During disease progression the cells that comprise solid malignancies undergo significant changes in gene copy number and chromosome structure. Colorectal cancer provides an excellent model to study this process. To indentify and characterize chromosomal abnormalities in colorectal cancer, we performed a statistical analysis of 299 expression and 130 SNP arrays profiled at different stages of the disease, including normal tissue, adenoma, stages 1–4 adenocarcinoma, and metastasis. We identified broad (> 1/2 chromosomal arm) and focal (< 1/2 chromosomal arm) events. Broad amplifications were noted on chromosomes 7, 8q, 13q, 20, and X and broad deletions on chromosomes 4, 8p, 14q, 15q, 17p, 18, 20p, and 22q. Focal events (gains or losses) were identified in regions containing known cancer pathway genes, such as VEGFA , MYC , MET , FGF6 , FGF23 , LYN , MMP9 , MYBL2 , AURKA , UBE2C , and PTEN . Other focal events encompassed potential new candidate tumor suppressors (losses) and oncogenes (gains), including CCDC68 , CSMD1 , POLR1D , and PMEPA1 . From the expression data, we identified genes whose expression levels reflected their copy number changes and used this relationship to impute copy number changes to samples without accompanying SNP data. This analysis provided the statistical power to show that deletions of 8p, 4p, and 15q are associated with survival and disease progression, and that samples with simultaneous deletions in 18q, 8p, 4p, and 15q have a particularly poor prognosis. Annotation analysis reveals that the oxidative phosphorylation pathway shows a strong tendency for decreased expression in the samples characterized by poor prognosis.

Several studies have verified the existence of multiple chromosomal abnormalities in colon cancer. However, the relationships between DNA copy number and gene expression have not been adequately explored nor globally monitored during the progression of the disease. In this work, three types of array-generated data (expression, single nucleotide polymorphism, and comparative genomic hybridization) were collected from a large set of colon cancer patients at various stages of the disease. Probes were annotated to specific chromosomal locations and coordinated alterations in DNA copy number and transcription levels were revealed at specific positions. We show that across many large regions of the genome, changes in expression level are correlated with alterations in DNA content. Often, large chromosomal segments, containing multiple genes, are transcriptionally affected in a coordinated way, and we show that the underlying mechanism is a corresponding change in DNA content. This implies that whereas specific chromosomal abnormalities may arise stochastically, the associated changes in expression of some or all of the affected genes are responsible for selecting cells bearing these abnormalities for clonal expansion. Indeed, particular chromosomal regions are frequently gained and overexpressed (e.g., 7p, 8q, 13q, and 20q) or lost and underexpressed (e.g., 1p, 4, 5q, 8p, 14q, 15q, and 18) in primary colon tumors, making it likely that these changes favor tumorigenicity. Furthermore, we show that these aberrations are absent in normal colon mucosa, appear in benign adenomas (albeit only in a small fraction of the samples), become more frequent as disease advances, and are found in the majority of metastatic samples. (Cancer Res 2006; 66(4): 2129-37)

Purpose: Aberrant promoter methylation and genomic instability occur frequently during colorectal cancer development. CpG island methylator phenotype (CIMP) has been shown to associate with microsatellite instability, and BRAF mutation and is often found in the right-side colon. Nevertheless, the relative importance of CIMP and chromosomal instability (CIN) for tumorigenesis has yet to be thoroughly investigated in sporadic colorectal cancers. Experimental Design: We determined CIMP in 161 primary colorectal cancers and 66 matched normal mucosae using a quantitative bisulfite/PCR/ligase detection reaction (LDR)/Universal Array assay. The validity of CIMP was confirmed in a subset of 60 primary tumors using MethyLight assay and five independent markers. In parallel, CIN was analyzed in the same study cohort using Affymetrix 50K Human Mapping arrays. Results: The identified CIMP-positive cancers correlate with microsatellite instability (P = 0.075) and the BRAF mutation V600E (P = 0.00005). The array-based high-resolution analysis of chromosomal aberrations indicated that the degree of aneuploidy is spread over a wide spectrum among analyzed colorectal cancers. Whether CIN was defined by copy number variations in selected microsatellite loci (criterion 1) or considered as a continuous variable (criterion 2), CIMP-positive samples showed a strong correlation with low-degree chromosomal aberrations (P = 0.075 and P = 0.012, respectively). Similar correlations were observed when CIMP was determined by MethyLight assay (P = 0.001 and P = 0.013, respectively). Conclusion: CIMP-positive tumors generally possess lower chromosomal aberrations, which may only be revealed using a genome-wide approach. The significant difference in the degree of chromosomal aberrations between CIMP-positive and the remainder of samples suggests that epigenetic (CIMP) and genetic (CIN) abnormalities may arise from independent molecular mechanisms of tumor progression.

