Bioinformatics analysis of the genes involved in the extension of prostate cancer to adjacent lymph nodes by supervised and unsupervised machine learning methods: The role of SPAG1 and PLEKHF2

The present study aimed to identify the genes associated with the involvement of adjunct lymph nodes of patients with prostate cancer (PCa) and to provide valuable information for the identification of potential diagnostic biomarkers and pathological genes in PCa metastasis. The most important candidate genes were identified through several machine learning approaches including K-means clustering, neural network, Naïve Bayesian classifications and PCA with or without downsampling.In total, 21 genes associated with lymph nodes involvement were identified. Among them, nine genes have been identified in metastatic prostate cancer, six have been found in the other metastatic cancers and four in other local cancers. The amplification of the candidate genes was evaluated in the other PCa datasets. Besides, we identified a validated set of genes involved in the PCa metastasis. The amplification of SPAG1 and PLEKHF2 genes were associated with decreased survival in patients with PCa.     

Clonal Architectures and Driver Mutations in Metastatic Melanomas

To reveal the clonal architecture of melanoma and associated driver mutations, whole genome sequencing (WGS) and targeted extension sequencing were used to characterize 124 melanoma cases. Significantly mutated gene analysis using 13 WGS cases and 15 additional paired extension cases identified known melanoma genes such as BRAF, NRAS, and CDKN2A, as well as a novel gene EPHA3, previously implicated in other cancer types. Extension studies using tumors from another 96 patients discovered a large number of truncation mutations in tumor suppressors (TP53 and RB1), protein phosphatases (e.g., PTEN, PTPRB, PTPRD, and PTPRT), as well as chromatin remodeling genes (e.g., ASXL3, MLL2, and ARID2). Deep sequencing of mutations revealed subclones in the majority of metastatic tumors from 13 WGS cases. Validated mutations from 12 out of 13 WGS patients exhibited a predominant UV signature characterized by a high frequency of C->T transitions occurring at the 3′ base of dipyrimidine sequences while one patient (MEL9) with a hypermutator phenotype lacked this signature. Strikingly, a subclonal mutation signature analysis revealed that the founding clone in MEL9 exhibited UV signature but the secondary clone did not, suggesting different mutational mechanisms for two clonal populations from the same tumor. Further analysis of four metastases from different geographic locations in 2 melanoma cases revealed phylogenetic relationships and highlighted the genetic alterations responsible for differential drug resistance among metastatic tumors. Our study suggests that clonal evaluation is crucial for understanding tumor etiology and drug resistance in melanoma.     

FGF‐8b induces growth and rich vascularization in an orthotopic PC‐3 model of prostate cancer

Fibroblast growth factor 8 (FGF‐8) is expressed at an increased level in a high proportion of prostate cancers and it is associated with a poor prognosis of the disease. Our aim was to study the effects of FGF‐8b on proliferation of PC‐3 prostate cancer cells and growth of PC‐3 tumors, and to identify FGF‐8b‐associated molecular targets. Expression of ectopic FGF‐8b in PC‐3 cells caused a 1.5‐fold increase in cell proliferation in vitro and a four‐ to fivefold increase in the size of subcutaneous and orthotopic prostate tumors in nude mice. Tumors expressing FGF‐8b showed a characteristic morphology with a very rich network of capillaries. This was associated with increased spread of the cancer cells to the lungs as measured by RT‐qPCR of FGF‐8b mRNA. Microarray analyses revealed significantly altered, up‐ and downregulated, genes in PC‐3 cell cultures (169 genes) and in orthotopic PC‐3 tumors (61 genes). IPA network analysis of the upregulated genes showed the strongest association with development, cell proliferation (CRIP1, SHC1), angiogenesis (CCL2, DDAH2), bone metastasis (SPP1), cell‐to‐cell signaling and energy production, and the downregulated genes associated with differentiation (DKK‐1, VDR) and cell death (CYCS). The changes in gene expression were confirmed by RT‐qPCR. In conclusion, our results demonstrate that FGF‐8b increases the growth and angiogenesis of orthotopic prostate tumors. The associated gene expression signature suggests potential mediators for FGF‐8b actions on prostate cancer progression and metastasis. J. Cell. Biochem. 107: 769–784, 2009. © 2009 Wiley‐Liss, Inc.     

