Correspondence: Kohei Miyazono, MD, PhD, Department of Molecular Pathology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan. Email: email@example.com
Bone morphogenetic proteins (BMPs) are multi-functional cytokines, which belong to the transforming growth factor-β (TGF-β) family. In some cancer tissues, aberrant expression of various BMP signal components has been detected. Here, we describe the divergent roles of BMPs during the progression of cancer. BMPs exhibit various effects on both cancer cells and on tumor microenvironments. BMPs inhibit the proliferation of cancer cells, with some exceptions. BMPs also induce the differentiation of certain cancer stem cells, and attenuate their aggressiveness. In parallel, BMPs play a critical role in the regulation of tumor angiogenesis and the metastasis of cancer cells. Some mouse xenograft models have revealed that cancer metastases are prevented by the inhibition of BMP signaling. Together, these findings imply that BMPs function as both suppressors and promoters of tumors in a context dependent manner. The bi-directional characteristics of BMPs in cancer are similar to those of TGF-β, which was previously described as a molecular ‘Jekyll and Hyde.’
Bone morphogenetic proteins (BMPs) are multi-functional proteins that were originally identified as factors that induce the differentiation of mesenchymal cells into osteoblastic and chondroblastic cells. BMPs were then found to exhibit versatile functions in the morphogenesis of various tissues and organs, including tooth, neuron, hair follicle, and vascular tissues. BMPs also play important roles in the maintenance of vascular homeostasis. BMPs and their orthologues are expressed in both vertebrates and invertebrates, such as Drosophila and Caenorhabditis elegans. In invertebrates, BMPs play pivotal roles in the development of early embryonic tissues, as well as in the morphogenesis of various organs.
BMPs are members of the transforming growth factor-β (TGF-β) family. It is now well-known that TGF-β displays bi-directional functions in the progression of cancer.[2, 3] TGF-β inhibits the growth of various epithelial cells, endothelial cells, hematopoietic cells, and immune cells. TGF-β also induces apoptosis of various epithelial cells. Thus, TGF-β functions as a tumor suppressor, and perturbations of TGF-β signaling result in the progression of certain types of cancer, including colorectal and pancreatic cancer.[2, 3] In contrast, TGF-β produced in cancer tissues induces tissue fibrosis, perturbations of immune and inflammatory reactions, and angiogenesis in the tumor microenvironment. Moreover, TGF-β stimulates the process of epithelial-mesenchymal transition (EMT) in cancer cells, as well as in normal epithelial cells, leading to the invasion and metastasis of cancer.[5, 6] Thus, TGF-β functions as a tumor-promoting factor in certain types of cancer.
The bi-directional roles of TGF-β have been well-documented by many investigators, with Bierie and Moses describing TGF-β to behave as a ‘molecular Jekyll and Hyde’ in the process of cancer progression. While BMPs have been reported to regulate the progression of various types of cancer, it has not been fully discussed whether BMPs exhibit uni- or bi-directional roles in the progression of cancer. In this review article, we discuss the multiple functions of BMPs in cancer cells as well as in the tumor microenvironment. The possibilities using BMPs or BMP antagonists for the regulation of cancer are also discussed.
In mammals, the TGF-β family includes 33 members, most of which are known to transduce signals through type I and type II serine/threonine kinase receptors and Smad proteins. While more than a dozen proteins were originally termed BMPs, some of them do not induce typical BMP-like signals; therefore, in this review article, we consider BMPs as factors that transduce signals through type I receptors for BMPs and BMP-specific Smad proteins (see below).
