Targeting the secreted RGDKGE collagen fragment reduces PD‑L1 by a proteasome‑dependent mechanism and inhibits tumor growth

Abstract

Structural alterations of collagen impact signaling that helps control tumor progression and the responses to therapeutic intervention. Integrins represent a class of receptors that include members that mediate collagen signaling. However, a strategy of directly targeting integrins to control tumor growth has demonstrated limited activity in the clinical setting. New molecular understanding of integrins have revealed that these receptors can regulate both pro‑ and anti‑tumorigenic functions in a cell type‑dependent manner. Therefore, designing strategies that block pro‑tumorigenic signaling, without impeding anti‑tumorigenic functions, may lead to development of more effective therapies. In the present study, evidence was provided for a novel signaling cascade in which β3‑integrin‑mediated binding to a secreted RGDKGE‑containing collagen fragment stimulates an autocrine‑like signaling pathway that differentially governs the activity of both YAP and (protein kinase‑A) PKA, ultimately leading to alterations in the levels of immune checkpoint molecule PD‑L1 by a proteasome dependent mechanism. Selectively targeting this collagen fragment, reduced nuclear YAP levels, and enhanced PKA and proteasome activity, while also exhibiting significant antitumor activity in vivo. The present findings not only provided new mechanistic insight into a previously unknown autocrine‑like signaling pathway that may provide tumor cells with the ability to regulate PD‑L1, but our findings may also help in the development of more effective strategies to control pro‑tumorigenic β3‑integrin signaling without disrupting its tumor suppressive functions in other cellular compartments.

Keywords: YAP; collagen; extracellular matrix; integrin αvβ3; programmed death‑ligand 1; protein kinase‑A; stroma.

Figure 1.

RGDKGE-containing collagen fragment in distinct tumor types. (A-C) Western blot analysis of LY and serum-free CM for the 16 kDa RGDKGE-containing collagen fragment or loading control tubulin in (A) Β16F10 melanoma, 4T1 mammary carcinoma and LLC; in (B) macrophages (RAW 264.7), endothelial cells (HUVEC), normal human melanocytes and HDF and in (C) Β16F10 melanoma, C32 melanoma, YUMM1.7 melanoma and A375 melanoma cells. (D and E) Western blot analysis of lysates from individual solid (D) 4T1 and (E) Β16F10 tumors growing in mice for the 16 kDa RGDKGE-containing collagen fragment or loading control actin. (F) Western blot analysis of lysates from individual biopsies of malignant human melanoma tumors for the 16 kDa RGDKGE-containing collagen fragment or loading control actin. (G and H) Quantification of (G) Β16F10 and (H) 4T1 cell binding to collagen peptide P2 in the presence of non-specific control antibody (Ab Cont) or anti-RGDKGE antibody (Mab XL313). Data bars represent the mean ± SEM cell binding from triplicate wells. (I) Example of changes in Β16F10 tumor size following treatment of mice with different doses of Mab XL313 or control antibody (Ab Cont) (scale bar, 1 cm). (J) Quantification of the dose-dependent effects of anti-RGDKGE collagen fragment antibody (Mab XL313) or non-specific control antibody (Ab Cont) on the growth Β16F10 tumors in vivo. Data bars indicate the mean ± SEM tumor volumes (n=4–6 per group). P-value represents comparison of each group to antibody control. (K) Example of changes in 4T1 tumor size following treatment of mice with Mab XL313 or control antibody (Ab Cont) (scale bar, 1 cm). (L) Quantification of the effects of anti-RGDKGE collagen fragment antibody (Mab XL313) or non-specific control antibody (Ab Cont) on the growth 4T1 tumors in vivo. Data bars indicate the mean ± SEM tumor volumes (n=7 per group). *P<0.05 vs. control at each time point. LY, whole cell lysates; CM, conditioned medium; LLC, Lewis Lung Carcinoma; HDF, human dermal fibroblasts; Mab, monoclonal antibody.

Figure 2.

