Emerging roles of the bone morphogenetic protein pathway in cancer: potential therapeutic target for kinase inhibition

Abstract

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor-β (TGF-β) family signalling pathway. Similar to TGF-β, the complex roles of BMPs in development and disease are demonstrated by their dichotomous roles in various cancers and cancer stages. Although early studies implicated BMP signalling in tumour suppressive phenotypes, the results of more recent experiments recognize BMPs as potent tumour promoters. Many of these complexities are becoming illuminated by understanding the role of BMPs in their contextual role in unique cell types of cancer and the impact of their surrounding tumour microenvironment. Here we review the emerging roles of BMP signalling in cancer, with a focus on the molecular underpinnings of BMP signalling in individual cancers as a valid therapeutic target for cancer prevention and treatment.

BMP signalling pathway: a primer

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) family. First described by their ability to induce ectopic bone formation [1,2], BMPs are now recognized as multifunctional cytokines essential for defining the vertebral embryonic axis during development and regulating key biological processes, such as endochondral bone formation, organogenesis and angiogenesis. To date, there are over 20 known BMP ligands (Figure 1). BMPs are synthesized as large inactive precursors (400–500 amino acids) comprise an N-terminal signal peptide, prodomain that ensures proper folding and a mature C-terminal peptide [3,4]. Sequential cleavage of two sites within the prodomain (R-X-K-R and R-X-X-R respectively) by furin and procollagen C-proteinase (also known as BMP1) yields the mature BMP monomer (50–100 amino acids) [5]. The mature BMP monomer has seven cysteines; six form intramolecular disulfide bonds, whereas the seventh dimerizes with another monomer to generate the biologically active BMP ligand, which exists as homo- and heterodimers [6,7].

Figure 1. Schematic representation of the canonical BMP signalling cascade.

Binding of the BMP ligand dimer to its type I and type II receptors forms the BMP transmembrane signalling complex. Within this complex, the type II receptor serine–threonine kinase phosphorylates and activates the type I receptor. The canonical signalling occurs when type I receptor serine–threonine kinase phosphorylates SMAD1/5/8, leading to the formation of the SMAD1/5/8–SMAD4 complex. The SMAD1/5/8–SMAD4 complex then translocates to the nucleus, where it activates the expression of BMP target genes such as Id1, −2 and −3. BMP signalling is regulated at multiple steps. Extracellular antagonists can complex with BMP ligand to prevent binding to its receptor. Smurf1/2 and SMAD6/7 can prevent phosphorylation of SMAD1/5/8 and binding to its co-factor SMAD4. Small molecule BMP inhibitors, such as dorsomorphin (DM) and DMH1, are competitive kinase inhibitors of BMP type I receptor. Inside the green insert are the non-canonical (SMAD-independent) signalling pathways, p38, LIMK, Rho and ROCK. Not shown are additional BMP pathway modulators such a co-receptors, which can modulate ligand-receptor binding affinity, and pseudoreceptors, which block receptor activation.

All members of the TGF-β superfamily, including BMPs, bind to and recruit two type I and two type II serine/threonine receptor kinases to form a transmembrane receptor complex [5,7,8] (Figures 1 and 2). Upon formation of this complex, the constitutively active type II receptor phosphorylates the type I receptor, activating its kinase domain [9] (Figure 2). Activated type I receptors then signal via the SMAD family of downstream mediators [9]. SMADs are divided into three distinct classes: (1) receptor regulated SMADs (R-SMADs); (2) common-mediator SMAD (Co-SMAD) and (3) inhibitory SMADs (I-SMADs). The R-SMADs (SMAD1, -2, -3, -5 and -8) are directly phosphorylated and activated by type I receptors. R-SMADs, in turn, active SMAD4, the sole Co-SMAD, and, together, translocate to the nucleus to activate downstream BMP target genes. The I-SMADs (SMAD6 and -7), in contrast, antagonize TGF-β and BMP signalling [9]. Independent of the canonical SMAD-mediated signalling pathway, BMPs can also participate in a non-canonical pathway that leads to activation of the mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) signalling cascade [10].

Figure 2. Partial list of TGF- β/BMP ligands, and respective receptor and R-SMAD activation.

TGF-β superfamily ligands are arranged according to phylogenetic analysis [5]. In some cases, involvement of co-receptors (Endoglin) are represented. The yellow box represents ligand–receptor interactions that trigger SMAD1/5/8 signalling cascade, and the blue box represents ligand–receptor interactions that trigger SMAD2/3 signalling cascade. The hatched box represents ligand–receptor interactions that trigger either cascade depending on the presence of a co-receptor Endoglin. This is a simplified representation of very complex sets of molecular interactions and not meant to be comprehensive. Modified from [5]: Mueller, T.D. and Nickel, J. (2012) Promiscuity and specificity in BMP receptor activation. FEBS Lett. 586, 1846–1859; and [8]: Yadin, D., Knaus, P. and Mueller, T.D. (2016) Structural insights into BMP receptors: specificity, activation and inhibition. Cytokine Growth Factor Rev. 27, 13–34.

As shown in Figure 1, TGF-β signalling is very complex, involving the engagement of multiple type I and type II receptors and R-SMADs [5]. In general, BMP ligands engage three type II receptors (Table 1): activin receptor, type IIA, (ActR-IIA); activin receptor, type IIB (ActR-IIB); and BMP receptor, type II (BMPR2) [5,8]. In addition, BMPs engage four type I receptors (Table 1): activin receptor like kinase-1 (ALK1), also known as activin A receptor type II-like 1; activin receptor like kinase-2 (ALK2), also known as activin A receptor, type I; activin receptor like kinase-3 (ALK3), also known as BMP receptor, type 1A; and activin receptor like kinase-6 (ALK6), also known as BMP receptor, type 1B [5]. BMP type I receptor activation leads to phosphorylation of SMAD1/5/8, which translocates to the nucleus to complex with SMAD4 and activate target genes, such as the inhibitors of DNA binding/differentiation (Id) family [5] (Figure 2). The Id proteins, comprise Id1, Id2, Id3 and Id4, are key regulators of cell proliferation and are implicated in tumorigenesis through their pro-apoptotic and oncogenic properties.

Table 1. Nomenclature of TGFβ/BMP type I and type II receptors.

Although each receptor is known by multiple names, accepted symbol for the gene encoding each BMP receptor is italicized in the right column. Protein symbol for each receptor used in the present review is bold faced.

	Alternative names
Type I receptors
Activin receptor-like kinase 1	ACVRL1, ALK1
Activin A receptor, type I	ACVR1, ALK2, ACTRI, ACVR1A, SKR1, TSR1, ACVRLK2
Bone morphogenetic protein receptor, type IA	BMPRIA, ALK3, CD292, SKR5, ACVRLK3
Bone morphogenetic protein receptor, type IB	BMPRIB, ALK6
Activin receptor, type IB	ACVR1B, ALK4, ACTRIB; ACVRLK1; ALK4; SKR2
Transforming growth factor-beta receptor 1	TGFBR1, ALK5, ACVRLK4
Activin A receptor, type IC	ACVR1C, ALK7, ACVRLK7
BMP type II receptors
Bone morphogenetic protein receptor, type II	BMPR2, BMPR2, BMPR-II, BMR2, PPH1, BMPR3, BRK-3, POVD1, T-ALK
Activin A receptor, type IIA	ACVR2A, ActR-IIA, ACTRII, ACVR2
Activin A receptor, type IIB	ACVR2B, ActR-IIB, ACTRIIB, HTX4
Transforming growth factor-beta receptor II	TGFRB2, TGFR2, TGF-β-RII

BMP signalling is regulated at multiple levels, including tissue distribution of ligands and receptors, assembly of distinct transmembrane receptor complexes by numerous ligand homo- and heterodimer combinations [5], and extracellular protein antagonists that directly bind to BMP ligands with distinct affinities. The extracellular BMP antagonists are classified into three subfamilies: 1) DAN family, which includes Gremlin, DAND5 (COCO) and Sclerostin (SOST1); 2) Twisted gastrulation (TSG) and 3) Chordin and Noggin [11]. BMP signalling is also regulated by intracellular factors such as the I-SMADs, SMAD ubiquitination regulator factors (Smurfs), and serine/threonine phosphatases, such as PP1A [12]. The sheer complexity of BMP-TGF-β signalling, involving multiple extracellular antagonists, multiple forms of ligand homo-heterodimers, complex ligand–receptor interactions, and overlap between the canonical BMP and TGF-β signalling pathways, poses a significant challenge towards identifying appropriate targets for therapeutic development.

