Lymph node metastasis, an early and prognostically important event in the progression of many human cancers, is associated with expression of VEGF-D. Changes to lymph node vasculature that occur during malignant progression may create a metastatic niche capable of attracting and supporting tumor cells. In this study, we sought to characterize molecules expressed in lymph node endothelium that could represent therapeutic or prognostic targets. Differential mRNA expression profiling of endothelial cells from lymph nodes that drained metastatic or nonmetastatic primary tumors revealed genes associated with tumor progression, in particular bone morphogenetic protein-4 (BMP-4). Metastasis driven by VEGF-D was associated with reduced BMP-4 expression in high endothelial venules, where BMP-4 loss could remodel the typical high-walled phenotype to thin-walled vessels. VEGF-D expression was sufficient to suppress proliferation of the more typical BMP-4–expressing high endothelial venules in favor of remodeled vessels, and mechanistic studies indicated that VEGF receptor-2 contributed to high endothelial venule proliferation and remodeling. BMP-4 could regulate high endothelial venule phenotype and cellular function, thereby determining morphology and proliferation responses. Notably, therapeutic administration of BMP-4 suppressed primary tumor growth, acting both at the level of tumor cells and tumor stromal cells. Together, our results show that VEGF-D–driven metastasis induces vascular remodeling in lymph nodes. Furthermore, they implicate BMP-4 as a negative regulator of this process, suggesting its potential utility as a prognostic marker or antitumor agent. Cancer Res; 71(20); 6547–57. ©2011 AACR.

Lymphatic dissemination is considered to be an early and crucial route of metastasis for many cancers (1, 2). Blind-ending lymphatic capillaries drain fluid, cells, and macromolecules from tissue interstitium into a hierarchy of vessels punctuated by lymph nodes (LN), which provide immunologic surveillance for a particular lymphatic drainage basin (3). The presence of metastatic tumor cells in the “sentinel” LN draining a tumor site is a key factor in disease management: substantial clinical data indicates adverse prognostic significance of tumor-positive LNs for many tumor types (4, 5). However, a clear understanding of the mechanistic role of LNs in tumor progression is still lacking.

VEGF-D and VEGF-C are important inducers of the growth and differentiation of blood vessels and lymphatics. When overexpressed in experimental tumors these growth factors elicit angiogenesis and lymphangiogenesis, and are furthermore associated with increased metastasis to LNs and distant organs (1). VEGF-D and VEGF-C expression is also associated with metastasis to LNs in many human cancers, and is independently associated with poor prognosis (6). Recently, it has emerged that modulation of lymphatics and blood vessels—including high endothelial venules (HEV), vessels specialized for leukocyte trafficking (7, 8)—also occurs in draining LNs of some tumors (9, 10). Such alterations can precede the arrival of metastatic cells (7, 11–13), and members of the VEGF family have been implicated in these changes (12–15). The importance of alterations to LN endothelium is highlighted by studies of human breast cancer: lymphangiogenesis or angiogenesis within metastatic tumor deposits in sentinel LNs was found to be associated with, and sometimes independently predictive of, distant metastasis or survival (9, 16, 17).

Here, we sought to characterize changes to the vasculature within tumor-draining LNs, to identify molecules with prognostic or therapeutic potential. We compared the molecular profiles of enriched endothelial cell (EC) populations from LNs draining nonmetastatic tumors with those from LNs draining metastatic (VEGF-D–overexpressing) tumors. BMP-4 was downregulated in the HEVs of LNs draining metastatic tumors. This observation was linked with the remodeling of HEVs induced by VEGF-D–driven metastasis, thus implicating BMP-4 as a regulator of HEV morphology and cell function. Furthermore, therapeutically applied BMP-4 protein inhibited primary tumor growth. This study indicates that VEGF-D's prometastatic activity includes remodeling of specialized LN endothelium, and identifies new roles for BMP-4 in cancer and vascular biology.

Lists of antibodies, primers and detailed protocols are contained in the Supplementary Methods section.

Metastatic and nonmetastatic xenograft tumor models

293 EBNA-1 tumor cell lines stably expressing full-length human VEGF-D (293-VEGF-D), human VEGF-C (293-VEGF-C), or vector alone (293-Apex) were established in SCID/NOD mice as described (18). 293 EBNA-1 cells were a gift from Kari Alitalo, University of Helsinki, Finland. Regular growth and morphology of transfected cell lines was monitored routinely and growth factor expression verified by Western blot prior to each experiment. LNs were analyzed within the timeframe that metastasis typically occurs in this model; that is, 2 to 4 weeks postimplantation. All animal experiments were carried out with the approval of the Institutional Animal Ethics Committee.

Enrichment of LN EC populations

Draining LNs of metastatic or nonmetastatic tumors pooled from 1 to 5 mice were enzymatically digested, then tumor cells and leukocytes were depleted using immunomagnetic selection (Miltenyi Biotec) for class I HLA and CD16/CD32. The remaining cells were cultured in EGM-2 MV media (Lonza) before enrichment for ECs by selection for podoplanin (19). See Supplementary Fig. S1 for detailed procedure.

Microarray analysis

Duplicate samples of LN EC total RNA (RNeasy Plus kit, Qiagen) were applied to Affymetrix expression arrays (430 2.0; Australian Genome Research Facility). Raw intensity data were analyzed using GeneChip Operating Software (Affymetrix), and profiles compared via Robust Multiarray Analysis and linear modeling using AffylmGUI software (20). Microarray data are deposited in NCBI's Gene Expression Omnibus; series accession number GSE31123 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE31123).

Human LNs

Breast cancer-associated LNs with or without histologically identifiable metastases (n = 7 patients, 22 LNs), or control nontumor-associated LNs collected during cardiac surgery (n = 3 patients), were obtained as a pilot study. Access to deidentified tissue (formalin fixed, paraffin embedded) was provided by the Pathology Department, Royal Melbourne Hospital, with permission from the Melbourne Health Human Research Ethics Committee.

Immunostaining and image quantitation

For BMP-4/MECA-79 quantitation, 2 to 3 sections of each tumor-draining LN (∼6 per group) were immunostained (18). For HEV morphometry, the luminal and basal edges of HEVs were traced using Metamorph Premier (Molecular Devices), to determine lumen area, average vessel wall width and endothelial area using Integrated Morphometry Analysis parameters (journal available on request). HEVs with 50% or more of their circumference staining for BMP-4 were designated BMP-4high; or otherwise BMP-4low. Data were analyzed according to a linear mixed model (Supplementary Methods).

