Purpose PI3K-mTOR pathway activation is often associated with altered expression or mutations of PIK3CA, TP53/p73, PTEN and TGFβR in head and neck squamous cell carcinomas (HNSCC). However, little is known about how these alterations affect response to PI3K-mTOR targeted agents. Experimental Design In this preclinical study, PI3K-Akt-mTOR signaling was characterized in 9 HNSCC (UM-SCC) cell lines and Human Oral Keratinocytes (HOK). We investigated the molecular and anti-cancer effects of dual PI3K/mTOR inhibitor PF-04691502(PF-502) in UM-SCC expressing PIK3CA with decreased wtTP53, mtTP53-/+mtTGFβR2, and in HNSCC of a conditional Pten/Tgfbr1 double knockout (2cKO) mouse model, displaying PI3K-Akt-mTOR activation. Results UM-SCC showed increased PIK3CA expression and Akt/mTOR activation, and PF-502 inhibited PI3K/mTORC1/2 targets. In human HNSCC expressing PIK3CA and decreased wtTP53 and p73, PF-502 reciprocally enhanced TP53/p73 expression and growth inhibition, which was partially reversible by p53 inhibitor pifithrin-α. Most UM-SCC with wtTP53 exhibited a lower IC50 than those with mtTP53 status. PF-502 blocked growth in G0/G1 and increased apoptotic subG0 DNA. PF502 suppressed tumorigenesis and showed combinatorial activity with radiation in a wtTP53 UMSCC xenograft model. PF-502 also significantly delayed HNSCC tumorigenesis and prolonged survival of Pten/Tgfbr1 deficient mice. Significant inhibition of p-Akt, p-4EBP1, p-S6, Ki67, as well as increased p53 and TUNEL were observed in tumor specimens. Conclusions PI3K-mTOR inhibition can enhance TP53/p73 expression and significantly inhibit tumor growth alone or when combined with radiation in HNSCC with wtTP53. PIK3CA, TP53/p73, PTEN and TGFβ alterations are potential modifiers of response and merit investigation in future clinical trials with PI3K-mTOR inhibitors.
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and has a 5-year survival rate of ~50%. Therapy for HNSCC has only improved marginally over the past four decades, due in part to treatment resistance because of tumor heterogeneity. As such, effort is being directed toward personalizing treatment regimens for individual HNSCC patients using tumor genotyping and targeted small molecule inhibitors of the most activated and deleterious tumor pathways. The PI3K-Akt-mTOR axis is one of the most frequently altered signaling cascades in HNSCC, with amplifications or mutations in >90% of HNSCC tumors, and plays an important role in its pathogenesis. Recent evidence also implicates TP53 underexpression or mutation in over 80% of HNSCC tumor samples. As such, PI3K/mTOR and TP53 pathways are important in HNSCC development and could serve as useful therapeutic targets. In this study, we investigate the in vitro and in vivo effects of a novel dual molecular antagonist of PI3K/mTOR, PF-04691502 (PF-502), on PI3K/mTOR targets, TP53 activity, and HNSCC tumorigenesis. A panel of HNSCC (UM-SCC) cell lines was surveyed, the majority of which exhibited elevated expression of PI3K/mTOR pathway proteins when compared to normal human oral keratinocytes. These proteins include PI3K p110α, pAKT(S473), pAKT(T308), pmTOR, p4EBP1(S65, T37/46), and pS6(S240/244). PF-502 inhibits phosphorylation of these PI3K/mTOR targets and induces TP53 and p73 expression in UM-SCC cell lines. PI3K/mTOR target inhibition and TP53/p73 induction is associated with decreased cellular proliferation by MTT assay and increased apoptosis by flow cytometry. TP53 inhibition by pifithrin-α or siRNA knockdown partially attenuates the effects of PF-502, supporting a role of TP53 in growth inhibition. The effects of PF-502 on tumorigenicity were examined in human HNSCC xenografts in SCID mice and in conditional double Pten/Tgfbr1 knockout mice (2cKO), which develop HNSCC spontaneously. PF-502 inhibited or prevented tumor growth and prolonged host survival. PF-502 decreased pAKT(S473), p4EBP1(T37/46), pS6(S240/244) and proliferation marker, Ki67, as well as increased expression of TP53, p73, and TUNEL staining for apoptosis in tumor specimens by immunohistochemistry. Thus, PF-502 improves survival and inhibits the development and progression of