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Comparative Study
. 2012 May 1;18(9):2558-68.
doi: 10.1158/1078-0432.CCR-11-2824. Epub 2012 Mar 12.

mTOR as a molecular target in HPV-associated oral and cervical squamous carcinomas

Alfredo A Molinolo  1 , Christina Marsh, Mohamed El Dinali, Nitin Gangane, Kaitlin Jennison, Stephen Hewitt, Vyomesh Patel, Tanguy Y Seiwert, J Silvio Gutkind
Affiliations
Comparative Study

mTOR as a molecular target in HPV-associated oral and cervical squamous carcinomas

Alfredo A Molinolo et al. Clin Cancer Res. .

Abstract

Purpose: The incidence of head and neck squamous cell carcinomas (HNSCC) associated with human papillomavirus (HPV) infection has increased over the past decades in the United States. We aimed at examining the global impact of HPV-associated HNSCC and whether the established key role of mTOR activation in HNSCC is also observed in HPV(+) HNSCC lesions, thereby providing novel treatment options for HPV-associated HNSCC patients.

Experimental design: An international HNSCC tissue microarray (TMA) was used to analyze the expression of p16(INK4A), a surrogate for HPV infection, and Akt-mTOR pathway activation. Results were confirmed in a large collection of HPV(-) and HPV(+) HNSCC cases and in a cervical cancer (CCSCC) TMA. Observations were validated in HNSCC and CCSCC-derived cell lines, which were xenografted into immunodeficient mice for tumorigenesis assays.

Results: Approximately 20% of all HNSCC lesions could be classified as HPV(+), irrespective of their country of origin. mTOR pathway activation was observed in most HPV(+) HNSCC and CCSCC lesions and cell lines. The preclinical efficacy of mTOR inhibition by rapamycin and RAD001 was explored in HPV(+) HNSCC and CCSCC tumor xenografts. Both mTOR inhibitors effectively decreased mTOR activity in vivo and caused a remarkable decrease in tumor burden. These results emphasize the emerging global impact of HPV-related HNSCCs and indicate that the activation of the mTOR pathway is a widespread event in both HPV(-) and HPV-associated HNSCC and CCSCC lesions.

Conclusions: The emerging results may provide a rationale for the clinical evaluation of mTOR inhibitors as a molecular targeted approach for the treatment of HPV-associated malignancies.

