Characterization of residual cancer by comparison of a pair of organoids established from a patient with esophageal squamous cell carcinoma before and after neoadjuvant chemotherapy.

Neoadjuvant chemotherapy (NAC) followed by surgery is a standard approach for management of locally advanced esophageal squamous cell carcinoma (ESCC). Patients who do not respond well to NAC have a poor prognosis. Despite extensive research, the mechanisms of chemoresistance in ESCC remain largely unknown. Here, we established paired tumor organoids-designated as PreNAC-O and PostNAC-O-from one ESCC patient before and after NAC, respectively. Although the two organoids did not exhibit significant differences in proliferation, morphology or drug sensitivity in vitro, the tumorigenicity of PostNAC-O in vivo was significantly higher than that of PreNAC-O. Xenografts from PreNAC-O tended to exhibit keratinization, while those from PostNAC-O displayed conspicuous necrotic areas. The tumorigenicity of PostNAC-O xenografts during the chemotherapy was comparable to that of PreNAC-O without treatment. Furthermore, the gene expression profiles of the xenografts suggested that expression of genes involved in the EMT and/or hypoxia response might be related to the tumorigenicity of PostNAC-O. Our data suggested that the tumorigenicity of residual cancer had been enhanced, outweighing the effects of chemotherapy, rather than being attributable to intrinsic chemoresistance. Further studies are required to clarify the extent to which residual cancers share a common mechanism similar to that revealed here.

Overexpression of VSNL1 Enhances Cell Proliferation in Colorectal Carcinogenesis.

INTRODUCTION: We have previously reported that overexpression of visinin-like protein 1 (VSNL1) is frequently observed in advanced colorectal adenocarcinomas and correlates with poorer prognosis. In this study, we determined the levels of VSNL1 expression in the earlier stages of colorectal tumors including adenomas and adenocarcinomas, and attempted to clarify the functional significance of VSNL1 overexpression in colorectal carcinogenesis. METHODS: Levels of VSNL expression in colorectal tumor tissues were analyzed using immunohistochemistry. The effects of VSNL1 downregulation and overexpression on cell proliferation, resistance to apoptosis, and invasiveness were determined using two VSNL1-overexpressing colorectal cancer cell lines, CW-2 and HCT-116 and VSNL1 inducibly expressing SNU-C5, respectively. Gene expression signatures in VSNL1-downregulated CW-2 and HCT-116 were identified using transcriptome and gene set enrichment analyses. RESULTS: VSNL1 expression was restricted to only a few crypt cells in the non-tumorous epithelium, whereas it became enhanced in adenomas and adenocarcinomas with the progression of tumorigenesis. Downregulation of VSNL1 in CW-2 and HCT-116 cells suppressed their proliferation through induction of apoptosis. Conversely, overexpression of VSNL1 in SNU-C5 cells enhanced resistance to anoikis. Transcriptome and gene set enrichment analyses revealed that downregulation of VSNL1 altered the expression level of the apoptosis-related gene set in CW-2 and HCT-116 cells. CONCLUSION: VSNL1 plays a role in both the development and progression of colorectal tumors by enhancing cell viability.

Association of immune-related expression profile with sensitivity to chemotherapy in esophageal squamous cell carcinoma.

Neoadjuvant chemotherapy (NAC) followed by surgery is one of the standard therapeutic approaches in Japan for patients with locally advanced esophageal carcinoma. Recently, the JCOG1109 study revealed that NAC with docetaxel, cisplatin and 5-fluorouracil (5-FU) (DCF-NAC) is superior to NAC with cisplatin and 5-FU, and has now become the standard preoperative chemotherapy. Using a microarray system, we have previously investigated the expression profiles of endoscopic biopsy samples from patients with esophageal squamous cell carcinoma (ESCC) before DCF-NAC (preNAC) and identified 17 molecules as biomarkers predictive of a pathologically complete response to DCF-NAC. Here, we re-grouped our previous dataset based on the histopathological response grade with the addition of several microarray profiles and conducted a re-analysis using bioinformatic web tools including DAVID, GSEA, UALCAN, and CIBERSORTx. We identified 204 genes that were differentially expressed between the highly resistant and sensitive groups. Some of these differentially expressed genes (DEGs) were related to the immune response and showed higher expression in the sensitive group. UALCAN showed that high expression of 28 of the top 50 DEGs was associated with a favorable prognosis (p < 0.25), and that this reached a significant (p < 0.05) level for 18 of them, suggesting that patients with high expression of these genes might have benefited from chemotherapy and thus had a better outcome. In preNAC biopsy tissues from a DCF-sensitive case, we demonstrated the presence of cells expressing mRNA for CXCL9, one of the prognosis-related DEGs. Our results highlight the association of immune-related expression profile in preNAC ESCC with the DCF-NAC efficacy.