Like TGF-βs, BMPs bind to two different serine/threonine kinase receptors (specifically, type I and type II receptors), both of which are essential for the transduction of BMP signals (Fig. 1a). Certain BMPs bind to type I receptors alone; however, binding affinities to type I receptors increase in the presence of type II receptors. Type II receptor serine/thereonine kinases are constitutively active, and activate type I receptor kinases through the phosphorylation of the GS (Gly-Ser-rich) domain of type I receptors. Among the five different type II receptors in mammals, BMPs bind to BMP type II receptor (BMPR-II) and activin type II and type IIB receptors (ActR-II and ActR-IIB, respectively). Among the seven type I receptors, BMPs bind to activin receptor-like kinase (ALK)-1, -2, -3, and -6. BMPs may be classified into several subclasses by their structural similarities and abilities to bind to certain type I receptors. BMP-2 and BMP-4 are structurally similar to each other, and preferentially bind to ALK-3 and ALK-6. BMP-5, -6, -7, and -8 bind to ALK-2 and ALK-6. Growth and differentiation factor (GDF)-5, -6, and -7 preferentially bind to ALK-6, but do not bind to other BMP type I receptors. BMP-9 and BMP-10 bind to ALK-1, and weakly bind to ALK-2 (Fig. 1b). The availability and activity of BMPs are modulated by various extracellular agonists and antagonists. Structurally diverse secreted proteins, such as noggin and chordin, bind directly to BMPs, and regulate the diffusion and binding to their cognate receptors.
Type I receptors that are activated by type II receptors phosphorylate and activate receptor-regulated Smads (R-Smads) specific for BMP signaling, i.e., Smad1 and Smad5 (Smad1/5) in mammals (Fig. 2). Smad8 is structurally similar to Smad1 and 5; however, its roles in BMP signaling have not been fully determined. TGF-βs, activins and nodal activate another set of R-Smads, i.e., Smad2 and Smad3. Activated R-Smads form complexes with common-partner Smad (co-Smad; Smad4 in mammals), and move into the nucleus. Complexes containing R-Smads and co-Smad bind to various transcription factors and transcriptional co-activators (p300, CBP and GCN5) or co-repressors (Ski and SnoN) in the nucleus, and regulate the transcription of target genes. Smad6 and Smad7 are inhibitory Smads (I-Smads), which repress TGF-β family signaling through multiple mechanisms. Smad6 preferentially inhibits BMP signaling activated by ALK-3.[10, 11] In contrast, Smad7 inhibits most TGF-β family signaling by interacting with all type I receptors. In addition to the activation of Smad signaling pathways, BMPs activate various non-Smad signaling pathways, including ERK, JNK, and p38 MAP kinases, phosphatidyl inositol-3 (PI3) kinase-Akt and small GTPase pathways. These non-Smad pathways cooperate with Smad signaling pathways and regulate various cellular responses in target cells.
BMPs as Growth Inhibitors
TGF-β inhibits epithelial cell proliferation through induction of cyclin-dependent kinase (CDK) inhibitors, e.g., p21CIP1/WAF1 and p15INK4B. TGF-β also represses the expression of c-Myc and cell division cycle 25C (Cdc25), which inhibit cell proliferation. BMPs have also been reported to inhibit cell proliferation in a cell-type and context-dependent manner,[12-14] and serve as tumor suppressors of certain types of cancer, including breast, prostate and thyroid carcinomas. In contrast to TGF-β, BMPs induce the expression of p21CIP1/WAF1, but not that of other CDK inhibitors or c-Myc, in certain epithelial cells.
Recent data have revealed that BMP-2 and BMP-4 inhibit the proliferation of gastric epithelial cells, and function as tumor suppressors of diffuse-type gastric carcinoma (an aggressive type of gastric carcinoma) cells. Shirai and colleagues evaluated the correlation between BMP signaling and proliferation of gastric epithelial cells in normal gastric epithelium and intestinal metaplasia (Fig. 3a). In these tissues, strong staining for phospho-Smad1/5/8 (pSmad1/5/8) was mainly detected in the nuclei of surface epithelial cells located in the gastric pit. The majority of pSmad1/5/8-positive cells co-expressed p21CIP1/WAF1 in their nuclei. Conversely, MIB-1-positive Ki67-expressing cells were not frequently observed in pSmad1/5/8- and p21CIP1/WAF1-positive cells in these tissues. MIB-1-positive cells were mainly distributed in the lower parts of the gastric pit, where weak or negative staining for pSmad1/5/8 was frequently observed. Thus, BMPs may primarily act on differented epithelial cells, but not on rapidly dividing cells in the gastric pit.