Development and Validation of a Novel Platform-Independent Metastasis Signature in Human Breast Cancer

PurposeThe molecular drivers of metastasis in breast cancer are not well understood. Therefore, we sought to identify the biological processes underlying distant progression and define a prognostic signature for metastatic potential in breast cancer.ResultsWe identified a broad range of metastatic potential that was independent of intrinsic breast cancer subtypes. 146 genes were significantly associated with metastasis progression and were linked to cancer-related biological functions, including cell migration/adhesion, Jak-STAT, TGF-beta, and Wnt signaling. These genes were used to develop a platform-independent gene expression signature (M-Sig), which was trained and subsequently validated on 5 independent cohorts totaling nearly 1800 breast cancer patients with all p-values < 0.005 and hazard ratios ranging from approximately 2.5 to 3. On multivariate analysis accounting for standard clinicopathologic prognostic variables, M-Sig remained the strongest prognostic factor for metastatic progression, with p-values < 0.001 and hazard ratios > 2 in three different cohorts.ConclusionM-Sig is strongly prognostic for metastatic progression, and may provide clinical utility in combination with treatment prediction tools to better guide patient care. In addition, the platform-independent nature of the signature makes it an excellent research tool as it can be directly applied onto existing, and future, datasets.     

Genetic variation in nucleotide excision repair pathway genes influence prostate and bladder cancer susceptibility in North Indian population

BACKGROUND:

Inherited polymorphisms of XPD and XPC genes may contribute to subtle variations in NER DNA repair capacity and genetic susceptibility to development of urological cancer such as prostate and bladder cancer.

MATERIALS AND METHODS:

We genotyped four Single Nucleotide Polymorphs (SNPs) of the DNA repair gene XPD and XPC in 195 prostate cancer (PCa) and 212 bladder cancer (BC) patients and 250 healthy controls from the same area. XPD Exon 10 (G>A) by amplification refractory mutation system and Exon 23 (A>C), XPC Intron 9 (Ins/Del) and Exon 15 (A>C) were genotyped by PCR-RFLP.

RESULTS:

Variant genotype of XPC demonstrated association with PCa as well as in BC (P, 0.013; P, 0.003). Combined genotype (GA+AA) revealed association with PCa and in BC (P, 0.012, P, 0.002). Variant allele also demonstrated risk in both the cancer. Diplotype of XPD and XPC was associated with a significant increase in PCa and BC risk. Variant (+/+) genotype of XPC intron 9 shown increased risk with PCa and in BC (P, 0.012; P, 0.032). CC genotype of XPC exon 15 revealed increase risk (P, 0.047) with PCa not in BC. In clinopathological grade variant allele of XPC intron 9 and 15 demonstrated risk with high grade of tumor and bone metastasis of PCa. In BC variant allele of XPD exon 10 and 15 also shown association with tumor grade. XPC intron 9 influences the risk of BC in former tobacco users in BC.

CONCLUSIONS:

Our result support that SNPs in XPD and XPC gene may reduce NER repair capacity and play a major role for PCa and BC in North India.     

Tumor‐propagating cells and Yap/Taz activity contribute to lung tumor progression and metastasis

Metastasis is the leading cause of morbidity for lung cancer patients. Here we demonstrate that murine tumor propagating cells (TPCs) with the markers Sca1 and CD24 are enriched for metastatic potential in orthotopic transplantation assays. CD24 knockdown decreased the metastatic potential of lung cancer cell lines resembling TPCs. In lung cancer patient data sets, metastatic spread and patient survival could be stratified with a murine lung TPC gene signature. The TPC signature was enriched for genes in the Hippo signaling pathway. Knockdown of the Hippo mediators Yap1 or Taz decreased in vitro cellular migration and transplantation of metastatic disease. Furthermore, constitutively active Yap was sufficient to drive lung tumor progression in vivo. These results demonstrate functional roles for two different pathways, CD24‐dependent and Yap/Taz‐dependent pathways, in lung tumor propagation and metastasis. This study demonstrates the utility of TPCs for identifying molecules contributing to metastatic lung cancer, potentially enabling the therapeutic targeting of this devastating disease.     