Secreted RGDKGE-containing collagen fragment is generated by a cathepsin-dependent process. (A) Western blot analysis of LY and serum-free CM for the 16 kDa RGDKGE-containing collagen fragment or loading control tubulin in non-attached Β16F10 melanoma cells over a time course. (B) Western blot analysis of LY and serum-free CM for the 16 kDa RGDKGE-containing collagen fragment or loading control tubulin in non-attached Β16F10 cells treated with Brefeldin-A. (C) Quantification of the mean ± SEM fold change in Coll-1(α2) mRNA in Coll-1 (α2) specific shRNA transfected and non-targeting control shRNA-transfected Β16F10 cells. (D) Western blot analysis of whole cell lysates for the 16 kDa RGDKGE-containing collagen fragment or loading control actin in non-targeting (Cont shRNA) and Coll-1 (α2) knockdown Β16F10 cells. (E) Western blot analysis of whole cell lysates for the 16 kDa RGDKGE-containing collagen fragment or loading control tubulin in non-attached Β16F10 cells treated with Cath-In or control DMSO. LY, whole cell lysates; CM, conditioned medium; shRNA, shorth hairpin RNA; Cath-In, cathepsin inhibitor. *P<0.05.

Figure 3.

Selective targeting of the secreted RGDKGE-containing collagen fragment reduces PD-L1 levels in tumor cells. (A-D) Western blot analysis of whole cell lysates for PD-L1 or loading control tubulin in non-attached (A) Β16F10, (B) YUMM1.7, (C) C32 and (D) A375 tumor cells treated for 1 h with anti-RGDKGE collagen fragment antibody (Mab XL313) or non-specific control (Ab Cont). (E) Western blot analysis of whole cell lysates for PD-L1 or loading control tubulin in non-attached Β16F10 cells treated with vehicle (Cont) or Bref-A. (F) Western blot analysis of whole cell lysates for PD-L1 or loading control tubulin in non-attached Β16F10 cells treated with vehicle (Cont), Bref-A, Bref-A plus the RGDKGE-containing collagen peptide P2 (Bref-A + P2) or Bref-A plus control peptide (Bref-A + CP). PD-L1, programmed death-ligand 1; Bref-A, Brefeldin-A.

Figure 4.

Reduction of PD-L1 levels in Β16F10 cells by selectively targeting the RGDKGE collagen fragment depends on YAP. (A) Western blot analysis of nuclear extracts for YAP or loading control TBP in Β16F10 cells treated with anti-RGDKGE collagen fragment antibody (Mab XL313) or non-specific control (Ab Cont). (B) Western blot analysis of cell whole cell lysates for YAP or loading control tubulin in control non-targeting shRNA (NT-shRNA) and Yap-shRNA knock down Β16F10 cells. (C) Western blot analysis of whole cell lysates for PD-L1 or loading control tubulin in non-attached control transfected Β16F10 cells (Β16F10-NT) treated with anti-RGDKGE collagen fragment antibody (Mab XL313) or non-specific control (Ab Cont). (D) Western blot analysis of whole cell lysates for PD-L1 or loading control tubulin in non-attached Yap-knock down transfected Β16F10 cells (Β16F10-YAP/K/D) treated with anti-RGDKGE collagen fragment antibody (Mab XL313) or non-specific control (Ab Cont). PD-L1, programmed death-ligand 1; shRNA, short hairpin RNA; Mab, monoclonal antibody.

Figure 5.

Reduction of PD-L1 levels in Β16F10 cells by selectively targeting the RGDKGE collagen fragment is mediated by proteasome. (A) Quantification of the mean ± SEM fold change in PD-L1 mRNA levels in non-attached Β16F10 cells treated for 15 min with non-specific control antibody (Ab Cont) or anti-RGDKGE collagen fragment antibody (Mab XL313) from 3 independent experiments. (B) Western blot analysis of whole cell lysates for PD-L1 or control tubulin in non-attached Β16F10 cells pre-treated with either control buffer DMSO or the proteasome inhibitor MG132, then treated with anti-RGDKGE collagen fragment antibody (Mab XL313) or non-specific control (Ab Cont) for 15 min. (C and D) Quantification of the mean ± SEM fold from 4 independent experiments change in the relative levels of proteasome activity in Β16F10 cells (C) treated with either vehicle control or Bref-A and (D) treated with control peptide CP or the RGDKGE-containing collagen peptide P2. (E) Quantification of the mean ± SEM fold change in the relative levels of proteasome activity in Β16F10 cells treated with either anti-RGDKGE collagen fragment antibody (Mab XL313) or non-specific control (Ab Cont) from 3 independent experiments. (F) Quantification of the mean ± SEM fold change in the levels of proteasome activity in Β16F10 cells treated with anti-β3 integrin antibody (Anti-β3) or non-specific control (Ab Cont) from 3 independent experiments. *P<0.05. PD-L1, programmed death-ligand 1; Bref-A, Brefeldin-A.

