| Backgrounds and purpose:In men, cancer of the prostate gland is the most commonly diagnosed non-cutaneous malignancy, accounting for29%of all cancer cases and the second most common cause of death by cancer in the USA. In2012, an estimated241,740men were diagnosed with prostate cancer and28,170men died of prostate cancer. The majority of cancer-associated deaths and essentially all prostate cancer deaths are due to metastases rather than primary tumor burden. Thus, decreasing mortality of prostate cancer depends on understanding the biology that underlies metastasis such as identification of genes involved in cancer metastasis that would benefit the design of more effective clinical intervention strategies. There is a wealth of evidence indicating that the acquisition of malignant progression and aggressive traits of cancer can be promoted or inhibited by a set of functional genes known as metastasis-regulatory genes in various cancers. These can be broadly categorized as pro-metastasis or metastasis-suppressor genes. Pro-metastasis genes drive conversion from non-metastatic to metastatic cells. Metastasis-suppressor genes can suppress metastases without affecting primary tumor growth.To identify candidate metastasis-regulatory genes in prostate cancer, a common and straightforward method is to elucidate a differential gene list (signature) derived from analysis of a transcriptional study on prostate cancer samples with groups correlated with poor prognosis. However, the single study-based signature is often underpowered, truncated, and low quality. These limitations can be overcome by combining related independent studies into a meta-analysis for larger sample size and smaller false discovery. There are a limited number of published prostate cancer gene-expression studies having clinical survival outcome data for meta-analysis. Sowe implemented and performed a large meta-analysis of breast cancer gene expression profiles from223datasets containing10,581human breast cancer samples using a novel data similarity-based approach (iterative EXALT). Cancer gene expression signatures extracted from individual datasets were clustered by data similarity and consolidated into a meta-signature with a recurrent and concordant gene expression pattern. A retrospective survival analysis was performed to evaluate the predictive power of a novel meta-signature deduced from transcriptional profiling studies of human breast cancer. Validation cohorts consisting of6,011breast cancer patients from21different breast cancer datasets and1,110patients with other malignancies (lung and prostate cancer) were used to test the robustness of our findings.The certification standard is the predictive accuracy for patients’ survival of the new gene signature. In this meta-analysis, based on data similarity,633tumor markers were integrated into121tumor markers. From these tumor markers, we found11of them represent the behavior of a highly malignant tumor. Then we identified50genes from them, named BRmet50. BRmet50was proved to be a better tumor gene signature than the ones in the past with a more accurate prediction of prognosis of breast cancer patients and this prediction result is independent with commonly used clinical and pathological parameters. In addition, the gene signature is not only limited to the predictability accuracy for breast cancer prognosis, and prostate cancer, lung cancer prognosis also accurately predicted. BRmet50contains11candidate tumor metastasis suppressor genes and39candidate tumor metastasis promoting genes.This study intends to use bioinformatics methods to screen the11candidate tumor metastasis suppressor genes, and then to identify the biological behavior of the target candidate genes in prostate cancer in order to discover new prostate cancer metastasis suppressor gene.Materials and methods:1. The screening of the candidate prostate cancer metastasis suppressor gene1.1To determine the association between a recently defined50-gene expression signature and prostate cancer.We first examined whether the signature could be applied to predict prognosis in published prostate cancer datasets. Various prostate cancer outcomes (relapse, distant metastasis, or death) from957prostate cancer patients were used as clinical end points in the survival analyses.1.2To screen the best candidate prostate cancer metastasis suppressor gene.Eleven of50genes were downregulated in aggressive tumors. We used a bioinformatic approach to determine if any of the11suppressor candidate genes exhibited downregulation in human prostate cancer. We surveyed gene expression of the11candidates among data from10published transcriptional profiling studies performed on normal or diseased prostate tissues. Differential gene expression (up or down) was determined by a comparison between an experimental group and a control group within each dataset.Then we will take the best candidate gene to do further