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. Author manuscript; available in PMC: 2014 Jan 15.
Published in final edited form as: Clin Cancer Res. 2013 May 2;19(14):3808–3819. doi: 10.1158/1078-0432.CCR-12-2716

PI3K-mTOR inhibitor PF-04691502 anti-tumor activity is enhanced with induction of wild-type TP53 in human xenograft and murine knockout models of head and neck cancer

Amanda Herzog 1,2,*, Yansong Bian 1,*, Robert Vander Broek 1,2, Bradford Hall 3, Jamie Coupar 1, Hui Cheng 1, Anastasia L Sowers 4, John D Cook 4, James B Mitchell 4, Zhong Chen 1, Ashok B Kulkarni 3, Carter VanWaes 1,*,*
PMCID: PMC3715575  NIHMSID: NIHMS475965  PMID: 23640975

Abstract

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.

Keywords: PF-04691502, PI3K/mTOR inhibition, TP53, Pten Tgfbr1 knockout mice, head and neck cancer

Introduction

The Phosphatidyl Inositol-3-Kinase, viral-akt homologue, and mammalian Target of Rapamycin (PI3K/Akt/mTOR) kinases act as key regulators of a cascade of signal and effector molecules that are aberrantly activated in a broad variety of human cancers (1,2). The PI3K/Akt/mTOR pathway is emerging as important in oncogenesis of head and neck squamous cell carcinomas (HNSCC), which arise from the upper aerodigestive tract (3, 4). We previously showed that aberrant PI3K-mediated Akt phosphorylation may be induced by Epidermal Growth Factor Receptor (EGFR), Hepatocyte Growth Factor Receptor (c-MET), and their ligands, which are often overexpressed by HNSCC (5,6). Genetic alterations in the PIK3CA gene encoding the key catalytic subunit of PI3K were also subsequently found to be prevalent, with amplification reported in ~ 55% and activating mutations in ~6–8% of HNSCC tumors (7–9).

Increase in PI3K-Akt activation has also been linked to dysregulation of severaltumor suppressors prevalent in HNSCC, including PIP3 phosphatase PTEN, TGF-β receptors (TGFβR) 1 or 2, and the TP53/p73 family. PTEN expression is decreased in ~60%, and mutated in ~7% of HNSCC (9–11). We recently detected increased phospho-Akt concurrent with decrease in PTEN and TGFβR1 expression in 8/20 (40%) human HNSCC (11). Conditional double knockout of Pten/Tgfbr1 enhanced development of HNSCCs with increased expression and phosphorylation of the EGFR-Akt-mTOR axis in mice (11,12). Similarly, knockout of Tgfbr2 in combination with activating mutation of Kras also enhanced tumorigenesis of HNSCC with increased EGFR (13). In human HNSCC, we found that TGFBR2 may be inactivated infrequently by mutation, or repressed by mtTP53 (14). Mutation or inactivation of TP53 and/or inactivation of related family member p73 occurs in >80% of HNSCC (15–18). Altogether, widespread activation of PI3K-Akt-mTOR, and downstream mediators has been demonstrated in >90% of human HNSCC, implicating it as a key oncogenic pathway overlapping loss of these tumor suppressors in pathogenesis of HNSCC (19–21).

Inhibitors of PI3K, Akt or mTOR individually have demonstrated anti-tumor activity in human and murine HNSCC models. We reported that a selective PI3K inhibitor strongly attenuated Akt phosphorylation, cell growth, and angiogenesis factor expression by HNSCC in vitro, indicating PI3K is important in pathway activation, promotion of the malignant phenotype, and as a potential therapeutic target (5,6). An Akt inhibitor attenuated growth and migration of HNSCC lines in vitro, and tumorigenesis and metastasis in an orthotopic HNSCC model in vivo (22). Targeting mTOR by rapamycin or analogs was found to potently inhibit tumorigenesis of human HNSCC tumor xenografts (21, 23, 24). Rapamycin also inhibited development of carcinogen-induced SCC of the oral cavity and skin (25,26), and HNSCC that develop in genetically engineered KrasG12D/p53−/−,Pten−/−,orPten−/−/Tgfbr1−/− mice in vivo (27–30). In a pilot pharmacodynamic clinical study with rapalog temsirolimus for 3 weeks, evidence for tumor reduction was observed in 8/14 patients (31). In that study, p-S6 was strongly inhibited, but p-AKT was only weakly inhibited, suggesting that combined targeting of PI3K-mTORC1/2 warrants investigation in HNSCC.

While the aforementioned studies indicate PI3K-mTOR activation occurs via multiple mechanisms together with frequent alterations in tumor suppressor TP53 and other genes, little is yet known about how these alterations may be related, or affect response to PI3K-mTOR targeted agents. Interestingly, we observed decreased expression of wild type (wt)TP53, p73 and its co-factor YAP, in HNSCC tumors and cell lines displaying enhanced p-AKT, suggesting a potential inverse link between these alterations (16–18). Further, an anti-inflammatory drug, quinacrine, which restored TP53 expression, growth arrest, and sensitivity to DNA damaging therapy with cisplatin (16), was later reported to be an inhibitor of PI3K-Akt signaling (32). Quinacrine or an Akt inhibitor were also found to enhance nuclear expression and pro-apoptotic function of cofactor YAP, important in stabilization and pro-apoptotic function of p73 (18, R. Ehsanian, unpublished observations). These findings suggest the hypothesis that oncogenic PI3K-AKT-mTOR activation may be linked to repression of TP53/p73 expression and function, and potentially reversible by inhibitors of PI3K-AKT-mTOR signaling in HNSCC. Such agents establishing reexpression of TP53/p73 could potentially enhance response in combination with DNA damaging radiation or chemotherapeutic modalities, important in therapy of HNSCC.

