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Supplementary table2 Differentially methylated regions identified by bumphunter

Increasing evidence suggests that left-sided colon cancer (LCC) and right-sided colon cancer (RCC) are emerging as two different colorectal cancer types with distinct clinical characteristics. However, the discrepancy in the underlying molecular event between these types of cancer has not been thoroughly elucidated to date and warrants comprehensive investigation. To this end, an integrated dataset from The Cancer Genome Atlas was used to compare and contrast LCC and RCC, covering mutation, DNA methylation, gene expression, and miRNA. Briefly, the signaling pathway cross-talk is more prevalent in RCC than LCC, such as RCC-specific PI3K pathway, which often exhibits cross-talk with the RAS and P53 pathways. Meanwhile, methylation signatures revealed that RCC was hypermethylated relative to LCC. In addition, differentially expressed genes (n = 253) and differentially expressed miRNAs (n = 16) were determined between LCC and RCC. Especially for Prostate Cancer Susceptibility Candidate 1 (PRAC1), a gene that was closely associated with hypermethylation, was the top significantly downregulated gene in RCC. Multi-omics comparison of LCC and RCC suggests that there are more aggressive markers in RCC and that tumor heterogeneity occurs within the location-based subtypes of colon cancer. These results clarify the debate regarding the conflicting prognosis between LCC and RCC, as proposed by different studies.Implications: The underlying molecular features present in LCC and RCC identified in this study are beneficial for adopting reasonable therapeutic approaches to prolong overall survival and progression-free survival in colorectal cancer patients. Mol Cancer Res; 16(3); 476–85. ©2017 AACR.

IHC staining of selected LCC (A~C) and RCC (D~F) of PRAC

Background. Hypoxia is one of the driving forces of cancer progression, recurrence, and metastasis. However, the association between the tumor hypoxic tumor microenvironment and the tumor mutation burden (TMB) is poorly understood in gastrointestinal cancer. Methods. Approximately 2,000 samples from colorectal cancer (CRC) and stomach adenocarcinoma (STAD) patients were obtained from the gene expression omnibus database and the cancer genome Atlas databases and were clustered and subtyped by nonnegative matrix factorization. Significant differentially expressed genes that were possibly related to survival differences between the hypoxic and normoxic groups were subjected to multivariate Cox regression. Results. Gastrointestinal cancer patients with CRC and STAD were further divided into two subgroups, namely, the hypoxia group and the normoxia group, and hypoxia was correlated with unfavorable outcomes. Notably, hypoxic tumors had lower TMB but significantly higher levels of immune and stromal infiltration. A signature of HEYL and NRP1 selected by LASSO classified gastrointestinal cancer patients into either a low or high-risk group, allowing for the combination of TMB status with markers of hypoxia in future clinical applications. Conclusions. Hypoxia is an independent prognostic factor and a strong immune infiltration indicator in gastrointestinal tumors of different organs, especially for cancers with low TMB.

Background Complete remission is observed in less than half of hypermutated (HM) tumours after immune checkpoint blockade therapy, indicating that HM tumours are very heterogeneous. Thus, there is an urgent requirement to decipher the unknown intrinsic HM tumour subtypes. Methods Statistical analysis was performed on somatic mutation data from 5519 tumours across 11 cancer types obtained from The Cancer Genome Atlas and 338 colorectal cancer (CRC) samples obtained from an Asian cohort. Samples with a tumour mutation burden >10 mut/Mb were classified as HM. A total of 1040 HM samples harbouring corresponding transcriptomes were used for non-negative matrix factorisation clustering. Tumour mutational burden, neoantigens, T cell receptor (TCR) diversity, stromal score and immune score were compared between the subtypes. Results HM tumours fell into three distinct immune subtypes: HM1, HM2 and HM3. HM3 tumours were correlated with increased CD8 T cell infiltration, high TCR diversity, a high immune score and prolonged survival. HM2 tumours were correlated with an abundant stromal component, epithelial–mesenchymal transition, TGFβ, angiogenesis hallmarks and poor outcomes. The infiltration of more CD8 T cells and increased chemokine expression in HM3 were validated in CRC by immunofluorescence. Conclusions These findings will facilitate the development of a subtype-oriented therapy strategy to enhance the treatment effect in the near future.

Patients with right-sided colon cancer (RCC) generally have a poorer prognosis than those with left-sided colon cancer (LCC). We previously found that homeobox C6 (HOXC6) was the most significantly upregulated gene in RCC compared to LCC. However, it remains unclear whether HOXC6 plays a role in tumor proliferation and metastasis. Our study aimed to explore the potential oncogenic role and the detailed molecular mechanism of HOXC6 in RCC. In this study, HOXC6 was validated to be overexpressed in RCC and associated with poor prognosis. Furthermore, overexpression of HOXC6 promoted the migration and invasion of colon cancer cells through inducing EMT by activating the Wnt/β-catenin signaling pathway and inhibition of DKK1 secretion. Lastly, we preliminary explored the translational effect of HOXC6 and found that silencing of HOXC6 made HCT116 and HT29 cells more sensitive to irinotecan.

