Palmitoyltransferase ZDHHC3 Aggravates Nonalcoholic Steatohepatitis by Targeting S-Palmitoylated IRHOM2.

Underestimation of the complexity of pathogenesis in nonalcoholic steatohepatitis (NASH) significantly encumbers development of new drugs and targeted therapy strategies. Inactive rhomboid protein 2 (IRHOM2) has a multifunctional role in regulating inflammation, cell survival, and immunoreaction. Although cytokines and chemokines promote IRHOM2 trafficking or cooperate with partner factors by phosphorylation or ubiquitin ligases-mediated ubiquitination to perform physiological process, it remains unknown whether other regulators induce IRHOM2 activation via different mechanisms in NASH progression. Here the authors find that IRHOM2 is post-translationally S-palmitoylated at C476 in iRhom homology domain (IRHD), which facilitates its cytomembrane translocation and stabilization. Fatty-acids challenge can directly promote IRHOM2 trafficking by increasing its palmitoylation. Additionally, the authors identify Zinc finger DHHC-type palmitoyltransferase 3 (ZDHHC3) as a key acetyltransferase required for the IRHOM2 palmitoylation. Fatty-acids administration enhances IRHOM2 palmitoylation by increasing the direct association between ZDHHC3 and IRHOM2, which is catalyzed by the DHHC (C157) domain of ZDHHC3. Meanwhile, a metabolic stresses-triggered increase of ZDHHC3 maintains palmitoylated IRHOM2 accumulation by blocking its ubiquitination, consequently suppressing its ubiquitin-proteasome-related degradation mediated by tripartite motif containing 31 (TRIM31). High-levels of ZDHHC3 protein abundance positively correlate with the severity of NASH phenotype in patient samples. Hepatocyte-specific dysfunction of ZDHHC3 significantly inhibits palmitoylated IRHOM2 deposition, therefore suppressing the fatty-acids-mediated hepatosteatosis and inflammation in vitro, as well as NASH pathological phenotype induced by two different high-energy diets (HFHC & WTDF) in the in vivo rodent and rabbit model. Inversely, specific restoration of ZDHHC3 in hepatocytes markedly provides acceleration over the course of NASH development via increasing palmitoylation of IRHOM2 along with suppression of ubiquitin degradation. The current work uncovers that ZDHHC3-induced palmitoylation is a novel regulatory mechanism and signal that regulates IRHOM2 trafficking, which confers evidence associating the regulation of palmitoylation with NASH progression.

A preliminary study on the association between epithelial-mesenchymal transition and circulating tumor cells in renal cell carcinoma.

Background: Circulating tumor cells (CTCs) are considered useful prognostic factors for various cancers, and in 2014, our research group conducted a comparative experiment of CTC detection in patients with renal cell cancer (RCC). However, the reason for the low detection rate of CTCs in cancer patients using the CellSearch® system is still unknown, although it has been hypothesized to be attributed to the likelihood that CTCs undergoing epithelial-mesenchymal transition (EMT) do not express the CTC biomarkers cytokeratin (CK)8/18/19 or epithelial cell adhesion molecule (EpCAM). The overall aim of the current study was to investigate the expression levels of CK8/18/19 and EpCAM in relation to the EMT biomarkers vimentin and E-cadherin in patients with RCC. Methods: Patients with RCC who had undergone radical nephrectomy or partial resection between May 2014 and December 2014 were initially recruited. Results: Among 34 RCC patients, nine co-expressed EpCAM and CK8/18/19 in primary tumor tissues. The CellSearch® results showed that CK8/18/19 was expressed in 5 of 6 patients (5/6) and EpCAM was expressed in 6 patients (6/6). However, the isolation by size of tumor cells (ISET) technique showed these were co-expressed in only four of the 10. The expression of CK8/18/19, EpCAM, vimentin, and E-cadherin was distributed unequally in different enumeration groups of CTCs (all P>0.05), and the positive expression of CK8/18/19 was correlated with neutrophil number and tumor size (P<0.05). The positive expression of vimentin was correlated with the Karnofsky Performance Status (KPS) score and clinical stage of renal cancer patients (P<0.05). Conclusions: Our results indirectly proved the occurrence of EMT in the formation of CTCs by comparing and analyzing the expression of CK8/18/19 and EpCAM in renal cancer tissues and the detection results of CTCs.