Oncosuppressor methylation: a possible key role in colon metastatic progression

K‐RAS and BRAF gene mutations are mandatory to set anti‐EGFR therapy in metastatic colorectal cancer (mCRC) patients. Due to the relationship of these mutations with tumor epigenotype, we hypothesized the potential role of oncosuppressor methylation of genes involved in K‐RAS/BRAF pathway (CDKN2A, RASSF1A, and RARbeta suppressor genes) in inhibiting EGFR signaling cascade. Primary tumor and synchronous liver metastatic tissues of 75 mCRC patients were characterized for promoter methylation by QMSP and for K‐RAS and BRAF mutations. RARbeta, RASSF1A, and CDKN2A genes were methylated in 82%, 35%, and 26% of primary tumors, respectively. RASSF1A resulted significantly more frequently methylated in liver metastasis than in primary site (P = 0.015), while RARbeta was significantly lower methylated in distant metastasis (P = 1.2 × 10^?6). As regards methylation content, RASSF1A methylation status was significantly higher in liver metastasis with respect to primary tumor (P = 0.000) underlying the role of this gene in liver metastatic progression. In our series K‐RAS and BRAF were mutated in 39% and 4% of cases, respectively. Methylation frequencies seemed to be unrelated to gene mutations; on the other hand, RASSF1A mean content methylation resulted significantly higher in liver than in primary tumor (288.78 vs. 56.23, respectively, P = 0.05) only in K‐RAS wild‐type cases sustaining a specific role of this gene in metastatic site thus supporting its function in strengthening the apoptotic role of K‐RAS. These evidences held the role of oncosuppressor methylation in both colon tumorigenesis and progression and suggested that epigenetic events should be taken into account when biological therapies in mCRC patients have to be set. J. Cell. Physiol. 226: 1934–1939, 2011. © 2010 Wiley‐Liss, Inc.     

Exosomal long noncoding RNA HOXD-AS1 promotes prostate cancer metastasis via miR-361-5p/FOXM1 axis

Development of distant metastasis is the main cause of deaths in prostate cancer (PCa) patients. Understanding the mechanism of PCa metastasis is of utmost importance to improve its prognosis. The role of exosomal long noncoding RNA (lncRNA) has been reported not yet fully understood in the metastasis of PCa. Here, we discovered an exosomal lncRNA HOXD-AS1 is upregulated in castration resistant prostate cancer (CRPC) cell line derived exosomes and serum exosomes from metastatic PCa patients, which correlated with its tissue expression. Further investigation confirmed exosomal HOXD-AS1 promotes prostate cancer cell metastasis in vitro and in vivo by inducing metastasis associated phenotype. Mechanistically exosomal HOXD-AS1 was internalized directly by PCa cells, acting as competing endogenous RNA (ceRNA) to modulate the miR-361-5p/FOXM1 axis, therefore promoting PCa metastasis. In addition, we found that serum exosomal HOXD-AS1 was upregulated in metastatic PCa patients, especially those with high volume disease. And it is correlated closely with Gleason Score, distant and nodal metastasis, Prostatic specific antigen (PSA) recurrence free survival, and progression free survival (PFS). This sheds a new insight into the regulation of PCa distant metastasis by exosomal HOXD-AS1 mediated miR-361-5p/FOXM1 axis, and provided a promising liquid biopsy biomarker to guide the detection and treatment of metastatic PCa.Subject terms: Bone metastases, Prostate cancer     

A novel prognostic signature of immune‐related genes for patients with colorectal cancer

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers with an estimated 1.8 million new cases worldwide and associated with high mortality rates of 881 000 CRC‐related deaths in 2018. Screening programs and new therapies have only marginally improved the survival of CRC patients. Immune‐related genes (IRGs) have attracted attention in recent years as therapeutic targets. The aim of this study was to identify an immune‐related prognostic signature for CRC. To this end, we combined gene expression and clinical data from the CRC data sets of The Cancer Genome Atlas (TCGA) into an integrated immune landscape profile. We identified a total of 476 IRGs that were differentially expressed in CRC vs normal tissues, of which 18 were survival related according to univariate Cox analysis. Stepwise multivariate Cox proportional hazards analysis established an immune‐related prognostic signature consisting of SLC10A2, FGF2, CCL28, NDRG1, ESM1, UCN, UTS2 and TRDC. The predictive ability of this signature for 3‐ and 5‐year overall survival was determined using receiver operating characteristics (ROC), and the respective areas under the curve (AUC) were 79.2% and 76.6%. The signature showed moderate predictive accuracy in the validation and GSE38832 data sets as well. Furthermore, the 8‐IRG signature correlated significantly with tumour stage, invasion, lymph node metastasis and distant metastasis by univariate Cox analysis, and was established an independent prognostic factor by multivariate Cox regression analysis for CRC. Gene set enrichment analysis (GSEA) revealed a relationship between the IRG prognostic signature and various biological pathways. Focal adhesions and ECM‐receptor interactions were positively correlated with the risk scores, while cytosolic DNA sensing and metabolism‐related pathways were negatively correlated. Finally, the bioinformatics results were validated by real‐time RT?qPCR. In conclusion, we identified and validated a novel, immune‐related prognostic signature for patients with CRC, and this signature reflects the dysregulated tumour immune microenvironment and has a potential for better CRC patient management.     

