Bone morphogenetic protein signaling and growth suppression in colon cancer

Abstract

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta superfamily, which utilize BMP receptors and intracellular SMADs to transduce their signals to regulate cell differentiation, proliferation, and apoptosis. Because mutations in BMP receptor type IA (BMPRIA) and SMAD4 are found in the germline of patients with the colon cancer predisposition syndrome juvenile polyposis, and because the contribution of BMP in colon cancers is largely unknown, we examined colon cancer cells and tissues for evidence of BMP signaling and determined its growth effects. We determined the presence and functionality of BMPR1A by examining BMP-induced phosphorylation and nuclear translocation of SMAD1; transcriptional activity via a BMP-specific luciferase reporter; and growth characteristics by cell cycle analysis, cell growth, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide metabolic assays. These assays were also performed after transfection with a dominant negative (DN) BMPR1A construct. In SMAD4-null SW480 cells, we examined BMP effects on cellular wound assays as well as BMP-induced transcription in the presence of transfected SMAD4. We also determined the expression of BMPR1A, BMP ligands, and phospho-SMAD1 in primary human colon cancer specimens. We found intact BMP signaling and modest growth suppression in HCT116 and two derivative cell lines and, surprisingly, growth suppression in SMAD4-null SW480 cells. BMP-induced SMAD signaling and BMPR1A-mediated growth suppression were reversed with DN BMPR1A transfection. BMP2 slowed wound closure, and transfection of SMAD4 into SW480 cells did not change BMP-specific transcriptional activity over controls due to receptor stimulation by endogenously produced ligand. We found no cell cycle alterations with BMP treatment in the HCT116 and derivative cell lines, but there was an increased G1 fraction in SW480 cells that was not due to increased p21 transcription. In human colon cancer specimens, BMP2 and BMP7 ligands, BMPRIA, and phospho-SMAD1 were expressed. In conclusion, BMP signaling is intact and growth suppressive in human colon cancer cells. In addition to SMADs, BMP may utilize SMAD4-independent pathways for growth suppression in colon cancers.

Fig. 1

Bone morphogenetic protein (BMP) receptor type IA (BMPRIA) and SMAD protein expression. A: immunoprecipitation of BMPRIA in various colon cancer cell lines. The C2C12 mouse cell line is used as a positive control. FET and CaCo are additional colon cancer cell lines. B–D: nuclear extracts of HCT116 (B), HCT116 + chr2 (C), and HCT116 + chr3 (D) cell lines treated with BMP2 or BMP7. Membranes were blotted for phosphorylated (p)SMAD1, SMAD1, and SMAD4. Histone was used as a loading control for nuclear fractionations.

Fig. 2

BMP-induced transcriptional activity in colon cancer cells. A and B: fold induction of BMP2 (A) or BMP7 (B) treatment over no treatment (control, CNT) of BMP-induced SMAD transcriptional activity in HCT116 cell line derivatives. C: relative amount of SMAD-induced transcriptional activity in SW480 cells with and without SMAD4 transfection. D: RT-PCR of SW480 cells for basal (endogenous) BMP2 and BMP7 expression and after treatment with BMP2, BMP7, or Noggin. GAPDH was used as a control.

Fig. 3

Effect of BMP2 or BMP7 treatment on cell growth as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in HCT116, HCT116 + chr2, HCT116 + chr3, and SW480 cells. Two-factor with replications ANOVA was used to determine P values: *P < 0.05 and **P < 0.01.

Fig. 4

Effect of BMP2 or BMP7 treatment on cell growth as assessed by cell counting in HCT116 (A), HCT116 + chr2 (B), HCT116 + chr3 (C), and SW480 (D) cells after 2 and 4 days of BMP2 and BMP7 treatment.

Fig. 5

Relative amount of SMAD-induced transcriptional activity after BMP ligand treatment and the effect of dominant negative (DN) BMPR1A transfection. In both HCT116 + chr2 (A) and HCT116 + chr3 (B) cells, DN BMPR1A transfection reduced BMP-induced SMAD transcriptional activity. Constitutively active (CA) BMPR1A is shown as a positive control in the absence of ligand treatment. In SMAD4-null SW480 cells (C), DN BMPR1A reduced endogenous (CNT) and BMP2-induced SMAD transcriptional activity when cotransfected with SMAD4.

Fig. 6

DN BMPR1A transfection reversed BMP-induced growth suppression as assessed by MTT assay in HCT116 + chr2 (A), HCT116 + chr3 (B), and SW480 (C) cells. Two-factor with replications ANOVA was used to determine P values: *P < 0.05 and **P < 0.01.

Fig. 7

Wound closure assay of untreated (control) SW480 cells and those treated with BMP2, BMP7, or Noggin. A: representative pictures for days 0, 3, and 4. B: bar graph of SW480 wound closure at day 3.

Fig. 8

Effect of BMPs on p21 transcription. A: results of p21 luciferase assay using pWWP-luc plasmid on HCT116, HCT116 + chr2, HCT116 + chr3, and SW480 cell lines with BMP2 and BMP7 treatment. B–E: results of p21 mRNA expression in HCT116 (B), HCT116 + chr2 (C), HCT116 + chr3 (D), and SW480 (E) cells after treatment with BMP2, BMP7, and Noggin.

Fig. 9

Representative pictures of immunohistochemistry on 4 paired human colon cancer specimens stained for BMP2, BMP7, BMPRIA, and pSMAD1. Controls represent stained samples without primary antibody. Intestinal hamartomatous polyps with and without mutations in BMPR1A were used as additional controls (not shown). Magnification: ×40.