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Abstract Despite major progress in elucidating the pathobiology of head and neck squamous cell carcinoma (HNSCC), the high frequency of disease relapse correlates with unacceptably deficient patient survival. We previously showed that cancer stem-like cells (CSCs) drive tumorigenesis and progression of HNSCC. Although CSCs constitute only 2–5% of total tumor cells, CSCs contribute to tumor progression by virtue of their high tumorigenic potential and their resistance to chemo-, radio-, and immunotherapy. Not only are CSCs resistant to therapy, but cytotoxic agents actually enhance cancer stemness by activating transcription of pluripotency factors and by inducing expression of Bmi-1, a master regulator of stem cell self-renewal. We hypothesized therapeutic inhibition of interleukin-6 receptor (IL-6R) suppresses Bmi-1 to overcome intrinsic chemoresistance of CSCs. We observed that high Bmi-1 expression correlates with decreased (p = 0.04) recurrence-free survival time in HNSCC patients (n = 216). Blockade of IL-6R by lentiviral knockdown or pharmacologic inhibition with a humanized monoclonal antibody (Tocilizumab) is sufficient to inhibit Bmi-1 expression, secondary sphere formation, and to decrease the CSC fraction even in Cisplatin-resistant HNSCC cells. IL-6R inhibition with Tocilizumab abrogates Cisplatin-mediated increase in CSC fraction and induction of Bmi-1 in patient-derived xenograft (PDX) models of HNSCC. Notably, Tocilizumab inhibits Bmi-1 and suppresses growth of xenograft tumors generated with Cisplatin-resistant HNSCC cells. Altogether, these studies demonstrate that therapeutic blockade of IL-6R suppresses Bmi-1 function and inhibits cancer stemness. These results suggest therapeutic inhibition of IL-6R might be a viable strategy to overcome the CSC-mediated chemoresistance typically observed in HNSCC patients.

Abstract Background The skin is the largest organ of the human body. Upon injury, the skin triggers a sequence of signaling pathways that induce epithelial proliferation, migration, and ultimately, the re-establishment of the epithelial barrier. Our study explores the unknown epigenetic regulations of wound healing from a histone perspective. Posttranslational modifications of histones enhance chromatin accessibility and modify gene transcription. Methods Full-thickness wounds were made in the dorsal skin of twenty-four C57/B6 mice (C57BL/6J), followed by the use of ring-shaped silicone splints to prevent wound contraction. Tissue samples were collected at three time points (post-operatory day 1, 4, and 9), and processed for histology. Immunofluorescence was performed in all-time points using markers for histone H4 acetylation at lysines K5, K8, K12, and K16. Results We found well-defined histone modifications associated with the stages of healing. Most exciting, we showed that the epidermis located at a distance from the wound demonstrated changes in histone acetylation, particularly the deacetylation of histone H4K5, H4K8, and H4K16, and hyperacetylation of H4K12. The epidermis adjacent to the wound revealed the deacetylation of H4K5 and H4K8 and hyperacetylation of H4K12. Conversely, the migratory epithelium (epithelial tongue) displayed significant acetylation of H4K5 and H4K12. The H4K5 and H4K8 were decreased in the newly formed epidermis, which continued to display high levels of H4K12 and H4K16. Conclusions This study profiles the changes in histone H4 acetylation in response to injury. In addition to the epigenetic changes found in the healing tissue, these changes also took place in tissues adjacent and distant to the wound. Furthermore, not only deacetylation but also hyperacetylation occurred during tissue repair and regeneration.

Tissue integrity requires constant maintenance of a quiescent, yet responsive, population of stem cells. In the skin, hair follicle stem cells (HFSCs) that reside within the bulge maintain tissue homeostasis in response to activating cues that occur with each new hair cycle or upon injury. We found that PTEN, a major regulator of the PI3K-AKT pathway, controlled HFSC number and size in the bulge and maintained genomically stable pluripotent cells. This regulatory function is central for HFSC quiescence, where PTEN-deficiency phenotype is in part regulated by BMAL1. Furthermore, PTEN ablation led to downregulation of BMI-1, a critical regulator of adult stem cell self-renewal, and elevated senescence, suggesting the presence of a protective system that prevents transformation. We found that short- and long-term PTEN depletion followed by activated BMAL1, a core clock protein, contributed to accumulation of HFSC.

