AMIGO2 enhances the invasive potential of colorectal cancer by inducing EMT.

In our previous studies, we identified amphoterin-inducible gene and open reading frame 2 (AMIGO2) as a driver gene for liver metastasis and found that AMIGO2 expression in cancer cells worsens the prognosis of patients with colorectal cancer (CRC). Epithelial-mesenchymal transition (EMT) is a trigger for CRC to acquire a malignant phenotype, such as invasive potential, leading to metastasis. However, the role of AMIGO2 expression in the invasive potential of CRC cells remains unclear. Thus, this study aimed to examine AMIGO2 expression and elucidate the mechanisms by which it induces EMT and promotes CRC invasion. Activation of the TGFß/Smad signaling pathway was found involved in AMIGO2-induced EMT, and treatment with the TGFß receptor inhibitor LY2109761 suppressed AMIGO2-induced EMT. Studies using CRC samples showed that AMIGO2 expression was highly upregulated in the invasive front, where AMIGO2 expression was localized to the nucleus and associated with EMT marker expression. These results suggest that the nuclear translocation of AMIGO2 induces EMT to promote CRC invasion by activating the TGFß/Smad signaling pathway. Thus, AMIGO2 is an attractive therapeutic target for inhibiting EMT and metastatic CRC progression.

AMIGO2 is involved in the spread of peritoneal metastasis in serous ovarian cancer via promoting adhesion to the peritoneal mesothelial cells.

BACKGROUND: Amphoterin-induced gene and open reading frame 2 (AMIGO2) have been reported to be related to the prognosis of colorectal, gastric, and cervical cancer. However, their association with ovarian cancer remains unclear. This study aimed to elucidate the role of AMIGO2 in ovarian cancer. METHODS: AMIGO2 expression was evaluated using immunohistochemistry in patients with ovarian serous carcinoma. We validated in vitro studies using four serous ovarian cancer cell lines and in vivo studies using a murine model. RESULTS: The AMIGO2-high group had significantly shorter progression-free survival (PFS) than the AMIGO2-low group. The predictive index of the AMIGO2-high group was considerably higher than that of the AMIGO2-low group. The rate of complete cytoreductive surgery was lower in the AMIGO2-high group than in the AMIGO2-low group. Moreover, in vitro studies revealed that four serous ovarian cancer cell lines exhibited AMIGO2 expression and adhesion to mesothelial cells. Adhesion to mesothelial cells was attenuated by AMIGO2 knockdown in SKOV3 and SHIN3 cells. Furthermore, AMIGO2 downregulation in SKOV3 cells significantly suppressed peritoneal dissemination in the murine model. CONCLUSION: These results suggest that high AMIGO2 expression in serous ovarian carcinoma cells contributes to a poor prognosis by promoting peritoneal metastasis through enhanced cell adhesion to mesothelial cells.

Exosomal miR-493 suppresses MAD2L1 and induces chemoresistance to intraperitoneal paclitaxel therapy in gastric cancer patients with peritoneal metastasis.

Intraperitoneal (IP) chemotherapy with paclitaxel (PTX) for gastric cancer (GC) with peritoneal metastasis (PM) is considered a promising treatment approach, however, there are no useful biomarkers to predict the efficacy of IP therapy. We examined the association between intra-peritoneal exosomes, particularly exosomal micro-RNAs (exo-miRNAs), and IP-chemo sensitivity. MKN45 cells that were cultured with intra-peritoneal exosomes from patients who did not respond to IP therapy with PTX (IPnon-respond group) exhibited resistance to PTX compared with exosomes from responding patients (IPrespond group) (p = 0.002). A comprehensive search for exo-miRNAs indicated that miR-493 was significantly up-regulated in exosomes from the IPnon-respond group compared with those collected from the IPrespond group. The expression of miR-493 in PTX-resistant MKN45 cells (MKN45PTX-res) was higher compared with that in MKN45. In addition, MKN45PTX-res cells exhibited lower MAD2L1 gene and protein expression compared with MKN45. Finally, miR-493 enhancement by transfection of miR-493 mimics significantly down-regulated MAD2L1 expression in MKN45 cells and reduced PTX sensitivity. Our results suggest that intra-peritoneal exo-miR-493 is involved in chemoresistance to PTX by downregulating MAD2L1 in GC with PM. Exo-miR-493 may be a biomarker for chemoresistance and prognosis of GC patients with PM and may also be a promising therapeutic target.