Kailai Wang,1,* Yanmei Yang,2,* Shu Zheng,1 Wangxiong Hu1 1Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education), The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310009, People’s Republic of China; 2Key Laboratory of Reproductive and Genetics, Ministry of Education, Women’s Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310006, People’s Republic of China*These authors contributed equally to this workCorrespondence: Wangxiong Hu; Shu Zheng, Tel +86-571-87784606 ; +86-571-87784501, Fax +86-571-87214404, Email wxhu@zju.edu.cn; zhengshu@zju.edu.cnIntroduction: Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide. However, the driver genes that promote CRC metastasis remain poorly understood. Association mining mines and extracts the repeated correlations and relevance in a dataset to predict the appearance of other data items according to the appearance of one item.Methods: Here, the Apriori algorithm was used to find the frequent mutational gene sets (FMGSs) and hidden association rules (ARs) within these FMGSs from 383 CRCs with whole exome sequencing datasets. The weighted correlation network analysis (WGCNA) was used to identify the hub genes in CRC. CCK8, colony formation, cell migration and invasion assays were adopted to detect the roles of hub genes in CRC.Results: Intriguingly, we found that MAL2 (myelin and lymphocyte protein 2) was associated with TP53 and APC in stage IV of CRC, and further subnetwork exploration based on WGCNA identified MAL2 as a potent hub gene. To validate the metastasis-related role of MAL2 in CRC, a lentivirus-based overexpression system was utilized to construct MAL2-overexpressing human CRC LOVO cells. Overexpression of MAL2 remarkably inhibited CRC cell proliferation and invasion.Conclusion: Our results highlighted that MAL2 acts as a tumor suppressor in CRC and could serve as a potential therapeutic target.Keywords: Apriori, CRC, frequent mutational gene sets, MAL2

Aberrant DNA methylation is thought to be an early event in cancer development. Thus, identification of DNA methylation-based markers may provide valuable evidence in clinical decision-making. In this study, a DNA methylation dataset from 514 normal-tumor pairs is used to explore possible shared differentially methylated regions (DMRs) across 12 cancer types. Results showed that DMR in Dopamine receptor D5 (DRD5) promoter may be serviced as a good candidate biomarker across different cancer types. We further validated the extended DMR (292bp) in DRD5 promoter using SEQUENOM MassARRAY platform. Detection of DRD5 promoter dynamic methylation will allow rapid risk assessment at diagnosis, for suspicious tumor with the tissue biopsies.

POLE mutations, which lead to an ultramutated phenotype in colorectal cancer (CRC), have been reported as a promising marker in immunotherapy. We performed sequencing of CRC cases in Zhejiang University (ZJU) and extracted obtainable data from recently published results, including The Cancer Genome Atlas (TCGA), Japanese studies and clinical trials, to present clinical patterns of POLE driver‐mutated CRC and reveal its heterogeneity. The rate of somatic POLE driver mutations has been reported as 2.60% (ZJU cohort), 1.50% (TCGA cohort), 1.00% (Japan cohort), and 1.00% (Lancet cohort). POLE driver mutations show a clearly increased mutation burden (mean TMB: 217.98 mut/Mb in ZJU; 203.13 mut/Mb in TCGA). Based on pooled data, more than 70.00% of patients with POLE driver mutations were diagnosed before they were 55 years old and at an early disease stage (Stage 0–II >70.00%), and more than 70.00% were male. Among Asian patients, 68.40% developed POLE driver mutations in the left‐side colon, whereas 64.00% of non‐Asian patients developed them in the right‐side colon (p, POLE could be a promising marker for immunotherapy, and its heterogeneity should not be ignored. Here we present the clinical patterns and heterogeneity of POLE driver mutations so that investigators and physicians will be better equipped to design clinical trials and analyze their data.

Background Patients with microsatellite instability-high (MSI-H) colorectal cancer (CRC) generally have a better prognosis than patients with microsatellite stable (MSS) CRC. However, some MSI-H CRC patients do not gain overall survival benefits from immune checkpoint-blockade treatment. In other words, heterogeneity within the subgroup of MSI-H tumors remains poorly understood. Thus, an in-depth molecular characterization of MSI-H tumors is urgently required. Methods Here, we use nonnegative matrix factorization (NMF)-based consensus clustering to define CRC MSI-H subtypes in The Cancer Genome Atlas and a French multicenter cohort GSE39582. CIBERSORT was used to calculate the proportions of 22 lymphocytes in tumor tissue in MSI-H subtypes. Results MSI-H CRC samples basically clustered into two subgroups (MSI-H1 and MSI-H2). MSI-H1 was characterized by a lower BRAF mutational status, higher frequency of chromosomal instability, global hypomethylation, and worse survival than MSI-H2. Further examination of the immune landscape showed that macrophages of the M2 phenotype were enriched in MSI-H1, which may be associated with poor prognosis in this subgroup. Conclusions Our results illustrate the genetic heterogeneity in MSI-H CRCs and macrophages may serve as good targets for anticancer therapy in MSI-H1. Graphical abstract Electronic supplementary material The online version of this article (10.1186/s12964-019-0397-4) contains supplementary material, which is available to authorized users.