Adenoid cystic carcinoma (ACC) is an uncommon malignancy of the salivary glands that is characterized by local recurrence and distant metastasis due to its resistance to conventional therapy. Platinum-based therapies have been extensively explored as a treatment for ACC, but they show little effectiveness. Studies have shown that a specific group of tumor cells, harboring characteristics of cancer stem cells (CSCs), are involved in chemoresistance of myeloid leukemias, breast, colorectal and pancreatic carcinomas. Therapeutic strategies that target CSCs improve the survival of patients by decreasing the rates of tumor relapse, and epigenetic drugs, such as histone deacetylase inhibitors (HDACi), have shown promising results in targeting CSCs. In this study, we investigated the effect of the HDACi Suberoylanilide hydroxamic acid (Vorinostat), and cisplatin, alone or in combination, on CSCs and non-CSCs from ACC. We used CSCs as a biological marker for tumor resistance to therapy in patient-derived xenograft (PDX) samples and ACC primary cells. We found that cisplatin reduced tumor viability, but enriched the population of CSCs. Systemic administration of Vorinostat reduced the number of detectable CSCs in vivo and in vitro, and a low dose of Vorinostat decreased tumor cell viability. However, the combination of Vorinostat and cisplatin was extremely effective in depleting CSCs and reducing tumor viability in all ACC primary cells by activating cellular senescence. These observations suggest that HDACi and intercalating agents act more efficiently in combination to destroy tumor cells and their stem cells.

A frequent observation in several malignancies is the development of resistance to therapy that results in frequent tumor relapse and metastasis. Much of the tumor resistance phenotype comes from its heterogeneity that halts the ability of therapeutic agents to eliminate all cancer cells effectively. Tumor heterogeneity is, in part, controlled by cancer stem cells (CSC). CSC may be considered the reservoir of cancer cells as they exhibit properties of self-renewal and plasticity and the capability of reestablishing a heterogeneous tumor cell population. The endowed resistance mechanisms of CSC are mainly attributed to several factors including cellular quiescence, accumulation of ABC transporters, disruption of apoptosis, epigenetic reprogramming, and metabolism. There is a current need to develop new therapeutic drugs capable of targeting CSC to overcome tumor resistance. Emerging in vitro and in vivo studies strongly support the potential benefits of combination therapies capable of targeting cancer stem cell-targeting agents. Clinical trials are still underway to address the pharmacokinetics, safety, and efficacy of combination treatment. This review will address the main characteristics, therapeutic implications, and perspectives of targeting CSC to improve current anticancer therapeutics.

Cisplatin-based chemotherapy is the standard treatment of choice for head and neck squamous cell carcinoma (HNSCC). The efficiency of platinum-based therapies is directly influenced by the development of tumor resistance. Multiple signaling pathways have been linked to tumor resistance, including activation of nuclear factor kappa B (NFκB). We explore a novel mechanism by which NFκB drives HNSCC resistance through histone modifications. Post-translational modification of histones alters chromatin structure, facilitating the binding of nuclear factors that mediate DNA repair, transcription, and other processes. We found that chemoresistant HNSCC cells with active NFκB signaling respond to chemotherapy by reducing nuclear BRCA1 levels and by promoting histone deacetylation (chromatin compaction). Activation of this molecular signature resulted in impaired DNA damage repair, prolonged accumulation of histone γH2AX and increased genomic instability. We found that pharmacological induction of histone acetylation using HDAC inhibitors prevented NFκB-induced cisplatin resistance. Furthermore, silencing NFκB in HNSCC induced acetylation of tumor histones, resulting in reduced chemoresistance and increased cytotoxicity following cisplatin treatment. Collectively, these findings suggest that epigenetic modifications of HNSCC resulting from NFκB-induced histone modifications constitute a novel molecular mechanism responsible for chemoresistance in HNSCC. Therefore, targeted inhibition of HDAC may be used as a viable therapeutic strategy for disrupting tumor resistance caused by NFκB.