Based on the evidence that amplification of PIK3CA, upstream GFRs, or tumor suppressor inactivation may promote or complement PI3K-AKT-mTOR pathway activation in HNSCC, we explored the activity of a novel, selective competitive kinase inhibitor of both class I PI3Ks and mTORs (C1 and C2), PF-04691502 (PF-502; Pfizer) (33,34). The effects of PF-502 on PI3K-mTOR signaling, TP53/p73 expression, growth, apoptosis and sensitivity to DNA damaging radiation treatment were examined in human HNSCC cell lines, xenograft models with alterations in PI3KCA, TP53/p73, TGFBR2, and our newly developed Pten/Tgfbr1 double knockout (2cKO) mouse model.

Materials and Methods

Cell Lines

A panel of nine HNSCC cell lines from the University of Michigan squamous cell carcinoma (UM-SCC) series was obtained from Dr. T.E. Carey (University of Michigan; ref. 35, Supplemental Table 1). The origin of these UM-SCC cell lines were authenticated by genotyping with 9 markers as listed and described in 2010 (35), and preserved in frozen stocks that were used within 3 months of culture. These HNSCC are well characterized for status of TP53 and TGFBR tumor suppressor alterations (Supplemental Tables 2, 3) (14, 16, 17). The UM-SCC cells were cultured in MEM and 10% FCS. Human oral keratinocytes (HOK) obtained from oral gingival mucosa from Lonza (Allendale, NJ) were used as a control cell line.

Reagents

PF-04691502 was provided by Pfizer though a Materials Transfer Agreement with NIDCD, and described previously (33,34). The drug was resuspended to a stock solutionof 10 mM in DMSO for in vitro experiments and 2.35mM in 0.5% Methylcellulose (Sigma Aldrich) to deliver 5–10 mg/kg for in vivo experiments.

Western Blot

Western blots were performed as previously described (11,16,30), using whole cell lysates and antibodies as described in Supplemental Methods. The quantification of the protein expression detected by Western blots was performed by protein densitometry with ImageJ 1.45k software, and the calculation was performed using a formula described in Supplemental Methods.

MTT/XTTcell density assay and cell cycle analysis by DNA flow cytometry

Cell density were evaluated using MTT or XTT assay as indicated, and cell cycle effects were characterized by DNA flow cytometry as described previously (16, 18) and in Supplemental Methods.

HNSCC xenograft and radiation regrowth delay tumor models

All animal experiments were carried out under protocols approved by the Animal Care and Use Committee of the NIDCD or NCI, and were in compliance with the Guide for the Care and Use of Laboratory Animal Resource, (1996) National Research Council. Four-to six-week-old female BALB/c severe combined immunodeficient (SCID) (for xenografts) or nu/nu mice (for radiation experiments) were obtained from Frederick Cancer Research and Development Center (National Cancer Institute) and housed in a specific pathogen-free animal facility. Mice were injected subcutaneously (s.c.) in flanks with 5 × 106 UM-SCC 1 or 46 cells and treated 1–2 weeks later, as described in Supplemental Methods and Suppl. Fig. 5.

Tgfbr1/Pten 2cKO mouse model studies

The Tgfbr1/Pten 2cKO mice were generated from crosses between Tgfbr1 cKO mice (K14-CreERtam;Tgfbr1f/f) and Ptenf/f mice, and induced as recently reported (11), and treated as described in Supplemental Methods and Suppl. Fig. 5.

Immunohistochemical analyses of tumors

Immunohistochemical staining (IHC) and quantifications of slides with tumor from control or PF-502-treated mice were performed using a previously published method (11), and as described in Supplemental Methods.

Statistics

Statistical analyses used were as indicated in figure legends and Supplemental Methods.

Results

Differential expression and molecular effects of dual PI3K/mTOR inhibitor PF-502 on PI3K/Akt/mTOR pathway components and TP53/p73 in HNSCC cell lines

We examined the expression and phosphorylation of key PI3K/Akt/mTOR pathway proteins in a panel of nine human HNSCC (UM-SCC) cell lines, that have been previously genotyped and that demonstrate alterations prevalent in HNSCC tumors, including EGFR and PI3K-Akt activation (5,6), as well as inactivation or mutations ofTP53/p73 and TGFβRs (refs. 14, 16–17; Supplemental Tables 1–3). Expression and/or phosphorylation of several PI3K/Akt/mTOR pathway components were differentially increased in most of the UM-SCC lines when compared with control human oral keratinocytes (HOK, Fig. 1A) and normalized to β-actin and UM-SCC1. A marked increase in protein expression of the PI3K p110alpha catalytic subunit (PIK3CA), p-Akt (S473 and T308), p-S6 (S240/244) and p-4EBP1 (T37/46) was observed in 2/3 wtTP53 deficient (UM-SCC 1 & 6) and most mtTP53 (UM-SCC11A-46) lines. Thus, the majority of UM-SCC lines demonstrated differentially increased expression and/or phosphorylation of several PI3K-Akt-mTOR pathway components, consistent with previous studies in HNSCC tumors (7, 8, 19).

Figure 1. PI3K/Akt/mTOR pathway activation and inhibition by PF-502 in HNSCC.

