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Supplementary Figure Legends from Heterogeneous Nuclear Ribonucleoprotein G Shows Tumor Suppressive Effect against Oral Squamous Cell Carcinoma Cells

Purpose: Heterogeneous nuclear ribonucleoproteins (hnRNP) are nucleic acid binding proteins involved in RNA processing. We found that hnRNP G is expressed in normal human oral epithelial cells while frequently not found in the cells derived from human oral squamous cell carcinomas (HOSCC). The current study was designed to test the hypothesis that hnRNP G is a tumor suppressor.Experimental Design: We investigated the expression levels of hnRNP G protein in normal, precancerous, and malignant oral tissues by in situ immunohistochemistry. In addition, wild-type or mutant hnRNP G was ectopically overexpressed in HOSCC cells and their effects on cellular replication kinetics, colonogenic efficiency, anchorage-independent growth, and in vivo tumorigenicity were determined.Results:In situ immunohistochemical staining showed robust presence of hnRNP G in the basal cell layers of normal oral epithelium but the level of its staining was markedly reduced in dysplastic or cancerous tissues. Ectopic expression of wild-type hnRNP G in cancer cells lacking hnRNP G expression or containing mutant hnRNP G resulted in severe retardation of proliferation, reduction of colonogenic efficiency, loss of anchorage-independent growth, and reduction of in vivo tumorigenicity in immunocompromised mice. In addition, hnRNP G overexpression led to up-regulation of the expression of TXNIP, a cell cycle inhibitory gene, and significantly reduced the expression of the genes that promote cellular proliferation, such as EGR1, JUND, JUNB, FOS, FOSL1, ROS, and KIT.Conclusions: These results indicate that hnRNP G is a tumor suppressor against HOSCC but its mechanisms of action remain to be further investigated.

Supplementary Figure S1 from Heterogeneous Nuclear Ribonucleoprotein G Shows Tumor Suppressive Effect against Oral Squamous Cell Carcinoma Cells

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.

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.

Cancer stem‑like cells (CSCs; also referred to as tumor‑initiating cells) play crucial roles in tumor progression and aggressiveness. Recent studies have demonstrated the antitumor activity of zoledronic acid (ZA), a third‑generation 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 anchorage‑independent growth and epithelial thickening in organotypic raft cultures. Moreover, ZA treatment resulted in suppression of self‑renewal capacity, a key feature of CSCs. ZA also inhibited important CSC properties, such as migration and chemo‑radioresistance. Mechanistically, ZA exposure significantly decreased chemokine (C‑C 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 ZA‑treated 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.

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.

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.

Alcohol (ethanol, EtOH) consumption during pregnancy can result in fetal alcohol spectrum disorders (FASDs), which are characterized by prenatal and postnatal growth restriction and craniofacial dysmorphology. Recently, cell-derived extracellular vesicles, including exosomes and microvesicles containing several species of RNAs (exRNAs), have emerged as a mechanism of cell-to-cell communication. However, EtOH’s effects on the biogenesis and function of non-coding exRNAs during fetal development have not been explored. Therefore, we studied the effects of maternal EtOH exposure on the composition of exosomal RNAs in the amniotic fluid (AF) using rat fetal alcohol exposure (FAE) model. Through RNA-Seq analysis we identified and verified AF exosomal miRNAs with differential expression levels specifically associated with maternal EtOH exposure. Uptake of purified FAE AF exosomes by rBMSCs resulted in significant alteration of molecular markers associated with osteogenic differentiation of rBMSCs. We also determined putative functional roles for AF exosomal miRNAs (miR-199a-3p, miR-214-3p and let-7g) that are dysregulated by FAE in osteogenic differentiation of rBMSCs. Our results demonstrate that FAE alters AF exosomal miRNAs and that exosomal transfer of dysregulated miRNAs has significant molecular effects on stem cell regulation and differentiation. Our results further suggest the usefulness of assessing molecular alterations in AF exRNAs to study the mechanisms of FAE teratogenesis that should be further investigated by using an in vivo model.

Medication-related osteonecrosis of the jaw (MRONJ) is a rare but detrimental intraoral lesion that predominantly occurs in patients with long-term use of antiresorptive agents, such as bisphosphonate and denosumab, a human anti–receptor activator of NF-κB ligand (RANKL) monoclonal antibody (Ab). Surgical intervention, such as tooth extraction, is a known risk factor for MRONJ, which is often performed to eliminate preexiting pathologic inflammatory conditions, such as periodontal diseases. Nonetheless, it remains unknown whether pre-existing periodontal disease condition exacerbates, or removal of such condition ameliorates, MRONJ development after tooth extraction. In this study, we combined the ligature-induced periodontitis and the tooth extraction mouse models under the administration of zoledronic acid (ZOL) or anti-RANKL Ab, and provide experimental evidence that a pre-existing pathologic inflammatory condition exacerbates MRONJ development after tooth extraction in mice. Under ZOL administration, tooth extraction alone induced ONJ lesions; however, extraction of a ligature-placed tooth further exacerbated ONJ development. When the ligature was removed and the inflammatory condition was deescalated, ONJ development was ameliorated. Anti-RANKL Ab administration resulted in similar outcomes. Interestingly, unlike ZOL-administered mice, anti-RANKL Ab–administered mice exhibited complete absence of osteoclasts, suggesting that physical presence of osteoclasts is not directly involved in ONJ development. Collectively, our study demonstrated that periodontal disease is a functionally linked risk factor that predisposes ONJ development after tooth extraction in the presence of bisphosphonate and denosumab.