Terahertz technology has been widely used in biomedical research. Herein, terahertz time-domain attenuated total reflection (THz TD-ATR) spectroscopy was employed to characterize and discriminate human cancer cell lines (DLD-1 and HT-29). Terahertz responses of the cell lines were measured and Savitzky-Golay algorithm was applied to smooth the spectra of refractive index, absorption coefficient and dielectric loss tangent in terahertz regime. Principal component analysis (PCA) was then adopted for feature extraction and cell characterization. Based on the processed data, cancer cell lines were discriminated by applying random forests (RF) method to analyze three characteristic parameters separately and the results from them were compared. Results indicate that absorption coefficient was the most sensitive parameter for cancer cell discrimination. Our study suggests great potential for human cancer cell recognition and provides experimental basis for liquid biopsy.

Background: HOXC6 was the most significantly upregulated gene in right-sided colon cancer (RCC) compared to left-sided colon cancer (LCC) according to our previous study. It is known that RCC has a higher immunogenicity than LCC because much higher prevalence of MSI-H samples, however, the role of HOXC6 acted in MSI-H tumors remains poorly understood. Methods: Expression datasets from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus databases were used to analyze the differential expression and prognostic role of HOXC6 in CRC. ELISA and qRT-PCR were performed to examine the association of HOXC6 and CCL2. Immunohistochemistry and immunofluorescence were performed to evaluate the correlation of HOXC6 and M2 macrophage infiltration. CCK8 and Transwell were used to evaluate the proliferation and migration of tumor cells in vitro.Results: HOXC6 was overexpressed in MSI-H CRC and associated with poor prognosis. Upregulation of CCL2 by HOXC6 could attract more M2 macrophage infiltration. IL6 secreted by M2 macrophages could induce epithelial-mesenchymal transition (EMT) of tumor cells by upregulating HOXC6. Overexpression of HOXC6 promoted the migration and invasion of CRC cells in vitro. Finally, inhibition of IL6/JAK pathway using ruxolitinib downregulated HOXC6 and suppressed invasion of HCT116 cells.Conclusion: Our study revealed that overexpression of HOXC6 attracted more M2 macrophage infiltration and the positive crosstalk between M2 macrophages and HOXC6-highly expressed tumor led to EMT and enhanced the migration ability of CRC, which offered a promising therapic target for treatment of MSI-H CRC.

Background Patients with right-sided colon cancer (RCC) generally have a poorer prognosis than those with left-sided colon cancer (LCC). We previously found that homeobox C6 (HOXC6) was the most significantly upregulated gene in RCC compared to LCC. However, it remains unclear whether HOXC6 plays a role in tumor proliferation and metastasis. Our study aimed to explore the potential oncogenic role and the detailed molecular mechanism of HOXC6 in RCC. Methods Expression datasets from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were used to uncover the underlying oncogenic role of HOXC6 in RCC. CCK8, transwell, subcutaneous tumor cell injection and tail vein tumor cell injection assays were used to evaluate the proliferation, migration, and invasion of tumor cells in vitro and in vivo. Immunohistochemistry and immunofluorescence were performed to evaluate the correlation of HOXC6 and M2 macrophage infiltration. Western blot, qRT-PCR, ELISA, co-IP, and luciferase reporter assays were performed to examine the association of HOXC6 with epithelial-mesenchymal transition (EMT). Results HOXC6 was overexpressed in RCC and associated with poor prognosis. Overexpression of HOXC6 promoted the migration and invasion of CRC cells in vitro and in vivo. Increase in CCL2 expression by upregulation of HOXC6 could attract more infiltrating M2 macrophages. IL6 secreted by M2 macrophages could induce EMT in tumor cells by upregulating HOXC6 and activating the Wnt/β-catenin signaling pathway via inhibition of DKK1 secretion. Conclusions Our study indicates that overexpression of HOXC6 induces EMT by regulating the DKK1/Wnt/β-catenin axis. The positive crosstalk between M2 macrophages and HOXC6 in tumors led to poor prognosis of RCC.

Prognostic factors of synchronous bone metastatic colorectal cancer (CRC) are still undetermined. We aimed to investigate survival outcome and prognostic factors of patients with synchronous bone metastatic CRC. Information of patients with synchronous bone metastatic CRC were obtained from the Surveillance, Epidemiology, and End Results (SEER) and West China Hospital (WCH) databases. Cases from SEER database composed construction cohort, while cases from WCH database were used as validation cohort. A novel nomogram was constructed to predict individual survival probability based on Cox regression model. The performance of the nomogram was internally and externally validated using calibration curves and concordance index (C-index). Three hundred and eighty-one patients from SEER database were eligible. The median disease specific OS was 9.0 months (95% confidence interval [CI]: 7.3–10.7 months). Multivariate Cox analysis identified seven independent prognostic factors including histological type, differentiation grade, T stage of primary tumor, CEA level, systemic chemotherapy, combined with liver metastasis and combined with lung metastasis. A novel nomogram was established based on these variables. In the internal validation, the C-index (0.72, 95% CI 0.69–0.75) and calibration curve indicated well performance of this nomogram at predicting survival outcome in bone metastatic CRC. In the external validation, the C-index was 0.57 (95% CI 0.46–0.68). The prognosis of synchronous bone metastatic CRC is very poor. Histological type, differentiation grade, T stage of primary tumor, CEA level, systemic chemotherapy, combined with liver metastasis and combined with lung metastasis are independent prognostic factors. Further study is warranted to confirm the practicality of the prognostic nomogram.