Treatment of ear-draining LNs with recombinant VEGF-D

One microgram of purified VEGF-D dimers (0.05 μg/μL; Vegenics Ltd.) in PBS, or PBS alone as control, was injected intradermally into the ears of SCID/NOD mice for 3 consecutive days. On day 4, BrdU (Invitrogen) was injected intraperitoneally, and ear-draining (superficial parotid) LNs were harvested 2 days later.

Treatment of tumors with neutralizing antibodies

Mice bearing metastatic (VEGF-D overexpressing) tumors received 3 times weekly intraperitoneal injections of neutralizing antibodies (800 μg) to VEGF receptor-2 (VEGFR-2; DC101; Imclone) or VEGF-D (VD1; ref. 21), or PBS. For analysis of HEVs, sections of LNs draining nonmetastatic and antibody-treated metastatic tumors were used from one experiment. LNs of PBS-treated metastatic tumors where HEVs were not obscured by tumor infiltration were included from an identically conducted experiment as a control.

BMP-4 therapeutic model

Tumor-bearing mice were injected intraperitoneally from day 1, 3 times weekly, with 1.4 μg of human BMP-4 (R&D Systems) in 200 μL PBS with 0.652 mg/mL BSA, or a vehicle control of PBS with 0.32 mmol/L HCl and 1 mg/mL BSA, until day 12 or experiment termination. Serum was sampled 60 minutes posttreatment and BMP-4 quantified by ELISA (R&D).

Statistical analysis

Data were compared using a 2-tailed Student t test, or Fisher's exact test for comparison of proportions. Graphed data represent mean ± SE unless specified otherwise.

Enrichment of endothelial cells from tumor-draining LNs

A model of VEGF-D–driven tumor metastasis to regional LNs was used to examine molecular changes in LN endothelium during metastasis (Fig. 1A). Overexpression of VEGF-D in 293-EBNA-1 tumor cells drives metastasis to local LNs within 2 to 4 weeks of implantation in approximately 80% of cases. Vector-transfected tumor cells (no VEGF-D) served as a nonmetastatic control (18). Podoplanin (19) was used as a highly expressed, protease-resistant selection marker to derive cell populations enriched for lymphatic ECs and related EC types, which may respond to VEGF-D (Fig. 1B). Microarray analysis revealed expression of EC-characteristic genes, including VEGFR-2, neuropilin-1 and neuropilin-2, endothelial nitric oxide synthase, CD34 and TIE-2; while desmin and calponin-1, found in fibroblastic lineages, and chondroitin sulfate proteoglycan 4 (NG-2 antigen), characteristic of pericytes, were absent. These findings confirmed that the podoplanin+ve cells were enriched for ECs. The LN ECs heterogeneously expressed ICAM-1 and endoglin, markers of endothelial activation in inflammation and angiogenesis, respectively (Fig. 1C; Supplementary Methods).

Figure 1.

Isolation of ECs from tumor-draining LNs. A, schematic of approach to investigate differentially expressed genes in enriched ECs from LNs draining metastatic or nonmetastatic tumors. B, immunomagnetic selection for podoplanin-enriched populations of LN ECs, as confirmed by flow cytometry. Gray line, isotype control; percentages represent proportions within podoplanin+ve gate (isotype control proportion subtracted). C, enriched EC populations from LNs draining metastatic tumors were analyzed for ICAM-1 (green) and endoglin expression by immunofluorescence or flow cytometry.

Figure 1.

Isolation of ECs from tumor-draining LNs. A, schematic of approach to investigate differentially expressed genes in enriched ECs from LNs draining metastatic or nonmetastatic tumors. B, immunomagnetic selection for podoplanin-enriched populations of LN ECs, as confirmed by flow cytometry. Gray line, isotype control; percentages represent proportions within podoplanin+ve gate (isotype control proportion subtracted). C, enriched EC populations from LNs draining metastatic tumors were analyzed for ICAM-1 (green) and endoglin expression by immunofluorescence or flow cytometry.

Close modal

Identification of endothelial-expressed genes modulated during metastasis to LNs

LN ECs from metastatic and nonmetastatic tumor models were compared by microarray (Fig. 2A). Of the top 10 differentially expressed genes (ranked by adjusted P value), 9 were downregulated in LNs draining metastatic tumors compared with their nonmetastatic counterparts, and all 10 showed more than 2-fold difference in expression (Table 1; Fig. 2B). Candidates were subsequently selected for further analysis based on relevance to endothelial and cancer biology. qRT-PCR validated the downregulation of Bmp4, Unc5c, Cfh, Emcn, and Gpr39 in ECs from LNs draining metastatic tumors, and the upregulation of Nova1 (Fig. 2C). Bmp4 showed the greatest abundance and more than 5-fold difference in expression, and was thus selected for further investigation.

Figure 2.

Identification of differentially expressed genes in LN ECs. A, ECs from LNs draining metastatic or nonmetastatic tumors (labeled nonmetastatic or metastatic LN EC) were compared by microarray. B, a volcano plot of log odds of differential expression against fold change illustrates significantly differentially expressed genes. C, for selected genes, differential expression was validated by qRT-PCR. Shown are 2 representative examples (1 and 2) of pairwise comparisons. Data are mean ± SD of triplicate reactions. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 2.

Identification of differentially expressed genes in LN ECs. A, ECs from LNs draining metastatic or nonmetastatic tumors (labeled nonmetastatic or metastatic LN EC) were compared by microarray. B, a volcano plot of log odds of differential expression against fold change illustrates significantly differentially expressed genes. C, for selected genes, differential expression was validated by qRT-PCR. Shown are 2 representative examples (1 and 2) of pairwise comparisons. Data are mean ± SD of triplicate reactions. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Close modal
Table 1.

Genes differentially expressed in ECs of LNs draining nonmetastatic versus metastatic tumors—microarray analysis

Gene symbolNameFold changeaPb
Bmp4 Bone morphogenetic protein 4 6.03 0.014 
Cfh Complement component factor H 4.69 0.014 
Unc5c unc-5 homolog C (C. elegans) 2.69 0.014 
Emcn Endomucin 5.80 0.014 
Gpr39 G protein-coupled receptor 39 2.94 0.014 
Mbd1 Methyl-CpG binding domain protein 1 4.13 0.017 
Mylk Myosin, light polypeptide kinase 7.92 0.019 
Perp PERP, TP53 apoptosis effector 6.31 0.034 
Hs3st1 Heparan sulfate (glucosamine) 3-O-sulfotransferase 1 3.19 0.034 
Nova1 Neuro-oncological ventral antigen 1 −3.80 0.035 
Gene symbolNameFold changeaPb
Bmp4 Bone morphogenetic protein 4 6.03 0.014 
Cfh Complement component factor H 4.69 0.014 
Unc5c unc-5 homolog C (C. elegans) 2.69 0.014 
Emcn Endomucin 5.80 0.014 
Gpr39 G protein-coupled receptor 39 2.94 0.014 
Mbd1 Methyl-CpG binding domain protein 1 4.13 0.017 
Mylk Myosin, light polypeptide kinase 7.92 0.019 
Perp PERP, TP53 apoptosis effector 6.31 0.034 
Hs3st1 Heparan sulfate (glucosamine) 3-O-sulfotransferase 1 3.19 0.034 
Nova1 Neuro-oncological ventral antigen 1 −3.80 0.035 

aFold change in abundance: nonmetastatic over metastatic.

bBenjamini–Hochberg adjusted P value.

