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This study suggests that sesamin can restrain NF-κB p65 to activate HIF-1α transcription under hypoxic conditions, thereby inhibiting VEGFA expression and effectively suppressing colorectal tumor angiogenesis.

Figure S4 shows the cellular location of PTBP3 in breast cancer cells

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Purpose: To investigate the expression pattern and significance of DNA repair genes JWA and X-ray repair cross complement group 1 (XRCC1) in gastric cancer.Experimental Design: Expressions of JWA and XRCC1 were assessed by immunohistochemistry in a training cohort and they went into a second testing cohort and finally to a validating cohort. Prognostic and predictive role of JWA and XRCC1 expression status in cases treated with surgery alone or combined with adjuvant chemotherapy was evaluated, respectively.Results: JWA and XRCC1 protein levels were significantly downregulated in gastric cancer lesions compared with adjacent noncancerous tissues. Low tumoral JWA or XRCC1 expression significantly correlated with shorter overall survival (OS), as well as with clinicopathologic characteristics in patients without adjuvant treatment. Multivariate regression analysis showed that low JWA and XRCC1 expressions, separately and together, were independent negative markers of OS. Adjuvant fluorouracil-leucovorin-oxaliplatin (FLO) significantly improved OS compared with surgery alone (log-rank test, P = 0.01). However, this effect was evident only in the JWA or XRCC1 low expression group (HR = 0.44; 95% CI: 0.26–0.73; P = 0.002, and HR = 0.44, 95% CI: 0.26–0.75; P = 0.002, respectively); Adjuvant fluorouracil-leucovorin-platinol (FLP) did not improve OS, except in the patients with low JWA and XRCC1 expressions (P = 0.010 for JWA and 0.024 for XRCC1, respectively).Conclusions: JWA and XRCC1 protein expressions in tumor are novel candidate prognostic markers and predictive factors for benefit from adjuvant platinum-based chemotherapy (FLO or FLP) in resectable human gastric carcinoma. Clin Cancer Res; 18(10); 2987–96. ©2012 AACR.

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Supplemental figure legends and experimental procedures

The RNA polypyrimidine tract-binding protein PTBP3 is a little studied paralog of PTBP1, which has oncogenic properties. In this study, we demonstrate that PTBP3 induces epithelial–mesenchymal transition (EMT) in breast tumor cells and promotes their invasive growth and metastasis. Elevated expression of PTBP3 associated significantly with lymph node metastasis, advanced histology grade, TNM stage, and poor 5-year overall survival of patients. In human mammary epithelial cells, PTBP3 overexpression was sufficient to induce EMT and to enhance cell migration, invasion, and cancer stem-like cell properties. PTBP3 regulated expression of the EMT regulatory transcription factor ZEB1 by binding the 3′UTR of its mRNA, thereby preventing its degradation. Conversely, ZEB1 ablation blocked the ability of PTBP3 to induce EMT. Overall, our findings define PTBP3 as a regulator of EMT that acts by governing expression of ZEB1, and they establish an oncogenic function of PTBP3, suggesting its candidacy as a theranostic target.Significance: These findings define PTBP3 as a regulator of EMT that acts by governing expression of ZEB1, and they establish an oncogenic function of PTBP3, suggesting its candidacy as a theranostic target. Cancer Res; 78(2); 387–98. ©2017 AACR.