Aberrant DNA methylation is thought to be an early event in cancer development. Thus, identification of DNA methylation-based markers may provide valuable evidence in clinical decision-making. In this study, a DNA methylation dataset from 514 normal-tumor pairs is used to explore possible shared differentially methylated regions (DMRs) across 12 cancer types. Results showed that DMR in Dopamine receptor D5 (

Great mutational heterogeneity is observed both across cancer types (>1000‐fold) and within a given cancer type, with a fraction harboring >10 mutations per million bases, thus termed hypermutation. We determined the genome‐wide effects of high mutation load on the transcriptome and methylome of two cancer types; namely, colorectal cancer (CRC) and stomach adenocarcinoma (STAD). Briefly, hierarchical clustering of the expression and methylation profiles showed that the majority of CRC and STAD hypermutated samples were mixed and separated from their respective non‐hypermutated samples, exceeding the boundary of tissue specificity. Further in‐detailed exploration uncovered that the underlying molecular mechanism may be related to the perturbation of chromatin remodeling genes.

Background Angiopoietin-like protein 1 (ANGPTL1) has been reported to suppress migration and invasion in lung and breast cancer, acting as a novel tumor suppressor candidate. Nevertheless, its effects on colorectal cancer (CRC) remain poorly defined. In this study, we aim to demonstrate the biological function of ANGPTL1 in CRC cells. Methods We explored ANGPTL1 mRNA expression in human CRC tissues and its association with prognosis. CRC cell lines overexpressing ANGPTL1 or with ANGPTL1 knocked down were constructed and analyzed for changes in proliferation, colony formation, migration and invasion. ANGPTL1-regulated microRNAs were analyzed, and microRNA inhibitor and mimics were used to explore the role of microRNA in ANGPTL1-associated biological function. Results ANGPTL1 mRNA expression was down-regulated in CRC tissues, and high ANGPTL1 expression predicted better survival in CRC patients. ANGPTL1 overexpression resulted in suppressed migration and invasion in vitro, and it prolonged overall survival in mouse models. By contrast, its down-regulation enhanced migration and invasion of CRC cells. MicroRNA-138 expression was positively correlated with ANGPTL1 mRNA level in CRC tissues and up-regulated by ANGPTL1 in CRC cells. In addition, the microRNA-138 inhibitor or mimics could reverse or promote the ANGPTL1-mediated inhibition of the migratory capacity of CRC cells, respectively. Conclusions This study is the first to demonstrate the biological function of ANGPTL1 in CRC cells. ANGPTL1 expression was down-regulated in CRC tissues and inversely correlated with poor survival. ANGPTL1 repressed migration and invasion of CRC cells, and microRNA-138 was involved in this process. Electronic supplementary material The online version of this article (doi:10.1186/s13046-017-0548-7) contains supplementary material, which is available to authorized users.

// Wangxiong Hu 1, * , Yanmei Yang 2, * , Xiaofen Li 1 , Shu Zheng 1, 3 1 Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education), The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310009, China 2 Key Laboratory of Reproductive and Genetics, Ministry of Education, Women’s Hospital, Zhejiang University, Hangzhou, Zhejiang 310006, China 3 Research Center for Air Pollution and Health, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310009, China * These authors have contributed equally to this work Correspondence to: Shu Zheng, email: zhengshu@zju.edu.cn Keywords: gene expression, organ-specific genes, pan-cancer, weighted correlation network analysis Received: June 08, 2016 Accepted: December 05, 2016 Published: December 27, 2016 ABSTRACT It is widely accepted that some messenger RNAs are evolutionarily conserved across species, both in sequence and tissue-expression specificity. To date, however, little effort has been made to exploit the transcriptome divergence between cancer and adjacent normal tissue at the pan-organ level. In this work, a transcriptome sequencing dataset from 675 normal-tumor pairs, representing 21 solid organs in The Cancer Genome Atlas, is used to evaluate expression evolution. The results show that in most cancer types, gene expression divergence and organ-specificity are reduced in cancer tissue compared to adjacent normal tissue. Furthermore, we observe that all cancers share cell cycle dysregulation through interrogating differentially expressed protein coding genes. Meanwhile, weighted correlation network analysis is used to detect of the gene module structure variation between cancer and adjacent normal tissue. And modules consisting of tightly co-regulated genes in cancer change substantially compared with those in adjacent normal tissue. We thus assume that the destruction of a coordinated regulatory network might result in tumorigenesis and tumor progression. Our results provide new insights into the complex cancer biology and shed light on the mysterious regulation mode for cancer.