The functions of BMPs in this type of gastric carcinoma were also studied using three different human diffuse-type gastric carcinoma cell lines (OCUM-12, HSC-39, and OCUM-2MLN). Overexpression of the dominant-negative form of ALK-3 (lacking the intracellular domain) in OCUM-12 and HSC-39 cells enhanced their growth in vivo when subcutaneously transplanted into nude mice. BMP-4 induced cell cycle arrest in these cells via the induction of p21CIP1/WAF1 in a Smad-dependent manner (Fig. 3b). Regulation of the expression of p15INK4B or c-Myc was not observed in these diffuse-type gastric carcinoma cells. Overexpression of the constitutively active form of ALK-3, which contains a mutation in the GS domain, in HSC-39 and OCUM-2MLN cells, suppressed the proliferation of these cells in vitro and in vivo. It should be noted that TGF-β failed to inhibit the proliferation of OCUM-2MLN cells in vitro, although it suppressed the proliferation of these cells in vivo by regulating tumor angiogenesis. These findings suggest that BMP-2 and BMP-4 regulate the proliferation of diffuse-type gastric carcinoma through a mechanism different from that of TGF-β, and strongly support the hypothesis that BMPs suppress tumorigenesis in gastric epithelium.
In addition to diffuse-type gastric carcinoma, Miyazaki and colleagues reported that BMP-7 inhibits the proliferation of androgen-insensitive prostate cancer PC-3 and DU-145 cells under low serum conditions. BMP-7 induced the expression of p21CIP1/WAF1, leading to the hypophosphorylation of the retinoblastoma protein (RB). Analyses using PC-3 cells expressing a constitutively active ALK-6 in a tetracycline-regulated manner revealed that BMP signaling resulted in the reduction of the tumor sizes of the PC-3 cells subcutaneously injected into nude mice. These findings suggest that BMPs inhibit the growth and proliferation of certain tumor cells through induction of p21CIP1/WAF1. However, it should be noted that BMP-2 inhibited the proliferation of another prostate cancer cell line, LNCaP, in the presence of androgen, while BMP-2 stimulated its proliferation in the absence of androgen. The detailed mechanism of BMP-mediated growth regulation of prostate cancer cells still remains to be elucidated.
BMP Signaling in Juvenile Polyposis Syndrome
Juvenile polyposis syndrome is an autosomal dominant syndrome, in which multiple hamartomatous polyps and cancers occur in the gastrointestinal tract. In approximately half of patients with juvenile polyposis syndrome, a germline mutation in the MADH4 (encoding Smad4) or BMPR1A (encoding ALK-2) is found,[19-21] suggesting that BMP signaling plays a pivotal role in the development of juvenile polyposis syndrome. Accordingly, inhibition of BMP signaling by transgenic expression of Noggin leads to the development of intestinal polyposis.[22, 23]
In juvenile polyposis syndrome, stromal parts, but not epithelial parts, in the gastrointestinal tracts appear to be primarily involved. Histological examination reveals edematous lamina propria with inflammatory cells and cystically dilated glands lined by cuboidal to columnar epithelium. Beppu et al. reported that conditional knock-out of Bmpr2 in the colorectal stroma of mice resulted in epithelial hyperplasia in the colon, with an increased proliferation of epithelial cells. These findings suggest that gastrointestinal cancer in juvenile polyposis syndrome may occur via abnormalities in stromal tissues, which lead to the transformation of adjacent epithelial cells.
Bi-directional Functions of BMPs in Colorectal Cancer
Similar to juvenile polyposis syndrome, mutations in BMP signal components have been observed in the majority of colorectal cancers. Through analyses using tissue microarray and immunohistochemistry, Kodach and colleagues determined the expression of BMP receptors, Smad4 and phospho-Smad1/5/8 in 72 sporadic colorectal cancers. Impaired expression of BMPR-II was more frequently observed in microsatellite unstable colorectal cancers (85%) than in microsatellite stable cancers (29%), and showed a mutually exclusive expression pattern compared to Smad4. Phosphorylation of Smad1/5/8 was not detected in the majority of colorectal cancers (70%), and the absence of phospho-Smad1/5/8 was correlated with the loss of BMPR-II or Smad4. In addition, Park et al. reported the presence of somatic frameshift mutations of BMPR2 in 13.2% of colorectal cancers with microsatellite instability. Supporting these clinical findings, BMPs have been reported to inhibit the in vitro or in vivo proliferation of colon cancer cells;[28, 29] hence, BMPs are assumed to act as tumor suppressors in sporadic colon cancer.