The extraordinary invasiveness of human glioblastoma multiforme (GBM) contributes to treatment failure and the grim prognosis of patients diagnosed with this tumor. Consequently, it is imperative to define further the cellular mechanisms that control GBM invasion and identify promising novel therapeutic targets. Melanoma differentiation associated gene–9 (MDA-9/syntenin) is a highly conserved PDZ domain–containing scaffolding protein that promotes invasion and metastasis in vitro and in vivo in human melanoma models. To determine whether MDA-9/syntenin is a relevant target in GBM, we investigated its expression in tumor samples and involvement in GBM invasion and angiogenesis. We assessed MDA-9/syntenin levels in available databases, patient tumor samples, and human-derived cell lines. Through gain-of-function and loss-of-function studies, we analyzed changes in invasion, angiogenesis, and signaling in vitro. We used orthotopic xenografts with GBM6 cells to demonstrate the role of MDA-9/syntenin in GBM pathogenesis in vivo. MDA-9/syntenin expression in high-grade astrocytomas is significantly higher than normal tissue counterparts. Forced overexpression of MDA-9/syntenin enhanced Matrigel invasion, while knockdown inhibited invasion, migration, and anchorage-independent growth in soft agar. Moreover, overexpression of MDA-9/syntenin increased activation of c-Src, p38 mitogen-activated protein kinase, and nuclear factor kappa-B, leading to elevated expression of matrix metalloproteinase 2 and secretion of interleukin-8 with corresponding changes observed upon knockdown. GBM6 cells that stably express small hairpin RNA for MDA-9/syntenin formed smaller tumors and had a less invasive phenotype in vivo. Our findings indicate that MDA-9/syntenin is a novel and important mediator of invasion in GBM and a key regulator of pathogenesis, and we identify it as a potential target for anti-invasive treatment in human astrocytoma.

The oncogene astrocyte elevated gene-1 (AEG-1; MTDH) is highly expressed in glioblastoma multiforme (GBM) and many other types of cancer, where it activates multiple signaling pathways that drive proliferation, invasion, angiogenesis, chemoresistance, radioresistance, and metastasis. AEG-1 activates the Akt signaling pathway and Akt and c-Myc are positive regulators of AEG-1 transcription, generating a positive feedback loop between AEG-1 and Akt in regulating tumorigenesis. Here, we describe in GBM cells a direct interaction between an internal domain of AEG-1 and the PH domain of Akt2, a major driver in GBM. Expression and interaction of AEG-1 and Akt2 are elevated in GBM and contribute to tumor cell survival, proliferation, and invasion. Clinically, in silico gene expression and immunohistochemical analyses of patient specimens showed that AEG-1 and Akt2 expression correlated with GBM progression and reduced patient survival. AEG-1–Akt2 interaction prolonged stabilization of Akt2 phosphorylation at S474, regulating downstream signaling cascades that enable cell proliferation and survival. Disrupting AEG-1–Akt2 interaction by competitive binding of the Akt2-PH domain led to reduced cell viability and invasion. When combined with AEG-1 silencing, conditional expression of Akt2-PH markedly increased survival in an orthotopic mouse model of human GBM. Our study uncovers a novel molecular mechanism by which AEG-1 augments glioma progression and offers a rationale to block AEG-1–Akt2 signaling function as a novel GBM treatment. Cancer Res; 74(24); 7321–32. ©2014 AACR.

Purpose: The growth-related oncogene α (GROα) is a secreted interleukin-like molecule that interacts with the CXCR2 G-protein–coupled receptor. We found that the mRNA and protein products of GROα are more highly expressed in neoplastic than normal colon epithelium, and we studied potential mechanisms by which GROα may contribute to tumor initiation or growth. Experimental Design: Cell lines that constitutively overexpress GROα were tested for growth rate, focus formation, and tumor formation in a xenograft model. GROα expression was determined by Affymetrix GeneChip (241 microdissected colon samples), real-time PCR (n = 32), and immunohistochemistry. Primary colon cancer samples were also employed to determine copy number changes and loss of heterozygosity related to the GROα and fibulin-1 genes. Results: In cell cultures, GROα transfection transformed NIH 3T3 cells, whereas inhibition of GROα by inhibitory RNA was associated with apoptosis, decreased growth rate, and marked up-regulation of the matrix protein fibulin-1. Forced expression of GROα was associated with decreased expression of fibulin-1. Expression of GROα mRNA was higher in primary adenocarcinomas (n = 132), adenomas (n = 32), and metastases (n = 52) than in normal colon epithelium (P < 0.001). These results were confirmed by real-time PCR and by immunohistochemistry. Samples of primary and metastatic colon cancer showed underexpression of fibulin-1 when compared with normal samples. There were no consistent changes in gene copy number of GROα or fibulin-1, implying a transcriptional basis for these findings. Conclusion: Elevated expression of GROα is frequent in adenocarcinoma of the colon and is associated with down-regulation of the matrix protein fibulin-1 in experimental models and in clinical samples. GROα overexpression abrogates contact inhibition in cell culture models, whereas inhibition of GROα expression is associated with apoptosis. Importantly, coexpression of fibulin-1 with GROα abrogates key aspects of the transformed phenotype, including tumor formation in a murine xenograft model. Targeting GRO proteins may provide new opportunities for treatment of colon cancer.