Cigarette smoking greatly promotes the progression of kidney renal clear cell carcinoma (KIRC), however, the underlying molecular events has not been fully established. In this study, RCC cells were exposed to the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, nicotine-derived nitrosamine ketone) for 120 days, and then the soft agar colony formation, wound healing and transwell assays were used to explore characteristics of RCC cells. RNA-seq was used to explore differentially expressed genes. We found that NNK promoted RCC cell growth and migration in a dose-dependent manner, and RNA-seq explored 14 differentially expressed genes. In TCGA-KIRC cohort, Lasso regression and multivariate COX regression models screened and constructed a five-gene signature containing ANKRD1, CYB5A, ECHDC3, MT1E, and AKT1S1. This novel gene signature significantly associated with TNM stage, invasion depth, metastasis, and tumor grade. Moreover, when compared with individual genes, the gene signature contained a higher hazard ratio and therefore had a more powerful value for the prognosis of KIRC. A nomogram was also developed based on clinical features and the gene signature, which showed good application. Finally, AKT1S1, the most crucial component of the gene signature, was significantly induced after NNK exposure and its related AKT-mTOR signaling pathway was dramatically activated. Our findings supported that NNK exposure would promote the KIRC progression, and the novel cigarette smoke-related five-gene signature might serve as a highly efficient biomarker to identify progression of KIRC patients, AKT1S1 might play an important role in cigarette smoke exposure-induced KIRC progression.

Ovarian cancer is the fifth common cause of death in woman worldwide. The tripartite motif-containing (TRIM) proteins consist of more than 70 known protein members. Studies have showed that TRIM proteins are involved in cancer and play important roles in cancer cell proliferation, migration, adhesion and metastasis. Recent studies have indicated that TRIM59, as a putative ubiquitin ligase, is up-regulated in some cancers and associated with poor prognosis of gastric cancer. However, the exact roles of TRIM59 in ovarian cancer are still unknown. In this study, we found that TRIM59 expression was increased and positively associated with histological grades (P = 0.000), FIGO stages (P = 0.016), and metastasis (P = 0.027) in ovarian cancer. A integrative data analysis tool revealed that ovarian cancer patients with high TRIM59 expression were correlated with more unfavorable overall and progression-free survival than the rest patients with low TRIM59 expression (P = 0.0024 and P = 7.5×10-6, respectively). Based on the finding in the clinical data, we performed a series of cell line and animal experiments, and found that TRIM59 knockdown could significantly inhibit the ovarian cancer cell proliferation, clone formation, and invasion in vitro and the ovarian cancer growth of the subcutaneous and orthotopic implantation in vivo. Furthermore, TRIM59 was found to interact with Annexin A2 and induce Annexin A2 expression. Our data imply that TRIM59 can serve as a promising prognostic marker and a potential therapeutic target.

Increasing evidence indicates that inflammatory microenvironment facilitates tumor metastasis. Here, we found that LFG-500, a novel synthetic flavonoid, significantly inhibited epithelial–mesenchymal transition (EMT) in human lung adenocarcinoma A549 and H1299 cells co-cultured with LPS-challenged THP-1 cells or cultured in THP-1 cell-derived conditioned medium. Moreover, we found that TNF-α is a direct and decisive factor for promoting EMT and LFG-500 suppressed TNF-α-induced EMT and cell motility. NLRP3 knockdown inactivated NLRP3 inflammasome, which subsequently inhibited EMT and blocked cell migration, indicating that TNF-α-induced EMT requires the NLRP3 inflammasome. LFG-500 inhibited the activation of the NLRP3 inflammasome, thus inhibiting EMT. Moreover, LFG-500 treatment significantly inhibited metastasis in vivo by downregulating NLRP3 expression. Importantly, we found that NLRP3 was highly expressed in high-grade lung adenocarcinoma and that its expression was correlated with lymph node metastasis. NLRP3 and vimentin levels were significantly increased in matched metastatic lymph nodes. Moreover, a significant positive correlation was observed between their levels. Together, these results suggest that LFG-500 markedly suppresses EMT by inhibiting the NLRP3 inflammasome in the inflammatory microenvironment and that NLRP3 is a potential biomarker of lung adenocarcinoma metastasis.