GV1001 is a 16‑amino acid peptide derived from the human telomerase reverse transcriptase (hTERT) protein (616‑626; EARPALLTSRLRFIPK), which lies within the reverse transcriptase domain. Originally developed as an anticancer vaccine, GV1001 demonstrates diverse cellular effects, including anti‑inflammatory, tumor suppressive and antiviral effects. In the present study, the radioprotective and antifibrotic effects of GV1001 were demonstrated through suppressing transforming growth factor‑β (TGF‑β) signaling. Proliferating human keratinocytes underwent premature senescence upon exposure to ionizing radiation (IR), however, treatment of cells with GV1001 allowed the cells to proliferate and showed a reduction in senescent phenotype. GV1001 treatment notably increased the levels of Grainyhead‑like 2 and phosphorylated (p‑)Akt (Ser473), and reduced the activation of p53 and the level of p21/WAF1 in irradiated keratinocytes. It also markedly suppressed the level of TGF‑β signaling molecules, including p‑small mothers against decapentaplegic (Smad)2/3 and Smad4, and TGF‑β target genes, including zinc finger E‑box binding homeobox 1, fibronectin, N‑cadharin and Snail, in irradiated keratinocytes. Furthermore, GV1001 suppressed TGF‑β signaling in primary human fibroblasts and inhibited myofibroblast differentiation. Chromatin immunoprecipitation revealed that GV1001 suppressed the binding of Smad2 on the promoter regions of collagen type III α1 chain (Col3a1) and Col1a1. In a dermal fibrosis model in vivo, GV1001 treatment notably reduced the thickness of fibrotic lesions and the synthesis of Col3a1. These data indicated that GV1001 ameliorated the IR‑induced senescence phenotype and tissue fibrosis by inhibiting TGF‑β signaling and may have therapeutic effects on radiation‑induced tissue damage.

Cancer stem cells (CSCs; also known as tumor-initiating cells) are a small population of cancer cells that retain characteristics similar to those of normal stem cells. CSCs are known to be responsible for metastasis, drug resistance, and cancer recurrence. Thus, controlling CSCs may provide an effective therapeutic intervention that inhibits tumor growth and aggressiveness. Despite the importance of targeting CSCs in cancer therapy, the detailed nature of oral CSCs remains underexplored. This article reviews the current understanding of oral CSCs, with emphasis on recent advances in novel signaling pathways involved in their stemness regulation.

Oral mucosa provides the first line of defense against a diverse array of environmental and microbial irritants by forming the barrier of epithelial cells interconnected by multiprotein tight junctions (TJ), adherens junctions, desmosomes, and gap junction complexes. Grainyhead-like 2 (GRHL2), an epithelial-specific transcription factor, may play a role in the formation of the mucosal epithelial barrier, as it regulates the expression of the junction proteins. The current study investigated the role of GRHL2 in the Porphyromonas gingivalis ( Pg)–induced impairment of epithelial barrier functions. Exposure of human oral keratinocytes (HOK-16B and OKF6 cells) to Pg or Pg-derived lipopolysaccharides ( Pg LPSs) led to rapid loss of endogenous GRHL2 and the junction proteins (e.g., zonula occludens, E-cadherin, claudins, and occludin). GRHL2 directly regulated the expression levels of the junction proteins and the epithelial permeability for small molecules (e.g., dextrans and Pg bacteria). To explore the functional role of GRHL2 in oral mucosal barrier, we used a Grhl2 conditional knockout (KO) mouse model, which allows for epithelial tissue-specific Grhl2 KO in an inducible manner. Grhl2 KO impaired the expression of the junction proteins at the junctional epithelium and increased the alveolar bone loss in the ligature-induced periodontitis model. Fluorescence in situ hybridization revealed increased epithelial penetration of oral bacteria in Grhl2 KO mice compared with the wild-type mice. Also, blood loadings of oral bacteria (e.g., Bacteroides, Bacillus, Firmicutes, β- proteobacteria, and Spirochetes) were significantly elevated in Grhl2 KO mice compared to the wild-type littermates. These data indicate that Pg bacteria may enhance paracellular penetration through oral mucosa in part by targeting the expression of GRHL2 in the oral epithelial cells, which then impairs the epithelial barrier by inhibition of junction protein expression, resulting in increased alveolar tissue destruction and systemic bacteremia.

Introduction Surgical interventions such as tooth extraction increase the chances of developing osteonecrosis of the jaw in patients receiving bisphosphonates (BPs) for the treatment of bone-related diseases. Tooth extraction is often performed to eliminate preexisting pathological inflammatory conditions that make the tooth unsalvageable; however, the role of such conditions on bisphosphonate-related osteonecrosis of the jaw (BRONJ) development after tooth extraction is not clearly defined. Here, we examined the effects of periapical periodontitis on tooth extraction–induced BRONJ development in mice. Methods Periapical periodontitis was induced by exposing the pulp of the maxillary first molar for 3 weeks in C57/BL6 mice that were intravenously administered with BPs. The same tooth was extracted, and after an 3 additional weeks, the mice were harvested for histologic, histomorphometric, and histochemical staining analyses. Results Pulp exposure induced periapical radiolucency as shown by increased inflammatory cells, tartrate-resistant acid phosphatase–positive osteoclasts, and bone resorption. When BPs were administered, pulp exposure did not induce apical bone resorption despite the presence of inflammatory cells and tartrate-resistant acid phosphatase–positive osteoclasts. Although tooth extraction alone induced BRONJ lesions, pulp exposure further increased tooth extraction-induced BRONJ development as shown by the presence of more bone necrosis. Conclusions Our study demonstrates that a preexisting pathological inflammatory condition such as periapical periodontitis is a predisposing factor that may exacerbate BRONJ development after tooth extraction. Our study further provides a clinical implication wherein periapical periodontitis should be controlled before performing tooth extraction in BP users in order to reduce the risk of developing BRONJ.

Long-term administration of nitrogen-containing bisphosphonates can induce detrimental side effects such as bisphosphonate-related osteonecrosis of the jaw (BRONJ) in human. Although inflammation is known to be associated with BRONJ development, the detailed underlying mechanism remains unknown. Here, we report that the pro-inflammatory cytokine IL-36α is, in part, responsible for the BRONJ development. We found a notably higher level of IL-36α and lower level of collagen in the BRONJ lesions in mice. We also found that IL-36α remarkably suppressed TGF-β-mediated expression of Collα1 and α-Sma via the activation of Erk signaling pathway in mouse gingival mesenchymal stem cells. When IL-36 signaling was abrogated in vivo, development of BRONJ lesions was ameliorated in mice. Taken together, we showed the pathologic role of IL-36α in BRONJ development by inhibiting collagen expression and demonstrated that IL-36α could be a potential marker and a therapeutic target for the prevention and treatment of BRONJ. © 2016 American Society for Bone and Mineral Research.