Figure 6.

Reduction of PD-L1 levels in Β16F10 cells by selectively targeting the RGDKGE collagen fragment depends on PKA activity. (A) Quantification of the mean ± SEM fold change in the relative levels of PKA activity in non-attached control-transfected Β16F10 cells (Β16F10-NT) or Yap-shRNA knock down Β16F10 cells (Β16F10-YAP/K/D) from 3 independent experiments. (B) Quantification of the mean ± SEM fold change in the relative levels of PKA activity in non-attached Β16F10 cells treated with non-specific control peptide CP or RGDKGE-containing collagen peptide P2 for 15 min from 3 independent experiments. (C) Quantification of the mean ± SEM fold change in the relative levels of PKA activity in non-attached Β16F10 cells treated with non-specific control antibody (Ab Cont) or anti-RGDKGE collagen fragment antibody (Mab XL313) for 15 min from 3 independent experiments. (D) Quantification of the mean ± SEM fold change in the relative levels of PKA activity in non-attached Β16F10 cells treated with either non-specific control antibody (Ab Cont) or anti-β3 integrin antibody (Anti-β3) for 15 min from 3 independent experiments. (E) Western blot analysis of whole cell lysates for PD-L1 or control tubulin in non-attached Β16F10 cells pre-treated with either control buffer DMSO or the PKA inhibitor H89, then treated with anti-RGDKGE collagen fragment antibody (Mab XL313) or non-specific control (Ab Cont) for 15 min. *P<0.05. PD-L1, programmed death-ligand 1; PKA, protein kinase-A; Mab, monoclonal antibody.

Figure 7.

Inhibition of Β16F10 tumor growth by Mab XL313 depends on PD-L1 levels. (A) Example of changes in control non-targeting Β16F10 tumor size following treatment of mice with Mab XL313 or control antibody (Ab Cont) (scale bar, 1 cm). (B) Quantification of the effects of anti-RGDKGE collagen fragment antibody (Mab XL313) or non-specific control antibody (Ab Cont) on the growth of control non-targeting transfected Β16F10 tumor cells (Non-Targeting-Β16F10). Arrow indicates start of treatment with antibodies. Data points indicate the mean ± SEM tumor volumes (n=10–11 per group). P-value indicates comparison to control at each time point. (C) Example of changes in PD-L1-K/D Β16F10 tumor size following treatment of mice with Mab XL313 or control antibody (Ab Cont), (scale bar, 1 cm). (D) Quantification of the effects of anti-RGDKGE collagen fragment antibody (Mab XL313) or non-specific control antibody (Ab Cont) on the growth of Β16F10 tumor cells in which PD-L1 levels have been knocked down (PD-L1-K/D Β16F10). Arrow indicates start of treatment with antibodies. Data points indicate the mean ± SEM tumor volumes (n=10 per group). (E) Working model of a potential mechanism by which selectively targeting the endogenously generated RGDKGE collagen fragment may regulate PD-L1 levels. Blocking the secreted RGDKGE-containing collagen fragment with anti-XL313 Mab inhibits this collagen fragment from binding and signaling through integrin β3, thereby reducing the levels of nuclear YAP and enhancing PKA activity, which contributes to the reduction of PD-L1 by enhancing proteasomal-mediated degradation. *P<0.05 vs. control at each time point. PD-L1, programmed death-ligand 1; Mab, monoclonal antibody; PKA, protein kinase-A.