The Involvement Of Lgr5in Carcinogenesis And Progression Of Colorectal Cancer

BACKGROUND AND OBJECTIVEColorectal cancer (CRC) is one of the most common malignant tumors in China. Although the vast majority of patients with CRC undergo radical surgical resection of tumor and adjuvant chemoradiotherapy, many still die of CRC relapse or metastasis because of the presence of the minimal residual cancer cells. In recent years, studies about molecular targeted therapy with unique curative effect and high safety have been to be the hotpot in the research of medical treatment of tumor. As we well known that G protein-coupled receptors (GPCRs) constitute the largest superfamily of receptors in human genome, and can regulate a variety of physiological functions and pathological processes, such as involving the tumor genesis and metastasis. Therefore, the structure characteristic with seven transmembrane, important roles in the signal transduction and intrinsic activities of GPCRs determine themselves to be the excellent drug targets. At present,60%-70%of potential drug targeted proteins are GPCRs, many of these targeting drugs have been proved to possess excellent anti-tumor effects in some clinical trials. Thus, it is very important and necessary to find some potential GPCRs members that involve the carcinogenesis and development of CRC. Leucine-rich repeat-containing G-protein-coupled receptor5(Lgr5) is a member of the G protein coulpled receptor super-family and has been proved to be a downstream target gene of Wnt/p-catenin pathway. It can play an important role in the process of embryonic development and organ formation. Some recent studies showed that Lgr5appeared to be a stem cell marker for cells with intestinal differentiation, and its overexpression has been demonstrated in hepatocellular carcinoma, basal cell carcinoma, esophageal adenocarcinomas, CRC and ovarian cancers. Though R-spondin has been testified to be the ligand of Lgr5in2011, the mode of intracellular signalling is at present unclear, and little is known about Lgr5function and its underlying mechanisms in the carcinogenesis and progression of CRC. The confirmation of these issues that overexpression of Lgr5can involve the initiation, invasive and metastasis of CRC will be sure to provide solid evidence to the development of medicines targeted at Lgr5as well as the treatment and management of CRC patients. Thus, the Lgr5 expression status was investigated in normal mucosa tissue, primary tumor and/or metastasis tumor of102CRC staged as Ⅰ～Ⅳ,18adnoma,12normal mucosa tissue, and42CRC cases staged as Ⅳ, as well as the biological features of LoVo cells with the ablation Lgr5expression were explored. And the purposes of our study are as follows:(1) to better explore the potential relation between Lgr5overexpression and the carcinogenesis of CRC, and the correlation between Lgr5and β-catenin expression,(2) to study the potential relationship of Lgr5overexpression to the cancer invasion and metastasis, expression status of HER2、VEGF and Ki-67proliferation index in advanced CRC cases, and (3) to establish a study model of LoVo cells with reduced Lgr5expression, and to investigate the biological function of Lgr5in the field of proliferation, migration ability, VEGF expression and the phenotype of epithelial-mesenchymal transition (EMT), et al based on the cell model above.MATERIALS AND METHODSAll human samples in this study were obtained from the affiliated Drum Tower Hospital, Nanjing Univeristy Medical School. The patient private identification information was deleted and the study protocol was approved by the Medical Ethics Committee of the Hospital. The study was carried out in three major sections. In part1, tissue microarrays were constructed, which consisted of102CRC,18colon adenoma, and12colon normal mucosa cases collected between2002and2007. Then, immunohistochemistry staining (IHC) were performed according to the manufacturer’s instructions of the En Vision immunostaining system with primary anti-human monoclonal antibodies against Lgr5, β-catenin, and mutation-type p53antigens. Additionally, expression of Lgr5in tissue sections of tumor centre and invasive margins of21cases of CRC certified to be immunoreactive of Lgr5in TMA was evaluated and possible differences of Lgr5expression between them were analyzed. In part2, the expression status of Lgr5were study by fluorescent quantitation RT-PCR (qRT-PCR) and EnVision IHC staining in the normal mucosa, primary tumor, metastasis tumor in lymph nodes of resected specimens and biopsy tissue of distant metastasis in42CRC cases staged as phase IV and collected from2009to2011. Additionally, immunostaining against HER2, VEGF and Ki-67was performed by EnVision method as metioned above. Subsquently, the relationship between Lgr5expression and the survival time of patients, expression of HER2, VEGF and Ki-67was further studied, respectively. Moreover, additional comparation analysis of Lgr5expression between tumor centre and invasive margin or residual cancer cells around coagulative necrosis were performed in51 cancer tissue paraffin blocks in this section. In part3, to address the functional relevance of Lgr5expression in CRC cell lines, we reduced its expression by small interfering RNA (siRNA) in LoVo cells, which harbor the high hyperplasia and metastasis potential and have been proved to express Lgr5highly in this and the other studies previously, and established a cell model with the ablation Lgr5expression. Then, the ultrastructural features, proliferation index, cycle distribution, migration potential, as well as the expression status of VEGF and EMT associated proteins were detected in negative contral and contral LoVo cells as well as Lgr5siRNA LoVo cells by transmission electron microscope, MTT