Role of BMP signalling in cancer pathogenesis

Although a relationship between BMPs and cancer has been noted for over 20 years, the precise roles of BMP signalling in cancer development and progression are just beginning to be elucidated. Depending on the BMP ligand and cancer type, BMPs can either promote or inhibit tumorigenesis. This review serves to further elucidate the role of the BMP pathway and shed light on the recent development and use of small molecule BMP inhibitors for potential cancer therapy. Here, we present an updated review of BMP function in specific cancers (Table 2) and discuss the potential of the BMP pathway as a therapeutic target.

Table 2. Partial list of studies elucidating the role of BMP signalling in cancer.

Studies are listed based on different levels of evidence from human tumour data to in vitro cellular and in vivo preclinical data.

	Studies
Acute megakaryoblastic leukaemia (AMKL, non-Down syndrome)	Human somatic mutation: CBFA2T3-GLIS2 fusion protein identified in non-DS-AMKL patients. Overexpression of BMP2/4 observed in non-DS-AMKL patients expressing fusion gene (Gruber et al. [33]).
	In vitro: Increased Id1 expression and ability of cells to self-renew in murine hematopoietic cells transfected with chimeric transcript; effect inhibited by DM.
	In vivo: BMP gain-of-function phenotypes in Drosophila expressing CBFA2T3-GLIS2, e.g. shortened, blistered wings and ectopic wing veination.
Diffuse intrinsic pontine glioma (DIPG)	Human somatic mutations: 7 gain-of-function somatic mutations in ACVR1 (ALK2) were identified in patients with DIPG (R206H, Q207E, R258G, G328V, G328W, G328E and G356D) (St Jude Children’s Research Hospital-Washington University Pediatric Cancer Genome Project [155], Buczkowicz et al. [50], Taylor et al. [51], Fontebasso et al. [52]). 5 of the 7 mutations identical with germline mutations in fibrodysplasia ossificans progressive (FOP).
	In vitro: Increased levels of pSMAD1/5 in human DIPG samples with R206H and G328E mutations in ACVR1 gene. Increased expression of Id1 and Id2 in G328V mutant human astrocytes. Increased levels of pSMAD 1/5 in G328E, G328V, R258G, G356D and R206H mutant mouse astrocytes; SMAD phosphorylation inhibited by LDN-193189.
	In vivo: Disruption of dorsoventral patterning and loss of head and dorsal structures in zebrafish expressing G328V, G356D, R206H, G328W, G328E, R258G mutations; partial rescue of ventralization phenotype with LDN-193189.
High-grade glioma (HGG)	BMP signalling is active in certain high-grade gliomas (HGG) obtained from adult patients (Hover [55]). Deletion of BMPR1A (ALK3) in oncogenic mouse astrocytes resulted in decreased proliferation, and invasion, migration. In addition, in a mouse model of HGG involving orthotopic transplantation of transformed astrocytes, the genetic knockout of BMPR1A (ALK3) showed a dramatic increase in survival. In vitro effects of BMPR1A ablation was recapitulated with DMH1 [55].
Lung Cancer	Alterations in gene expression: 98 % of human NSCLC aberrantly overexpress BMP2 (Langenfeld [57]). Decreased 1-year survival rate in patients with elevated serum BMP2 (Choi et al. [59]). High BMP2 expression associated with lymph node metastasis and advanced tumour stage (Chu et al. [60]).
	In vitro: In A549 lung adenocarcinoma cells with increased expression of BMP2, LDN-193189 reduced cell growth and promoted cell death as measured by increased LDH release (Fontinos et al. [147]). BMP2 dose-dependent stimulation of cell migration and invasion in A549 and H7249 lung cancer cell lines; effect completely inhibited by Noggin (Langenfeld et al. [62]). Growth inhibition and cell death of H1229 and A549 lung cancer cell lines as well as decreased phosphorylation of SMAD1/5/8 and Id1-3 expression using small molecule BMP antagonists(Langenfeld et al. [64]). DMH1 reduced cell invasion and migration of A549 cells (Hao et al. [149]).
	In vivo: BMP2 enhanced tumour growth and angiogenesis in murine model of lung cancer (Langenfeld et al. [63]). DMH1 significantly reduced tumour growth in mouse model of NSCLC (Hao et al. [149]).
Colorectal cancer	Alterations in gene expression. Germline mutations in BMPR1A and SMAD4 in roughly 50 % of individuals with juvenile polyposis (Jass et al. [16], van Hattem et al. [17], Calva-Cequeria et al. [18]). Increased BMP7 expression associated with increased tumour aggression and worse clinical prognosis (Motoyama et al. [28]) in patients with colorectal cancer.
	In vitro: Enhanced migration and invasion of HCT116 cells with overexpression of BMP4 (Deng et al. [24]).
Pancreatic cancer	Alterations in gene expression: Increased expression of BMP2/4/7 in human pancreatic cancer specimens.
	In vitro: BMP-induced EMT leading to increased invasiveness and up-regulation of matrix metalloproteinase-2 (MMP-2) in Panc-1 cells (Gordon et al. [66]).
Ovarian cancer	Alterations in gene expression: Overexpression of BMP2/4 and Id1/3 in primary human ovarian cancer cell lines (Shepherd et al. [75,76]). Amplification of BMP ligands in human ovarian cancers, correlation of BMP receptor expression with poor prognosis, and active BMP signalling in human ovarian tissue (Hover et al. [156]).
	In vitro: DM or LDN-193189 decreased viability of EOC216 ovarian cancer cell line (Ali et al. [150]). DMH1 reduced ovarian tumour sphere growth and enhanced sensitivity of ovarian cancer cells to Cisplatin treatment (Hover et al. [156]).
	In vivo: In mouse model of serous ovarian cancer, use of recombinant BMP2 promoted significant tumour growth, an effect that was inhibited by DM (Peng et al. [80]). DM also significantly increased median survival time in a mouse xenograft model of epithelial ovarian cancer (Ali et al. [150]).
Osteosarcoma	Alterations in gene expression: Overexpression of BMP2/4 and BMP6 in high-grade osteosarcoma with malignant fibre histiosarcoma (MFH)-type pattern and chondroblastic osteosarcoma respectively (Yoshikawa et al. [83], Sulzbacher et al. [84]). Co-expression of BMP-2/4 and BMPR2 associated with reduced 5-year survival rate(Yoshikawa et al. [87]).
	In vitro: BMP2 enhanced heptotactic migration of murine osteosarcoma Dunn cells to several extracellular matrix ECM) components, including fibronectin, type I collagen, and laminin-1, and promoted cell migration, cell proliferation, and focal adhesion formation via modulation of fibronectin-integrin-β1 signalling. Noggin abrogated the stimulatory effects of BMP2 (Sotobori [90]).
Prostate cancer	Alterations in gene expression: BMP-6 expression detected in over 50 % of patients with metastatic prostate cancer; absent from non-metastatic or benign-prostate samples (Bentley et al. [92]). Higher-grade human prostate tumours (Gleason score of 6 or more) have increased BMP6 immunostaining than lower-grade human prostate tumours (Gleason score of 4 or less) (Barnes et al. [95]). BMP6 mRNA detected exclusively in malignant epithelial cells in metastatic prostate specimens, but undetectable in benign prostate samples (Hamdy et al. [94]). Positive correlation between epithelial staining for Id-1 and BMP-6 in prostate cancer samples (Darby et al. [96]).
	In vitro/in vivo: BMP6 enhanced rate of cell migration and invasion in PC3M and DU145 cell lines (Darby et al. [96]). Anti-BMP6 attenuated LuCaP 23.1-induced osteoblastic activity and reduced interosseous tumour size in mouse model of prostate cancer (Dai et al. [97]).
Breast cancer	Alterations in gene expression: Elevated expression of BMPR1A and BMPR2 associated with poor relapse-free survival time (Owens et al. [115]).
	Overexpressing BMP7 primary tumours associated with accelerated bony metastasis (Alarmo et al. [118]).
	In vitro: BMP2 enhanced cell migration in MCF-7 cells and increased expression of BCSG1, breast-cancer-specific gene (Clement et al. [106]).
	In vivo: In mouse model of breast cancer, BMP2 formed tumours with enhanced vasculature and increased Id1 and p38 MAPK activation (Clement et al. [106], Rada et al. [107]). DMH1 treated mice with breast cancer had reduced primary tumour burden and lung metastasis as well as decreased lymphatic vessel growth and expression of EMT markers e.g. Snail, Twist (Owens et al. [115]). Deletion of the BMP receptor BMPR1A impairs mammary tumour formation and metastasis in a mouse genetic model expressing MMTV.PyMT oncogene (Pickup et al. [117]).
Melanoma	Alterations in gene expression: Enhanced immunostaining of BMP4/7 in primary and metastatic melanoma specimens (Rothhammer et al. [123]).
	In vitro: Significant inhibition of cellular invasion in stably transfected chordin or BMP4 antisense cell lines.
	In vivo: BMP2 enhanced melanoma cell migration in chick embryos transplanted with B16-F1 cells; melanoma cell migration inhibited by Noggin (Busch et al [124])
Head and neck squamous cell cancer	Alterations in gene expression: 98 % of HNSCC express baseline BMP-2 expression. High levels of BMP2 expression associated with increased incidence of local failure rate (Sand et al. [132]). Overexpression of BMP2 associated with increased lymph node metastasis and extracapsular spread of metastatic lymph nodes (Zhou et al. [134]).
	In vitro: BMP4 promoted cell invasion and migration in Tu686 and Tu212 cell lines (Xu et al. [135]).
	In vivo: Mouse model of oral squamous cell carcinoma treated with rhBMP2 had reduced survival and rapid local tumour growth as well as poorly differentiated tumour morphology (Kokorina et al. [133]).

