Background:
Head and Neck Squamous Cell Carcinomas (HNSCC) constitute the eighth most common cancer globally, with the eighth highest mortality rate amongst all cancer types. TP53 is the most frequently mutated gene in HNSCC. TP53 mutant HNSCC displays a genetic aberration in the mammalian target of Rapamycin (mTOR) pathway, which is involved in various oncologic processes such as cell growth, proliferation, and angiogenesis. Compared to wild-type TP53, mutant TP53 HNSCC exhibits poor survival and prognosis, warranting an urgent need for effective therapy. Everolimus is an mTOR inhibitor that has been traditionally used as an immunosuppressant in solid organ transplantation. Recently, Everolimus has been investigated as a potential anticancer agent due to its ability to indirectly inhibit tumor angiogenesis and decrease vascularity, along with directly inhibiting tumor cell growth and proliferation. Additionally, a phase two clinical trial showed that HNSCC patients had a significantly improved 2-year progression-free survival when treated with Everolimus. This study aims to evaluate whether the antitumor activity of Everolimus is related to the modulation of angiogenesis and lymphangiogenesis in TP53 mutant HNSCC xenografts.

Methods: p53 mutant HNSCC cell lines were grown in regular culture conditions and treated with Everolimus (100 ng/ml) for 24 hours. Enzyme-Linked Immunosorbent Assay (ELISA) was used to measure VEGF in the cell culture supernatant quantitatively. TP53 mutant HNSCC cells (HN31 and FaDu) were injected into the flank of athymic nude mice. After five days, mice were treated with an oral suspension of Everolimus (5mg/kg in 1% CMC-Na) for three weeks, while control mice received 1% CMC-Na. Tumor volume and body weight were monitored three times a week. At the end of the experiment, tumors were harvested and analyzed by immunohistochemistry to examine tumor vascularization. Tumor lysates were analyzed by Western blot to determine protein levels of the mTOR, pS6/HIF-1α/VEGF-A signaling.

Results: Treatment with a daily 5mg/kg dose of Everolimus displayed a significant suppression of tumor growth and mTOR signaling as indicated by suppressed S6 phosphorylation. ELISA, Western blot analysis demonstrated that Everolimus downregulated levels of HIF1A and VEGF-A protein expression. Additionally, the immunohistochemical analysis showed that the antitumor activity of Everolimus was associated with anti-angiogenesis and anti-lymphangiogenesis, as indicated by a significantly decreased microvessel density of vascular and lymphatic vessels in HN31 and FaDu xenografts (p<0.05).

Conclusions: Our data suggest that Everolimus prevents TP53 mutant xenograft growth by inhibiting angiogenesis and lymphangiogenesis through downregulation of mTOR/HIF-1α/VEGF-A signaling. Our study suggests a promising role for mTOR inhibitors in the prevention of p53 mutant HNSCC recurrence.