Patient-Derived Ex Vivo Cultures and Endpoint Assays with Surrogate Biomarkers in Functional Testing for Prediction of Therapeutic Response.

Prediction of therapeutic outcomes is important for cancer patients in order to reduce side effects and improve the efficacy of anti-cancer drugs. Currently, the most widely accepted method for predicting the efficacy of anti-cancer drugs is gene panel testing based on next-generation sequencing. However, gene panel testing has several limitations. For example, only 10% of cancer patients are estimated to have druggable mutations, even if whole-exome sequencing is applied. Additionally, even if optimal drugs are selected, a significant proportion of patients derive no benefit from the indicated drug treatment. Furthermore, most of the anti-cancer drugs selected by gene panel testing are molecularly targeted drugs, and the efficacies of cytotoxic drugs remain difficult to predict. Apart from gene panel testing, attempts to predict chemotherapeutic efficacy using ex vivo cultures from cancer patients have been increasing. Several groups have retrospectively demonstrated correlations between ex vivo drug sensitivity and clinical outcome. For ex vivo culture, surgically resected tumor tissue is the most abundant source. However, patients with recurrent or metastatic tumors do not usually undergo surgery, and chemotherapy may be the only option for those with inoperable tumors. Therefore, predictive methods using small amounts of cancer tissue from diagnostic materials such as endoscopic, fine-needle aspirates, needle cores and liquid biopsies are needed. To achieve this, various types of ex vivo culture and endpoint assays using effective surrogate biomarkers of drug sensitivity have recently been developed. Here, we review the variety of ex vivo cultures and endpoint assays currently available.

Efficient Establishment of Bile-Derived Organoids From Biliary Cancer Patients.

Patient-derived tumor organoids have considerable potential as an in vitro diagnostic tool for drug susceptibility testing. In the present study, we investigated whether bile collected for diagnostic purposes could be a potential source for the establishment of biliary cancer organoids. Among 68 cases of biliary cancer, we successfully generated 60 bile-derived organoids (BDOs) from individual patients. Consistent with previous reports that described biliary cancer organoids from surgical tissues, the BDOs showed diverse morphologies such as simple cysts, multiloculated cysts, thick capsulated cysts, and solid masses. They also harbored mutations in KRAS and TP53 at frequencies of 15% and 55%, respectively. To enrich the cancer organoids by removing contaminated noncancerous components of BDOs, we attempted to verify the effectiveness of 3 different procedures, including repeat passage, xenografting, and selection with an MDM2 inhibitor for TP53 mutation-harboring BDOs. By monitoring the sequence and expression of mutated TP53, we found that all these procedures successfully enriched the cancer organoids. Our data suggest that BDOs can be established with minimal invasiveness from almost all patients with biliary cancers, including inoperable cases. Thus, despite some limitations with respect to the characterization of BDOs and methods for the enrichment of cancer cell-derived organoids, our data suggest that BDOs could have potential applications in personalized medicine.

Involvement of clusterin expression in the refractory response of pancreatic cancer cells to a MEK inhibitor.

Constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway is essential for tumorigenesis of pancreatic ductal adenocarcinoma (PDAC). To date, however, almost all clinical trials of inhibitor targeting this pathway have failed to improve the outcome of patients with PDAC. We found that implanted MIA Paca2, a human PDAC cell line sensitive to a MAPK inhibitor, PD0325901, became refractory within a week after treatment. By comparing the expression profiles of MIA Paca2 before and after acquisition of the refractoriness to PD0325901, we identified clusterin (CLU) as a candidate gene involved. CLU was shown to be induced immediately after treatment with PD0325901 or expressed primarily in more than half of PDAC cell lines, enhancing cell viability by escaping from apoptosis. A combination of PD0325901 and CLU downregulation was found to synergistically or additively reduce the proliferation of PDAC cells. In surgically resected PDAC tissues, overexpression of CLU in cancer cells was observed immunohistochemically in approximately half of the cases studied. Collectively, our findings highlight the mechanisms responsible for the rapid refractory response to MEK inhibitor in PDAC cells, suggesting a novel therapeutic strategy that could be applicable to patients with PDAC using inhibitor targeting the MAPK signaling pathway and CLU.

Enhanced phosphorylation of c-Jun by cisplatin treatment as a potential predictive biomarker for cisplatin response in combination with patient-derived tumor organoids.

Despite recent advances in sequencing technology and large-scale drug screenings employing hundreds of cell lines, the predictive accuracy of mutation-based biomarkers is still insufficient as a guide for cancer therapy. Therefore, novel types of diagnostic methods using alternative biomarkers would be highly desirable. We have hypothesized that sensitivity-specific changes in the phosphorylation of signaling molecules could be useful in this respect. Here, with the aim of developing a method for predicting the response of cancers to cisplatin using a combination of specific biomarker(s) and patient-derived tumor organoids (PDOs), we found that cisplatin-sensitive cell lines or PDOs showed enhanced phosphorylation of c-Jun (p-c-Jun) within 24 h after cisplatin treatment. We also compared the responses of 6 PDOs to cisplatin with the therapeutic effect of neoadjuvant chemotherapy (docetaxel/cisplatin/5-fluorouracil) in 6 matched patients. Mechanistically, the c-Jun induction was partly related to TNF signaling induced by cisplatin. Our data suggest that enhanced phosphorylation of c-Jun in response to cisplatin treatment could be a predictive biomarker for the efficacy of cisplatin in selected cancer patients.

Downregulation of ZNF395 Drives Progression of Pancreatic Ductal Adenocarcinoma through Enhancement of Growth Potential.

BACKGROUND: Progression of pancreatic intraepithelial neoplasia (PanIN) to invasive carcinoma is a critical factor impacting the prognosis of patients with pancreatic tumors. However, the molecular mechanisms involved are not fully understood. We have reported that the process frequently involves loss of chromosome 8p, causing downregulation of DUSP4, thus conferring invasive ability on cancer cells. Here, we focus on ZNF395, whose expression was also found to be decreased by 8p loss and was predicted to be a growth suppressor gene. METHODS: Pancreatic cancer cell lines inducibly expressing ZNF395 were established to assess the functional significance of ZNF395 in pancreatic carcinogenesis. Immunohistochemistry was also performed to analyze the expression levels of ZNF395 in pancreatic cancer tissues. RESULTS: Induction of ZNF395 in pancreatic cancer cells resulted in marked activation of JNK and suppression of their proliferation through a delay in cell cycle progression. Immunohistochemistry revealed that ZNF395 was expressed ubiquitously in both normal pancreatic ducts and PanINs but was significantly reduced in invasive cancers, especially those showing poor differentiation. CONCLUSION: ZNF395 acts as a novel tumor suppressor gene. Its downregulation caused by 8p loss in intraepithelial cells accelerates their proliferation through dysregulation of the cell cycle, leading to progression to invasive cancer.

Early response in phosphorylation of ribosomal protein S6 is associated with sensitivity to trametinib in colorectal cancer cells.

Mutations in RAS or BRAF are associated with poor prognosis and resistance to epidermal growth factor receptor (EGFR)-targeted therapy in colorectal cancer (CRC). Despite their common ability to activate downstream genes such as MEK and ERK, the therapeutic benefit of MEK inhibitors for patients with RAS/BRAF mutant CRC is limited, highlighting the need for biomarkers to predict the efficacy of MEK inhibition. Previously, we reported that a change in phosphorylation of ribosomal protein S6 (pS6) after MEK inhibition was significantly associated with sensitivity to MEK inhibition in gastric cancer cells. Here, we investigated the value of the response in pS6 for predicting the efficacy of trametinib, a MEK inhibitor, in patients with RAS/BRAF mutant CRC using patient-derived CRC organoids. We found that a subset of CRC cell lines and organoids were sensitive to trametinib. The change in phosphorylated ERK, a downstream molecule of the RAS/RAF/MEK pathway, was not significantly associated with trametinib sensitivity. On the other hand, only those with sensitivity showed a reduction of pS6 levels in response to trametinib. The change in pS6 after trametinib treatment was detectable by Western blotting, immunohistochemistry or immunocytochemistry. We also demonstrated an impact of MEK inhibition on pS6 in vivo using a xenograft model. Our data suggest that, in combination with patient-derived organoids, immunostaining-based detection of pS6 could be useful for prediction of trametinib sensitivity.