HNSCC in preclinical models. Inhibition of pAkt, p4EBP1, pS6, and induction of TP53 and p73 are linked with the antitumor effects of PF-502 in HNSCC in vitro and in vivo, warranting further investigation of PI3K/mTOR inhibitors in a clinical subset of HNSCC patients with overactivation of PI3K/mTOR pathway and repression of TP53. Supported by NIH Medical Research Scholars Program (RVB), NIDCD intramural project ZIA-DC-000073, 74 (CVW) and NIDCR intramural project ZIA-DE-000698 (ABK). Citation Format: Robert Vander Broek, Yansong Bian, Amanda Herzog, Bradford Hall, Jamie Coupar, Zhong Chen, Ashok B. Kulkarni, Carter Van Waes. Preclinical investigation of antitumor effects of dual PI3K/mTOR inhibitor PF-04691502 in human xenograft and murine Pten/Tgfbr1 deficient head and neck cancer models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-314. doi:10.1158/1538-7445.AM2013-LB-314
Purpose: The therapeutic potential of a novel dual inhibitor of PI3K and mTOR kinases, PF-04691502 (PF-502), was investigated in human head and neck squamous cell carcinomas (HNSCC), and a novel TGF-Receptor 1 and PTEN conditional knockout (2cKO) mouse model in which mice develop HNSCC with activation of the PI3K-mTOR pathway. Experimental Design: Human oral keratinocytes (HOK) and a panel of 9 UM-SCC cell lines were examined for activation of PI3K-mTOR signaling by Western blot. Cell proliferation was assessed by MTT assay. Human HNSCC xenografts were established utilizing 5×106 wtTP53 UM-SCC 1 and mtTP53 UM-SCC 46 cells implanted subcutaneously into the flank of SCID mice. A novel 2cKO transgenic mouse model was created by generating Tgfbr1/Pten 2cKO mice (K14-CreERtam;Tgfbr1f/f;Ptenf/f). PF-502 10 mg/kg in 0.5% methylcellulose vehicle or vehicle alone was administered by oral gavage daily for 21 days. Tumor volume, weight, and body conditioning score were measured on a M/W/F schedule. Tumor immunostaining for inhibition of PI3K-mTOR activation (pAKT, pS6) and proliferation (Ki67) was performed. Results: UM-SCC cell lines showed increased activation of the PI3K/Akt/mTOR pathway as compared with HOKs. Treatment of UM-SCC 1 and 46 with PF-502 decreased pAkt and pS6 and caused a significant decrease in cell density (IC50 1.9 uM and 0.60uM respectively). UM-SCC 1 xenograft mice treated with PF-502 showed a decreased average tumor volume of 0.96 ± 0.40 cm3 versus control, 2.49 ± 0.94 cm3 on day 21. Median survival was improved with PF-502 from 23 days to 32 days. PF-502 treatment in UM-SCC 46 xenograft mice decreased the average tumor volume of 0.36 ± 0.40 cm3 versus control at 0.55 ± 0.14 cm3 on day 21, but no survival advantage was found. In 2cKO mice, PF-502 decreased average tumor volume to 0.0036 ± 0.0005 cm3 as compared to 0.916 ± 0.060 cm3 tumor volume in control mice. HNSCC tumors in 2cKO mice on day 21 were reduced from an average of 3.23 tumors in control to 0.26 tumors in PF-502 treated mice. Median survival was also improved to 64 days with PF-502 as compared to 39 days in controls. Tumors harvested from UM-SCC 1 and 46 xenograft and 2cKO mice treated with PF-502 showed inhibition of the PI3K/Akt/mTOR pathway as evidenced by decreased pAkt and pS6, and decreased proliferation via Ki67. PF-502 caused limited or no significant weight loss or cachexia during treatment, or long term adverse effects up to 6 months after treatment. Conclusions: PI3K/mTOR pathway activation was observed in most HNSCC lines as compared with HOK cells. PF-502 inhibited the PI3K/mTOR signaling and cell density of human HNSCC cell lines in vitro. PF-502 blocked development of HNSCC in a novel 2cKO mouse model, and significantly inhibited tumor growth and improved survival in human UM-SCC 1 with wtTP53 when compared UM-SCC 46 mtTP53 xenograft tumors. PF-502 was tolerated without symptomatic weight loss at the dosage tested. These results suggest PI3K/mTOR activation is an important target for therapy in HNSCC. Supported by NIDCD project ZIA-DC-000016, NIDCR project ZIA-DE-000698, and Howard Hughes Medical Institute-NIH Scholars program (AH). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A248.
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