Increased chemokine ligand 26 expression and its involvement in epithelial-mesenchymal transition in the endometrium with adenomyosis.

OBJECTIVE: Adenomyosis is a gynecologic disorder characterized by symptoms of dysmenorrhea, abnormal uterine bleeding, and infertility. This study aimed to analyze the expression profiles of key inflammatory cytokines in the endometrium with adenomyosis and their involvement in epithelial-mesenchymal transition (EMT). STUDY DESIGN: Endometrial tissues collected from premenopausal women with (n = 3) or without (n = 3) adenomyosis during the secretory phase were subjected to DNA array analysis to examine inflammatory cytokines. The gene and protein expression levels were re-evaluated by reverse transcription-polymerase chain reaction (n = 19) and immunohistochemistry (n = 56). Immunohistochemical analysis using the Histo-scores of chemokine ligand 26 (CCL26) and EMT-related factors was performed with uterine tissues resected for adenomyosis (n = 37), including those from patients treated with gonadotropin-releasing hormone agonist (GnRHa). An invasion assay was also performed using endometrial epithelial cells. RESULTS: DNA array results showed that CCL26, IL-1B, and CCL3 were upregulated. CCL26 mRNA expression was markedly higher in the endometrium with adenomyosis than in that without adenomyosis. Immunohistochemical analysis revealed that CCL26 expression was elevated in the epithelial cells of the basal layer of the endometrium with adenomyosis than in that without adenomyosis regardless of GnRHa treatment. In the basal layer of the endometrium with adenomyosis, CCL26 expression was positively correlated with neural-cadherin and ZEB1 expression; additionally, the cases with intrinsic-type adenomyosis had high expression levels of CCL26 and ZEB1. Exogenous CCL26 promoted the invasive activity of endometrial epithelial cells. CONCLUSIONS: CCL26, an inflammatory mediator, may be involved in the pathogenesis of adenomyosis by inducing EMT in the basal layer of the endometrium.

AMIGO2 expression as a predictor of recurrence in cervical cancer with intermediate risk.

Patients with recurrent cervical cancer have limited treatment options and are often considered to be incurable. Since the expression of amphoterin-induced gene and open reading frame 2 (AMIGO2) in clinical samples is a prognostic factor for colorectal cancer and gastric cancer, the present aimed to elucidate whether it is also a prognostic factor for cervical cancer. Patients with primary cervical cancer who underwent radical hysterectomy or radical trachelectomy at our institution (Faculty of Medicine, Tottori University, Yonago, Japan) between September 2005 and October 2016 were retrospectively collected. Immunohistochemical analysis using a specific antibody against AMIGO2 was performed on 101 tumor samples, and the clinical characteristics, disease-free survival (DFS) and overall survival (OS) of the patients were examined. Patients in the AMIGO2-high group had a shorter 5-year DFS and OS than those in the AMIGO2-low group (P<0.001). Furthermore, AMIGO2 was an independent prognostic factor for DFS in multivariate analysis (P=0.0012). Patients in the AMIGO2-high group exhibited obvious recurrence compared with those in the AMIGO2-low group in the high-(P=0.03) and intermediate-risk groups (P=0.003). Positive lymph node metastasis, and parametrial, stromal and lymph vascular space invasion were significantly more common in AMIGO2-high patients. Taken together, AMIGO2 expression may be a predictive marker of recurrence for cervical cancer. In particular, it may be an indicator to determine the need for postoperative adjuvant therapy in intermediate-risk group patients.

Epstein-Barr virus reactivation in peripheral B lymphocytes induces IgM-type thyrotropin receptor autoantibody production in patients with Graves' disease.

