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The biological significance of micro (mi)RNAs has traditionally been evaluated according to their RNA expression levels based on the assumption that miRNAs recognize and regulate their targets in an unvarying fashion. Here we show that a fraction of mature miRNAs including miR-17-5p, -21-5p, and -200c-3p and let-7a-5p harbor methyl marks that potentially alter their stability and target recognition. Importantly, methylation of these miRNAs was significantly increased in cancer tissues as compared to paired normal tissues. Furthermore, miR-17-5p methylation level in serum samples distinguished early pancreatic cancer patients from healthy controls with extremely high sensitivity and specificity. These findings provide a basis for diagnostic strategies for early-stage cancer and add a dimension to our understanding of miRNA biology. In cancer it is assumed that microRNAs recognise and regulate their targets uniformly. Here, the authors show that in gastrointestinal cancers methylation of microRNAs may impact their stability, and that levels of microRNA methylation are distinct in pancreatic cancer patients compared to healthy controls with potential diagnostic implications.

The cancer drug gemcitabine (GEM) is a key drug for treating pancreatic ductal adenocarcinoma (PDAC), but PDAC cells develop chemoresistance after long-term administration. Since the tolerance was immediately spread to every PDAC tissue in a patient, it is assumed that some certain efficient mechanisms underlay in the development of chemoresistance. Changes in the levels of particular microRNAs or alterations in intercellular communication play a dominant role in chemoresistance development, and recent data also suggest that exosomes play an important role in this process. In this study, we revealed that the loop conferred chemoresistance in PDAC cells. The loop was as follows; 1, The long-term exposure of GEM increased miR-155 expression in PDAC cells. 2, The increase of miR-155 induced two different functions; exosome secretion and chemoresistance ability via facilitating the anti-apoptotic activity. 3, Exosome deliver the miR-155 into the other PDAC cells and induce the following function. The target therapy to miR-155 or the exosome secretion effectively attenuated the chemoresistance, and these results were validated with both clinical samples and in vivo experiments. This mechanism represents a novel therapeutic target in GEM treatment to PDAC.

Pyruvate kinase M2 (PKM2) is an alternatively spliced variant of the pyruvate kinase gene that is preferentially expressed during embryonic development and in cancer cells. PKM2 alters the final rate-limiting step of glycolysis, resulting in the cancer-specific Warburg effect (also referred to as aerobic glycolysis). Although previous reports suggest that PKM2 functions in nonmetabolic transcriptional regulation, its significance in cancer biology remains elusive. Here we report that stimulation of epithelial–mesenchymal transition (EMT) results in the nuclear translocation of PKM2 in colon cancer cells, which is pivotal in promoting EMT. Immunoprecipitation and LC-electrospray ionized TOF MS analyses revealed that EMT stimulation causes direct interaction of PKM2 in the nucleus with TGF-β–induced factor homeobox 2 (TGIF2), a transcriptional cofactor repressor of TGF-β signaling. The binding of PKM2 with TGIF2 recruits histone deacetylase 3 to the E-cadherin promoter sequence, with subsequent deacetylation of histone H3 and suppression of E-cadherin transcription. This previously unidentified finding of the molecular interaction of PKM2 in the nucleus sheds light on the significance of PKM2 expression in cancer cells. Significance Our study shows that pyruvate kinase M2 (PKM2), an alternatively spliced variant of the pyruvate kinase gene, mediates epithelial–mesenchymal transition (EMT), which is critical for cancer cells to acquire invasive potential. Our study demonstrates that EMT stimulates nuclear translocation of PKM2 and decreases epithelial cadherin transcription (a requirement for EMT induction). Our results also demonstrate that PKM2 interacts with the transcriptional factor TGF-β–induced factor homeobox 2, which induces the deacetylation of histone H3, resulting in repressed E-cadherin expression. The precise understanding of nuclear PKM2 function suggests the potential for a model preventing cancer metastasis.

PurposeIn this study, we aim to clarify whether exosomes secreted from hepatocellular carcinoma (HCC) cells under hypoxia affect angiogenesis in endothelial cells.MethodsExosomes derived from human liver cancer cell lines were cultured under hypoxic or normoxic conditions for 24 h, isolated using ExoQuick-TC®, and co-cultured with HUVECs to evaluate angiogenic activity. We also evaluated the expression of miR-155 in the exosomes from 40 patients with HCC.ResultsExosomes under hypoxia remarkably enhanced tube formation of HUVECs. Both cellular and exosomal miR-155 were significantly up-regulated under hypoxic conditions. Knockdown of miR-155 in HCC cells attenuated the promotion of tube formation by exosomes under hypoxia in HUVECs, and high expression of exosomal miR-155 in preoperative plasma was significantly correlated with early recurrence.ConclusionThese results suggest that exosomes derived from HCC cells under hypoxia induce tube formation of HUVECs and that exosomal miR-155 may affect angiogenic activity in HCC.

