Orai3 Calcium Channel Contributes to Oral/Oropharyngeal Cancer Stemness through the Elevation of ID1 Expression.

Emerging evidence indicates that intracellular calcium (Ca2+) levels and their regulatory proteins play essential roles in normal stem cell proliferation and differentiation. Cancer stem-like cells (CSCs) are subpopulations of cancer cells that retain characteristics similar to stem cells and play an essential role in cancer progression. Recent studies have reported that the Orai3 calcium channel plays an oncogenic role in human cancer. However, its role in CSCs remains underexplored. In this study, we explored the effects of Orai3 in the progression and stemness of oral/oropharyngeal squamous cell carcinoma (OSCC). During the course of OSCC progression, the expression of Orai3 exhibited a stepwise augmentation. Notably, Orai3 was highly enriched in CSC populations of OSCC. Ectopic Orai3 expression in non-tumorigenic immortalized oral epithelial cells increased the intracellular Ca2+ levels, acquiring malignant growth and CSC properties. Conversely, silencing of the endogenous Orai3 in OSCC cells suppressed the CSC phenotype, indicating a pivotal role of Orai3 in CSC regulation. Moreover, Orai3 markedly increased the expression of inhibitor of DNA binding 1 (ID1), a stemness transcription factor. Orai3 and ID1 exhibited elevated expression within CSCs compared to their non-CSC counterparts, implying the functional importance of the Orai3/ID1 axis in CSC regulation. Furthermore, suppression of ID1 abrogated the CSC phenotype in the cell with ectopic Orai3 overexpression and OSCC. Our study reveals that Orai3 is a novel functional CSC regulator in OSCC and further suggests that Orai3 plays an oncogenic role in OSCC by promoting cancer stemness via ID1 upregulation.

Chronic Alcohol Exposure Promotes Cancer Stemness and Glycolysis in Oral/Oropharyngeal Squamous Cell Carcinoma Cell Lines by Activating NFAT Signaling.

Alcohol consumption is associated with an increased risk of several cancers, including oral/oropharyngeal squamous cell carcinoma (OSCC). Alcohol also enhances the progression and aggressiveness of existing cancers; however, its underlying molecular mechanism remains elusive. Especially, the local carcinogenic effects of alcohol on OSCC in closest contact with ingestion of alcohol are poorly understood. We demonstrated that chronic ethanol exposure to OSCC increased cancer stem cell (CSC) populations and their stemness features, including self-renewal capacity, expression of stem cell markers, ALDH activity, and migration ability. The ethanol exposure also led to a significant increase in aerobic glycolysis. Moreover, increased aerobic glycolytic activity was required to support the stemness phenotype of ethanol-exposed OSCC, suggesting a molecular coupling between cancer stemness and metabolic reprogramming. We further demonstrated that chronic ethanol exposure activated NFAT (nuclear factor of activated T cells) signaling in OSCC. Functional studies revealed that pharmacological and genetic inhibition of NFAT suppressed CSC phenotype and aerobic glycolysis in ethanol-exposed OSCC. Collectively, chronic ethanol exposure promotes cancer stemness and aerobic glycolysis via activation of NFAT signaling. Our study provides a novel insight into the roles of cancer stemness and metabolic reprogramming in the molecular mechanism of alcohol-mediated carcinogenesis.

Indigenous microbiota protects development of medication-related osteonecrosis induced by periapical disease in mice.