Figure 1

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(A) Nine UM-SCC cell lines were assessed for PI3K/Akt/mTOR pathway activation via Western blot and showed varying levels of increased activation of the pathway as compared with control HOK cells. The relative protein density normalized to UM-SCC1 and individual β-actin loading controls are shown above each blot. To probe for all 11 proteins of interest, the top 6 panels of protein densities were normalized to the middle actin panel, while the densitometry of the bottom five proteins was normalized to the bottom actin panel. (B) UM-SCC1 (wtTP53) and UM-SCC46 (mtTP53) cell lines were treated with 1.25 μM PF-502 or 0.01% DMSO control and cell lysates were procured 6, 12, and 24 hours after treatment, and subjected to SDS-PAGE and Western blot. PF-502 treated samples showed decreased pAkt (S473 and T308), p-4EBP1 (S65 and T37/46), and p-S6 (S240/244) in UM-SCC 1 and 46. Baseline TP53 and p73 in wtTP53 UM-SCC1 was increased after PF-502 administration. The numbers above each blot correspond to relative protein density measured in respect to UM-SCC1 or 46 6h DMSO treatment condition and appropriate β-actin loading controls.

To compare the molecular effects of the PI3K-mTOR inhibitor PF-502 in UM-SCC with different molecular alterations, UM-SCC lines 1 and 46 were selected for initial study (Fig 1B). These lines demonstrated increased expression of PIK3CA, p-Akt, and mTOR targets, but either express low levels of wtTP53 (UM-SCC1), or express mtTP53 and mtTGFβR2 tumor suppressor proteins (UM-SCC46), previously observed in HNSCC subsets (30, 36). Based on differing IC50s for UM-SCC1 and 46 of ~ 2 and 0.6 μM, respectively (Fig. 2A, B), we selected an intermediate concentration of PF502 of 1.25 μM, which resulted in detectable inhibition of UM-SCC1 and was ~2X the IC50 for UMSCC46. Treatment of both UM-SCC cell lines with PF-502 strongly inhibited PI3K and TORC2 targets p-Akt (T308/S473), and mTORC1 targets p-4EBP1 (S65/T37/46 residues) and pS6 (S240/244) by 6 hours post-administration, with sustained inhibition of p-S6 or p-4EBP1 (S65) observed up to 24 hours post-administration (Fig. 1B). However, greater than baseline phosphorylation was seen for PI3K target p-Akt (T308) and mTORC2 target p-Akt (S473) in UM-SCC1 by 12–24h, while earlier rebound for mTORC1 target p-4EBP1 (T37/46) was observed in UM-SCC46. PF-502 treatment had no inhibitory effect on total PI3K p110 alpha or Akt expression, consistent with its action as an inhibitor of kinase activity.

Figure 2. PF-502 inhibitor effects on cell density of UM-SCC cell lines and partial rescue by TP53 inhibitor pifithrin-α.

Figure 2

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UM-SCC 1 (A) and UM-SCC46 (B) were treated with varying concentrations of PF-502 (25 nM to 10 μM PF-502), and the effect on cell density was assessed in five-day MTT assay. Decreasing cell density was seen in both UM-SCC1 and UM-SCC46 with increasing PF-502 concentrations, with IC50 values of ~1.9 μM and 600 nM respectively. (C) Boxplots of IC50s for 6 mtTP53 (red) and 5 wtTP53 lines (cyan) (Suppl Table2). IC50 levels were significantly higher for most mtTP53 relative to wtTP53 cell lines, *P = 0.049, robust least-squares regression analysis (Suppl. Methods). Each sample was assayed in 6 replicates and data are presented as the mean for each concentration. The y-axis is log2 scaling, transformed. Dark horizontal lines represent the median, with the box representing the 25th and 75th percentiles. The whiskers indicate the lowest datum still within 1.5 IQR (interquartile range) of the lower quartile, and the highest datum still within 1.5 IQR of the upper quartile, and outliers represented by dots that are either 3×IQR or more above the top box (third quartile) or 3×IQR or more below the bottom box (first quartile). (D) UM-SCC-1 cells were treated with 0.05% DMSO, PI3K-mTOR inhibitor PF-502 (1 μM), selective TP53 inhibitor pifithrin-α (10 μM), or with a combination of pifithrin pretreatment for 5 hours prior to PF-502 exposure. Quantitative comparison of cell density by XTT assay 48h (Statistical significance: ** P value ≤ 0.01, **** P value ≤ 0.0001). PF-502 reduced cell density and cell adherence, effects partially reversed by TP53 inhibitor Pifithrin-α.

As we have previously found evidence for repression of TP53 and p73 expression and tumor suppressor function in HNSCC (16, 17), we examined if PI3K-mTOR inhibition modulated expression of these proteins (Fig. 1B). Interestingly, PF-502 treatment induced an increase in expression of TP53 up to 50-fold in the UM-SCC1 line expressing minimal detectable levels of wtTP53, while a lesser fold increase in TP53 was seen in UM-SCC 46 line with already elevated basal levels of mtTP53 between 6–24h. A moderate increase in p73 was also observed in both UM-SCC1 and 46 after PF-502 treatment. To confirm these findings and control for experimental variability, protein loading, cell density, or cell line variation, we performed additional experiments (Supplemental Fig. 1). Reproducible inhibition of selected PI3K/mTOR targets, and a clearly inducible expression of TP53/p73 proteins was observed in replicate as well as independent experiments with time/density matched controls for UM-SCC1 and 46(Supplemental Fig. 1A-D). Additionally, inhibition of selected PI3K/mTOR targets and induced expression of TP53/p73 proteins was observed in an independent wtTP53 line UM-SCC6 expressing detectable TP53 and p73 (Suppl Fig. 1E). These findings indicate that dual PI3K-mTOR inhibitor PF-502 had similar inhibitory effects on PI3K-mTOR signaling, while enhancing expression of TP53 and p73 expression in multiple UM-SCC lines with wtTP53 or mt TP53.

