Rapalogs induce non‐apoptotic, autophagy‐dependent cell death in HPV‐negative TP53 mutant head and neck squamous cell carcinoma

Abstract

TP53 is the most frequently mutated gene in head and neck squamous cell carcinoma (HNSCC). Patients with HPV-negative TP53 mutant HNSCC have the worst prognosis, necessitating additional agents for treatment. Since mutant p53 causes sustained activation of the PI3K/AKT/mTOR signaling pathway, we investigated the effect of rapalogs RAD001 and CCI-779 on HPV-negative mutTP53 HNSCC cell lines and xenografts. Rapalogs significantly reduced cell viability and colony formation. Interestingly, rapalogs-induced autophagy with no effect on apoptosis.Pretreatment with autophagy inhibitors, 3-methyladenine (3-MA) and ULK-101 rescued the cell viability by inhibiting rapalog-induced autophagy, suggesting that both RAD001 and CCI-779 induce non-apoptotic autophagy-dependent cell death (ADCD). Moreover, rapalogs upregulated the levels of ULK1 and pULK1 S555 with concomitant downregulation of the mTORC1 pathway. However, pretreatment of cells with rapalogs prevented the ULK-101-mediated inhibition of ULK1 to sustained autophagy, suggesting that rapalogs induce ADCD through the activation of ULK1.To further translate our in vitro studies, we investigated the effect of RAD001 in HPV-negative mutTP53 (HN31 and FaDu) tumor cell xenograft model in nude mice.Mice treated with RAD001 exhibited a significant tumor volume reduction without induction of apoptosis, and with a concomitant increase in autophagy. Further, treatment with RAD001 was associated with a considerable increase in pULK1 S555 and ULK1 levels through the inhibition of mTORC1. 3-MA reversed the effect of RAD001 on FaDu tumor growth suggesting that RAD001 promotes ACDC in HPVnegative mutTP53 xenograft. This is the first report demonstrating that rapalogs promote non-apoptotic ADCD in HPV-negative mutTP53 HNSCC via the ULK1 pathway. Further studies are required to establish the promising role of rapalogs in preventing the regrowth of HPV-negative mutTP53 HNSCC.

Everolimus downregulates STAT3/HIF-1α/VEGF pathway to inhibit angiogenesis and lymphangiogenesis in TP53 mutant head and neck squamous cell carcinoma (HNSCC)

“…Treatment with everolimus significantly inhibited the growth of TP53 mutant cell lines and attenuated the growth kinetics of tumor-cell xenografts. In accordance with our previous studies [35], everolimus inhibited mTORC1 activity by downregulating P-mTOR S2448, P-S6 S235/236, and 4EBP1 T70. Moreover, both in cell lines and xenografts, P-STAT3, HIF-1α, VEGF-A and VEGF-C were downregulated with everolimus treatment.…”

“…The intervention of these oncological processes might also halt the progression of TP53 mutant HNSCC, which has not been explored in this current study. Previous work from our lab has shown that everolimus induces autophagy-dependent cell death (ADCD) in TP53 mutant HNSCC through tumor cell-intrinsic mechanisms [ 35 ]. This study demonstrated that everolimus inhibits tumor angiogenesis and lymphangiogenesis through the downregulation of HIF-1α within the tumor microenvironment.…”

“…Anderson Cancer Center, 2) FaDu (R248L) procured from American Type Culture Collection, and 3) UPCI-SCC114 (SCC114) (R248Q), kindly provided by Dr. Susanne Gollin at the University of Pittsburgh. Information on TP53 mutational status and disease background of cell lines are described previously [ 35 ]. These cell lines have Evolutionary Action (EA) scores of more than 75, therefore considered as high risk TP53 mutations [ 45 ].…”

Herbal NF-κB Inhibitors Sensitize Rituximab-Resistant B Lymphoma Cells to Complement-Mediated Cytolysis

“…Curcumin (20 mg/kg body weight) or POH (50 mg/kg body weight) diluted in olive oil was injected intraperitoneally daily on day 8 for a total of 12 days, and rituximab (4 mg/kg body weight) was injected intraperitoneally on day 9 on 3 occasions 4 days apart (Q4D). Tumor size was measured with calipers, and tumor volume was calculated by the following formula: (width) 2 × length/2 ( 13 , 36 ).…”

Targeted therapy for head and neck cancer: signaling pathways and clinical studies

“…37 RAD001 also prevented tumor growth of cells with HPV-negative TP53 mutation in vivo through autophagy activity. 317 The novel mTOR inhibitors CZ415, AZD8055, OSI-027 (ASP4876), and CC-223 monotherapy inhibited HNSCC cell growth in vivo. [318][319][320] mTOR monotherapy and combination with other treatment regimens all demonstrated promising effects on the HNSCC xenograft model.…”