Rogerio M. Castilho

Dysregulated molecular networks in head and neck carcinogenesis

Multiple genetic and epigenetic events, including the aberrant expression and function of molecules regulating cell signaling, growth, survival, motility, angiogenesis, and cell cycle control, underlie the progressive acquisition of a malignant phenotype in squamous carcinomas of the head and neck (HNSCC). In this regard, there has been a recent explosion in our understanding on how extracellular components, cell surface molecules, and a myriad of intracellular proteins and second messenger systems interact with each other, and are organized in pathways and networks to control cellular and tissue functions and cell fate decisions. This emerging ability to understand the basic mechanism controlling inter- and intra-cellular communication has provided an unprecedented opportunity to understand how their dysregulation contributes to the growth and dissemination of human cancers. Here, we will discuss the emerging information on how the use of modern technologies, including gene array and proteomic studies, combined with the molecular dissection of aberrant signaling networks, including the EGFR, ras, NFkappaB, Stat, Wnt/beta-catenin, TGF-beta, and PI3K-AKT-mTOR signaling pathways, can help elucidate the molecular mechanisms underlying HNSCC progression. Ultimately, we can envision that this knowledge may provide tremendous opportunities for the diagnosis of premalignant squamous lesions, and for the development of novel molecular-targeted strategies for the prevention and treatment of HNSCC.

mTOR Mediates Wnt-Induced Epidermal Stem Cell Exhaustion and Aging

Epidermal integrity is a complex process established during embryogenesis and maintained throughout the organism lifespan by epithelial stem cells. Although Wnt regulates normal epithelial stem cell renewal, aberrant Wnt signaling can contribute to cancerous growth. Here, we explored the consequences of persistent expressing Wnt1 in an epidermal compartment that includes the epithelial stem cells. Surprisingly, Wnt caused the rapid growth of the hair follicles, but this was followed by epithelial cell senescence, disappearance of the epidermal stem cell compartment, and progressive hair loss. Although Wnt1 induced the activation of beta-catenin and the mTOR pathway, both hair follicle hyperproliferation and stem cell exhaustion were strictly dependent on mTOR function. These findings suggest that whereas activation of beta-catenin contributes to tumor growth, epithelial stem cells may be endowed with a protective mechanism that results in cell senescence upon the persistent stimulation of proliferative pathways that activate mTOR, ultimately suppressing tumor formation.

Accelerated Wound Healing by mTOR Activation in Genetically Defined Mouse Models

Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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.

Chemoprevention and Treatment of Experimental Cowden's Disease by mTOR Inhibition with Rapamycin

Cowdens disease is an autosomal dominant disorder characterized by the development of multiple mucocutaneous lesions and benign tumors, and enhanced cancer predisposition. Most Cowdens disease patients harbor inactivating mutations in the PTEN tumor suppressor gene which encodes a lipid phosphatase, PTEN, which restrains the phosphatidylinositol 3-kinase-Akt signaling pathway. We observed that the epithelial-specific deletion of Pten in mice causes multiple hyperproliferative and tumor lesions that strikingly resemble Cowdens disease. This animal model system provided an opportunity to explore novel therapeutic approaches in Cowdens disease. Indeed, we show here that rapamycin administration, which inhibits a key downstream target of Akt, mammalian target of rapamycin (mTOR), promotes the rapid regression of advanced mucocutaneous lesions. Furthermore, when administered before disease manifestation, rapamycin can halt the development of Cowdens disease-like lesions, thereby prolonging animal survival. These findings suggest that mTOR inhibition with rapamycin may represent a suitable therapeutic option for the chemoprevention and treatment of Cowden disease patients and others tumor syndromes that involve defective PTEN function.

