Potential Role of PDGFRβ-Associated THBS4 in Colorectal Cancer Development

Abstract

Colorectal cancer is a significant cause of death since it frequently metastasizes to several organs such as the lung or liver. Tumor development is affected by various factors, including a tumor microenvironment, which may be an essential factor that leads to tumor growth, proliferation, invasion, and metastasis. In the tumor microenvironment, abnormal changes in various growth factors, enzymes, and cytokines can wield a strong influence on cancer. Thrombospondin-4 (THBS4), which is an extracellular matrix protein, also plays essential roles in the tumor microenvironment and mediates angiogenesis by transforming growth factor-β (TGFβ) signaling. Platelet-derived growth factor receptor β (PDGFRβ), which is a receptor tyrosine kinase and is also a downstream signal of TGFβ, is associated with invasion and metastasis in colorectal cancer. We identified that PDGFRβ and THBS4 are overexpressed in tumor tissues of colorectal cancer patients, and that PDGF-D expression increased after TGFβ treatment in the colon cancer cell line DLD-1. TGFβ and PDGF-D increased cellular THBS4 protein levels and secretion but did not increase THBS4 mRNA levels. This response was further confirmed by the inositol 1,4,5-triphosphate receptor (IP3R) and stromal interaction molecule 1 (STIM1) blockade as well as the PDGFRβ blockade. We propose that the PDGFRβ signal leads to a modification of the incomplete form of THBS4 to its complete form through IP3R, STIM1, and Ca²⁺-signal proteins, which further induces THBS4 secretion. Additionally, we identified that DLD-1 cell-conditioned medium stimulated with PDGF-D promotes adhesion, migration, and proliferation of colon myofibroblast CCD-18co cells, and this effect was intensified in the presence of thrombin. These findings suggest that excessive PDGFRβ signaling due to increased TGFβ and PDGF-D in colorectal tumors leads to over-secretion of THBS4 and proliferative tumor development.

Keywords: Ca2+; PDGFRβ; THBS4; colorectal cancer.

Figure 1

Co-immunofluorescence and immunoblots for Thrombospondin-4 (THBS4) and Platelet-derived growth factor receptor β (PDGFRβ) in normal and tumor tissues of colon cancer patients. (A) Immunofluorescence with an anti-THBS4 antibody (green) and anti-PDGFRβ antibody (red) on normal (N) and tumor (T) tissues of colon cancer patients. Scale bars = 100 µm. (B) Western blot with anti-THBS4 and anti-PDGFRβ antibodies on normal and tumor tissues of colon cancer patients. * p < 0.05, compared with normal tissue, t-test.

Figure 2

Effect of TGFβ on mRNA levels of THBS4, PDGFRβ, and PDGFRβ ligands. Relative mRNA expression levels as determined with real-time polymerase chain reaction (PCR) for (A) PDGFRβ, THBS4, (B) PDGF-A, PDGF-B, PDGF-C, and PDGF-D of DLD-1 cells cultured in the presence (TGFβ 10mM) or absence (control) of TGFβ for 12 h. (C) Western blot with anti-PDGF-D, p-PDGFRβ, and PDGFRβ antibodies in DLD-1 cells cultured in the presence (TGFβ 10 μM) or absence (control) of TGFβ for 12 h. Three independent experiments were performed in duplicate. * p < 0.05 when compared with the control t-test.

Figure 3

Effect of TGFβ or PDGF-D on THBS4 of lysate and cultured medium of DLD-1 cells. (A) Western blot with anti-THBS4 antibody in whole cell lysate (left panel) or cultured medium (right panel) of DLD-1 cells cultured in the presence of TGFβ (1, 2, 5, 10 and 20 μM) for 20 h or PDGF-D (1, 2, 5, 10, and 20 μM) for 8 h. (B) Relative mRNA expression levels as determined with real-time polymerase chain reaction (PCR) for THBS4 of DLD-1 cells cultured in the presence (black) or absence (gray) of PDGF-D (20 μM) for 8 h. Three independent experiments were performed in duplicate. * p < 0.05 when compared with the control t-test.

Figure 4

Effect of PDGF-D stimulation of THBS4 after blockage of PDGFRβ, IP3R, and STIM1. (A) Western blot with anti-THBS4 antibody in whole cell lysate (left panel) or cultured medium (right panel) of DLD-1 cells cultured with PDGF-D (20 μM) for 8 h in the presence of imatinib (0, 0.2, 0.5, 1, 2, and 5 μM) for 16 h, 2-APB (0, 5, 10, 20, 50, and 100 μM) for 16 h, or ML-9 (0, 5, 10, 20, 50, and 100 μM) for 16 h, respectively. (B) Western blot with anti-THBS4 antibody in whole cell lysate or cultured medium of DLD-1 cells transfected with siIP3R or siSTIM1 and cultured with PDGF-D (20 μM) for 8 h. (C) Relative mRNA expression levels as determined with real-time PCR for THBS4 of DLD-1 cells cultured with PDGF-D (20 μM) for 8 h in the presence of imatinib (5 μM) for 16 h, 2-APB (100 μM) for 16 h or ML-9 (100 μM) for 16 h, respectively. Three independent experiments were performed in duplicate. * p < 0.05 when compared with the control t-test.

Figure 5

Effect of imatinib and 2-APB on THBS4 regulation by TGFβ and PDGF-D in a time-dependent manner. (A) Western blot with anti-PDGF-D and anti-THBS4 antibodies in the presence of TGFβ 10 µM (0, 4, 8, 12, 16, 20, and 24 h) or with anti-THBS4 antibody in the presence of PDGF-D 20 µM (0, 4, 8 and 12 h) in DLD-1 cells. (B) Western blot with anti-PDGF-D and anti-THBS4 antibodies in the presence of TGFβ 10 µ M (0, 4, 8, 12, 16, 20, and 24 h) or with anti-THBS4 antibody in the presence of PDGF-D 20 µM (0, 4, 8, and 12 h) in DLD-1 cells cultured with imatinib 5 µM for 16 h. (C) Western blot with anti-PDGF-D and anti-THBS4 antibodies in the presence of TGFβ 10 µM (0, 4, 8, 12, 16, 20, and 24 h) or with anti-THBS4 antibody in the presence of PDGF-D 20 µM (0, 4, 8 and 12 h) in DLD-1 cells cultured with 2-APB 100 µM for 16 h. The graphs in the right panel represent the relative expression of PDGF-D (black line with closed circle). THBS4 (red line with open circle) in relation to exposure (time, h) with TGFβ and the relative expression of THBS4 after the addition of PDGF-D (green line with closed triangle) through 12 h. Three independent experiments were performed in duplicate.

Figure 6

Effect of DLD-1 cell conditioned medium (CM) stimulated with PDGF-D and additional thrombin treatment on CCD-18co cells. (A,B) Adhesion and migration of CCD-18co cells cultured with DLD-1 CM stimulated with PDGF-D (P), imatinib (I), I + P, 2-APB (A), A + P, ML-9 (M), and M + P. DLD-1 cells were treated with imatinib 5 M, 2-APB 100 or ML-9 100 for 16 h, and then PDGF-D 20 additionally treated for 8 h. (C,D,E) CCD-18co cells were cultured with CM or CM with thrombin (1 U/mL) (CM + T) of DLD-1 cells stimulated with PDGF-D 20 for 8 h and subjected to an adhesion assay, migration assay, and an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. All experiments were performed in duplicate in three independent experiments. * p < 0.05, compared with normal or CM t-test.

Figure 7

Model representing possible molecular pathways by which PDGFRβ and THBS4 cause the development of colorectal cancer.