TRIM37 promotes gallbladder cancer proliferation by activating the Wnt/β-catenin pathway via ubiquitination of Axin1

BackgroundGallbladder cancer (GBC) is among the most lethal malignancies in the world, with a prognosis that is extremely poor. The results of previous studies suggest that tripartite motif containing 37 (TRIM37) contributes to the progression of numerous types of cancer. Nevertheless, there is little knowledge about the molecular mechanisms and functions of TRIM37 in GBC.MethodsA clinical significance assessment was conducted on TRIM37 following its detection by immunohistochemistry. In vitro and in vivo functional assays were performed to investigate the role of TRIM37 in GBC.ResultsIn this study, TRIM37 is upregulated in GBC tissues, which is associated with decreased histological differentiation, advanced TNM stage, and shorter overall survival rates. In vitro, TRIM37 knockdown inhibited cell proliferation and promoted apoptosis, and in vivo, TRIM37 knockdown suppressed GBC growth. Contrary to this, cell proliferation is increased in GBC cells when overexpression of TRIM37 is expressed. Mechanistic investigations revealed that TRIM37 promotes GBC progression through activation of the Wnt/β‑catenin signaling pathway via degradation of Axin1.ConclusionThe present study suggests that TRIM37 contributes to the development of GBC and thus provides an important biomarker for predicting GBC prognosis and an effective target for therapeutic intervention.     

Identification of phosphorylation sites on the E3 ubiquitin ligase UBR5/EDD

UBR5 (ubiquitin protein ligase E3 component n-recognin 5)/EDD (E3 ligase identified by differential display) is an E3 ubiquitin ligase that is a potential biomarker for poor prognosis for recurrent, platinum-resistant ovarian cancer. UBR5 has a role in the DNA damage response and many such proteins are regulated by phosphorylation. UBR5 is a 309 kDa nuclear phosphoprotein that we previously identified as a substrate of the MAP kinase ERK2. With its 477 potential phosphorylation sites, little is known about UBR5 phosphorylation and how it may regulate protein function. Currently, thirty-four sites of phosphorylation on UBR5 have been reported in the literature, mostly identified by large scale proteomics studies of tissues or of cells after various treatments; however, no studies have specifically targeted the identification of UBR5 phosphorylation sites. In this study, we used Liquid Chromatography-Mass Spectrometry (LC-MS/MS) to obtain a total sequence coverage of 64.3% from combining tryptic and GluC digests on UBR5 isolated from transfected COS-1 cells. We identified 24 sites of phosphorylation, 18 of which are novel sites. This data enhances our knowledge of UBR5 phosphorylation and provides a framework for the study of how phosphorylation affects UBR5 function.     

Forkhead Box L1 Is Frequently Downregulated in Gallbladder Cancer and Inhibits Cell Growth through Apoptosis Induction by Mitochondrial Dysfunction

Background

Forkhead box L1 (FOXL1), considered as a novel candidate tumor suppressor, suppresses proliferation and invasion in certain cancers. However, the regulation and function of FOXL1 in gallbladder cancer (GBC) remains unclear.

Methods

FOXL1 expression at mRNA and protein levels in GBC tissues and cell lines were examined by RT-PCR, immunohistochemistry and western blot assay. FOXL1 expression in GBC cell lines was up-regulated by transfection with pcDNA-FOXL1. The effects of FOXL1 overexpression on cell proliferation, apoptosis, migration and invasion were evaluated in vitro or in vivo. In addition, the status of mediators involved in migration, invasion and apoptosis was examined using western blot after transfection with pcDNA-FOXL1.

Results

FOXL1 was frequently downregulated in GBC tissues and cell lines. Its higher expression is associated with better prognosis, while its lower expression is correlated with advanced TNM stage and poor differentiation. FOXL1 overexpression in NOZ cells significantly suppresses cell proliferation, migration and invasion in vitro and tumorigenicity in nude mice. FOXL1 overexpression disrupted mitochondrial transmembrane potential and triggered mitochondria-mediated apoptosis in NOZ cells. In addition, FOXL1 overexpression suppressed ZEB1 expression and induced E-cadherin expression in NOZ cells.

