Purpose: The growth-related oncogene a (GROa) is a secreted interleukin-like molecule that interacts with the CXCR2 G-protein^coupled receptor. We found that the mRNA and protein products of GROa are more highly expressed in neoplastic than normal colon epithelium, and we studied potential mechanisms by which GROa may contribute to tumor initiation or growth. Experimental Design: Cell lines that constitutively overexpress GROa were tested for growth rate, focus formation, and tumor formation in a xenograft model. GROa 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 GROa and fibulin-1 genes. Results: In cell cultures, GROa transfection transformed NIH 3T3 cells, whereas inhibition of GROa by inhibitory RNA was associated with apoptosis, decreased growth rate, and marked up-regulation of the matrix protein fibulin-1. Forced expression of GROa was associated with decreased expression of fibulin-1. Expression of GROa 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 GROa or fibulin-1, implying a transcriptional basis for these findings. Conclusion: Elevated expression of GROa 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. GROa overexpression abrogates contact inhibition in cell culture models, whereas inhibition of GROa expression is associated with apoptosis. Importantly, coexpression of fibulin-1 with GROa 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.

Metformin and aspirin have been studied extensively as cancer preventative and therapeutic agents. However, the underlying molecular mechanisms for the inhibitory effects of pancreatic cancer development remain undefined. To gain further insight into their biological function in pancreatic cancer, we conducted a transcriptomic analysis using RNA sequencing to assess the differential gene expression induced by metformin (5 mM) and aspirin (2 mM), alone or in combination, after treatment of PANC-1 cells for 48 hours. Compared to an untreated control, metformin down-regulated 58 genes and up-regulated 91 genes, aspirin down-regulated 12 genes only, while metformin plus aspirin down-regulated 656 genes and up-regulated 449 genes (fold-change > 2, P < 10−5). Of the top 10 genes (fold-change > 10, P < 10−10) regulated by metformin plus aspirin, PCDH18, CCL2, RASL11A, FAM111B and BMP5 were down-regulated ≥ 20-fold, while NGFR, NPTX1, C7orf57, MRPL23AS1 and UNC5B were up-regulated ≥ 10-fold. Ingenuity Pathway Analysis (IPA) revealed that the pathways, “cholesterol biosynthesis”, “cell cycle: G1/S checkpoint regulation”, and “axonal guidance signaling” were the most statistically significant pathways modulated by metformin plus aspirin. Although the results need further functional validation, these data provide the first evidence for the synergistic action between metformin and aspirin in modulating the transcriptional profile of pancreatic cancer cells.

BackgroundBone morphogenetic proteins (BMP) are embryonic morphogens that are aberrantly expressed in lung cancer. BMPs mediate cell fate decisions and self-renewal of stem cells, through transcription regulation of inhibitor of differentiation protein/DNA binding proteins (Id1-3). Inhibition of BMP signaling decreases growth and induces cell death of lung cancer cells lines by downregulating the expression of Id proteins. It is not known whether the BMP signaling cascade regulates growth and the expression of Id proteins of lung cancer cells expressing the stem cell markers Oct4 and/or nestin.MethodsLung cancer cells expressing Oct4 or nestin were isolated from lung cancer cell lines by stably transfecting the Oct4 promoter or nestin promoter expression vectors that induce expression of the green fluorescent protein reporter.ResultsOur studies suggest that lung cancer cells expressing Oct4 or nestin are different cell populations. Microarray and quantitative RT-PCR demonstrated that the expression of specific stem cell markers were different between isolated Oct4 and nestin cells. Both the Oct4 and nestin populations were more tumorigenic than controls but histologically they were quite different. The isolated Oct4 and nestin cells also responded differently to inhibition of BMP signaling. Blockade of BMP signaling with the BMP receptor antagonist DMH2 caused significant growth inhibition of both the Oct4 and nestin cell populations but only increased cell death in the nestin population. DMH2 also induced the expression of nestin in the Oct4 population but not in the nestin cells. We also show that BMP signaling is an important regulator of Id1 and Id3 in both the Oct4 and nestin cell populations. Furthermore, we show that NeuN is frequently expressed in NSCLC and provide evidence suggesting that Oct4 cells give rise to cancer cells expressing nestin and/or NeuN.ConclusionThese studies show that although biologically different, BMP signaling is growth promoting in cancer cells expressing Oct4 or nestin. Inhibition of BMP signaling decreases expression of Id proteins and suppresses growth of cancer cells expressing Oct4 or Nestin. Small molecule antagonists of the BMP type I receptors represent potential novel drugs to target the population of cancer cells expressing stem cell markers.

Transforming Growth Factor-(TGF ) maintains keratinocyte homeostasis, and induces epithelial-tomesenchymal transition (EMT) in response to tissue injury. To investigate how these two TGF responses might become uncoupled during malignant transformation, we examined the TGF -regulated gene expression programs and cellular responses in human keratinocytes as a function of TGF type I receptor (T R-I) kinase activity and TGF level. The TGFmediated homeostatic gene response program and cellular growth arrest were extremely sensitive to a reduction in receptor kinase activity, while much stronger inhibition of TGF receptor activity was needed to inhibit the tissue injury response gene expression program and EMT. Both endogenous TGF and high exogenous levels of TGF induced the homeostatic response, while only high levels of TGF induced EMT. These results suggest that a reduction in receptor signaling activity may be sufficient for keratinocytes to escape from TGF 's tumor suppressor function, while higher levels of TGF associated with tumor progression are pro-invasive/metastatic. These findings have important implications for the optimal use of TGF /Smad signaling antagonists as anti-cancer therapeutics.

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