CD44s regulates the TGF-β-mediated mesenchymal phenotype and is associated with poor prognosis in patients with hepatocellular carcinoma

The prognosis for individuals diagnosed with hepatocellular carcinoma (HCC) remains poor because of the high frequency of invasive tumor growth, intrahepatic spread, and extrahepatic metastasis. Here, we investigated the role of the standard isoform of CD44 (CD44s), a major adhesion molecule of the extracellular matrix and a cancer stem cell marker, in the TGF-β-mediated mesenchymal phenotype of HCC. We found that CD44s was the dominant form of CD44 mRNA expressed in HCC cells. Overexpression of CD44s promoted tumor invasiveness and increased the expression of vimentin, a mesenchymal marker, in HCC cells. Loss of CD44s abrogated these changes. Also in the setting of CD44s overexpression, treatment with TGF-β1 induced the mesenchymal phenotype of HCC cells, which was characterized by low E-cadherin and high vimentin expression. Loss of CD44s inhibited TGF-β-mediated vimentin expression, mesenchymal spindle-like morphology, and tumor invasiveness. Clinically, overexpression of CD44s was associated with low expression of E-cadherin, high expression of vimentin, a high percentage of phospho-Smad2-positive nuclei, and poor prognosis in HCC patients, including reduced disease-free and overall survival. Together, our findings suggest that CD44s plays a critical role in the TGF-β-mediated mesenchymal phenotype and therefore represents a potential therapeutic target for HCC.     

Co-expression of CXCL8 and HIF-1α is associated with metastasis and poor prognosis in hepatocellular carcinoma

Hypoxia inducible factor-1α (HIF-1α), induces cytokines such as CXCL8 and tumor dissemination, chemo- and radio-resistance. We analyzed correlation between HIF-1α and CXCL8 levels, tumor characteristics and overall survival in 102 hepatocellular carcinoma (HCC) patients. Levels of HIF-1α and CXCL8 were increased in HCC tissues and cell lines. Patients with high levels of HIF-1α and CXCL8 had worse outcome and poorer prognosis than those with lower levels. Co-overexpression of HIF-1α and CXCL8 was an independent negative prognostic factor for overall and disease-free survival. HIF-1α silencing and CXCL8 siRNA decreased migration under hypoxic conditions in vitro. Hypoxia-induced activation of AKT/mTOR/STAT3 pathways was reversed by depletion of CXCL8. We conclude that HIF-1α and CXCL8 induce HCC progression and metastasis, associated with activation of AKT/mTOR/STAT3. Co-expression of HIF-1α and CXCL8 is a prognostic marker and a potential therapeutic target in HCC.     

A high HIF-1α expression genotype is associated with poor prognosis of upper aerodigestive tract carcinoma patients

The aim of the present study was to evaluate the role of HIF-1α genetic polymorphisms and protein expression in the development of metastasis in upper aerodigestive tract cancer (UADTC) patients. The expression of pro-angiogenic markers was also evaluated. Protein expression was analysed using immunohistochemistry, and RFLP analysis was used to investigate HIF-1α C1779T and G1790A polymorphisms in 52 patients with UADTC. Primary lesions were divided into 2 groups according to the absence or presence of metastasis. Lymph node samples were divided into 3 groups: metastatic lymph nodes, non-metastatic lymph nodes (both derived from patients with metastatic disease), and control lymph nodes, which were obtained from patients without any metastasis. The allele T was more frequently found in patients with metastatic disease. HIF-1α protein expression in the lymph nodes was increased in the presence of the T allele. Metastatic lymph nodes showed lower levels of HIF-1α, VEGFR1, and MMP-9 proteins compared to lymph nodes without metastasis, while VEGFR2 protein levels were increased. In agreement, HIF-1α expression was correlated with MMP-9. Cox regression analysis demonstrated that higher HIF-1α and MMP-9 protein expression levels and GA and GG genotypes were associated with poor survival. Our findings show that the C1772T and G1790A polymorphisms of the HIF-1α gene are associated with increased expression of the HIF-1α protein in UADTC. The present data indicate that non-metastatic tissues express higher levels of HIF-1α, VEGFR1, and MMP-9, while in metastatic lymph nodes, VEGFR2 protein expression is elevated. The present study also shows that the HIF-1α G1790A polymorphism and its protein expression have an impact on the prognosis of UADTC patients.     