Epstein-Barr virus (EBV) is a human herpes virus that latently infects B lymphocytes. When EBV is reactivated, host B cells differentiate into plasma cells and produce IgM-dominant antibodies as well as many progeny virions. The aims of the present study were to confirm the IgM dominance of thyrotropin-receptor antibodies (TRAbs) produced by EBV reactivation and investigate the roles of TRAb-IgM in Graves' disease. Peripheral blood mononuclear cells (PBMCs) containing TRAb-producing cells were stimulated for EBV reactivation, and TRAb-IgM and TRAb-IgG were measured by ELISA. TRAb-IgM were purified and TSH-binding inhibitory activities were assessed using a radio-receptor assay. Porcine thyroid follicular epithelial cells were cultured with TRAb-IgM and/or complements to measure the intracellular levels of cAMP and the amount of LDH released. TRAb-IgM/TRAb-IgG (the MG ratio) was examined in sequential serum samples of Graves' disease and compared among groups of thyroid function. The results obtained showed that IgM-dominant TRAb production was induced by EBV reactivation. TRAb-IgM did not inhibit TSH binding to TSH receptors and did not transduce hormone-producing signals. However, it destroyed thyroid follicular epithelial cells with complements. The MG ratio was significantly higher in samples of hyperthyroidism or hypothyroidism than in those with normal function or in healthy controls. A close relationship was observed between TRAb-IgM produced by EBV reactivation and the development and exacerbation of Graves' disease. The present results provide novel insights for the development of prophylaxis and therapeutics for Graves' disease.

Pivotal role for S-nitrosylation of DNA methyltransferase 3B in epigenetic regulation of tumorigenesis.

DNA methyltransferases (DNMTs) catalyze methylation at the C5 position of cytosine with S-adenosyl-L-methionine. Methylation regulates gene expression, serving a variety of physiological and pathophysiological roles. The chemical mechanisms regulating DNMT enzymatic activity, however, are not fully elucidated. Here, we show that protein S-nitrosylation of a cysteine residue in DNMT3B attenuates DNMT3B enzymatic activity and consequent aberrant upregulation of gene expression. These genes include Cyclin D2 (Ccnd2), which is required for neoplastic cell proliferation in some tumor types. In cell-based and in vivo cancer models, only DNMT3B enzymatic activity, and not DNMT1 or DNMT3A, affects Ccnd2 expression. Using structure-based virtual screening, we discovered chemical compounds that specifically inhibit S-nitrosylation without directly affecting DNMT3B enzymatic activity. The lead compound, designated DBIC, inhibits S-nitrosylation of DNMT3B at low concentrations (IC50 ≤ 100 nM). Treatment with DBIC prevents nitric oxide (NO)-induced conversion of human colonic adenoma to adenocarcinoma in vitro. Additionally, in vivo treatment with DBIC strongly attenuates tumor development in a mouse model of carcinogenesis triggered by inflammation-induced generation of NO. Our results demonstrate that de novo DNA methylation mediated by DNMT3B is regulated by NO, and DBIC protects against tumor formation by preventing aberrant S-nitrosylation of DNMT3B.

Liver Metastasis Formation Is Defined by AMIGO2 Expression via Adhesion to Hepatic Endothelial Cells in Human Gastric and Colorectal Cancer Cells.

The adhesion of circulating cancer cells to vascular endothelial cells is an initial and critical step in distant metastases. Amphoterin-induced gene and open reading frame 2 (AMIGO2) was found to regulate tumor cell adhesion to hepatic endothelial cells and act as a driver gene for liver metastasis in mouse cell lines. However, whether the role of AMIGO2 observed in mouse tumor cells can be extrapolated to human cancer cells in vivo has not been verified. In this study, AMIGO2 expression in various human gastric and colorectal cancer cells was found to be closely associated with their adhesion to human hepatic sinusoidal endothelial cells (HHSECs). Constitutive AMIGO2-knockdown clones of human gastric (MKN-45) and colorectal cancer cell lines (DLD-1) were established to examine whether AMIGO2 expression in cancer cells is involved in the adhesion to HHSECs in vitro and the formation of liver metastasis in vivo. All AMIGO2-knockdown cells showed significantly attenuated adhesion to HHSECs. In vivo analysis revealed that intrasplenic inoculation of AMIGO2-knockdown clones could engraft in the spleen but significantly suppressed liver metastasis in nude mice. This study demonstrated that the role of AMIGO2 as a driver gene of liver metastasis in mouse tumor cells can be extrapolated to human cancer cells.