researchs.1.3To choose the best candidate prostate cancer metastasis suppressor gene in1.2step and then determine whether its protein expression is lost in prostate cancer samples, we evaluated the candidate gene levels by Western blot in protein lysates from prostate cancer samples, non-malignant tissue and prostate cancer cells to identify it preplemently as a metastasis suppressor.2. The identifying of the candidate prostate cancer metastasis suppressor gene2.1Choose the proper prostate cancer cell lines with bioluminescence marker.2.2Identifying over expression vector for candidate gene and then to establish a system for stable candidate gene overexpression in prostate cancer cell lines; we used retroviral vector, which can carry the purpose gene to recombinant with host cell genome, so as to achieve the purpose of stable expression.2.3Standard in vitro studies to research the effect of the candidate gene’s over-expression in prostate cancer cell lines PC3and ARCaPM on proliferation, migration and invasiveness (each experiment was repeated at least three times)2.3.1Cell proliferation assay2.3.2Soft agar colony formation assay2.3.3Wound healing assay2.3.4Transwell chamber migration assay2.3.5Transwell chamber invasion assay2.4Animal models in vivo studies to research the effect of the candidate gene’s over-expression in PC3cell lines on the grown and metastasis in vivo in experimental animals (Any animal metastasis model experiment was repeated at least twice):Severe combined immunodeficient (SCID) male mice were randomized into two groups:PC3-luc/EV and PC3-luc/Candidate gene. After PC3xenografting, mice were imaged biweekly for bioluminescence using an in vivo Imaging System (MS) to monitor tumor growth. Upon sacrifice, the prostate and other organs were removed for imaging and histological examination. Survival data were plotted on a Kaplan-Meier curve, and two different groups were compared using the Log-rank (Mantel-Cox) test.For orthotopic xenografting (OX) experiments,10-week-old SCID mice were randomized into two groups:PC3-luc/EV and PC3-luc/candidate gene. PC3-luc/EV or PC3-luc/candidate cells (3x105in30u.1) mixing with neutralized collagen gel were implanted into the mouse anterior prostate (AP) lobe through a lower midline laparotomy incision. The liquid collagen gel containing PC3cells became solid in vivo. We then closed the incision of AP to let the solid gel piece stay inside of the lumen of the AP lobe. There was no cell suspension left in the space surrounding space of the prostatic ducts.For Intracardiac (IC) injections, in brief,6-8-week-old SCID male mice were randomized into two groups. PC3-luc/EV or PC3-luc/candidate gene cells (5×105) were re-suspended in100μl PBS and slowly injected into the left ventricle of the mice. IVIS and radiographs (Faxitron) were used to confirm successful injections into the mouse body and to monitor metastasis formation.Results:1. Discovery of candidate metastasis suppressor gene1.1Our results demonstrated that the50-gene signature successfully predicted clinical outcomes in all three prostate cancer datasets (p<0.001)1.2Out of the11candidate genes. SPARC-like1(SPARCL1) displayed the most consistent profile among the10datasets. Upregulation of SPARCL1was found in nonmalignant (benign) and normal prostate tissues. Downregulation of SPARCL1was observed in prostate cancer samples, tumors with high grade (T3B), high Gleason scores (GS>7), androgen independent (AI) status, or metastatic prostate tumors.1.3Protein SPARCL1levels were downregulated in prostate cancer samples in comparison to the non-malignant sample. SPARCL1expression is undetectable in LNCaP, ARCaPM, and PC3cancer cells. In contrast, high expression of SPARCL1was observed in benign human prostate tissue and benign prostate cell lines (NHPrE1).2. Identifying of the candidate metastasis suppressor geneThe preparation of the cell lines for the experiment and the establishment of the candidate gene over-expression:2.1The bioluminescent human prostate carcinoma cell line (PC3-Luc) from Dr. K. Pienta (University of Michigan Medical Center)) has bioluminent marker that has Luciferase can effect with luciferin and can be detected by IVIS.2.2pBMN-l-GFP retroviral transfection vector was applied to construct pBMN-I-SPARCL1-GFP vector and SPARCL1gene open reading frame fragment was successfully restructured and integrated with the host cells, such as PC3-Luc, ARCaPM. So they became PC3-LUC/SPARCL1, ARCaPM/SPARCL1cells and PC3-Luc/EV, ARCaPM/EV cell lines were the control cells. PC3-Luc/SPARCL1and ARCaPM/SPARCL1cells can over-express protein SPARCL1stably.Resuls of in vitro studies:2.3.1Proliferation rates of the PC3-luc/EV and PC3-luc/SPARCL1cells were similar in24-and48-hour measurements;2.3.2There were similar numbers of PC3-luc/EV and PC3-luc/SPARCL1colonies after3weeks culture.2.3.3At12hours, PC3-luc/SPARCL1cells showed significantly less motility and