Dual PI3K/mTOR inhibitor PF-04691502 differentially modulates cell growth and survival of HNSCC in vitro

UM-SCC1 and 46 cell lines treated with increasing PF-502 concentrations between 25 nM to 10 μM over a five-day MTT assay, exhibited a dose-dependent decrease in cell density (Fig. 2A, B). UM-SCC1 had a higher IC50 value of ~2 μM as compared with an IC50 of ~0.6 μM for UM-SCC46. As we observed PF-502 modulated TP53, we examined an expanded panel of UM-SCCs which included 5 wtTP53 and 6 mtTP53 lines (Suppl Tables 1,2) for ICs of PF-502 (Suppl Fig. 2). For IC50s, most mtTP53 lines appeared to be relatively more resistant, except for two with IC50s overlapping those of wtTP53 lines, while wtTP53 line UM-SCC1 was a more resistant outlier by boxplot analysis (Fig. 2C). When compared using robust least-squares regression analysis, the3IC50 levels were significantly higher for most mtTP53 relative to wtTP53 cell lines (P =0.049, Suppl. Methods). As we observed PF-502 increased TP53 in UM-SCC1, we examined if the decrease in cell density was attributable in part to TP53 by pretreatment with TP53 inhibitor, pifithrin-α (36), as observed previously with pifithrin and TP53 shRNA upon quinacrine induced TP53 activation (16). PF-502 treatment alone decreased cell density of UM-SCC1 (Fig. 2D), and was associated with a change in cellular morphology (rounding, detachment) (Suppl Fig. 3). While pifithrin alone had no appreciable effect on cell density, significantly higher cell density was observed when pifithrin was combined with PF-502, consistent with partial contribution of TP53 to growth inhibition (Fig. 2D, Suppl Fig. 3).

To further evaluate the effects of PF-502 on cell cycle and death, DNA cytofluorometric analysis of UM-SCC1 and 46 cells was compared after treatment with PF-502 at twice their established IC50 values (Fig. 3). UM-SCC1 cells treated with PF-502 (4 μM) exhibited increased G0/G1 accumulation over 24–48h, and sub-G0 DNA over 24–72h, consistent with the G0/G1 growth arrest and cell death expected with the PI3K/Akt/mTOR signal inhibition, and increased TP53/p73 protein expression observed with PF-502 above. By contrast, treatment of UM-SCC 46 cells with 1.2 μM PF-502 showed a lesser increase in G0/G1, and no further increase in the elevated sub-G0fraction observed with late growth. Because the differences in DNA fraction could be related to lower concentration used for UM-SCC46, we also compared the effects after treating both at the same concentration of ~IC50 for UM-SCC1 (2μM). Under these conditions, PI3K/Akt/mTOR inhibition still had a differentially greater effect on G0/G1 cell cycle arrest in UM-SCC1 than 46, and similar enhancement of sub-G0 DNA in both lines required ~3X the IC50 for UM-SCC46 (Suppl. Fig. 4). Cell cycle analysis performed for additional wtTP53/TGFBR2 lines UMSCC 6 (IC50~0.2μM), UM-SCC-9 (IC50~0.15μM) and mtTP53/wtTGFBR2 line UMSCC 38 (IC50~0.5μM) also demonstrated PF-502-induced G0/G1 accumulation (Suppl. Fig. 4A). Wt TP53 UM-SCC 6 and 9 showed relatively higher baseline and PF-502 inducible sub-G0 fraction by 24h, while PF-502 induced increased in subG0 DNA was delayed until 48h in UM-SCC38 with mtTP53/wtTGFBR2 (Suppl. Fig. 4B). Together, the above findings suggest that TP53 status as well as other factors contribute to the sensitivity of UM-SCC to PF-502.

Figure 3. Effects of PF-502 treatment on cell cycle and death in UM-SCC 1 and UM-SCC46 by DNA cytofluorometry.

Figure 3

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UM-SCC 1 and 46 cells were plated in monolayer and treated with PF-502 at a concentration equal to approximately two times the IC50 values (final concentration of 4 μM and 1.2 μM respectively). Cells were harvested for cell cycle analysis at 24, 48, and 72 hours post treatment. (A) UM-SCC1 cells showed increased G0/G1 arrest and sub-G0 DNA fragmentation. (B) UM-SCC46 cells showed limited increase in G0/G1 or sub-G0 fraction with PF-502 treatment compared to matched controls.

PF-04691502 differentially affects tumor growth, survival and delays tumor growth with radiation in a human HNSCC xenograft model with wtTP53

In order to evaluate the activity and tolerability of PF-502 in HNSCC in vivo, pilot dosage studies were performed using the UM-SCC1 (wtTP53) and UM-SCC46 (mtTP53 and mtTGFβR2) as human HNSCC xenograft models. Tumor cells were implanted subcutaneously into the flank of SCID mice, and treatment was initiated beginning ~14 days after implantation when tumors were palpable. PF-502 (5, 7.5, and 10 mg/kg) or control vehicle were given via oral gavage for 21 consecutive days (see schema, Suppl. Fig. 5). After establishing PF-502 10 mg/kg as the most effective tolerated dose (data not shown), we conducted a larger trial (n>12 per group) in UM-SCC1 and UM-SCC46 xenograft mice, and the effects on tumor growth and survival were compared (Fig. 4A–D). While UM-SCC1 demonstrated more rapid tumor growth than UM-SCC46 in vivo, PF-502 had a greater inhibitory effect on the tumor growth in the wtTP53 UM-SCC1 xenograft mouse model, than in UM-SCC46 with mtTP53 and mtTGFβR2 (Fig. 4A). At the completion of treatment on day 21, the difference in tumor volume versus control was highly statistically significant (p value <0.0001) in the UM-SCC1 model, and associated with an improvement in median survival of ~10 days (Fig. 4B). By contrast, PF-502 treatment in UM-SCC 46 xenograft mice exhibited a less potent anti-tumor effect (Fig. 4A), without significant improvement in survival (Fig. 4B).