However, there are many conflicting data regarding the role of BMPs in the progression of colorectal cancer. Expression of BMP signal components and the transduction of BMP signaling appear to remain intact in colorectal cancer cells used in previous reports. Moreover, Deng et al. reported that the expression levels of BMP-4 in colorectal cancer cells were higher than those in normal epithelium or in adenoma. BMPs promote the invasive activity and in vivo tumor formation of some colorectal cancer cells;[32-34] hence, BMPs function as tumor promoters in colorectal cancer under certain conditions. These findings suggest that context-dependent factors in colon cancer might determine the role of BMPs.
BMPs Induce the Differentiation of Certain Cancer Stem Cells
Cancer stem (or cancer-initiating) cells are a small subpopulation of tumor cells that exhibit high tumor-forming abilities. Cancer stem cells have stem cell-like properties, such as the ability to self-renew, differentiate into multi-lineage cells, and express some stem cell markers. Recent studies have also revealed pivotal roles of TGF-β signaling in the maintenance of stem-cell-like properties of certain cancer stem cells. Autocrine TGF-β signaling plays a critical role in maintaining the stem-cell–like properties and tumorigenic activity of glioma-initiating cells (GICs),[35, 36] and an ALK-5 inhibitor, SB431542, efficiently reduced these abilities in GICs (Fig. 4). SB431542 was shown to dramatically stimulate the differentiation of GICs, and induce the appearance of cells expressing neural or glial cell markers. Ikushima and colleagues reported that the TGF-β–Sox4–Sox2 pathway is essential for retaining the stem-cell-like properties of GICs. In addition, Peñuelas et al. reported that TGF-β induces the expression of leukemia inhibitory factor (LIF) in a Smad-dependent fashion, and activates the downstream JAK-STAT pathway, leading to an increase in the tumorigenesis of GICs. TGF-β inhibitors may thus be useful for the treatment of glioblastoma by targeting GICs.
In contrast to the TGF-β-Smad signaling pathway, the BMP-Smad signaling pathway appears to induce the differentiation of GICs (Fig. 4). Piccirillo et al. reported that BMPs activated BMP receptors and triggered the Smad signaling pathway in cells obtained from human glioblastoma, resulting in the inhibition of cell proliferation and an increase in the expression of neural differentiation markers. BMPs reduced the size of the cell population expressing CD133, suggesting that BMPs reduce the tumor-initiating cell pool of glioblastoma. In vivo administration of BMP-4 blocked the tumor growth in immune-compromised mice after the intracerebral inoculation of human glioblastoma cells. Lee et al. reported that the epigenetic silencing of ALK-6 in GICs impaired their differentiation abilities, leading to an increase in their tumorigenic ability.
BMPs have also been reported to regulate the differentiation of other types of cancer stem cells.[17, 39-43] Lombardo et al. demonstrated that BMP-4 promoted the terminal differentiation of CD133+ colorectal cancer stem cells, and attenuated tumorigenic capacity of them. In the case of breast cancer, Gao et al. reported that a secreted antagonist of BMPs, Coco, sustained the expression of stem cell transcription factors, Nanog and Sox2, and induced cancer stem cell traits and cancer colonization in the lungs. These findings suggest that the BMP-Smad signaling pathway, which is known to regulate the activity of normal stem cells, acts as a key negative regulator of cancer stem cells, and that BMPs may be used to prevent the growth and recurrence of certain types of cancers in humans.[17, 41-43]
BMP Signaling in Vascular Tissues and Angiogenesis
Tumor angiogenesis is essential for the growth and metastasis of tumor cells. TGF-β potently inhibits the growth of endothelial cells in vitro, while it stimulates angiogenesis in vivo. Increased expression of TGF-β is correlated with increased vascular density in some types of tumors; however, it sometimes suppresses tumor angiogenesis, as observed in diffuse-type gastric carcinoma models.[16, 44] Controversial data have been reported regarding the effects of BMPs on the growth and differentiation of endothelial cells in vitro. It is possible that BMPs have a highly context-dependent effect on endothelial cells; hence, BMPs may function as pro-angiogenic and anti-angiogenic factors in vivo.