Colorectal cancer

BMP ligands and their receptors are found in mesenchymal and epithelial cells within the colon and small intestine and are actively involved in apoptosis of mature colonic epithelial cells and maturation of small intestinal secretory cells [13]. Consistent with the important role of BMP signalling in gastrointestinal homoeostasis, specific gene mutations encoding components of the BMP pathway are associated with gastrointestinal cancer [14,15].

Juvenile Polyposis Syndrome (JPS, OMIM#174900) is an autosomal dominant syndrome that predisposes individuals to upper gastrointestinal polyps and a significantly increased lifetime risk of developing colorectal cancer. Germline mutations in BMPR1A (encoding ALK3) and SMAD4 genes account for nearly half of all JPS cases [16–18]. In histological tissue analysis of polyps from patients with JPS and a SMAD4 germline mutation, epithelial SMAD4 protein expression was absent from polyps, supporting an important role of BMP signalling in the development of epithelial hamartomas [19]. In in vivo studies of transgenic mice, forced expression of the BMP antagonist Noggin resulted in the formation of intestinal hamartomas that were morphologically similar to those found in human JPS [20]. Similarly, BMPR1A mutations in mice also induced intestinal hamartomatous polyps [21]. Taken together, both human and murine studies suggest that the germline loss of ALK3 and SMAD4 increases the risk of spontaneous gastrointestinal cancer.

The role of BMPs in sporadic colorectal cancer (CRC), however, is less well defined. In in vitro models, BMP2, BMP3 and BMP7 were tumour suppressive, and inactivation of the BMP3 gene through promoter methylation was associated with microsatellite instability and BRAF oncogene mutation [22,23,24]. In addition, several studies demonstrated a widespread loss of BMP function in epithelial cells of CRC. For instance, using phosphorylated-SMAD1, -5, -8 (p-SMAD 1/5/8) as a marker of BMP pathway activation, Kodach et al. [25] demonstrated that BMP signalling was inactivated in 70% of CRC specimens. From their study, Kodach et al. [25] hypothesized two pathways by which BMP loss-of-function leads to CRC: (1) loss of epithelial SMAD4 epithelial expression and 2) loss of epithelial BMPR2 expression. In CRC cell lines, loss of BMPR2 expression correlated with microsatellite instability and disruption of the 3′-untranslated region (3′UTR) of the BMPR2 gene [26]. At the same time, however, BMPR2 activation also correlated with more advanced tumour grade [26]. In addition, increased expression levels of BMP4 and BMP7 were associated with tumour progression as well as poorer clinical prognosis [27,28]. Interestingly, in contrast with the association of germline BMPR1A mutation with polyp formation, ALK3 (BMPR1A) loss-of-function did not lead to CRC [23]. In summary, loss of BMP signalling appears to be associated with early stages of tumorigenesis at the transition from adenoma to carcinoma, whereas BMP signal activation is associated with high-grade advanced tumours.

Recent finding that loss of SMAD4 in CRC cells switches BMP signalling from tumour suppressive to metastasis promoting may reconcile of some of the divergent roles of BMP signalling in CRC [14]. In CRC patients, loss of SMAD4 in the presence of normal BMP expression is associated with reduced survival times. In CRC cells lacking SMAD4, activation of BMP signalling promotes metastatic behaviour via activation of Rho signalling via ROCK and LIM domain kinase (LIMK). These results indicate the role of SMAD-independent non-canonical BMP pathway in CRC pathogenesis, and suggest that pharmaceuticals that block this pathway, such as ROCK inhibitors, might be efficacious for CRC [14].

Acute megakaryoblastic leukaemia (AMKL)

Acute megakaryoblastic leukaemia (AMKL; M7) is a clinically heterogeneous disease that represents 5–10% of all paediatric acute myeloid leukaemia (AML) and 1–2% of adult AML [29–31]. In children, AMKL is classified into two major subgroups: leukaemia in patients with Down syndrome (DS-AMKL) and leukaemia in patients without Down syndrome (non-DS-AMKL). DS-AMKL carries a very favourable prognosis with an event-free survival rate close to 80% [29,32]. Patients with non-DS-AMKL, however, fare considerably worse and, until recently, little was known about the genetic aberrations that underlie non-DS-AMKL.

Using transcriptome sequencing, Gruber et al. [33] found that 27% of patients with non-DS-AMKL had a cryptic inversion on chromosome 16, inv(16) (p13.3q24.3) that resulted in the fusion of CBFA2T3 to GLIS2. In addition, patients with CBFA2T3-GLIS2 had significantly reduced survival compared with patients without the chimeric transcript (28% compared with 42% 5-year survival) [33]. CBFA2T3, a member of the ETO family of transcriptional corepressors, is normally expressed in the hematopoietic system and is involved in maintaining hematopoietic stem cell quiescence [34]. Unlike CBFA2T3, GLIS2 is not normally found in the hematopoietic system; rather, GLIS2 is a member of the Kruppel-like zinc finger transcription factor family that is implicated in the regulation of normal renal development and function [35,36]. Interestingly, gene expression profiling showed that non-DS-AMKL patients with the CBFA2T3-GLIS2 fusion had marked overexpression of BMP2 and BMP4 [33].