Conclusion

Our findings suggested that dysregulated FOXL1 is involved in tumorigenesis and progression of GBC and may serve as a predictor of clinical outcome or even a therapeutic target for patients with GBC.     

Glycochenodeoxycholate promotes the metastasis of gallbladder cancer cells by inducing epithelial to mesenchymal transition via activation of SOCS3/JAK2/STAT3 signaling pathway

The incidence of gallbladder cancer (GBC) is relatively rare but a high degree of malignancy. The migration and invasion potential of GBC severely affects the prognosis of patients with GBC. Glycochenodeoxycholate (GCDC) is one of the most important components in GBC-associated microenvironment. However, the role of GCDC in the metastatic feature of GBC cells is not fully understood. First, the results of this study found that GCDC could effectively enhance the metastasis of GBC cells. Furthermore, GCDC could lead to the enhancement of epithelial to mesenchymal transition (EMT) phenotype in GBC cells, which is concerned to be an important mechanism of tumor metastasis. Further studies showed that GCDC treatment induced the upregulation of matrix metalloproteinase-3 (MMP3), MMP9, and SOCS3/JAK2/p-STAT3 signal pathway in GBC cells, which could regulate the level of EMT. Beside that, we also found the positive expression of farnesoid X receptor (FXR) in GBC cells and inhibition of FXR could significantly block the effect of GCDC on the metastasis of GBC cells. These results indicated that GCDC promoted GBC cells metastasis by enhancing the level of EMT and inhibition of FXR could significantly block the effect of GCDC. On one hand, FXR might be an indicator for predicting the metastasis of patient with GBC. On the other hand, FXR might serve as a potential antimetastasis target in GBC therapy.     

OLFM4 depletion sensitizes gallbladder cancer cells to cisplatin through the ARL6IP1/caspase-3 axis

BackgroundGallbladder cancer (GBC) is a highly lethal malignancy that carries an extremely poor prognosis due to its chemoresistant nature. Cisplatin (CDDP) is a first-line chemotherapeutic for GBC; however, patients experienced no benefit when treated with CDDP alone. The underlying mechanisms of CDDP resistance in GBC remain largely unknown.MethodsAgilent mRNA microarray analysis was performed between paired GBC and paracarcinoma to explore differentially expressed genes that might underlie drug resistance. Gene Set Enrichment Analysis (GSEA) was employed to identify key genes mediating CDDP resistance in GBC, and immunohistochemistry was performed to validate protein expression and test correlations with clinicopathological features. In vitro and in vivo functional assays were performed to investigate the proteins’ roles in CDDP resistance.ResultsOlfactomedin 4 (OLFM4) was differentially expressed between GBC and paracarcinoma and had the highest rank metric score in the GSEA. OLFM4 expression was increasingly upregulated from chronic cholecystitis to GBC in clinical tissue samples, and OLFM4 depletion decreased GBC cell proliferation and invasion. Interestingly, downregulation of OLFM4 reduced ARL6IP1 (antiapoptotic factor) expression and sensitized GBC cells to CDDP both in vitro and in vivo. The evidence indicated that CDDP could significantly increase Bax and Bad expression and activate caspase-3 cascade in OLFM4-depleted GBC cells through ARL6IP1. Clinically, lower OLFM4 expression was associated with good prognosis of GBC patients.ConclusionsOur results suggest that OLFM4 is an essential gene that contributes to GBC chemoresistance and could serve as a prognostic biomarker for GBC. Importantly, OLFM4 could be a potential chemotherapeutic target.     