Current therapies for pancreatic ductal cancer (PDAC) do not sufficiently control distant metastasis. Thus, new therapeutic targets are urgently needed. Numerous studies have suggested that the epithelial‐mesenchymal transition (EMT) is pivotal for metastasis of carcinomas. The fact that the EMT is reversible suggests the possibility that it is induced by an epigenetic mechanism. In this study, we aimed to investigate the role of histone deacetylase 1 (HDAC1), which is an epigenetic mechanism on distant metastasis of PDAC. We investigated the HDAC1 expression in 103 resected PDAC specimens obtained from patients who were treated with/without preoperative therapy using immunohistochemistry. To validate the findings in the clinical samples, we evaluated the HDAC1 activity, the EMT‐associated genes and the migration/invasion ability in vitro, and performed an HDAC1 inhibitor assay. The high expression of HDAC1 in clinical samples was significantly associated with poor progression‐free survival, especially distant metastasis‐free survival. In vitro, HDAC1 inhibitors decreased the invasion ability and reversed the EMT change; the only factor to show a concomitant decrease was the expression of SNAIL. We confirmed that the HDAC1 expression was associated with the SNAIL expression in clinical samples. Moreover, the resistant cells and parental cells did not show any significant differences in the expression of HDAC1; this was consistent with the finding that preoperative therapy did not alter the HDAC1 expression in clinical samples. The targeting of HDAC1, which could suppress metastasis by inhibiting the EMT, is a promising treatment option for PDAC. The expression of histone deacetylase 1 (HDAC1) in pancreatic ductal carcinoma (PDAC) indicates a higher migration ability, and a lack of response to chemotherapy/radiotherapy. Therapy targeting HDAC1, which might suppress metastasis, would be a promising treatment option for PDAC.

Background Fatty pancreas (FP) was recently recognized as a risk factor for pancreatic ductal adenocarcinoma (PDAC). It is unclear whether computed tomography (CT) can be used to make a FP diagnosis. This study investigated whether CT could provide a predictive value for PDAC by diagnosing FP. Methods The study included 183 consecutive patients who underwent distal pancreatectomy from February 2007 to January 2017, including 75 cases of PDAC and 108 cases of other pancreatic disease. Pancreatic CT density (pancreatic index; PI) at the initial diagnosis was calculated by dividing the CT number in the pancreas by the number in the spleen. To assess whether CT could be used to detect FP, 43 cases were evaluated pathologically for FP. We investigated the correlation between FP and PI, and determined the optimal PI cutoff value for detecting FP using receiver operating characteristics analysis. We then investigated whether the PI value could be used as a predictor for PDAC. Results Fourteen cases (32.6%) were pathologically diagnosed with FP. PI was significantly lower in the FP group versus the non-FP group (0.51 vs. 0.83; p  = 0.0049). ROC analysis indicated that the PI had good diagnostic accuracy for FP diagnosis (cutoff value 0.70; sensitivity 0.79, specificity 0.79). Low PI (≤0.70) was identified in the multivariate analysis as an independent risk factor for PDAC (odds ratio 2.31; p  = 0.023). Conclusions PI was strongly associated with pathological FP, which was independently associated with PDAC. PI shows promise as an imaging predictor for PDAC.

Preoperative chemoradiation therapy (CRT) for pancreatic ductal adenocarcinoma (PDAC) has emerged as a reasonable strategy that shows good prognostic impact. However, after preoperative CRT, resected specimens show remnant tumor cells, which indicate that some tumor cells had acquired or were selected for resistance to CRT. Recently, two oncological mechanisms, the EMT and the presence of CSCs, were reported to be associated with resistance in various cancers. Previous reports showed that HGF could induce EMT in PDAC cells; moreover, the HGF receptor, c‐Met, was identified as a dominant pancreatic CSC marker. However, the clinical significance of c‐Met expression remains unclear. So, we hypothesized that remnant PDAC tissue after CRT might harbor cells with high c‐Met expression, and these cells may exacerbate patients’ prognosis. In the immunohistochemical analysis, we showed that preoperative CRT was significantly associated with high c‐Met expression; moreover, high c‐Met expression was a significant marker of a dismal prognosis. Next, we investigated mechanisms of c‐Met upregulation in PDAC cells. We established GEM‐resistant and radioresistant PDAC cells to analyze the transcriptome involved in c‐Met expression. The microarray data for the established radiation‐resistant PDAC cells indicated miR‐181b‐5p downregulation, which targets ETS1, one of the transcription factors for c‐Met, and it was shown that radiation exposure induced c‐Met expression through ETS1 increase by the suppression of miR‐181b‐5p. These results suggested that targeting these mechanisms may promote the development of a novel multidisciplinary treatment strategy for improving preoperative CRT efficiency. Patients with high c‐Met expression exhibited a distinctly shorter survival time than those with low c‐Met expression. c‐Met expression in PDAC was significantly associated with the presence of preoperative CRT. Radiation exposure induced c‐Met expression in PDAC cells by inhibiting miR‐181b‐5p, which released its suppression of the transcription factor, ETS1; in turn, increased ETS1 levels enhanced c‐Met transcription.