Table S2. Multvariate Cox regression analysis of PTBP3 expression on 5-year overall and disease specific survival of CRC patients. (DOCX 15 kb)

Table S1. Univariate Cox regression analysis of PTBP3 expression and clinicopathologic variables predicting the survival of CRC patients. (DOCX 16 kb)

Several studies have been conducted to examine the association between CD44 expression and the prognosis of gastric cancer (GC). However, the conclusions remain controversial. We conducted a meta-analysis study of 16 published studies with 2403 patients to evaluate the correlation between CD44 expression and clinicopathological characteristics and overall survival of the GC patients. Pooled odds ratios (ORs) and their 95% confidence intervals (CIs) were used to assess the correlation of CD44 expression with the clinicopathological features of GC patients. Hazard ratios (HRs) with their 95% confidence intervals (CIs) were used to assess the association between CD44 and prognosis of GC patients. Total CD44 expression was detected in ten studies, and CD44v5 and CD44v6 expressions were detected in one and five papers, respectively. The results revealed that CD44 expression was associated with some clinicopahological features, such as lymph node metastasis (pooled OR = 1.81, 95% CI = 1.44–2.34, P = 0.000), distant metastasis (pooled OR = 3.29, 95% CI = 1.90–5.67, P = 0.001) and TNM stage (Pooled OR = 1.84, 95% CI = 1.13–2.99, P = 0.014). Moreover, we also found that GC patients with positive CD44 expression had a worse prognosis than the ones with negative CD44 expression (HR = 1.93, 95% CI = 1.54–2.42, P = 0.000). In stratified analysis, the combined HR with CD44 and CD44v6 was 2.20 (95% CI = 1.81–2.67) and 1.70 (95% CI = 1.00–2.90), respectively. These results suggested that positive CD44 expression could predict a lower overall survival rate and could be an independent dangerous prognostic factor in GC patients.

CD133 has been identified as a potential cancer stem cell (CSC) marker in non-small cell lung cancer (NSCLC). However, the clinical and prognostic significance of CD133 in NSCLC remains controversial. In this study, a meta-analysis with a total number of 13 studies was performed to clarify the association between CD133 expression and clinical outcomes in publications up to June 2013. Odds ratios (ORs) and their 95 % confidence intervals (CIs) were used to assess the association between CD133 expression and the clinicopathological characteristics of NSCLC. Hazard ratios (HRs) and their 95 % CI were used to quantify the predictive ability of CD133 on NSCLC prognosis. Analysis of these data showed that CD133 expression was not associated with any clinicopathological parameters except for histology (pooled OR = 1.35, 95%CI = 1.04-1.76, P = 0.024) and tumor differentiation (pooled OR = 3.19, 95%CI = 1.10-9.21, P = 0.032). Simultaneously, we also found that positive CD133 expression was not associated with disease-free survival (DFS) (pooled HR = 1.76, 95 % CI = 0.87-3.57, P = 0.114, random-effect) but was associated with overall survival (OS) (pooled HR = 2.06, 95 % CI = 1.08-3.91, P = 0.027, random-effect). Overall, it is appropriate to regard CD133 expression as a potential prognostic factor for the OS of NSCLC patients.

To investigate the potential role of SOX2 in gastric cancer (GC) metastasis. The SOX2 expression was detected using immunohistochemistry on a GC tissue microarray. The correlations of SOX2 expression with clinicopathological factors and 5-year survival were evaluated. To test the role of SOX2 in inhibiting GC metastasis, the cell transwell assay was performed. Real-time PCR and Western blot were used to explore the possible mechanism that SOX2 inhibits GC metastasis. In the present study, SOX2 expression was downregulated in GC tissues when compared to matching normal tissues. Moreover, patients with high SOX2 expression in cancerous tissues had less lymph node metastasis and better treatment outcome. At the subcellular level, SOX2 inhibited the GC cell migration and invasion by upregulating p21 expression. Moreover, SOX2 was determined to associate with the nuclear p21 expression. GC patients with high SOX2 and nuclear p21 expression had synergistically less lymph node metastasis and the better overall survival. Our results suggest that SOX2 is a promising and favorable metastatic biomarker for GC.