The Histone Acetyltransferase MOF Regulates SIRT1 Expression to Suppress Renal Cell Carcinoma Progression.

BACKGROUND: Renal cell carcinoma (RCC) is one of the most common and lethal human urological malignancies around the world. Although many advancements in diagnostic and therapeutic strategies have been acquired, the prognosis of patients with metastatic RCC was poor. Thus, there is an urgent need to understand the molecular mechanism of RCC. METHODS: The quantitative real-time PCR (qRT-PCR) was used to detect the RNA expression of MOF in human RCC tissues and cell lines. The protein expression of MOF was analyzed with immunohistochemistry (IHC) and Western blot. To understand the regulatory mechanism of MOF in liver cancer, ChIP-qPCR assay and dual-luciferase assay were performed. Moreover, a series of in vivo and in vitro experiments were conducted to evaluate the effect of MOF on renal cell carcinoma progression. RESULTS: In the present study, we found that Males absent on the first (MOF), a histone acetyltransferase involved in transcription activation, was significantly decreased in both RCC tissues and RCC cells compared to normal tissues and non-cancer cells. Moreover, MOF downregulation was associated with advanced histological grade, pathologic stage and distant metastasis of RCC patients. Ectopic expression of MOF could significantly attenuate cell proliferation and promote cell apoptosis. Besides, MOF overexpression also suppressed migration of RCC cells through inhibiting epithelial-mesenchymal transition (EMT). Importantly, the inhibition of tumor growth by MOF was further confirmed by in vivo studies. Mechanism dissection revealed that MOF could transcriptionally upregulate the expression of SIRT1, leading to attenuated STAT3 signaling, which was involved in cell proliferation and migration. Moreover, SIRT1 knockdown could restore the biological function induced by MOF overexpression. CONCLUSIONS: Our findings indicated that MOF serves as a tumor suppressor via regulation of SIRT1 in the development and progression of RCC, and MOF might be a potent biomarker for diagnosis and prognosis prediction of RCC patients.

The E3 ubiquitin-protein ligase Trim31 alleviates non-alcoholic fatty liver disease by targeting Rhbdf2 in mouse hepatocytes.

Systemic metabolic syndrome significantly increases the risk of morbidity and mortality in patients with non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). However, no effective therapeutic strategies are available, practically because our understanding of its complicated pathogenesis is poor. Here we identify the tripartite motif-containing protein 31 (Trim31) as an endogenous inhibitor of rhomboid 5 homolog 2 (Rhbdf2), and we further determine that Trim31 directly binds to Rhbdf2 and facilitates its proteasomal degradation. Hepatocyte-specific Trim31 ablation facilitates NAFLD-associated phenotypes in mice. Inversely, transgenic or ex vivo gene therapy-mediated Trim31 gain-of-function in mice with NAFLD phenotypes virtually alleviates severe deterioration and progression of steatohepatitis. The current findings suggest that Trim31 is an endogenous inhibitor of Rhbdf2 and downstream cascades in the pathogenic process of steatohepatitis and that it may serve as a feasible therapeutical target for the treatment of NAFLD/NASH and associated metabolic disorders.

LncRNA RCAT1 promotes tumor progression and metastasis via miR-214-5p/E2F2 axis in renal cell carcinoma.