BackgroundMicrosatellite instability-high (MSI-H) tumour patients generally have a better prognosis than microsatellite-stable (MSS) ones due to the large number of non-synonymous mutations. However, an increasing number of studies have revealed that less than half of MSI-H patients gain survival benefits or symptom alleviation from immune checkpoint-blockade treatment. Thus, an in-depth inspection of heterogeneous MSI-H tumours is urgently required.MethodsHere, we used non-negative matrix factorisation (non-NMF)-based consensus clustering to define stomach adenocarcinoma (STAD) MSI-H subtypes in samples from The Cancer Genome Atlas and an Asian cohort, GSE62254.ResultsMSI-H STAD samples are basically clustered into two subgroups (MSI-H1 and MSI-H2). Further examination of the immune landscape showed that immune suppression factors were enriched in the MSI-H1 subgroup, which may be associated with the poor prognosis in this subgroup.ConclusionsOur results illustrate the genetic heterogeneity within MSI-H STADs, with important implications for cancer patient risk stratification, prognosis and treatment.

Background Metastasis is the major cause of cancer. Previously much effort has been made in identifying the metastasis-specific highly or low expressed genes, often with little success. We thus postulate that tumor immune microenvironment (TIME) disorder is the fundamental reason associated with cancer progression. Here, we try to determine the candidates associated with cancer progression in colorectal cancer (CRC). Methods CRC mRNA expression data sets were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO), respectively. CIBERSORT was used to decipher the proportions of 22 tumor-infiltrating lymphocytes (TILs). TILs that correlated with patient survival time in the multivariate Cox regression analysis were determined using the least absolute shrinkage and selection operator (LASSO) method. Survival differences between good- and poor-prognosis based on the prognostic index (PI) were tested by the Kaplan-Meier method and analyzed with the log-rank test with functions survfit and survdiff in the survival package for R. A P value Results Tumor-associated macrophages (TAMs) of the M2 phenotype was significantly enriched in stage IV CRCs than in other stages. CRC patients could be separated into two groups with high or low PI based on the proportion of mast cell and M2 macrophages. In addition, a negative correlation of M2 macrophages and CD8 T cell was observed. Conclusions Targeting pro-metastatic immune cells such as macrophages (e.g., transform into M1-like macrophages) may be a possible therapeutic strategy for CRC.

Background Prostate-derived Ets factor (PDEF) belongs to the Ets family of transcription factors. It plays an important role in tumorigenesis and progression of many tumors such as prostate cancer, breast cancer, and gastric cancer. However, its biological function in colorectal cancer (CRC) is still unclear. Methods A lentivirus vector overexpressing PDEF was constructed and transfected into SW620 cells with lipofectine. In order to knock down the expression of PDEF, PDEF-custom si-RNA was transfected into sw620 cells. The expression level of PDEF was detected by western blot and real time-polymerase chain reaction. The proliferation, invasion, migration and cell cycle of SW620 cells were investigated after transfection. Meanwhile, transfected SW620 cells were subcutaneously injected into nude mouse, and the specimens were harvested from the injection site for histological analysis after six-weeks injection. Results Cell proliferation was significantly inhibited when PEDF was overexpressed. Transwell tests showed that PDEF overexpression could suppress the migration and invasion of SW620 cells. In contrast, the ability of proliferation, migration and invasion became stronger when PDEF was knocked down. Further flow cytometry showed that overexpression of PDEF could reduce the ratio of cells at the G2/M phase. In addition, subcutaneous transplanted tumors overexpressing PDEF in the xenograft model were significantly smaller than the control group. Conclusions Overexpression of PDEF could inhibit the proliferation and invasion of CRC cell in vitro and in vivo. Our study suggested that PDEF could serve as a potential tumor biomarker and drug target in CRC.

Background HomeoboxC6 (HOXC6) belongs to the homeobox family, members of which encode a highly conserved family of transcription factors that play an important role in morphogenesis in all multicellular organisms. It was found to be upregulated in multiple-cancers, such as breast cancer, prostate cancer, liver cancer, colorectal cancer and so on. Upregulation of HOXC6 also contributed to poor prognosis in colorectal cancer. However, there were very few researches about HOXC6 in colorectal cancer and it was still unknown how it plays its role in tumor cell proliferation and metastasis in colorectal cancer. Methods Cell culture and HOXC6 overexpressed HCT116 cell line constructionHCT116 was cultured with 1640 medium added with 10% FBS and 1% Penicillin-Streptomycin Solution with general culture condition. Clinical patient tumor samples7 patients including 4 left-sided patients and 3 right-sided colorectal patients were collected for HOXC6 expression analysis compared to their para-tumor samples Total protein and plasma & nuclear protein extraction and Western blot analysis CCK8 and transwell with or without matrigel analysis RNASeq and Co-IP analysis Total mRNA extraction and qPCR analysis Survival analysis of gene expression in UALCAN database Results HOXC6 was upregulated in right-sided colorectal cancer (figure 1A) Upregulation of HOXC6 contributed to migration and invasion but not in proliferation (figure 1D, E) Wnt/b-catenin and P53 pathway were activated by HOXC6 upregulation (figure 1F, G, H) DKK1 was upregulated by HOXC6 and could combined to HOXC6 (figure 1I) EMT was induced by HOXC6 upregulation (figure 1J) Upregulation of HOXC6 contributed to poor prognosis in colorectal cancer (figure 1K) Conclusions HOXC6 could upregulate DKK1, Wnt/b-catenin pathways and induce EMT which contributed to metastasis in colorectal cancer. Further, we will explore the mechanisms of how upregulation of DKK1 could activate Wnt/b-catenin pathway.

