Intrahepatic cholangiocellular carcinoma (ICC) has poor prognosis, especially with lymph node (LN) metastasis (LNM). Lysine methyltransferase SET and MYND domain-containing 2 (SMYD2) has critical roles and is reportedly associated with poor prognosis in various cancers, but the role of SMYD2 in ICC is unclear. Here, we examined the role of SMYD2 in the progression of LNM of ICC. We immunohistochemically analyzed SMYD2 expression using resected specimens first in primary ICC and then in metastatic LNs derived from ICC. For functional analyses, we then performed cell proliferation assay following SMYD2 inhibition using two ICC cell lines such as HuCCT-1 and HuH28. To explore the detailed mechanisms of LNM, immunocytochemistry and western blotting related to epithelial-mesenchymal transition (EMT) were first performed. Moreover, we performed clinically immunohistochemical staining of C-C motif chemokine receptor 7 (CCR7) in metastatic LNs, immunofluorescence staining and western blotting for functional analyses to evaluate the relationship between SMYD2 and CCR7. Finally, p65 of nuclear factor-kappa B (NF-κB) and phospho-p65 (Ser536) status were checked as the regulator of CCR7. Protein expression levels of SMYD2 in the primary ICC lesion were not associated with any clinicopathological characteristics or prognosis. However, SMYD2 was expressed in metastatic LNs of all cases and LNM was strongly associated with poor disease-free and overall survival also as previously reported. This study therefore aimed to clarify the molecular-biological roles for LNM by SMYD2. SMYD2 knockdown significantly inhibited ICC cell proliferation. According to subsequent EMT-related research, SMYD2 knockdown did not induce morphological changes or cadherin switching. Next, chemotaxis assay revealed that SMYD2 knockdown reduced chemotaxis in HuCCT-1 from metastatic ascites but not in HuH28 from the primary lesion. CCR7 expression in metastatic LNs was immunologically confirmed in all cases and had a positive correlation with SMYD2 expression. Decreased expression of CCR7 was observed in SMYD2-suppressed HuCCT-1 cells by immunofluorescence staining and western blotting simultaneously with the decreasing phosphorylation of p65S536. SMYD2 could be demonstrated to express in all metastatic LNs derived from ICC and may regulate this metastatic mechanism through CCR7-dependent chemotaxis rather than EMT via the SMYD2-phospho-p65S536-CCR7 pathway.

Abstract Many factors including ionizing radiation and iodine deficiency are known to increase thyroid carcinogenesis risk. Our dataset analysis of The Cancer Genome Atlas (TCGA) showed that lower mRNA expression of NK2 homeobox 1 ( NKX2-1 ) transcription factor, a master regulator of genesis, homeostasis, and function of thyroid, is linked to poor prognosis of papillary thyroid cancer patients. Here we provide the findings that thyroid-specific Nkx2-1 conditional knockout ( Nkx2-1 ΔT ) mice develop thyroid adenoma and carcinoma in higher frequency with combined exposure to radiation and iodine deficiency than control Nkx2-1 fl/fl mice. Iodine deficiency caused oxidative stress, which subsequently resulted in DNA damage, leading to transformation of thyroid follicular cells. RNA-seq gene set enrichment analysis indicated higher production of reactive oxygen species (ROS) in the thyroids of Nkx2-1 ΔT as compared to Nkx2-1 fl/fl mice with combined exposure to radiation and iodine deficiency. This was accompanied by a feedback induction of SOD3 (superoxide dismutase 3) and GPX2 (glutathione peroxidase 2). These antioxidants were naturally expressed at higher levels in the thyroids of Nkx2-1 ΔT than Nkx2-1 fl/fl mice without iodine deficiency or radiation. Nkx2-1 ΔT thyroids exhibited abnormal follicle architecture and up-regulation of Acox2 (encoding acyl-CoA oxidase 2), which produces hydrogen peroxide. These results suggest that loss of NKX2-1 may contribute to excess ROS production, which elevates basal oxidative stress resulting in the promotion of ROS-induced carcinogenesis. We propose a role for NKX2-1 as a regulator of ROS production homeostasis in the thyroid. Its disturbance would dispose thyroid follicular cells more vulnerable to the ROS-producing carcinogens.

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is one of the highly lethal malignancies, with no established and effective cure. Therefore, identifying novel molecular targets for this cancer remains an urgent priority. Methods and results We employed a syngeneic orthotopic inoculation model using murine pancreatic cancer Panc02 cells to better recapitulate the pancreatic tumor microenvironment. We established highly metastatic derivatives through three serial transplantation cycles, designated Panc02-3P cells. These cells exhibited enhanced tumorigenicity and metastatic potential both in vitro and in vivo. The RNA-sequence analysis revealed that Panc02-3P cells exhibited mesenchymal-like gene expression changes, compared with parental Panc02 cells, despite no significant changes in EMT-related transcription factor expression. In addition, we found that members of the lysyl oxidase (LOX) family, including LOX, LOXL1, and LOXL3, were markedly upregulated in Panc02-3P cells. The loss-of-function assays using siRNAs targeting LOX and LOXL1 demonstrated that the suppression of these genes significantly attenuated the migratory ability of Panc02-3P cells. Conclusion Collectively, our findings suggest that the LOX family members are associated with the acquisition of a highly migratory and mesenchymal-like phenotype in metastatic pancreatic cancer cells and may contribute to tumor progression.