Emerging evidence indicates that Orai1, a key calcium channel for store-operated Ca2+ entry, is associated with human cancer. However, the underlying mechanism by which Orai1 regulates cancer progression remains unknown. Here we report that intracellular level of Orai1 is increased in a stepwise manner during oral/oropharyngeal carcinogenesis and highly expressed in cancer stem-like cell (CSC)-enriched populations of human oral/oropharyngeal squamous cell carcinoma (OSCC). Ectopic Orai1 expression converted non-tumorigenic immortalized oral epithelial cells to malignant cells that showed CSC properties, e.g., self-renewal capacity, increased ALDH1HIGH cell population, increased key stemness transcription factors, and enhanced mobility. Conversely, inhibition of Orai1 suppressed tumorigenicity and CSC phenotype of OSCC, indicating that Orai1 could be an important element for tumorigenicity and stemness of OSCC. Mechanistically, Orai1 activates its major downstream effector molecule, NFATc3. Knockdown of NFATc3 in the Orai1-overexpressing oral epithelial cells abrogates the effect of Orai1 on CSC phenotype. Moreover, antagonist of NFAT signaling also decreases CSC phenotype, implying the functional importance of Orai1/NFAT axis in OSCC CSC regulation. Our study identifies Orai1 as a novel molecular determinant for OSCC progression by enhancing cancer stemness, suggesting that inhibition of Orai1 signaling may offer an effective therapeutic modality against OSCC.

Orai1 is a pore-subunit of store-operated Ca(2+) release-activated Ca(2+) (CRAC) channel that mediates Ca(2+) influx in most non-excitable cells via store-operated Ca(2+) entry (SOCE) mechanism. We previously demonstrated that Orai1 is involved in mediating osteogenic potential of mesenchymal stem cells (MSCs), but the underlying mechanism of this function remains unknown. Here, we report that Orai1 mediates osteogenic differentiation via bone morphogenic protein (BMP) signaling pathway in bone marrow MSCs (BMSCs). In osteogenic conditions, BMSCs derived from wild-type mice underwent osteoblastic differentiation and induced mineralization as demonstrated by increased alkaline phosphatase activity and alizarin red S staining, respectively. The expression of Runx2, a master regulator of osteoblast differentiation, and osteogenic differentiation markers were markedly increased in wild-type BMSCs under osteogenic conditions. In contrast, osteogenic conditions failed to induce such effects in BMSCs derived from Orai1-deficient (Orai1(-/-)) mice, indicating that Orai1 is, in part, necessary for osteogenic differentiation of MSCs. We also found that BMP2 successfully induced phosphorylation of Smad1/5/8, the immediate effector molecules of BMP signaling, in wild-type BMSCs, but failed to do so in Orai1(-/-) BMSCs. Downstream target genes of BMP signaling pathway were consistently increased by osteogenic conditions in wild-type BMSCs, but not in Orai1(-/-) BMSCs, suggesting a novel molecular link between Orai1 and BMP signaling pathway in the osteogenic differentiation process. Further functional studies demonstrated that activation of BMP signaling rescues osteogenic differentiation capacity of Orai1(-/-) BMSCs. In conclusion, Orai1 regulates osteogenic differentiation through BMP signaling, and the Orai1-BMP signaling may be a possible therapeutic target for treating bone-related diseases.

Grainyhead-like 2 (GRHL2) is one of the three mammalian homologues of Drosophila Grainyhead involved in epithelial morphogenesis. We recently showed that GRHL2 also controls normal epithelial cell proliferation and differentiation. In this study, we investigated the role of GRHL2 in oral carcinogenesis and the underlying mechanism. GRHL2 expression was elevated in cells and tissues of oral squamous cell carcinomas (OSCCs) compared with normal counterparts. Knockdown of GRHL2 resulted in the loss of in vivo tumorigenicity, cancer stemness and epithelial phenotype of oral cancer cells. GRHL2 loss also inhibited oral cancer cell proliferation and colony formation. GRHL2 regulated the expression of miR-200 family and Octamer-binding transcription factor 4 (Oct-4) genes through direct promoter DNA binding. Overexpression of miR-200 genes in the oral cancer cells depleted of GRHL2 partially restored the epithelial phenotype, proliferative rate and cancer stemness, indicating that miR-200 genes in part mediate the functional effects of GRHL2. Taken together, this study demonstrates a novel connection between GRHL2 and miR-200, and supports protumorigenic effect of GRHL2 on OSCCs.

Grainyhead-Like 2 (GRHL2) is an epithelial-specific transcription factor that regulates epithelial morphogenesis and differentiation. Prior studies suggested inverse regulation between GRHL2 and TGF-β in epithelial plasticity and potential carcinogenesis. Here, we report the role of GRHL2 in oral carcinogenesis in vivo using a novel Grhl2 knockout (KO) mouse model and the underlying mechanism involving its functional interaction with TGF-β signaling. We developed epithelial-specific Grhl2 conditional KO mice by crossing Grhl2 floxed mice with those expressing CreER driven by the K14 promoter. After induction of Grhl2 KO, we confirmed the loss of GRHL2 and its target proteins, while Grhl2 KO strongly induced TGF-β signaling molecules. When exposed to 4-nitroquinoline 1-oxide (4-NQO), a strong chemical carcinogen, Grhl2 wild-type (WT) mice developed rampant oral tongue tumors, while Grhl2 KO mice completely abolished tumor development. In cultured oral squamous cell carcinoma (OSCC) cell lines, TGF-β signaling was notably induced by GRHL2 knockdown while being suppressed by GRHL2 overexpression. GRHL2 knockdown or KO in vitro and in vivo, respectively, led to loss of active p-Erk1/2 and p-JNK MAP kinase levels; moreover, ectopic overexpression of GRHL2 strongly induced the MAP kinase activation. Furthermore, the suppressive effect of GRHL2 on TGF-β signaling was diminished in cells exposed to Erk and JNK inhibitors. These data indicate that GRHL2 activates the Erk and JNK MAP kinases, which in turn suppresses the TGF -β signaling. This novel signaling represents an alternative pathway by which GRHL2 regulates carcinogenesis, and is distinct from the direct transcriptional regulation by GRHL2 binding at its target gene promoters, e.g., E-cadherin, hTERT, p63, and miR-200 family genes. Taken together, the current study provides the first genetic evidence to support the role of GRHL2 in carcinogenesis and the underlying novel mechanism that involves the functional interaction between GRHL2 and TGF-β signaling through the MAPK pathways.