Renal cell carcinoma is the second malignant tumors in the urinary system with high mortality and morbidity. Increasing evidence suggests that long non-coding RNAs (lncRNAs) play critical roles in tumor development and progression. In the current study, based on the publicly available data obtained from GEO and TCGA database, we identified five prognosis-related lncRNAs with the ability to predict the prognosis of patients with renal cell carcinoma. Among them, the uncharacterized and upregulated lncRNA RCAT1 (renal cancer-associated transcript 1) was identified as the key lncRNA. Our data further revealed that the expression of lncRNA RCAT1 was significantly upregulated in renal cell carcinoma tissues and cells. Gain-of-function and loss-of-function studies showed that lncRNA RCAT1 promoted cell proliferation, migration, and invasion in vitro and in vivo. Furthermore, we verified that lncRNA RCAT1 could abundantly sponge miR-214-5p, which served as a tumor suppressor in renal cell carcinoma. Significantly, miR-214-5p overexpression could attenuate the promotion of cell proliferation and metastasis induced by lncRNA RCAT1. Moreover, we found that E2F2 was a direct target of miR-214-5p, and lncRNA RCAT1 could protect E2F2 from miR-214-5p-mediated degradation. Taken together, our findings suggested that lncRNA RCAT1 could enhance the malignant phenotype of renal cell carcinoma cells by modulating miR-214-5p/E2F2 axis, and lncRNA RCAT1 might be a novel prognostic biomarker and a potential therapeutic target for renal cell carcinoma.

Comparison of CellSearch and Circulating Tumor Cells (CTC)-Biopsy Systems in Detecting Peripheral Blood Circulating Tumor Cells in Patients with Gastric Cancer.

BACKGROUND The purpose of this study was to compare circulating tumor cells (CTCs)/circulating tumor microemboli (CTM) detection rates of the CellSearch and CTC-Biopsy systems in patients with gastric cancer (GC). We also investigated potential correlations between clinicopathological characteristics and prognosis in patients with GC. MATERIAL AND METHODS This prospective study was conducted at the Shandong Institute of Cancer Prevention and Control in China. Fifty-nine patients with GC and 22 healthy volunteers were recruited and their peripheral blood samples were examined by the CTC-Biopsy system and CellSearch system for CTC. RESULTS The rate of detection of CTCs/CTM was significantly higher with the CTC-Biopsy system than with the CellSearch system (59.32% vs. 27.12%, P<0.001). The Kappa value was 0.179, indicating poor consistency. CTCs detected with the CellSearch system in patients with stage III/IV GC was significantly correlated with neutrophil count (P=0.020), neutrophil/lymphocyte ratio (N/L ratio) (P=0.009), CA19-9 (P=0.049), tumor size (P=0.026), and the extent of vascular invasion (P=0.007). CTCs detected with the CTC-Biopsy system correlated with tumor differentiation (P=0.010). CTM in patients with stage I/II GC and stage II/IV GC correlated with CEA (P=0.004) and tumor differentiation (P=0.030), respectively. A CTC count >3 detected with the CellSearch system, and not the CTC-Biopsy system, correlated with reduced progression-free survival and overall survival. CONCLUSIONS The CTC-Biopsy system was superior to the CellSearch system for detecting CTCs in GC patients. CTM were detected with the CTC-Biopsy system but not with the CellSearch system. CTCs detected with the CellSearch system correlated with various clinicopathological factors and long-term survival outcomes.

Comparison of two detection systems for circulating tumor cells among patients with renal cell carcinoma.

BACKGROUND/AIMS: Detection of circulating tumor cells (CTCs) in cancer patients has diagnostic and prognostic importance. However, the clinical implications of CTC detection in patients with renal cell carcinoma (RCC) are still unclear. In this study, we investigated the clinical significance of CTCs using two detection systems, the CellSearch system (CSS) and isolation by size of epithelial tumor cells (ISET), among RCC patients. METHODS: We recruited 36 RCC patients and 22 healthy volunteers as controls. Blood was drawn before treatment. Samples were analyzed using the CSS and ISET. We prospectively followed the RCC patients to determine overall and progression-free survival. RESULTS: We did not detect CTCs in the control group using either the CSS or ISET. CTCs were detected in 7/36 patients (19.4%) using the CSS and in 13/36 patients (36.1%) using ISET, while circulating microemboli (CTMs) were detected in three patients (8.3%). The presence of ISET-detected CTCs correlated with clinical tumor node metastasis (TNM) stages, while the CSS-detected CTCs did not. After 36 months (median), CTCs detected by both methods failed to correlate with overall and progression-free survival among RCC patients. CONCLUSION: We discovered that ISET is more suitable than the CSS for detecting CTCs in RCC patients. The presence of CTCs/CTMs in RCC patients correlated with higher TNM stages, suggesting that the presence of CTCs could be a prognostic marker in RCC patients.