The development of head and neck squamous cell carcinoma (HNSCC) involves the accumulation of genetic and epigenetic alterations in tumor-suppressor proteins, together with the persistent activation of growth-promoting signaling pathways. The activation of epidermal growth factor receptor (EGFR) is a frequent event in HNSCC. However, EGFR-independent mechanisms also contribute to the activation of key intracellular signaling routes, including signal transducer and activator of transcription-3 (STAT3), nuclear factor κB (NFκB), and Akt. Indeed, the autocrine activation of the gp130 cytokine receptor in HNSCC cells by tumor-released cytokines, such as IL-6, can result in the EGFR-independent activation of STAT3. In this study, we explored the nature of the molecular mechanism underlying enhanced IL-6 secretion in HNSCC cells. We found that HNSCC cells display an increased activity of the IL-6 promoter, which is dependent on the presence of an intact NFκB site. Furthermore, NFκB inhibition downregulated IL-6 gene and protein expression, and decreased the release of multiple cytokines. Interestingly, interfering with NFκB function also prevented the autocrine/paracrine activation of STAT3 in HNSCC cells. These findings demonstrate a cross-talk between the NFκB and the STAT3 signaling systems, and support the emerging notion that HNSCC results from the aberrant activity of a signaling network.

The sequencing of the head and neck cancer has provided a blueprint of the most frequent genetic alterations in this cancer type. They include inactivating mutations in Notch, p53, and p16ink4a tumor suppressor genes, in addition to nonoverlapping activating mutations of the PIK3CA and RAS oncogenes or inactivation of the tumor suppressor gene PTEN. Notably, these genetic alterations, along with epigenetic changes, result in increased activity of phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, which is present in most head and neck squamous cell carcinomas (HNSCCs). Moreover, we show here that approximately 30% of HNSCCs exhibit reduced PTEN expression. We challenged the biologic relevance of this finding by combining the intraoral administration of a tobacco surrogate, 4-nitroquinoline 1-oxide, with a genetically defined animal model displaying reduced PTEN expression, achieved by the conditional deletion of Pten using the keratin promoter 14 CRE-lox system. This provided a specific genetic and environmentally defined animal model for HNSCC that resulted in the rapid development of oral-specific carcinomas. Under these experimental conditions, control mice did not develop HNSCC lesions. In contrast, most mice harboring Pten deficiency developed multiple SCC lesions in the lateral border and ventral part of the tongue and floor of the mouth, which are the preferred anatomic sites for human HNSCC. Overall, our study highlights the likely clinical relevance of reduced PTEN expression and/or inactivation in HNSCC progression, while the combined Pten deletion with exposure to tobacco carcinogens or their surrogates may provide a unique experimental model system to study novel molecular targeted treatments for HNSCC patients.

Current knowledge about Periostin biology has expanded from its recognized functions in embryogenesis and bone metabolism to its roles in tissue repair and remodeling and its clinical implications in cancer. Emerging evidence suggests that Periostin plays a critical role in the mechanism of wound healing; however, the paracrine effect of Periostin in epithelial cell biology is still poorly understood. We found that epithelial cells are capable of producing endogenous Periostin that, unlike mesenchymal cell, cannot be secreted. Epithelial cells responded to Periostin paracrine stimuli by enhancing cellular migration and proliferation and by activating the mTOR signaling pathway. Interestingly, biomechanical stimulation of epithelial cells, which simulates tension forces that occur during initial steps of tissue healing, induced Periostin production and mTOR activation. The molecular association of Periostin and mTOR signaling was further dissected by administering rapamycin, a selective pharmacological inhibitor of mTOR, and by disruption of Raptor and Rictor scaffold proteins implicated in the regulation of mTORC1 and mTORC2 complex assembly. Both strategies resulted in ablation of Periostin-induced mitogenic and migratory activity. These results indicate that Periostin-induced epithelial migration and proliferation requires mTOR signaling. Collectively, our findings identify Periostin as a mechanical stress responsive molecule that is primarily secreted by fibroblasts during wound healing and expressed endogenously in epithelial cells resulting in the control of cellular physiology through a mechanism mediated by the mTOR signaling cascade.