ΔNp63 isoform-mediated β-defensin family up-regulation is associated with (lymph)angiogenesis and poor prognosis in patients with squamous cell carcinoma

Beside a role in normal development/differentiation, high p63 immunoreactivity is also frequently observed in squamous cell carcinoma (SCC). Due to the complexity of the gene, the role of each p63 isotype in tumorigenesis is still confusing. Constitutively produced or induced in inflammatory conditions, human beta-defensins (HβDs) are cationic peptides involved in host defenses against bacteria, viruses and fungi. Here, we investigated both the role of p63 proteins in the regulation of HβDs and the implication of these antimicrobial peptides in tumor (lymph)angiogenesis. Thus, in contrast to TAp63 isotypes, we observed that ΔNp63 proteins (α, β, γ) induce HβD1, 2 and 4 expression. Similar results were observed in cancer tissues and cell lines. We next demonstrated that ΔNp63-overexpressing SCC are associated with both a poor prognosis and a high tumor vascularisation and lymphangiogenesis. Moreover, we showed that HβDs exert a chemotactic activity for (lymphatic) endothelial cells in a CCR6-dependent manner. The ability of HβDs to enhance (lymph)angiogenesis in vivo was also evaluated. We observed that HβDs increase the vessel number and induce a significant increase in relative vascular area compared to negative control. Taken together, the results of this study suggest that ΔNp63-regulated HβD could promote tumor (lymph)angiogenesis in SCC microenvironment.     

Hypermethylation of RARβ2 correlates with high COX-2 expression and poor prognosis in patients with colorectal carcinoma

Silencing of gene expression by aberrant methylation at the CpG islands is common in human tumors, including colorectal cancer. This epigenetic alteration affects promoter of genes having crucial cellular functions such as tumor suppressor, DNA repair, apoptosis, cell adhesion, etc. We investigated the methylation status in the promoter regions of the RARβ2, RASSF1A, DAPKinase, and CDH1 genes in 73 colorectal carcinoma and 43 paired normal tissues of Tunisian patients using methylation-specific PCR assays. The association between methylation status and the clinicopathological features was evaluated. To determine whether aberrant methylation affects gene expression, we performed immunohistochemistry analysis for E-cadherin and COX-2, a target gene of RARβ2. The methylation frequencies vary from 80.8% for RARβ2 to 35.6% for RASSF1A while in non-tumor-paired samples; the frequencies of methylation are significantly lower for all the fourth genes tested. The methylation status did not correlate with any of the clinical features considered; however, aberrant methylation of RARβ2 was associated with a shortened overall patients’ survival (p logrank?=?0.026); nevertheless, it needs to be confirmed on larger sample size. Moreover, a significant inverse association was observed between methylation status of RARβ2 and COX-2 protein expression in tumor specimen (p?=?0.014). On the other hand, we found that loss of E-cadherin expression was significantly associated with aberrant methylation of the CDH1 promoter (p?=?0.005). Our findings showed that RARβ2 was frequently methylated in colorectal cancer and correlated with a worse prognosis and high expression of COX-2 suggesting a link between these two proteins in colorectal carcinogenesis. We also showed that epigenetic alteration of CDH1 is a major mechanism of the loss of E-cadherin protein expression in primary colorectal tumors.     

High expression of Cdc25B and low expression of 14-3-3σ is associated with the development and poor prognosis in urothelial carcinoma of bladder

Cdc25 dual-specicity phosphatases are essential regulators at critical stages of cell cycle. Cdc25B is overexpressed in several human tumor types. The activity of Cdc25B is regulated by 14-3-3 dimer. To investigate the roles of Cdc25B and 14-3-3σ in bladder carcinoma, we examined expressions of Cdc25B and 14-3-3σ proteins in bladder carcinoma and cell lines and analyzed their roles in the development and prognosis of urinary bladder carcinoma. Immunohistochmistry was used to detect the expressions of Cdc25B and 14-3-3σ in 105 bladder carcinomas. Moreover, expressions of Cdc25B and 14-3-3σ were analyzed by real-time PCR and Western blot in 40 bladder carcinomas and 20 normal epithelial tissues. Specific siRNA was used to knockdown the expression of Cdc25B or 14-3-3σ. Wild-type plasmid was used to overexpress 14-3-3σ. MTT assay and Flow cytometry were used to examine proliferation and cell cycle of bladder cancer cells. There were higher Cdc25B expression and lower 14-3-3σ expression in carcinomas than in the adjacent normal tissues (P?<?0.05), positive and negative correlations being noted with clinical stage and histopathologic grade. Cdc25B expression was positively correlated with recurrence and poor prognosis. Downregulation of Cdc25B resulted in slower growth, more G2/M cells and 14-3-3σ increasing. However, upregulation and downregulation of 14-3-3σ did not affect cell growth and Cdc25B expression. It showed that Cdc25B upregulation and 14-3-3σ downregulation might promote development of bladder cancer and suggested a poor prognosis. Moreover, Cdc25B could play an important role on the bladder cancer cell proliferation and cell cycle progression and regulate expression of 14-3-3σ.     