Figure 4. Effects of PF-502 treatment on tumorigenesis and survival in two HNSCC xenograft mouse models.

Figure 4

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Human HNSCC xenografts were established utilizing 5×106wtTP53 UM-SCC1 or mtTP53 UM-SCC46 cells implanted subcutaneously into the flank of SCID mice (n=13 or 12 mice per treatment group respectively). Treatment was initiated when tumors were palpable, 14 days after implantation. Mice were treated with 10 mg/kg PF-502 by oral gavage (o.g.) for 21 consecutive days. (A). Tumor volume was reduced by PF-502 for UM-SCC1 on day 21 (p<0.0001). Tumor volume reduction in the UM-SCC46 model at day 21 was not significant (P>0.05). (B) Survival of mice bearing UM-SCC1 xenografts treated with PF-502 showed a median survival advantage of ~10 days, while UM-SCC46 xenograft mice did not show improved survival with PF-502 treatment. (C) The effects of control (0.5% methyl cellulose/PBS), PF-502 (10 mg/kg), 15 Gy radiation, and PF-502 plus radiation on the growth of UM-SCC1 xenografts. PF-502 was administered by o.g. once daily for 5 days, and radiation treatment was delivered on the fourth day, 2 hr after PF-502 gavage (arrows). Error bars = SEM (n=5 mice/group).

In both HNSCC xenograft mouse models, tumor growth resumed after daily PF-502 treatment was withdrawn on day 21. This growth rate was relatively consistent with that seen in vehicle treated control mice. In the UM-SCC46 xenograft model, this tumor growth increase after treatment cessation led to equalization of tumor volume by about day 32, accounting for the lack of effect on survival. Regarding the drug toxicity, PF-502 10 mg/kg was well tolerated, with all mice having normal body conditioning scores of 3. While weight increase was delayed in UM-SCC 1 as compared to UM-SCC-46 xenograft mice treated with PF-502, this approximated the difference in tumor volume and mass of ~2.25g between treated and control mice for days 18–21 (Suppl. Fig. 6A, B).

As PF-502 enhanced expression of wtTP53 and p73 in UM-SCC1, we examined if combining PF502 with DNA-damaging therapy with radiation further delayed tumor regrowth. As shown in Figure 4C, five daily doses of PF-502 or 15 Gy single dose radiation inhibited tumor growth compared to control. The combination of PF-502 and radiation further delayed tumor growth. The time for tumors to reach 4 times the initially measured tumor volume relative to the control for PF-502 alone, 15 Gy, and PF-502 plus 15 Gy was 23.2 (p = 0.20), 23.6 (p = 0.04), and 29.4 (p = 0.02) days, respectively.

PF-04691502 delays HNSCC tumor development, growth and improves survival in Pten/Tgfbr1 double knockout mice

Decreased PTEN and TGFβR1 has been detected in ~40% of human HNSCC and implicated in PI3K/Akt/mTOR activation and tumor development (10–12). As we recently established that increased PI3K-Akt-mTOR activation and HNSCC tumorigenesis with 100% penetrance is a consequence in Pten/Tgfbr1 double knockout (2cKO) mice (11), we examined the potential of PF-502 to inhibit development and growth of HNSCC in this defined transgenic mouse model. After a pilot dosage escalation study, PF-502 10 mg/kg for 3 weeks was found to be well-tolerated and without significant weight loss or toxicity for up to six months (data not shown). Treatment of larger groups of Pten/Tgfbr1 2cKO mice (n>18) with PF-502 was then initiated 4 weeks after deletion of Pten and Tgfbr1 via tamoxifen-induced Cre expression, and 2 weeks before full development of HNSCC, when ~50% of mice exhibit small visible lesions. 2cKO mice were treated with 10 mg/kg PF-502 for 21 consecutive days by oral gavage. PF-502 treated 2cKO mice exhibited a significant decrease in development and growth of external head and neck and oral tongue tumors (Fig. 5A–D; p<0.05), as compared with control mice. Delayed tumor development and growth were seen after treatment cessation, requiring ~20 days after treatment cessation before reaching similar tumor volumes as controls (Fig. 5D). This PF-502 mediated tumor growth delay in 2cKO mice corresponded to an improvement in median survival of ~20 days, from a median survival of ~40 days in control mice to >60 days in PF-502 treated mice (Fig. 5E). PF-502 was well tolerated, without significant weight loss in treated versus control mice (Suppl. Fig. 6C).

Figure 5. PF-502 delays HNSCC development in Tgfbr1/Pten 2cKO mice.