Bacterial infection is a common finding in patients, who develop medication-related osteonecrosis of the jaw (MRONJ) by the long-term and/or high-dose use of anti-resorptive agents such as bisphosphonate (BPs). However, pathological role of bacteria in MRONJ development at the early stage remains controversial. Here, we demonstrated that commensal microbiota protects against MRONJ development in the pulp-exposed periapical periodontitis mouse model. C57/BL6 female mice were treated with intragastric broad-spectrum antibiotics for 1 week. Zoledronic acid (ZOL) through intravenous injection and antibiotics in drinking water were administered for throughout the experiment. Pulp was exposed on the left maxillary first molar, then the mice were left for 5 weeks after which bilateral maxillary first molar was extracted and mice were left for additional 3 weeks to heal. All mice were harvested, and cecum, maxilla, and femurs were collected. ONJ development was assessed using µCT and histologic analyses. When antibiotic was treated in mice, these mice had no weight changes, but developed significantly enlarged ceca compared to the control group (CTL mice). Periapical bone resorption prior to the tooth extraction was similarly prevented when treated with antibiotics, which was confirmed by decreased osteoclasts and inflammation. ZOL treatment with pulp exposure significantly increased bone necrosis as determined by empty lacunae and necrotic bone amount. Furthermore, antibiotics treatment could further exacerbate bone necrosis, with increased osteoclast number. Our findings suggest that the commensal microbiome may play protective role, rather than pathological role, in the early stages of MRONJ development.

DYRK1A is required for maintenance of cancer stemness, contributing to tumorigenic potential in oral/oropharyngeal squamous cell carcinoma.

DYRK1A, one of the dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs), plays an important role in various biological processes by regulating downstream targets via kinase-dependent and independent mechanisms. Here, we report a novel role of DYRK1A in maintaining tumor growth and stemness of oral/oropharyngeal squamous cell carcinoma (OSCC) cells. Deletion of DYRK1A from OSCC cells abrogated their in vivo tumorigenicity and self-renewal capacity, the key features of cancer stem-like cells (CSCs; also referred to as tumor-initiating cells). The DYRK1A deletion also induced the suppression of CSC populations and properties, such as migration ability and chemoresistance. Conversely, ectopic expression of DYRK1A in OSCC cells augmented their CSC phenotype. Among five DYRK members (DYRK1A, 1B, 2, 3, and 4), DYRK1A is the most dominantly expressed kinase, and its expression is upregulated in OSCC compared to normal oral epithelial cells. More importantly, DYRK1A was highly enriched in various CSC-enriched OSCC populations compared to their corresponding non-CSC populations, indicating its pivotal role in cancer progression and stemness. Further, our study revealed that fibroblast growth factor 2 (FGF2) is a key regulator in the DYRK1A-mediated CSC regulation. Functional studies demonstrated that the loss of DYRK1A inhibits CSC phenotype via reduction of FGF2. Overexpression of DYRK1A promotes CSC phenotype via upregulation of FGF2. Our study delineates a novel mechanism of cancer stemness regulation by DYRK1A-FGF2 axis in OSCC. Thus, inhibition of DYRK1A would lead to a potential novel therapeutic option for targeting CSCs in OSCC.

Zoledronic acid impairs oral cancer stem cells by reducing CCL3.

Cancer stem­like cells (CSCs; also referred to as tumor­initiating cells) play crucial roles in tumor progression and aggressiveness. Recent studies have demonstrated the antitumor activity of zoledronic acid (ZA), a third­generation bisphosphonate, in various types of human cancer. However, its effect on oral CSCs and the underlying mechanism remain obscure. The present study demonstrated that ZA suppresses the growth and stemness properties of oral/oropharyngeal squamous cell carcinoma (OSCC) cells. ZA inhibited the malignant characteristics of OSCC cells, such as anchorage­independent growth and epithelial thickening in organotypic raft cultures. Moreover, ZA treatment resulted in suppression of self­renewal capacity, a key feature of CSCs. ZA also inhibited important CSC properties, such as migration and chemo­radioresistance. Mechanistically, ZA exposure significantly decreased chemokine (C­C motif) ligand 3 (CCL3) expression in OSCC cells. It was further demonstrated that CCL3 signaling via its receptor is crucial for supporting the CSC phenotype in OSCC cells. Moreover, an antagonist of the CCL3 receptor reversed the effect of CCL3 on CSC properties, and exogenous CCL3 rescued the suppressaed CSC phenotype in ZA­treated OSCC cells. These results demonstrated that ZA suppresses the CSC phenotype in OSCC cells by reducing CCL3 expression, suggesting that ZA may be an effective therapeutic agent for oral cancer by targeting CSCs.

