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. 2013 Jun;15(6):620-30.
doi: 10.1593/neo.13432.

Improved clearance during treatment of HPV-positive head and neck cancer through mTOR inhibition

Joseph D Coppock  1 , Bryant G Wieking, Alfredo A Molinolo, J Silvio Gutkind, W Keith Miskimins, John H Lee
Affiliations

Improved clearance during treatment of HPV-positive head and neck cancer through mTOR inhibition

Joseph D Coppock et al. Neoplasia. 2013 Jun.

Abstract

Human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) incidence is increasing at a near epidemic rate. We investigated whether the mammalian (or mechanistic) target of rapamycin (mTOR) inhibitor, rapamycin, can be used as a concurrent agent to standard-of-care cisplatin/radiation therapy (CRT) to attenuate tumor lactate production, thus enhancing CRT-induced immune-mediated clearance of this antigenic tumor type. A C57Bl/6-derived mouse oropharyngeal epithelial cell line retrovirally transduced with HPV type 16 E6/E7 and human squamous cell carcinoma cell lines were evaluated for their response to rapamycin in vitro with proliferation assays, Western blots, and lactate assays. Clonogenic assays and a preclinical mouse model were used to assess rapamycin as a concurrent agent to CRT. The potential of rapamycin to enhance immune response through lactate attenuation was assessed using quantitative tumor lactate bioluminescence and assessment of cell-mediated immunity using E6/E7-vaccinated mouse splenocytes. Rapamycin alone inhibited mTOR signaling of all cancer cell lines tested in vitro and in vivo. Furthermore, rapamycin administered alone significantly prolonged survival in vivo but did not result in any long-term cures. Given concurrently, CRT/rapamycin significantly enhanced direct cell killing in clonogenic assays and prolonged survival in immunocompromised mice. However, in immunocompetent mice, concurrent CRT/rapamycin increased long-term cures by 21%. Preliminary findings suggest that improved survival involves increased cell killing and enhanced immune-mediated clearance in part due to decreased lactate production. The results may provide rationale for the clinical evaluation of mTOR inhibitors concurrent with standard-of-care CRT for treatment of HPV-positive HNSCC.

