To help identify genes, which may regulate metastasis in lung cancer, we performed representational difference analysis between a patient-derived non-small cell lung carcinoma (NSCLC) and immortalized normal human bronchial epithelial cells. This analysis revealed that bone morphogenetic proteins-2/4 (BMP) mRNA was expressed in the lung carcinoma. BMP-2/4 are known to induce pluripotent cell differentiation, enhance cell migration and stimulate proliferation during embryonic development. Despite being powerful morphogens it is not known whether BMP-2/4 have significant biological activity in human carcinomas. Furthermore, it has not been established whether the mature active BMP-2/4 protein is aberrantly expressed in patient-derived tumors. The purpose of this study was to determine whether the expression of the mature BMP-2/4 protein is disregulated in human lung carcinomas and to establish whether it has adverse biological activity. This study reveals that the mature BMP-2 protein, but not BMP-4, is highly over-expressed in human NCSLC with little to no expression in normal lung tissue or benign lung tumors. The expression of BMP-2 localized specifically to the cancer cells. Recombinant BMP-2 stimulated in vitro, the migration and invasiveness of the A549 and H7249 human lung cancer cell lines. In vivo, recombinant BMP-2 enhanced the growth of tumors formed from A549 cells injected subcutaneously into nude mice. Furthermore, inhibition of BMP-2 activity with either recombinant noggin or anti-BMP-2 antibody resulted in a significant reduction in tumor growth. This study shows that expression of the mature BMP-2 protein is disregulated in the majority of NSCLC. BMP-2 enhancement of tumor cell migration and invasion, as well as stimulating tumor growth in vivo, suggests it has important biological activity in lung carcinomas.

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.

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Bone morphogenetic proteins (BMPs) are highly conserved morphogens that are essential for normal development. BMP-2 is highly expressed in the majority of non-small cell lung carcinomas (NSCLC) but not in normal lung tissue or benign lung tumors. The effects of the BMP signaling cascade on the growth and survival of cancer cells is poorly understood. We show that BMP signaling is basally active in lung cancer cell lines, which can be effectively inhibited with selective antagonists of the BMP type I receptors. Lung cancer cell lines express alk2, alk3, and alk6 and inhibition of a single BMP receptor was not sufficient to decrease signaling. Inhibition of more than one type I receptor was required to decrease BMP signaling in lung cancer cell lines. BMP receptor antagonists and silencing of BMP type I receptors with siRNA induced cell death, inhibited cell growth, and caused a significant decrease in the expression of inhibitor of differentiation (Id1, Id2, and Id3) family members, which are known to regulate cell growth and survival in many types of cancers. BMP receptor antagonists also decreased clonogenic cell growth. Knockdown of Id3 significantly decreased cell growth and induced cell death of lung cancer cells. H1299 cells stably overexpressing Id3 were resistant to growth suppression and induction of cell death induced by the BMP antagonist DMH2. These studies suggest that BMP signaling promotes cell growth and survival of lung cancer cells, which is mediated through its regulation of Id family members. Selective antagonists of the BMP type I receptors represents a potential means to pharmacologically treat NSCLC and other carcinomas with an activated BMP signaling cascade.

BackgroundBone morphogenetic proteins (BMP) are embryonic proteins that are part of the transforming growth factor (TGFβ) superfamily, which are aberrantly expressed in many carcinomas. Inhibition of BMP receptors with small molecule inhibitors decreases growth and induces death of lung cancer cells, which involves the downregulation of Id1 and Id3 by a Smad dependent mechanism. Developmentally, BMP and TGFβ signaling utilizes Smad-1/5 independent mechanisms to stabilize the expression of X-linked inhibitor of apoptosis protein (XIAP) and activate TGFβ activated kinase 1 (TAK1), which are known to be potent inhibitors of apoptosis. The role of BMP signaling in regulating XIAP and TAK1 in cancer cells is poorly understood. Furthermore, the interaction between the BMP and TGFβ signaling cascades in regulating the activation of TAK1 in cancer cells has not been elucidated.MethodsFeedback regulation between the BMP and TGFβ signaling pathways and their regulation of XIAP, TAK1, and Id1 were examined in lung cancer cells utilizing siRNA and inhibitors targeting BMP type I receptors, inhibitors of BMP and TGFβ type I receptors, and an inhibitor of BMP and TGFβ type I and type II receptors.ResultsWe show that upon inhibition of BMP signaling in lung cancer cells, the TGFβ signaling cascade is activated. Both the BMP and TGFβ pathways activate TAK1, which then increases the expression of Id1. Inhibition of TGFβ signaling increased Id1 expression except when BMP signaling is suppressed, which then causes a dose-related decrease in the expression of Id1. Inhibition of both BMP and TGFβ signaling enhances the downregulation of TAK1. Our data also suggests that the blockade of the BMP type II receptor enhances the downregulation XIAP, which is important in decreasing the activity of TAK1. Knockdown studies demonstrate that both XIAP and TAK1 regulate the survival of lung cancer cells.ConclusionsThis paper highlights that targeting the BMP and TGFβ type I and type II receptors causes a downregulation of XIAP, TAK1, and Id1 leading to cell death of lung cancer cells. Small molecule inhibitors targeting the BMP and TGFβ receptors represents a potential novel means to treat cancer patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0511-9) contains supplementary material, which is available to authorized users.