Cellular Effect And Mechanism Of Action Of A Novel-type PI3K/mTOR Dual Inhibitor,GDC-0084,on Cutaneous Squamous Cell Carcinoma(cSCC)

Title:GDC-0084,a novel PI3K/m TOR dual inhibitor,inhibits cutaneous squamous cell carcinoma cell growth in vitro and in vivo.Aims: Cutaneous squamous cell carcinoma(c SCC)is the malignant tumor derived from epidermal keratinocytes.It is an important contributor of cancer-associated human mortalities.With its incidence rate rising recently,c SCC accounts for 15-20% of all cutaneous malignancies.Clinical treatments with the combination of surgery,radiotherapy,and/or chemotherapy show efficiency only in early-stage and well-defined c SCC.The prognosis of high-grade and/or metastatic c SCC is still poor.Molecularly-targeted therapy has shown great potential for c SCC treatment.Overactivation of PI3K(phosphatidylinositol 3-kinase)-Akt-m TOR(mammalian target of rapamycin)signaling cascade plays a pivotal role in tumorigenesis and progression of c SCC.GDC-0084 is a novel and potent small-molecule PI3K-m TOR dual inhibitor.The present study examined its potential activity and underlying signaling mechanisms in human c SCC cells.Methods:By CCK-8 assay,cell colony formation assay was carried out to test the potential effect of GDC-0084 on c SCC cell survival.Brd U ELISA and Ed U staining assays were utilized to detect the effects of GDC-0084 on the proliferation of c SCC cells.PI-FACS method was employed to detect the effect of GDC-0084 on c SCC cell cycle progressionCaspase-3/9 kit,Annexin V-PI FACS assay and histone DNA ELISA kit were utilized to detect the effect of GDC-0084 on the apoptosis of c SCC cells.Western blot assay was employed to detect the expression of apoptotic proteins(cleaved-Caspase-3/9)after GDC-0084 treatment.Western blot assay was employed to test PI3K-Akt-m TOR,MEK-ERK and DNA-dependent protein kinase(DNA-PKcs)catalytic subunit(DNA-PKcs)signalings after GDC-0084 treatment in c SCC cells.SCID mouse bearing A431 xenograft model was established to observe the anti-c SCC activity by GDC-0084 administration.A431 xenograft tumor tissues were isolated,and the changes of PI3K-Akt-m TOR and DNA-PKcs signalings after GDC-0084 administration were detected.Results:GDC-0084 at nanomole concentrations potently inhibited survival and proliferation of established(A431,SCC-13 and SCL-1 lines)and primary human c SCC cells.GDC-0084 induced apoptosis activation in human c SCC cells.GDC-0084 induced G1-S cell cycle arrest in human c SCC cells.GDC-0084 was non-cytotoxic to the normal human skin fibroblasts/keratinocytes.GDC-0084 blocked PI3K-Akt-m TOR cascade in human c SCC cells.GDC-0084 had no significant effect on MEK-ERK signaling in c SCC cells.GDC-0084 blocked DNA-PKcs activation in human c SCC cells.Restoring DNA-PKcs activation by a constitutively active-DNA-PKcs(S2056D)partially inhibited GDC-0084-induced cell death and apoptosis in A431 cells.In vivo,GDC-0084 daily gavage potently inhibited A431 xenograft tumor growth in SCID mice.In GDC-0084-treated tumor tissues,PI3K-Akt-m TOR and DNA-PKcs activation was significantly inhibited.Conclusion:GDC-0084 inhibits human c SCC cell growth in vitro and in vivo through blocking PI3K-Akt-m TOR and DNA-PKcs signalings.