MicroRNA-98 acts as a tumor suppressor in hepatocellular carcinoma via targeting SALL4.

MicroRNAs (miRs) are involved in the development and progression of hepatocellular carcinoma (HCC), but the regulatory mechanism of miR-98 in HCC still remains unclear. Here we found that miR-98 was significantly downregulated in HCC tissues compared to matched adjacent normal tissues (ANTs). Low miR-98 expression was associated with tumor size, metastasis, portal vein tumor embolus, and poor overall survival. Ectopic expression of miR-98 decreased the proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of HCC cells. SALL4 was identified as a novel target of miR-98, and the protein expression of SALL4 was inhibited by miR-98 in HCC cells. Overexpression of SALL4 reversed the suppressive effects of miR-98 on the malignant phenotypes of HCC cells. Besides, SALL4, upregulated in HCC tissues compared to the matched ANTs, was inversely correlated to the miR-98 levels in HCC tissues. In addition, overexpression of miR-98 markedly suppressed the tumor growth as well as tumor-induced death in nude mice. In summary, miR-98 plays a suppressive role in the proliferation, migration, invasion and EMT of HCC cells, partly at least, via directly inhibition of SALL4. Therefore, the miR-98/SALL4 axis may become a promising therapeutic target for HCC.

Survivin mRNA-circulating tumor cells are associated with prostate cancer metastasis.

Detection of circulating tumor cells (CTCs) has been made to develop reliable assays for early diagnosis of various cancers. Overexpression of survivin in cancer cells is strongly associated with tumor progression. Although upregulation of survivin is observed in various tumors, its expression profile in the peripheral blood of prostate cancer (PCa) patients has not yet been investigated. In this study, we validated the application of survivin as the tumor marker to detect CTC and assessed its utility for diagnosis of PCa distant metastasis. Immunohistochemistry and quantitative real-time PCR (QRT-PCR) were performed to confirm the levels of surviving expression in PCa tissues. In addition, CTC values in 3 mL of peripheral blood from PCa patients, benign prostate hyperplasia (BPH) patients, and normal controls were also measured by the survivin-targeted PCR. Our results showed that surviving was overexpressed in PCa tissues. The median levels of blood surviving mRNA of PCa patients, BPH patients, and normal controls were 5.67 (range from 0 to 12.46), 2.24 (range from 0 to 6.55), and 1.85 (range from 0 to 3.82), respectively. The levels of survivin are positively associated with PCa distant metastasis. Our results concluded that quantitation of CTCs through survivin-PCR could be a promising marker for diagnosis of PCa metastasis.

Circulating Tumor Cell Analyses in Patients With Esophageal Squamous Cell Carcinoma Using Epithelial Marker-Dependent and -Independent Approaches.