JWA, a multifunctional microtubule-binding protein, plays an important role in regulating tumor metastasis via inhibition of matrix metalloproteinase-2 (MMP-2). Recent investigations suggest that MMP-2 is an angiogenesis-associated molecule. In this study, we provide novel evidence that JWA inhibits tumor angiogenesis in gastric cancer (GC). In two independent retrospective GC cohorts, we found that the expression of JWA was downregulated and that of MMP-2 was upregulated in GC tissues compared with the same in normal gastric mucosa. For patients treated with surgery alone, a strong and independent negative prognostic value was shown for low JWA and high MMP-2 expressions separately, which was even stronger when combined (hazard ratio = 7.75, P < 0.001, in the training cohort; hazard ratio = 2.31, P < 0.001, in the validation cohort). Moreover, we found that loss of JWA expression was strongly correlated with increased GC angiogenesis. In vitro, JWA inhibited MMP-2 at both messenger RNA and protein levels by modulating Sp1 activity. Knockdown of endogenous JWA resulted in enhanced human umbilical vein endothelial cell tube formation and MMP-2 expression. Furthermore, JWA was found to inhibit Sp1 activity via an ubiquitin-proteasome-dependent mechanism and to downregulate the expression of the proangiogenic MMP-2. Our findings imply that JWA and MMP-2 may serve as promising prognostic markers in resectable GC, with JWA as a useful biomarker of angiogenesis in GC and a potential therapeutic target by MMP-2 modulation.

Previous studies have revealed that expression of allograft inflammatory factor-1 (AIF-1) protein appears to be increased in malignancies and is correlated with a poorer prognosis in cervical cancer, while its role in gastric cancer has not been reported. We analyzed the expression of AIF-1 in 78 cancer lesions and the corresponding non-cancerous tissues by immunohistochemistry. In contrast with other cancers, we found that AIF-1 protein levels were significantly decreased in 53 of the 78 (67.9%) gastric cancer tissues when compared with the matched normal tissues. This was further confirmed using 7 pairs of fresh gastric cancer tissues and matched adjacent normal tissues. Low tumoral AIF-1 expression was significantly correlated with less favorable clinicopathological characteristics, as well as with reduced overall survival (P

Protein p21Cip1/Waf1 is a cyclin-dependent kinase inhibitor, which plays important roles in cell cycle arrest, senescence, and apoptosis. Interestingly, the nuclear and cytoplasmic p21 executes various functions in the cell. In this study, we investigated the prognostic impact of subcellular p21 expression in gastric cancer (GC). Expressions of subcellular p21 was assessed by immunohistochemistry using a tissue microarray in a training cohort and it went into a second testing cohort and finally to a validating cohort. Prognostic and predictive role of subcellular p21 expression status was evaluated. We also studied the roles of subcellular p21 in GC cell migration and invasion. Nuclear and cytoplasmic p21 protein levels were significantly reduced and increased in GC lesions compared with adjacent non-cancerous tissues, respectively. Low nuclear p21 or high cytoplasmic p21 expression significantly correlated with shorter overall survival (OS), as well as with clinicopathologic characteristics in patients. Multivariate regression analysis showed that low nuclear and high cytoplasmic p21 expression, separately and together, were independent negative markers of OS. Finally, we found that nuclear p21 inhibits but cytoplasmic p21 promotes cell migration and invasion abilities. These findings suggest that nuclear and cytoplasmic p21 protein expression in tumor are novel candidate prognostic markers in resectable human gastric carcinoma, and they exert distinct roles in cell migration and invasion.