The impact of AMIGO2 on prognosis and hepatic metastasis in gastric cancer patients.

BACKGROUND: Gastric cancer (GC) is one of the most common malignancies, and the liver is the most common site of hematogenous metastasis of GC. AMIGO2 is a type I transmembrane protein that has been implicated in tumour cell adhesion in adenocarcinomas; however, its importance in GC remains undetermined. METHODS: We analyzed AMIGO2 expression by immunohistochemistry using the specific monoclonal antibody for human AMIGO2 in 128 patients who underwent GC surgery to evaluate its relationship between various metastatic and clinical outcomes in GC. RESULTS: Immunohistochemistry revealed that AMIGO2 expression was an independent prognostic factor for overall survival, disease-specific survival, and liver metastasis in GC patients. CONCLUSIONS: This study showed that AMIGO2 is induced in GC tissues and can mediate hepatic metastasis. Determining AMIGO2 expression in GC will help predict patient prognosis and the incidence of liver metastasis.

Panel of human cell lines with human/mouse artificial chromosomes.

Human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) are non-integrating chromosomal gene delivery vectors for molecular biology research. Recently, microcell-mediated chromosome transfer (MMCT) of HACs/MACs has been achieved in various human cells that include human immortalised mesenchymal stem cells (hiMSCs) and human induced pluripotent stem cells (hiPSCs). However, the conventional strategy of gene introduction with HACs/MACs requires laborious and time-consuming stepwise isolation of clones for gene loading into HACs/MACs in donor cell lines (CHO and A9) and then transferring the HAC/MAC into cells via MMCT. To overcome these limitations and accelerate chromosome vector-based functional assays in human cells, we established various human cell lines (HEK293, HT1080, hiMSCs, and hiPSCs) with HACs/MACs that harbour a gene-loading site via MMCT. Model genes, such as tdTomato, TagBFP2, and ELuc, were introduced into these preprepared HAC/MAC-introduced cell lines via the Cre-loxP system or simultaneous insertion of multiple gene-loading vectors. The model genes on the HACs/MACs were stably expressed and the HACs/MACs were stably maintained in the cell lines. Thus, our strategy using this HAC/MAC-containing cell line panel has dramatically simplified and accelerated gene introduction via HACs/MACs.

AMIGO2 contained in cancer cell-derived extracellular vesicles enhances the adhesion of liver endothelial cells to cancer cells.

Adhesion of cancer cells to vascular endothelial cells in target organs is an initial step in cancer metastasis. Our previous studies revealed that amphoterin-induced gene and open reading frame 2 (AMIGO2) promotes the adhesion of tumor cells to liver endothelial cells, followed by the formation of liver metastasis in a mouse model. However, the precise mechanism underlying AMIGO2-promoted the adhesion of tumor cells and liver endothelial cells remains unknown. This study was conducted to explore the role of cancer cell-derived AMIGO2-containing extracellular vesicles (EVs) in the adhesion of cancer cells to human hepatic sinusoidal endothelial cells (HHSECs). Western blotting indicated that AMIGO2 was present in EVs from AMIGO2-overexpressing MKN-28 gastric cancer cells. The efficiency of EV incorporation into HHSECs was independent of the AMIGO2 content in EVs. When EV-derived AMIGO2 was internalized in HHSECs, it significantly enhanced the adhesion of HHSECs to gastric (MKN-28 and MKN-74) and colorectal cancer cells (SW480), all of which lacked AMIGO2 expression. Thus, we identified a novel mechanism by which EV-derived AMIGO2 released from AMIGO2-expressing cancer cells stimulates endothelial cell adhesion to different cancer cells for the initiate step of liver metastasis.

Establishment of an antibody specific for AMIGO2 improves immunohistochemical evaluation of liver metastases and clinical outcomes in patients with colorectal cancer.