Figure 5

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Treatment was initiated 4 weeks after 5 day Tamoxifen administration to induce Cre expression, and approximately 2 weeks before development of HNSCC. Tgfbr1/Pten 2cKO mice were treated with PF-502 10 mg/kg or 0.5% methylcellulose vehicle alone by o.g. for 21 days. (A) Representative HNSCC in 4 control mice on day 21. (B) Representative PF-502 treated mice showed visibly reduced tumor burden on day 21. (C) PF-502 treated mice had fewer tongue tumors as compared with control mice (30% versus 55.6% respectively) on day 21 despite similar initial tongue tumor incidence. Tongue tumors also visibly covered a surface in PF-502 mice (9.8%) as compared with control mice (21.3%) on day 21 (P<0.0001). (D) 2cKO mice treated with PF-502 (n = 19) showed decreased tumor volume versus control vehicle (n = 18) during 21 days of PF-502 treatment. (E) PF-502 treated 2cKO mice showed improved median survival of about >60 days as compared with 40 days for control mice (median survival advantage of >20 days; n = 19 and n = 18 respectively).

PF-502 inhibited PI3K/Akt/mTOR and Ki-67, and increased TP53 and TUNEL staining in xenograft and 2cKO models in vivo

Effects of PF-502 treatment for 21 days on PI3K/Akt/mTOR, TP53/p73, and related proliferation (Ki67) and apoptosis (TUNEL) markers were examined by immunostaining in HNSCC tumors from UM-SCC1 and 46 xenografts and Pten/Tgfbr1 2cKO mice (Fig. 6A). Tumor immunostaining was quantified by histoscores (Fig. 6B). PF-502 treatment for 21 days significantly decreased PI3K/Akt/mTOR pathway signaling as measured by decreased staining of pAkt (S473) and downstream p-4EBP1 (T37/46) and p-S6 (S240/244) in tumor specimens from all three models (Fig. 6). Conversely, TP53 was significantly increased in tumors from wtTP53 UM-SCC1 and mtTP53 UM-SCC46 xenografts, but did not reach significance in 2cKO mice. p73 staining was slightly increased, but also did not reach statistical significance in the three tumor models. There was a statistically significant decrease in proliferation (Ki67) and increase in apoptosis (TUNEL) marker staining in tumor specimens from all three models, with greater relative increase in TP53 and TUNEL staining observed in UM-SCC1 with wtTP53. TP53 inducible cell cycle dependent kinase inhibitor p21Cip1 gene expression was increased in wtTP53 UM-SCC-1, but not in mtTP53 UM-SCC-46 tumors, providing further evidence for functional activation of TP53 (Suppl. Fig. 7).

Figure 6. Effects of PF-502 on PI3K/Akt/mTOR signaling, TP53/p73, Ki67 proliferation and TUNEL apoptosis marker expression in UM-SCC 1 and 46 xenograft and 2cKO mouse models of HNSCC.

Figure 6

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Immunostaining was performed on tumors harvested approximately 4 hours after final PF-502 administration on day 21. (A) Representative IHC staining. Bars, 100 μM. (B) Staining histoscores. Yellow: untreated; red: PF502 treated samples. PF-502 treated mice showed a reduction in the levels of p-AktS473, p-4EBP1T37/46, p-S6S240/244 and Ki67 proliferation marker staining in all three models. HNSCC from PF-502 treated wtTP53 UM-SCC 1 xenograft mice showed a greater relative increase in TP53 and TUNEL apoptosis marker staining as compared with mtTP53 UM-SCC46 PF-502 treated 2cKO tumors. Statistical analysis was performed by t-test(* p<0.05; **p<0.01; ***p<0.001). The increase in p73 in PF-503 treated mice did not reach significance p<0.05).

Discussion

Here, we show that PI3K/mTOR antagonist PF-04691502 has anti-tumor activity in PIK3CA-overexpressing/TP53-deficient human and Pten/Tgfbr1-deficient murine head and neck cancer models, which reflect alterations found concurrently with PI3K-Akt-mTOR activation in human HNSCC subsets. Notably, in HNSCC from the subset in which we previously demonstrated decreased wtTP53/p73 expression (16, 17), PF-502 inhibited PI3K/Akt/mTOR signaling, and reciprocally enhanced TP53 and p73 expression, supporting a model whereby oncogenic activation of the PI3K/Akt/mTOR pathway can repress TP53 and p73 expression (Suppl Fig. 8). PF-502 significantly delayed HNSCC tumorigenesis and prolonged survival of mice bearing wtTP53 UM-SCC-1 xenografts, and in Pten/Tgfbr1 deficient mice, in which PI3K/Akt activation is a major driver of HNSCC (11,12). More limited activity was observed in human UM-SCC46 with PIK3CA overexpression, Akt/mTOR activation, but mutations of both TP53 and TGFβR2 (14, 16). Our findings comparing IC50s in a wider panel of UM-SCC in vitro suggest that TP53 status as well as other factors contribute to the sensitivity of UM-SCC to PF-502. Preliminary findings from the TCGA have detected amplifications or putative activating mutations in PIK3CA in ~30% of HNSCC, which overlap alterations in TP53, and other less prevalent candidate genes (37). Together, these observations underscore the importance of evaluating molecular and anti-tumor effects of PI3K-Akt-mTOR pathway targeted therapy in experimental models and clinical subsets with different genetic and molecular alterations of biological relevance in HNSCC.