Dysregulation of protein tyrosine phosphatase, receptor type B (PTPRB) correlates with the development of a variety of tumors. Here we show that PTPRB promotes metastasis of colorectal cancer (CRC) cells via inducing epithelial-mesenchymal transition (EMT). We find that PTPRB is expressed at significantly higher levels in CRC tissues compared to adjacent nontumor tissues and in CRC cell lines with high invasion. PTPRB knockdown decreased the number of invasive CRC cells in an in vitro wound healing model, and also reduced tumor metastasis in vivo. Conversely, PTPRB overexpression promoted CRC cell invasion in vitro and metastasis in vivo. PTPRB overexpression decreased vimentin expression and promoted E-cadherin expression, consistent with promotion of EMT, while PTPRB knockdown had the opposite effect. Hypoxic conditions induced EMT and promoted invasion in CRC cells, but these effects were eliminated by PTPRB knockdown. EMT blockade via TWIST1 knockdown inhibited the migration and invasiveness of CRC cells, and even increased PTPRB expression could not reverse this effect. Altogether, these data support the conclusion that PTPRB promotes invasion and metastasis of CRC cells via inducing EMT, and that PTPRB would be a novel therapeutic target for the treatment of CRC.

Introduction: Abnormal status of gene expression plays an important role in tumorigenesis, progression and metastasis of breast cancer. Mechanisms of gene silence or activation were varied. Methylation of genes may contribute to alteration of gene expression. This study aimed to identify differentially expressed hub genes which may be regulated by DNA methylation and evaluate their prognostic value in breast cancer by bioinformatic analysis. Methods: GEO2R was used to obtain expression microarray data from GSE54002, GSE65194 and methylation microarray data from GSE20713, GSE32393. Differentially expressed-aberrantly methylated genes were identified by FunRich. Biological function and pathway enrichment analysis were conducted by DAVID. PPI network was constructed by STRING and hub genes was sorted by Cytoscape. Expression and DNA methylation of hub genes was validated by UALCAN and MethHC. Clinical outcome analysis of hub genes was performed by Kaplan Meier-plotter database for breast cancer. IHC was performed to analyze protein levels of EXO1 and Kaplan-Meier was used for survival analysis. Results: 677 upregulated-hypomethylated and 361 downregulated-hypermethylated genes were obtained from GSE54002, GSE65194, GSE20713 and GSE32393 by GEO2R and FunRich. The most significant biological process, cellular component, molecular function enriched and pathway for upregulated-hypomethylated genes were viral process, cytoplasm, protein binding and cell cycle respectively. For downregulated-hypermethylated genes, the result was peptidyl-tyrosine phosphorylation, plasma membrane, transmembrane receptor protein tyrosine kinase activity and Rap1 signaling pathway (All p< 0.05). 12 hub genes (TOP2A, MAD2L1, FEN1, EPRS, EXO1, MCM4, PTTG1, RRM2, PSMD14, CDKN3, H2AFZ, CCNE2) were sorted from 677 upregulated-hypomethylated genes. 4 hub genes (EGFR, FGF2, BCL2, PIK3R1) were sorted from 361 downregulated-hypermethylated genes. Differential expression of 16 hub genes was validated in UALCAN database (p

Figure S6. Survival status of MSI-H CRC subtypes. (PDF 175 kb)

Figure S1. Clustering of 77 MSI-H CRCs in GSE39582 by NMF. Correlation matrix heatmaps correspond to rank 2 to 6. (PDF 830 kb)

Figure S4. Clustering of 53 stage II MSI-H CRCs in GSE39582 by NMF. Correlation matrix heatmaps correspond to rank 2 to 6. (PDF 557 kb)

Figure S2. MSI-H CRC patients (GSE39582) fell into two gene expression-based subtypes. (PDF 2907 kb)

Figure S7. Boxplot distribution of CCL2 and CCL5 expression level between MSI-H1 and MSI-H2 by using TCGA and GSE39582 data. (PDF 412 kb)

Figure S3. Clustering of 48 stage II MSI-H CRCs in TCGA by NMF. Correlation matrix heatmaps correspond to rank 2 to 6. (PDF 488 kb)