Manifestations of gene expression profiles in human right atrial myocardium caused by mechanical stretch.

This investigation was aimed to identify gene profiles in human atrial myocardium in response to chronic mechanical stretch. Right atrial appendages from 21 patients were divided into 2 groups based on the size of right atrial volume. The microarray DATA analyses differentially identified 335 genes (> 2.0-fold, corrected P < 0.05) including "functionally unknown genes". This study identified 26 up-regulated genes (natriuretic peptide B, G protein subunit gamma 13, thyroid stimulating hormone beta, etc.) and 23 down-regulated genes (oligodendrocyte transcription factor 1, carbonic anhydrase 12, etc.), which could be responsible for chronic stretch-mediated structural remodeling in the atrium.

DUSP4 is involved in the enhanced proliferation and survival of DUSP4-overexpressing cancer cells.

Dual-specificity phosphatase 4 (DUSP4), a MAP kinase phosphatase, has been regarded as a tumor suppressor gene in several cancers. However, high-level expression of DUSP4 is occasionally observed in specific cancers and its functional significance in carcinogenesis is not fully understood. In the present study, we showed that downregulation of DUSP4 suppressed the proliferation of cancer cell lines exhibiting high expression of DUSP4 by inducing apoptosis and cell cycle arrest at G2/M phase. Expression microarray analyses and pathway analyses revealed that downregulation of DUSP4 activated the p53 signaling pathway, and might be involved in cell growth suppression. Aberrant accumulation of p53 and induction of p53 downstream target genes were further investigated. Furthermore, cell growth suppression following downregulation of DUSP4 was markedly attenuated in p53-deleted cells established using the CRISPR/Cas9 system. These findings suggest that constitutive expression of DUSP4 in cancer cells contributes to enhanced proliferation through escape from apoptosis and cell cycle arrest. We propose that DUSP4 could be a novel therapeutic target for cancers overexpressing it.

A transgenic mouse expressing miR-210 in proximal tubule cells shows mitochondrial alteration: possible association of miR-210 with a shift in energy metabolism.

Previously we reported that the microRNA miR-210 is aberrantly upregulated in clear cell renal cell carcinoma (ccRCC) via deregulation of the VHL-HIF pathway. In the present study, to investigate the biological impact of miR-210 in ccRCC tumorigenesis, we developed a transgenic mouse line expressing miR-210 in proximal tubule cells under control of the mouse SGLT2/Slc5a2 promoter. Light microscopy revealed desquamation of the tubule cells and regeneration of the proximal tubule, suggesting that miR-210 expression led to damage of the proximal tubule cells. Electron microscopy revealed alterations to the mitochondria in proximal tubule cells, with marked reduction of the mitochondrial inner membrane, which is the main site of ATP production via oxidative phosphorylation (OxPhos). An additional in vitro study revealed that this loss of the inner membrane was associated with downregulation of Iscu and Ndufa4, the target genes of miR-210, suggesting that the miR-210-ISCU/NDUFA4 axis may affect mitochondrial energy metabolism. Furthermore, metabolome analysis revealed activation of anaerobic glycolysis in miR-210-transfected cells, and consistent with this the secretion of lactate, the final metabolite of anaerobic glycolysis, was significantly increased. Lactate concentration was higher in the kidney cortex of transgenic mice relative to wild-type mice, although the difference was not significant (p = 0.070). On the basis of these findings, we propose that miR-210 may induce a shift of energy metabolism from OxPhos to glycolysis by acting on the mitochondrial inner membrane. In addition to activation of glycolysis, we observed activation of the pentose phosphate pathway (PPP) and an increase in the total amount of amino acids in miR-210-transfected cells. This may help cells synthesize nucleotides and proteins for building new cells. These results suggest that miR-210 may be involved in the metabolic changes in the early stage of ccRCC development, helping the cancer cells to acquire growth and survival advantages. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Downregulation of dual-specificity phosphatase 4 enhances cell proliferation and invasiveness in colorectal carcinomas.