High-risk human papillomavirus (HPV) is a major risk factor for oral and pharyngeal cancers (OPCs), yet the detailed mechanisms by which HPV promotes OPCs are not understood. Forkhead box M1B (FoxM1B) is an oncogene essential for cell cycle progression and tumorigenesis, and it is aberrantly overexpressed in many tumors. We previously showed that FoxM1B was the putative target of an epithelial-specific transcription factor, Grainyhead-like 2 (GRHL2). In the current study, we demonstrate that HPV type 16 (HPV-16) E6 induces FoxM1B in human oral keratinocytes (HOKs) and tonsillar epithelial cells (TECs) in part through GRHL2. FoxM1B was barely detectable in cultured normal human oral keratinocytes (NHOKs) and progressively increased in immortalized HOKs harboring HPV-16 genome (HOK-16B) and tumorigenic HOK-16B/BaP-T cells. Retroviral expression of HPV-16 E6 and/or E7 in NHOKs, TECs, and hypopharyngeal carcinoma cells (FaDu) revealed induction of FoxM1B and GRHL2 by the E6 protein but not E7. Both GRHL2 and FoxM1B were strongly induced in the epidermis of HPV-16 E6 transgenic mice and HPV+ oral squamous cell carcinomas. Ectopic expression of FoxM1B led to acquisition of transformed phenotype in HOK-16B cells. Loss of FoxM1B by lentiviral short hairpin RNA vector or chemical inhibitor led to elimination of tumorigenic characteristics of HOK-16B/BaP-T cells. Luciferase reporter assay revealed that GRHL2 directly bound and regulated the FoxM1B gene promoter activity. Using epithelial-specific Grhl2 conditional knockout mice, we exposed wild-type (WT) and Grhl2 KO mice to 4-nitroquinolin 1-oxide (4-NQO), which led to induction of FoxM1B in the tongue tissues and rampant oral tumor development in the WT mice. However, 4-NQO exposure failed to induce tongue tumors or induction of FoxM1B expression in Grhl2 KO mice. Collectively, these results indicate that HPV-16 induces FoxM1B in part through GRHL2 transcriptional activity and that elevated FoxM1B level is required for oropharyngeal cancer development.

High-risk human papillomaviruses (e. g., HPV16, HPV18) are closely associated with the development of head and neck cancers including oral/oropharyngeal squamous cell carcinoma (OSCC). We previously demonstrated immortalization of normal human oral keratinocytes by introducing high-risk HPV whole genome, suggesting that HPV infection plays an important role in the early stage of oral carcinogenesis. Although HPV infection may occur in different stages of cancer development, roles of HPV in exacerbating malignant phenotypes in already-transformed cells in the context of cancer stemness are not clearly defined. In this study, we investigated the role of HPV16 in promoting the virulence of HPV-negative OSCC. Introducing HPV16 whole genome in HPV-negative OSCC increased malignant growth and self-renewal capacity, a key characteristic of cancer stem cells (CSCs). HPV16 also enhanced other CSC properties, including aldehyde dehydrogenase 1 (ALDH1) activity, migration/invasion, and CSC-related factor expression. Mechanistically, we found that HPV16 inhibited the expression of miR-181a and miR-181d (miR-181a/d) at the transcriptional level. Ectopic expression of miR-181a/d decreased anchorage independent growth and CSC phenotype of HPV16-transfected OSCC. Furthermore, silencing of miR-181a/d target genes, i. e., K-ras and ALDH1, abrogated the effects of HPV16 in HPV16-transfected OSCC, supporting the functional importance of HPV16/miR-181a/d axis in HPV-mediated oral carcinogenesis. Our study suggests that high-risk HPV infection further promotes malignancy in HPV-negative OSCC by enhancing cancer stemness via miR-181a/d regulation. Consequently, miR-181a/d may represent a novel therapeutic agent for the treatment of HPV-positive OSCC., Highlights • HPV16 directs malignant progression of OSCC by enhancing stemness properties of OSCC. • HPV16 represses miR-181 by inhibiting its promoter activity. • The HPV-induced phenotypes are epigenetically mediated by miR-181a/d. • miR-181a/d may represent a novel therapeutic agent for HPV-positive OSCC.

p53, the guardian of the genome, is a tumor suppressor protein and critical for the genomic integrity of the cells. Many studies have shown that intracellular level of p53 is enhanced during replicative senescence in normal fibroblasts, and the enhanced level of p53 is viewed as the cause of senescence. Here, we report that, unlike in normal fibroblasts, the level of intracellular p53 reduces during replicative senescence and oncogene-induced senescence (OIS) in normal human keratinocytes (NHKs). We found that the intracellular p53 level was also decreased in age-dependent manner in normal human epithelial tissues. Senescent NHKs exhibited an enhanced level of p16(INK4A) , induced G2 cell cycle arrest, and lowered the p53 expression and transactivation activity. We found that low level of p53 in senescent NHKs was due to reduced transcription of p53. The methylation status at the p53 promoter was not altered during senescence, but senescent NHKs exhibited notably lower level of acetylated histone 3 (H3) at the p53 promoter in comparison with rapidly proliferating cells. Moreover, p53 knockdown in rapidly proliferating NHKs resulted in the disruption of fidelity in repaired DNA. Taken together, our study demonstrates that p53 level is diminished during replicative senescence and OIS and that such diminution is associated with H3 deacetylation at the p53 promoter. The reduced intracellular p53 level in keratinocytes of the elderly could be a contributing factor for more frequent development of epithelial cancer in the elderly because of the loss of genomic integrity of cells.

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

Bisphosphonates (BPs) are anti-resorptive agents commonly used to treat bone-related diseases; however, soft tissue-related side-effects are frequently reported in some BP users, such as oral or gastrointestinal (GI) ulcerations. BPs are stable analogs of pyrophosphate and have high affinity to hydroxyapatite, allowing them to bind to the bone surfaces and exert suppressive effects on osteoclast functions. However, the underlying mechanisms as to how bone-seeking BPs also exert cytotoxic effects on soft tissue remain unknown. In the present study, we investigated the localization of nitrogen-containing BPs (N-BPs) in hard and soft tissue using fluorescently-labeled N-BPs in vitro. We developed osteomucosal tissue constructs in vitro to recapitulate the hard and soft tissue of the oral cavity. A histological examination of the osteomucosal tissue constructs revealed a differentiated epithelium over the bone containing osteocytes and the periosteum, similar to that observed in the rat palatal tissues. Following treatment with the fluorescently-labeled bisphosphonate, AF647-ZOL, the osteomucosal constructs exhibited fluorescent signals, not only in the bone, but also in the epithelium. No fluorescent signals were observed from the control- or ZOL-treated constructs, as expected. Collectively, the data from the present study suggest that N-BPs localize to epithelial tissue and that such a localization and subsequent toxicity of N-BPs may be associated, at least in part, with soft tissue-related side-effects.

