The accumulation of sphingosine kinase 2 disrupts the DNA damage response and promotes resistance to genotoxic agents.

There is a known correlation between cancer and changes in sphingolipids, specifically the production of sphingosine-1-phosphate (S1P) by sphingosine kinases 1 and 2 (SPHK1/SPHK2). However, a potential relationship between bioactive lipid molecules, SPHK, and the response to DNA damage is unknown. We aimed to evaluate the response of oral keratinocytes and head and neck cancer cell lines with SPHK2/S1P alterations to DNA damage. This assessment is intended to establish a link between these alterations and tumorigenesis as well as resistance to therapy. SPHK2 overexpression in oral squamous cells promoted evasion of apoptosis and cell cycle control, which increased the resistance to genotoxic agents (chemical and physical). Cells that have SPHK2 overexpression are more prone to DNA damage, which allows those damaged cells to survive and multiply. This is associated with a decrease in overall DNA methylation. These discoveries help to clarify the connection between SPHK2 and the response to DNA damage, as well as its capacity to aid in the resistance against genotoxic agents, including those used in cancer treatment.

SET protein as an epigenetics target.

The SET gene has four transcripts reported in NCBI, coding two isoforms of SET proteins. The most known function of SET protein is inhibiting protein phosphatase 2A, a tumor suppressor, which has been associated with different biological processes. In this review, our focus was on exploring the other SET functions related to epigenetic mechanisms, which impact cellular migration, cell cycle and apoptosis.

Up-regulation of TNF-alpha/NFkB/SIRT1 axis drives aggressiveness and cancer stem cells accumulation in chemoresistant oral squamous cell carcinoma.

Tumor resistance remains an obstacle to successfully treating oral squamous cell carcinoma (OSCC). Cisplatin is widely used as a cytotoxic drug to treat solid tumors, including advanced OSCC, but with low efficacy due to chemoresistance. Therefore, identifying the pathways that contribute to chemoresistance may show new possibilities for improving the treatment. This work explored the role of the tumor necrosis factor-alpha (TNF-alpha)/NFkB signaling in driving the cisplatin resistance of OSCC and its potential as a pharmacological target to overcome chemoresistance. Differential accessibility analysis demonstrated the enrichment of opened chromatin regions in members of the TNF-alpha/NFkB signaling pathway, and RNA-Seq confirmed the upregulation of TNF-alpha/NFkB signaling in cisplatin-resistant cell lines. NFkB was accumulated in cisplatin-resistant cell lines and in cancer stem cells (CSC), and the administration of TNF-alpha increased the CSC, suggesting that TNF-alpha/NFkB signaling is involved in the accumulation of CSC. TNF-alpha stimulation also increased the histone deacetylases HDAC1 and SIRT1. Cisplatin-resistant cell lines were sensitive to the pharmacological inhibition of NFkB, and low doses of the NFkB inhibitors, CBL0137, and emetine, efficiently reduced the CSC and the levels of SIRT1, increasing histone acetylation. The NFkB inhibitors decreased stemness potential, clonogenicity, migration, and invasion of cisplatin-resistant cell lines. The administration of the emetine significantly reduced the tumor growth of cisplatin-resistant xenograft models, decreasing NFkB and SIRT1, increasing histone acetylation, and decreasing CSC. TNF-alpha/NFkB/SIRT1 signaling regulates the epigenetic machinery by modulating histone acetylation, CSC, and aggressiveness of cisplatin-resistant OSCC and the NFkB inhibition is a potential strategy to treat chemoresistant OSCC.

Interplay between EZH2/ß-catenin in stemness of cisplatin-resistant HNSCC and their role as therapeutic targets.

The Wnt/ß-catenin signaling pathway is associated with the regulation of cancer stem cells, and it can be driven by epigenetic modifications. Here, we aim to identify epigenetic modifications involved in the control of the Wnt/ß-catenin signaling and investigate the role of this pathway in the accumulation of cancer stem cells (CSC) and chemoresistance of Head and Neck Squamous Cell Carcinoma (HNSCC). Quantitative-PCR, western blot, shRNA assay, viability assay, flow cytometry assay, spheres formation, xenograft model, and chromatin immunoprecipitation were employed to evaluate the Wnt/ß-catenin pathway and EZH2 in wild-type and chemoresistant oral carcinoma cell lines, and in the populations of CSC and non-stem cells. We demonstrated that ß-catenin and EZH2 were accumulated in cisplatin-resistant and CSC population. The upstream genes of the Wnt/ß-catenin signaling (APC and GSK3ß) were decreased, and the downstream gene MMP7 was increased in the chemoresistant cell lines. The inhibition of ß-catenin and EZH2 combined effectively decreased the CSC population in vitro and reduced the tumor volume and CSC population in vivo. EZH2 inhibition increased APC and GSK3ß, and the Wnt/ß-catenin inhibition reduced MMP7 levels. In contrast, EZH2 overexpression decreased APC and GSK3ß and increased MMP7. EZH2 and ß-catenin inhibitors sensitized chemoresistant cells to cisplatin. EZH2 and H3K27me3 bounded the promoter of APC, leading to its repression. These results suggest that EZH2 regulates ß-catenin by inhibiting the upstream gene APC contributing to the accumulation of cancer stem cells and chemoresistance. Moreover, the pharmacological inhibition of the Wnt/ß-catenin combined with EZH2 can be an effective strategy for treating HNSCC.