Background The ubiquitous use of dibutyl phthalate (DBP), one of the most widely used plasticizers, results in extensive exposure to humans and the environment. DBP and its major metabolite, monobutyl phthalate (MBP), may alter steroid biosynthesis and their exposure may lead to damage to male reproductive function. Low-doses of DBP/MBP may result in increased steroidogenesis in vitro and in vivo. However, the mechanisms of possible effects of low-dose MBP on steroidogenesis remain unclear. The aim of present study was to elaborate the role of transcription factors and steroidogenic acute regulatory protein in low-dose MBP-induced distruption of steroidogenesis in mouse Leydig tumor cells (MLTC-1 cells). Methods In the present study, MLTC-1 cells were cultured in RPMI 1640 medium supplemented with 2 g/L sodium bicarbonate. Progesterone level was examined by I125-pregesterone Coat-A-Count radioimmunoassay (RIA) kits. mRNA and protein levels were assessed by reverse transcription-polymerase chain reaction (RT-PCR) and western blot, respectively. DNA-binding of several transcription factors was examined by electrophoretic mobility shift assay (EMSA). Results In this study, various doses of MBP (0, 10(−9), 10(−8), 10(−7), or 10(−6) M) were added to the medium followed by stimulation of MLTC-1 cells with human chorionic gonadotrophin (hCG). The results showed that MBP increased progesterone production and steroidogenic acute regulatory protein (StAR) mRNA and protein levels. However, the protein levels of cytochrome P450scc and 3 beta-hydroxy-steroid dehydrogenase (3 beta-HSD) were unchanged after MBP treatment. EMSA assay showed that DNA-binding of steroidogenic factors 1(SF-1), GATA-4 and CCAAT/enhancer binding protein-beta (C/EBP-beta) was increased in a dose-dependent manner after MBP exposure. Western blot tests were next employed and confirmed that the protein levels of SF-1, GATA-4 and C/EBP-beta were also increased. Additionally, western blot tests confirmed the expression of DAX-1, negative factor of SF-1, was dose-dependently down regulated after MBP exposure, which further confirmed the role of SF-1 in MBP-stimulated steroid biosynthesis. Conclusions In conclusion, we firstly delineated the regulation of StAR by transcription factors including SF-1, GATA-4 and C/EBP-beta maybe critical mechanism involved in low-dose MBP-stimulated steroidogenesis.

Expression of MDM2 protein appears to be increased in malignancy and correlated to prognosis of tumors, but its role in gastric cancer remains controversial. Our recent investigations indicated that JWA was a novel candidate biomarker for gastric cancer. To evaluate the impact of MDM2 protein expression alone, and in combination with JWA, on the prognostic and predictive of patients with resectable gastric cancer, expression of MDM2 and JWA were examined by immunohistochemistry in three large cohorts (total n = 1131) of patient with gastric cancer. We found that MDM2 protein levels were significantly upregulated in gastric cancer (70.4%, 57 of 81) compared with adjacent non‐cancerous tissues. High tumoral MDM2 expression significantly correlated with clinicopathologic characteristics, as well as with shorter overall survival (OS; P < 0.001 for all cohorts) in patients without adjuvant treatment. The effect of adjuvant fluorouracil–leucovorin–oxaliplatin (FLO) in improving OS compared with surgery alone was evident only in the high MDM2 group (hazard ratio = 0.57; 95% confidence interval, 0.37–0.89; P = 0.013). Furthermore, knockdown of MDM2 and overexpression of JWA had a synergistic effect on suppression of gastric cancer cell proliferation and migration. Patients with low MDM2 and high JWA expression had a better outcome of survival compared with the other groups (P < 0.001 for all cohorts). For the first time, our data suggest that MDM2 is a potent prognostic and predictive factor for benefit from adjuvant fluorouracil–leucovorin–oxaliplatin chemotherapy in resectable gastric cancer. The combination of MDM2 expression and JWA could serve as a more effective candidate prognostic biomarker for gastric cancer. Open Access article. Published by Wiley Open Access 2013.