The frequent activation of PI3K/Akt/mTOR pathway by upstream receptor tyrosine kinases or intrinsic alterations (3–12), provides an important rationale for investigation of dual PI3K/mTOR inhibitors in HNSCC. PI3K-Akt-mTOR signaling pathway is activated in most UM-SCC lines, and involves increased expression of the PIK3CA (p110alpha) catalytic subunit, p-Akt, p-mTOR, p-4EBP1 and p-S6 ( Fig. 1A; Suppl. Fig. 8), as often observed in human tumors (7, 19). Treatment with PF-502 blocked downstream targets of PI3K and mTORC2 (pAkt T308/S473), mTORC1 (p-S6(S240/244) and p-4EBP1(Thr37/46), consistent with dual PI3K/mTORC1/2 inhibitor activity. However, the duration of blockade was limited for p-Akt (S473 and T308) and some other downstream components in UM-SCC 1 and 46 by 12–24h, as also observed using lower concentrations of PF-502 in other tumor types in vitro and in vivo (34). Reactivation of PI3K-Akt with mTOR inhibition has been reported to result from loss of feedback inhibitory loop involving p-S6 and IRS-1 (Suppl. Fig. 8) (2). The greater than baseline increase in p-Akt (S473 and T308) by 24h for UM-SCC1 (Fig. 1B) could potentially explain the requirement for markedly higher concentrations of PF-502 to inhibit proliferation of UM-SCC 1 relative to UM-SCC 46 in vitro (Fig. 2A, B). However, these p-Akt markers were similarly inhibited in all 3 tumor models in mice receiving 10mg/kg every 24h in vivo, indicating that steady state concentrations of PF-502 achieved were adequate to inhibit p-Akt and p-S6 in vivo. A phase I clinical trial with daily PF-502 for recurrent cancers has recently been completed, and p-Akt as a candidate marker of PF-502 activity is planned (R. Millham, Pfizer, Personal Communication). Preliminary analysis of this phase I clinical trial provides evidence for clinical activity, with prolonged stable disease in 12/36 (33%) patients (R. Millham, Pfizer, Personal Communication), similar to tumoristatic effects we observed in UM-SCC1 and 2cKO models. These data also suggest that a subset of tumors are sensitive to clinically achievable concentrations, while alterations in others may attenuate the anti-tumor effects of PI3K-mTOR inhibition.

In this regard, the reciprocal enhancement in expression of TP53/p73 tumor suppressor proteins, and apparent relationship between TP53 status and IC50 for the majority of UM-SCC lines with blockade of the PI3K-Akt-mTOR pathway, are important findings of this study. Increase in TP53 and p73 was detected in wtTP53 UM-SCC1 and 6 lines in vitro, and mtTP53 and p73 was also further increased in UM-SCC46. Pifithrin partially attenuated PF-502 induced growth inhibition in wtTP53 line UM-SCC1, consistent with contribution of TP53 to growth inhibition. We further show that UM-SCC1 with inducible wtTP53 exhibits greater G0/G1 growth arrest, and that concentrations ~3X IC50 for UM-SCC46 with mtTP53 are required to induce a comparable increase in subG0 DNA (Fig. 3 and Suppl. Fig. 4). Further, wtTP53 UM-SCC 6 and 9 showed relatively higher baseline and PF-502 inducible sub-G0 fraction by 24h, while PF-502 did not induce increased subG0 DNA until 48h in UM-SCC38 with mtTP53/wtTGFBR2 (Suppl. Fig. 4, right panels). Together these data suggest that TP53 status as well as other factors contribute to the sensitivity of UM-SCC to PF-502.

Similar effects of PF502 associated with genotype were observed in vivo (Fig. 6). We found that UM-SCC1, which is wtTP53 genotype and exhibits greater differential induction of TP53 and growth arrest than mtTP53/TGFBR2 line UMSCC46, shows a greater reduction in proliferation (Ki67) and induction of apoptosis (TUNEL), consistent with the higher sensitivity to tumor inhibition observed. Further, PF-502 induced TP53 target and growth arrest gene p21 in UM-SCC1 tumors, which was not observed in mtTP53 line UM-SCC46 (Supplemental Fig. 7). These data support that wtTP53 genotype and function contribute to the difference observed. The capability of PI3K-mTOR inhibition to enhance TP53 expression and effects when combined with radiation, has broader implications for combination with radiation or chemotherapies that enhance the TP53 DNA damage response.

We previously showed that wtTP53 mRNA and protein expression is repressed in UM-SCC1 and a subset of HNSCC, and that the repression of TP53 mRNA, protein, and TP53-dependent growth arrest and cytotoxicity was reversible by treatment with quinacrine (16), but its target of action was unclear. Subsequent studies suggested that quinacrine enhances TP53 re-expression via an Akt-dependent mechanism (32). We also reported that Akt mediates phosphorylation and cytoplasmic inactivation of p73 cofactor YAP in the same subset, which is important in p73 stabilization and transcription (18). Recently, mTOR inhibitor rapamycin was implicated in modulating p73 genes involved in mesenchymal differentiation and tumorigenesis in rhabdomysarcoma (38). Our current results demonstrating that a dual PI3K-mTOR inhibitor increases TP53, and to lesser extent, p73 expression, further support the hypothesis that this pathway contributes to inactivation of TP53/p73 in this HNSCC subset.

Our 2cKO transgenic mouse model enabled evaluation of the effect of PF-502 on HNSCC with defined genetic alterations in PTEN and TGF-β signaling, which we have shown enhance PI3K-Akt activation (11). Treatment of the novel Tgfbr1/Pten 2cKO HNSCC mouse model with PF-502 inhibited p-Akt and p-S6, and significantly inhibited tumor development and prolonged survival. These effects were also associated with an increase in TP53, marked decrease in proliferation marker Ki67, and increase in TUNEL apoptosis marker immunostaining in PF-502 treated 2cKO tumors. Clinical studies will be needed to determine how individual and combined TP53 and TGFβ pathway alterations affect sensitivity to PI3K-mTOR inhibitors. Additionally, inclusion of HPV+ HNSCC, which also demonstrate concurrent PIK3CA activation and TP53 inactivation (37), and sensitivity to mTOR inhibitor rapamycin in preclinical models (39), is warranted.