Further functional studies confirmed activation of the BMP signalling pathway via the canonical SMAD-dependent pathway through in vitro expression of downstream BMP targets and in vivo BMP gain-of-function phenotypes [33]. Transfection of CBFA2T3-GLIS2 in murine hematopoietic cells increased expression of Id1 and promoted cell self-renewal, both of which were inhibited by the small molecule BMP inhibitor dorsomorphin (DM) [33] (Figure 3). AMKL cell lines that expressed CBFA2T3-GLIS2 also had reduced cell viability and proliferation in the presence of DM compared with AMKL cell lines that lacked the fusion transcript [33]. Finally, expression of CBFA2T3-GLIS2 in Drosophila led to increased expression of endogenous dpp, the fly homologue of BMP4, resulting in shortened, blistered wings and ectopic wing veination, phenotypes consistent with dpp gain-of-function [33,37]. Because BMPs are known to be involved in hematopoietic differentiation, and increased BMP signalling has been shown to induce CD34 + progenitor differentiation into megakaryocytes [38], the critical importance of BMP signalling in AMKL pathogenesis is not surprising.

Figure 3.

Chemical structure of published small molecule BMP inhibitors

Chronic myelogenous leukaemia (CML) and other leukaemias

CML is driven by the oncogenic tyrosine kinase fusion protein BCR (breakpoint cluster region)-Abl (Abelson murine leukaemia viral oncogene homologue 1) [39]. Treatment of CML with small molecule tyrosine kinase inhibitors (TKIs) that inhibit BCR-Abl, such as Imatinib, has been transformative in controlling CML for the majority of patients [40]. However, currently available TKIs do not eliminate quiescent leukemic stem cell populations [41]. Consequently, long-term suppressive TKI treatment is required to sustain remission, and tumour recurrence occurs as leukemic cells acquire resistance to BCR-Abl inhibitors [42]. Therefore, recent attention has focused on discovery of new therapeutic approaches to target additional signalling pathways in the leukemic stem cell population.

The BMP pathway plays an important role in hematopoietic stem cell (HSC) renewal within the bone marrow (BM) niche, and deregulated BMP signalling has been observed in various hematologic malignancies, including CML [42]. Although exact details of mechanisms are yet to be fully elucidated, recent studies indicate that alterations in the BMP pathway act in a paracrine fashion to enhance proliferation and survival of primitive leukemic stem cells and more mature myeloid progenitors [42,43]. In addition, BMP signalling appears to have oncogenic properties for promyelocytic leukaemia and acute myelogenous leukaemia, presumably via similar effects on leukemic stem cells [44,45]. Taken together, emerging evidence suggest that BMP inhibition may represent a novel approach for leukaemia treatment by targeting leukemic stem cells, perhaps in conjunction with existing targeted therapies.

Diffuse intrinsic pontine glioma and other high-grade gliomas

Diffuse intrinsic pontine glioma (DIPG) is the leading cause of death in children with brain tumours and carries a poor prognosis with a median survival time of less than 1 year [46]. Surgical resection is not feasible due to the challenging anatomical position of the tumour and, to date, chemotherapy, radiation and targeted agents have not been effective [47], prompting efforts to better understand the molecular genetics underlying DIPG.

BMPs are actively involved in neurogenesis [48] and activation of the BMP signalling pathway has recently been implicated in the pathogenesis of DIPG. Four concurrent, independent studies identified recurrent somatic mutations in the ACVR1 gene, which encodes ALK2, in 46 of 195 DIPG samples [49–52]. These gain-of-function mutations are due to seven different amino acid substitution: R206H, Q207E, R258G, G328V, G328W, G328E and G356D. Interestingly, with the exception of R258G and G328V, the ACVR1 mutations in DIPG are identical with the germline mutations found in fibrodysplasia ossificans progressiva (FOP), an autosomal dominant disorder characterized by heterotopic ossification of soft tissues [53]. Since the FOP ACVR1 mutations promote BMP signalling through increased phosphorylation of R-SMADs and increased expression of downstream transcriptional targets [54], DIPG tumour growth and resistance to therapy could be driven by aberrant BMP signalling in a cell autonomous fashion.

Indeed, in vitro and in vivo studies demonstrated activation of BMP signalling in ACVR1 mutant DIPG through increased levels of phosphorylated SMADs and Id1 and Id2 expression. Human glioma specimens with ACVR1 mutations expressed higher levels of p-SMAD1/5 compared with wild type controls [49,50]. Transfection of mutant versions of ACVR1 in mouse astrocytes also resulted in increased levels of p-SMAD1/5 [49]. Furthermore, treatment of the same mutant ACVR1 astrocytes with the small molecule BMP inhibitor LDN-193189 (Figure 3) inhibited SMAD phosphorylation [49]. ACVR1 mutant human astrocytes also expressed increased levels of Id1 and Id2, confirming downstream activation of the BMP pathway, and increased proliferation of cultured brainstem progenitor cells [50]. In in vivo zebrafish experiments, expression of mutant ACVR1 disrupted embryologic dorsoventral patterning and induced ventralization and loss of head and dorsal structures. Moreover, addition of LDN-193189 partially rescued the ventralization phenotype, providing further evidence that the DIPG-associated mutations activate ALK2 [49].

Interestingly, although these studies demonstrated that ACVR1 mutations drive DIPG tumorigenesis, these same somatic mutations that are seen in FOP patients do not lead to cancer. To date, there are no reported cases of DIPG in FOP patients. Furthermore, when expressed in mouse astrocytes, mutations in ACVR1 associated with DIPG do not induce tumorigenesis [49], suggesting that whereas activation of BMP signalling is necessary for development of the oncogenic phenotype, additional molecular aberrancies are also required.

Recently, we demonstrated that BMP signalling is active in the majority of high-grade glioma (HGG) cells obtained from adult patients [55]. Moreover, consistent with a role of BMP signalling in human HGG, deletion of BMPR1A (ALK3) in oncogenic mouse astrocytes resulted in decreased proliferation, invasion and migration. In addition, in a mouse model of HGG involving orthotopic transplantation of transformed astrocytes, the genetic knockout of BMPR1A (ALK3) showed a dramatic increase in survival. Finally, much of the in vitro effects of BMPR1A ablation were recapitulated using a small molecule BMP inhibitor DMH1. Taken together, these results suggest that BMP signalling may play an important role in the pathogenesis of HGG and that pharmacological agents such as DMH1 may be efficacious for treatment of a subset of adult HGG driven by BMP signalling.

Lung cancer

Lung cancer is the leading cause of cancer-related deaths in the United States. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. Although BMP2 is essential for lung development, normal mature lung cells do not produce BMP2 [56]. BMP2 is aberrantly overexpressed in 98% of NSCLC, and increased levels of p-SMAD 1/5 and Id1 are reported in NSCLC biopsies [57]. As such, the role of BMP signalling in NSCLC has been the subject of much research.

Of the >20 BMP ligands, BMP2 has an established prooncogenic role in the development of NSCLC [58]. Patients with advanced NSCLC have significantly elevated BMP2 levels compared with that of health controls as well as higher metastatic burden and decreased survival [58,59]. Patients with lymph node metastasis and more advanced tumour stages also have higher levels of BMP2 gene expression [60]. In genomic studies, single nucleotide polymorphisms (SNPs) in the BMP2 gene associated with unfavourable response to therapy and decreased survival. For example, homozygous carriers of the SNP rs235756 had a significantly shorter mean survival time compared with that of either wild type or heterozygous carriers [61]. In addition, in vitro studies demonstrated that BMP2 can stimulate cell growth and invasion in A549 and H7240 lung cancer cell lines in a dose-dependent manner, an effect that can be completely inhibited by the BMP2 antagonist, Noggin [62]. Furthermore, BMP2 increased tumour growth [62] and enhanced tumour angiogenesis in murine lung cancer models [63].

Small molecule BMP inhibitors have been utilized to further explore the role of BMP signalling in lung cancer. In the H1299 and A549 lung cancer cell lines, which express endogenous levels of BMP, small molecule BMP antagonists (Figure 3) significantly decreased phosphorylation of SMAD1/5/8 and expression of Id1–3 [64]. Importantly, inhibition of BMP type I receptors by small molecule inhibitors resulted in significant growth inhibition and cell death in H1229 and A549 cells [64]. Thus, the use of selective small molecule BMP inhibitors demonstrated the therapeutic potential of blocking the canonical SMAD-dependent pathway in lung cancer.