Bone metastases in patients with metastatic renal cell carcinoma: are they always associated with poor prognosis?

Purpose

Aim of this study was to investigate for the presence of existing prognostic factors in patients with bone metastases (BMs) from RCC since bone represents an unfavorable site of metastasis for renal cell carcinoma (mRCC).

Materials and methods

Data of patients with BMs from RCC were retrospectively collected. Age, sex, ECOG-Performance Status (PS), MSKCC group, tumor histology, presence of concomitant metastases to other sites, time from nephrectomy to bone metastases (TTBM, classified into three groups: <1 year, between 1 and 5 years and >5 years) and time from BMs to skeletal-related event (SRE) were included in the Cox analysis to investigate their prognostic relevance.

Results

470 patients were enrolled in this analysis. In 19 patients (4%),bone was the only metastatic site; 277 patients had concomitant metastases in other sites. Median time to BMs was 16 months (range 0 − 44y) with Median OS of 17 months. Number of metastatic sites (including bone, p = 0.01), concomitant metastases, high Fuhrman grade (p < 0.001) and non-clear cell histology (p = 0.013) were significantly associated with poor prognosis. Patients with TTBM >5 years had longer OS (22 months) compared to patients with TTBM <1 year (13 months) or between 1 and 5 years (19 months) from nephrectomy (p < 0.001), no difference was found between these two last groups (p = 0.18). At multivariate analysis, ECOG-PS, MSKCC group and concomitant lung or lymph node metastases were independent predictors of OS in patients with BMs.

Conclusions

Our study suggest that age, ECOG-PS, histology, MSKCC score, TTBM and the presence of concomitant metastases should be considered in order to optimize the management of RCC patients with BMs.     

Overexpression of nuclear β-catenin at invasive front in rectal carcinoma is associated with lymph node metastasis and poor prognosis

Background

This study aims to investigate whether nuclear β-catenin overexpression at invasive front in rectal carcinoma is associated with lymph node metastasis and prognosis.

Methods

Immunohistochemistry was adopted to detect the expression of β-catenin in rectal carcinoma and lymph node metastatic lesions. Spearman’s rank correlation analysis and Tukey’s test were used to evaluate the association between nuclear β-catenin expression at invasive front in rectal carcinoma and lymph node metastasis. Kaplan–Meier method and multivariate Cox regression model were used to evaluate the prognostic value of nuclear β-catenin overexpression at invasive front in rectal carcinoma for disease-free survival (DFS) and overall survival (OS).

Results

Overexpression of nuclear β-catenin at the invasive front in rectal carcinoma in stage III-N₂ was significantly higher than that in stage III-N₁ (73.4 vs. 40.4 %, P < 0.001). Nuclear β-catenin expression at the invasive front in rectal carcinoma was associated with the expression of nuclear β-catenin in corresponding lymph node metastatic lesions (r = 0.297, P < 0.001). Overexpression of nuclear β-catenin at the invasive front in rectal carcinoma was correlated with the number of metastatic lymph nodes (P < 0.001). Patients with nuclear β-catenin overexpression at the invasive front in rectal carcinoma had poor DFS (P = 0.002) and OS (P = 0.003). Moreover, overexpression of nuclear β-catenin at the invasive front was an independent prognosticator for unfavorable DFS and OS (P = 0.002 and 0.001).

Conclusions

Our findings suggest that overexpression of nuclear β-catenin at the invasive front in rectal carcinoma may be a useful marker to evaluate lymph node metastasis, as well as a promising predictor of poor prognosis.     

Circulating TGF-β1-related biomarkers in patients with hepatocellular carcinoma and their association with HCC staging scores

TGF-β1 was inversely correlated with E-cadherin but significantly correlated with VEGF. VEGF and AFP had a low coefficient value but statistically significant. A significant correlation was found between E-cadherin and MMP2. In conclusion, TGF-β and E-cadherin are inversely correlated in HCC patients' sera and not related to the BCLC classification nor survival but rather to the biological properties of the tumor.     

