Purpose The incidence of head and neck squamous cell carcinomas (HNSCC) associated with papillomavirus (HPV) infection has increased over the past decades in the US. 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 p16INK4A, 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 pre-clinical 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.

Despite our improved understanding of cancer, the 5-year survival rate for head and neck squamous cell carcinomas (HNSCC) patients remains relatively unchanged at 50% for the past three decades. HNSCC often metastasize to locoregional lymph nodes, and lymph node involvement represents one of the most important prognostic factors of poor clinical outcome. Among the multiple dysregulated molecular mechanism in HNSCC, emerging basic, preclinical, and clinical findings support the importance of the mTOR signaling route in HNSCC progression. Indeed, we observed here that the activation of mTOR is a widespread event in clinical specimens of HNSCC invading locoregional lymph nodes. We developed an orthotopic model of HNSCC consisting in the implantation of HNSCC cells into the tongues of immunocompromised mice. These orthotopic tumors spontaneously metastasize to the cervical lymph nodes, where the presence of HNSCC cells can be revealed by histological and immunohistochemical evaluation. Both primary and metastatic experimental HNSCC lesions exhibited elevated mTOR activity. The ability to monitor and quantitate lymph node invasion in this model system enabled us to explore whether the blockade of mTOR could impact on HNSCC metastasis. We found that inhibition of mTOR with rapamycin and the rapalog RAD001 diminished lymphangiogenesis in the primary tumors and prevented the dissemination of HNSCC cancer cells to the cervical lymph nodes, thereby prolonging animal survival. These findings may provide a rationale for the future clinical evaluation of mTOR inhibitors, including rapamycin and its analogs, as part of a molecular-targeted metastasis preventive strategy for the treatment of HNSCC patients.