Pancreatic cancer

Pancreatic cancer is an aggressive tumour with a high mortality rate and limited treatment options, due in part to the late diagnosis for non-specific symptoms and poor response to chemotherapy. Despite current therapies for pancreatic cancer, the cure rate is less than 5% [65]. Given the importance of BMP in the pathogenesis of other solid tumours, there has been growing interest in studying BMP signalling in pancreatic cancer.

BMPs have a complex role in pancreatic cancer. In primary pancreatic cancer specimens, BMP2, BMP4 and BMP7 are elevated, and BMP treatment increased invasiveness of Panc-1 pancreatic cancer cells via SMAD1-mediated activation of matrix metalloproteinase (MMP)-2, the latter of which promotes tumour invasion by degradation of the extracellular matrix [66]. Of note, in the BMP pathway, SMAD4 is a key mediator of BMP target gene transcription [15] (Figure 2). Mutations in the SMAD4 (also known as depleted in pancreatic cancer locus 4; DPC4) gene occurs in >50% of pancreatic cancers, suggesting a tumour suppressor role for SMAD4 [67–69]. Inactivation of SMAD4 is also associated with poor clinical prognosis, and restoration of SMAD4 in SMAD4-depleted cancer cell lines reduced cell growth [69–71]. In SMAD4-positive pancreatic ductal adenocarcinoma (PDAC), reduction in BMPR1A (ALK3) expression increased cell viability, Matrigel invasion, activation of angiogenesis markers and matrix modification [72]. Treatment with BMP2 decreased the viability of SMAD4-positive PDAC cells, but, paradoxically, increased the viability of SMAD4-negative cells. Conversely, treatment with LDN-193189 increased viability of SMAD4-positive cells but decreased viability of SMAD4-negative cells. Together, these results suggest that BMPs have divergent roles in pancreatic cancer pathogenesis depending on the presence or absence of SMAD4: when SMAD4 is present, BMP acts as a tumour suppressor; whereas in its absence, BMP becomes a tumour promoter [72]. Thus, small molecule BMP inhibitors may be selectively therapeutic for SMAD4-negative pancreatic cancer.

Ovarian cancer

Ovarian cancer is the most lethal gynaecologic malignancy. Epithelial ovarian cancer (EOC) accounts for the majority of ovarian cancer and has the highest mortality rate for ovarian cancer [73]. Treatment for EOC includes surgical debulking and/or chemotherapy. Although the vast majority of patients are responsive to chemotherapy, up to 75% of patients will relapse within the first 18 months [74]. Unfortunately, recurrent disease is not curable.

A number of studies indicate that BMP signalling has protumorigenic roles in ovarian cancer. In ovarian cancer cell lines, autocrine stimulation by BMP4 up-regulated Id1 and Id3 expression [75,76]. In addition, human ovarian cancer cells had a nearly four-fold increase in Id3 expression compared with normal primary human ovarian surface epithelial cells [75]. Consistent with the tumour promoting role of BMP, BMP2 expression in high-grade EOC was associated with a poorer prognosis and a significantly shorter survival period [77]. Studies on mesenchymal stem cells (MSCs), multipotent progenitor cells that display tropism towards tumours, [78] have also shed further light into the role of BMP in ovarian cancer. In patients with primary ovarian cancer, >90% of tumour samples expressed carcinoma-associated MSCs (CA-MSCs). In addition, MSCs isolated from human tumour samples promoted tumorigenesis in immune deficient mice [79]. Interestingly, CA-MSCs had increased BMP2 expression compared with control MSCs [79]. Ovarian tumours grown with CA-MSCs also had increased levels of p-SMAD1/5 compared with tumours grown with adipose-MSCs. Similarly, addition of BMP2 to tumours cells increased levels of p-SMAD1/5, an effect that was attenuated by the BMP antagonist Noggin [79]. Taken together, these results suggest that disrupting both autocrine and paracrine BMP signalling may be a therapeutic approach for EOC.

More recently, the functional activation of the BMP signalling pathway was examined in patients with serous ovarian cancer [80]. Gene expression profiling demonstrated that up-regulation of SMAD5 was associated with poorer prognosis. In addition, SMAD5, BMPR1A, BMPR1B and BMPR2 expression all positively correlated with MKI67 (marker of proliferation Ki-67). Furthermore, immunohistochemistry studies demonstrated that nuclear p-SMAD5 level inversely correlated with clinical prognosis: overall survival of patients with high nuclear p-SMAD5 levels was significantly lower than that of patients with low nuclear p-SMAD5. In a mouse model of serous ovarian cancer, administration of recombinant BMP2 also promoted significant tumour growth compared with that of control mice, an effect that was inhibited by DM. Nuclear p-SMAD5 level was also highest in tumour tissue from mice treated with recombinant BMP2 and lowest in tumour tissue from mice treated with DM. A significant positive correlation between p-SMAD5 levels and expression of Ki67 was also observed. [80] These studies highlight the potential therapeutic use of small molecule BMP inhibitors in ovarian cancers.

Osteosarcoma

Osteosarcomas are the most common primary bone malignancies in children and young adults and accounts for more than 50% of bone cancers in individuals under the age of 20 years [81]. Because of their importance in bone and cartilage development, BMPs have been studied in the pathogenesis of osteosarcoma.

BMP ligands and receptors have been detected in the majority of osteosarcoma cell lines. Moreover, human osteosarcoma samples demonstrated activated BMP signalling as measured by phosphorylated SMAD1 levels [82]. Interestingly, human osteosarcoma specimens expressed different levels of BMP expression depending on the subtype of osteosarcoma. For example, high-grade osteosarcoma with malignant fibrous histiosarcoma (MFH)-type pattern had increased BMP2 and BMP4 expression [83], whereas chondroblastic osteosarcoma had increased BMP6 expression [84].

Studies found that osteosarcomas with increased BMP signalling were more chemo-resistant and had a higher likelihood of metastasis, resulting in a reduced 5-year survival rate [85]. For instance, increased expression of BMPR2 in osteosarcoma tumours was associated with a higher tendency for tumour metastasis [86], and patients with increased co-expression of BMP2, BMP4 and BMPR2 had a poorer prognosis and significantly reduced 5-year survival rate [87]. In genomic studies, individuals homozygous for the SNPs rs3178250 and rs1005464 in the BMP2 gene were at increased risk for osteosarcoma [88]. In addition to the human observational studies, functional studies demonstrated the role of BMP signalling in osteosarcoma disease progression. In human osteosarcoma cell lines, BMP2 or BMP9 overexpression promoted tumour cell growth, suggesting that BMPs are pro-mitogenic in osteosarcomas [89]. Moreover, BMP2 treatment promoted osteosarcoma cell migration and invasion whereas the BMP antagonist Noggin abrogated the stimulatory effects of BMP2. Taken together, these studies suggest that BMP signalling plays an important role in osteosarcoma pathogenesis and disease progression [90].