INSTRUCTION: The human amphoterin-induced gene and open reading frame (AMIGO) was identified as a novel cell adhesion molecule of type I transmembrane protein. AMIGO2 is one of three members of the AMIGO family (AMIGO1, 2, and 3), and the similarity between them is approximately 40% at the amino acid level. We have previously shown that AMIGO2 functions as a driver of liver metastasis. Immunohistochemical analysis of AMIGO2 expression in colorectal cancer (CRC) using a commercially available anti-AMIGO2 mouse monoclonal antibody clone sc-373699 (sc mAb) correlated with liver metastasis and poor prognosis. However, the sc mAb was found to be cross-reactive with all three molecules in the AMIGO family. METHODS: We generated a rat monoclonal antibody clone rTNK1A0012 (rTNK mAb) for human AMIGO2. The rTNK mAb was used to re-evaluate the association between AMIGO2 expression and liver metastases/clinical outcomes using the same CRC tissue samples previously reported with sc mAb. RESULTS: Western blot analysis revealed that a rTNK mAb was identified as being specific for AMIGO2 protein and did not cross-react with AMIGO1 and AMIGO3. The rTNK mAb and sc mAb showed higher AMIGO2 expression, which correlates with a high frequency of liver metastases (65.3% and 47.5%, respectively), while multivariate analysis showed that AMIGO2 expression was an independent prognostic factor for liver metastases (p = 7.930E-10 and p = 1.707E-5). The Kaplan-Meier analyses showed that the rTNK mAb (p = 0.004), but not sc mAb (p = 0.107), predicted worse overall survival in patients with high AMIGO2 expression. The relationship between AMIGO2 expression and poor disease-specific survival showed a higher level of significance for rTNK mAb (p = 0.00004) compared to sc mAb (p = 0.001). CONCLUSIONS: These results indicate that the developed rTNK1A0012 mAb is an antibody that specifically recognizes AMIGO2 by immunohistochemistry and can be a more reliable and applicable method for the diagnostic detection of liver metastases and worse prognosis in patients with high AMIGO2-expressing CRC.

MTA1, a metastasis­associated protein, in endothelial cells is an essential molecule for angiogenesis.

Our previous study revealed that metastasis­associated protein 1 (MTA1), which is expressed in vascular endothelial cells, acts as a tube formation promoting factor. The present study aimed to clarify the importance of MTA1 expression in tube formation using MTA1­knockout (KO) endothelial cells (MTA1­KO MSS31 cells). Tube formation was significantly suppressed in MTA1­KO MSS31 cells, whereas MTA1­overexpression MTA1­KO MSS31 cells regained the ability to form tube­like structures. In addition, western blotting analysis revealed that MTA1­KO MSS31 cells showed significantly higher levels of phosphorylation of non­muscle myosin heavy chain IIa, which resulted in suppression of tube formation. This effect was attributed to a decrease of MTA1/S100 calcium­binding protein A4 complex formation. Moreover, inhibition of tube formation in MTA1­KO MSS31 cells could not be rescued by stimulation with vascular endothelial growth factor (VEGF). These results demonstrated that MTA1 may serve as an essential molecule for angiogenesis in endothelial cells and be involved in different steps of the angiogenic process compared with the VEGF/VEGF receptor 2 pathway. The findings showed that endothelial MTA1 and its pathway may serve as promising targets for inhibiting tumor angiogenesis, further supporting the development of MTA1­based antiangiogenic therapies.

Inflammation-Related Carcinogenesis: Lessons from Animal Models to Clinical Aspects.

Inflammation-related carcinogenesis has long been known as one of the carcinogenesis patterns in humans. Common carcinogenic factors are inflammation caused by infection with pathogens or the uptake of foreign substances from the environment into the body. Inflammation-related carcinogenesis as a cause for cancer-related death worldwide accounts for approximately 20%, and the incidence varies widely by continent, country, and even region of the country and can be affected by economic status or development. Many novel approaches are currently available concerning the development of animal models to elucidate inflammation-related carcinogenesis. By learning from the oldest to the latest animal models for each organ, we sought to uncover the essential common causes of inflammation-related carcinogenesis. This review confirmed that a common etiology of organ-specific animal models that mimic human inflammation-related carcinogenesis is prolonged exudation of inflammatory cells. Genotoxicity or epigenetic modifications by inflammatory cells resulted in gene mutations or altered gene expression, respectively. Inflammatory cytokines/growth factors released from inflammatory cells promote cell proliferation and repair tissue injury, and inflammation serves as a "carcinogenic niche", because these fundamental biological events are common to all types of carcinogenesis, not just inflammation-related carcinogenesis. Since clinical strategies are needed to prevent carcinogenesis, we propose the therapeutic apheresis of inflammatory cells as a means of eliminating fundamental cause of inflammation-related carcinogenesis.