Rosuvastatin Prevents the Exacerbation of Atherosclerosis in Ligature-Induced Periodontal Disease Mouse Model.

Periodontitis is a local and systemic inflammatory condition and a risk factor of atherosclerosis, but no studies investigated the effect of a statin on atherogenesis affected by severe periodontitis. In this study, we investigated the effect of rosuvastatin (RSV) on atherogenesis in Apolipoprotein E-deficient mice receiving silk ligature placement around the maxillary second molars. Mice with the ligature placement developed severe periodontitis and vascular inflammation. RSV significantly inhibited the development of periodontitis and vascular inflammation and remarkably blocked the increased lipid deposition and the atherogenic gene expression in the arterial wall and aortic sinus induced by severe periodontitis. To understand the mechanistic effect of RSV on periodontitis-associated atherogenesis, we investigated the in vitro effect of RSV on various effect of TNF-α, a major proinflammatory cytokine for periodontitis and atherogenesis. We found that RSV notably inhibited the TNF-α-induced osteoclast formation, endothelial cell phenotypic changes, foam cell formation, and the expression of CD47 and other oncogenes in arterial smooth muscle cells. Taken together, our study indicates that RSV prevents the exacerbation of atherosclerosis induced periodontitis by inhibiting local, systemic and vascular inflammation, as well as the expression of CD47 from arterial smooth muscle cells in mice.

Genetic and Epigenetic Characterization of Pulpal and Periapical Inflammation.

Pulpal and periapical diseases affect a large segment of the population. The role of microbial infections and host effector molecules in these diseases is well established. However, the interaction between host genes and environmental factors in disease susceptibility and progression is less well understood. Studies of genetic polymorphisms in disease relevant genes have suggested that individual predisposition may contribute to susceptibility to pulpal and periapical diseases. Other studies have explored the contribution of epigenetic mechanisms to these diseases. Ongoing research expands the spectrum of non-coding RNAs in pulpal disease to include viral microRNAs as well. This review summarizes recent advances in the genetic and epigenetic characterization of pulpal and periapical disease, with special emphasis on recent data that address the pathogenesis of irreversible pulpal pathosis and apical periodontitis. Specifically, proinflammatory and anti-inflammatory gene expression and gene polymorphism, as well as recent data on DNA methylation and microRNAs are reviewed. Improved understanding of these mechanisms may aid in disease prevention as well as in improved treatment outcomes.

Proinflammatory cytokine TNFα promotes HPV-associated oral carcinogenesis by increasing cancer stemness.

High-risk human papillomaviruses (HPVs) are involved in the development of several human cancers, including oropharyngeal squamous cell carcinomas. However, many studies have demonstrated that HPV alone is not sufficient for the oncogenic transformation of normal human epithelial cells, indicating that additional cofactors are required for the oncogenic conversion of HPV-infected cells. Inasmuch as chronic inflammation is also closely associated with carcinogenesis, we investigated the effect of chronic exposure to tumor necrosis factor α (TNFα), the major proinflammatory cytokine, on oncogenesis in two immortalized oral keratinocyte cell lines, namely, HPV16-immortalized and human telomerase reverse transcriptase (hTERT)-immortalized cells. TNFα treatment led to the acquisition of malignant growth properties in HPV16-immortalized cells, such as (1) calcium resistance, (2) anchorage independence, and (3) increased cell proliferation in vivo. Moreover, TNFα increased the cancer stem cell-like population and stemness phenotype in HPV16-immortalized cells. However, such transforming effects were not observed in hTERT-immortalized cells, suggesting an HPV-specific role in TNFα-promoted oncogenesis. We also generated hTERT-immortalized cells that express HPV16 E6 and E7. Chronic TNFα exposure successfully induced the malignant growth and stemness phenotype in the E6-expressing cells but not in the control and E7-expressing cells. We further demonstrated that HPV16 E6 played a key role in TNFα-induced cancer stemness via suppression of the stemness-inhibiting microRNAs miR-203 and miR-200c. Overexpression of miR-203 and miR-200c suppressed cancer stemness in TNFα-treated HPV16-immortalized cells. Overall, our study suggests that chronic inflammation promotes cancer stemness in HPV-infected cells, thereby promoting HPV-associated oral carcinogenesis.