Prostate cancer

Prostate cancer is the most common noncutaneous malignancy in men in the United States. Bones of the axial skeleton are the predominant site of prostate cancer metastasis and are primarily osteoblastic lesions [91]. Complications from bone metastasis include pathologic fractures, spinal cord compression and bone pain. Unfortunately, bony metastasis portends advance disease and treatment is often only symptomatic. Because BMPs are potent osteoinductive factors and bone metastasis is predominantly osteoblastic, expression of BMP signalling in prostate cancer has been the subject of much investigation.

mRNA levels of BMP1–6 have been measured in prostatic tissue from patients with benign prostatic hyperplasia (BPH), non-metastatic prostatic adenocarcinoma and metastatic prostatic adenocarcinoma [92]. BMP6 expression was present in over half of prostate tissue specimens from patients with metastatic prostatic carcinoma. However, BMP6 expression was notably absent from patients with non-metastatic prostate carcinoma or BPH [92]. Subsequent studies demonstrated up-regulation of BMP6 mRNA and protein expression in bone metastasis as well as high-grade tumours [93,94]. In addition, higher-grade prostate tumours, characterized by a Gleason score of 6 or more, exhibited more intense BMP6 immunostaining compared with lower-grade prostate tumours with Gleason scores of 4 or less [95]. BMP6-positive prostate cancer samples also had increased epithelial staining for Id1, indicating activation of BMP signalling [96]. Interestingly, although BMP6 did not alter prostate tumour cell growth in vitro, BMP6 did increase tumour invasiveness [97]. In addition, bone metastasis in prostate cancer appears to mediate cross-talk between the Wnt and BMP signalling. Wnt signalling up-regulated both BMP4 and BMP6 expression in prostate cancer cells and promoted pro-osteoblast activity in both BMP-dependent and BMP-independent mechanisms [97].

Unlike that of BMP6, the expression levels of BMP2, BMP4 and BMP7 in the same study were low to absent from primary prostate cancers [92]. In addition, expression of BMP receptors was decreased in more aggressive prostate cancers [98,99]. However, subsequent in vitro studies demonstrated that individual BMP or BMPR expression did not always correlate with functional activity. For example, although BMP2 expression was down-regulated in advanced prostate cancer [100], addition of BMP2 promoted cell invasiveness of C4-2B, LnCaP and PC-3 prostate cell lines [97,101]. In addition, although BMP7 expression was low to absent from primary tumours, BMP7 expression was increased in bone metastatic prostate cancer and overexpression of Noggin inhibited growth of BMP7 positive osteolytic cancer lesions [101]. Moreover, BMP7 enhanced PC-3 tumour cell motility and growth in vitro [102]. Although these studies highlight the complexity of BMP signalling in prostate cancer, they also identify the need for further investigation to better elucidate the exact role of BMPs in prostate cancer.

Breast cancer

Breast cancer is the most commonly diagnosed cancer in the United States and is a leading cause of cancer death in women. The propensity of breast cancer to metastasize to bone along with multiple studies demonstrating aberrant BMP expression in breast cancer have prompted further study into the potential role of BMP signalling in the pathogenesis of breast cancer.

Of the BMPs, BMP2 is most studied in breast cancer. In breast cancer cell lines and primary tumours, BMP2 expression levels are consistently low, similar to levels found in normal mammary tissue. Functional studies, however, suggest a dual role for BMP2 in breast cancer: suppressing cancer cell proliferation while also promoting cancer cell invasion and migration.

The antiproliferative effect of BMP2 on breast cancer is associated with up-regulation of SMAD1/4, p21, and the tumour suppressor PTEN (phosphatase and tensin homologue). In MCF-7 human breast cancer cells, BMP2 inhibited oestradiol-induced proliferation of tumour cells through up-regulation of p21, a cyclin-dependent kinase inhibitor, and led to SMAD1/4-mediated arrest of cells in the G1 phase of the cell cycle [103]. BMP2 treatment of MCF-7 cells also increased, in a dose-dependent manner, protein levels of PTEN by inhibiting its degradation [104]. Furthermore, at very high concentrations of up to 1 μg/ml, BMP2 also directly inhibited cellular proliferation of human breast tumour cells [105].

At the same time, BMP2 also promotes the motility and invasiveness of breast cancer cells. Brief exposure of MCF-7 cells to BMP2 enhanced tumour cell migration and MCF-7 cells transfected with BMP2 had increased expression of BCSG1, a breast cancer-specific gene. In addition, in a mouse xenograft model, BMP2 overexpressing MCF-7 breast cancer cells formed tumours that had increased neovascularization as well as increased Id1 expression and p38 MAPK activation [106,107]. BMP2 also stimulated tumour migration and invasion by inducing expression of an extracellular matrix glycoprotein, tenascin-W, in the tumour stroma [108]. Higher expression of tenascin-W in the stroma around breast cancer lesions portended increased likelihood of metastasis and more aggressive tumour behaviour [108]. BMP2 also induced microcalcification, an important diagnostic marker on mammograms for breast cancer patients, in a dose-dependent manner in a rat xenograft model of breast cancer [109,110].

High BMP4 expression is frequently found in breast cancer cell lines and primary breast tumours [111]. Functional studies indicate that BMP4, like BMP2, has a dual role in breast cancer. For example, in MDA-231 breast cancer cells, BMP4 reduced cell migration and invasion by inhibiting matrix metalloproteinase (MMP) 9 expression [112], and, in a larger panel of breast cancer cell lines and primary breast tumours, BMP4 expression correlated with cell cycle arrest and lower tumour grade [113]. By contrast, BMP4 was found to promote tumour migration and invasion [113], although only in certain cell-lines. BMP4 also enhanced tumour cell invasion by stimulating stromal fibroblasts, an effect inhibited by the BMP small molecule inhibitor DMH1 [114]. Taken together, these studies demonstrate the context dependence of the role of BMP ligands in breast cancer and the challenges in selectively targeting individual BMPs.

In contrast with the dual roles of BMP2 and BMP4 in breast cancer, the receptors ALK3 (BMPR1A), ALK6 (BMPR1B) and BMPR2 appear to promote cancer progression. In breast cancer patients, elevated ALK3 and BMPR2 expression was associated with a reduced relapse-free survival time [115]. In addition, in a tissue microarray analysis, BMPR1B overexpression and SMAD1 phosphorylation were associated with higher tumour grade, more aggressive cancer progression and poorer prognosis [116]. Consistent with the oncogenic role of BMPR1A, in a mouse genetic model expressing MMTV.PyMT oncogene, deletion of BMPR1A impaired mammary tumour formation and metastasis [117]. Finally, treatment with DMH1 reduced lung metastasis in MMTV.PyVmT expressing mice, and the tumours were less proliferative and more apoptotic. In addition, DMH1 treatment altered fibroblasts, lymphatic vessels and macrophages within tumour microenvironment to be less tumour promoting [115]. These results raise the exciting possibility that inhibiting BMP signalling may successfully target both the tumour and the tumour microenvironment to reduce tumour burden and metastasis. Finally, DMH1’s effects on tumour infiltration by immune cells suggest that BMP inhibition could enhance the effectiveness of cancer immunotherapies [115].

Like prostate cancer, breast cancer has a tropism to metastasize to the bone. BMP7, which is highly expressed in primary breast cancers, is associated with accelerated bony metastasis in patients with BMP7 positive tumours [118]. Furthermore, the BMP-SMAD transcriptional pathway is activated in bone metastasis from breast cancer. In bone metastasis samples from breast cancer patients and in a mouse xenograft model, high levels of p-SMAD1/5/8 were observed in comparison with primary tumour cells or lymph node metastasis, suggesting that BMPs specifically mediate metastasis of breast cancer cells to bone [119]. In addition, functional studies demonstrated that expression of dominant negative ALK3 in MDA-231-D prostate cancer cells inhibited phosphorylation of SMAD1/5/8 as well as transcription of Id1. Finally, in a mouse xenograft model, expression of the dominant negative ALK3 significantly reduced the size of bony metastasis and prolonged mean survival time [119].

Melanoma

Melanoma is the most commonly fatal cutaneous cancer and is a major public health burden, increasing at a rate unrivalled by any other cancer [120]. Despite extensive research and the use of chemo- and radiotherapy, the prognosis for metastatic melanoma remains poor.