AMIGO2 as a novel indicator of liver metastasis in patients with colorectal cancer.

Our previous study showed that adhesion molecule with immunoglobulin like domain 2 (AMIGO2) is a pivotal driver gene of liver metastasis via regulating tumor cell adhesion to liver endothelial cells in mouse models. The aim of the present study was to clarify the role of AMIGO2 in liver metastasis in patients the colorectal cancer (CRC). Two human CRC cell lines, Caco-2 (AMIGO2-low) and HCT116 (AMIGO2-high), were used in this study. AMIGO2-overexpressing Caco-2 and AMIGO2-knockdown HCT116 cells were generated by transfection with an AMIGO2 expression vector or AMIGO2 small interfering RNA, respectively. Cell proliferation, invasion and adhesion to human liver endothelial cells were examined in in vitro studies. Immunohistochemical analysis was also performed to evaluate the association between AMIGO2 expression and liver metastasis in patients with CRC. In vitro studies revealed that cell proliferation, invasion and adhesion to liver endothelial cells were accelerated by upregulation of AMIGO2 expression, but suppressed by downregulation of AMIGO2 expression in human CRC cells. Immunohistochemical analysis using clinical CRC specimens revealed that AMIGO2 expression was associated with the frequency of liver metastasis (P<0.01), but not that of pulmonary metastasis (P=0.611) and peritoneal dissemination (P=0.909). In addition, AMIGO2 expression levels in tumor cells were significantly higher in liver metastatic foci than primary lesions (P=0.012). In conclusion, the present results indicated that AMIGO2 expression may contribute to the formation of liver metastasis in CRC.

Splice variants of lysosome­associated membrane proteins 2A and 2B are involved in sunitinib resistance in human renal cell carcinoma cells.

Sunitinib, a tyrosine kinase inhibitor, is among the first­line treatments for metastatic or advanced stage renal cell carcinoma (RCC). However, patients with RCC develop resistance to sunitinib. We have previously demonstrated that lysosome­associated membrane protein 2 (LAMP­2), which has three splice variants with different functions (LAMP­2A, LAMP­2B, and LAMP­2C), is involved in RCC. In the present study, we examined which splice variants of LAMP­2 contributed to sunitinib resistance in RCC cells. In vitro analysis using ACHN, human RCC cell line, revealed that the IC50 of sunitinib was significantly increased by overexpression of LAMP­2A and LAMP­2B, but not LAMP­2C (P<0.01). Kaplan­Meier survival analysis using clinical samples revealed an association between shorter survival and high expression of LAMP­2A and LAMP­2B, but not LAMP­2C, in patients with RCC treated with sunitinib (P=0.01). Furthermore, high expression of LAMP­2A and LAMP­2B in RCC revealed a weak to moderate inverse correlation with the tumor shrinkage rate and progression­free survival, respectively. Thus, high expression of LAMP­2A and LAMP­2B contributed to the acquisition of sunitinib resistance, indicating that the expression of these two variants can predict the efficacy of sunitinib treatment in patients with RCC.

Loss of the cystine/glutamate antiporter in melanoma abrogates tumor metastasis and markedly increases survival rates of mice.