Histone Lys demethylase KDM3C demonstrates anti-inflammatory effects by suppressing NF-κB signaling and osteoclastogenesis.

Histone Lys-specific demethylases (KDMs) play a key role in many biological processes through epigenetic mechanisms. However, the role of KDMs in inflammatory responses to oral bacterial infection is poorly understood. Here, we show a novel regulatory role of KDM3C in inflammatory responses to oral bacterial infection. KDM3C expression is transiently suppressed in human and mouse macrophages exposed to LPS from Porphyromonas gingivalis (Pg LPS). Loss of KDM3C in both human and mouse macrophages led to notable induction of proinflammatory cytokines in response to Pg LPS stimulation. Also, KDM3C depletion led to strong induction of p65 phosphorylation and accelerated nuclear translocation in cells exposed to Pg LPS. Kdm3C knockout (KO) in mice led to increased alveolar bone destruction upon induction of experimental periodontitis or pulp exposure compared with those of the wild-type (WT) littermates. The Kdm3C KO mice also revealed an increased number of osteoclasts juxtaposed to the bony lesions. We also confirmed enhanced osteoclastogenesis by bone marrow-derived macrophages isolated from the Kdm3C KO compared with the WT controls. These findings suggest an anti-inflammatory function of KDM3C in regulating the inflammatory responses against oral bacterial infection through suppression of NF-κB signaling and osteoclastogenesis.-Lee, J. Y., Mehrazarin, S., Alshaikh, A., Kim, S., Chen, W., Lux, R., Gwack, Y., Kim, R. H., Kang, M. K. Histone Lys demethylase KDM3C demonstrates anti-inflammatory effects by suppressing NF-κB signaling and osteoclastogenesis.

NFATc3 plays an oncogenic role in oral/oropharyngeal squamous cell carcinomas by promoting cancer stemness via expression of OCT4.

Nuclear factor of activated T cells (NFATc1-c4), a family of transcription factors, is involved in many biological processes by regulating various downstream target genes. However, their role in cancer progression remains controversial. We here report that NFATc3 is the dominant isoform of NFAT in human oral epithelial cells, and its expression was increased in a stepwise manner during the progression of oral/oropharyngeal squamous cell carcinoma (OSCC). More importantly, NFATc3 was highly enriched in self-renewing cancer stem-like cells (CSCs) of OSCC. Increased expression of NFATc3 was required for the maintenance of CSC self-renewal, as NFATc3 inhibition suppressed tumor sphere formation in OSCC cells. Conversely, ectopic NFATc3 expression in non-tumorigenic immortalized oral epithelial cells resulted in the acquisition of self-renewal and increase in CSC phenotype, such as enhanced ALDH1HIGH cell population, mobility and drug resistance, indicating the functional role of NFATc3 in the maintenance of CSC phenotype. NFATc3 expression also converted the non-tumorigenic oral epithelial cells to malignant phenotypes. Mechanistic investigations further reveal that NFATc3 binds to the promoter of OCT4, a stemness transcription factor, for its activation, thereby promoting CSC phenotype. Moreover, suppression of OCT4 abrogated CSC phenotype in the cell with ectopic NFATc3 overexpression and OSCC, and ectopic OCT4 expression sufficiently induced CSC phenotype. Our study indicates that NFATc3 plays an important role in the maintenance of cancer stemness and OSCC progression via novel NFATc3-OCT4 axis, suggesting that this axis may be a potential therapeutic target for OSCC CSCs.