In several epithelial malignancies, detection of circulating tumor cells (CTCs) in the peripheral blood has diagnostic, prognostic, and therapeutic implications. However, the clinical relevance of CTCs in esophageal squamous cell carcinoma (ESCC) has not yet been ascertained. The study was conducted with the aim of determining the clinical significance of CTCs in patients with ESCC by using 2 CTC detection systems, one epithelial marker-dependent and the other epithelial marker-independent. Paired peripheral blood samples were prospectively obtained from 61 ESCC patients before treatment and were analyzed for CTCs isolated by the CellSearch system (CS) and the method of isolation by size of epithelial tumor (ISET). Blood samples from 22 healthy volunteers were used as controls. Out of 61 study subjects, CTCs were detected in 20 patients (32.8%) by the ISET method and in only 1 patient (1.6%) by the CS method. Circulating tumor microemboli (CTM) were observed in 3 of 61 (4.9%) patients using ISET, but were undetectable in any of the patient by CS method. No CTCs/CTM were detected by either method in control groups. By ISET method, the presence of CTCs appeared to correlate with the stage of ESCC and with the baseline median platelet levels. No correlation with any other relevant clinicopathological variables was observed. Our results clearly indicate the ability of both CS and ISET methods to detect CTCs in peripheral blood samples from ESCC patients. However, the CellSearch system appears to have a poorer sensitivity as compared with the ISET method. Further studies are essential for assessing the role of such technologies in ESCC.

Neoadjuvant therapy combined with a BMP regimen for treating penile cancer patients with lymph node metastasis: a retrospective study in China.

BACKGROUND AND AIM: Combination chemotherapy is emerging in the management of advanced penile cancer. However, evidence-based chemotherapeutic regimens in the current guidelines are lacking. The aim of this study was to evaluate the efficacy of preoperative neoadjuvant chemotherapy combined with a BMP regimen including bleomycin (BLM), methopterin (MTX) and cisplatin (DDP) for treating advanced penile cancer patients. METHODS: We retrospectively audited the clinical and follow-up data of 24 penile cancer patients with fixed inguinal lymph node metastasis that were admitted in our hospital from 2001 to 2010 and received preoperative neoadjuvant chemotherapy. RESULTS: A total of 24 patients with advanced penile cancer (pN3) were recruited in this study. All patients received preoperative neoadjuvant chemotherapy combined with a BMP regimen. The average cycle of chemotherapy was two cycles (range 1-4 cycles). Among 24 adjuvant cases, 15 patients that responded to neoadjuvant chemotherapy underwent penectomy and inguinal lymphadenectomy. In contrast, nine cases did not respond to chemotherapy and received palliative local radiotherapy. Overall, the 1-, 2- and 5-year survival rates were 70.8, 50.0 and 45.8 %, respectively. The 5-year survival rate between the responder and non-responder groups was statistically significant (73.3 vs. 0 %, P < 0.001). CONCLUSION: Neoadjuvant chemotherapy combined with a BMP regimen followed by surgery is beneficial to patients with advanced penile cancer.

A comparative proteomic study identified calreticulin and prohibitin up-regulated in adrenocortical carcinomas.

BACKGROUND: Identifying novel tumor biomarkers to develop more effective diagnostic and therapeutic strategies for patients with ACC is urgently needed. The aim of the study was to compare the proteomic profiles between adrenocortical carcinomas (ACC) and normal adrenocortical tissues in order to identify novel potential biomarkers for ACC. METHODS: The protein samples from 12 ACC tissues and their paired adjacent normal adrenocortical tissues were profiled with two-dimensional electrophoresis; and differentially expressed proteins were identified by mass spectrometry. Expression patterns of three differently expressed proteins calreticulin, prohibitin and HSP60 in ACC, adrenocortical adenomas (ACA) and normal adrenocortical tissues were further validated by immunohistochemistry. RESULTS: In our proteomic study, we identified 20 up-regulated and 9 down-regulated proteins in ACC tissues compared with paired normal controls. Most of the up-regulated proteins were focused in protein binding and oxidoreductase activity in Gene Ontology (GO) molecular function classification. By immunohistochemistry, two biomarkers calreticulin and prohibitin were validated to be overexpressed in ACC compared with adrenocortical adenomas (ACA) and normal tissues, but also calreticulin overexpression was significantly associated with tumor stages of ACC. CONCLUSION: For the first time, calreticulin and prohibitin were identified to be novel candidate biomarkers for ACC, and their roles during ACC carcinogenesis and clinical significance deserves further investigation. VIRTUAL SLIDES: The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1897372598927465.