Identification of RECQ1-regulated transcriptome uncovers a role of RECQ1 in regulation of cancer cell migration and invasion

The RECQ protein family of helicases has critical roles in protecting and stabilizing the genome. Three of the 5 known members of the human RecQ family are genetically linked with cancer susceptibility syndromes, but the association of the most abundant human RecQ homolog, RECQ1, with cellular transformation is yet unclear. RECQ1 is overexpressed in a variety of human cancers, indicating oncogenic functions. Here, we assessed genome-wide changes in gene expression upon knockdown of RECQ1 in HeLa and MDA-MB-231 cells. Pathway analysis suggested that RECQ1 enhances the expression of multiple genes that play key roles in cell migration, invasion, and metastasis, including EZR, ITGA2, ITGA3, ITGB4, SMAD3, and TGFBR2. Consistent with these results, silencing RECQ1 significantly reduced cell migration and invasion. In comparison to genome-wide annotated promoter regions, the promoters of genes downregulated upon RECQ1 silencing were significantly enriched for a potential G4 DNA forming sequence motif. Chromatin immunoprecipitation assays demonstrated binding of RECQ1 to the G4 motifs in the promoters of select genes downregulated upon RECQ1 silencing. In breast cancer patients, the expression of a subset of RECQ1-activated genes positively correlated with RECQ1 expression. Moreover, high RECQ1 expression was associated with poor prognosis in breast cancer. Collectively, our findings identify a novel function of RECQ1 in gene regulation and indicate that RECQ1 contributes to tumor development and progression, in part, by regulating the expression of key genes that promote cancer cell migration, invasion and metastasis.     

Chromothripsis is a common mechanism driving genomic rearrangements in primary and metastatic colorectal cancer

Background

Structural rearrangements form a major class of somatic variation in cancer genomes. Local chromosome shattering, termed chromothripsis, is a mechanism proposed to be the cause of clustered chromosomal rearrangements and was recently described to occur in a small percentage of tumors. The significance of these clusters for tumor development or metastatic spread is largely unclear.

Results

We used genome-wide long mate-pair sequencing and SNP array profiling to reveal that chromothripsis is a widespread phenomenon in primary colorectal cancer and metastases. We find large and small chromothripsis events in nearly every colorectal tumor sample and show that several breakpoints of chromothripsis clusters and isolated rearrangements affect cancer genes, including NOTCH2, EXO1 and MLL3. We complemented the structural variation studies by sequencing the coding regions of a cancer exome in all colorectal tumor samples and found somatic mutations in 24 genes, including APC, KRAS, SMAD4 and PIK3CA. A pairwise comparison of somatic variations in primary and metastatic samples indicated that many chromothripsis clusters, isolated rearrangements and point mutations are exclusively present in either the primary tumor or the metastasis and may affect cancer genes in a lesion-specific manner.

Conclusions

We conclude that chromothripsis is a prevalent mechanism driving structural rearrangements in colorectal cancer and show that a complex interplay between point mutations, simple copy number changes and chromothripsis events drive colorectal tumor development and metastasis.     

Integrative Analysis of Somatic Mutations Altering MicroRNA Targeting in Cancer Genomes

Determining the functional impact of somatic mutations is crucial to understanding tumorigenesis and metastasis. Recent sequences of several cancers have provided comprehensive lists of somatic mutations across entire genomes, enabling investigation of the functional impact of somatic mutations in non-coding regions. Here, we study somatic mutations in 3′UTRs of genes that have been identified in four cancers and computationally predict how they may alter miRNA targeting, potentially resulting in dysregulation of the expression of the genes harboring these mutations. We find that somatic mutations create or disrupt putative miRNA target sites in the 3′UTRs of many genes, including several genes, such as MITF, EPHA3, TAL1, SCG3, and GSDMA, which have been previously associated with cancer. We also integrate the somatic mutations with germline mutations and results of association studies. Specifically, we identify putative miRNA target sites in the 3′UTRs of BMPR1B, KLK3, and SPRY4 that are disrupted by both somatic and germline mutations and, also, are in linkage disequilibrium blocks with high scoring markers from cancer association studies. The somatic mutation in BMPR1B is located in a target site of miR-125b; germline mutations in this target site have previously been both shown to disrupt regulation of BMPR1B by miR-125b and linked with cancer.     