A role for COX2-derived PGE2 and PGE2-receptor subtypes in head and neck squamous carcinoma cell proliferation

The overexpression of cyclooxygenase (COX)-2 is a frequent event in squamous cell carcinomas of the head and neck (HNSCC), and non-steroidal anti-inflammatory drugs, which are potent inhibitors of COX-1 and COX-2, exert chemopreventive effects on HNSCC cancer development. COX-2 promotes the release of the pro-inflammatory mediator prostaglandin E2 (PGE2), which acts on its cell surface G protein-coupled receptors EP1, EP2, EP3, and EP4. Here, we investigated the role of PGE2 and its receptors in cellular proliferation in HNSCC. The expression of COX-2 and EP1-4 was examined in immortalized oral epithelial cells and in a representative panel of HNSCC cell lines, and based on these data EP1-EP3 and COX-2 expression were evaluated by immunohistochemistry in a large clinical sample collection using HNSCC tissue microarrays. The ability of selective COX-2 inhibition to block PGE2 secretion was measured by ELISA specific assays. The effects of PGE2 on cell proliferation were evaluated using PGE2, its stable analog, and EP2 and EP3-specific synthetic agonists. The results presented here show that HNSCC tumoral lesions and their derived cell lines constitutively express COX-2 and the EP1, EP2 and EP3 receptors for PGE2. HNSCC cells secrete PGE2, which can be suppressed by low concentrations of COX-2 selective inhibitors, without inhibiting cell proliferation. Exogenously added stable PGE2 and EP3-specific agonists induce DNA synthesis in all HNSCC cell lines tested. Overall, our study supports the emerging notion that PGE2 produced in the tumor microenvironment by the overexpression of COX-2 in tumoral and inflammatory cells may promote the growth of HNSCC cells in an autocrine and paracrine fashion by acting on PGE2 receptors that are widely expressed in most HNSCC cancer cells. In particular, our findings suggest that EP3 receptor may play a more prominent role in HNSCC cell growth promotion, thus providing a rationale for the future evaluation of this PGE2 receptor as a target for HNSCC prevention strategies.

Inhibition of Histone Deacetylase Impacts Cancer Stem Cells and Induces Epithelial-Mesenchyme Transition of Head and Neck Cancer

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.

Rac1 Is Required for Epithelial Stem Cell Function during Dermal and Oral Mucosal Wound Healing but Not for Tissue Homeostasis in Mice

Background The 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 Findings Here 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/Significance Together, 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.

Requirement of Rac1 distinguishes follicular from interfollicular epithelial stem cells

Epithelial stem cells in the bulge region within the hair follicle maintain the cyclic hair growth, but whether these stem cells also contribute to the epidermal renewal remains unclear. Here, we observed that the conditional deletion of the Rac1 gene in the mouse skin, including the potential follicular and epidermal stem cell compartments, results in alopecia owing to defective hair development. Surprisingly, mice lacking the expression of this Rho GTPase do not display major alterations in the interfollicular skin. Furthermore, Rac1 excision from primary epithelial keratinocytes results in the inability to reconstitute hair follicles and sebaceous glands when grafted onto mice, but epithelial cells lacking Rac1 can nonetheless form a healthy epidermis. Together, these findings support the emerging view that the epidermis and the hair follicles are maintained by different epithelial stem cells, and provide evidence that the requirement for Rac1 function can distinguish these distinct stem cells populations.

TRIP13 promotes error-prone nonhomologous end joining and induces chemoresistance in head and neck cancer

Head and neck cancer (SCCHN) is a common, aggressive, treatment-resistant cancer with a high recurrence rate and mortality, but the mechanism of treatment-resistance remains unclear. Here we describe a mechanism where the AAA-ATPase TRIP13 promotes treatment-resistance. Overexpression of TRIP13 in non-malignant cells results in malignant transformation. High expression of TRIP13 in SCCHN leads to aggressive, treatment-resistant tumors and enhanced repair of DNA damage. Using mass spectrometry, we identify DNA-PKcs complex proteins that mediate non homologous end joining (NHEJ), as TRIP13 binding partners. Using repair-deficient reporter systems, we show that TRIP13 promotes NHEJ, even when homologous recombination is intact. Importantly, overexpression of TRIP13 sensitizes SCCHN to an inhibitor of DNA-PKcs. Thus, this study defines a new mechanism of treatment resistance in SCCHN and underscores the importance of targeting NHEJ to overcome treatment failure in SCCHN and potentially in other cancers that overexpress TRIP13.

NFκB mediates cisplatin resistance through histone modifications in head and neck squamous cell carcinoma (HNSCC)

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.