// Chunwen Tan 1, 2, * , Wangxiong Hu 1, * , Yanqin Huang 1 , Jiaojiao Zhou 1 , Shu Zheng 1 1 Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education, Key Laboratory of Molecular Biology in Medical Sciences, Zhejiang Province, China), Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China 2 Zhejiang Provincial Hospital of Traditional Chinese Medicine, Hangzhou, China * These authors have contributed equally to this work Correspondence to: Shu Zheng, email: zhengshu@zju.edu.cn Keywords: risk, genetic variants, colorectal cancer, colorectal adenoma, Chinese Received: December 13, 2015 Accepted: October 01, 2016 Published: October 19, 2016 ABSTRACT Background : Recent genome-wide association studies (GWAS) identified eighteen single-nucleotide polymorphisms (SNPs) to be significantly associated with the risk of colorectal cancer (CRC). However, overall results of the following replications are inconsistent and little is known about whether these associations also exit in colorectal adenomas (CRA). Methods: The SNP genotyping was performed using a Sequenom MassARRAY to investigate the association of these eighteen SNPs with colorectal neoplasm in a case-control study consisted of 1049 colorectal cancers, 283 adenomas, and 1030 controls. Results : Two of these SNPs, rs10505477 and rs719725, showed evidence of an association in both CRC and CRA in our study population. Besides, seven SNPs (rs10808555, rs7014346, rs7837328, rs704017, rs11196172, rs4779584, and rs7229639) were significantly associated with CRC, and another one SNP rs11903757 was over-represented in CRA compared with controls. The strongest association was provided by rs11196172 (OR = 2.02, 95% CI = 1.66 - 2.46, P < 0.0001) and rs11903757 (OR = 1.96, 95% CI = 1.28 - 3.00, P = 0.0026). Conclusion : These results suggest that some previously reported SNP associations also have impact on CRC and CRA predispositions in the Han Chinese population. A part of genetic risk to CRC is possibly mediated by susceptibility to adenomas.

// Jiekai Yu 1, * , Xiaofen Li 1, * , Chenhan Zhong 1 , Dan Li 1 , Xiaohui Zhai 1 , Wangxiong Hu 1 , Cheng Guo 1 , Ying Yuan 2 , Shu Zheng 1 1 Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education), the Second Affiliated Hospital, Zhejiang University School of Medicine, China 2 Department of Medical Oncology, The Second Affiliated Hospital, Zhejiang University School of Medicine, Zhejiang, China * These authors have contributed equally to this work Correspondence to: Shu Zheng, email: zhengshu@zju.edu.cn Keywords: iTRAQ-MS, gene microarray, colorectal cancer, biomarkers Received: April 05, 2016 Accepted: August 10, 2016 Published: September 20, 2016 ABSTRACT Proteins, as executives of genes’ instructions, are responsible for cellular phenotypes. Integrating proteomics with gene microarray, we conducted this study to identify potential protein biomarkers of colorectal cancer (CRC). Isobaric tags with related and absolute quantitation (iTRAQ) labeling mass spectrometry (MS) was applied to screen and identify differentially expressed proteins between paired CRC and adjacent normal mucosa. Meanwhile, Affymetrix U133plus2.0 microarrays were used to perform gene microarray analysis. Verification experiments included immunohistochemistry (IHC), western blot and enzyme-linked immunosorbent assay (ELISA) of selected proteins. Overall, 5469 differentially expressed proteins were detected with iTRAQ-MS from 24 matched CRC and adjacent normal tissues. And gene microarray identified 39859 differential genes from 52 patients. Of these, 3083 differential proteins had corresponding differentially expressed genes, with 245 proteins and their genes showed >1.5-fold change in expression level. Gene ontology enrichment analysis revealed that up-regulated proteins were more involved in cell adhesion and motion than down-regulated proteins. In addition, up-regulated proteins were more likely to be located in nucleus and vesicles. Further verification experiments with IHC confirmed differential expression levels of 5 proteins (S100 calcium-binding protein A9, annexin A3, nicotinamide phosphoribosyltransferase, carboxylesterase 2 and calcium activated chloride channel A1) between CRC and normal tissues. Besides, western blot showed a stepwise increase of annexin A3 abundance in normal colorectal mucosa, adenoma and CRC tissues. ELISA results revealed significantly higher serum levels of S100 calcium-binding protein A9 and annexin A3 in CRC patients than healthy controls, validating diagnostic value of these proteins. Cell experiments showed that inhibition of annexin A3 could suppress CRC cell proliferation and aggressiveness. S100 calcium-binding protein A9, annexin A3, nicotinamide phosphoribosyltransferase, carboxylesterase 2 and calcium activated chloride channel A1 were probably potential biomarkers of colorectal cancer. Annexin A3 was a potentially valuable therapeutic target of CRC.

