In this issue of Cell Stem Cell, Jia et al. (2020) identify residual cancer stem cells (CSCs) as a mechanism of immunotherapy resistance in head and neck squamous cell carcinoma (HNSCC). Remarkably, targeting this population of CSCs can be exploited to potentiate immunotherapy and reduce tumor recurrence and metastasis.

G-protein coupled receptors (GPCRs) and their associated heterotrimeric G proteins impinge on pathways that control epithelial cell self-renewal and differentiation. Although it is known that Gαs protein signaling regulates skin homeostasis in vivo, the role of GPCR-coupled Gαi proteins in the skin is unclear. Here, by using a chemogenetic approach, we demonstrate that GPCR-Gαi activation can regulate keratinocyte proliferation and differentiation and that overactivation of Gαi-signaling in the basal compartment of the mouse skin can lead to epidermal hyperplasia. Our results expand our understanding of the role of GPCR-cAMP signaling in skin homeostasis and reveal overlapping and divergent roles of the cAMP-regulating heterotrimeric Gαs and Gαi proteins in keratinocytes.

BACKGROUND: Cetuximab, a monoclonal blocking antibody against the epidermal growth factor receptor EGFR, has been approved for the treatment of squamous cell carcinomas of the head and neck (HNSCC). However, only few patients display long-term responses, prompting the search for cetuximab resistance mechanisms and new therapeutic options enhancing cetuximab effectiveness. METHODS: Cetuximab-sensitive HNSCC cells were retro-engineered to express PIK3CA and RAS oncogenes. These cells and HNSCC cells harboring endogenous PIK3CA and RAS oncogenes were xenografted into mice (n = 10 per group) and studied for their biochemical, antitumor, antiangiogenic, and antilymphangiogenic responses to cetuximab and mTOR targeting agents. All P values are two-sided. RESULTS: Cetuximab treatment of PIK3CA- and RAS-expressing HNSCC xenografts promoted an initial antitumor response, but all tumors relapsed within few weeks. In these tumors, cetuximab did not decrease the activity of mTOR, a downstream signaling target of EGFR, PIK3CA, and RAS. The combined administration of cetuximab and mTOR inhibitors exerted a remarkably increased antitumor activity, particularly in HNSCC cells that are resistant to cetuximab as a single agent. Indeed, cotargeting mTOR together with cetuximab caused a rapid tumor collapse of both PIK3CA- and RAS-expressing HNSCC xenografts (P < .001), concomitant with reduced proliferation (P < .001) and lymphangiogenesis (P < .001). CONCLUSION: The presence of PIK3CA and RAS mutations and other alterations affecting the mTOR pathway activity in HNSCC could be exploited to predict the potential resistance to cetuximab, and to select the patients that may benefit the most from the concomitant administration of cetuximab and PI3K and/or mTOR inhibitors as a precision molecular therapeutic option for HNSCC patients.

Radiotherapy is commonly used in patients with oral cavity and pharyngeal cancers, usually resulting in irreversible salivary hypofunction. Currently management of radiation damage to salivary glands still remains a great challenge. Recent studies show that activation of mammalian target of rapamycin (mTOR) occurs in salivary gland lesions, making it possible to apply mTOR inhibitor for treatment. Our results indicate inhibition of mTOR by rapamycin significantly alleviated irradiation-induced salivary hypofunction by restoring 46% salivary flow rate and protecting histological structures in swine. Furthermore, rapamycin protected human submandibular gland cell line (HSG) from irradiation-induced cell depletion and loss of cell proliferation capacity. These findings lay the foundation for a new clinical application of rapamycin to prevent irradiation-induced salivary hypofunction.

The rising incidence of human papillomavirus (HPV)-associated malignancies, especially for oropharyngeal cancers, has highlighted the urgent need to understand how the interplay between high-risk HPV oncogenes and carcinogenic exposure results in squamous cell carcinoma (SCC) development. Here, we describe an inducible mouse model expressing high risk HPV-16 E6/E7 oncoproteins in adults, bypassing the impact of these viral genes during development. HPV-16 E6/E7 genes were targeted to the basal squamous epithelia in transgenic mice using a doxycycline inducible cytokeratin 5 promoter (cK5-rtTA) system. After doxycycline induction, both E6 and E7 were highly expressed, resulting in rapid epidermal hyperplasia with a remarkable expansion of the proliferative cell compartment to the suprabasal layers. Surprisingly, in spite of the massive growth of epithelial cells and their stem cell progenitors, HPV-E6/E7 expression was not sufficient to trigger mTOR activation, a key oncogenic driver in HPV-associated malignancies, and malignant progression to SCC. However, these mice develop SCC rapidly after a single exposure to a skin carcinogen, DMBA, which was increased by the prolonged exposure to a tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Thus, only few oncogenic hits may be sufficient to induce cancer in E6/E7 expressing cells. All HPV-E6/E7 expressing SCC lesions exhibited increased mTOR activation. Remarkably, rapamycin, an mTOR inhibitor, abolished tumor development when administered to HPV-E6/E7 mice prior to DMBA exposure. Our findings revealed that mTOR inhibition protects HPV-E6/E7 expressing tissues form SCC development upon carcinogen exposure, thus supporting the potential clinical use of mTOR inhibitors as a molecular targeted approach for prevention of HPV-associated malignancies.