The multifunctional protein JWA was previously identified as a novel regulator of focal adhesion kinase (FAK/PTK2) in suppressing cancer cell adhesion, invasion and metastasis. JWA is downregulated in gastric cancer (GC) and a prognostic and predictive biomarker for resectable GC. However, the value of FAK combined with JWA for GC patients as a biomarker has not been studied. Here we evaluated the roles of FAK alone and combined with JWA in GC patients treated with surgery alone or combined with adjuvant platinum-based chemotherapy. Two tissue microarrays were constructed of specimens from resected GC (n = 709 in total) for detection of FAK and JWA expression by immunohistochemistry. Correlations between both proteins and clinicopathological features as well as prognostic and predictive values were evaluated. Compared with adjacent non-cancerous tissues, FAK protein levels were remarkably up-regulated in GC lesions (P

Objective CHIP (carboxy terminus of Hsc70 interacting protein) is an E3 ubiquitin ligase that can induce ubiquitination and degradation of several tumour related proteins, and acts as a suppressor of tumour metastasis. This study explored the biological function and clinical significance of CHIP in gastric cancer (GC). Methods The prognostic value of CHIP expression was evaluated using tissue microarray and immunohistochemical staining in two independent human GC cohorts. The role of CHIP on tumorigenicity and angiogenesis was determined in vitro and in vivo. Results CHIP expression was significantly decreased in GC lesions compared with paired non-cancerous tissues. Low tumoral CHIP expression significantly correlated with clinicopathological characteristics in patients, as well as with shorter overall survival in both cohorts. Multivariate Cox regression analysis revealed that CHIP expression was an independent prognostic factor for human GC patients. Moreover, CHIP overexpression impeded the formation of anchorage independent colonies in soft agar, suppressed the growth of xenografts in nude mice and inhibited endothelial cell growth and tube formation by suppressing nuclear factor κB (NF-κB) mediated interleukin 8 (IL-8) expression in vitro. In vivo studies also confirmed that CHIP inhibited blood vessel formation and recruitment of CD31 positive cells in matrigel plugs. Also, CHIP interacted with NF-κB/p65 and promoted its ubiquitination and degradation by proteasome, terminating NF-κB activity and inhibiting IL-8-induced angiogenesis, which correlated with subsequent tumour metastasis. Conclusions Decreased CHIP expression in GC resulted in increased angiogenesis and contributed to GC progression and poor prognosis. CHIP expression is a GC candidate clinical prognostic marker and a putative treatment target.

Purpose: To investigate the expression pattern and significance of DNA repair genes JWA and X-ray repair cross complement group 1 (XRCC1) in gastric cancer. Experimental Design: Expressions of JWA and XRCC1 were assessed by immunohistochemistry in a training cohort and they went into a second testing cohort and finally to a validating cohort. Prognostic and predictive role of JWA and XRCC1 expression status in cases treated with surgery alone or combined with adjuvant chemotherapy was evaluated, respectively. Results: JWA and XRCC1 protein levels were significantly downregulated in gastric cancer lesions compared with adjacent noncancerous tissues. Low tumoral JWA or XRCC1 expression significantly correlated with shorter overall survival (OS), as well as with clinicopathologic characteristics in patients without adjuvant treatment. Multivariate regression analysis showed that low JWA and XRCC1 expressions, separately and together, were independent negative markers of OS. Adjuvant fluorouracil-leucovorin-oxaliplatin (FLO) significantly improved OS compared with surgery alone (log-rank test, P = 0.01). However, this effect was evident only in the JWA or XRCC1 low expression group (HR = 0.44; 95% CI: 0.26–0.73; P = 0.002, and HR = 0.44, 95% CI: 0.26–0.75; P = 0.002, respectively); Adjuvant fluorouracil-leucovorin-platinol (FLP) did not improve OS, except in the patients with low JWA and XRCC1 expressions (P = 0.010 for JWA and 0.024 for XRCC1, respectively). Conclusions: JWA and XRCC1 protein expressions in tumor are novel candidate prognostic markers and predictive factors for benefit from adjuvant platinum-based chemotherapy (FLO or FLP) in resectable human gastric carcinoma. Clin Cancer Res; 18(10); 2987–96. ©2012 AACR.