Background Hepatocellular carcinoma has poor prognosis due to its high recurrence rate, even after curative surgery. Epigenetic regulators are known to play a critical role in cancer progression, and the histone methyltransferase SETD8/KMT5A has been reported to be overexpressed in various malignancies. In this study, we aimed to elucidate the role of SETD8/KMT5A in hepatocellular carcinoma. Methods We investigated SETD8/KMT5A expression in 345 primary hepatocellular carcinoma resection specimens through immunohistochemical staining. For functional analyses, we conducted a loss-of-function study of SETD8/KMT5A using hepatocellular carcinoma cell lines, including in vitro assays for proliferation, cell cycle, invasion, and RNA sequencing with gene ontology analysis. Additionally, we performed xenograft experiments in mice and performed similar experiments using the SETD8/KMT5A inhibitor UNC0379. Results All cases were divided into either the SETD8 high-expression (n = 197) or low-expression (n = 148) groups. The high-expression group exhibited significantly poorer 5-year overall survival and 2- and 5-year disease-free survival compared with the low-expression group (both p < 0.001). Multivariate analysis indicated that high SETD8 expression was an independent poor prognosis factor in overall (p = 0.0255) and disease-free (p = 0.0051) survival. SETD8/KMT5A knockdown suppressed proliferation by inhibition of G1 to S phase transition (p < 0.001). Gene ontology terms were related to cancer progression, including cell adhesion, MAPK-related signaling and chromatin remodeling. SETD8/KMT5A knockout using CRISPR/Cas9 inhibited tumor growth (p < 0.01) in vivo. Conclusions SETD8/KMT5A overexpression was associated with poor prognosis and was an independent prognostic factor in hepatocellular carcinoma. In vitro and in vivo analysis, the inhibition of SETD8 could repress hepatocellular carcinoma progression through the regulation of cell activity and cell cycle transition.

Overcoming chemoresistance is a major challenge in ovarian cancer, where impaired apoptosis and mitochondrial dysfunction play key roles. Ubiquinol-cytochrome c reductase hinge protein (UQCRH), a component of mitochondrial complex III, is involved in intrinsic apoptosis, but its role in ovarian cancer remains unclear. We investigated UQCRH expression, regulation, and function in ovarian cancer. Pan-cancer analyses showed UQCRH downregulation across multiple malignancies, with particularly low expression in ovarian cancer. Immunohistochemistry confirmed reduced expression in clinical specimens, including complete loss in a subset of clear cell carcinomas. Functionally, UQCRH overexpression suppressed tumor growth and peritoneal dissemination in mouse models. While it did not affect proliferation or migration, it significantly enhanced apoptotic sensitivity under stress and in response to apoptotic stimuli. Mechanistically, UQCRH promoter hypermethylation was observed in ovarian cancer and inversely correlated with mRNA expression, as assessed by nanopore sequencing, indicating epigenetic silencing. Treatment with a DNA demethylating agent restored UQCRH expression and enhanced cisplatin-induced apoptosis. These findings suggest that epigenetic suppression of UQCRH may contribute to apoptotic resistance and chemoresistance. UQCRH may represent a potential biomarker for epigenetic priming therapy, particularly in clear cell ovarian cancer.

Melanoma tissues exhibit an acidic microenvironment compared with that of surrounding normal tissues. However, the effects of acidic conditions on the lymphatic metastasis, a crucial prognostic factor for patients with melanoma, are unclear. In the present study, we aimed to investigate the role of the acidic microenvironment in the function of lymphatic endothelial cells. We first conducted gene expression profiling using human dermal lymphatic endothelial cells (HDLECs) treated with low pH media. Based on these results, we focused on Thy-1/CD90, whose expression increased in a time-dependent manner in HDLECs under acidic conditions. Immunohistochemical analysis of primary tumor tissues in a mouse melanoma model revealed an increased expression of Thy-1 in lymphatic endothelial cells. The expression of integrin αvβ3, a receptor for Thy-1, was also up-regulated in melanoma cells under acidic conditions. The adhesion of HDLECs to melanoma cells was accelerated under acidic conditions, which was reduced by Thy-1 knockdown in HDLECs. Furthermore, lymphatic metastasis was significantly attenuated in a mouse melanoma metastasis model when inoculated with integrin αv-silenced melanoma cells. These results suggest that acid-induced Thy-1 in lymphatic endothelial cells, as well as integrin αvβ3 in melanoma cells, may promote their mutual cellular adhesion, contributing to lymphatic metastasis.