Although efavirenz (EFV) is efficacious as an anti-retroviral therapy when combined with other antiretroviral drugs, it may cause adverse clinical effects, including skin and mucosal eruptions, central nervous system complications, hepatotoxicity, renal failure and pulmonary complications. The present study investigated the phenotypic alterations caused by EFV in normal human keratinocytes (NHKs) and determined the cell death pathways leading to the lack of epithelial proliferation and regeneration. Replication kinetics, cellular morphology, and protein and mRNA levels of cell cycle regulatory genes and cell death markers were compared between the EFV-exposed cells and the untreated control. EFV treatment led to cell proliferation arrest and cell death of the NHKs by inducing autophagy mediated by proteasome-dependent degradation of p53. EFV also reduced the levels of mTOR and active ERK signaling in NHKs. Chemical inhibition of p53 degradation with a proteasome inhibitor led to reduced autophagic response of NHKs to EFV. In addition, EFV triggered terminal differentiation of NHKs by inducing the expression of involucrin, filaggrin, loricrin and genes involved in cornified envelope formation. Inhibition of autophagy in the EFV-treated NHKs with 3-methylalanine reduced the levels of involucrin and the extent of cell death. Our data indicate that EFV elicits cytotoxic effects on NHKs in part through induction of autophagy and aberrant differentiation of cells.

Stem cells from human exfoliated deciduous tooth (SHED) offer several advantages over other stem cell sources. Using SHED, we examined the roles of zinc and the zinc uptake transporter ZIP8 (Zrt- and irt-like protein 8) while inducing SHED into insulin secreting β cell-like stem cells (i.e., SHED-β cells). We observed that ZIP8 expression increased as SHED differentiated into SHED-β cells, and that zinc supplementation at day 10 increased the levels of most pancreatic β cell markers—particularly Insulin and glucose transporter 2 (GLUT2). We confirmed that SHED-β cells produce insulin successfully. In addition, we note that zinc supplementation significantly increases insulin secretion with a significant elevation of ZIP8 transporters in SHED-β cells. We conclude that SHED can be converted into insulin-secreting β cell-like cells as zinc concentration in the cytosol is elevated. Insulin production by SHED-β cells can be regulated via modulation of zinc concentration in the media as ZIP8 expression in the SHED-β cells increases.

Introduction Mesenchymal stem cells (MSCs) are typically cultured as adherent monolayer using a conventional tissue culture technique. However, this technique incompletely reproduces an in vivo microenvironment of stem cells and results in the loss of stemness properties. Three-dimensional (3D) sphere culture is one of the most widely used 3D culture techniques that have been developed to recapitulate the in vivo microenvironment. However, the stemness and multilineage differentiation capacity of spheres derived from dental pulp stem cells (DPSCs) have not been well investigated. Methods DPSCs were cultured and examined for the sphere-forming ability in serum-free, nonadherent conditions. The expression of pluripotency transcription factors was assayed by reverse transcription quantitative polymerase chain reaction and Western blot analysis. The expression of MSC-associated markers was determined by flow cytometry. Multilineage differentiation capacity was examined by alkaline phosphatase, alizarin red S, and oil red O assays. Subcutaneous transplantation in nude mice was used to examine the in vivo mineralized tissue-forming ability of sphere and adherent monolayer cells derived from DPSCs. Results We showed that DPSCs form spheres. DPSC spheres exhibited a distinct stem cell phenotype characterized by robust expression of pluripotency transcription factors and decreased expression of MSC-associated markers compared with their corresponding adherent monolayer cells. Functionally, DPSC spheres exhibited enhanced in vitro multilineage differentiation capacity. The expression of multilineage differentiation-related genes was also highly increased in DPSC spheres. Furthermore, DPSC sphere cells possessed higher in vivo mineralized tissue-forming ability than adherent monolayer cells. Conclusions Our findings indicate that sphere-forming cells are unique multipotent cell populations in DPSCs. Our study further suggests that DPSC spheres may provide a unique opportunity for pulp tissue regeneration.

Hereditary multiple exostoses (EXT) is an autosomal dominantly inherited disease characterized by the formation of cartilage-capped prominences (exostoses) that develop from the juxtaepiphyseal regions of the long bones. Recently, EXT1 and EXT2 genes were cloned and germline mutations of EXT1 and EXT2 were identified in EXT families. In this study, we performed a mutational analysis of EXT1 and EXT2 genes in eight unrelated Korean EXT families by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis followed by direct DNA sequencing. As a result, we were able to identify one family (SNU-OC3) with the EXT1 mutation and another family (SNU-OC15) with the EXT2 mutation. The EXT1 mutation was a 10-bp deletion at the 3' end of exon 5 (CTAATTTAGg) including the splice site of this exon. The EXT2 mutation identified in the SNU-OC15 family was a missense mutation at codon 85 of exon 2 (TGC-->CGC), resulting in an amino acid change from cysteine to arginine. This missense mutation cosegregated with the disease phenotype in this family, suggesting that it is the disease-causing mutation. These two mutations identified in EXT1 and EXT2 are novel ones.

Nitrogen-containing bisphosphonates (N-BPs) are the most widely used anti-resorptive agents in the treatment of bone-related diseases. N-BPs inhibit bone resorption by specifically targeting osteoclasts, bone-resorbing cells. However, soft tissue toxicity, such as oral or gastrointestinal (GI) ulcerations has frequently been reported in N-BP users, suggesting that N-BPs may also directly target cells other than osteoclasts. Previously, we reported that BPs inhibit proliferation without inducing the apoptosis of normal human oral keratinocytes (NHOKs). However, the molecular mechanisms through which N-BPs inhibit the proliferation of NHOKs are not yet fully understood. In this study, we performed gene expression profiling in N-BP-treated NHOKs and identified cyclin A2 as one of the most commonly downregulated genes. When the NHOKs were treated with N-BPs, we found that the level of cyclin A2 was suppressed in a dose- and time-dependent manner. In addition, the protein level of cyclin A2 was also significantly lower in oral epithelial cells in N-BP-treated oral mucosal tissue constructs. Cyclin A2 promoter reporter assay revealed that N-BPs inhibited the luciferase activity, indicating that the inhibition of cyclin A2 expression occurs at the transcriptional level. Furthermore, N-BPs did not alter the expression of cyclin A2 in normal human oral fibroblasts (NHOFs), suggesting that the effect of N-BPs on cyclin A2 expression may be cell-type specific. Thus, the findings of our study demonstrate that the inhibition of NHOK proliferation by N-BPs is mediated, at least in part, by the suppression of cyclin A2 expression at the transcriptional level, which may explain the underlying mechanisms of soft tissue toxicity by N-BPs.

