Morphogenetic protein 2 (BMP-2) is normally expressed in the embryo promoting the development of several organs. Aberrant expression of BMP-2 occurs in approximately 98% of lung carcinomas, however, its role in regulating tumor growth is poorly understood. We show that BMP-2 induces Id-1 expression in lung cancer cell lines through its activation of Smad-1/5, which is dependent on cell culture conditions. A549 cells in DMEM/5% FCS BMP-2 activated Smad-1/5 and caused a transient increase in proliferation. In serum-free medium, BMP-2 induced significantly less Smad-1/5 activation and Id-1 expression, and produced significant growth inhibition. The affect of BMP-2 on tumor growth in vivo was substantially more significant. Recombinant BMP-2 coinjected with A549 cells, into nude mice increased proliferation and produced an increase in Id-1 expression. Forced expression of BMP-2 in A549 cells significantly enhanced tumor growth in the lungs following intravenous injection but not of subcutaneous tumors. Tumors in the lung were found to have an activated Smad-1/5 and expressed Id-1. Subcutaneous tumors expressed less activated Smad-1/5 and Id-1 than that of controls. Human lung carcinomas were also found to express an activated Smad-1/5 and Id-1. We provide evidence that BMP-2 promotes tumor growth. This paper highlights that cell culture experiments may not reveal the full biological affects of BMP-2, and its activity varies depending of the local environment.

The mechanism by which gastroesophageal reflux promotes metaplasia→dysplasia→ carcinoma is unknown. The aim of the study is to determine if repeated exposure to acid and bile confers a tumorigenic phenotype in a telomerase (hTERT)-immortalized benign Barrett’s cell line, BAR-T. BAR-T cells were exposed to acid (pH 4) (A) and bile salt (200µM glycochenodeoxycholic acid) (B) daily for 5 min up to 65+ wks. The control cells were grown in parallel without any A or B treatment. Cell morphology, proliferation, transformation, and molecular changes in the gene expression for COX-2, TC22, p53 and p53 target genes were analyzed at 8–12 wks intervals. At 46 wks BAR-T cells exposed to (A+B) showed distinct phenotypic changes: forming clusters and acini, and at 65 wks displayed foci in monolayer, and formed distinct colonies in soft agar. Untreated cells did not show any such changes. In A+B treated BAR-T cells, COX-2 mRNA increased 10-20-fold, TC22 mRNA increased by 2-3-fold at 22 – 65 wks, p53, MDM2, PERP and p21mRNA increased 2.5-, 6.4-, 4- and 2.6- fold respectively when compared to untreated cells at 34 weeks. However, at 58 wks onward, there was a sharp decline of p53 and its target genes to the baseline level. At 65 weeks A+B treated BAR-T cells formed tumor in nude mice whereas untreated cells did not. We demonstrate a novel in-vitro model of transformation of a benign Barrett’s cell line following repeated exposure to A+B over the course of 65 wks.

Bone morphogenetic protein-2 (BMP-2) is an evolutionary conserved protein that is essential for embryonic development. BMP-2 is highly expressed in f98% of human lung carcinomas with little expression in normal lung tissues. BMP-2 has been shown to enhance mobility, invasiveness, and metastasis of cancer cell lines. During development, BMP-2 induces the proto-oncogene phosphoinositide 3-kinase (PI3K)/ mammalian target of rapamycin (mTOR) signaling pathway to regulate stem cell differentiation. We show that BMP-2 induces the phosphorylation of mTOR in A549 and H1299 lung cancer cell lines, which is attenuated by the PI3K antagonists LY-294002 and wortmannin. p70S6 kinase, which is a direct downstream target of mTOR, is also regulated by BMP-2 in lung cancer cell lines. We find that BMP-2 induces cyclin E in A549 and H1299 cells, which is mediated by the PI3K/ mTOR signaling pathway. The regulation of cyclin E by BMP-2 occurs through a Smad 1/5 -independent mechanism. Forced expression of BMP-2 in A549 cells (A549/BMP-2) induces transformation as shown by an increase in foci formation. The mTOR antagonist, rapamycin, prevented foci formation of the A549/BMP-2 cells. This study provides evidence that BMP-2-mediated transformation of lung cancer cells involves the activation of the PI3K/mTOR signaling pathway. (Mol Cancer Res 2005;3(12):679 -84)

Although a protective role for mesalamine against colon cancer in ulcerative colitis has been shown epidemiologically, its molecular mechanism is unknown. We cloned and sequenced a novel human tropomyosin (hTM) isoform, TC22, which is an alternatively spliced variant of normal epithelial hTM isoform 5 (hTM5), identical apart from 25 C-terminal amino acids. TC22 is expressed in 100% of colorectal carcinoma but is not expressed in normal colon epithelial cells. To explore a molecular mechanism of chemoprevention, we examined the effect of mesalamine on TC22 expression using LS180 colon cancer cells. Expression of hTM5 and TC22 was investigated at the protein and gene levels by fluorescence-activated cell sorting and real-time reverse transcription-polymerase chain reaction. Small interference RNA (siRNA) against the TC22 variant were transfected into LS180 colon cancer cells, reducing protein and transcript levels by 45 to 50%. Mesalamine or sulfasalazine (2 mM), but not sulfapyridine, significantly (p Ͻ 0.02-0.006) reduced the expression of the TC22 transcript and significantly (p Ͻ 0.05 to Ͻ0.0002) reduced the expression of TC22 protein in a dose-dependent and reversible manner. Rosiglitazone, a specific peroxisome proliferator-activated receptor-␥ (PPAR␥) agonist, similarly and significantly (p Ͻ 0.002) reduced TC22 protein expression. A polymerase chain reaction array of 84 cancer-related genes performed on TC22 siRNA-transfected cells demonstrated a significant (more than two times) change in targets involved in apoptosis, adhesion, angiogenesis, and tissue remodeling. We conclude that mesalamine, sulfasalazine, and rosiglitazone significantly reduced the cellular expression of TC22, implicating PPAR␥ in this modulation. Similar suppression of TC22 by siRNA produced gene level changes on several critical carcinogenic pathways. These findings suggest a novel antineoplastic molecular effect of mesalamine.

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