Senescence-associated tissue microenvironment promotes colon cancer formation through the secretory factor GDF15

Abstract

The risk of colorectal cancer (CRC) varies between people, and the cellular mechanisms mediating the differences in risk are largely unknown. Senescence has been implicated as a causative cellular mechanism for many diseases, including cancer, and may affect the risk for CRC. Senescent fibroblasts that accumulate in tissues secondary to aging and oxidative stress have been shown to promote cancer formation via a senescence-associated secretory phenotype (SASP). In this study, we assessed the role of senescence and the SASP in CRC formation. Using primary human colon tissue, we found an accumulation of senescent fibroblasts in normal tissues from individuals with advanced adenomas or carcinomas in comparison with individuals with no polyps or CRC. In in vitro and ex vivo model systems, we induced senescence using oxidative stress in colon fibroblasts and demonstrated that the senescent fibroblasts secrete GDF15 as an essential SASP factor that promotes cell proliferation, migration, and invasion in colon adenoma and CRC cell lines as well as primary colon organoids via the MAPK and PI3K signaling pathways. In addition, we observed increased mRNA expression of GDF15 in primary normal colon tissue from people at increased risk for CRC in comparison with average risk individuals. These findings implicate the importance of a senescence-associated tissue microenvironment and the secretory factor GDF15 in promoting CRC formation.

Keywords: GDF15; colon organoids; colorectal cancer; microenvironment; senescence.

Figure 1

Increased number of senescent fibroblasts in normal colon from individuals with advanced adenomas or CRC. (a) Representative sample of human colon mucosa subjected to immunofluorescence for γ‐H2A.X and Ki67. Senescent cells were identified by being γ‐H2A.X (green) positive and Ki67 (red) negative. Lower panels are magnified images of the boxed regions. Arrows denote the senescent cells. (Scale bar = 50 µm for upper panel, scale bar = 10 µm for lower panel.) (b) Percentage of senescent fibroblasts in stroma of normal colon mucosa from individuals with no adenomas or CRC (n = 19, median age = 59), advanced adenomas (n = 10, median age = 60), or CRC (n = 20, median age = 59). The statistical significance was determined using a Student's t test. Statistically significant differences are indicated: *p < 0.05, **p < 0.01, ***p < 0.001

Figure 2

Senescent fibroblasts have oncogenic effects on colon epithelial cell lines and colon organoids. (a) Senescent CCD‐18Co enhance cell proliferation in colorectal adenoma cell lines (LT97 and AA/C1) and CRC cell lines (Caco‐2 and HT‐29) compared with normal CCD‐18Co fibroblasts (n = 3 independent experiments). (b, c) Senescent CCD‐18Co promote cell proliferation in human colon organoids. Cell proliferation was measured by immunofluorescence staining for Ki67. Representative images of Ki67 staining in colon organoids (b). Quantification of Ki67‐positive cells in colon organoids co‐cultured with senescent CCD‐18Co vs. normal CCD‐18Co (c). All quantification used TissueQuest software and is based on three independent experiments run on 20 organoids. (d, e) Senescent CCD‐18Co significantly promoted cell migration (d) and cell invasion (e) in the LT97, AA/C1, Caco‐2, and HT‐29 cell lines (n = 3 independent experiments). Statistical significance was determined using a Student's t test. Statistically significant differences are indicated: *p < 0.05, **p < 0.01, ***p < 0.001

Figure 3

Identification and validation of GDF15 as a component of the SASP in senescent colon fibroblasts. (a) Representative heat map of microarray analysis of senescent fibroblasts. The most highly expressed genes in the senescent colon fibroblast cell line CCD‐18Co (left column) and primary colon fibroblast s1005395 (right column) are shown. GDF15 was up‐regulated in both fibroblast lines after the induction of senescence. (b) The mRNA expression of GDF15 was confirmed to be increased in senescent CCD‐18Co (left panel) and senescent s1005395 (right panel) using RT–PCR (n = 3 independent experiments). (c) Western blot analysis of GDF15 expression shows GDF15 was highly induced in senescent fibroblasts compared to normal fibroblast cells in CCD‐18Co (left panel) and s1005395 (right panel) (N=normal, S=senescent). (d) Secreted GDF15 is increased in the conditioned media of the senescent fibroblast cell lines. ELISAs were run on conditioned medium collected from senescent fibroblasts. Increased GDF15 concentrations compared to the control cell lines for both CCD‐18Co (left panel) and s1005395 (right panel) were observed (n = 3 independent experiments). The statistical significance was assessed using a Student's t test. Statistically significant differences are indicated: *p < 0.05, **p < 0.01, ***p < 0.001