Melanocytes are derived from neural crest cells and BMP2 and BMP4 are known to promote neural crest cell migration [121] Compared with that of normal melanocytes, increased mRNA expression of BMP2, BMP6, BMP7 and BMP8 has been observed in 14 different melanoma cell lines. BMP7 expression, in particular, correlated with more aggressive melanoma cell lines [122]. In addition, primary and metastatic melanoma tissue specimens showed increased expression of BMP4 and BMP7 [123]. Functional studies demonstrated that BMP2 and BMP4 play an important role in melanoma cell invasion. In chick embryos transplanted with B16-F1 melanoma cells, BMP2 treatment facilitated cell migration, which was abrogated by treatment with Noggin [124]. In addition, specific inhibition of BMP4 through antisense RNA resulted in significant inhibition (70%) of cellular invasion [123]. In melanoma cell lines, BMP4 also acted as a chemoattractant and promoted angiogenesis, features that were in part mediated by up-regulation of Id-1 [125]. Additional studies showed that BMP2 and BMP4 mediated tumour cell invasion and migration by increasing fibroblast production of matrix metalloproteinases (MMPs) 1, 2, 3 and 13 [126]. Further studies are warranted, but these preliminary studies suggest that the use of small molecule BMP inhibitors may offer a novel therapeutic strategy for this highly aggressive and fatal disease.

Head and neck cancer

Head and neck squamous cell carcinoma (HNSCC) is one of the leading causes of cancer-related deaths. Advanced HNSCC is characterized by aggressive destruction of surrounding tissues and, despite multimodal treatment, prognosis remains poor [127].

BMP2 expression has been detected in tongue and gingival squamous cell carcinoma cell lines as well as benign and malignant salivary gland tumours [128,129]. Compared with normal mucosa, human oral squamous cell carcinoma (SCC) tumours overexpress BMP2, BMP4, BMP5 and BMPR1A (ALK3), and metastatic lymph nodes retain strong BMP2, BMP4 and BMP5 expression [130,131]. A recent tissue microarray of HNSCC found that the majority of samples [132] (98%) expressed BMP2. Furthermore, high levels of BMP2 expression were associated with increased tumour recurrence, a finding consistent with prior studies that demonstrated that treatment of oral carcinoma cell lines with BMP2 resulted in more aggressive cell invasion and metastasis [132,133]. In addition, by using global expressional profiles, Zhou et al. [134] found that increased BMP2 expression was associated with increased lymph node metastasis and extracapsular spread of metastatic lymph nodes.

Activation of the BMP signalling pathway has also been demonstrated in HNSCC. In HNSCC primary specimens, BMP4 expression and SMAD1 phosphorylation were increased, and higher levels of each were associated with poorer clinical prognosis [135]. In in vitro studies, stimulation of HNSCC with BMP4 induced epithelial–mesenchymal transition (EMT), which is thought to be an important step in tumour invasion. In addition, in vitro knockdown of SMAD1 in Tu686 and Tu212 HNSCC cell lines inhibited EMT and migration and invasiveness of the cancer cell lines [135]. In a 3D model of cancer cell migration, suppression of BMP4 expression by the microRNA let-7i not only altered cell morphology, but also reduced mesenchymal cell migration [136]. In addition, the same study reported elevated expression of BMP4 and decreased expression of let-7i in the majority of HNSCC specimens, consistent with BMP signal activation [136]. Similar studies have also found that higher levels of BMP6 and Id1 expression in oesophageal SCC were also associated with lower 2- and 5-year survival rates [137,138], and that high Id1 expression was an independent prognostic factor for poor prognosis in oesophageal SCC patients [139]. These studies suggest that BMP signalling is pro-tumorigenic in HNSCC and that further investigation of small molecule BMP inhibitor use is warranted, as they may offer promising primary or adjunctive treatment for HNSCC patients.

Small molecule BMP inhibitors

First generation small molecule BMP inhibitors: dorsomorphin, LDN-193189

In zebrafish embryos, BMP signalling functions to establish dorsoventral axis by specifying the ventral fates. In an in vivo zebrafish screen, dorsomorphin (DM; Figure 3; Table 3) was found to recapitulate in a dose-dependent manner the dorsalized phenotype associated with mutations in the BMP pathway [140]. DM, a pyrazolopyrimidine derivative (6-[4-(2-piperidin-1-ylethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-a]pyrimidine), was the first identified small molecule inhibitor of the BMP signalling pathway that had minimal effects on TGF-β and activin. For example, DM inhibited BMP-induced phosphorylation of SMAD1/5/8, but had no effect on MAPK p38 activation or TGF-β1-induced activation of SMAD2. Furthermore, whereas DM inhibited the activity of constitutively active ALK2/3/6, BMP type I receptors, it had no effect on the activity of ALK4/5/7, the TGF-β and activin type I receptors [140]. In addition, DM did not cause cyclopia in zebrafish, a phenotype associated with disruption of TGF-β and activin signalling [141].

Table 3. Inhibition of selected kinases in vitro by small molecule BMP inhibitors.

Results of in vitro kinases assays for ALK1-6, BMPR2, TGF-β type II receptor (TGFR2) and VEGF receptor (KDR) are shown as IC50 (concentration at 50 % inhibition). IC50s for in vitro kinase assay are not identical with the published IC50s for cell based studies described in the text. *Results obtained by CCH, which may vary from published reports based on experimental conditions. n.d.: not determine. NO: no inhibition at highest concentration tested (>30–100 μM).

IC50 (nM)	Dorsomorphin (DM)* [140]	LDN-193189 [145, 157]	DMH1* [158]	DMH2* [158]	DMH3* [158]	ML347* [144]	K02288 [145]	LDN-212854* [159]	LDN-214117 [146]
ALK1	100	1	25	10	5	45	2	55	27
ALK2	60	1	100	40	25	30	1	15	27
ALK3	95	5	<5	<5	<5	>10000	34	120	1171
ALK6	230	17	48	<5	10	>9000	6	220	3000
ALK4	>20000	100	>9000	1400	640	NO	302	>9000	n.d.
ALK5	>17000	350	NO	2400	2000	NO	321	>11000	n.d.
BMPR2	70	n.d.	NO	>3800	2200	NO	n.d.	n.d.	n.d.
TGFR2	100	n.d.	NO	86	246	NO	n.d.	n.d.	n.d.
KDR	20	556	NO	2400	2000	>19000	>10000	2770	n.d.

The potential pharmacological use of DM, however, has been tempered by its moderate inhibition of BMP signalling in cells (IC50 ∼0.5 μM) and metabolic instability in mouse liver (t_1/2 = 10.4 min) as well as its significant off-target effects [27]. Because DM is structurally homologous with known inhibitors of the vascular endothelial growth factor type II receptor, (VEGF-2/KDR), DM, not surprisingly, significantly inhibits not only KDR but also platelet-derived growth factor receptor-β (PDGFR-β) signalling at a potency level close to that of BMP (IC50 ∼0.8 μM) [142].

A follow-up structure–activity relationship (SAR) studies led to the identification of a DM derivative, LDN-193189 (4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline hydrochloride; Figure 3; Table 3) [27]. DM and LDN-193189 share the same pyrazolo[1,5-a]pyrimidine scaffold but LDN-193189 is more potent and selective for BMP signalling (∼100 fold) and also more metabolically stable. LDN-193189 also has less activity against PDGFR-β (∼10 fold). Moreover, in a mouse model of FOP, LDN-193189 dramatically reduced heterotopic ossification [140]. However, in vivo assays using DM and LDN-193189 in zebrafish resulted in early lethality at higher concentrations, presumably from simultaneous dual inhibition of BMP and TGF-β/nodal signalling during the critical stages of early embryonic development [143].