The cystine/glutamate antiporter, system xc- , is essential for the efficient uptake of cystine into cells. Interest in the mechanisms of system xc- function soared with the recognition that system xc- presents the most upstream node of ferroptosis, a recently described form of regulated necrosis relevant for degenerative diseases and cancer. Since targeting system xc- hold the great potential to efficiently combat tumor growth and metastasis of certain tumors, we disrupted the substrate-specific subunit of system xc- , xCT (SLC7A11) in the highly metastatic mouse B16F10 melanoma cell line and assessed the impact on tumor growth and metastasis. Subcutaneous injection of tumor cells into the syngeneic B16F10 mouse melanoma model uncovered a marked decrease in the tumor-forming ability and growth of KO cells compared to control cell lines. Strikingly, the metastatic potential of KO cells was markedly reduced as shown in several in vivo models of experimental and spontaneous metastasis. Accordingly, survival rates of KO tumor-bearing mice were significantly prolonged in contrast to those transplanted with control cells. Analyzing the in vitro ability of KO and control B16F10 cells in terms of endothelial cell adhesion and spheroid formation revealed that xCT expression indeed plays an important role during metastasis. Hence, system xc- emerges to be essential for tumor metastasis in mice, thus qualifying as a highly attractive anticancer drug target, particularly in light of its dispensable role for normal life in mice.

Bisphosphonate-induced reactive oxygen species inhibit proliferation and migration of oral fibroblasts: A pathogenesis of bisphosphonate-related osteonecrosis of the jaw.

BACKGROUND: The onset mechanism for bisphosphonate-related osteonecrosis of the jaw (BRONJ) has been reported, with a focus on bone remodeling, biofilm formation, and epithelial cell proliferation and migration. However, the involvement of stromal cells, especially fibroblasts, in the oral cavity is unclear. Therefore, this study was focused on how bisphosphonates (BPs) affect orthotopic periodontal ligament fibroblasts from the viewpoint of oxidative stress compared with ectopically obtained fibroblasts. METHODS: Normal human periodontal ligament fibroblasts (HPdLFs) and normal human dermal fibroblasts (NHDFs) were used to gain insight into the functional differences in sensitivity and reactions to BPs. Cell growth assay, measurement of reactive oxygen species (ROS) and nitric oxide (NO) production, and wound-healing assay in vitro were performed. Maxillary first molars were extracted in C57BL/6 mice and either BP, N-acetyl-cysteine (NAC), and BP or saline were administered. RESULTS: BP-induced IC50 values were significantly lower in HPdLFs (30.6 µM) than in NHDFs (109.7 µM). BP resulted in an increase in ROS, but not NO generation in HPdLFs. BPs also inhibited proliferation and migration of HPdLFs but not NHDFs, while the addition of a ROS inhibitor, NAC, reversed those inhibitions. A BRONJ mouse model in which BP was administered and then the tooth was extracted, impaired wound healing of the socket was observed. When NAC was administered before tooth extraction, wound healing was significantly improved. CONCLUSION: These results suggest that BP causes fibroblasts obtained from the oral cavity but not from skin to generate ROS and that the subsequent ROS-mediated inhibition of fibroblast growth and migration definitely delays wound healing, thereby contributing to BRONJ pathogenesis.

PITX1 protein interacts with ZCCHC10 to regulate hTERT mRNA transcription.

Telomerase is a ribonucleoprotein ribonucleic enzyme that is essential for cellular immortalization via elongation of telomere repeat sequences at the end of chromosomes. Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase holoenzyme, is a key regulator of telomerase activity. Telomerase activity, which has been detected in the majority of cancer cells, is accompanied by hTERT expression, suggesting that this enzyme activity contributes to an unlimited replication potential of cancer cells via regulation of telomere length. Thus, hTERT is an attractive target for cancer-specific treatments. We previously reported that pared-like homeodomain 1 (PITX1) is a negative regulator of hTERT through direct binding to the hTERT promoter. However, the mechanism by which the function of PITX1 contributes to transcriptional silencing of the hTERT gene remains to be clarified. Here, we show that PITX1 and zinc finger CCHC-type containing 10 (ZCCHC10) proteins cooperate to facilitate the transcriptional regulation of the hTERT gene by functional studies via FLAG pull-down assay. Co-expression of PITX1 and ZCCHC10 resulted in inhibition of hTERT transcription, in melanoma cell lines, whereas mutate-deletion of homeodomain in PITX1 that interact with ZCCHC10 did not induce similar phenotypes. In addition, ZCCHC10 expression levels showed marked decrease in the majority of melanoma cell lines and tissues. Taken together, these results suggest that ZCCHC10-PITX1 complex is the functional unit that suppresses hTERT transcription, and may play a crucial role as a novel tumor suppressor complex.