Localization of BMP-4 protein in HEVs and differential expression in metastasis

Immunohistochemistry showed that BMP-4 protein was localized to HEVs (Fig. 3A), confirmed by costaining for the specific MECA-79 epitope (22). BMP-4 protein was present in a subset of HEVs in LNs draining both nonmetastatic and metastatic tumors (Fig. 3A), and in LNs from nontumor-bearing SCID/NOD and immunocompetent mice (Fig. 3A, data not shown). HEVs did not endogenously express podoplanin (Supplementary Fig. S2), suggesting podoplanin probably became upregulated in HEV ECs during the brief culturing between extraction from LN and purification for microarray analysis (23, 24). Although MECA-79 stained the surface of HEV ECs, BMP-4 seemed primarily in the cytoplasm (Fig. 3A inset), implying that HEV ECs express BMP-4 protein. No other sites of BMP-4 localization were observed in the LN or primary tumor. This supported the conclusion that HEV ECs are the main source of BMP-4 mRNA and protein in LNs.

Figure 3.

Localization and differential expression of BMP-4 protein in LNs. Draining LNs of metastatic and nonmetastatic tumors (labeled metastatic and nonmetastatic LNs) or axillary LNs of immunocompetent mice were sectioned and stained with BMP-4 and MECA-79 antibodies by immunofluorescence (A) or by standard immunohistochemistry in serial sections (B). Scale bar is 50 μm (A) or 100 μm (B). In (B), arrows denote BMP-4high and arrowheads BMP-4low HEVs. Proportional BMP-4 expression in HEVs was quantitated as BMP-4–stained/total MECA-79–stained endothelial area, and the proportion of BMP-4high HEVs. n = 16–17 sections; ***, P < 0.001 (C).

Figure 3.

Localization and differential expression of BMP-4 protein in LNs. Draining LNs of metastatic and nonmetastatic tumors (labeled metastatic and nonmetastatic LNs) or axillary LNs of immunocompetent mice were sectioned and stained with BMP-4 and MECA-79 antibodies by immunofluorescence (A) or by standard immunohistochemistry in serial sections (B). Scale bar is 50 μm (A) or 100 μm (B). In (B), arrows denote BMP-4high and arrowheads BMP-4low HEVs. Proportional BMP-4 expression in HEVs was quantitated as BMP-4–stained/total MECA-79–stained endothelial area, and the proportion of BMP-4high HEVs. n = 16–17 sections; ***, P < 0.001 (C).

Close modal

Quantitation of staining revealed that HEV-expressed BMP-4 was significantly reduced (by ∼50%), in LNs draining metastatic versus nonmetastatic tumors (P < 0.001; Fig. 3B and C). This illustrated a shift from predominately BMP-4high to predominately BMP-4low HEVs in LNs draining nonmetastatic versus metastatic tumors respectively; however, both LN types contained some BMP-4high and some BMP-4low HEVs (Fig. 3B and C). Therefore, the downregulation of BMP-4 mRNA was reflected at the protein level in vivo.

BMP-4 loss marks HEV remodeling in cancer

We examined LNs for evidence of tumor-induced HEV remodeling (7), and explored whether VEGF-D or BMP-4 was associated with this process (Fig. 4A). In LNs draining nonmetastatic tumors, BMP-4high HEVs had significantly smaller lumen areas than BMP-4low HEVs (P = 0.0017; Fig. 4B). In LNs draining metastatic tumors, however, the BMP-4high HEVs were more dilated than in the nonmetastatic context (P = 0.028; Fig. 4B). Significantly, BMP-4high HEVs had thicker vessel walls than BMP-4low HEVs in all LNs (P < 0.001; Fig. 4B), suggesting that BMP-4 expression was closely linked with HEV morphology. Although the remaining BMP-4high HEVs in LNs draining metastatic tumors largely retained their greater vessel wall width, there was a strong trend suggesting reduced width compared with those in LNs draining nonmetastatic tumors, indicating that VEGF-D–driven metastasis could affect the endothelial width of BMP-4high HEVs (P = 0.064; Fig. 4B). We also observed remodeled HEVs in a pilot study of human breast cancer-associated LNs with or without histologically identifiable metastasis (Fig. 4E), confirming its occurrence in human disease (7).

Figure 4.

HEV remodeling in VEGF-D–driven tumor metastasis. Luminal and basolateral edges of immunostained HEVs were traced manually to create binary masks (A; scale bar, 20 μm), allowing morphometric quantitation of lumen area and vessel wall width in draining LNs of metastatic and nonmetastatic tumors (metastatic and nonmetastatic LNs; B). n = 18–20 for lumen area and n = 6 for vessel wall width; *, P < 0.05; **, P < 0.01; ***, P < 0.001. To measure proliferating HEV ECs, LNs (n = 5) were costained for PCNA (C). Serial sections of LNs were stained for VEGFR-2 and VEGFR-3 (D). Arrows, HEVs; arrowheads, lymphatics; scale bar, 50 μm. E, tumor-associated LNs from breast cancer patients or control LNs were immunostained with MECA-79 antibody. Arrows denote examples of remodeled HEVs. Scale bar: 20 μm.

Figure 4.

HEV remodeling in VEGF-D–driven tumor metastasis. Luminal and basolateral edges of immunostained HEVs were traced manually to create binary masks (A; scale bar, 20 μm), allowing morphometric quantitation of lumen area and vessel wall width in draining LNs of metastatic and nonmetastatic tumors (metastatic and nonmetastatic LNs; B). n = 18–20 for lumen area and n = 6 for vessel wall width; *, P < 0.05; **, P < 0.01; ***, P < 0.001. To measure proliferating HEV ECs, LNs (n = 5) were costained for PCNA (C). Serial sections of LNs were stained for VEGFR-2 and VEGFR-3 (D). Arrows, HEVs; arrowheads, lymphatics; scale bar, 50 μm. E, tumor-associated LNs from breast cancer patients or control LNs were immunostained with MECA-79 antibody. Arrows denote examples of remodeled HEVs. Scale bar: 20 μm.