Second and third-generation small molecule BMP inhibitors

A large in vivo SAR study of dorsomorphin analogues identified DMH1 (4-[6-[4-(1-methylethoxy) phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), DMH2 (4-(2-(4-(3-(quinolin-4-yl)pyrazolo[1,5-a]pyrimidin-6-yl)phenoxy)ethyl)morpholine) and DMH3 (N,N-dimethyl-3-(4-(3-(quinolin-4-yl)pyrazolo[1,5-a]pyrimidin-6-yl)phenoxy)propan-1-amine) as synthetic compounds with the highest selectivity for BMP pathway based on specificity for causing dorsalization without additional effects (Figure 3; Table 3) [143]. In comparison with DM and LDN-193189, DMH1 is very selective for ALK2 with no discernable activity against ALK5 (TGFBR-1), AMPK, KDR (VEGFR2), PDGFR-beta, or BMP-4 induced p38 MAPK phosphorylation [143]. A further refinement led to the synthesis of ML347 (5-(6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinolone; Figure 3; Table 3), a potent inhibitor (IC50 = 152 nM) that can discriminate between type I BMP receptors: >300-fold selectivity for ALK2 compared with ALK3 [144]. A recent independent screen also identified a highly selective 2-aminopyridine compound K02288 (3-[6-amino-5-(3,4,5-trimethoxy-phenyl)-pyridin-3-yl]-phenol; Figure 3; Table 3) that inhibits BMP-stimulated phosphorylation of SMAD1/5/8 without affecting TGF-β signalling [145]. Another novel compound, LDN-214117 (1-(4-(6-methyl-5-(3,4,5-trimethoxyphenyl)pyridin-3-yl)phenyl)piperazine; Figure 3; Table 3) has a higher degree of selectivity for ALK2 than LDN-193189 or K02288 and a lower cytotoxic profile [146].

The use of small molecule BMP inhibitors in preclinical models of cancer therapy

The emergence of small molecule BMP inhibitors as in vitro and in vivo chemical tools has prompted recent studies on the therapeutic potential of pharmacological inhibition of BMP signalling for various cancers. Although the number of cancer types in which BMP inhibitors show therapeutic potential is increasing, the first such studies have focused on the effects of BMP inhibitors on lung cancer, epithelial ovarian cancer and breast cancer.

Lung cancer

The application of small molecule BMP inhibitors to lung cancer therapy has been rigorously studied and shown to be effective in suppressing lung cancer tumorigenesis in vitro and in vivo. In the highly malignant A549 lung adenocarcinoma cells, which have increased expression of BMP2, LDN-193189 reduced cell growth and promoted cell death [147]. In a separate study, LDN-193189, along with DMH1 and DMH2, decreased cell growth, proliferation and clonogenicity of both the A549 and H1299 cell lines [64]. In addition, DMH2, which is highly selective for ALK2, was the most potent inhibitor of BMP signalling in both the A549 and H1299 cell lines and resulted in significant attenuation of cell growth and Id expression [64]. DMH2 also significantly inhibited cancer cell growth of A549 and H1229 cell lines expressing Oct4 or Nestin, stem cell markers that promote cell survival and are associated with increased chemo-resistance [148]. More recently, DMH1, which is more selective for ALK-2 than DMH2, significantly reduced tumour growth in a mouse xenograft model of NSCLC [149].

Epithelial ovarian cancer

DM and LDN offer promising adjuvant therapy for chemo-resistant epithelial ovarian cancer (EOC). DM and LDN-193189 increased cancer survival and decreased cell viability in a dose-dependent manner in a mouse intraperitoneal xenograft model of EOC [150]. Furthermore, in cisplatin (CP)-sensitive and CP-resistant EOC cell lines, DM and LDN-193189 worked synergistically through different mechanisms to resensitize platinum-resistant EOC cells to platinum agents-DM induced cell autophagy whereas LDN-193189 induced cell apoptosis [150]. Similarly, the more selective DMH1 has been recently demonstrated to be an effective neoadjuvant to cisplatin. Together with cisplatin, DMH1 reduced ovarian sphere growth and enhanced cisplatin response of the chemo-resistant cell lines OVCAR8 and NCI-RES [151].

Breast cancer

Recent studies have suggested that inhibition of BMP signalling in breast cancer can provide therapeutic benefit both in vitro and in vivo. In A17 breast cancer cells, DM reduced cell proliferation and reduced expression of EMT markers Vimentin, Snail and Slug [152]. In mammary epithelial cell lines and primary murine tumour cells, LDN-193189 inhibited clonogenicity and cell migration [153]. In a more recent study using a murine model of breast cancer, DMH1 reduced not only primary tumour burden but also lung metastasis [115]. DMH-1 treated tumours also had reduced lymphatic vessel growth and decreased expression of EMT markers, such as Snail and Twist [115].

Taken together, these studies on lung, epithelial ovarian and breast cancer suggest that small molecule BMP inhibitors may represent a novel class of therapeutic agents. However, further studies are necessary to investigate both potential off-target and clinical side effects.

Concluding remarks

Although the role of BMP signalling in cancer has been less studied compared with the closely related TGF-β pathway, it is now clear that BMP signalling plays many important roles in cancer biology. In many cancers, BMPs appear to have multifunctional and often seemingly opposing roles in promoting or inhibiting cancer, prompting additional studies to further clarify the BMP signalling context. Different BMP ligands and receptors as well as the canonical SMAD-dependent and SMAD-independent pathways probably play distinct roles in different cancers and different cancer processes. In addition, BMP signalling affects not just cancer cells directly but also the surrounding tumour microenvironment, including tumour microvasculature and immune cells. Against the backdrop of complex cancer biology, the discovery of dorsomorphin and subsequent generations of BMP small molecule inhibitors has spurred a growing number of studies that suggest that BMP signalling may indeed be a promising therapeutic target for a variety of cancers. For instance, using DMH1, the most selective known inhibitor of ALK1, ALK2 and ALK3, we showed that a systemic inhibition of BMP signalling not only reduced primary tumour burden in a spontaneous mouse model of metastatic breast cancer, but also reduced metastasis via its effects on the stromal compartments of fibroblasts, lymphatic vessel drainage of tumours and polarization of the immune system [154]. Development of newer and more selective compounds with improved pharmacokinetic characteristics will not only advance mechanistic studies of cancers in vivo but also lead to potentially effective cancer therapies.

From the translational medicine perspective, the complexity of BMP signalling will require a detailed understanding of the involvement of specific BMP signalling components in individual tumours. The status of BMP signal activation, and its effects on cancer cells and tumour microenvironment can be evaluated in a patient’s primary tumours as well as recurrent or metastatic tumours in order to formulate individualized therapeutic strategy. Such analysis may also affect the choice of specific BMP inhibitors, with varying selectivity, should these agents become available in the clinic. From a mechanistic standpoint, we have focused on developing small molecule inhibitors that are exquisitely selective for the type I BMP receptors, even at the level of subtype-specificity, yet some cancers may benefit from a ‘pan’ BMP inhibitor or off-target effects on other pathways. In the near future, BMP inhibitors may be useful as stand-alone agent, as an adjuvant to the standard of care, or as a component of a cocktail of successful targeted therapies, including cancer immunotherapies. In conclusion, a more detailed molecular understanding of the role of BMP signalling in a variety of cancer contexts may herald an era of new class of pharmaceuticals that target BMP signalling in the armamentarium of precision medicines in oncology.

Abbreviations

ALK1

activin receptor like kinase-1

AMKL

acute megakaryoblastic leukaemia

BMP

bone morphogenetic protein

BPH

benign prostatic hyperplasia

CA-MSC

carcinoma-associated MSC

CML

chronic myelogenous leukaemia

CRC

colorectal cancer

DIPG

diffuse intrinsic pontine glioma

DM

dorsomorphin

EOC

epithelial ovarian cancer

FOP

fibrodysplasia ossificans progressiva

HNSCC

head and neck squamous cell carcinoma

HSC

hematopoietic stem cell

LIMK

LIM domain kinase

mTOR

mammalian target of rapamycin

MAPK

mitogen-activated protein kinase

MFH

malignant fibrous histiosarcoma

MMP

matrix metalloproteinase

MSC

mesenchymal stem cell

NSCLC

non-small cell lung cancer

PDAC

pancreatic ductal adenocarcinoma

PI3K

phosphoinositide 3-kinase

SAR

structure–activity relationship

SNP

single nucleotide polymorphism

TGF-β

transforming growth factor-β

TKI

tyrosine kinase inhibitor

TSG

twisted gastrulation