| As one of the most frequently occurring human malignancy, colorectal cancer (CRC) has its incidence and mortality rate listed the 3rd and 4th respectively among all kinds of cancers globally. Every year, there are about 1,400,000 newly diagnosed cases and more than 693,900 patients die from CRC. The incidence of CRC in China has increased rapidly in recent years. According to Cancer Statistics in China,2015,376,300 patients are diagnosed with CRC in China, and 191,000 patients died from the disease. TNM classification provides valuable prognostic information and instruction on standardized treatment, however, it fails to meet the requirement of individual patients’therapy, the fact being most of the patients in stage II or stage III did not benefit from adjuvant chemotherapy based on TNM classification. Therefore, it’s of great clinical value to identify molecular markers related to prognosis and treatment, which can be used to assist screening high-risk patients with early stage CRC that might experience tumor metastasis or recurrence, so that they can benefit from subsequent adjuvant treatment. Pathological mechanism research is also imperative.Clinically, more than 90% of cancer deaths are caused by tumor recurrence or distant metastais. Around 20% of patients with newly diagnosed CRC present with distant metastases,40-50% of CRC patients with no metastases at diagnosis will also have distant metastases eventually incurred after treatment. It is well recognized that tumor metastasis is a complex, multi-step process. In recent years, compelling evidence indicates that Epithelial-mesenchymal transition (EMT) is a major mechanism of cancer cell invasion and metastasis by undergoing a phenotypic conversion.EMT is a highly conserved cellular process through which polarized, immotile epithelial cells convert to motile mesenchymal cells under physiological or pathological stimuli. During EMT, cells lose their epithelial features of polarity and adherent and tight junctions, gain mesenchymal properties, including characteristic gene expression changes, and exhibit increased motility. Biochemically, epithelial markers such as the adherens junction molecules E-cadherin and zonula occludens-1 (ZO-1) are repressed and mesenchymal markers such as N-cadherin, Vimentin and Fibronectin are upregulated, together with increased matrix metalloproteinase-9 secretion to degrade the extracellular matrix. In addition, several transcription factors such as members of the Snail (SNAIL and SLUG), ZEB (ZEB1 and SIP1), and bHLH (E47 and TWIST) families also contribute to the occurrence of EMT by directly or indirectly binding to the E-boxes at the E-cadherin promoter to silence its expression. Thus, we aim to evaluate the impact of EMT markers on the clinicopathologic features or prognosis, and identify a subset of patients who are at high risk of death in CRC.A variety of extracellular signals can trigger EMT, including TGF-β, Notch, Hedgehog, Matrix, Wnt, etc. Among these, TGF-β is considered a classic inducer of EMT in an autocrine or paracrine manner. There are comprehensive reporting regarding to TGF-β signaling pathway. TGF-β binds to its receptors and activates Smad2 and Smad3, then phosphorylated SMAD2/3 partner with Smad4 to translocate into the nucleus where SMAD complexes control target gene transcription, including EMT- and motility-related genes. In addition, TGF-β signaling can occur via Smad-independent pathways related to cell movement and apoptosis, such as the activation of phosphatidylinositol 3-kinase (PI3K)/Akt, mitogen-activated protein kinase (MAPK), and small GTPases of the Rho family.Apart from the classic EMT markers, more and more reports reveal that lots of novel molecules play important roles in EMT. In our laboratory, insulin-like growth factor binding protein-related protein 1 (IGFBP-rPl) was screened from adenocarcinoma-nomal mucosa cDNA library using suppression subtractive hybridization (SHH), which was overexpressed in colorectal adenocarcinoma tissue. Our previous studies clearly revealed that IGFBP-rPl inhibits cell proliferation and induces apoptosis in CRC cell lines. Moreover, the abundance of IGFBP-rPl is inversely correlated with a poor prognosis. Our previous studies clearly revealed that IGFBP-rPl could block EMT process in vitro. Therefore, we used patient samples and several in vivo models of EMT and metastasis to reveal a unique role for IGFBP-rPl in inhibiting EMT and metastasis. Although IGFBP-rP1 has been reported to inhibit tumor cell migration abilities, its functional role in EMT and metastasis remain largely unknown. Due to IGFBP-rPl binding IGFs or insulin with low affinity, it indicates that IGFBP-rPl may function in IGFs/insulin-independent manner. Our previous microarray analysis provided evidence that IGFBP-rPl has a close relationship with TGF-β, and further influences Smad3 expression.Therefore, to confirm whether IGFBP-rPl inhibits EMT and metastasis via a TGF-β/smad-dependent pathway warrants further explore.Three hundred forty-six colorectal tumor samples were obtained from Runrun Shao hospital, Zhejiang Province, China, from 2004 to 2008. RNA extraction for investigating the messenger RNA (mRNA) expression of EMT markers, including E-cadherin, Vimentin, Fibronectin, Snail, Slug, Twist, Zebl and Zeb2, was conducted in CRC tissues by real-time PCR. All follow-up ended on Sep.30th 2013. Postoperative follow-up data were obtained from 167 patients, and the median follow-up duration was 72 months (range from 3 