MicroRNA-361-5p slows down gliomas development through regulating UBR5 to elevate ATMIN protein expression

MicroRNA (miR)-361-5p has been studied to suppress gliomas development. Based on that, an insight into the regulatory mechanism of miR-361-5p in gliomas was supplemented from ubiquitin protein ligase E3 component N-recognin 5 (UBR5)-mediated ubiquitination of ataxia-telangiectasia mutated interactor (ATMIN). miR-361-5p, ATMIN, and UBR5 levels were clinically analyzed in gliomas tissues, which were further validated in gliomas cell lines. Loss/gain-of-function method was applied to determine the roles of miR-361-5p and UBR5 in gliomas, as to cell viability, migration, invasion, colony formation ability, and apoptosis in vitro and tumorigenesis in vivo. The relationship between miR-361-5p and UBR5 was verified and the interaction between UBR5 and ATMIN was explored. It was detected that reduced miR-361-5p and ATMIN and enhanced UBR5 levels showed in gliomas. Elevating miR-361-5p was repressive in gliomas progression. UBR5 was directly targeted by miR-361-5p. UBR5 can ubiquitinate ATMIN. miR-361-5p suppressed gliomas by regulating UBR5-mediated ubiquitination of ATMIN. Downregulating UBR5 impeded gliomas tumor growth in vivo. Upregulating miR-361-5p targets UBR5 to promote ATMIN protein expression, thus to recline the malignant phenotype of gliomas cells.Subject terms: Cell biology, Diseases     

The E3 ligase UBR2 regulates cell death under caspase deficiency via Erk/MAPK pathway

Escape from cell death is a key event in cancer establishment/progression. While apoptosis is often considered as the main cell death pathway, upon caspase inhibition, cell death is rather delayed than blocked leading to caspase-independent cell death (CICD). Although described for years, CICD’s underlying mechanism remains to be identified. Here, we performed a genome-wide siRNA lethality screening and identified the RING-Type E3 Ubiquitin Transferase (UBR2) as a specific regulator of CICD. Strikingly, UBR2 downregulation sensitized cells towards CICD while its overexpression was protective. We established that UBR2-dependent protection from CICD was mediated by the MAPK/Erk pathway. We then observed that UBR2 is overexpressed in several cancers, especially in breast cancers and contributes to CICD resistance. Therefore, our work defines UBR2 as a novel regulator of CICD, found overexpressed in cancer cells, suggesting that its targeting may represent an innovative way to kill tumor cells.Subject terms: Cancer, Cell death     

Loss of desmoglein-2 promotes gallbladder carcinoma progression and resistance to EGFR-targeted therapy through Src kinase activation

Gallbladder carcinoma (GBC) exhibits poor prognosis due to local recurrence, metastasis, and resistance to targeted therapies. Using clinicopathological analyses of GBC patients along with molecular in vitro and tumor in vivo analysis of GBC cells, we showed that reduction of Dsg2 expression was highly associated with higher T stage, more perineural, and lymphatic invasion. Dsg2-depleted GBC cells exhibited significantly enhanced proliferation, migration, and invasiveness in vitro and tumor growth and metastasis in vivo through Src-mediated signaling activation. Interestingly, Dsg2 binding inhibited Src activation, whereas its loss activated cSrc-mediated EGFR plasma membrane clearance and cytoplasmic localization, which was associated with acquired EGFR-targeted therapy resistance and decreased overall survival. Inhibition of Src activity by dasatinib enhanced therapeutic response to anti-EGFR therapy. Dsg2 status can help stratify predicted patient response to anti-EGFR therapy and Src inhibition could be a promising strategy to improve the clinical efficacy of EGFR-targeted therapy.Subject terms: Tumour biomarkers, Tumour-suppressor proteins     