AZGP1 is androgen responsive and involved in AR-induced prostate cancer cell proliferation and metastasis

Alpha-2-glycoprotein 1, zinc-binding (AZGP1), known as zinc-alpha-2-glycoprotein (ZAG), is a multifunctional secretory glycoprotein and relevant to cancer metastasis. Little is known regarding the underlying mechanisms of AZGP1 in prostate cancer (PCa). In the present study, we report that AZGP1 is an androgen-responsive gene, which is involved in AR-induced PCa cell proliferation and metastasis. In clinical specimens, the expression of AZGP1 in PCa tissues is markedly higher than that in adjacent normal tissues. In cultures, expression of AZGP1 is upregulated by the androgen-AR axis at both messenger RNA and protein levels. Furthermore, Chip-Seq assay identifies canonical androgen-responsive elements (AREs) at AZGP1 enhancer; and dual-luciferase reporter assays reveal that the AREs is highly responsive to androgen whereas mutations of the AREs abolish the reporter activity. In addition, AZGP1 promotes G1/S phase transition and cell cycle progress by increasing cyclin D1 levels in PCa cells. Functional studies demonstrate that knocking down endogenous AZGP1 expression in LNCaP and CWR22Rv1 cells largely weaken androgen/AR axis-induced cell migration and invasion. In vivo xenotransplantation tumor experiments also show that AZGP1 involves in androgen/AR axis-mediated PCa cell proliferation. Taken together, our study implicates for the first time that AZGP1 is an AR target gene and is involved in androgen/AR axis-mediated cell proliferation and metastasis in primary PCa.     

Identification of a novel cell cycle-related gene signature predicting survival in patients with gastric cancer

Gastric cancer (GC) is one of the most fatal cancers in the world. Thousands of biomarkers have been explored that might be related to survival and prognosis via database mining. However, the prediction effect of single gene biomarkers is not specific enough. Increasing evidence suggests that gene signatures are emerging as a possible better alternative. We aimed to develop a novel gene signature to improve the prognosis prediction of GC. Using the messenger RNA (mRNA)-mining approach, we performed mRNA expression profiling in a large GC cohort (n = 375) from The Cancer Genome Atlas (TCGA) database. Gene Set Enrichment Analysis (GSEA) was performed, and we recovered genes related to the G2/M checkpoint, which we identified with a Cox proportional regression model. We identified a set of five genes (MARCKS, CCNF, MAPK14, INCENP, and CHAF1A), which were significantly associated with overall survival (OS) in the test series. Based on this five-gene signature, the test series patients could be classified into high-risk or low-risk subgroups. Multivariate Cox regression analysis indicated that the prognostic power of this five-gene signature was independent of clinical features. In conclusion, we developed a five-gene signature related to the cell cycle that can predict survival for GC. Our findings provide novel insight that is useful for understanding cell cycle mechanisms and for identifying patients with GC with poor prognoses.     

Development and validation of hub genes for lymph node metastasis in patients with prostate cancer

Lymph node metastasis is one of the most important independent risk factors that can negatively affect the prognosis of prostate cancer (PCa); however, the exact mechanisms have not been well studied. This study aims to better understand the underlying mechanism of lymph node metastasis in PCa by bioinformatics analysis. We analysed a total of 367 PCa cases from the cancer genome atlas database and performed weighted gene co‐expression network analysis to explore some modules related to lymph node metastasis. Gene Ontology analysis and pathway enrichment analysis were conducted for functional annotation, and a protein‐protein interaction network was built. Samples from the International Cancer Genomics Consortium database were used as a validation set. The turquoise module showed the most relevance with lymph node metastasis. Functional annotation showed that biological processes and pathways were mainly related to activation of the processes of cell cycle and mitosis. Four hub genes were selected: CKAP2L, CDCA8, ERCC6L and ARPC1A. Further validation showed that the four hub genes well‐distinguished tumour and normal tissues, and they were good biomarkers for lymph node metastasis of PCa. In conclusion, the identified hub genes facilitate our knowledge of the underlying molecular mechanism for lymph node metastasis of PCa.