| Background/Objective:Colorectal cancer is one of the most common malignant tumors, more than 1 million new cases per year on average were diagnosed, incidence of the disease in the world ranks 3rd all over the world. Although the incidence of colorectal cancer in our country is slightly lower than the western developed countries, but in recent years as people living standard is rising and the change of the diet, the incidence of colorectal cancer is also on the rise year by year in our country.Tumor heterogeneity is one of the characteristics in a variety of cancers including colorectal tumors, and it includes heterogeneity between tumors (different genetic and phenotypic differences between tumor cells) and heterogeneity within the tumors (different genetic and phenotypic differences existwithin the same tumor cells). Heterogeneity within the tumors can be divided into spatial heterogeneity in heterogeneity (in the same tumor, different area has different tumor cells) and time heterogeneity (the original tumor and metastatic tumor have different tumor cells).In fact, Phenotypic and functional heterogeneity arise among cancer cells within the same tumour as a consequence of genetic change, environmental differences and reversible changes in cell properties. Recent studies have revealed extensive genetic diversity both between and within tumours. This heterogeneity affects key cancer pathways, driving phenotypic variation, and poses a significant challenge to personalized cancer medicine. A major cause of genetic heterogeneity in cancer is genomic instability. This instability leads to an increased mutation rate and can shape the evolution of the cancer genome through a plethora of mechanisms. Recent therapeutic advances in oncology have been driven by the identification of tumour genotype variations between patients, called interpatient heterogeneity, that predict the response of patients to targeted treatments. Subpopulations of cancer cells with unique genomes in the same patient may exist across different geographical regions of a tumour or evolve over time, called intratumour heterogeneity. Sequencing technologies can be used to characterize intratumour heterogeneity at diagnosis, monitor clonal dynamics during treatment and identify the emergence of clinical resistance during disease progression. Genetic interpatient and intratumour heterogeneity can pose challenges for the design of clinical trials that use these data. Colorectal cancer molecular targets including the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs) and cyclooxygenase 2 (COX-2), etc., in which EGFR and VEGF have been well studied. In recent years, more and more studies show that PI3K/Akt/mTOR signaling pathway in colorectal cancer exist high expression. So this topic that further study of mTOR signaling pathway will help deepen the understanding the mechanism of colorectal cancer occurrence and development, and provides a certain basis of molecular biology for colorectal cancer gene therapy and drug therapy.Present research indicate the dysregulation of mammalian target of rapamycin (mTOR) and mTOR signaling pathway was frequently observed in a variety of human cancers. mTOR exists in two functionally distinct complexes:mTORC1 (containing mTOR, Raptor, etc.) and mTORC2 (containing mTOR, Rictor, etc.). The mTORCl is sensitive to rapamycin and responds to multiple stimuli, such as energy status, growth factors, amino acids, and inflammation. The mTORC2 affecting cell morphology and actin polymerization mainly promotes cell proliferation and survival through phosphorylation of Akt and SGK. Rapamycin insensitive companion of mTOR (Rictor) which is insensitive to rapamycin forms mTORC2 by binding with mLST8, mSinl and Protor. Currently, only a limited number of reports indicate Rictor has some biological functions in malignant tumors. For example, it is reported that miR-152 is a tumor suppressor by targeting Rictor in gynecological cancers. Research shows that mir-424/503 could inhibit the occurrence and development of a variety of human tumors, including prostate and colon cancer, by inhibiting the expression of Rictor protein. Although Rictor might be involved in cancer progression, its expression in CRC remains unclear. Thus in our experiments, we tested the expression levels of Rictor between colorectal cancer tissue (experimental group) vs para-carcinoma tissue (control group) by using immunohistochemistry in 62 colorectal cancer paraffin tissue samples excised during operations. We further verified the expression difference in cellular level by comparing the expression between CRC cells and human normal liver cells.Methods:Samples Collection:62 colorectal cancer paraffin tissue samples excised during operations were collected from our hospital affiliated to Nanfang Medical University during June 2008 and Augast 2010. Patients we chose did not accept any chemical treatment or other anti-cancer therapies before surgery, and we excluded patients who did not aceept complete follow-up and clinicopathologic data, and those without the ethics committee’s consent. Adjacent normal mucosa tissues were taken from tumor cut edge 5 cm or more by pathology examination based on the rules of clean and no cancer cell invasion. Tumour biopsies conventional hematoxylin-eosin (HE) staining for pathological diagnosis and pathological classification were used to observe the surgical specimens of colorectal cancer tumor differentiation degree and pathological type, and tumor tissue biopsy was classified with reference to the international union of anti-cancer discriminant standard TNM staging. Tumor tissue VS corresponding normal tissue adjacent to carcinoma were divided into groups, carcinoma tissue section was treated as the experimental group compared to normal tissue adjacent to carcinoma. Immunohistochemical staining was used to analyse Rictor protein expression in the slices in each group. Statistical analysis:We conducted statistical analyses by using SPSS 20.0 software (IBM SPSS, Armonk, NY, USA), Fisher exact test or x2 test was used for the comparison of the rate between