Release of TGFβig-h3 by gastric myofibroblasts slows tumor growth and is decreased with cancer progression

Abstract

Tumor progression has been linked to changes in the stromal environment. Myofibroblasts are stromal cells that are often increased in tumors but their contribution to cancer progression is not well understood. Here, we show that the secretomes of myofibroblasts derived from gastric cancers [cancer-associated myofibroblasts (CAMs)] differ in a functionally significant manner from those derived from adjacent tissue [adjacent tissue myofibroblasts (ATMs)]. CAMs showed increased rates of migration and proliferation compared with ATMs or normal tissue myofibroblasts (NTMs). Moreover, conditioned medium (CM) from CAMs significantly stimulated migration, invasion and proliferation of gastric cancer cells compared with CM from ATMs or NTMs. Proteomic analysis of myofibroblast secretomes revealed decreased abundance of the extracellular matrix (ECM) adaptor protein like transforming growth factor-β-induced gene-h3 (TGFβig-h3) in CAMs, which was correlated with lymph node involvement and shorter survival. TGFβig-h3 inhibited IGF-II-stimulated migration and proliferation of both cancer cells and myofibroblasts, and suppressed IGF-II activation of p42/44 MAPkinase; TGFβig-h3 knockdown increased IGF-II- and CM-stimulated migration. Furthermore, administration of TGFβig-h3 inhibited myofibroblast-stimulated growth of gastric cancer xenografts. We conclude that stromal cells exert inhibitory as well as stimulatory effects on tumor cells; TGFβig-h3 is a stromal inhibitory factor that is decreased with progression of gastric cancers.

Fig. 1.

Increased migration and proliferation of cultured gastric cancer-associated myofibroblasts. (A) Positive α-SMA (green) and vimentin (red) staining in cultured myofibroblasts (nuclear staining with 4ʹ,6-diamidino-2-phenylindole, blue); top, CAMs; middle, ATMs; bottom, NTMs. (B) Increased migration of CAMs compared with ATMs and NTMs (left) in Boyden chambers, and individual pair-wise comparisons of CAMs versus their corresponding ATMs (right). (C) Increased BrdU labeling (left), but not apoptosis indicated by cleaved caspase-3 staining (right), in CAMs compared with ATMs and NTMs. (D) Shorter G₁ phase in CAMs compared with ATMs. (E) Individual pair-wise comparison of BrdU labeling and cleaved caspase-3 staining in CAMs versus their corresponding ATMs. Horizontal arrows, P < 0.05, ANOVA and n = 10–14.

Fig. 2.

Increased epithelial–mesenchymal transition, migration, invasion and proliferation of AGS cancer cells treated with CM from CAMs compared with ATMs. (A) Example of epithelial–mesenchymal transition characterized by scattering, α-SMA (green) and phalloidin staining (f-actin, red; nuclear staining with 4ʹ,6-diamidino-2-phenylindole, blue) in AGS cells treated with CM from NTMs (top) compared with CAMs (bottom). (B) Quantification of α-SMA-positive AGS cells treated with CM from CAMs compared with ATMs and NTMs; a: P < 0.05 versus control, b: P < 0.05 versus NTM, c: P < 0.05 versus ATM (ANOVA). (C) Comparison of AGS cell migration (left) and invasion (right) in response to CM from paired samples of CAMs and ATMs. (D) ³[H]-thymidine incorporation in AGS cells treated with CM from CAMs compared with their matched ATMs. (E) ³[H]-thymidine incorporation in CAMs from patients with high (pN2-4) versus low or no (pN0-1) lymph node involvement; see Supplementary Methods, available at Carcinogenesis Online for details of the TNM classification. Horizontal arrows, ANOVA or t-test, *P < 0.05 and n = 10–14.

Fig. 3.

Identification of TGFβig-h3 as differentially expressed in CAMs. (A) Representative spectra showing, top, identification of one of the precursor peptides for a typical tryptic fragment (GDELADSALEIFK); middle, identification of fragments of the precursor peptide; bottom, isobaric tagging for relative and absolute quantitation reporter ions for this identification. (B) The sequence of TGFβig-h3 showing in red the coverage of tryptic peptides identified in a representative sample; functional domains of the protein are underlined. (C) Representative western blots of TGFβig-h3 in media of CAMs and ATMs from patients with high (pN2-4) (right side) versus low or no (pN0-1) (left side) lymph node involvement showing depressed abundance in CAMs from the former.

Fig. 4.

Inhibition of myofibroblast and cancer cell proliferation and migration by TGFβig-h3 and stimulation of apoptosis. (A) Concentration-dependent inhibition of IGF-II (100ng/ml)-stimulated myofibroblast and AGS cell migration by TGFβig-h3. (B) TGFβig-h3 (1 µg/ml) inhibition of IGF-II-stimulated myofibroblast, AGS and MKN45 cell proliferation determined by EdU incorporation. (C) TGFβig-h3 increased caspase-3 staining of myofibroblast, AGS and MKN45 cells. (D) TGFβig-h3 increased Bax and Bim in myofibroblasts detected by western blot and decreased Bcl-2. Horizontal arrows, ANOVA or t-test, *P < 0.05 and n = 3.

Fig. 5.

Enhanced migration of myofibroblasts and AGS cells after knockdown of TGFβig-h3. (A) TGFβig-h3 siRNA knockdown (TGFβig-h3-KO) increased IGF-II (100ng/ml)-stimulated myofibroblast migration. (B) CM from TGFβig-h3 siRNA-treated myofibroblasts increased AGS cell migration compared with CM from cells treated with control oligonucleotides. (C) IGF-II-stimulated myofibroblast migration is inhibited by U0126 (UO, 10 µM) and SB202190 (SB, 3 µM) but not LY294002 (LY, 50 µM) and JNK-II (JNK, 50 µM). (D) Representative western blot showing phosphorylation of p42/44 kinase was inhibited by TGFβig-h3. Horizontal arrows, *P < 0.05 and n = 3.

Fig. 6.

Inhibitory effects of TGFβig-h3 in a xenograft model of stromal-stimulated tumor growth. (A) Representative images of α-SMA localization in xenografts and (B) quantification and statistical analysis of tumor volume. Mice were treated with TGFβig-h3 (1 µg per mouse per day) either for the whole duration of the experiment (4 weeks) or after 2 weeks of tumor growth. Treated xenografts were compared with untreated xenografts with and without co-injection of CAMs with MKN45 cells as appropriate. n = 5 per group, *P < 0.05 and Dunnett for multiple comparisons; all data are represented as mean ± standard error of the mean.