ALK-1 is specifically expressed in vascular endothelial cells. Mutations in the human ALK1 gene are responsible for the development of hereditary hemorrhagic telangiectasia (Osler-Weber-Rendu disease). While natural ligands for ALK-1 had not been elucidated for a long period, Brown et al. reported that BMP-9 and BMP-10 bind to ALK-1 in endothelial cells with high affinities. More recently, the type II receptor for BMP-9 was reported to be ActR-IIB.
There have been conflicting observations regarding the effects of BMP-9 on endothelial cells. Some reports demonstrated that the BMP-9/ALK-1 signaling pathway inhibits the proliferation of endothelial cells.[47, 48] In contrast, Suzuki and colleagues reported that BMP-9, as well as BMP-4 and BMP-6, induced the proliferation of in-vitro-cultured mouse embryonic-stem-cell-derived endothelial cells (MESECs) by inducing the expression of vascular endothelial growth factor receptor (VEGFR) 2 and Tie2, which is a receptor for angiopoietin-1.[49, 50] Importantly, the effects of BMP-9 on endothelial cells are observed at concentrations as low as 1 ng/mL, whereas the effects of BMP-4 or BMP-6 are observed at concentrations higher than 60 ng/mL. Reduced expression of ALK-1 in MESECs abrogated the effects of BMP-9 on the growth of MESECs. Moreover, in vivo angiogenesis was promoted by BMP-9 in some animal models.
Taken together, the BMP-9/ALK-1 signaling pathway activates the proliferation of endothelial cells under certain conditions, and plays an important role in angiogenesis in vivo and the maintenance of vascular homeostasis. Supporting these observations, several reports suggested that ALK-1 is a new therapeutic target for tumor angiogenesis.[51-53] Cunha and colleagues demonstrated that silencing the expression of the ALK1 gene, or systemic treatment with the ALK1-Fc fusion protein (RAP-041), suppressed the growth and progression of tumors through inhibition of angiogenesis in a transgenic mouse model (Rip-tag model). Thus, BMP-9 functions as a tumor promoting factor; hence, blocking the BMP-9/ALK-1 pathway by trapping BMP-9 by the ALK1-Fc fusion protein, or by other methods (e.g., ALK-1 antibodies), may prove useful for the treatment of cancer.
Regulation of Cancer Metastasis by BMPs
During the metastasis of cancer, cancer cells and tumor microenvironments interact with each other through various growth factors. Among them, the pro-metastatic roles of TGF-β during the progression of breast cancer have been well-documented.[54, 55] In the case of bone metastasis of breast cancer, TGF-β stimulates the production of parathyroid hormone-related protein (PTHrP) and interleukin (IL)-11 by breast cancer cells, which in turn enhance the osteoclast activity via the receptor activator of nuclear factor-κB (RANK)-RANK ligand (RANKL) system. Therapeutic benefits of TGF-β signal inhibition by various strategies have been highlighted in many types of cancer.[3, 56]
A number of recent studies have investigated the possible involvement of BMP signaling in cancer metastasis. Intensity of BMP signaling appears to be positively correlated with the degree of cancer malignancy and clinical stage in cancer patients.[58-60] Increasing evidence suggests that BMPs promote the motility and invasiveness of various types of cancer cells, such as prostate cancer, colon cancer, and malignant melanoma.[32, 61-63] In addition, Katsuno and colleagues reported that both TGF-β and BMP-2 promoted the motility and invasiveness of highly metastatic human breast cancer cells MDA-231-D in vitro, and demonstrated the involvement of TGF-β and BMP signaling in bone metastasis of breast cancer using functional in vivo bioluminescence imaging techniques. Furthermore, Langenfeld et al. reported that forced expression of BMP-2 in A549 lung adenocarcinoma cells significantly enhanced tumor growth in the lungs after intravenous injection of the cells.