Zipper-interacting protein kinase promotes epithelial-mesenchymal transition,invasion and metastasis through AKT and NF-κB signaling and is associated with metastasis and poor prognosis in gastric cancer patients

Zipper-interacting Protein Kinase (ZIPK) belongs to the death-associated protein kinase family. ZIPK has been characterized as a tumor suppressor in various tumors, including gastric cancer. On the other hand, ZIPK also promotes cell survival. In this study, both in vitro and in vivo assays indicated that ZIPK promoted cell growth, proliferation, migration, invasion, tumor formation and metastasis in nude mice. ZIPK induced epithelial-mesenchymal transition (EMT) with increasing expression of β-catenin, mesenchymal markers, Snail and Slug, and with decreasing expression of E-cadherin. Furthermore, ZIPK activated the AKT/IκB/NF-κB pathway, which can promote EMT and metastasis. Additionally, ZIPK expression was detected in human primary gastric cancer and their matched metastatic lymph node samples by immunohistochemistry. Increased expression of ZIPK in lymph node metastases was significantly associated with stage VI and abdominal organ invasion. Survival analysis revealed that patients with increased ZIPK expression in metastatic lymph nodes had poor disease-specific survival. Taken together, our study reveals that ZIPK is a pro-oncogenic factor, which promotes cancer metastasis.     

CIP2A expression is associated with altered expression of epithelial–mesenchymal transition markers and predictive of poor prognosis in pancreatic ductal adenocarcinoma

CIP2A has been regarded as a novel potential therapeutic target for multiple cancers. The aim of this study was to detect CIP2A expression in pancreatic ductal adenocarcinoma (PDA) and to analyze its association with prognosis of PDA patients. The expression of CIP2A and three epithelial–mesenchymal transition (EMT)-related proteins (E-cadherin, N-cadherin, and vimentin) was examined in 96 PDA tissue samples by immunohistochemistry. Fifty-four cases (56.3 %) were defined as positive for CIP2A expression. Immunohistochemistry showed that CIP2A expression was correlated with poor tumor differentiation, TNM stage, and lymph node metastasis. Kaplan–Meier survival analysis showed that patients with CIP2A-positive expression showed lower overall survival rate than those with CIP2A-negative expression. Multivariate analysis showed that CIP2A expression was an independent prognostic factor for PDA patients. Furthermore, positive expression of CIP2A was strongly associated with loss of the epithelial marker E-cadherin and acquisition of the expression of the mesenchymal markers N-cadherin and vimentin. These findings suggest that CIP2A might promote EMT and progression in PDA, and thus may be a potential therapeutic target for patients with PDA.     

High expression of IL-13 receptor α2 in colorectal cancer is associated with invasion, liver metastasis, and poor prognosis

Autocrine secretion of cytokines by metastatic colorectal cancer cells and their role during invasion and liver homing has been poorly characterized. In this study, we used cytokine arrays to analyze the secretomes of poorly and highly metastatic colorectal cancer cells. Compared with poorly metastatic cancer cells, highly metastatic cells expressed increased levels of the immunosuppressive cytokines interleukin (IL)-4 and IL-13 in addition to increased surface expression of the high affinity IL-13 receptor IL-13Rα2, suggesting that IL-13Rα2 mediates IL-13 effects in colorectal cancer cells. Silencing of IL-13Rα2 in highly metastatic cells led to a decrease in adhesion capacity in vitro and a reduction in liver homing and increased survival in vivo, revealing a role for this receptor in cell adhesion, migration, invasion, and metastatic colonization. In support of this, IL-13 signaling activated the oncogenic signaling molecules phosphoinositide 3-kinase, AKT, and SRC in highly metastatic cells. Clinically, high expression of IL-13Rα2 was associated with later stages of disease progression and poor outcome in patients with colorectal cancer. Our findings therefore support a critical role for IL-13Rα2 expression in colon cancer invasion and metastasis.     

The expression of Survivin and NF-κB associated with prognostically worse clinicopathologic variables in hepatocellular carcinoma

Expressions of Survivin and nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) are associated with a poor prognosis in many malignancies. However, their relationship in hepatocellular carcinoma remains unclear. To investigate the protein expression of Survivin and NF-κB, determine their role in the pathogenesis of hepatocellular carcinoma, and correlate expression with patient survival outcome, immunohistochemistry was used to detect the protein expression of Survivin and NF-κB in 305 cases of hepatocellular carcinoma. Statistical analysis was performed to determine the relationship between the protein expression of Survivin and NF-κB and clinicopathological parameters, survival time, and prognosis. Survivin was expressed predominantly in the cytoplasm, and NF-κB was expressed mostly in the nucleolus. Survivin and NF-κB are expressed at significantly higher rates in hepatocellular carcinoma compared with benign tissue (75.7 vs 13.4 %, P?P?NF-κB expression levels are associated with poor prognostic factors, including tumor size, capsular invasion, tumor thrombus of the portal vein, metastasis of the lymph node, and clinical staging. There was an obvious positive correlation between the expression of Survivin and NF-κB in hepatocellular carcinoma (r?=?0.23, P?NF-κB had significantly shorter survival compared with patients negative for protein expression (P?NF-κB are associated with worse survival outcome in patients with hepatocellular carcinoma. Thus, these proteins could be used as negative prognostic indicators.     