Pharmacological inhibition of HDAC6 overcomes cisplatin chemoresistance by targeting cancer stem cells in oral squamous cell carcinoma.

BACKGROUND: Chemoresistance is associated with recurrence and metastasis in oral squamous cell carcinoma (OSCC). The cancer stem cell (CSC) subpopulation is highly resistant to therapy, and they are regulated by epigenetic mechanisms. HDACs are histone deacetylase enzymes that epigenetically regulate gene expression. HDAC6 acts on several physiological processes, including oxidative stress, autophagy and DNA damage response, and its accumulation is associated with cancer. Here, we investigate the role of HDAC6 in CSC-mediated chemoresistance in oral carcinoma in addition to its application as a therapeutic target to reverse chemoresistance. METHODS: Wild-type oral carcinoma cell lines (CAL27 WT and SCC9 WT), cisplatin-resistant (CAL27 CisR and SCC9 CisR), and the subpopulations of cancer stem cells (CSC+) and non-stem (CSC-) derived from CisR cells were investigated. HDAC6 accumulation was analyzed by Western blot and immunofluorescence; DNA damage was evaluated by immunofluorescence of phospho-H2A.X; the qPCR for PRDX2, PRDX6, SOD2, and TXN and ROS assay assessed oxidative stress. Apoptosis and CSC accumulation were investigated by flow cytometry. RESULTS: We identified the accumulation of HDAC6 in CisR cell lines and CSC. Cisplatin-resistant cell lines and CSC demonstrated a reduction in DNA damage and ROS and elevated expression of PRDX2. The administration of tubastatin A (a specific HDAC6 inhibitor) increased oxidative stress and DNA damage and decreased PRDX2. Tubastatin A as a monotherapy induced apoptosis in CisR and CSC and reduced the stemness phenotype. CONCLUSION: High levels of HDAC6 sustain CSC subpopulation and chemoresistance in OSCC, suggesting HDAC6 as a pharmacological target to overcome resistance and perhaps prevent recurrence in OSCC.

Epigenetic modifications control loss of adhesion and aggressiveness of cancer stem cells derived from head and neck squamous cell carcinoma with intrinsic resistance to cisplatin.

OBJECTIVES: The aims of this study were to investigate the epigenetic mechanisms and biological changes implicated in intrinsic and acquired resistance to cisplatin, a chemotherapy commonly used to treat head and neck squamous cell carcinoma. DESIGN: Intrinsic resistance (IR) was established in CAL-27 and acquired resistance (AR) in SCC-9 cell lines. Changes in the phenotype were evaluated by immunofluorescence, colony assay, invasion and spheres formation. Epigenetic regulation were assessed by quantitative PCR and western blot. RESULTS: Changes DNA damage accumulation, and a decrease of reactive oxygen species in cisplatin-resistant cell lines suggest a protection mechanism against cell death. Increases in aggressiveness, observed by clonogenic and invasive potentials, were more pronounced on the CAL-27 IR cell line. Cancer stem cells (CSC) were increased in cisplatin-resistant cells, and the administration of cisplatin increases CSC accumulation in CAL-27 IR. The loss of adhesion was noticed in CSC from IR cells. The upregulation of the genes HDAC2, HDAC9, SIRT1, KAT2B, KAT6A, KAT6B, and BRD4, the HDAC1 nuclear distribution and the decrease of the acetylated proteins H3K9, H3K36, H3K79, and H4K5 indicate that the IR mobilizes epigenetic modifications in acetylation levels, favoring the aggressiveness phenotype. Therefore, the treatment of CSC derived from CAL-27 IR with the histone deacetylase inhibitor, Vorinostat, partially recovered the CSC adhesion ability by up-regulating the levels of FAK, ß3 integrin, and Vinculin proteins. CONCLUSIONS: Our findings indicate that intrinsic-resistant cells are regulated by epigenetic modifications, which could be a potential target to treat resistant head and neck squamous cell carcinoma.