Luciana O. Almeida

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.

Periostin Responds to Mechanical Stress and Tension by Activating the MTOR Signaling Pathway

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.

Epigenetic Modifications and Head and Neck Cancer: Implications for Tumor Progression and Resistance to Therapy

Head and neck squamous carcinoma (HNSCC) is the sixth most prevalent cancer and one of the most aggressive malignancies worldwide. Despite continuous efforts to identify molecular markers for early detection, and to develop efficient treatments, the overall survival and prognosis of HNSCC patients remain poor. Accumulated scientific evidences suggest that epigenetic alterations, including DNA methylation, histone covalent modifications, chromatin remodeling and non-coding RNAs, are frequently involved in oral carcinogenesis, tumor progression, and resistance to therapy. Epigenetic alterations occur in an unsystematic manner or as part of the aberrant transcriptional machinery, which promotes selective advantage to the tumor cells. Epigenetic modifications also contribute to cellular plasticity during tumor progression and to the formation of cancer stem cells (CSCs), a small subset of tumor cells with self-renewal ability. CSCs are involved in the development of intrinsic or acquired therapy resistance, and tumor recurrences or relapse. Therefore, the understanding and characterization of epigenetic modifications associated with head and neck carcinogenesis, and the prospective identification of epigenetic markers associated with CSCs, hold the promise for novel therapeutic strategies to fight tumors. In this review, we focus on the current knowledge on epigenetic modifications observed in HNSCC and emerging Epi-drugs capable of sensitizing HNSCC to therapy.

Laser phototherapy triggers the production of reactive oxygen species in oral epithelial cells without inducing DNA damage

Abstract. Laser phototherapy (LPT) is widely used in clinical practice to accelerate healing. Although the use of LPT has advantages, the molecular mechanisms involved in the process of accelerated healing and the safety concerns associated with LPT are still poorly understood. We investigated the physiological effects of LPT irradiation on the production and accumulation of reactive oxygen species (ROS), genomic instability, and deoxyribose nucleic acid (DNA) damage in human epithelial cells. In contrast to a high energy density (20  J/cm2), laser administered at a low energy density (4  J/cm2) resulted in the accumulation of ROS. Interestingly, 4  J/cm2 of LPT did not induce DNA damage, genomic instability, or nuclear influx of the BRCA1 DNA damage repair protein, a known genome protective molecule that actively participates in DNA repair. Our results suggest that administration of low energy densities of LPT induces the accumulation of safe levels of ROS, which may explain the accelerated healing results observed in patients. These findings indicate that epithelial cells have an endowed molecular circuitry that responds to LPT by physiologically inducing accumulation of ROS, which triggers accelerated healing. Importantly, our results suggest that low energy densities of LPT can serve as a safe therapy to accelerate epithelial healing.

Sensitizing mucoepidermoid carcinomas to chemotherapy by targeted disruption of cancer stem cells

Mucoepidermoid carcinoma (MEC) is the most common malignancy of salivary glands. The response of MEC to chemotherapy is unpredictable, and recent advances in cancer biology suggest the involvement of cancer stem cells (CSCs) in tumor progression and chemoresistance and radioresistance phenotype. We found that histone acetyltransferase inhibitors (HDACi) were capable of disrupting CSCs in MEC. Furthermore, administration of HDACi prior to Cisplatin (two-hit approach) disrupts CSCs and sensitizes tumor cells to Cisplatin. Our findings corroborate to emerging evidence that CSCs play a key role in tumor resistance to chemotherapy, and highlights a pharmacological two-hit approach that disrupts tumor resistance to conventional therapy.

Unlocking the chromatin of adenoid cystic carcinomas using HDAC inhibitors sensitize cancer stem cells to cisplatin and induces tumor senescence

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.

PI3K-PTEN dysregulation leads to mTOR-driven upregulation of the core clock gene BMAL1 in normal and malignant epithelial cells.

Dysfunctional clock signaling is observed in a variety of pathological conditions. Many members of the clock gene family are upregulated in tumor cells. Here, we explored the consequences of a commonly disrupted signaling pathway in head and neck cancer on the regulation of circadian clock genes. PTEN is a key molecular controller of the PI3K signaling, and loss of PTEN function is often observed in a variety of cancers. Our main goal was to determine whether PTEN regulates circadian clock signaling. We found that oxidation-driven loss of PTEN function resulted in the activation of mTOR signaling and activation of the core clock protein BMAL1 (also known as ARNTL). The PTEN-induced BMAL1 upregulation was further confirmed using small interference RNA targeting PTEN, and in vivo conditional depletion of PTEN from the epidermis. We observed that PTEN-driven accumulation of BMAL1 was mTOR-mediated and that administration of Rapamycin, a specific mTOR inhibitor, resulted in in vivo rescue of normal levels of BMAL1. Accumulation of BMAL1 by deletion of PER2, a Period family gene, was also rescued upon in vivo administration of mTOR inhibitor. Notably, BMAL1 regulation requires mTOR regulatory protein Raptor and Rictor. These findings indicate that mTORC1 and mTORC2 complex plays a critical role in controlling BMAL1, establishing a connection between PI3K signaling and the regulation of circadian rhythm, ultimately resulting in deregulated BMAL1 in tumor cells with disrupted PI3K signaling.

