Platelet-derived growth factor (PDGF) cross-signaling via non-corresponding receptors indicates bypassed signaling in colorectal cancer

Abstract

Platelet-derived growth factor (PDGF) signaling, besides other growth factor-mediated signaling pathways like vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF), seems to play a crucial role in tumor development and progression. We have recently provided evidence for upregulation of PDGF expression in UICC stage I-IV primary colorectal cancer (CRC) and demonstrated PDGF-mediated induction of PI3K/Akt/mTOR signaling in CRC cell lines. The present study sought to follow up on our previous findings and explore the alternative receptor cross-binding potential of PDGF in CRC. Our analysis of primary human colon tumor samples demonstrated upregulation of the PDGFRβ, VEGFR1, and VEGFR2 genes in UICC stage I-III tumors. Immunohistological analysis revealed co-expression of PDGF and its putative cross-binding partners, VEGFR2 and EGFR. We then analyzed several CRC cell lines for PDGFRα, PDGFRβ, VEGFR1, and VEGFR2 protein expression and found these receptors to be variably expressed amongst the investigated cell lines. Interestingly, whereas Caco-2 and SW480 cells showed expression of all analyzed receptors, HT29 cells expressed only VEGFR1 and VEGFR2. However, stimulation of HT29 cells with PDGF resulted in upregulation of VEGFR1 and VEGFR2 expression despite the absence of PDGFR expression and mimicked the effect of VEGF stimulation. Moreover, PDGF recovered HT29 cell proliferation under simultaneous treatment with a VEGFR or EGFR inhibitor. Our results provide some of the first evidence for PDGF cross-signaling through alternative receptors in colorectal cancer and support anti-PDGF therapy as a combination strategy alongside VEGF and EGF targeting even in tumors lacking PDGFR expression.

Keywords: EGFR; PDGF; VEGFR; bypassed signaling; colorectal cancer.

Figure 1. PDGF receptors, their binding ligands, and downstream signaling pathways with diverse cellular effects.

PDGF receptor/ligand binding can activate the PI3K/Akt and MAPK pathways and influences cell survival, angiogenesis, proliferation, and migration.

Figure 2. Increased PDGFR and VEGFR gene expression in early and advanced stage human colon cancer.

RT-qPCR analysis of (A) PDGFRα, (B) PDGFβ, (C) VEGFR1, and (D) VEGFR2 in normal mucosa and human colon cancers (UICC stage I/II, n = 20 and UICC stage III/IV, n = 22). The relative quantification value is expressed as 2^−ΔΔCq. Results are presented as mean ± SD; ^*** p < 0.001.

Figure 3. Co-expression of PDGF with VEGFR2 and EGFR in human colon cancer.

Representative immunofluorescence double staining of colon cancer tissue (20 patient tissues were stained) exhibiting positive co-expression of PDGF (Cy3, red) and VEGFR2 or EGFR (Alexa 488, green). Arrows indicate regions demonstrating positive dual expression. Magnification 200×. Scale Bar = 50 uM.

Figure 4. Variable expression of PDGFR and VEGFR in established CRC cell lines.

(A) Representative Western blot analysis of PDGFRα, PDGFRβ, VEGFR1, and VEGFR2 in HT29, Caco-2, and SW480 CRC cells. β-Actin was used as loading control. Representative immunostaining of PDGFRα, PDGFRβ, VEGFR1, and VEGFR2 in (B) HT29 and (C) Caco-2 cells. Magnification: 200×. Scale Bar = 20 uM.

Figure 5. Effects of PDGF and VEGF on corresponding receptor expression in Caco-2 and HT29 CRC cells.

RT-qPCR analysis of (A) PDGFRα, (B) PDGFRβ, (C) VEGFR1, and (D) VEGFR2 expression following stimulation of Caco-2 cells with PDGF or VEGF. RT-qPCR analysis of (E) VEGFR1 and (F) VEGFR2 expression following stimulation of HT29 cells with PDGF or VEGF. The relative quantification value is expressed as 2^−ΔΔCq. Results are presented as mean ± SD, ^* p < 0.05, n = 3.

Figure 6. Pro-proliferative effects of PDGF on HT29 CRC cells under simultaneous inhibition of VEGFR2 or EGFR.

PDGF recovered proliferation of cells treated with (A) the VEGFR2 inhibitor, ramucirumab, and (B) the EGFR inhibitor, cetuximab. Neither PDGF nor VEGF were able to recover proliferation of cells treated with (C) the broad range TKI, regorafenib. Results are presented as ± SD, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, n = 3.

Figure 7. Suggested crosstalk of PDGF with alternative binding partners on tumor cell surface.

Flexible binding of PDGF to alternative receptors like VEGFR or EGFR bypassing corresponding receptor signaling may result in ineffective outcome of therapies with monoclonal antibodies (anti-VEGFR or -EGFR) or tyrosine kinase inhibitors with limited target spectrum efficacy.