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Figures

Figure 1
Figure 1
Inhibition of cell proliferation of human and mouse HNSCC cell lines by rapamycin is associated with decreases in mTOR signaling. (A) Cell proliferation assay. Primary HTEs are compared to four tonsillar SCC lines, two HPV- (SCC1 and SCC84) and two HPV+ (SCC47 and SCC90), after 72 hours of rapamycin treatment at the indicated concentrations. (B) Western blot analysis of downstream targets of mTOR. Whole-cell lysates of the cell lines described in A after 24 hours of 10 nM rapamycin treatment were analyzed for protein levels downstream of mTOR. (C) Mouse correlate to A. A retrovirally transduced and immortalized HPV+ mouse oropharyngeal epithelial (MOE) cell line possessing the E6 and E7 oncogenes (E6/E7) is compared to an empty vector containing control line (LXSN). (D) Corresponding mouse correlate Western blots of downstream targets of mTOR. All cell lines treated with rapamycin showed a dose-dependent decrease in cell proliferation (all lines at 10–100 nM rapamycin, P ≤ .047 to control). Regarding Western blots, with rapamycin treatment, total S6K migrates slightly faster, indicating dephosphorylation. Phosphorylation of S6K (P-S6K) is nearly completely inhibited, and 4EBP1 shows decreases in the slower migrating, hyperphosphorylated bands and increases in the faster migrating, hypophosphorylated bands. Blots have been cropped for presentation and those with dark backgrounds have been enhanced in contrast and brightness in entirety using Microsoft PowerPoint before cropping. Full-length blots can be seen in Figure W1.
Figure 2
Figure 2
Rapamycin enhances the direct CRT-induced cytotoxicity in clonogenic assays. E6/E7/Ras MOEs allowed to establish for 24 hours were treated with rapamycin at the indicated concentrations for an additional 24 hours. Cells were then dosed with cisplatin and radiation with rapamycin treatment continued concurrently. After 1 week, colonies were fixed, stained, and counted. Representative images of the colonies formed at each rapamycin concentration with equal cisplatin and radiation dosing in all conditions are shown in A. Average colony number at each concentration is shown in B, and average colony size is shown in C. Rapamycin was seen to enhance the direct CRT-induced cytotoxicity to this model HPV+ HNSCC cell line, with fewer (*P < .03, ** P < .01) and smaller (*P < .03, **P < .03) colonies forming in the presence of rapamycin.
Figure 3
Figure 3
Rapamycin inhibits mTOR signaling at the tumor level to prolong survival and enhance CRT-induced tumor clearance in wild-type mice. (A) Kaplan-Meier survival analysis with log-rank significance test in wild-type male C57Bl/6 mice with E6/E7/Ras MOE tumors of the right flank receiving daily i.p. rapamycin or vehicle (0 mg/kg, n = 7; 1 mg/kg, n = 6; 5 mg/kg, n = 6; 0 to 1 mg/kg, NS; 1 to 5 mg/kg, NS; 0 to 5mg/kg, P < .0004). (B) Phospho-S6 immunohistochemical staining of tumor sections harvested at end point showing inhibited mTOR signaling with rapamycin treatment (1 mg/kg). (C) Survival analysis with log-rank significance test in wild-type male C57Bl/6 mice receiving daily rapamycin or vehicle for 21 days concurrent with once weekly cisplatin (0.132 mg/mouse) and radiation (8 Gy) for 3 weeks. The combined overall survival for three separate replicates of the experiment is shown (0 mg/kg, n = 30; 1 mg/kg, n = 28; 5 mg/kg, n = 25; 0 to 1 mg/kg, NS; 1 to 5 mg/kg, NS; 0 to 5 mg/kg, P ≤ .055). A rapamycin dose-dependent increase in tumor-free survival concurrent with equal dosing of CRT was observed, 27% with CRT alone, 39% at 1 mg/kg rapamycin + CRT, and 48% at 5 mg/kg rapamycin + CRT. The highest dose of rapamycin concurrent with CRT showed a 21% overall increase in tumor-free survival in wild-type mice compared to vehicle.
Figure 4
Figure 4
Rapamycin prolongs survival and enhances CRT in immunocompromised mice but does not lead to any long-term cures. (A) Kaplan-Meier survival analysis with log-rank significance test in immunocompromised, Rag-1-deficient male C57Bl/6 mice with E6/E7/Ras MOE tumors receiving daily rapamycin treatment or vehicle (for all groups, n = 5; 0 to 1 mg/kg, P < .006; 1 to 5 mg/kg, NS; 0 to 5 mg/kg, P < .004). (B) Survival analysis with log-rank significance test in Rag-1-deficient, immunocompromised male C57Bl/6 mice receiving daily rapamycin or vehicle for 21 days concurrent with CRT as described above (0 mg/kg, n = 10; 1 mg/kg, n = 9; 5 mg/kg, n = 9; 0 to 1 mg/kg, P < .0003; 1 to 5 mg/kg, P < .002; 0 to 5 mg/kg, P < .00001).
Figure 5
Figure 5
Rapamycin attenuates tumor cell lactate production. (A) In vitro lactate assay. E6/E7/Ras MOEs or HPV+ SCCs plated to 100% confluence were incubated for 4 hours in the presence of the indicated doses of rapamycin. The media was then subject to a commercially available colorimetric lactate assay, the concentration determined through standard curve, and the results plotted as average lactate concentration per dosage group (n = 3). Rapamycin significantly decreased lactate production in both HPV+ SCC (SCC47: 1 nM, P < .01; 10 nM, P < .05; 100 nM, P < .02; SCC90: 10 nM, P < .02; 100 nM, P < .02) and E6/E7/Ras MOE (10 nM, P < .02; 100 nM, P < .05) cell lines. (B) Quantitative lactate bioluminescence. Lactate levels were visualized in end-point tumor sections of E6/E7/Ras tumors from daily vehicle or rapamycintreated C57Bl/6 mice using a lactate-dependent luciferase-containing buffer system as described by Broggini-Tenzer et al. [34]. Intensities of bioluminescent images of six (n = 6) tumor sections from multiple mice at each dosage group were averaged. A representative image is shown alongside tabulated mean bioluminescent intensity and P values compared to control per dosage group (1 to 5 mg/kg, NS).
Figure 6
Figure 6
Lactic acid inhibits CMI and decreases lymphocyte perforin levels specific to our HPV+ HNSCC tumor model. (A) CMI assay. CFSE-stained E6/E7/Ras cells were irradiated (30 Gy) and co-cultured for 24 hours with isolated mixed lymphocytes from the spleens of mice vaccinated with Ad5 E6/E7 [6] at 0, 5, and 10 mM lactic acid in media (RPMI) followed by dead cell staining (TO-PRO-3) and flow cytometry. CFSE-positive cells were gated and mean TO-PRO-3 signal averaged (n = 3) for each dosage group. In all dosage groups, the presence of lymphocytes led to significantly increased dead cell staining (P < .02), while lactic acid in the absence of lymphocytes did not itself induce any cell death (splenocytes: 0, 5, and 10 mM, NS). A trend of decreasing dead cell staining with increasing lactic acid concentration was observed. At 10 mM lactic acid, cell-mediated cytotoxicity was significantly decreased as indicated by decreased dead cell staining (*P < .005). (B) Lymphocytes cultured alone in lactic acid for 24 hours showed a dose-dependent decrease by Western blot in total perforin, the primer of CMI. Concentrations of lactic acid at 10 mM and higher were tested as significant inhibition of CMI was not seen at less than 10 mM. The blot has been cropped for presentation and canbe seen infull in Figure W2.
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