It is widely accepted that aberrant activation of the Wnt signaling pathway is responsible for the development of precursor lesions of colorectal cancer (CRC). However, the molecular mechanisms involved in the process of progression from these precursor lesions to invasive lesions of CRC are not fully understood. Recently, we reported that constitutive activation of MAPK accompanied by downregulation of dual-specificity phosphatase 4 (DUSP4), a MAPK phosphatase, contributes to the progression of precursor lesions in the pancreas. In this study, we found that downregulation of DUSP4 was related to constitutive activation of ERKs in CRC cells. Restoration of DUSP4 resulted in inactivation of ERKs, leading to suppression of both proliferation and invasiveness, as shown by treatment with an MEK inhibitor. Furthermore, immunohistochemistry revealed that DUSP4 expression was upregulated in the superficial region of CRC tissue, whereas it was significantly downregulated in the deep region. In contrast, ERKs in the deep region were markedly hyperactivated compared to those in the superficial region. These results suggest that activation of the MAPK signaling pathway caused by downregulation of DUSP4 is responsible for progression of CRCs and would be a promising therapeutic target.

Overexpression of cannabinoid receptor 1 in esophageal squamous cell carcinoma is correlated with metastasis to lymph nodes and distant organs, and poor prognosis.

In patients with esophageal squamous cell carcinoma (ESCC), the status of metastasis to lymph nodes is strongly associated with prognosis. Consequently, development of a biomarker to detect the presence of metastasis would be clinically valuable. In this study, we found that overexpression of cannabinoid receptor 1 (CB1R) was applicable as a marker for prediction of metastasis in ESCC. CB1R overexpression was detected immunohistochemically in 54 of 88 cases (61.4%). The intensity of CB1R expression was uniform in both intraepithelial and invasive regions in each case, and was significantly correlated with the status of metastasis to lymph nodes (P = 0.046) and distant organs (P = 0.047). Furthermore, multivariate analysis revealed that CB1R overexpression was independently associated with poor prognosis (P = 0.019). Biological analysis of CB1R overexpression using ESCC cell lines revealed that CB1R activation appeared to promote cell proliferation and invasion. On the basis of these findings, we propose that evaluation of CB1R expression status in biopsy specimens of ESCC using immunohistochemistry might be clinically useful for prediction of metastasis to lymph nodes and distant organs.

Reduced phosphorylation of ribosomal protein S6 is associated with sensitivity to MEK inhibition in gastric cancer cells.

Gastric cancer (GC) is characterized by amplifications of receptor tyrosine kinases (RTK) and KRAS, therefore, targeting of the RTK/KRAS downstream pathways could help to broaden the applicability of molecular targeted therapy for GC. We assembled a panel of 48 GC cell lines and screened predictors of responsiveness to inhibition of the RAF/MEK/ERK pathway, one of the RTK/KRAS downstream pathways. We found that GC cells with MET amplification or KRAS mutation, but not amplification, tended to be sensitive to MEK inhibition. However, several cell lines without RTK/KRAS alterations also showed high sensitivity to MEK inhibition. We then focused on the phosphorylation of RTK/KRAS downstream molecules to screen for predictors' sensitivity to MEK inhibition. We found that the phosphorylation level of mammalian target of rapamycin complex 1 (mTORC1) downstream molecules, including p70S6K, 4EBP1, and S6, was significantly associated with sensitivity to MEK inhibition in GC cells (P < 0.05), suggesting that mTORC1 activity is related to the sensitivity to MEK inhibition. Furthermore, the change in mTORC1 activity after MEK inhibition was also significantly associated with this sensitivity (P < 0.001). Among the mTORC1 downstream molecules, the change in S6 phosphorylation (pS6) showed the most significant correlation with sensitivity. Using xenograft models derived from highly sensitive and resistant cell lines, we found specific reduction of pS6 in xenografts from highly sensitive cell lines after 6 h of treatment with an MEK inhibitor. Thus, our data suggest the potential clinical applicability of an MEK inhibitor for a proportion of GC patients who could be selected on the basis of pS6 change after MEK inhibition.