Long-term administration of nitrogen-containing bisphosphonates can induce detrimental side effects such as bisphosphonate-related osteonecrosis of the jaw (BRONJ) in human. Although inflammation is known to be associated with BRONJ development, the detailed underlying mechanism remains unknown. Here, we report that the pro-inflammatory cytokine IL-36α is, in part, responsible for the BRONJ development. We found a notably higher level of IL-36α and lower level of collagen in the BRONJ lesions in mice. We also found that IL-36α remarkably suppressed TGF-β-mediated expression of Collα1 and α-Sma via the activation of Erk signaling pathway in mouse gingival mesenchymal stem cells. When IL-36 signaling was abrogated in vivo, development of BRONJ lesions was ameliorated in mice. Taken together, we showed the pathologic role of IL-36α in BRONJ development by inhibiting collagen expression and demonstrated that IL-36α could be a potential marker and a therapeutic target for the prevention and treatment of BRONJ. © 2016 American Society for Bone and Mineral Research.

Cancer stem-like cell (CSC; also known as tumor initiating cell) is defined as a small subpopulation of cancer cells within a tumor and isolated from various primary tumors and cancer cell lines. CSCs are highly tumorigenic and resistant to anticancer treatments. In this study, we found that prolonged exposure to tumor necrosis factor alpha (TNFα), a major proinflammatory cytokine, enhances CSC phenotype of oral squamous cell carcinoma (OSCC) cells, such as an increase in tumor sphere-forming ability, stem cell-associated genes expression, chemo-radioresistance, and tumorigenicity. Moreover, activation of Notch1 signaling was detected in the TNFα-exposed cells, and suppression of Notch1 signaling inhibited CSC phenotype. Furthermore, we demonstrated that inhibition of a Notch downstream target, Hes-1, led to suppression of CSC phenotype in the TNFα-exposed cells. We also found that Hes1 expression is commonly upregulated in OSCC lesions compared to precancerous dysplastic lesions, suggesting the possible involvement of Hes1 in OSCC progression and CSC in vivo. In conclusion, inflammatory cytokine exposure may enhance CSC phenotype of OSCC, in part by activating the Notch-Hes1 pathway.

Normal human keratinocytes (NHKs) undergo premature senescence following exposure to ionizing radiation (IR). This study investigates the effect of Bmi-1, a polycomb group protein, on radiation-induced senescence response. When exposed to IR, NHK transduced with Bmi-1 (NHK/Bmi-1) showed reduced senescent phenotype and enhanced proliferation compared with control cells (NHK/B0). To investigate the underlying mechanism, we determined the production of reactive oxygen species (ROS), expression of ROS-generating enzymes, and DNA repair activities in cells. ROS level was increased upon irradiation but notably reduced by Bmi-1 transduction. Irradiation led to strong induction of oxidase genes, e.g., Lpo (lactoperoxidase), p22-phox, p47-phox, and Gp91, in NHK/B0 but their expression was almost completely silenced in NHK/Bmi-1. Induction of oxidase genes upon irradiation was linked with loss of trimethylated histone 3 at lysine 27 (H3K27Me3), but NHK/Bmi-1 expressed a higher level of H3K27Me3 compared with NHK/B0. Bmi-1 transduction suppressed IR-associated induction of jumanji domain containing 3 while enhancing the expression of EZH2, thereby preventing the loss of H3K27Me3 in the irradiated cells. Furthermore, NHK/Bmi-1 demonstrated increased repair of IR-induced DNA damage compared with NHK/B0. These results indicate that Bmi-1 elicits radioprotective effects on NHK by mitigating the genotoxicity of IR through epigenetic mechanisms.

MicroRNAs (miRNAs) are epigenetic regulators of gene expression, and their deregulation plays an important role in human cancer, including oral squamous cell carcinoma (OSCC). Recently, we found that miRNA-181a (miR-181a) was upregulated during replicative senescence of normal human oral keratinocytes. Since senescence is considered as a tumor suppressive mechanism, we thus investigated the expression and biological role of miR-181a in OSCC. We found that miR-181a was frequently downregulated in OSCC. Ectopic expression of miR-181a suppressed proliferation and anchorage independent growth ability of OSCC. Moreover, miR-181a dramatically reduces the growth of OSCC on three dimensional organotypic raft culture. We also identified K-ras as a novel target of miR-181a. miR-181a decreased K-ras protein level as well as the luciferase activity of reporter vectors containing the 3'-untranslated region of K-ras gene. Finally, we defined a minimal regulatory region of miR-181a and found a positive correlation between its promoter activity and the level of miR-181a expression. In conclusion, miR-181a may function as an OSCC suppressor by targeting on K-ras oncogene. Thus, miR-181a should be considered for therapeutic application for OSCC.

We previously demonstrated that Bmi-1 extended the in vitro life span of normal human oral keratinocytes (NHOK). We now report that the prolonged life span of NHOK by Bmi-1 is, in part, due to inhibition of the TGF-beta signaling pathway. Serial subculture of NHOK resulted in replicative senescence and terminal differentiation and activation of TGF-beta signaling pathway. This was accompanied with enhanced intracellular and secreted TGF-beta1 levels, phosphorylation of Smad2/3, and increased expression of p15(INK4B) and p57(KIP2). An ectopic expression of Bmi-1 in NHOK (HOK/Bmi-1) decreased the level of intracellular and secreted TGF-beta1 induced dephosphorylation of Smad2/3, and diminished the level of p15(INK4B) and p57(KIP2). Moreover, Bmi-1 expression led to the inhibition of TGF-beta-responsive promoter activity in a dose-specific manner. Knockdown of Bmi-1 in rapidly proliferating HOK/Bmi-1 and cancer cells increased the level of phosphorylated Smad2/3, p15(INK4B), and p57(KIP2). In addition, an exposure of senescent NHOK to TGF-beta receptor I kinase inhibitor or anti-TGF-beta antibody resulted in enhanced replicative potential of cells. Taken together, these data suggest that Bmi-1 suppresses senescence of cells by inhibiting the TGF-beta signaling pathway in NHOK.