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Figures

Figure 1
Figure 1
Distribution of p16 positive cases in squamous cell carcinoma of the head and neck (HNSCC) and uterine cervix (Cervical SCC). A, the upper figure shows representative cores of HNSCC lesions (HNSCC) stained with H&E and p16 (p16 IHC), with strong nuclear and cytoplasm staining (top panel). A negative HNSCC lesion is shown below. B, a uterine cervical carcinoma (Cervical SCC) TMA was used as positive control for p16 staining. C, about 18% of the HNSCC cases were positive for p16 by immunohistochemistry, while only 1 of the 24 cases of cervical SCC studied of was negative for p16. C, the distribution of p16+ HNSCC was similar in all 8 countries analyzed, although South Africa and Thailand had a relatively higher number of cases.
Figure 2
Figure 2
The expression of p16, pAktS473, pS6, EGFR and pEGFR was evaluated by immunohistochemistry in HNSCC TMAs, and this information was used as input to generate a heat-map. The right panel shows a series of representative immunostaining in p16+ and p16− negative samples. As shown in the heat map, cases positive for p16 display an increased pAktS473 and pS6 expression. Elevated levels of EGFR but not of pEGFR were detected in p16+ samples.
Figure 3
Figure 3
HNSCC and cervical SCCs cases were analyzed by IHC for p16, pAktS473, and pS6 expression. The figure shows a representative squamous cell carcinoma (H&E) of each group. All cervical SCC and HNSCC HPV+ cases were strongly reactive for p16 in both nuclei and cytoplasm, and this was absent in the HNSCC HPV− cases (p16). Strong immunodetection of pS6 and pAktS473, with preferential cytoplasmic and membranous distribution, respectively, were observed in both cervical SCC and HNSCC HPV+ and HPV− cases, while non neoplastic oral mucosa shows very limited pS6 and pAktS473 immunostaining (bottom right). Quantification of IHC staining based on the % of stained cells in each tumoral lesion shows remarkable activation of the mTOR pathway, as reflected by the pS6 staining in a high percentage of the cervical cancers and HNSCC, the latter in both HPV+ and HPV− groups. For pAktS473, however, the fraction of immunostained cases is smaller in cervical SCC and HNSCC HPV+ lesions. The graphical representation of each individual HNSCC case (right graphs) demonstrates that both HPV+ (n=28) and HPV− (n=21) exhibit a highly significant increase in pS6 and pAktS473 with respect to the levels of the phosphorylated forms of S6 and Akt in non-neoplastic oral tissues (C) (n=19 and n=20, respectively). No significant differences were observed in the level of activation of Akt-mTOR pathway molecules when comparing HPV− and HPV+ HNSCC lesions (ns).
Figure 4
Figure 4
A, A representative series of HNSCC cell lines were assessed by PCR for the presence of HPV DNA; 4 out of 9 HNSCC cell lines were positive, using cervical cancer HeLa cells as a positive control. B, cells were evaluated in parallel by Western blot for expression levels of Akt, pAktS473, S6, pS6, and p16, as indicated. α-Tubulin was used as a loading control. The Akt and mTOR pathways were significantly activated in the vast majority of the cell lines investigated, irrespective of their p16 status. Immortalized oral keratinocytes, NOKSI cells, representative of a normal phenotype, were used as control cells to simulate (EGF) and inhibit (EGF + LY) the Akt-mTOR pathway. C, two HPV-associated HNSCC and cervical cancer-derived cell lines, UDSCC2 and HeLa, respectively, were used to validate the biochemical activity of clinically relevant inhibitors allosteric mTOR inhibitors, rapamycin and RAD001, on the Akt-mTOR pathway. As seen, exposure of cells to 100 nM of these inhibitors caused a time dependent reduction in the levels of pS6, while they increased the levels of pAktS473 in HeLa but not in HNSCC cells.
Figure 5
Figure 5
Excised tumors from athymic mice treated for 3 days with rapamycin, RAD001 or control diluent, were processed for histological evaluation. A, UDSCC2 tumors grow in control treated animals (upper left) as moderately differentiated squamous cell carcinomas, with solid sheets of cells occasionally forming keratinized clumps of cells. In animals treated with rapamycin or RAD001, edema and areas of initial tissue disruption were observed (upper, middle and right). The expression of both pS6 and pAktS473 was significantly decreased by treatment with both mTOR inhibitors, as seen in individual tissues by immunohistochemistry (A, middle and bottom row) and the quantification of multiple treated tumors (B). C, A very similar profile was observed with HeLa xenografts, which are poorly differentiated squamous cell carcinomas (left). The treated tumors showed features similar to those described for UDSCC2 (upper row, middle and right). A decrease in the expression of pS6 and pAktS473 is evident upon treatment with both mTOR inhibitors (C, middle and bottom row) as further confirmed by quantification of the immunoreactions (D). (** p<0.01; ***p<0.001).
Figure 6
Figure 6
Athymic mice were transplanted with either UDSCC2 or HeLa cells. When the tumor volume reached approximately 200 mm3, the animals were randomized and treated daily ip with control diluent, or 5 mg/kg of rapamycin or RAD001. Tumor growth curves show significant size decrease upon RAD001 and rapamycin treatment with respect to control treated mice in both UDSCC2 and HeLa xenografts (n=20 xenografts in each treatment arm; *** p<0.001). A representative histological picture of each treatment group and cell lines is shown in the rights panels. The antitumor effect of rapamycin and RAD001 was further confirmed by evaluating tumor weights (bottom) at the end of the treatment period (n=20, ** p<0.01; ***p<0.001).
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