c-Src在上皮性卵巢癌组织中的表达及意义

目的:探讨上皮性卵巢癌组织中细胞肉瘤蛋白(Cell sarcoma,c-Src)的表达及其其临床意义。方法:通过Western blotting方法检测4例正常卵巢组织及21例上皮性卵巢组织中c-Src蛋白的表达。同时,通过免疫组化的方法检测26例正常卵巢组织和134例上皮性卵巢癌组织中c-Src的表达。统计分析c-Src的表达与上皮性卵巢肿瘤的临床病理因素之间的关系。结果:与正常卵巢组织相比,上皮性卵巢癌组织中c-Src蛋白的表达明显增高。c-Src蛋白的过表达与上皮性卵巢癌患者的临床病理分期、组织学分化程度、淋巴结转移情况及肿瘤复发之间差异存在统计学意义(P0.05),但与患者的年龄等因素均无显著相关性(P0.05)。此外,c-Src蛋白的高表达,与上皮性卵巢癌患者的预后密切相关(P0.05)。结论:c-Src在上皮性卵巢癌组织中过表达,且与肿瘤的恶性生物学行为密切相关,可能作为评估上皮性卵巢癌恶性程度、病情进展的重要指标及分子靶向疗法的新靶点。     

UBR5 targets tumor suppressor CDC73 proteolytically to promote aggressive breast cancer

UBR5, a HECT-domain E3 ubiquitin ligase, is an attractive therapeutic target for aggressive breast cancers. Defining the substrates of UBR5 is crucial for scientific understanding and clinical intervention. Here, we demonstrate that CDC73, a component of the RNA polymerase II-associated factor 1 complex, is a key substrate that impedes UBR5’s profound tumorigenic and metastatic activities in triple-negative breast cancer (TNBC) via mechanisms of regulating the expression of β-catenin and E-cadherin, tumor cell apoptosis and CD8⁺ T cell infiltration. Expression of CDC73 is also negatively associated with the progression of breast cancer patients. Moreover, we show that UBR5 destabilizes CDC73 by polyubiquitination at Lys²⁴³, Lys²⁴⁷, and Lys²⁵⁷ in a non-canonical manner that is dependent on the non-phosphorylation state of CDC73 at Ser⁴⁶⁵. CDC73 could serve as a molecular switch to modulate UBR5’s pro-tumor activities and may provide a potential approach to developing breast cancer therapeutic interventions.Subject terms: Breast cancer, Ubiquitylation, Tumour-suppressor proteins     

LAPTM4B Allele *2 Is a Marker of Poor Prognosis for Gallbladder Carcinoma

Background

Lysosomal protein transmembrane 4 beta (LAPTM4B) is a novel cancer-related gene which has two alleles designated LAPTM4B*1 and LAPTM4B*2. In this study we investigated the correlation of LAPTM4B genotype with prognosis and clinicopathologic features in patients who had undergone curative resection for gallbladder carcinoma (GBC).

Methodology/Principal Findings

PCR assay was performed to determine the LAPTM4B genotype in 85 patients. The correlation of LAPTM4B genotype with clinicopathologic parameters was assessed with the Chi-squared test. Differences in patient survival were determined by the Kaplan–Meier method. Multivariate analysis of prognostic factors was carried out with Cox regression analysis. Patients with LAPTM4B *2 had both significantly shorter overall survival (OS) and shorter disease-free survival (DFS) (both P<0.001). Multivariate analysis showed that LAPTM4B genotype is a prognostic factor for OS and DFS (both P<0.001).

Conclusions/Significance

LAPTM4B allele *2 is a risk factor associated with poor prognosis in patients with resected GBC, and LAPTM4B status may be therefore be useful preoperatively as an adjunct in evaluation of the operability of GBC.     

Combined p53/Bax mutation results in extremely poor prognosis in gastric carcinoma with low microsatellite instability

Gastric cancer is highly refractory to DNA-damaging therapies. We therefore studied both gene mutation and protein expression of p53 and Bax in a cohort of 116 patients with gastric cancer who underwent R0-resection with a curative intent. Bax mutation was independent from severe microsatellite instability (MSI), that is, global mismatch repair deficiency as determined by analysis of BAT-25/BAT-26 microsatellite markers. Thus, Bax-frameshift mutation is a feature of tumors with low MSI. In contrast and as expected, no p53 mutations were observed in the microsatellite instable tumors. p53 Mutation or p53 overexpression did not have an impact on disease prognosis. p53-Inactivation was, however, associated with an extremely poor prognosis in the subgroup of patients with Bax-mutated tumors. Thus, we show for the first time that the combined mutation of p53 and Bax, two key regulators of the mitochondrial apoptosis pathway, results in an extremely aggressive tumor biology and poor clinical prognosis.     