test, flow cytometry, transwell assays and western blot trial, respectively. Immunoreactivity of neoplastic cells to each antibody was double-blindly semi-quantified by two pathologists in the study of part1and part2. SPSS16.0software packet (SPSS, Chicago, IL) was used for various statistical analyses. Differences in measurement data among groups were tested by using of the one way ANOVA, and enumeration data by using t inspection or x2inspection and Fisher’s exact tests. The Spearman rank test was used for correlation analysis. Survival analysis was performed by Kaplan-Meier analysis with Log-rank test. The P value less than0.05was defined as statistically significant.RESULTSIn part1, Lgr5immunoreactivity was observed only in single cells of normal colon mucosal crypts but high in28%(5/18) adenomas, and significantly higher in54%(55/102, P<0.05) CRC cases. In normal control colonic crypts, scattered, rare single-crypt epithelial cells in the crypt base were Lgr5-immunoreactive, which was deemed negative because of the paucity of immunopositive cells, according to the IHC scoring criteria. In contrast, the number of Lgr5immunoreactive cells with cytoplasmic localization was increased in adenomas, spreading from the crypt base to the surface in a patchy distribution pattern. And in CRCs, the immunostaining patterns were local and patchy in most Lgr5expression positive cases (51/55), and diffuse in four cases. In normal mucosa, adenoma, and CRC, β-catenin expression (cytoplasmic and nuclear staining) was seen in25%(3/12),27%(5/18), and81%(83/102) cases, respectively, in contrast to0,0, and40%(41/102) for p53expression, respectively. In CRC, Lgr5expression was more common in women than men (P<0.0001), and positively correlated with β-catenin expression (P<0.001), but not with patients’ages, tumor sizes, nodal status, TNM stages, and p53expression. There were no statistical difference in Lgr5expression between invasive margin and tumor centre in21cases of CRC staged as Ⅱ-Ⅳ. In part2, based on qRT-PCR examination, the higher mRNA expression was found in71.4%(30/42) CRC cases than normal mucosa tissue of colorectum (P<0.01) In stage IV CRC cases with Lgr5immunoreactivity, both carcinoma in invasive margin and residual cancer cells around or among the coagulative necrosis presented stronger immunostaining against Lgr5than those in tumor centre (P<0.05) based on51cancer tissue paraffin blocks in this study cohort. And strong positive staining was often observed in tumor budding cells of the carcinoma. Additionally, the mRNA expression of Lgr5increased in distant metastasis than it in primary tumor of CRC (P<0.05) by qRT-PCR examination, and so do the Lgr5protein in metastasis cancer tissue tested by IHC in both lymph nodes and distant area (P<0.05). Interestingly, the immunoreactivity of Lgr5was more common in cases staged as IV (92.9%,39/42) than those staged as Ⅰ-Ⅳ (54%,55/102)(P<0.001).In stage IV CRC cases, HER2overexpression was detected in28.9%(IHC3+) and42.1%cases (IHC3+/2+) cases. However, neither Lgr5nor HER2related statistically with the prognosis of the CRC patients, though there was a positive correlation between Lgr5and HER2or Ki-67(P<0.05). Besides,93.9%(31/33) cases were positive for VEGF immunostaining, and most of which (71%,22/31) were scored as IHC2+or3+. In part3, the knockdown efficiency of Lgr5expression monitored by qRT-PCR was very efficient (about90%) and specific. Subsequent experiments revealed that knockdown of Lgr5expression level did not affect the growth of LoVo cells and cell cycle distribution as adherent monolayers. However, loss of Lgr5had striking and positive effects on the migration ability of the LoVo cells because of the decreased number of LoVo cells in the underside of transwell filters. The western blot results displayed that upon knockdown of Lgr5in LoVo cells, VEGF protein was markedly downregulated (P<0.01), and the expression of EMT associated proteins also changed obviously (P<0.01), such as the upregulation of the epithelial marker E-cadherin and downregulation of mesenchymal marker a-SMA. In addition, the ultrastructure features of LoVo cells with reduced Lgr5expression presented much less microvilli, microfilament and microtubule bundles, and more lysosomes and swell mitochondrias.CONCLUSIONSOur results highlight the importance of Lgr5overexpression in the carcinogenesis and progression of CRC and some possible mechanisms through a serial of assays in human CRC tissue and LoVo cells in vitro. Both the number and location of Lgr5and β-catenin positive cells were different among the normal mucosa tissue, adnoma and cancer in colon and rectum. These results suggested that up-regulation of Lgr5expression, especially in female patients, might play an important role in colorectal carcinogenesis, probably through the Wnt/β-catenin pathway. In stage Ⅳ CRC cases, higher expression of Lgr5could be seen in metastasis cancer, invasive margin or residul tumor cells around neoplastic coagulative necrosis than in primary tumor and tumor centre, respectively. These results indicated that upregulation of Lgr5expression could involve the invasion, distant and regional metastasis, and death control of CRC cells. Spearman rank correlation showed positive correlations between Lgr5and HER2overexpression, Ki-67proliferation index respectively in CRC staged as Ⅳ, but both Lgr5and HER2were not related with the prognosis of the CRC patients in this study. Knockdown the expression of Lgr5could weaken the migration ability, ablate the VEGF protein expression, transform the EMT phenotype such as upregulate the epithelial marker E-cadherin and downregulate the mesenchymal marker a-SMA, and alter the ultrastructure features of microvilli, microfilament and microtubule bundles, mitochondria and so on.