Figure 4

The pro‐oncogenic effects of co‐cultured senescent fibroblasts on colon epithelial cells are abolished by GDF15 knockdown in senescent fibroblast cell lines. (a) The mRNA of GDF15 was reduced in senescent CCD‐18Co (left panel) and s1005395 (right panel) transfected with shGDF15 (shGDF15‐1 and shGDF15‐2) compared to cells transfected with scrambled shRNA and to parental cells (n = 2 independent experiments). (b) The protein expression of GDF15 was decreased in senescent CCD‐18Co (left panel) and s1005395 (right panel) transfected with shGDF15 compared to cells transfected with scrambled shRNA and to parental cells (S=senescent, N=normal). (c–e) Cell proliferation (c), cell migration (d), and cell invasion (e) were suppressed in colon epithelial cells (including LT97, AA/C1, Caco‐2, and HT‐29) co‐cultured with senescent CCD‐18Co with shGDF15 knockdown (n = 2–3 independent experiments). (f–h) Cell proliferation (f), migration (g), and invasion (h) abilities were reduced in colon epithelial cells (including LT97, AA/C1, Caco‐2, and HT‐29) co‐cultured with senescent primary fibroblast s1005395 with shGDF15 knockdown (n = 2–3 independent experiments). (i, j) Cell proliferation of colon organoids was repressed when organoids were co‐cultured with senescent fibroblasts with shGDF15 knockdown. Cell proliferation was measured by immunofluorescent staining of Ki67 (red). Representative images of Ki67 staining in colon organoids (i). Quantification of Ki67‐positive cell in colon organoids (j). All quantification is based on two independent experiments run on 20 organoids and was done using TissueQuest software. The statistical significance was determined using a Student's t test. Statistically significant differences are indicated: *p < 0.05, **p < 0.01, ***p < 0.001

Figure 5

Secreted GDF15 from senescent fibroblasts mediates pro‐oncogenic effects on epithelial cells through activating the MAPK and PI3K pathways. (a, b) The phosphorylation of p38, ERK, and AKT was induced in colon epithelial cells (LT97, AA/C1, Caco‐2, and HT‐29) that were co‐cultured with senescent CCD‐18Co(A) or s1005395(B) cells compared to the epithelial cells grown with normal fibroblasts (S=senescent, N=normal). (c, f) The phosphorylation of p38, ERK, and AKT was reduced in colon epithelial cells, including LT97(C), AA/C1(D), Caco‐2(E), and HT‐29(F) when they were co‐cultured with senescent fibroblast cell line CCD‐18Co after shGDF15 knockdown (S=senescent, N=normal). In all experiments, colon epithelial cell lines and colon fibroblasts were co‐cultured for three days. (g) Human recombinant GDF15 (rhGDF15) induced the phosphorylation of p38, ERK, and AKT in all four colon epithelial cells (LT97, AA/C1, Caco‐2, and HT‐29). Colon epithelial cell lines were treated with rhGDF15 (20 ng/ml) for three days. The medium with rhGDF15 was refreshed every day. DPBS (0.1%) was used as a vehicle control treatment. (h–j) Cell proliferation (h), migration (i), and invasion (j) were reduced in colon epithelial cells (LT97, AA/C1, Caco‐2, and HT‐29) treated with U0126 (ERK inhibitor), SB203580 (p38 inhibitor), and LY294002 (AKT inhibitor). In cell proliferation, migration, and invasion assay, colon epithelial cell lines and senescent fibroblasts were co‐cultured in the presence of inhibitors, including U0126 (5 µM), SB203580 (10 µM), and LY294002 (10 µM). DMSO (0.1%) served as the vehicle control treatment (n = 2 independent experiments). The statistical significance was determined using a Student's t test. Statistically significant differences are indicated: *p < 0.05, **p < 0.01, ***p < 0.001

Figure 6

The expression of GDF15 in the colon of patients with no adenomas or CRC, with advanced adenoma(s), and with CRC. As measured by RT–PCR, the mRNA expression of GDF15 was elevated in the normal colon mucosa from subjects with advanced adenoma(s) (n = 11) or CRC (n = 22) in comparison with subjects with no polyps or CRC (n = 11). The clinical characteristics of the study subjects are provided in Table S2 . Statistically significant differences are indicated: *p < 0.05, **p < 0.01, ***p < 0.001