The genome is organized and packed into the nucleus through interactions with core histone proteins. Emerging evidence suggests that tumors are highly responsive to epigenetic alterations that induce chromatin-based events and dynamically influence tumor behavior. We examined chromatin organization in head and neck squamous cell carcinoma (HNSCC) using acetylation levels of histone 3 as a marker of chromatin compaction. Compared to control oral keratinocytes, we found that HNSCC cells are hypoacetylated and that microenvironmental cues (e.g., microvasculature endothelial cells) induce tumor acetylation. Furthermore, we found that chemical inhibition of histone deacetylases (HDAC) reduces the number of cancer stem cells (CSC) and inhibits clonogenic sphere formation. Paradoxically, inhibition of HDAC also induced epithelial-mesenchymal transition (EMT) in HNSCC cells, accumulation of BMI-1, an oncogene associated with tumor aggressiveness, and expression of the vimentin mesenchymal marker. Importantly, we observed co-expression of vimentin and acetylated histone 3 at the invasion front of human HNSCC tumor tissues. Collectively, these findings suggest that environmental cues, such as endothelial cell-secreted factors, modulate tumor plasticity by limiting the population of CSC and inducing EMT. Therefore, inhibition of HDAC may constitute a novel strategy to disrupt the population of CSC in head and neck tumors to create a homogeneous population of cancer cells with biologically defined signatures and predictable behavior.

BackgroundThe regenerative capacity of the skin, including the continuous replacement of exfoliated cells and healing of injuries relies on the epidermal stem cells and their immediate cell descendants. The relative contribution of the hair follicle stem cells and the interfollicular stem cells to dermal wound healing is an area of active investigation. Recent studies have revealed that the small GTPase Rac1, which regulates cell migration and nuclear gene expression, is required for hair follicle stem function but not for the normal homeostasis of the interfollicular skin.Methodology/principal findingsHere we explored whether Rac1 contributes to wound healing in the skin and in the oral mucosa, the latter an anatomical site that presents similar architecture to that of the skin but is devoid of any hair follicle structures, and hence lacks hair follicle stem cells. Epidermal Rac1 gene excision led to the clearly delayed closure of cutaneous wounds. Remarkably, genetic ablation of Rac1 from the oral mucosa resulted in the complete inability of oral wounds to heal. We present evidence that the lack of oral mucosal re-epithelization may result from the reduced migratory capacity of cells lacking Rac1 together with altered expression of injury-induced proliferative and cellular stress-related expression programs.Conclusions/significanceTogether, these observations support that while the normal development and homeostasis of the interfollicular skin and oral mucosa do not require Rac1 function, the interfollicular and oral epithelial stem cells may require a Rac1-dependent program to orchestrate the tissue response to injury and ultimate for wound closure. Ultimately, these findings may enable the molecular characterization of the acute tissue regenerative response of these stem cell populations, thus facilitating the identification of novel molecular-targeted strategies aimed at accelerating wound closure.

The management of slow or non-healing ulcerations constitutes an increasing clinical challenge in the developed world because of the ageing of the population and the pandemic rise in type II diabetes. Recent studies suggest that molecular circuitries deployed by tumor cells to promote cancerous growth may also contribute to tissue regeneration. Here, we exploited this emerging information to search for novel molecular targets to accelerate wound healing.We found that the activation of the PI3K-Akt-mTOR pathway, whose aberrant function is a frequent event in human neoplasia, represents an integral component of the normal wound healing process. By the use of genetically defined approaches, including the epithelial-specific ablation of Pten and Tsc1, we show that mTOR activation can dramatically increase epithelial cell proliferation, migration, and cutaneous wound healing, while pharmacological inhibition of mTOR with rapamycin delays wound closure.Overall, our findings indicate that the transient pharmacologic activation of the PI3K-Akt-mTOR signaling axis may represent a novel clinical intervention strategy to accelerate the healing of debilitating and life-threatening wounds.