groups, and we analyzed the correlation between survival time and variables using the Kaplan-Meier method and compared survival curves using the log-rank test for survival analyses. Statistical significance was set at 0.05.The expression levels of Rictor and mTOR in HCT116, SW480, LoVo and HCoEpiC cells were detected by indirect immunofluorescence and western blot.62 colorectal cancer paraffin tissue samples were excised during operation to verify the expression difference of Rictor between colorectal cancer tissue (experimental group) vs para-carcinoma tissue was detected by using immunohistochemistry (control group). The relationship between expression levels of Rictor protein and clinical pathologic features of two groups was compared, and the relationship between the expression difference of Rictor and the overall survival rate of colorectal cancer patients was analyzed.Results:1. Between June 2008 to August 2010 surgery for 62 cases of patients with colorectal cancer, in which male 34 cases, female 28 cases, aged 30-88, and the median age is 66. All of the patients has not been treated by preoperative radiotherapy or chemotherapy; All cases of biopsy were reviewed under two experienced pathologists. Conventional hematoxylin-eosin (HE) staining was used for pathological diagnosis and pathological classification, and to observe the tumor differentiation degree in each surgical specimens of colorectal cancer cases, the low differentiation case is 41 and high differentiation case is 21, and with reference to the international union of anti-cancer discriminant standard, including 28 patients within stage I/Ⅱ, III/IV degree 34 cases.2. Immunohistochemical results displayed that Rictor protein mainly existed in the cytoplasm (in figure 1). Positive expression rate of Rictor in colorectal cancer tissues was 58.1%(36/62), and in the normal tissue adjacent to carcinoma, positive expression rate of Rictor was 14.5%(9/62). Rictor protein expression level in colorectal cancer tissue is significantly higher than normal tissues adjacent to colorectal carcinoma, the difference was statistically significant (P< 0.05).3. To investigate the correlation between Rictor expression and clinicopathological characteristics, we collected 62 CRC samples to detect the expression of Rictor protein by using immunochemistry method. Positive rate of Rictor protein expression and Chi-square values were listed in table 1. Statistical correlation analysis results show:the expression level of Rictor protein in colorectal cancer organizations was associated with the Ducks stages and lymph node metastasis of tumor (P< 0.05), but patients’ age, gender, tumor size, differentiation degree, depth of invasion and histological type (P> 0.05).4. Rictor protein expression and survival analysis:Kaplan and Meier survival analysis results:the survival time of patients with colorectal cancer was correlated with Rictor protein expression levels. Patients whose Rictor protein expression is positive had a shorter survival time than those expressing negative, log-rank test showed the difference was statistically significant (P< 0.05).5. Confocal fluorescence microscope observation results show that the immunofluorescence double labeling staining HCT116, SW480, LoVo and HCoEpiC cells, and the expression of mTOR and Rictor located in the cytoplasm in four types of cells, and two kinds of proteins expressed in the cytoplasm showed no significant difference between each cell lines. By this western blotting experiments were used to make a quantitative comparison.6. In order to identify the expression and significance of Rictor protein in CRC formerly in cellular level, we firstly detected Rictor protein expression in HCT116, SW480, LoVo and HCoEpiC cells by using western blotting method. Rictor protein expression in CRC cell lines is higher than that of normal liver cell line HCoEpiC, so in vitro study also suggested that Rictor overexpression existed in CRC, which meant Rictor might be involved in tumorigenesis and cancer progression in CRC.In a word, the expression level of Rictor in colorectal cancer tissues was significantly higher than that of para-carcinoma tissues(P<0.05). In cellular experiments, we’ve also noticed that the expression level of Rictor and mTOR in colon cancer cell lines was higher than that of human normal colon epithelialcell line HCoEpiC. The expression of Rictor was relevant to Ducks stage and lymphatic metastasis, whereas it had no no statistical significance in other clinicopathological parameter (P>0.05). Patients with Rictor expression had a lower overall survival rate than those without Rictor expression.Conclusion:This study found that Rictor expression might have relation to CRC initiation and progression; we also confirmed that Rictor expression is associated with Ducks stages and lymph node metastasis; moreover, we verified that Rictor had correlation to prognosis of patients with CRC. As thus, Rictor would play a crucial role in directing treatment and prognosis evaluation for CRC patients. The overexpression of Rictor was relevant to the initiation and development of colorectal cancer. Rictor can be regarded as an independent indicator for judging the prognosis of colorectal cancer.Since this study is a retrospective analysis, the study population is from China, so there are still many limitations. In addition, more work about the potential role of Rictor in the origin of colorectal cancer was needed to test, such as prospective studies and Rictor and mTOR protein molecular mechanism research. To sum up, this study verified Rictor and mTOR protein high expression in colorectal cancer, including Rictor protein expression level is closely related to the Ducks stages and lymph node metastasis of the tumor. Rictor protein can be treated as an independent prognostic indicator of colorectal cancer. The above work for further research on the function of Rictor and mTOR protein in colorectal cancer provides a certain theoretical basis, for the prognosis and treatment of tumor provides a new train of thought. |
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