Radiotherapy is commonly used in patients with oral cavity and pharyngeal cancers, usually resulting in irreversible salivary hypofunction. Currently management of radiation damage to salivary glands still remains a great challenge. Recent studies show that activation of mammalian target of rapamycin (mTOR) occurs in salivary gland lesions, making it possible to apply mTOR inhibitor for treatment. Our results indicate inhibition of mTOR by rapamycin significantly alleviated irradiation-induced salivary hypofunction by restoring 46% salivary flow rate and protecting histological structures in swine. Furthermore, rapamycin protected human submandibular gland cell line (HSG) from irradiation-induced cell depletion and loss of cell proliferation capacity. These findings lay the foundation for a new clinical application of rapamycin to prevent irradiation-induced salivary hypofunction.

Mutually exclusive activating mutations in the GNAQ and GNA11 oncogenes, encoding heterotrimeric Gαq family members, have been identified in ∼ 83% and ∼ 6% of uveal and skin melanomas, respectively. However, the molecular events underlying these GNAQ-driven malignancies are not yet defined, thus limiting the ability to develop cancer-targeted therapies. Here, we focused on the transcriptional coactivator YAP, a critical component of the Hippo signaling pathway that controls organ size. We found that Gαq stimulates YAP through a Trio-Rho/Rac signaling circuitry promoting actin polymerization, independently of phospholipase Cβ and the canonical Hippo pathway. Furthermore, we show that Gαq promotes the YAP-dependent growth of uveal melanoma cells, thereby identifying YAP as a suitable therapeutic target in uveal melanoma, a GNAQ/GNA11-initiated human malignancy.

Genomic alterations in GNAS, the gene coding for the Gαs heterotrimeric G protein, are associated with a large number of human diseases. Here, we explored the role of Gαs on stem cell fate decisions by using the mouse epidermis as a model system. Conditional epidermal deletion of Gnas or repression of PKA signalling caused a remarkable expansion of the stem cell compartment, resulting in rapid basal-cell carcinoma formation. In contrast, inducible expression of active Gαs in the epidermis caused hair follicle stem cell exhaustion and hair loss. Mechanistically, we found that Gαs-PKA disruption promotes the cell autonomous Sonic Hedgehog pathway stimulation and Hippo signalling inhibition, resulting in the non-canonical activation of GLI and YAP1. Our study highlights an important tumour suppressive function of Gαs-PKA, limiting the proliferation of epithelial stem cells and maintaining proper hair follicle homeostasis. These findings could have broad implications in multiple pathophysiological conditions, including cancer.

mTOR inhibition has emerged as a promising strategy for head and neck squamous cell carcinomas (HNSCC) treatment. However, most targeted therapies ultimately develop resistance due to the activation of adaptive survival signaling mechanisms limiting the activity of targeted agents. Thus, co-targeting key adaptive mechanisms may enable more effective cancer cell killing. Here, we performed a synthetic lethality screen using shRNA libraries to identify druggable candidates for combinatorial signal inhibition. We found that the ERK pathway was the most highly represented. Combination of rapamycin with trametinib, a MEK1/2 inhibitor, demonstrated strong synergism in HNSCC-derived cells in vitro and in vivo, including HNSCC cells expressing the HRAS and PIK3CA oncogenes. Interestingly, cleaved caspase-3 was potently induced by the combination therapy in PIK3CA+ cells in vitro and tumor xenografts. Moreover, ectopic expression of PIK3CA mutations into PIK3CA- HNSCC cells sensitized them to the pro-apoptotic activity of the combination therapy. These findings indicate that co-targeting the mTOR/ERK pathways may provide a suitable precision strategy for HNSCC treatment. Moreover, PIK3CA+ HNSCC are particularly prone to undergo apoptosis after mTOR and ERK inhibition, thereby providing a potential biomarker of predictive value for the selection of patients that may benefit from this combination therapy.

Oral mucosal wound healing has long been regarded as an ideal system of wound resolution. However, the intrinsic characteristics that mediate optimal healing at mucosal surfaces are poorly understood, particularly in humans. We present a unique comparative analysis between human oral and cutaneous wound healing using paired and sequential biopsies during the repair process. Using molecular profiling, we determined that wound-activated transcriptional networks are present at basal state in the oral mucosa, priming the epithelium for wound repair. We show that oral mucosal wound-related networks control epithelial cell differentiation and regulate inflammatory responses, highlighting fundamental global mechanisms of repair and inflammatory responses in humans. The paired comparative analysis allowed for the identification of differentially expressed SOX2 (sex-determining region Y-box 2) and PITX1 (paired-like homeodomain 1) transcriptional regulators in oral versus skin keratinocytes, conferring a unique identity to oral keratinocytes. We show that SOX2 and PITX1 transcriptional function has the potential to reprogram skin keratinocytes to increase cell migration and improve wound resolution in vivo. Our data provide insights into therapeutic targeting of chronic and nonhealing wounds based on greater understanding of the biology of healing in human mucosal and cutaneous environments.