Heat Shock Protein 60 Overexpression Is Associated with the Progression and Prognosis in Gastric Cancer

Background

Heat shock protein 60 (HSP60) is a chaperonin with essential functions for cell physiology and survival, and its expression correlates with prognosis in a number of malignancies. The aim of this study is to determine the relationship of HSP60 status with clinicopathological parameters and prognosis in gastric cancer.

Methods

The levels of HSP60 and matrix metallopeptidase 9 (MMP-9) antigen was evaluated by immunohistochemistry in 223 gastric carcinoma samples. The association between HSP60 and MMP-9, clinicopathological parameters, and prognosis of gastric cancer was examined.

Results

The level of HSP60 protein was significantly associated with depth invasion, lymph node metastasis and stage of disease (all P<0.05). Both univariate and multivariate analyses revealed that HSP60 was an independent prognostic factor for both overall survival (OS) and recurrence-free survival (RFS) (both P<0.05). Furthermore, HSP60 overexpression was associated with a poor prognosis in patients with advanced gastric cancer in different risk groups. Moreover, HSP60 was significantly correlated with MMP-9 among 223 gastric cancer tissues (P<0.001). Patients who had HSP60 overexpression, in which tumor cells displayed high invasiveness, had poor OS and shorter RFS.

Conclusion

HSP60 plays an important role on tumor aggressiveness and prognosis, and may act as a promising target for prognostic prediction.     

SP/NK‐1R promotes gallbladder cancer cell proliferation and migration

Aberrant substance P/neurokinin‐1 receptor (SP/NK‐1R) system activation plays a critical role in various disorders, however, little is known about the expression and the detailed molecular mechanism of the SP and NK‐1R in gallbladder cancer (GBC). In this study, we firstly analyzed the expression and clinical significance of them in patients with GBC. Then, cellular assays were performed to clarify their biological role in GBC cells. Moreover, we investigated the molecular mechanisms regulated by SP/NK‐1R. Meanwhile, mice xenografted with human GBC cells were analyzed regarding the effects of SP/NK1R complex in vivo. Finally, patient samples were utilized to investigate the effect of SP/NK‐1R. The results showed that SP and NK‐1R were highly expressed in GBC. We found that SP strongly induced GBC cell proliferation, clone formation, migration and invasion, whereas antagonizing NK‐1R resulted in the opposite effects. Moreover, SP significantly enhanced the expression of NF‐κB p65 and the tumor‐associated cytokines, while, Akt inhibitor could reverse these effects. Further studies indicated that decreasing activation of NF‐κB or Akt diminished GBC cell proliferation and migration. In consistent with results, immunohistochemical staining showed high levels of Akt, NF‐κB and cytokines in tumor tissues. Most importantly, the similar conclusion was obtained in xenograft mouse model. Our findings demonstrate that NK‐1R, after binding with the endogenous agonist SP, could induce GBC cell migration and spreading via modulation of Akt/NF‐κB pathway.     

MicroRNA-326 Functions as a Tumor Suppressor in Glioma by Targeting the Nin One Binding Protein (NOB1)

Malignant glioma is the most common type of primary brain tumor in adults, characterized by rapid tumor growth and infiltration of tumor cells throughout the brain. Alterations in the activity of the 26S proteasome have been associated with malignant glioma cells, although the specific defects have not been identified. Recently, microRNA-326 (miR-326) was shown to play an important role in glioblastoma and breast cancer, but the underlying molecular mechanisms remain unclear. In the present study, the human Nin one binding protein (NOB1) was identified as a direct target of miR-326 and a potential oncogene in human glioma. Similar to NOB1 silencing by shRNA, overexpression of miR-326 in human glioma cell lines (A172 and U373) caused cell cycle arrest at the G1 phase, delayed cell proliferation and enhanced apoptosis. MiR-326 inhibited colony formation in soft agar and decreased growth of a xenograft tumor model, suggesting that miR-326 and NOB1 are required for tumorigenesis in vitro and in vivo. Furthermore, these processes were shown to involve the MAPK pathway. NOB1 overexpression in human glioma samples was detected by Affymetrix array analysis, and NOB1 mRNA and protein levels were shown to be increased in high-grade glioma compared to low-grade glioma and normal brain tissue. Furthermore, high levels of NOB1 were associated with unfavorable prognosis of glioma patients. Taken together, these results indicate that miR-326 and NOB1 may play an important role in the development of glioma.     