Many studies have demonstrated that some genes (e.g. APC, BRAF, KRAS, PTEN, TP53) are frequently mutated in cancer, however, underlying mechanism that contributes to their high mutation frequency remains unclear. Here we used Apriori algorithm to find the frequent mutational gene sets (FMGSs) from 4,904 tumors across 11 cancer types as part of the TCGA Pan-Cancer effort and then mined the hidden association rules (ARs) within these FMGSs. Intriguingly, we found that well-known cancer driver genes such as BRAF, KRAS, PTEN, and TP53 were often co-occurred with other driver genes and FMGSs size peaked at an itemset size of 3~4 genes. Besides, the number and constitution of FMGS and ARs differed greatly among different cancers and stages. In addition, FMGS and ARs were rare in endocrine-related cancers such as breast carcinoma, ovarian cystadenocarcinoma, and thyroid carcinoma, but abundant in cancers contact directly with external environments such as skin melanoma and stomach adenocarcinoma. Furthermore, we observed more rules in stage IV than in other stages, indicating that distant metastasis needed more sophisticated gene regulatory network.

Background It is well-known that tumour is caused by somatic mutations. However, great mutational heterogeneity is observed both across cancer types (>1000 fold) and with a given cancer type, with a fraction of them harbour >10 mutations per Mb, thus termed hypermutation. Hypermutated patients are suitable for PD-1 blockade with favourable prognosis. Nevertheless, other omics such as transcriptome and methylome in hypermutated samples remain poorly understood. Here, we try to determine the genome-wide effects of high mutation loads on transcriptome and methylome across two cancer types, namely colorectal cancer (CRC) and stomach adenocarcinoma (STAD). Methods All tumour mRNA expression datasets (RNASeqV2) and DNA methylation data (HumanMethylation450) were obtained from The Cancer Genome Atlas. Known batch effects were corrected using the ComBat function implemented in the Bioconductor sva package. Differentially expressed gene (DEGs, false discovery rate (FDR) adjusted P-value 2 ) analysis between hypermutated and non-hypermutated was performed by DEGSeq package for R/Bioconductor. Significantly differentially methylated site (DMS, FDR adjusted P-value 0.2) was performed by limma package for R/Bioconductor. Results Most hypermutated cases were driven by microsatellite instability-high (MSI-H). Meanwhile, 935 and 1,047 DEGs and 1604 and 53 DMSs were identified in CRC and STAD (hypermutated vs. non-hypermutated), respectively. A well-conceived five-gene signature (DNAI2, EPHA5, GAS2L1, RNH1, TAGLN3) was identified that could predict prognosis of STAD hypermutated patients with high performance (C-index: 0.84). Additionally, hierarchical clustering of expression and methylation profiles show that majority of CRC and STAD hypermutated samples were mixed and separated from their respective non-hypermutated samples, exceeding the boundary of tissue-specificity. Further in-detail exploration uncovered that the underlying molecular mechanism was related to perturbation of chromatin remodelling genes (ARID1A, NCOR1, and MLL1 ~4) both in CRC and STAD hypermutated samples. Conclusions We thus concluded that MSI-H->chromatin remodelling genes inactivation->DNA methylation/expression variation axis shared in hypermutated samples was responsible for the consistent expression and methylation patterns across cancer types.

Liver metastases develop in more than half of the patients with colorectal cancer (CRC) and are associated with a poor prognosis. The factors influencing liver metastasis of CRC are poorly characterized, but this information is urgently needed. We have now discovered that small extracellular vesicles (sEVs; exosomes) derived from CRC can be specifically targeted to liver tissue and induce liver macrophage polarization toward an interleukin-6 (IL-6)-secreting proinflammatory phenotype. More importantly, we found that microRNA-21-5p (miR-21) was highly enriched in CRC-derived sEVs and was essential for creating a liver proinflammatory phenotype and liver metastasis of CRC. Silencing either miR-21 in CRC-sEVs or Toll-like receptor 7 (TLR7) in macrophages, to which miR-21 binds, abolished CRC-sEVs' induction of proinflammatory macrophages. Furthermore, miR-21 expression in plasma-derived sEVs was positively correlated with liver metastasis in CRC patients. Collectively, our data demonstrate a pivotal role of CRC-sEVs in promoting liver metastasis by inducing an inflammatory premetastatic niche through the miR-21-TLR7-IL-6 axis. Thus, sEVs-miR-21 represents a potential prognostic marker and therapeutic target for CRC patients with liver metastasis.

Chloroplasts are essential organelles, in which genes have widely been used in the phylogenetic analysis of green plants. Here, we took advantage of the breadth of plastid genomes (cpDNAs) sequenced species to investigate their dynamic changes. Our study showed that gene rearrangements occurred more frequently in the cpDNAs of green algae than in land plants. Phylogenetic trees were generated using 55 conserved protein-coding genes including 33 genes for photosynthesis, 16 ribosomal protein genes and 6 other genes, which supported the monophyletic evolution of vascular plants, land plants, seed plants, and angiosperms. Moreover, we could show that seed plants were more closely related to bryophytes rather than pteridophytes. Furthermore, the substitution rate for cpDNA genes was calculated to be 3.3 × 10 − 10 , which was almost 10 times lower than genes of nuclear genomes, probably because of the plastid homologous recombination machinery.