Autotaxin expression and its connection with the TNF-alpha-NF-κB axis in human hepatocellular carcinoma

Background  

Autotaxin (ATX) is an extracellular lysophospholipase D that generates lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). Both ATX and LPA have been shown to be involved in many cancers. However, the functional role of ATX and the regulation of ATX expression in human hepatocellular carcinoma (HCC) remain elusive.     

KIN17 promotes cell migration and invasion through stimulating the TGF-β/Smad2 pathway in hepatocellular carcinoma

KIN17 DNA and RNA binding protein (Kin17) is involved in the regulation of tumorigenesis of diverse human cancers. However, its role in the cancer progression and metastasis in hepatocellular carcinoma (HCC) remains largely unknown. Bioinformatics and immunohistochemistry staining were used to investigate the expression pattern of KIN17 and its prognostic value in HCC patients. The transwell, wound-healing assay was employed to determine the effects of KIN17 on migration and invasion of HCC cells in vitro. The tail veins model was employed to determine the effects of KIN17 on lung metastasis in vivo. The biological mechanisms involved in cell migration and invasion regulated by KIN17 were determined with Western blot analysis method. KIN17 expression was significantly increased in HCC tissues compared with adjacent normal tissues, with particularly higher in portal vein tumor thrombus and intrahepatic metastasis tissues. Patients with higher KIN17 expression experienced poor overall and disease free survival. KIN17 knockdown in HuH7 and HepG2 cells significantly reduced cell migration and invasion abilities, whereas its overexpression promoted migration and invasion in MHCC-97L and HepG2 cells in vitro and in vivo. In HuH7 and HepG2 cells, KIN17 knockdown inhibited the TGF-β/Smad2 pathway. In contrast, KIN17 overexpression stimulated TGF-β/Smad2 pathway in MHCC-97L and HepG2 cells, along with the genes involved in the epithelial-mesenchymal transition. These findings suggest that KIN17 promotes migration and invasion in HCC cells by stimulating the TGF-β/Smad2 pathway. KIN17 could be a promising prognostic biomarker, as well as a potential therapeutic target in HCC.     

PKCα-induced drug resistance in pancreatic cancer cells is associated with transforming growth factor-β1

Background

Drug resistance remains a great challenge in the treatment of pancreatic cancer. The goal of this study was to determine whether TGF-β1 is associated with drug resistance in pancreatic cancer.

Methods

Pancreatic cancer BxPC3 cells were stably transfected with TGF-β1 cDNA. Cellular morphology and cell cycle were determined and the suppressive subtracted hybridization (SSH) assay was performed to identify differentially expressed genes induced by TGF-β1. Western blotting and immunohistochemistry were used to detect expression of TGF-β1-related genes in the cells and tissue samples. After that, the cells were further treated with an anti-cancer drug (e.g., cisplatin) after pre-incubated with the recombinant TGF-β1 plus PKCα inhibitor Gö6976. TGF-β1 type II receptor, TβRII was also knocked down using TβRII siRNA to assess the effects of these drugs in the cells. Cell viability was assessed by MTT assay.

Results

Overexpression of TGF-β1 leads to a markedly increased invasion potential but a reduced growth rate in BxPC3 cells. Recombinant TGF-β1 protein increases expression of PKCα in BxPC3 cells, a result that we confirmed by SSH. Moreover, TGF-β1 reduced the sensitivity of BxPC3 cells to cisplatin treatment, and this was mediated by upregulation of PKCα. However, blockage of PKCα with Gö6976 and TβRII with siRNA reversed the resistance of BxPC3 cells to gemcitabine, even in the presence of TGF-β1. Immunohistochemical data show that pancreatic cancers overexpress TGF-β1 and P-gp relative to normal tissues. In addition, TGF-β1 expression is associated with P-gp and membranous PKCα expression in pancreatic cancer.

Conclusions

TGF-β1-induced drug resistance in pancreatic cancer cells was associated with PKCα expression. The PKCα inhibitor Gö6976 could be a promising agent to sensitize pancreatic cancer cells to chemotherapy.