胃癌中RhoC和MMP-9的表达及其临床意义

目的探讨RhoC基因和基质金属蛋白酶9(matrixmetalloproteinases-9,MMP-9)基因在胃癌中的表达及其与临床病理因素之间的关系。方法采用免疫组化SP法检测120例胃癌组织中RhoC和MMP-9的表达。结果120例胃癌组织中RhoC和MMP-9阳性率分别为80.00%(96/120)、85.83%(103/120),两者表达存在相关性(P<0.05)。RhoC和MMP-9表达与胃癌的浸润深度、淋巴结转移和肿瘤临床分期有明显差异(P<0.05)。结论RhoC和MMP-9蛋白在胃癌中过量表达与胃癌的发生发展密切相关,检测RhoC和MMP-2蛋白表达对于评估胃癌的预后有一定意义。     

Overexpression of leucine-rich repeat-containing G protein-coupled receptor 5 predicts poor prognosis in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignancies and the fifth leading cause of cancer-related death worldwide. Novel prognostic biomarkers are urgently needed for patients with HCC. Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) overexpression may promote tumor metastasis in HCC. However, few studies investigate the prognosis predictive role of LGR5 in patients with HCC. Herein, we aimed to examine the expression level of LGR5 in tumors and its correlation with clinical characteristics and survivals of patients with HCC. LGR5 expression in tumor specimens and adjacent tissue resected from 66 patients were detected by immunohistochemistry. The results showed that the expression of LGR5 was markedly higher in HCC than in normal adjacent tissues (P?=?.006). High expression of LGR5 was significantly correlated with later disease stage (P?=?.009). In addition, high LGR5 expression was remarkably correlated with short overall survival than those with low LGR5 expression (P?<?.05). The median overall survival of patients with high LGR5 expression was 12?months, whereas that of patients with low LGR5 expression was still not reached (longer than 70?months). Notably, in our limited cases, we did not detect any difference in tumor size, lymphatic invasion, or metastasis in patients with high or low expression of LGR5. In conclusion, high protein level of LGR5 was associated with poor prognosis of these patients. LGR5 appears to be a valuable prognostic predictor clinically and a potential target in HCC therapy.     

Role of Sciellin in gallbladder cancer proliferation and formation of neutrophil extracellular traps

Apart from primary tumor development and metastasis, cancer-associated thrombosis is the second cause of cancer death in solid tumor malignancy. However, the mechanistic insight into the development of gallbladder cancer (GBC) and cancer-associated thrombosis remains unclear. This study aimed to investigate the mechanistic role of Sciellin (SCEL) in GBC cell proliferation and the development of venous thromboembolism. The expression level of SCEL was determined by immunohistochemical staining. Roles of SCEL in gallbladder cancer cell were determined by molecular and cell biology methods. SCEL was markedly upregulated in GBC and associated with advanced TNM stages and a poor prognosis. Furthermore, SCEL interacted with EGFR and stabilized EGFR expression that activates downstream PI3K and Akt pathway, leading to cell proliferation. In addition, SCEL induces tumor cell IL-8 production that stimulates the formation of neutrophil extracellular traps (NETs), accelerating thromboembolism. In xenografts, SCEL-expressing GBCs developed larger tumors and thrombosis compared with control cells. The present results indicate that SCEL promotes GBC cell proliferation and induces NET-associated thrombosis, thus serving as a potential therapeutic target.Subject terms: Cancer microenvironment, Tumour biomarkers