Other inhibitors of the PI3K-Akt-mTOR pathway have demonstrated varying levels of activity in HNSCC and lung SCC models associated with status of PI3K, PTEN, TP53, or p73/YAP1. In a recent report, lung SCC lines harboring receptor tyrosine kinase activation, PI3K mutation or amplification, and PTEN loss were most sensitive to a PI3K inhibitor, GDC-0941. Further, activity of this inhibitor was enhanced by combination with chemotherapy (40). PI3K inhibitor BKM120 and PI3K-mTOR inhibitor BEZ235 showed activity in lung SCC with wtPI3K (41). In contrast BKM120 and BEZ235 showed more limited cytotoxicity alone in HNSCC line SCC25 (42), which we note was previously reported to lack TP53 resulting from deletion of two base pairs in codon 209 (43). BKM120 also lacked antiproliferative effect in cell lines without high PI3K p85-subunit expression (44). Dual PI3K/mTOR inhibitor, BEZ235, was recently reported to show preclinical activity in a HNSCC xenograft model with reduction in tumor volume (44). An Akt inhibitor was shown to reduce metastasis in an orthotopic model of wtTP53 HNSCC line SCC-1 (22). We previously showed that an Akt inhibitor can enhance YAP in wtTP53 lines, an important cofactor for p73-mediated tumor suppression (18). However, the suppression of tumorigenesis but lack of complete regression observed with PI3K/Akt/mTOR inhibition by monotherapy with PF-502 or these agents suggests that combination with other therapies merits investigation.

As PF-502 induced TP53 and or p73, and showed evidence of increased activity with radiation, combination of PI3K-mTOR inhibition with DNA damaging therapies such as radiation or cisplatin active in HNSCC holds promise. Reduction of radiation toxicity by mTOR inhibitor rapamycin has been reported (45). Combination of PI3K/Akt/mTOR inhibition with other targeted agents could also potentially lead to improved anti-tumor effects with lower doses of each specific inhibitor, thereby limiting toxicity while improving efficacy (2). As we showed that MEK-ERK signaling is often coactivated with PI3K in HNSCC (5, 6), combined inhibition of these pathways could help improve on the tumoristatic effects observed with PI3K-mTOR inhibition alone.

Supplementary Material

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Translational Relevance.

While PI3K/mTOR activation is prevalent in cancers, we found that activity of PI3K/mTOR targeted agents differed among HNSCC models displaying distinct molecular alterations affecting TP53 and TGFβtumor suppressor function. Notably, in HNSCC in which we demonstrated repression of wild-type TP53, PF-502 reciprocally enhanced TP53 expression, apoptosis, and demonstrated significant therapeutic activity alone and in combination with radiation. PF-502 also demonstrated anti-tumor activity in a Pten/Tgfbr1 deficient murine head and neck cancer model, but not in a human HNSCC with mtTP53/TGFBR2. These findings provide evidence for activity of PI3K/mTOR inhibitor PF-502 in HNSCCs in which PI3K-Akt activation is driven by distinct underlying alterations, and contributes to repression of TP53. Evaluation of PIK3CA as well as TP53 and TGFBR alterations prevalent in HNSCC may be important to include in future clinical trials in order to identify selective factors for the broader class of PI3K-mTOR inhibitors.

Acknowledgments

Grant information: Supported by NIDCD intramural projects ZIA-DC-000073 and ZIA-DC-000074 (C. Van Waes), NIDCR intramural project ZIA-DE-000698 (A.B. Kulkarni), and ZIA-SC-006321 (J.B. Mitchell).

The authors wish to thank Drs. Barbara Conley and Eva Szabo for their reading and helpful comments on the manuscript; Drs. Anthony Saleh, Xinping Yang, Chris Silvin, and Julie Kan for their technical support.

Footnotes

Disclosure of Potential Conflicts of Interest

C. Van Waes is recipient of Pfizer pharmaceuticals under a Material Transfer Agreement with NIDCD, but otherwise holds no financial interests. A.B. Kulkarni and Y. Bian are co-inventors of Pten/Tgfbr1 knockout mice, for which a patent application is pending (#20120114640).

Author Contributions

Conception and design: C. Van Waes, Y. Bian, A. Herzog, Z. Chen, B. Hall, A.B. Kulkarni, J.B. Mitchell

Development of methodology: A. Herzog, Z. Chen, B. Hall, Y. Bian, R. Vander Broek

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A. Herzog, Y. Bian, R. Vander Broek, J. Coupar, B. Hall, A. L. Sowers, J.A. Cook, A.B. Kulkarni, C. Van Waes

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A. Herzog, Y. Bian, R. Vander Broek, H. Cheng, B. Hall, A.L. Sowers, J.A. Cook, J.B. Mitchell, A.B. Kulkarni, C. Van Waes

Writing, review, and/or revision of the manuscript: A. Herzog, Y. Bian, R. Vander Broek, H. Cheng, B.Hall, Z. Chen, J.B. Mitchell, A.B. Kulkarni, C. Van Waes

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A. Herzog, Y. Bian, R. Vander Broek, J. Coupar, A. Sowers, B. Hall

Study supervision: C. Van Waes, A.B. Kulkarni, J.B. Mitchell

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

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NIHMS475965-supplement-1.docx (23.3KB, docx)
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NIHMS475965-supplement-7.tif (120.2KB, tif)
8
NIHMS475965-supplement-8.tif (303.5KB, tif)
9
NIHMS475965-supplement-9.tif (15.8MB, tif)

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