Atrial Fibrillation-Mediated Upregulation of miR-30d Regulates Myocardial Electrical Remodeling of the G-Protein-Gated K(+) Channel, IK.ACh.

BACKGROUND: Atrial fibrillation (AF) begets AF in part due to atrial remodeling, the molecular mechanisms of which have not been completely elucidated. This study was conducted to identify microRNA(s) responsible for electrical remodeling in AF. METHODS AND RESULTS: The expression profiles of 1205 microRNAs, in cardiomyocytes from patients with persistent AF and from age-, gender-, and cardiac function-matched control patients with normal sinus rhythm, were examined by use of a microRNA microarray platform. Thirty-nine microRNAs differentially expressed in AF patients' atria were identified, including miR-30d, as a candidate responsible for ion channel remodeling by in silico analysis. MiR-30d was significantly upregulated in cardiomyocytes from AF patients, whereas the mRNA and protein levels ofCACNA1C/Cav1.2 andKCNJ3/Kir3.1, postulated targets of miR-30d, were markedly reduced.KCNJ3/Kir3.1 expression was downregulated by transfection of the miR-30 precursor, concomitant with a reduction of the acetylcholine-sensitive inward-rectifier K(+)current (IK.ACh).KCNJ3/Kir3.1 (but notCACNA1C/Cav1.2) expression was enhanced by the knockdown of miR-30d. The Ca(2+)ionophore, A23187, induced a dose-dependent upregulation of miR-30d, followed by the suppression ofKCNJ3mRNA expression. Blockade of protein kinase C signaling blunted the [Ca(2+)]i-dependent downregulation of Kir3.1 via miR-30d. CONCLUSIONS: The downward remodeling ofIK.AChis attributed, at least in part, to deranged Ca(2+)handling, leading to the upregulation of miR-30d in human AF, revealing a novel post-transcriptional regulation ofIK.ACh. (Circ J 2016; 80: 1346-1355).

Genomic Loss of DUSP4 Contributes to the Progression of Intraepithelial Neoplasm of Pancreas to Invasive Carcinoma.

The progression from precursor lesions of pancreatic cancer, including pancreatic intraepithelial neoplasia and intraductal papillary mucinous neoplasm (IPMN), to invasive disease is characterized by stepwise accumulation of genetic alterations. However, it remains unclear whether additional alterations are required for the progression of high-grade neoplasms to invasive pancreatic carcinoma. We compared the genomic profiles of paired noninvasive and invasive carcinoma tissues collected from patients with IPMN. We demonstrate that the frequency of genomic copy-number aberrations significantly increased during the course of invasion, and the loss of 8p11.22-ter was more often associated with invasive tissues. Expression profiling in pancreatic cancer cell lines with and without 8p11.22-ter revealed that DUSP4, an MAPK phosphatase, was significantly downregulated in cells lacking 8p11.22-ter as well as in invasive carcinomas due to genomic loss. Restoration of DUSP4 expression in pancreatic cancer cells significantly suppressed invasiveness and anoikis resistance via ERK inactivation. Accordingly, we found that blockade of ERK signaling by MEK inhibition was effective in an orthotopic xenograft model and significantly extended survival. Collectively, our findings demonstrate a genetic mechanism by which pancreatic precursor lesions progress to invasive carcinomas and highlight DUSP4 as a novel invasion suppressor that can be therapeutically exploited through manipulation of ERK signaling. Cancer Res; 76(9); 2612-25. ©2016 AACR.

Kidney-specific knockout of Sav1 in the mouse promotes hyperproliferation of renal tubular epithelium through suppression of the Hippo pathway.

