Carcinoma associated fibroblasts derived from oral squamous cell carcinoma promote lymphangiogenesis via c-Met/PI3K/AKT in vitro

Abstract

Carcinoma-associated fibroblasts (CAFs) are dominant components of the tumor microenvironment (TME) that promote the development, progression and metastasis of cancer. c-Met is a receptor of the hepatocyte growth factor (HGF), which is involved in lymphangiogenesis. Currently, the roles of CAFs during lymphangiogenesis are unknown. It has been hypothesized that CAFs contribute to lymphangiogenesis of oral squamous cell carcinoma (OSCC) via a HGF/c-Met complex. The expression of HGF in OSCC was determined using CAFs derived from OSCC tissue and it was demonstrated that HGF is overexpressed in OSCC-derived CAFs. It was also revealed that c-Met was highly expressed in human lymphatic endothelial cells (HLECs) when co-cultured with CAFs. Furthermore, it was demonstrated that recombinant human HGF significantly enhanced the proliferation, migration, invasion and tube formation of HLECs. By contrast, the inhibition of c-Met expression suppressed the aforementioned biological activities and also downregulated the expression of c-Met, phosphoinositide 3-kinase and phosphorylated protein kinase B. Taken together, these data demonstrate that c-Met is associated with the regulation of lymphangiogenesis. Thus, the results of the present study indicate that c-Met may be a promising novel therapeutic target to treat patients with OSCC.

Keywords: c-Met; carcinoma-associated fibroblasts; hepatocyte growth factor; lymphangiogenesis; oral squamous cell carcinoma.

Figure 1.

Chemiluminescent immunoassay to determine HGF levels in the supernatant of CAFs and NFs from oral squamous cell carcinoma tissues, and western blots analysis of c-Met and p-c-Met expression in HLECs co-cultured with CAFs and NFs. (A) Representative HGF levels from the supernatant of CAFs and NFs. (B) Representative western blots of c-Met and p-c-Met expression in HLECs co-cultured with CAFs and NFs for 24 and 48 h. *** P<0.001. HGF, hepatocyte growth factor; CAFs, carcinoma-associated fibroblasts; NFs, normal fibroblasts; HLECs, human lymphatic endothelial cells.

Figure 2.

rhHGF promotes the proliferation, migration and invasion of HLECs. (A) Cell Counting kit-8 assay for the growth curve of HLECs treated with 1.5625–200 ng/ml rhHGF for 12 or 24 h; (B) Images of migrating and invading cells and (C) the number of migrating and invading HLECs treated with 0, 0.3, 3.0 or 30 ng/ml rhHGF for 24 h. Cells were identified using an inverted phase contrast microscope (magnification, ×100). Results are the representative of three independent experiments. ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. NC after 12 h; **P<0.01 and ***P<0.001 vs. NC after 24 h. rhHGF, recombinant human hepatocyte growth factor; HLECs, human lymphatic endothelial cells; NC, negative control; OD, optical density; HGF, hepatocyte growth factor.

Figure 3.

rhHGF promotes the tube formation of HLECs. (A) Matrigel in vitro HLEC tube formation assay of cells treated with 3.0 or 30 ng/ml rhHGF for 14 h. Images were visualized using an inverted phase contrast microscope (magnification, ×100). (B) Quantification of tube formation assay. The number of tubes was standardized to relative tube formation ability based on the NC group. Assays were conducted independently in triplicate. ***P<0.001. rhHGF, recombinant human hepatocyte growth factor; HLECs, human lymphatic endothelial cells; NC, negative control; HGF, hepatocyte growth factor.

Figure 4.

Inhibition of c-Met downregulates the proliferation, migration, invasion and tube formation of HLECs. (A) Cell Counting kit-8 assay measuring the proliferation of HLECs, treated with and without 400 nM JNJ for 24 h. ***P<0.001. (B) Images of migrating and invading cells and the tube formation assays. Number of (C) migrating and (D) invading cells. (E) Relative tube formation ability of HLECs treated with and without 400 nM JNJ. Images were visualized using an inverted phase contrast microscope (magnification, ×100). All quantitative data were averaged from triple independent results. **P<0.01 and ***P<0.001. HLECs, human lymphatic endothelial cells; NC, negative control; CAFs, carcinoma-associated fibroblasts; OD, optical density; JNJ, JNJ-38877605.

Figure 5.

Inhibition of c-Met suppresses the expression of c-Met, PI3K and AKT in HLECs. (A) Reverse transcription-quantitative polymerase chain reaction measuring levels of c-Met mRNA in HLECs co-cultured with CAFs supplemented with or without 400 nM JNJ for 48 h. (B) Western blot assay of PI3K, p-AKT and T-AKT in HLECs co-cultured with CAFs supplemented with or without 400 nM JNJ for 48 h. These data are representative of three independent experiments. *P<0.05. PI3K, phosphoinositide 3-kinase; HLECs, human lymphatic endothelial cells; CAFs, carcinoma-associated fibroblasts; JNJ, JNJ-38877605; T-AKT, total protein kinase B; p-AKT, phosphorylated protein kinase B.