Close modal

We next investigated whether HEV remodeling involved EC proliferation. Interestingly, BMP-4high HEV ECs in LNs draining metastatic tumors had a significantly lower proliferation rate than those from the nonmetastatic model (P = 0.026; Fig. 4C). Furthermore, BMP-4low HEV ECs in LNs draining metastatic tumors had a significantly higher proliferation rate than the BMP-4high HEV ECs (P = 0.015; Fig. 4C). These results indicated that the proliferation response of HEV ECs to tumor-secreted VEGF-D may be modulated by BMP-4; another way in which VEGF-D–driven metastasis may induce remodeling of HEV characteristics via reduction of BMP-4 expression.

The role of VEGF-D and VEGFR-2 in HEV remodeling

To determine whether HEVs could respond directly to tumor-secreted human VEGF-D, we examined VEGFR-2 and VEGFR-3 expression in LNs. VEGFR-2 was expressed on most HEVs, blood vessel capillaries and lymphatics (Fig. 4D). VEGFR-3 was strongly expressed on lymphatics, but was essentially absent from HEVs. Thus HEVs are capable of responding to VEGFR-2 ligands.

In vivo approaches were utilized to investigate the specific pathways controlling HEV remodeling. Injection of VEGF-D into the mouse ear mimics tumor-secreted growth factor draining to regional LNs. After 3 days of VEGF-D treatment, proliferation of BMP-4high HEV ECs was decreased (P = 0.034; Fig. 5A), suggesting VEGF-D was responsible for the effect observed in tumor-draining LNs (Fig. 4C), and that suppression of proliferation in BMP-4high HEVs by VEGF-D may occur early in the metastatic process. Alteration of lumen area, vessel wall width and BMP-4 expression may require a longer stimulation period as neither was affected in this experiment (Fig. 5A and B); however, BMP-4high HEVs again exhibited significantly thicker vessel walls (Fig. 5A).

Figure 5.

Involvement of VEGF-D and VEGFR-2 in HEV remodeling. Mouse ears were injected with recombinant VEGF-D protein or PBS. Draining LNs were analyzed using MECA-79/BMP-4 (n = 10) and MECA-79/BrdU (n = 5) immunofluorescence for effects on HEV lumen area, vessel wall width, and proliferation (A) and BMP-4 expression (B). Mice bearing metastatic tumors were treated with antibodies to VEGFR-2 or VEGF-D. HEV morphology and proliferation (C) and BMP-4 expression (D) were assessed as above in tumor-draining LNs (n = 5–6). *, P < 0.05; **, P < 0.01; ***, P < 0.001. E, schematic of the proposed mechanisms of HEV remodeling.

Figure 5.

Involvement of VEGF-D and VEGFR-2 in HEV remodeling. Mouse ears were injected with recombinant VEGF-D protein or PBS. Draining LNs were analyzed using MECA-79/BMP-4 (n = 10) and MECA-79/BrdU (n = 5) immunofluorescence for effects on HEV lumen area, vessel wall width, and proliferation (A) and BMP-4 expression (B). Mice bearing metastatic tumors were treated with antibodies to VEGFR-2 or VEGF-D. HEV morphology and proliferation (C) and BMP-4 expression (D) were assessed as above in tumor-draining LNs (n = 5–6). *, P < 0.05; **, P < 0.01; ***, P < 0.001. E, schematic of the proposed mechanisms of HEV remodeling.

Close modal

In addition, mice bearing metastatic tumors were treated with neutralizing antibodies to VEGF-D or VEGFR-2. These antibodies can reduce rates of VEGF-D–driven metastasis to LNs (M. Matsumoto and colleagues; unpublished data). Anti–VEGFR-2 treatment of metastatic tumors significantly reduced the lumen area of BMP-4high HEVs (vs. metastatic + PBS, P = 0.039; vs. nonmetastatic tumors, P = 0.049; Fig. 5C). Vessel wall width of BMP-4high HEVs was again reduced in LNs draining metastatic tumors (P = 0.019; Fig. 5C). Importantly, both anti–VEGFR-2 and anti–VEGF-D treatments effectively blocked this remodeling (P = 0.049 for anti–VEGFR-2 and P = 0.008 for anti–VEGF-D treatments), returning the vessel wall width to that of the nonmetastatic control. VEGFR-2 blockade also increased the vessel wall width of BMP-4low HEVs (vs. metastatic + PBS, P = 0.007; vs. nonmetastatic, P = 0.053; Fig. 5C), whereas BMP-4high HEVs still had significantly thicker walls than BMP-4low HEVs in all conditions (P < 0.001). Proliferation rates of both BMP-4high (P = 0.032) and BMP-4low (P = 0.026) HEV ECs were reduced by anti–VEGFR-2 treatment, whereas anti–VEGF-D treatment reduced proliferation of BMP-4low HEVECs particularly (P = 0.018; Fig. 5C). These analyses implicate VEGFR-2 in mediating HEV EC dilation and proliferation, and reiterate that BMP-4 expression modulates the proliferation response of HEV ECs to VEGF-D. Interestingly, anti–VEGFR-2 treatment did not restore the reduced BMP-4 expression in LNs draining metastatic tumors; in contrast, BMP-4 expression under anti–VEGF-D treatment was very close to that of LNs draining nonmetastatic tumors (Fig. 5D). This suggests that VEGF-D may induce BMP-4 downregulation, via a VEGFR-2–independent mechanism.

Effects of exogenous BMP-4 on tumor progression

As this study was designed to identify and analyze molecular targets with prognostic and/or therapeutic potential, we established a therapeutic model to determine the effects of exogenously-administered BMP-4. Activity and stability of recombinant human BMP-4 were verified by bioassay (Supplementary Fig. S3A; Supplementary Methods). As shown in Fig. 6A, BMP-4 inhibited the exponential growth of VEGF-D–overexpressing primary tumors by approximately 50% (day 20, P = 0.056; day 22, P = 0.036; day 24, P = 0.080). In addition, similar tumors overexpressing VEGF-C were reduced in size by approximately 56% by BMP-4 treatment (day 15, P = 0.067; day 18, P = 0.021; day 23, P = 0.026). BMP-4 could thus inhibit tumor growth driven by 2 different lymphangiogenic/angiogenic growth factors. ELISA results confirmed that injected BMP-4 reached the systemic circulation at approximately 1,200 pg/mL in serum after 60 minutes (Fig. 6B). Interestingly, under the conditions and timecourse of these experiments the BMP-4 treatment did not seem to affect metastasis to LNs or HEV morphology (Fig. 6C and data not shown). Analysis of HEVs did reveal a trend suggesting that in metastasis-positive LNs draining VEGF-D–overexpressing tumors, more BMP-4high HEVs were observed under BMP-4 treatment than for the control (mean ± SE: BMP-4, 40.9 ± 10.1; vehicle, 29.5 ± 10.0; n = 5, P = 0.16). Furthermore, BMP-4high HEVs again exhibited thicker vessel walls than BMP-4low HEVs in both treatment conditions (P < 0.001; Supplementary Fig. S3B), confirming the importance of endogenous BMP-4 expression.