We have previously reported that Salvador homologue 1 (SAV1), a component of the Hippo pathway, is significantly down-regulated in high-grade clear cell renal cell carcinoma (ccRCC) due to 14q copy number loss, and that this down-regulation contributes to the proliferation and survival of renal tubular epithelial cells through activation of Yes-associated protein 1 (YAP1), a downstream target of the Hippo pathway. However, the impact of SAV1 loss on the proliferation and survival of kidney cells in vivo remained to be determined. To address this issue, we generated kidney-specific Sav1-knockout (Cdh16-Cre;Sav1(fl/fl) ) mice. Sav1 deficiency enhanced the proliferation of renal tubular epithelial cells in Cdh16-Cre;Sav1(fl/fl) mice, accompanied by nuclear localization of Yap1, suggesting suppression of the Hippo pathway. Sav1 deficiency in renal tubules also caused structural and cellular abnormalities of the epithelial cells, including significant enlargement of their nuclei. Furthermore, Cdh16-Cre;Sav1(fl/fl) mice developed both glomerular and tubular cysts. Although lining cells of the glomerular cysts showed no atypia, those of the tubular cysts showed variations in cell size and nuclear shape, which became more severe as the mice aged. In aged Cdh16-Cre;Sav1(fl/fl) mice, we observed focal disruption of proximal tubules and perivascular lymphocytic infiltration. In conclusion, Sav1 is required for the maintenance of growth, nuclear size and structure of renal tubules under physiological conditions, and its deficiency leads to the acquisition of enhanced proliferation of renal epithelial cells through suppression of Hippo signalling.

Downregulation of WDR20 due to loss of 14q is involved in the malignant transformation of clear cell renal cell carcinoma.

Previously, we reported that genomic loss of 14q occurs more frequently in high-grade than in low-grade clear cell renal cell carcinomas (ccRCCs), and has a significant impact on the levels of expression of genes located in this region, suggesting that such genes may be involved in the malignant transformation of ccRCCs. Here, we found that six of the genes located in the minimal common region of 14q loss were significantly downregulated in high-grade ccRCCs due to copy number loss. Using a dataset from The Cancer Genome Atlas Research Network, we found that downregulation of one of these six genes, WDR20, was significantly associated with poorer outcome in patients with ccRCC, suggesting that WDR20 downregulation may be involved in the malignant transformation of ccRCCs. In functional assays, exogenous WDR20 significantly inhibited the growth of RCC cell lines and induced apoptosis. Interestingly, the phosphorylation levels of ERK and protein kinase B/AKT, which reportedly contribute to the malignant phenotype of RCC cells, were clearly reduced by exogenous expression of WDR20. Thus, our data suggest that downregulation of WDR20 due to 14q loss may be involved in the malignant transformation of ccRCCs, in part through activation of the ERK and protein kinase B/AKT pathways.

Glucose Fluctuations Aggravate Cardiac Susceptibility to Ischemia/Reperfusion Injury by Modulating MicroRNAs Expression.

BACKGROUND: The influence of glucose fluctuations (GF) on cardiovascular complications of diabetes mellitus (DM) has been attracting much attention. In the present study, whether GF increase susceptibility to ischemia/reperfusion in the heart was investigated. METHODS AND RESULTS: Male rats were randomly assigned to either a control, DM, and DM with GF group. DM was induced by an injection of streptozotocin, and glucose fluctuation was induced by starvation and insulin injection. One sequential program comprised 2 hypoglycemic episodes during 4 days. The isolated hearts were subjected to 20-min ischemia/30-min reperfusion. The infarct size was larger in hearts with GF than those with sustained hyperglycemia. Activities of catalase and superoxide dismutase were decreased, and expressions of NADPH oxidase and thioredoxin-interacting protein were upregulated by GF accompanied by an increase of reactive oxygen species (ROS). Swollen mitochondria with destroyed cristae were observed in diabetic hearts; they were further devastated by GF. Microarray analysis revealed that the expressions of microRNA (miRNA)-200c and miRNA-141 were abundant in those hearts with GF. Overexpression of miRNA-200c and miRNA-141 decreased mitochondrial superoxide dismutase and catalase activities, and increased ROS levels. Meanwhile, knockdown of miRNA-200c and miRNA-141 significantly decreased ROS levels in cardiomyocytes exposed to GF. CONCLUSIONS: GF increased ROS generation and enhanced ischemia/reperfusion injury in the diabetic heart. Upregulated miRNA-200c and miRNA-141 may account for the increased ROS.