Triple-negative breast cancer (TNBC) is a malignant type of breast cancer. Owing to the lack of expression of receptors that serve as molecular targets for standard therapy for breast cancer, conventional cytotoxic chemotherapy is the primary treatment option for TNBC. However, TNBC exhibits a high degree of genomic heterogeneity, rendering it resistant to chemotherapy. Therefore, there is an urgent need to identify novel therapeutic targets for the treatment of TNBC. Advances in massively parallel sequencing technology have enabled the identification of cancer cell-specific gene expression patterns and epigenetic alterations that regulate their expression. Cancer cell-specific super-enhancers (SEs) have been identified as effective therapeutic targets for cancer. In this study, we identified the functional roles of epigenetic changes and their regulatory mechanisms in TNBC cells. TNBC cell-specific SEs were formed near several genes that contribute to malignant cancer cell acquisition. We found that the transcription factor OCT-2 (encoded by POU2F2) was responsible for the formation of SEs and the expression of genes encoded in the vicinity of the SE regions. Overexpression of POU2F2 enhances the metastasis of TNBC cells in mice, and its expression is highly correlated to poor prognosis of TNBC patients. Our findings provide a new insight into cancer cell-specific epigenetic changes induced by OCT-2, which trigger the progression of TNBC, and suggest possible candidates that could be targeted for the treatment of TNBC.

Cancer tissues exhibit an acidic microenvironment owing to the accumulation of protons and lactic acid produced by cancer and inflammatory cells. To examine the role of an acidic microenvironment in lymphatic cancer metastasis, gene expression profiling was conducted using human dermal lymphatic endothelial cells (HDLECs) treated with a low pH medium. Microarray and gene set enrichment analysis revealed that acid treatment induced the expression of inflammation-related genes in HDLECs, including genes encoding chemokines and adhesion molecules. Acid treatment-induced chemokines C-X3-C motif chemokine ligand 1 (CX3CL1) and C-X-C motif chemokine ligand 6 (CXCL6) autocrinally promoted the growth and tube formation of HDLECs. The expression of vascular cell adhesion molecule 1 (VCAM-1) increased in HDLECs after acid treatment in a time-dependent manner, which, in turn, enhanced their adhesion to melanoma cells. Among various acid-sensing receptors, HDLECs basally expressed G protein-coupled receptor 4 (GPR4), which was augmented under the acidic microenvironment. The induction of chemokines or VCAM-1 under acidic conditions was attenuated by GPR4 knockdown in HDLECs. In addition, lymph node metastases in a mouse melanoma model were suppressed by administering an anti-VCAM-1 antibody or a GPR4 antagonist. These results suggest that an acidic microenvironment modifies the function of lymphatic endothelial cells via GPR4, thereby promoting lymphatic cancer metastasis. Acid-sensing receptors and their downstream molecules might serve as preventive or therapeutic targets in cancer.

Hepatocellular carcinoma (HCC) has a high rate of recurrence and poor prognosis, even after curative surgery. Multikinase inhibitors have been applied for HCC patients, but their effect has been restricted. This study aims to clarify the clinical impact of SUV420H1/KMT5B, one of the methyltransferases for histone H4 at lysine 20, and elucidate the novel mechanisms of HCC progression. We retrospectively investigated SUV420H1 expression using HCC clinical tissue samples employing immunohistochemical analysis (n = 350). We then performed loss-of-function analysis of SUV420H1 with cell cycle analysis, migration assay, invasion assay and RNA sequence for Gene Ontology (GO) pathway analysis in vitro, and animal experiments with xenograft mice in vivo. The SUV420H1-high-score group (n = 154) had significantly poorer prognosis for both 5-year overall and 2-year/5-year disease-free survival than the SUV420H1-low-score group (n = 196) (p < 0.001 and p < 0.05, respectively). The SUV420H1-high-score group had pathologically larger tumor size, more tumors, poorer differentiation, and more positive vascular invasion than the SUV420H1-low-score group. Multivariate analysis demonstrated that SUV420H1 high score was the poorest independent factor for overall survival. SUV420H1 knockdown could suppress cell cycle from G1 to S phase and cell invasion. GO pathway analysis showed that SUV420H1 contributed to cell proliferation, cell invasion, and/or metastasis. Overexpression of SUV420H1 clinically contributed to poor prognosis in HCC, and the inhibition of SUV420H1 could repress tumor progression and invasion both in vitro and in vivo; thus, further analyses of SUV420H1 are necessary for the discovery of future molecularly targeted drugs.

U0126 accelerates apoptosis of CRC cells by U0126 (related to Figure 6).