Figure 6.

Therapeutic administra-tion of BMP-4. A, BMP-4 or vehicle control was administered to mice from day 1 until day 12 or experiment termination, and tumor volume measured (n = 9–11). B, detection of BMP-4 in serum by ELISA (n = 3). C, LNs were scored histologically positive or negative for metastatic cells. D, immunohistochemistry detecting BMPR-II expression on multiple tumor cell types including blood vessels, inset. E, Western blot detecting BMPR-II in cultured tumor cells and metastatic (VEGF-D) tumor lysates, and densitometric quantitation of expression (n = 3; full-length blot, Supplementary Fig. S5).

Figure 6.

Therapeutic administra-tion of BMP-4. A, BMP-4 or vehicle control was administered to mice from day 1 until day 12 or experiment termination, and tumor volume measured (n = 9–11). B, detection of BMP-4 in serum by ELISA (n = 3). C, LNs were scored histologically positive or negative for metastatic cells. D, immunohistochemistry detecting BMPR-II expression on multiple tumor cell types including blood vessels, inset. E, Western blot detecting BMPR-II in cultured tumor cells and metastatic (VEGF-D) tumor lysates, and densitometric quantitation of expression (n = 3; full-length blot, Supplementary Fig. S5).

Close modal

Mechanisms of BMP-4–induced tumor growth suppression

To clarify the mechanism by which BMP-4 suppressed primary tumor growth, we first examined the distribution of its receptors. BMPs bind a heterodimeric complex of type I and type II receptors (25). Immunohistochemistry for BMPR-II revealed broad expression on multiple cell types including tumor cells, stroma, and endothelium of large blood vessels (Fig. 6D). Microarray analysis indicated that the VEGF-D–overexpressing tumor cells expressed BMPR2, as well as BMPRIA and ACTR1A, but not BMPR1B (Supplementary Table S2), whereas immunocytochemistry confirmed expression of BMPR-IA and BMPR-II protein on tumor cells and tumor-derived fibroblasts (Supplementary Fig. S4A; Supplementary Methods). Interestingly, Western blotting revealed that BMPR-II protein was more abundant in BMP-4–treated than control-treated VEGF-D–overexpressing metastatic tumors (P = 0.048; Fig. 6E and Supplementary Methods), potentially representing a feedback loop that could contribute to tumor suppression.

In vitro stimulation showed that proliferation of VEGF-D–overexpressing tumor cells was unaffected by BMP-4 (Supplementary Fig. S4B). Conversely, preliminary experiments suggested that 100 ng/mL BMP-4 could induce detectable death in flow cytometric and microscopy-based assays (6.9% ± 0.3% vs. control 4.3% ± 1.0% 7-aminoactinomycin-D–positive cells, P = 0.036; 396 ± 22 vs. control 270 ± 23 propidium iodide-positive cells/field, P = 0.002). Tumor-derived fibroblasts proliferated significantly when stimulated with BMP-4 (Supplementary Fig. S4C). Interestingly, BMP-4–stimulated human lymphatic ECs showed increased Ki-67 expression while being slightly reduced in number, suggesting a short-term apoptosis effect followed by a slower proliferation response (Supplementary Fig. S4D). These data collectively suggest that BMP-4–induced tumor growth suppression is enacted via multiple cell types.

Changes to the blood or lymphatic vasculature in tumor-draining LNs have prognostic significance in cancer (9, 16, 17, 26), and may facilitate metastasis (11–13). Understanding the mechanisms and functional consequences of these alterations will be critical in determining the overall role of LN metastasis in tumor progression, and could advance prognostication and treatment for cancer patients. Here, we have identified molecules involved in the remodeling of HEVs in tumor-draining LNs, and an additional role for BMP-4 in suppressing primary tumor growth.

Microarray analysis of enriched LN EC populations revealed differential expression of several genes with significance to endothelial and tumor biology. Analysis of isolated EC subtypes has enabled identification of important functional molecules (ref. 27 and manuscript submitted). Although our isolation strategy utilized podoplanin, commonly used to distinguish lymphatic endothelium, immunohistochemical validation revealed BMP-4 to be differentially expressed in HEVs, a specialized venous endothelial type that did not express podoplanin in vivo. Subsequent to observations that blood vascular ECs cocultured with lymphatic ECs could spontaneously acquire expression of lymphatic-characteristic molecules including podoplanin (23), it has been shown that substantial plasticity exists between arterial, venous, and lymphatic EC lineages, controlled by specific transcription factors and reflecting their common embryonic origin (24). Our observations provide further confirmation of this plasticity and relatedness. Another similar study used microarray analysis of isolated lymphatic ECs from primary tumors, which were briefly cultured, to identify novel markers with prognostic significance (28). Our study advances upon this by examining the endothelium of tumor-draining LNs.

The morphologic changes we observed to be associated with VEGF-D–driven metastasis and BMP-4 reduction—that is, remodeling of the normally “high” -walled HEVs into flat walled, more dilated vessels with altered proliferation responses—were consistent with those observed in mouse models and human breast cancer (7). Other investigators observed suppression of the HEV-expressed lymphotactic chemokine CCL21 and reduced lymphocyte recruitment in tumor-draining LNs (29). Such physical and molecular features of HEVs endothelium are integral to their role in trafficking leukocytes into the LN to facilitate immune responses (8). Although these investigators analyzed total HEVs, we identified HEV subtypes (BMP-4high and BMPlow) which can respond differentially to tumor-associated stimuli. Although the functional significance of HEV height is poorly understood, flattening of HEV ECs seems to reduce leukocyte transmigration rates (30). Lower branching-order HEVs were observed to support lower rates of lymphocyte adhesion than higher-order HEVs (29); interestingly, in our studies lower-order HEVs tended to have flatter endothelium and lower BMP-4 expression than higher-order HEVs. It is possible that HEV remodeling may echo homeostatic differences in the morphologic, molecular, and functional characteristics of different branching-order HEVs. Ultimately, tumor-induced HEV remodeling could assist in generating a metastatic niche (31): proliferating, dilated blood vessels derived from remodeled HEVs could enrich the nutrient and oxygen supply to a LN, whereas impaired immune function would promote tumor cell survival. The proximity of remodeled HEVs and lymphatic vessels could provide a shortcut for metastatic cells into the blood vasculature and thus systemic circulation (31, 32).

