Stromal DDR2 Promotes Ovarian Cancer Metastasis through Regulation of Metabolism and Secretion of Extracellular Matrix Proteins

Abstract

Ovarian cancer is the leading cause of gynecologic cancer-related deaths. The propensity for metastasis within the peritoneal cavity is a driving factor for the poor outcomes associated with this disease, but there is currently no effective therapy targeting metastasis. In this study, we investigate the contribution of stromal cells to ovarian cancer metastasis and identify normal stromal cell expression of the collagen receptor, discoidin domain receptor 2 (DDR2), that acts to facilitate ovarian cancer metastasis. In vivo, global genetic inactivation of Ddr2 impairs the ability of Ddr2-expressing syngeneic ovarian cancer cells to spread throughout the peritoneal cavity. Specifically, DDR2 expression in mesothelial cells lining the peritoneal cavity facilitates tumor cell attachment and clearance. Subsequently, omentum fibroblast expression of DDR2 promotes tumor cell invasion. Mechanistically, we find DDR2-expressing fibroblasts are more energetically active, such that DDR2 regulates glycolysis through AKT/SNAI1 leading to suppressed fructose-1,6-bisphosphatase and increased hexokinase activity, a key glycolytic enzyme. Upon inhibition of DDR2, we find decreased protein synthesis and secretion. Consequently, when DDR2 is inhibited, there is reduction in secreted extracellular matrix proteins important for metastasis. Specifically, we find that fibroblast DDR2 inhibition leads to decreased secretion of the collagen crosslinker, LOXL2. Adding back LOXL2 to DDR2 deficient fibroblasts rescues the ability of tumor cells to invade. Overall, our results suggest that stromal cell expression of DDR2 is an important mediator of ovarian cancer metastasis.

Implications: DDR2 is highly expressed by stromal cells in ovarian cancer that can mediate metastasis and is a potential therapeutic target in ovarian cancer.

Figure 1.. High DDR2 expression in ovarian cancer stroma correlates with poor survival

(A) Representative images of DDR2 immunohistochemical staining (IHC) of tumor and stroma of ovarian cancer patients living <3 years and >5 years. Images were captured at 15X magnification. (B) DDR2 IHC score in patients who lived <3 years (n=50) compared to patients who lived >5 years (n=61). Data presented as mean ± SD. ****P < 0.0001. (C) Kaplan-Meier survival curves showing differences in overall survival between ovarian cancer patients tumor and/or stromal DDR2 expression. Positive tumor/positive stroma n=25 (red), negative tumor/positive stroma n=11 (purple), positive tumor/negative stroma n=17 (blue), negative tumor/negative stroma n=41 (black).

Figure 2.. Loss of stromal Ddr2 inhibits ovarian cancer metastasis and results in decreased intratumoral collagen deposition

(A) Intraperitoneal tumor spread as quantified by tumor nodules, ascites, and tumor weight in Ddr2 wild type (WT) and Ddr2 knockout (KO) mice (n=12; 6 KO, 6 WT). Graphs depict indicated measures of tumor growth. Data are represented as mean ± SD. * P < 0.05. Representative images of the abdominal cavity taken 43 days after injection of ID8 Trp53^−/−Brca2^−/− cells into Ddr2 WT and Ddr2 KO mice. Arrows indicate tumor deposits on the diaphragm and throughout the small bowel mesentery. (B) Representative trichrome-stained images and quantification of fibrillar collagen of tumors from Ddr2 wild type (WT) or Ddr2 knockout (KO) mice. Data are represented as mean ± SD. **** P < 0.0001. (C) Representative picro-sirius red-stained images and quantification of total collagen of tumors from Ddr2 wild type (WT) or Ddr2 knockout (KO) mice. Data are represented as mean ± SD. ** P < 0.01.

Figure 3.. DDR2 depletion in human mesothelial cells decreases ovarian cancer cell attachment and clearance

(A) Western blot of DDR2 expression in HPMC 141 transfected with siControl and siDDR2. (B) Quantification of ovarian cancer cell (ES2) attachment to human mesothelial cells (HPMC 141) with or without DDR2 expression (siControl or siDDR2). Data presented as mean ± SD from three independent experiments, * P < 0.05. (C) DDR2 expression in HPMC 234 and 226 by western blot. (D) Representative images of clearance by ES2 spheroids (red) in the presence of HPMC 234 (left) or 226 (right) (green). (E) Representative images of clearance by ES2 spheroids (red) in the presence of mesothelial cells (HPMC 261) with siRNA knockdown (siControl or siDDR2) (green). (F) Quantification of clearance relative to spheroid area at t = 0 of mesothelial cells with or without DDR2 expression (siControl or siDDR2) by ES2 cancer cells. Data are presented as mean ± SD from three independent experiments, ** P < 0.01. (G) Representative images of clearance of mesothelial cells (green) by ES2 spheroids (red) in the presence of CR13452 (DDR2 inhibitor) or DMSO (vehicle). (H) Quantification of clearance relative to spheroid area at t = 0 of mesothelial cells treated with CR13452 (DDR2 inhibitor) or DMSO (vehicle). Data are presented as mean ± SD from three independent experiments, *** P < 0.001. (I) Representative images of clearance of mouse mesothelial cells (green) by BPPNM spheroids (red) in the presence of CR13452 (DDR2 inhibitor) or DMSO (vehicle). (J) Quantification of clearance relative to spheroid area at t = 0 of mouse mesothelial cells treated with CR13452 (DDR2 inhibitor) or DMSO (vehicle). Data are presented as mean ± SD from three independent experiments, * P < 0.05.