Profiling the Behavior of Distinct Populations of Head and Neck Cancer Stem Cells.

Cancer stem cells (CSCs) are a subpopulation of tumor cells endowed with self-renewal properties and the capacity to dynamically adapt to physiological changes that occur in the tumor microenvironment. CSCs play a central role in resistance to therapy and long-term disease recurrence. Better characterization and understanding of the available in vitro tools to study the biology of CSCs will improve our knowledge of the processes underlying tumor response to therapy, and will help in the screening and development of novel strategies targeting CSCs. We investigated the behavior of different populations of head and neck CSCs grown under ultra-low adhesion conditions. We found that invasion and adhesion differ among tumorsphere subtypes (holospheres, merospheres and paraspheres), and their tumor cell progeny also harbor distinct self-renewal and clonogenic potentials. Furthermore, holospheres contained higher numbers of head and neck CSCs, as detected by the CD44 cancer stem cell marker and aldehyde dehydrogenase (ALDH) enzymatic activity. In addition, holospheres showed reduced proliferation (Ki67), hypoacetylation of histones, and increased expression of the BMI-1 epithelial stem cell marker, suggesting activation of stem cell programs. Collectively, our results suggest that holospheres enrich a specific population of CSCs with enhanced “stemness” and invasive potential.

Epigenetic Modifications and Accumulation of Dna Double-strand Breaks in Oral Lichen Planus Lesions Presenting Poor Response to Therapy

Abstract Epigenetics refers to changes in cell characteristics that occur independently of modifications to the deoxyribonucleic acid (DNA) sequence. Alterations mediated by epigenetic mechanisms are important factors in cancer progression. Although an exciting prospect, the identification of early epigenetic markers associated with clinical outcome in premalignant and malignant disorders remains elusive. We examined alterations in chromatin acetylation in oral lichen planus (OLP) with distinct clinical behavior and compared the alterations to the levels of DNA double-strand breaks (DSBs). We analyzed 42 OLP patients, who had different responses to therapy, for acetyl-histone H3 at lys9 (H3K9ac), which is associated with enhanced transcription and nuclear decondensation, and the presence of DSBs, as determined by accumulation of phosphorylated &ggr;H2AX foci. Patients with high levels of H3K9ac acetylation failed to respond to therapy or experienced disease recurrence shortly after therapy. Similar to H3K9ac, patients who responded poorly to therapy had increased accumulation of DNA DSB, indicating genomic instability. These findings suggest that histone modifications occur in OLP, and H3K9ac and &ggr;H2AX histones may serve as epigenetic markers for OLP recurrence.

SET oncoprotein accumulation regulates transcription through DNA demethylation and histone hypoacetylation

Epigenetic modifications are essential in the control of normal cellular processes and cancer development. DNA methylation and histone acetylation are major epigenetic modifications involved in gene transcription and abnormal events driving the oncogenic process. SET protein accumulates in many cancer types, including head and neck squamous cell carcinoma (HNSCC); SET is a member of the INHAT complex that inhibits gene transcription associating with histones and preventing their acetylation. We explored how SET protein accumulation impacts on the regulation of gene expression, focusing on DNA methylation and histone acetylation. DNA methylation profile of 24 tumour suppressors evidenced that SET accumulation decreased DNA methylation in association with loss of 5-methylcytidine, formation of 5-hydroxymethylcytosine and increased TET1 levels, indicating an active DNA demethylation mechanism. However, the expression of some suppressor genes was lowered in cells with high SET levels, suggesting that loss of methylation is not the main mechanism modulating gene expression. SET accumulation also downregulated the expression of 32 genes of a panel of 84 transcription factors, and SET directly interacted with chromatin at the promoter of the downregulated genes, decreasing histone acetylation. Gene expression analysis after cell treatment with 5-aza-2′-deoxycytidine (5-AZA) and Trichostatin A (TSA) revealed that histone acetylation reversed transcription repression promoted by SET. These results suggest a new function for SET in the regulation of chromatin dynamics. In addition, TSA diminished both SET protein levels and SET capability to bind to gene promoter, suggesting that administration of epigenetic modifier agents could be efficient to reverse SET phenotype in cancer.