Our study provides an important contribution to understanding the molecular mechanisms driving tumor-induced HEV remodeling (Fig. 5E). The effects of BMP-4 and VEGF-D–driven metastasis on HEV vessel wall width were strongly evident, whereas differences in lumen area and proliferation were more dynamic and may be sensitive to other factors. The differing impacts of VEGFR-2 and VEGF-D blockade suggest involvement of another VEGFR-2 ligand. Several studies have implicated VEGF-A in stimulating HEV growth and remodeling in immune responses (33, 34); thus endogenous VEGF-A could contribute to VEGFR-2–mediated HEV dilation in tumor-draining LNs. In addition, VEGF-A might be involved in the differential proliferative response of BMP-4high and BMP-4low HEVs to VEGF-D. BMP-4 can increase expression and phosphorylation of VEGFR-2 in ECs, thus enhancing responsiveness to autocrine or paracrine VEGF-A (35). BMP-4 itself could signal to ECs in an autocrine manner (36), and might upregulate VEGF-A expression by LN stromal cells (34, 37), thus potentiating a VEGF-A/VEGFR-2 signaling loop. VEGF-D may then inhibit proliferation of BMP-4high HEV ECs in the tumor context by competing with VEGF-A for binding to VEGFR-2. In contrast, HEVs expressing less BMP-4 may have less dependency on endogenous VEGF-A/VEGFR-2 signaling, and be sensitive to proangiogenic stimulation by tumor-derived VEGF-D (Supplementary Fig. S6). VEGF-D might induce this sensitization via downregulation of BMP-4 over longer exposure periods. Preferential proliferation of BMP-4low HEVs earlier during VEGF-D–driven metastasis could also contribute to the preponderance of these vessels over time.

Signaling through HEV-expressed VEGFR-2 by VEGF-D (potentially also VEGF-A) likely contributes partially to altering HEV vessel wall width, and the physical presence of tumor cells in LNs could also affect HEV remodeling. Notably, the difference in vessel wall width corresponding to BMP-4 expression is larger than that attributable to VEGF-D and VEGFR-2 blockade in the metastatic tumor context. BMP-4 may thus act as a key regulator of HEVs, controlling EC morphology and responsiveness to angiogenic factors. BMP-4 induces a columnar epithelial phenotype in premalignant Barrett's oesophagus (38), and could play a similar role in maintaining high endothelium. Loss of BMP-4 may enable completion of the remodeling process induced by VEGF-D–driven metastasis (Fig. 5E). Although the mechanism controlling BMP-4 expression remains unclear, the restorative effect of VEGF-D blockade suggests a VEGF-D–driven mechanism independent of VEGFR-2. Lymphatics and HEVs are regulated in a coordinated manner during immunization responses, via crosstalk involving lymphotoxin β receptor (32); similarly, modulation of lymphatics in tumor-draining LNs (e.g., via VEGF-D/VEGFR-3 signaling) may influence HEV characteristics.

As a member of the TGF-β superfamily of multipotent cytokines, the role of BMP-4 in tumor progression can be complex and highly context specific (25, 39). We showed that while endogenously expressed BMP-4 regulates HEVs, exogenous BMP-4 can restrict primary tumor growth. BMP-4 is also known to induce apoptosis of other tumor cell lines (40, 41) and microvascular ECs (42), although in other studies proangiogenic responses were observed, possibly due to potentiation of VEGF-A/VEGFR-2 signaling (35). Our data suggest that lymphatic ECs may respond to BMP-4 in a similar way. An increase in proliferation of tumor-derived fibroblasts stimulated with BMP-4 in vitro is intriguing considering that cancer-associated fibroblasts are commonly implicated in promoting tumorigenesis (43). The upregulation of BMPR-II expression in BMP-4–treated tumors recapitulates a similar observation in Xenopus embryos indicating that Bmpr2 is a target gene of BMP-4 signaling (44). Expression of several other regulators of BMP-4 signaling is also induced by BMP-4, raising the possibility that blockade of relevant signaling inhibitors might enhance the efficacy of BMP-4 treatment. Previous in vivo studies have described antitumorigenic effects of BMP-4 for several tumor types (40, 45–47)—as well as protumorigenic effects for some—but thus far only one other study, using a model of glioblastoma multiforme, has demonstrated an antitumor effect of therapeutically administered recombinant BMP-4 (48). Although the authors identified a prodifferentiation effect on tumor stem cells, we noted that VEGF-D is highly expressed in glioblastoma multiforme (49). Our study adds weight to the potential of BMP-4 as an antitumor agent by showing that it can inhibit tumor growth driven by 2 different lymphangiogenic/angiogenic factors through action on both tumor cells and stroma.

The context-specific nature of BMP-4 signaling does compel careful tuning of BMP-4 targeting and dosage to ensure a robust antitumor effect. A more constant dosage of BMP-4, or a delivery system more targeted to the LN, may help clarify whether therapeutically administered BMP-4 can reverse HEV remodeling or inhibit metastasis. Nevertheless, reduction of BMP-4 expression in HEVs is an important early molecular indicator of remodeling, as it precedes loss of MECA-79 upon incorporation into the vasculature of the tumor deposit (7). Clinical studies will establish whether BMP-4 may represent a convenient surrogate marker of HEV remodeling in cancer. Furthermore, BMP-4 or HEV remodeling may serve as indicators of systemic or distant effects of prometastatic tumor-derived factors such as VEGF-D, and provide prognostic information relevant to metastasis, treatment response, or patient outcome. Our data further highlight the need to better understand the functional and prognostic significance of the LN, and in particular its vasculature, to cancer metastasis, as well as the potential of BMP-4 as a multipotent antitumor agent.

M.G. Achen and S.A. Stacker: commercial research grant, Imclone; ownership interest, Circadian Technologies; consultant/advisory board, Vegenics. R. Shayan: ownership interest, Circadian Technologies. The other authors disclosed no potential conflicts of interest.

The authors thank You-Fang Zhang, Rachael Inder, Kathryn Ardipradja, Sally Roufail, and Janna Taylor for technical support. The authors also thank Gordon Smyth and Keith Satterley of the Walter and Eliza Hall Institute for consultation.

