Despite therapeutic advancements, there has been little change in the survival of patients with head and neck squamous cell carcinoma (HNSCC). Recent results suggest that cancer-associated fibroblasts (CAF) drive progression of this disease. Here, we report that autophagy is upregulated in HNSCC-associated CAFs where it is responsible for key pathogenic contributions in this disease. Autophagy is fundamentally involved in cell degradation, but there is emerging evidence that suggests it is also important for cellular secretion. Thus, we hypothesized that autophagy-dependent secretion of tumor-promoting factors by HNSCC-associated CAFs may explain their role in malignant development. In support of this hypothesis, we observed a reduction in CAF-facilitated HNSCC progression after blocking CAF autophagy. Studies of cell growth media conditioned after autophagy blockade revealed levels of secreted IL-6, IL-8 and other cytokines were modulated by autophagy. Notably, when HNSCC cells were co-cultured with normal fibroblasts they upregulated autophagy through IL-6, IL-8 and bFGF. In a mouse xenograft model of HNSCC, pharmacological inhibition of Vps34, a key mediator of autophagy, enhanced the antitumor efficacy of cisplatin. Our results establish an oncogenic function for secretory autophagy in HNSCC stromal cells that promotes malignant progression.

Head and neck squamous cell carcinoma (HNSCC) accounts for 300,000 deaths per year worldwide, and overall survival rates have shown little improvement over the past three decades. Current treatment methods including surgery, chemotherapy, and radiotherapy leave patients with secondary morbidities. Thus, treatment of HNSCC may benefit from exploration of natural compounds as chemopreventive agents. With excellent safety profiles, reduced toxicities, antioxidant properties, and general acceptance for use as dietary supplements, natural compounds are viewed as a desirable area of investigation for chemoprevention. Though most of the field is early in development, numerous studies display the potential utility of natural compounds against HNSCC. These compounds face additional challenges such as low bioavailability for systemic delivery, potential toxicities when consumed in pharmacologic doses, and acquired resistance. However, novel delivery vehicles and synthetic analogues have shown to overcome some of these challenges. This review covers 11 promising natural compounds in the chemoprevention of HNSCC including vitamin A, curcumin, isothiocyanate, green tea, luteolin, resveratrol, genistein, lycopene, bitter melon, withaferin A, and guggulsterone. The review discusses the therapeutic potential and associated challenges of these agents in the chemopreventive efforts against HNSCC. .

As the fifth most common cancer worldwide, head and neck squamous cell carcinoma (HNSCC) is a leading global health burden. Despite therapeutic advancements, the survival rate has remained relatively unchanged for the last fifty years. To improve treatment and survival, there is a need to better understand the underlying biology of this disease. The tumor microenvironment’s role in promoting cancer progression and resistance to therapy has gathered great attention. In HNSCC, the predominant microenvironment cell type is tumor associated fibroblasts (TAFs). Studies in our lab and others have demonstrated the significant tumor promoting role of TAFs; however, little is understood of the underlying biology of TAFs. In this study, we identify that TAFs have upregulated an unconventional pathway of secretion: secretory autophagy. Although paradigmatically a degradation pathway, there has been a growing appreciation for a novel role of autophagy in cellular secretion. We hypothesized HNSCC induces secretory autophagy in the TAFs, modulating secreted factors responsible for tumor progression. We assessed the role of autophagy inhibition in alleviating TAF-facilitated HNSCC progression, and uncovered a significant reduction in proliferation, migration, and invasion of the cancer cells. This was achieved by both siRNA knockdown of Beclin-1, and therapeutic inhibition of the lysosome through chloroquine. We discovered that NFs placed in co-culture with HNSCC had a significantly upregulated level of autophagy marker LC3-II. By characterizing the role of known HNSCC secreted factors in inducing TAF autophagy, we identified basic fibroblast growth factor is responsible for the LC3-II accumulation. To understand which TAF factors are secreted by an autophagy dependent mechanism, we assessed TAF-conditioned media in the presence of autophagy knockdown. Key cytokines, such as IL-6 and IL-8, were identified as autophagy dependent. Rescue of these cytokines in autophagy inhibited TAF-conditioned media restored HNSCC migration. Although autophagy has been identified as upregulated in a variety of cancer types, no one has previously characterized the role of autophagy in HNSCC, and both early pre-clinical and clinical studies of autophagy inhibition as a cancer therapy have been limited by the lack of a specifically targeted inhibitor. We demonstrate a significant decrease in HNSCC progression in preclinical models by using a new and highly specific small molecule inhibitor of autophagy, SAR405, which inhibits Vps34, an upstream autophagy pathway kinase. In summary, we uncover a novel role for secretory autophagy in the tumor microenvironment which promotes tumor progression, and can be uniquely targeted for therapy. Citation Format: Jacob New, Levi Arnold, Megha Ananth, Sameer Alvi, Wen-Xing Ding, Sufi M. Thomas. Secretory autophagy in tumor associated fibroblasts promotes head and neck squamous cell carcinoma progression and emerges as a novel therapeutic target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2977. doi:10.1158/1538-7445.AM2017-2977

<div>Abstract<p>Despite therapeutic advancements, there has been little change in the survival of patients with head and neck squamous cell carcinoma (HNSCC). Recent results suggest that cancer-associated fibroblasts (CAF) drive progression of this disease. Here, we report that autophagy is upregulated in HNSCC-associated CAFs, where it is responsible for key pathogenic contributions in this disease. Autophagy is fundamentally involved in cell degradation, but there is emerging evidence that suggests it is also important for cellular secretion. Thus, we hypothesized that autophagy-dependent secretion of tumor-promoting factors by HNSCC-associated CAFs may explain their role in malignant development. In support of this hypothesis, we observed a reduction in CAF-facilitated HNSCC progression after blocking CAF autophagy. Studies of cell growth media conditioned after autophagy blockade revealed levels of secreted IL6, IL8, and other cytokines were modulated by autophagy. Notably, when HNSCC cells were cocultured with normal fibroblasts, they upregulated autophagy through IL6, IL8, and basic fibroblast growth factor. In a mouse xenograft model of HNSCC, pharmacologic inhibition of Vps34, a key mediator of autophagy, enhanced the antitumor efficacy of cisplatin. Our results establish an oncogenic function for secretory autophagy in HNSCC stromal cells that promotes malignant progression. <i>Cancer Res; 77(23); 6679–91. ©2017 AACR</i>.</p></div>

<p>Examples of Electron Microscopy Autophagosomes</p>

<p>Includes legends for all supplementary figures. S1: CAFs exhibit increased LC3 puncta per cell compared to NF. S2: CAF autophagy inhibition significantly decreases HNSCC in vitro progression. S3: IL-6 and IL-8 induce fibroblast autophagy. S4: bFGF treatment increases autophagy regulated secreted factors. S5: Autophagy inhibition reduces HNSCC in combination therapy, and does not affect CAF proliferation.</p>

<p>Autophagy inhibition reduces HNSCC in combination therapy, and does not affect CAF proliferation</p>

<p>Examples of Electron Microscopy Autophagosomes</p>