| Colorectal cancer (CRC) is one of the most common malignant tumors which severely threats the public health. The incidence and mortality of CRC has ranked third malignant tumor according to the latest statistics data in America in 2015. The incidence in China is rising year by year because of the changes of lifestyle and diet elements. Although there has been a remarkable improvement in the diagnosis and treatment of CRC recently, the mortality rate of CRC is still high, which is severely destructive to human health. Accordingly, there is an important clinical value and social significance to further study the mechanism of CRC initiation, development and metastasis and to search for the biomarker of diagnosis and prognosis.The theory of cancer stem cells (CSCs) states that a subset of cells are present in the tumor and have the capacity of self-renewal and multiple differentiation and, thereby endow such qualities as heterogenicity and metastasis. These cells are named CSCs. Numerous researches have been done in this area by researchers, but the theory face many challenges. A formidable difficulty is the lack of good and specific markers to isolate CSCs. And most of the proved CSCs markers derive from cell lines and animal experiments. However, their expression in the progression of colorectal cancer is not clear, and it is not established whether these markers can be seen as markers for prognosis. Therefore, we chosed ALDH1A1. CD44v6, Lgr5, and Nanog as candidate CSCs markers and studied their role in the progression of colorectal cancer.PTEN, an anti-oncogene, plays an important role in cancer stem cells (CSCs). Bone morphogenetic protein 4 (BMP4) or thymosin β4 (Tβ4) can inhibit the oncogenicity of the CSCs from colorectal cancer and induce their differentiation, and PTEN expression is upregulated in this process. PTEN-Long is a isoform protein of PTEN. It was first reported in the journal Science in 2013, which was followed by several similar reports. PTEN gene encodes two isoform proteins,55kd PTEN and 75kd PTEN-Long. PTEN-Long is an amino-terminal extension of the 55kd PTEN and is synthesized by alternative translation initiation at an upstream CTG codon, PTEN-Long can be detected in breast cancer. And when the PTEN-Long was overexpressed in breast cancer and brain glioblastoma cells, the PI3K signal pahtway was inhibited, which resulted in the inhibiton of proliferation, a similar effect as PTEN exerted. Besides, PTEN-Long has a leader sequence of signal peptide. So the PTEN-Long protein can be secreted out of the cell and get inside of other cells to play its anti-oncogenic role. This study is so interesting that we aim to study its roles in colorectal cancer.The objectives of the present study are to investigate the significance of expressions of several candidate CSCs markers in the progression of colorectal cancer and to explore the function of PTEN-Long in colorectal cancer.1. Expressions of ALDH1A1, CD44v6, Lgr5 and Nanog and their significances in the progression of colorectal cancerImmunohistochemistry was applied to detect expressions of ALDH1A1, CD44v6, Lgr5 and Nanog, and independent samples from two groups were prepared. The first group included 419 samples of tissue microarray (TMA). The relationship between the afore-mentioned four porteins and clinicopathological features and its influence on prognosis were analyzed. The second group was histopathological sections, including 217 adenomas (low grade:105 cases; high grade:112 cases) and 72 primary tumors in colorectum with correspondent metastastic lesions in the liver. Based on the molecular model of the initiation and development of colorectal cancer, we systematically studied the expression patterns of the afore-mentioned four porteins in the course of normal mucosa-adenoma-primary colorectal cancer- metastatic lesion in liver. A total of 193 cases of colorectal cancer samples was added to the first group to validate the conclusions from the studies on ALDH1A1.1). The expression patterns and clinical significances of ALDH1A1 in the progression of CRCALDH1A1 expression in the colorectal tissues was assessed in the cytoplasm as well as nucleus. In the progression of CRC, ALDH1A1 expression in low-grade adenoma was predominant over high-grade adenoma, primary CRC and the correpsonding liver metastasis, there was no difference among latter three [low-grade adenoma> (high-grade adenoma= primary carcinoma= liver metastasis)]. Stromal ALDH1A1 expression was just found in primary CRC and liver metastasis, and positive expression of ALDH1A1 in the nucleus was frequently accompanied by its positive expression in the stroma. Univariate Kaplan-Meier survival analysis indicated that no significant association was found between the cytoplasmic expression of ALDH1A1 and the survival of CRC patients; and the patients with nuclear staining of ALDH1A1 have a better prognosis. Multivariate COX survival analysis demonstrated that positive expression of ALDH1A1 in the nucleus is an independent prognostic factor.2). The expression patterns and clinical significances of CD44v6 in the progression of CRCCD44v6 was expressed in the membrane and cytoplasm in the colorectal tissues. During the development of CRC, CD44v6 had a highest level expression in adenoma, followed by primary carcinoma and liver metastasis, while no difference was found between the low-grade adenoma and high-grade adenoma, and no difference was found between the primary carcinoma and liver metastasis [normal mucosa< (low-grade adenoma= high-grade adenoma)> primary carcinoma and liver metastasis]. CD44v6 expression in the front area of the carcinoma was lower than that in the central area. Univariate Kaplan-Meier survival analysis suggested no significantly statistical association between CD44v6 expression and prognosis, but found a marginal P value. Lower expression of CD44v6 is indicative of a worse prognosis, which is coincident with the fact that patients with lymph node metastasis have low expression of CD44v6. Spearman analysis found a positive association between