This work was supported by the NHMRC of Australia and Operational Infrastructure Support Program, Victorian Government, Australia. The Pfizer Fellowship (S.A. Stacker). NHMRC fellowships (S.A. Stacker and M.G. Achen). R.H. Farnsworth received a Melbourne Research Scholarship from the University of Melbourne. R. Shayan received a Surgical Scientist Scholarship from the Royal Australasian College of Surgeons.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Achen
MG
,
Stacker
SA
. 
Tumor lymphangiogenesis and metastatic spread-new players begin to emerge
.
Int J Cancer
2006
;
119
:
1755
60
.
2.
Sleeman
J
,
Schmid
A
,
Thiele
W
. 
Tumor lymphatics
.
Semin Cancer Biol
2009
;
19
:
285
97
.
3.
Shayan
R
,
Achen
MG
,
Stacker
SA
. 
Lymphatic vessels in cancer metastasis: bridging the gaps
.
Carcinogenesis
2006
;
27
:
1729
38
.
4.
Tanis
PJ
,
Nieweg
OE
,
Valdes
Olmos RA
,
Th Rutgers
EJ
,
Kroon
BB
. 
History of sentinel node and validation of the technique
.
Breast Cancer Res
2001
;
3
:
109
12
.
5.
Leong
SP
,
Cady
B
,
Jablons
DM
,
Garcia-Aguilar
J
,
Reintgen
D
,
Jakub
J
, et al
Clinical patterns of metastasis
.
Cancer Metastasis Rev
2006
;
25
:
221
32
.
6.
Thiele
W
,
Sleeman
JP
. 
Tumor-induced lymphangiogenesis: a target for cancer therapy?
J Biotechnol
2006
;
124
:
224
41
.
7.
Qian
CN
,
Berghuis
B
,
Tsarfaty
G
,
Bruch
M
,
Kort
EJ
,
Ditlev
J
, et al
Preparing the “soil”: the primary tumor induces vasculature reorganization in the sentinel lymph node before the arrival of metastatic cancer cells.
Cancer Res
2006
;
66
:
10365
76
.
8.
Girard
JP
,
Springer
TA
. 
High endothelial venules (HEVs): specialized endothelium for lymphocyte migration
.
Immunol Today
1995
;
16
:
449
57
.
9.
Guidi
AJ
,
Berry
DA
,
Broadwater
G
,
Perloff
M
,
Norton
L
,
Barcos
MP
, et al
Association of angiogenesis in lymph node metastases with outcome of breast cancer
.
J Natl Cancer Inst
2000
;
92
:
486
92
.
10.
Hirakawa
S
. 
From tumor lymphangiogenesis to lymphvascular niche
.
Cancer Sci
2009
;
100
:
983
9
.
11.
Harrell
MI
,
Iritani
BM
,
Ruddell
A
. 
Tumor-induced sentinel lymph node lymphangiogenesis and increased lymph flow precede melanoma metastasis
.
Am J Pathol
2007
;
170
:
774
86
.
12.
Hirakawa
S
,
Kodama
S
,
Kunstfeld
R
,
Kajiya
K
,
Brown
LF
,
Detmar
M
. 
VEGF-A induces tumor and sentinel lymph node lymphangiogenesis and promotes lymphatic metastasis
.
J Exp Med
2005
;
201
:
1089
99
.
13.
Hirakawa
S
,
Brown
LF
,
Kodama
S
,
Paavonen
K
,
Alitalo
K
,
Detmar
M
. 
VEGF-C-induced lymphangiogenesis in sentinel lymph nodes promotes tumor metastasis to distant sites
.
Blood
2007
;
109
:
1010
7
.
14.
Kawai
H
,
Minamiya
Y
,
Ito
M
,
Saito
H
,
Ogawa
J
. 
VEGF121 promotes lymphangiogenesis in the sentinel lymph nodes of non-small cell lung carcinoma patients
.
Lung Cancer
2008
;
59
:
41
7
.
15.
Van den Eynden
GG
,
Van der Auwera
I
,
Van Laere
SJ
,
Trinh
XB
,
Colpaert
CG
,
van Dam
P
, et al
Comparison of molecular determinants of angiogenesis and lymphangiogenesis in lymph node metastases and in primary tumours of patients with breast cancer
.
J Pathol
2007
;
213
:
56
64
.
16.
Van den Eynden
GG
,
Vandenberghe
MK
,
van Dam
PJ
,
Colpaert
CG
,
van Dam
P
,
Dirix
LY
, et al
Increased sentinel lymph node lymphangiogenesis is associated with nonsentinel axillary lymph node involvement in breast cancer patients with a positive sentinel node
.
Clin Cancer Res
2007
;
13
:
5391
7
.
17.
van der Schaft
DW
,
Pauwels
P
,
Hulsmans
S
,
Zimmermann
M
,
van de Poll-Franse
LV
,
Griffioen
AW
. 
Absence of lymphangiogenesis in ductal breast cancer at the primary tumor site
.
Cancer Lett
2007
;
254
:
128
36
.
18.
Stacker
SA
,
Caesar
C
,
Baldwin
ME
,
Thornton
GE
,
Williams
RA
,
Prevo
R
, et al
VEGF-D promotes the metastatic spread of tumor cells via the lymphatics
.
Nat Med
2001
;
7
:
186
91
.
19.
Breiteneder-Geleff
S
,
Soleiman
A
,
Kowalski
H
,
Horvat
R
,
Amann
G
,
Kriehuber
E
, et al
Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium
.
Am J Pathol
1999
;
154
:
385
94
.
20.
Wettenhall
JM
,
Simpson
KM
,
Satterley
K
,
Smyth
GK
. 
affylmGUI: a graphical user interface for linear modeling of single channel microarray data
.
Bioinformatics
2006
;
22
:
897
9
.
21.
Achen
MG
,
Roufail
S
,
Domagala
T
,
Catimel
B
,
Nice
EC
,
Geleick
DM
, et al
Monoclonal antibodies to vascular endothelial growth factor-D block its interactions with both VEGF receptor-2 and VEGF receptor-3
.
Eur J Biochem
2000
;
267
:
2505
15
.
22.
Streeter
PR
,
Rouse
BT
,
Butcher
EC
. 
Immunohistologic and functional characterization of a vascular addressin involved in lymphocyte homing into peripheral lymph nodes
.
J Cell Biol
1988
;
107
:
1853
62
.
23.
Groger
M
,
Loewe
R
,
Holnthoner
W
,
Embacher
R
,
Pillinger
M
,
Herron
GS
, et al
IL-3 induces expression of lymphatic markers Prox-1 and podoplanin in human endothelial cells
.
J Immunol
2004
;
173
:
7161
9
.
24.
Oliver
G
,
Srinivasan
RS
. 
Endothelial cell plasticity: how to become and remain a lymphatic endothelial cell