Heterogeneous nuclear ribonuclearproteins (hnRNPs) are nucleic acid-binding proteins and have critical roles in DNA repair, telomere regulation, and transcriptional gene regulation. Previously, we showed that hnRNP G has tumor-suppressive activity in human oral squamous cell carcinoma cells. Therefore, the identification of hnRNP G target genes is important for understanding the function of hnRNP G and its tumor suppressive activity. In this study, we identify a known tumor suppressor gene, thioredoxin-interacting protein (Txnip) gene as a novel target of hnRNP G. Expression of Txnip is upregulated by wild-type (wt) hnRNP G but not by a suppression-defective mutant hnRNP G (K22R) in human squamous cell carcinoma. Wt hnRNP G binds and transactivates the Txnip promoter in vivo, whereas the K22R mutant does not. Furthermore, overexpression of Txnip alone in cancer cells leads to the inhibition of anchorage-independent growth and in vivo tumorigenicity in immunocompromised mice, suggesting a reversion of the transformation phenotype. These studies indicate that hnRNP G promotes the expression of Txnip and mediates its tumor suppressive effect.

Many studies have suggested the involvement of wild-type (wt) p53 in the repair of DNA double-strand breaks (DSBs) via DNA end-joining (EJ) process. To investigate this possibility, we compared the capacity and fidelity of DNA EJ in RKO cells containing wt p53 and RKO cells containing no p53 (RKO cells with p53 knockdown). The p53 knockdown cells showed lower fidelity of DNA EJ compared to the control RKO cells. The DNA end-protection assay revealed the association of a protein complex including heterogeneous nuclear ribonucleoprotein G (hnRNP G) with the DNA ends in RKO cells containing wt p53, but not with the DNA ends in RKO cells with p53 knockdown. Depletion of endogenous hnRNP G notably diminished the fidelity of EJ in RKO cells expressing wt p53. Moreover, an ectopic expression of hnRNP G significantly enhanced the fidelity of DNA EJ and the protection of DNA ends in human cancer cells lacking hnRNP G protein or containing mutant hnRNP G. Finally, using recombinant hnRNP G proteins, we demonstrated the hnRNP G protein is able to bind to and protect DNA ends from degradation of nucleases. Our results suggest that wt p53 modulates DNA DSB repair by, in part, inducing hnRNP G, and the ability of hnRNP G to bind and protect DNA ends may contribute its ability to promote the fidelity of DNA EJ.

Bmi-1 is a member of the polycomb group (PcG) transcription repressors and is implicated in human carcinogenesis. In normal human oral keratinocytes (NHOK), we found that exogenous Bmi-1 expression significantly extended the replicative life span without causing cellular immortalization. Immortalization of NHOK occurs only in combination with human papillomavirus type 16 E6 (HPV-16 E6) but not with E7. During immortalization of NHOK by sequential expression of exogenous Bmi-1 and E6, telomerase activation was observed only after the cells had overcome crisis. Genetic analysis with E6 deletion mutants revealed that the intact second zinc finger domain (amino acids 118-122) was necessary for its cooperative effects with Bmi-1 in the immortalization process. Using these mutants, we found that the increased telomerase activity was closely associated with cell immortalization by Bmi-1 and E6, whereas p53 degradation was not. Using microarray analysis, we identified genes that are immortalization-specific and may participate in the process of NHOK immortalization by Bmi-1 and HPV-16 E6. Our results provide new information on the roles of Bmi-1 and HPV-16 E6 in the multi-step process of oral epithelial carcinogenesis.

Cancer stem cells (CSCs) are defined as a small subpopulation of cancer cells within a tumor and responsible for initiation and maintenance of tumor growth. Thus, understanding of molecular regulators of CSCs is of paramount importance for the development of effective cancer therapies. Here, we identified jumonji domain-containing protein 6 (JMJD6) as a novel molecular regulator of oral CSCs. JMJD6 is highly expressed in CSC-enriched populations of human oral squamous cell carcinoma (OSCC) cell lines. Moreover, immunohistochemical staining revealed significantly high level of JMJD6 in OSCC tissues compared to normal human oral epithelia, suggesting that expression of JMJD6 positively correlates with oral carcinogenesis. Subsequent functional analysis showed that knockdown of endogenous JMJD6 in OSCC strongly suppressed self-renewal capacity, a key characteristic of CSCs, and anchorage-independent growth. Conversely, ectopic expression of JMJD6 enhanced CSC characteristics including self-renewal, ALDH1 activity, migration/invasion and drug resistance. Expression of CSC-related genes was also markedly affected by modulating JMJD6 expression. Mechanistically, JMJD6 induces interleukin 4 (IL4) transcription by binding to its promoter region. IL4 rescues self-renewal capacity in JMJD6- knocked down OSCC cells, suggesting the importance of JMJD6-IL4 axis in oral CSCs. Our studies identify JMJD6 as a molecular determinant of CSC phenotype, suggesting that inhibition of JMJD6 may offer an effective therapeutic modality against oral cancer.

Purpose: Heterogeneous nuclear ribonucleoproteins (hnRNP) are nucleic acid binding proteins involved in RNA processing. We found that hnRNP G is expressed in normal human oral epithelial cells while frequently not found in the cells derived from human oral squamous cell carcinomas (HOSCC). The current study was designed to test the hypothesis that hnRNP G is a tumor suppressor. Experimental Design: We investigated the expression levels of hnRNP G protein in normal, precancerous, and malignant oral tissues by in situ immunohistochemistry. In addition, wild-type or mutant hnRNP G was ectopically overexpressed in HOSCC cells and their effects on cellular replication kinetics, colonogenic efficiency, anchorage-independent growth, and in vivo tumorigenicity were determined. Results: In situ immunohistochemical staining showed robust presence of hnRNP G in the basal cell layers of normal oral epithelium but the level of its staining was markedly reduced in dysplastic or cancerous tissues. Ectopic expression of wild-type hnRNP G in cancer cells lacking hnRNP G expression or containing mutant hnRNP G resulted in severe retardation of proliferation, reduction of colonogenic efficiency, loss of anchorage-independent growth, and reduction of in vivo tumorigenicity in immunocompromised mice. In addition, hnRNP G overexpression led to up-regulation of the expression of TXNIP, a cell cycle inhibitory gene, and significantly reduced the expression of the genes that promote cellular proliferation, such as EGR1, JUND, JUNB, FOS, FOSL1, ROS, and KIT. Conclusions: These results indicate that hnRNP G is a tumor suppressor against HOSCC but its mechanisms of action remain to be further investigated.