to 115 months). Next, to valiadate the results of qPCR, immunohistochemical staining (IHC) was performed for five markers (E-cadherin, Fibronectin, Snail, Slug and Twist) in 415 CRC specimens preserved in our laboratory. The cohort was obtained from the patients enrolled between 1991 and 2006 at the Xiaoshan 1st hospital, Zhejiang Province, China. The survival data was provided by the Xiaoshan Center for Disease Control. All follow-up ended on Sep.31st 2006. Postoperative follow-up data were obtained from all patients, and the median follow-up duration was 33 months (range from 1 to 186 months).IBM SPSS Statistics 20.0 for windows was used for statistical analysis. The significance of mRNA levels between different clinical phenotypes was determined by the Mann-Whitney test. Unpaired Student’s t tests were used to compare the significance of transcription factors expression between epithelial phenotype and mesenchymal phenotype. Comparisons of clinicopathological parameters in different EMT markers protein levels groups were made by the x2 test or Fisher’s exact test. Correlations were analyzed by Spearman correlation. Kaplan-Meier method was used for categorical variables and univariate Cox’s proportional hazard model was used for continuous variables to evaluate overall survival. Overall survival curves were constructed by the Kaplan-Meier method, whereas differences were analyzed using log-rank tests. Multivariate survival analysis was investigated with Cox’s proportional hazard model. Significance was set at P< 0.05.Here, we evaluated the impact of EMT markers on the clinicopathologic features or prognosis at both mRNA and protein levels in two CRC corhorts. At mRNA levels, we identified the differences in the expression of each gene according to the clinicopathological features. Transcription factors were more highly expressed in mesenchymal group as compared to epithelial group. A positive correlation existed between any two transcription factors. With the univariate and multivariate Cox proportional hazard models, Snail was associated unfavorably with patient overall survival (OS), and Snail expression was an unfavorable independent prognostic factor in stages I-II, but not stages Ⅲ-Ⅳ CRC. In addition, high Slug and Twist expression was correlated with a poor prognosis. Based on the results of qPCR, IHC was performed for five markers (E-cadherin, Fibronectin, Snail, Slug and Twist). IHC ananlysis was similar to the results of qPCR, but also had new findings. Positive Snail or Twist expression was a marker of bad prognosis. Moreover, patients with combined reduced E-cadherin expression and positive Snail or Twist expression survived shorter than those of other subgroups. Snail expression remained as an independent unfavourable prognostic factor in this cohort. Consistent with the result of qPCR, Snail expression was proven to be an independent prognostic factor within stages I-II patients. Similarly, survival curves revealed that positive Snail or Twist expression predicted an adverse prognosis only in TNM stages I-II. By comparing patients with different p53 status, we found that positive Snail expression worsened overall survival rates in a wild-type p53 dependent manner.In 217 colorectal specimens, the staining intensity of IGFBP-rPl was inversely correlated with histological grade, lymph-node metastasis and TNM stage. Immunostaining of IGFBP-rPl and the epithelial markers E-cadherin and P-catenin revealed a positive correlation in membrane expression. Conversely, we found a negative correlation between IGFBP-rPl and fibronectin expression. To explore the biological role of IGFBP-rPl in tumor formation in vivo, we inoculated CRC cell lines with and without IGFBP-rPl expression as xenografts in nude mice. IGFBP-rPl inhibited tumor formation of CRC cells in vivo. To characterize the EMT features of IGFBP-rPl-induced CRC tumors, we performed western blot analyses on all xenograft tumors. Consistent with the in vivo findings, we noted that IGFBP-rPl overexpression increased the epithelial marker ZO-1 and reduced the mesenchymal markers N-cadherin, vimentin, and MMP9, whereas silencing of IGFBP-rPl had the contrary effect. The functional role of IGFBP-rPl in inhibiting EMT in CRC is further strengthened. An immunocompetent mouse model of distant metastasis uncovered that IGFBP-rPl inhibited colorectal cancer metastasis.IGFBP-rPl decreased TGF-β receptor expression, with subsequent inactivation of TGF-β receptor pathways. To further validate whether IGFBP-rPl modulated the TGF-β/Smad signaling pathway, we used SB431542, which is a selective inhibitor of TGF-β/ALK5/Smad2 signaling. Western blots confirmed the inhibition of TGF-β R II and smad2/3 phosphorylation by SB431542 in two clones of IGFBP-rPl knockdown SW480 cells. SB431542 rescued the EMT status induced by IGFBP-rPl knockdown. Our data demonstrated that IGFBP-rPl involved in EMT program required TGF-β/Smad signaling. Moreover, exogenous rhIGFBP-rP1 protein decreased the activation of TGF-β signaling and restored the upregulation of TGF-β1-induced EMT-related markers by attenuating Smad pathways.From our data, it could be concluded that Snail expression worsens overall survival rates in a wild-type p53 dependent manner, and Snail expression is an independent prognostic factor within stages I-II CRC patients. Our data collectively provide evidence that IGFBP-rPl suppresses EMT and metastasis by attenuation of TGF-β/Smad signaling in CRC. |
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