.
Development
2010
;
137
:
363
72
.
25.
ten Dijke
P
,
Korchynskyi
O
,
Valdimarsdottir
G
,
Goumans
MJ
. 
Controlling cell fate by bone morphogenetic protein receptors
.
Mol Cell Endocrinol
2003
;
211
:
105
13
.
26.
Hirakawa
S
,
Detmar
M
,
Kerjaschki
D
,
Nagamatsu
S
,
Matsuo
K
,
Tanemura
A
, et al
Nodal lymphangiogenesis and metastasis: Role of tumor-induced lymphatic vessel activation in extramammary Paget's disease
.
Am J Pathol
2009
;
175
:
2235
48
.
27.
Farnsworth
RH
,
Achen
MG
,
Stacker
SA
. 
Lymphatic endothelium: an important interactive surface for malignant cells
.
Pulm Pharmacol Ther
2006
;
19
:
51
60
.
28.
Clasper
S
,
Royston
D
,
Baban
D
,
Cao
Y
,
Ewers
S
,
Butz
S
, et al
A novel gene expression profile in lymphatics associated with tumor growth and nodal metastasis
.
Cancer Res
2008
;
68
:
7293
303
.
29.
Carriere
V
,
Colisson
R
,
Jiguet-Jiglaire
C
,
Bellard
E
,
Bouche
G
,
Al Saati
T
, et al
Cancer cells regulate lymphocyte recruitment and leukocyte-endothelium interactions in the tumor-draining lymph node
.
Cancer Res
2005
;
65
:
11639
48
.
30.
Fossum
SS
,
ME Ford
WL
. 
The migration of lymphocytes across specialized vascular endothelium VII: the migration of T and B lymphocytes from the blood of the athymic, nude rat
.
Scand J Immunol
1983
;
17
:
539
49
.
31.
Qian
CN
,
Resau
JH
,
Teh
BT
. 
Prospects for vasculature reorganization in sentinel lymph nodes
.
Cell Cycle
2007
;
6
:
514
7
.
32.
Liao
S
,
Ruddle
NH
. 
Synchrony of high endothelial venules and lymphatic vessels revealed by immunization
.
J Immunol
2006
;
177
:
3369
79
.
33.
Webster
B
,
Ekland
EH
,
Agle
LM
,
Chyou
S
,
Ruggieri
R
,
Lu
TT
. 
Regulation of lymph node vascular growth by dendritic cells
.
J Exp Med
2006
;
203
:
1903
13
.
34.
Chyou
S
,
Ekland
EH
,
Carpenter
AC
,
Tzeng
TC
,
Tian
S
,
Michaud
M
, et al
Fibroblast-type reticular stromal cells regulate the lymph node vasculature
.
J Immunol
2008
;
181
:
3887
96
.
35.
Suzuki
Y
,
Montagne
K
,
Nishihara
A
,
Watabe
T
,
Miyazono
K
. 
BMPs promote proliferation and migration of endothelial cells via stimulation of VEGF-A/VEGFR2 and angiopoietin-1/Tie2 signalling
.
J Biochem
2008
;
143
:
199
206
.
36.
Sorescu
GP
,
Sykes
M
,
Weiss
D
,
Platt
MO
,
Saha
A
,
Hwang
J
, et al
Bone morphogenic protein 4 produced in endothelial cells by oscillatory shear stress stimulates an inflammatory response
.
J Biol Chem
2003
;
278
:
31128
35
.
37.
Deckers
MM
,
van Bezooijen
RL
,
van der Horst
G
,
Hoogendam
J
,
van Der Bent
C
,
Papapoulos
SE
, et al
Bone morphogenetic proteins stimulate angiogenesis through osteoblast-derived vascular endothelial growth factor A
.
Endocrinology
2002
;
143
:
1545
53
.
38.
Milano
F
,
van Baal
JW
,
Buttar
NS
,
Rygiel
AM
,
de Kort
F
,
DeMars
CJ
, et al
Bone morphogenetic protein 4 expressed in esophagitis induces a columnar phenotype in esophageal squamous cells
.
Gastroenterology
2007
;
132
:
2412
21
.
39.
Thawani
JP
,
Wang
AC
,
Than
KD
,
Lin
CY
,
La Marca
F
,
Park
P
. 
Bone morphogenetic proteins and cancer: review of the literature
.
Neurosurgery
2010
;
66
:
233
46
.
40.
Nishanian
TG
,
Kim
JS
,
Foxworth
A
,
Waldman
T
. 
Suppression of tumorigenesis and activation of Wnt signaling by bone morphogenetic protein 4 in human cancer cells
.
Cancer Biol Ther
2004
;
3
:
667
75
.
41.
Glozak
MA
,
Rogers
MB
. 
Specific induction of apoptosis in P19 embryonal carcinoma cells by retinoic acid and BMP2 or BMP4
.
Dev Biol
1996
;
179
:
458
70
.
42.
Kiyono
M
,
Shibuya
M
. 
Inhibitory Smad transcription factors protect arterial endothelial cells from apoptosis induced by BMP4
.
Oncogene
2006
;
25
:
7131
7
.
43.
Kalluri
R
,
Zeisberg
M
. 
Fibroblasts in cancer
.
Nat Rev Cancer
2006
;
6
:
392
401
.
44.
Karaulanov
E
,
Knochel
W
,
Niehrs
C
. 
Transcriptional regulation of BMP4 synexpression in transgenic Xenopus
.
Embo J
2004
;
23
:
844
56
.
45.
Blessing
M
,
Nanney
LB
,
King
LE
,
Hogan
BL
. 
Chemical skin carcinogenesis is prevented in mice by the induced expression of a TGF-beta related transgene
.
Teratog Carcinog Mutagen
1995
;
15
:
11
21
.
46.
Giacomini
D
,
Paez-Pereda
M
,
Theodoropoulou
M
,
Labeur
M
,
Refojo
D
,
Gerez
J
, et al
Bone morphogenetic protein-4 inhibits corticotroph tumor cells: involvement in the retinoic acid inhibitory action
.
Endocrinology
2006
;
147
:
247
56
.
47.
Buckley
S
,
Shi
W
,
Driscoll
B
,
Ferrario
A
,
Anderson
K
,
Warburton
D
. 
BMP4 signaling induces senescence and modulates the oncogenic phenotype of A549 lung adenocarcinoma cells
.
Am J Physiol Lung Cell Mol Physiol
2004
;
286
:
L81
6
.
48.
Piccirillo
SG
,
Reynolds
BA
,
Zanetti
N
,
Lamorte
G
,
Binda
E
,
Broggi
G
, et al
Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells
.
Nature
2006
;
444
:
761
5
.
49.
Debinski
W
,
Slagle-Webb
B
,
Achen
MG
,
Stacker
SA
,
Tulchinsky
E
,
Gillespie
GY
, et al
VEGF-D is an X-linked/AP-1 regulated putative onco-angiogen in human glioblastoma multiforme
.
Mol Med
2001
;
7
:
598
608
.