We determined the mitochondrial membrane status, presence of reactive oxygen species (ROS), and oxidative DNA adduct formation in normal human oral keratinocytes (NHOK) during senescence. The senescent cells showed accumulation of intracellular ROS and 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG), a major oxidative DNA adduct. Exposure of cells to H2O2 induced 8-oxo-dG accumulation in cellular DNA, which was rapidly removed in replicating NHOK. However, the 8-oxo-dG removal activity was almost completely abolished in the senescing culture. Both replicating and senescing NHOK expressed readily detectable 8-oxo-dG DNA glycosylase (hOGG1), the enzyme responsible for glycosidic cleavage of 8-oxo-dG. After exposure to H2O2, however, the intranuclear level of the hOGG1-alpha isoform was decreased in senescing but not in replicating NHOK. These results indicated that senescing NHOK accumulated oxidative DNA lesions in part due to increased level of endogenous ROS and impaired intranuclear translocation of hOGG1 enzyme upon exposure to oxidative stress.

Repair of DNA double-strand breaks (DSBs) is critical for the maintenance of cellular genetic integrity. DSBs are repaired by cellular end joining activity, which could proceed with varying degrees of accuracy. Abnormal end joining may lead to an accumulation of mutations and contribute to genetic instability and cellular aging. In the present study, we compared the efficiency and accuracy of end joining activities in exponentially replicating and senescing normal human oral keratinocytes (NHOK). We developed an in vitro end joining assay utilizing a plasmid linearized with a unique EcoR I or EcoR V restriction site. The efficiency of end joining was determined by PCR with primers that could amplify the fragment containing the end joining site. The accuracy of end joining was assessed by determining whether the original EcoR I site was restored after end joining. Both replicating and senescing cultures of NHOK yielded a similar level of end joining efficiency, which was noted by the similar intensity of PCR amplification. However, the frequency of end joining errors was significantly elevated in NHOK during replicative senescence. Senescing NHOK could thus accumulate abnormal end joining products, which might contribute to cellular aging and cancer.

Purpose: From numerous reports on proteins involved in DNA repair and telomere maintenance that physically associate with human telomerase reverse transcriptase (hTERT), we inferred that hTERT/telomerase might play a role in DNA repair. We investigated this possibility in normal human oral fibroblasts (NHOF) with and without ectopic expression of hTERT/telomerase. Experimental Design: To study the effect of hTERT/telomerase on DNA repair, we examined the mutation frequency rate, host cell reactivation rate, nucleotide excision repair capacity, and DNA end-joining activity of NHOF and NHOF capable of expressing hTERT/telomerase (NHOF-T). NHOF-T was obtained by transfecting NHOF with hTERT plasmid. Results: Compared with parental NHOF and NHOF transfected with empty vector (NHOF-EV), we found that (a) the N-methyl-N′-nitro-N-nitrosoguanidine-induced mutation frequency of an exogenous shuttle vector was reduced in NHOF-T, (b) the host cell reactivation rate of N-methyl-N′-nitro-N-nitrosoguanidine-damaged plasmids was significantly faster in NHOF-T; (c) the nucleotide excision repair of UV-damaged DNA in NHOF-T was faster, and (d) the DNA end-joining capacity in NHOF-T was enhanced. We also found that the above enhanced DNA repair activities in NHOF-T disappeared when the cells lost the capacity to express hTERT/telomerase. Conclusions: These results indicated that hTERT/telomerase enhances DNA repair activities in NHOF. We hypothesize that hTERT/telomerase accelerates DNA repair by recruiting DNA repair proteins to the damaged DNA sites.

PURPOSE: This study was designed to investigate the frequency of mutations in DNA mismatch repair genes in sporadic colorectal cancers. METHODS: Genomic DNAs procured from paraffin blocks of the pathologic specimens from 230 consecutive patients with colorectal cancer were examined for their microsatellite instability status using a mononucleotide microsatellite marker, BAT-26, and also evaluated expressions of hMLH1, hMSH2, and hMSH6 proteins by immunohistochemical staining. Any of these 230 patients did not have family histories of hereditary nonpolyposis colorectal cancer, familial adenomatous polyposis, colorectal cancer, or hereditary nonpolyposis colorectalrelated cancers, such as endometrial, small bowel, and ureteral and renal pelvic cancers. When microsatellite instability was positive, mutations in the simple repeated sequences of TGF-βRII, BAX, IGF IIR, hMSH3, and hMSH6 genes were examined. In microsatellite instability–positive or staining-negative cases, polymerase chain reaction–single- strand conformation polymorphism and DNA sequencing detected mutations of hMLH1, hMSH2, and hMSH6 genes. If mutations were found in tumor tissue samples, we tested for a germline mutation with a microdissected corresponding normal tissue. RESULTS: Among 230 cases of sporadic colorectal cancer, 21 (9.1 percent) manifested microsatellite instability. In the immunohistochemical staining, 20 (8.6 percent) showed loss of expressions. All 20 staining-negative cases were microsatellite instability–positive. Only 1 of 21 (4.8 percent) microsatellite instability– positive cases showed intact staining for three proteins. The frame-shift mutations of the simple repetitive sequences were found in 17 cases (81.0 percent) in TGF-βRII, 11 (52.4 percent) in BAX, 5 (23.8 percent) in IGF IIR, 7 (33.3 percent) in hMSH3, and 8 (38.1 percent) in hMSH6 genes. Germline mutation was observed in only one case, which accounts for 4.8 percent among positive microsatellite instability and 0.4 percent of total patients, and was found in hMSH2. Five somatic mutations (2 in hMLH1, 2 in hMSH2, and 1 in hMSH6) also were found. CONCLUSION: The results indicated that a germline mutation of DNA mismatch repair gene was a rare event in sporadic colorectal cancers.