migration (wound-healing closure percentage,15%) compared to the control (PC3-luc/EV,33%)(P=3.2×10-7);2.3.4Recombinant SPARCL1significantly inhibited both PC3and ARCaPM cells across a membrane in a transwell migration assay (inhibition rates,64%and67%, respectively)2.3.5In a48-hour invasion assay, more PC3-luc/EV cells invaded through the basement membrane compared to PC3-luc/SPARCL1cells. In comparison to the empty vector control (PC3-luc/EV) cells, SPARCL1significantly decreased the invasiveness of PC3cells by2.7-fold in a transwell Matrigel invasion assay (P=0.008). We found that recombinant SPARCL1could also reduce invasiveness of ARCaPM cells (P=0.022).Results of in vivo studies:2.4.1Total body tumor buden and the invasiveness of organs:In both OX (8mice in experimental group and10mice in control group) and IC models (8mice in experimental group and10mice in control group), mice implanted with PC3-luc/SPARCLl cells displayed smaller areas and less intense luciferase activities in either the whole body or the dissected organs compared to the empty vector control mice (PC3-luc/EV)2.4.2Confirmation of the OX tumor and metastasis sites.To confirm this PC3cell identity in xenografted tumors, IHC staining of GFP (PC3-luc cell marker, was earned out to show that these tumors originated from xenografted PC3cells (PC3-luc/EV and PC3-luc/SPARCL1). Histological analysis of orthotopically xenografted PC3-luc/EV and PC3-luc/SPARCLl tumors indicated that they had a similar pattern of invasive tumor cell growth in the prostate and other metastatic sites.2.4.3The effect of protein SPARCL1on the size of OX tumorTo determine whether SPARCL1had an effect on suppressing orthotopic tumor growth, final orthotopic tumor volumes from PC3-luc/EV and PC3-luc/SPARCL1mice were measured and compared. Data revealed similar tumor volumes in EV and SPARCL1groups (p>0.05)2.4.4The comparation of the total body tumor burden between the experimental group and control groupWe computed the whole body luminescence index from normalized whole-body photon emission rates (reflective of total tumor burden). The whole body luminescence indexes from PC3-luc/SPARCL1group were consistently lower by approximately4-to7-fold than those from PC3-luc/EV group (P<0.001). Consistent with the whole body tumor burden, the total bone luminescence indexes in the PC3-luc/SPARCL1group at week5post-injection were also significantly lower than PC3-luc/EV group (P=0.04)2.4.5The effect of protein SPARCL1on the survive time of the miceKaplan-Meier curves were generated to evaluate overall survival for control mice (PC3-luc/EV) and mice xenografted with SPARCL1overexpression cells (PC3-luc/SPARCL1). The results indicated a significant difference in overall survival between the mice inoculated with PC3-luc/SPARCL1and mice inoculated with PC3-luc/EV (P<0.0001). Overall, mice inoculated with PC3-luc/SPARCL1cells survived about two weeks longer. Mice bearing PC3-luc/SPARCL1tumors had a better prognosis, as demonstrated by100%survival at60days compared to0%for mice harboring control cells.2.4.6SPARCL1expression suppresses metastasisTo gain insight into whether SPARCL1had an effect on the development and tropism of metastases, we evaluated the incidences of metastasis and tissue distribution of metastatic lesions in PC3-luc/EV and PC3-luc/SPARCL1groups using both OX and IC models. The numbers of metastatic sites were determined via bioluminescence imaging of anesthetized mice, bioluminescence ex vivo of isolated organs and X-ray imaging of the mouse skeletal system. When compared to PC3-luc-EV group, the total numbers of final visceral metastasis for the PC3-luc/SPARCL1group were statistically significantly lower in both in vivo models, more specifically,27%lower in the OX model (P=0.01) and45%lower in the IC model (P<0.001). In the IC model, metastatic lesions among main long bone skeletal sites decreased in PC3-luc/SPARCL1group significantly between PC3-luc/EV and PC3-luc/SPARCL1(P=0.04).Conclusions:1. We have screened SPARCL1gene as the candidate prostate cancer metastasis suppressor gene by bioinformatics method;2. SPARCL1protein had no effect on cell proliferation or anchorage independent growth of prostate cancer cell lines in vitro and had no significant effect on the growth of primary orthotopic tumors in vivo animal studies.3. SPARCL1could suppress motility and invasion of prostate cancer cell lines in vitro and could suppress the metastasis of prostate cancer in vivo animal studies as well as prolong the survive time of the animals with tumor.In conclusion, we have provided evidence that SPARCL1is a new metastasis-suppressor gene in prostate cancer. This study sets the stage for further investigations of the basic mechanisms that underlie cancer metastasis. Additional studies on SPARCL1will be valuable for determining its mechanisms of metastasis suppression in cancer. |
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