing PANC-1cells, above results indicated thattargeting of mt K-Ras can eliminate the everolimus-induced ERK feedback loop and thusenhances the therapeutic efficacy of everolimus in treating K-Ras mt pancreatic cancerthrough its inhibition of mTORC1pathway in vivo.Conclusion:In conclusion, the findings presented here provide the rationale for a combination ofK-Ras and mTORC1inhibitors in the treatment of pancreatic cancer where K-Ras mutationsare nearly universal. K-Ras mutations contribute to everolimus-induced feedback activationof IGF-1-ERK pathway and thus targeting of mt K-Ras blocks the feedback ERK signal,reduced everolimus resistance and thus enhances therapeutic efficacy of rapalogs in treatinghuman pancreatic cancer. New cancer therapeutic agents are indeed currently under development for the combination therapies that simultaneously target the multiple corepathways in cancers.