BackgroundRepeat liver resection is an effective treatment, with long-term surgical outcomes for recurrent hepatocellular carcinoma and colorectal liver metastasis. However, the efficacy of a minimally invasive surgical approach for recurrent liver cancer is not yet confirmed. The purpose of this study is to examine the efficacy of minimally invasive repeat liver resection (MISRLR) compared with open repeat liver resection (ORLR) for primary and metastatic liver cancer. Here, we retrospectively analyzed the clinicopathological features and short-term surgical outcomes of patients undergoing MISRLR and ORLR.MethodsFrom 2005 to 2016, 97 patients with liver cancer underwent repeat hepatectomy. Of these patients, 68 patients receiving macroscopically curative resection and only hepatectomy, without other additional operations, were selected. Twenty patients underwent MISRLR and 48 patients underwent ORLR. We compared the clinicopathological and surgical parameters in the MISRLR group with those in the ORLR group.ResultsThere were no statistically significant differences in patients’ gender, age, viral infection status, Child–Pugh classification, tumor size, tumor number, tumor location, or the presence of liver cirrhosis in the two groups. The operative times were similar, but blood loss was significantly lower in MISRLR group (159 vs. 502 ml, P = 0.0035). The length of the postoperative hospital stay was significantly shorter in the MISRLR group (14.2 vs. 19.2 days, P = 0.0275). Postoperative complications were observed only in the ORLR group, with a complication rate of 19%.ConclusionsWe demonstrate that MISRLR for primary and metastatic liver cancer reduces blood loss and postoperative complications compared with ORLR. MISRLR might be a feasible and effective procedure for the selected patients.

Cancer stem cells (CSCs) were reported to be involved in resistance to chemo/radiation therapy. We previously reported that CD13 was both a marker of CSCs and a candidate therapeutic target in HCC. In the present study, we explored the antitumor effect of a combined therapy, where ubenimex, a CD13 inhibitor, was combined with conventional anticancer drugs, fluorouracil (5-FU), cisplatin (CDDP), doxorubicin (DXR) and sorafenib (SOR), and we elucidated the mechanism of these combination therapies. We evaluated changes in the expression of CD13 before and after treatment with anticancer drugs and with or without ubenimex in the human HCC cell lines HuH7 and PLC/PRF/5. The interactions between the anticancer drugs and ubenimex were determined with isobologram analyses. We analyzed cell cycle, apoptosis, and intracellular reactive oxygen species (ROS) levels to explore the mechanisms of the combination therapies. In both cell lines, the expression of CD13 increased after a 72-h exposure to each anticancer drug alone (P<0.05), and the expression of CD13 decreased with ubenimex administration (P<0.05). Isobologram analyses indicated that ubenimex had synergistic effects with 5-FU, CDDP and DXR, and an additive effect with SOR. Cell cycle analyses showed that ubenimex decreased the proportion of cells in G0/G1. Ubenimex enhanced the effects of 5-FU, CDDP and DXR by increasing apoptosis and intracellular ROS levels. In combination therapies, ubenimex synergistically enhanced the antitumor effects of 5-FU, CDDP and DXR on cell cycle regulation and apoptosis induction in HCC cell lines. The effects of ubenimex were due to increased intracellular ROS levels.

After the initial use of anti-folates for treatment of malignancies, folate metabolism has emerged as a rational diagnostic and therapeutic target in gastrointestinal cancer. The one-carbon metabolic pathway, which comprises three critical reactions (i.e., folate and methionine cycles), underlies this effect in conjunction with the trans-sulfuration pathway. Understanding of the one-carbon metabolism pathway has served to unravel the link between the causes and effects of cancer phenotypes leading to several seminal discoveries such as that of diadenosine tri-phosphate hydrolase, microRNAs, 5-FU and, more recently, trifluridine. In the folate cycle, glycine and serine fuel the mitochondrial enzymes SHMT2, MTHFD2 and ALDH1L2, which play critical roles in the cancer survival and proliferation presumably through purine production. In the methionine cycle, S-adenocyl methionine serves hydrocarbons and polyamines that are critical for the epigenetic controls. The trans-sulfuration pathway is a critical component in the synthesis of glutathione, which is involved in the production of reactive oxygen species in cancer stem cells. Therefore, characterization of one-carbon metabolism is indispensable to the development of precision medicine in the context of cancer diagnostics and therapeutics. In the present study, we review the historical issues associated with one-carbon metabolism and highlight the recent advances in cancer research.