Figure 4. DDR2 depletion in omental fibroblasts decreases ovarian cancer cell attachment and invasion

(A) Quantitation of ovarian cancer cell (ES2) attachment to omental fibroblasts (OF) with or without DDR2 expression (siControl or siDDR2). Data presented as mean ± SD from three independent experiments, ** P < 0.01. (B) Western blot depicting DDR2 protein expression in OF206 and OF174 with siRNA knockdown (siControl and siDDR2). (C) Matrigel invasion assay of ES2 cells in the presence of OF 206 with siControl, siDDR2#1 and siDDR2#2. Images were taken after 24h at 20x magnification. Graph depicts number of tumor cells that invaded per high power field (hpf). Representative images are below graph. (D) Matrigel invasion assay of ES2 cells in the presence of OF 174 with siControl, siDDR2#1 and siDDR2#2. Images were taken after 24h at 20x magnification. Graph depicts number of tumor cells that invaded per high power field (hpf). Representative images are below graph. (E) Matrigel invasion assay of ES2 cells in the presence of OF 206 and OF 174 treated with either DMSO (vehicle) or CR13452 (DDR2 inhibitor). Images were taken after 24h at 20x magnification. Graphs depict number of tumor cells that invaded per high power field (hpf). Representative images are below graph. Data presented as mean ± SD from three independent experiments. **** P < 0.0001. (F) Matrigel invasion assay of TYK-nu cells in the presence of OF 206 treated with either DMSO (vehicle) or CR13452 (DDR2 inhibitor). Images were taken after 48h at 20x magnification. Graphs depict number of tumor cells that invaded per high power field (hpf). Representative images are below graph.** P < 0.005.

Figure 5.. DDR2 depletion results in marked changes in stromal cell metabolism

(A) Metabolite set enrichment analysis with the top ten altered pathways shown. P-values provided are one-tailed after adjusting for multiple comparisons. (B) Quantification of changes in intracellular metabolites in OF 206 cells with and without DDR2 expression (siControl or siDDR2). The x and y axis represent experimental groups (siControl, dark grey; siDDR2, light grey) and metabolite mass spectrometry peak intensity. Data presented as mean ± SD. * P < 0.05, ** P < 0.01. (C) Quantification of changes in amino acids in OF 206 cells with and without DDR2 expression (siControl or siDDR2). The x and y axis represent experimental groups (siControl, dark grey; siDDR2, light grey) and metabolite mass spectrometry peak intensity. Data presented as mean ± SD. * P < 0.05, ** P < 0.01.

Figure 6.. DDR2 depletion alters glycolysis and protein synthesis

(A) Western blot of indicated proteins in OF 206 transfected with siControl or siDDR2.Note that molecular weights on the Jess Automatic Western differ slightly than traditional western blot. (B) DDR2, SNAI1, fructose-1,6-bisphosphatase 2 (FBP2), HIF-1α and hexokinase 2 (HK2) mRNA levels by quantitative RT-PCR from OF 206 transfected with siControl or siDDR2. ** P < 0.01, *** P < 0.001 and ***** P < 0.00001. (C) Hexokinase activity depicted as representative kinetic measurement (top) and hexokinase specific activity (bottom) in lysates prepared from OF 206 transfected with siControl or siDDR2 using PicoProbe Hexokinase Activity Assay Kit. Data are mean ± SD from two experiments performed in triplicate. *** P < 0.001. (D) Representative images are presented as synthetic bands from Jess capillary-based immunoassay in OF 206 transfected with siControl or siDDR2. Each lane corresponds to an individual capillary electrophoresis protein sample. phospho-mTOR and total mTOR protein levels were measured and quantified (p-mTOR/mTOR) in two independent experiments, data are presented as ratio relative to siControl level. (E) Protein synthesis quantification in OF 206 siControl and siDDR2 using Protein Synthesis Assay Kit. Data presented as percentage relative to control with mean ± SD. Protein synthesis quantification represents experiment performed in triplicate. ** P < 0.01, *** P < 0.001. (F) Quantification of total protein in conditioned media collected from OF 206 siControl and siDDR2. Data are mean ± SD and presented relative to controls from three independent experiments. *** P < 0.001.

Figure 7.. LOXL2 secretion from omental fibroblasts mediates ovarian cancer cell invasion

(A) Invasion assay of ES2 cells (left) or COV362 cells (middle) or TYK-nu (right) in the presence of conditioned media (CM) from OF206 cells with and without DDR2 expression (siControl or siDDR2). (B) Invasion assay of TYK-nu (left) or OVCAR8 (right) cells in the presence of conditioned media (CM) from OF244 cells with and without DDR2 expression (siControl or siDDR2). (C) Invasion assay in the presence of filtered, size-fractionated conditioned media from OF 206 cells with and without DDR2 expression (siControl or siDDR2). (D) Volcano Plot depicting significantly altered proteins from proteomic analysis of conditioned media of OFs with and without DDR2 expression (siControl or siDDR2). (E) Quantitative RT-PCR results of LOXL2 and DDR2 mRNA level in OF 206 siControl and siDDR2. n=3, ** P<0.01, *** P<0.001. (F) Western Blot of LOXL2 expression in SiControl or siDDR2 OF 206 cells. (G) LOX enzyme activity in conditioned media expressed as percentage of activity compared to OF206siControl. Data represent two independent experiments each performed in duplicate. (H) Invasion assay of ES2 cells in the presence of conditioned media (CM) from OF 206 cells treated with siControl, siDDR2, siLOXL2, or siDDR2 and siLOXL2. (I) Invasion assay of ES2 cells (left) and COV362 cells (right) in the presence of conditioned media from OF 206 cells treated with siControl, siControl with recombinant LOXL2 added to conditioned media (CM) or siDDR2 and siDDR2 with recombinant LOXL2 added to conditioned media.