Accordingly, inhibition of BMP signaling in cancer cells has been applied in several types of mouse xenograft models. A reduction of bone metastatic lesions of MDA-231-D cells was achieved by the forced expression of dominant-negative receptors for TGF-β and/or BMPs (TβR-II lacking the intracellular domain and ALK-3 lacking the intracellular domain, respectively) in cancer cells. Furthermore, overexpression of BMP antagonist noggin in cancer cells inhibited osteolytic bone metastasis of lung and prostate cancers in mouse models.[66, 67]
Recent evidence demonstrated that prostate cancer cells and osteoblasts in bone microenvironments interact with each other through BMP-mediated signaling. Thus, BMPs exert ‘pro-metastatic functions,’ implying that BMP is a putative target in the treatment of cancer metastasis. Specific inhibitors of BMP signaling, i.e., dorsomorphin and its related compounds, have been obtained.[69-71] These inhibitors appear to be useful for the treatment of metastasis in certain types of cancer.
In contrast to these findings, several reports have suggested that BMPs might function as inhibitors of cancer metastasis. Buijs et al. showed that BMP-7 reduces the formation of bone metastases in mouse xenograft models of breast cancer and prostate cancer.[73, 74] Furthermore, noggin positively regulated the bone metastatic abilities of several breast cancer cell lines. Hence, BMPs function as both pro-metastatic and anti-metastatic factors. Alarmo and Kallioniemi mentioned that the bi-directional character of BMPs might be explained by differences in the affinities of each BMP receptor to ligands.
Conclusion and Perspectives
Here, we described the multiple roles of BMPs during the progression of cancer (Table 1). Aberrant expression of BMP ligands has been detected in many types of cancers. BMPs influence various types of cancer cells, and regulate their proliferation and invasiveness. In parallel, BMPs also exert influence on tumor microenvironments and regulate the tumor angiogenesis. However, many conflicting data have been assimilated on BMPs. For instance, while the proliferation of some cancer cells is inhibited by BMPs, that of others is promoted by the same ligands. Though we have not discussed the relationship between BMPs and EMT in detail, recent reports have suggested that BMPs display bi-directional roles in the induction of EMT in certain types of epithelial cells.[76, 77] Similar to TGF-β, BMPs might also act initially as tumor suppressors, and later as tumor promoters.
Table 1. Multiple roles of BMPs during the progression of cancer
There are numerous reports available about BMPs and cancers. However, Thawani et al. suggested that most reports are based on results obtained from only a few types of cancer cell lines, and thus their findings are, in most cases, difficult to generalize. To elucidate the physiological roles of BMPs in cancer, it is extremely important to analyze a large number of materials simultaneously by using the latest high-throughput technologies. Recently, chromatin immunoprecipitation (ChIP)-sequencing facilitated genome-wide analyses of Smad-binding sites in many types of cells.[79-82] These high-throughput analyses may prove valuable for elucidating the context-dependent roles of BMPs in a comprehensive manner.
While BMPs exhibit bi-directional roles in cancer, we suggest the following possibilities for using BMPs and BMP antagonists in the treatment of cancer: (i) BMP ligands may be used to induce the differentiation of certain types of cancer stem cells, including glioblastoma stem cells; (ii) the BMP-9/ALK-1 signaling pathway may be an interesting target for the regulation of tumor angiogenesis; and (iii) the effects of BMP inhibitors (e.g., small molecule BMP receptor inhibitors and noggin) on the regulation of metastasis of certain types of cancer should be studied in detail in the future.
This work was supported by KAKENHI (Grant-in-Aid for scientific research on Innovative Area (Integrative Research on Cancer Microenvironment Network; grant number 22112002)) and for Young Scientists (B) (grant number 22700876); the Global Center of Excellence Program (Integrative Life Science Based on the Study of Biosignaling Mechanisms) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan; a Grant-in-Aid for Cancer Research for the Third-Term Comprehensive 10-Year Strategy for Cancer Control (H22-013) from the Ministry of Health, Labour and Welfare of Japan; and a grant from Swedish Cancer Society (grant number 10 0452). K.M. was supported by a Research Grant from the Takeda Science Foundation.
K.M. was supported by a research fund from Antisense Pharma GmbH (Germany).