CD44v6 expression and E-Cadherin expression.3). The expression patterns and clinical significances of Lgr5 in the progression of CRCLgr5 was expressed in the membrane and cytoplasm in the colorectal tissues. During the development of CRC, Lgr5 had a highet expression level in high-grade adenoma, followed by low-grade adenoma, primary carcinoma and liver metastasis, normal mucosa [normal mucosa< (low-grade adenoma< high-grade adenoma)> primary carcinoma and liver metastasis], with no statistical difference in Lgr5 expression between primary carcinoma and liver metastasis. Lgr5 expression was associated with tumor sites, histopathological grading and infiltration depth, with rectal tumor, low grade tumor and superficial infiltrating tumor had high expression of Lgr5. Univariate Kaplan-Meier survival analysis indicated that the patients with high Lgr5 expression have a better prognosis, and multivariate COX survival analysis showed Lgr5 expression is an independent prognostic factor. Lgr5 expression is not associated with P-catenin nuclear expression.4). The expression patterns and clinical significances of Nanog in the progression of CRCNanog was expressed in the cytoplasm rather than the nucleus in the colorectal tissues in our study. Nanog expression in high-grade adenoma was higher than that of low-grade adenoma, and its expression in primary carcinoma was higher than that of liver metastasis (normal mucosa< low-grade adenoma< high-grade adenoma> primary carcinoma> liver metastasis). Nanog expression was associated with tumor sites, histopathological grading and infiltration depth, with rectal tumor, low grade tumor and superficial infiltrating tumor had high expression of Nanog. Kaplan-Meier survival curves indicated that the patients with high Nanog expression have a better prognosis, and COX regression model showed Nanog is an independent prognostic factor.5) Correlation analysis among four markersThe expression of ALDH1 has no significant relevance to the expression of Lgr5 and Nanog in CRC. The CD44v6 expression is associated with ALDH1A1, Lgr5 and Nanog expression. However, due to the broad expression of CD44v6, its clinical significance needs to be further considered. The exprssion of Lgr5 and Nanog has a positive relation, which also displays consistent expression during the adenoma-primary cancer lesion-hepatic metastasis progression. Furthermore, by combing Lgr5 and Nanog together, it demonstrates that patients of Nanog+Lgr5+group have better prognosis than those of Nanog-Lgr5-group.6) Establishing a hazard model for survival status based on prognostic index and its values in prognosisBased on randomization principle,80% TMA samples were selected as training set to establish prognosis model, others were chosen as validation set to check the established model. Metastasis in lymph node, Distant metastasis, Lgr5 expressions and nuclear β-catenin expression are independent prognostic factors for survival in colorectal cancer, the prognostic index (PI) was calculated based on the four factors in the training set. And PI was proved to be more accurate than the TNM stage in estimating the 1-,3-,5-year survival status of CRC patients. The hazard model for predicting 1-,3-,5-year survival status was established with PI as variable, which could evaluate the individual survival status, and the hazard model was validated in the validation set.2. The biological function of PTEN-Long in colorectal cancerWe found expressions of PTEN and PTEN-Long in human colorectal cancer tissues and cell lines using commercial PTEN antibody, indicating there is alternate translation in human bodies. We obtained the full-length sequence of PTEN-Long starting initiation codon (CTG), inserted this sequence into p3xFLAG-CMVTM-14 vector, and established the expression vector PTEN-Long-CTG/ATG. In order to express the higher levels of PTEN-Long, we mutant CTG into ATG, and obtained the PTEN-Long-ATG/ATG. The two vectors were transfected into 293FT cell line which do not expresses PTEN. And the present study found expression of these two vectors in 75kd band, but the expression is higher in ATG/ATG than CTG/ATG, indicating that ATG is the predominant initiation codon and CTG is the subordination initiation codon. Interestingly, we also detected exogenous 55kd PTEN in the group of ATG/ATG vector, which is not coincident with the traditional view which consider the second ATG as a methionine. This indicates the alternate translation also exists in exogenous expression. We also produce the rabbit antibody directing at PTEN-Long. Because of the less effectiveness of the polyclonal antibody, we just testify its activity in the PTEN-Long overexpression. PTEN-Long-ATG/ATG expression vector was transfected into human colorectal cell lines DLD1, SW480 and RKO to obtain the stable positive cell lines.Alternate translation also exists in RKO cell line, which was accordant with 293FT. Transwell cell migration assay indicated that PTEN-Long can inhibit migration of CRC cells.On the basis of the aboving studies on expressions of ALDH1A1, CD44v6, Lgr5 and Nanog in colorectal cancer, the following conclusions could be maded.(1) ALDH1A1, CD44v6, Lgr5 and Nanog can be seen as prognosis markers for colorectal cancer, with high expressions of these proteins suggesting a relatively good prognosis. Positive ALDH1A1 staining in nucleus, Lgr5 expression and Nanog expression are all independent prognostic factors. Combinational analysis of Nanog and Lgr5 is better for determining prognosis than analyzed alone, with Nanog -Lgr5- patients with colorectal cancer has the worst prognosis. Based on metastasis in lymph node, distant metastasis, Lgr5 expressions and nuclear β-catenin expression, we obtained the PI. The hazard model based on variable PI could be used to predict individual hazard at the end of 1-,3-, and 5-year, with good repeatability.(2) The alternate translation of PTEN and its isoform PTEN-Long is present in the human body, and PTEN-Long has an inhibitory role in CRC. |
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