Hepatocyte Growth Factor/cMET Pathway Activation Enhances Cancer Hallmarks in Adrenocortical Carcinoma

Abstract

Adrenocortical carcinoma is a rare malignancy with poor prognosis and limited response to chemotherapy. Hepatocyte growth factor (HGF) and its receptor cMET augment cancer growth and resistance to chemotherapy, but their role in adrenocortical carcinoma has not been examined. In this study, we investigated the association between HGF/cMET expression and cancer hallmarks of adrenocortical carcinoma. Transcriptomic and immunohistochemical analyses indicated that increased HGF/cMET expression in human adrenocortical carcinoma samples was positively associated with cancer-related biologic processes, including proliferation and angiogenesis, and negatively correlated with apoptosis. Accordingly, treatment of adrenocortical carcinoma cells with exogenous HGF resulted in increased cell proliferation in vitro and in vivo while short hairpin RNA-mediated knockdown or pharmacologic inhibition of cMET suppressed cell proliferation and tumor growth. Moreover, exposure of cells to mitotane, cisplatin, or radiation rapidly induced pro-cMET expression and was associated with an enrichment of genes (e.g., CYP450 family) related to therapy resistance, further implicating cMET in the anticancer drug response. Together, these data suggest an important role for HGF/cMET signaling in adrenocortical carcinoma growth and resistance to commonly used treatments. Targeting cMET, alone or in combination with other drugs, could provide a breakthrough in the management of this aggressive cancer.

Figure 1

High expression and activation of HGF/cMET signaling in ACC patients. (A) HGF/cMET signaling pathway activation triggers a number of downstream oncogenic signaling cascades, leading to cell proliferation and tumor growth. (B) Representative hematoxylin and eosin (H&E) staining and HGF, cMET and phospho-cMET immunohistochemical analyses of tissue microarray samples from 55 ACC and 15 adrenal adenoma tissue samples (derived from 28 chemotherapy naïve ACCs and 15 patients with adrenal adenomas). (C,D) Immunohistochemical (IHC) analysis results for phosphorylated cMET Y1234/1235 (C) or HGF (D) in ACC tumors (n=55) compared with data for adrenal adenoma samples (n=15). a.u., arbitrary units. (E) Serum HGF concentration levels for ACC patients (n=22) compared to samples obtained from controls (n=7). The error bars represent 95% confidence intervals.

Figure 2

cMET signaling is activated in ACC. (A) Western blot analysis of frozen tumor tissues revealed higher levels of cMET in ACC (n=5) than in adrenocortical adenomas (n=5). (B) Multiplex immunoanalysis analysis of the cMET signaling downstream effectors phospho-STAT3, phospho-ATF2 and phospho-cJUN revealed activation of the cMET signaling in frozen tumor tissues from ACC (n=5) than in adrenocortical adenomas (n=5). The error bars represent 95% confidence intervals.

Figure 3

cMET is associated with enhancement of cancer hallmarks in ACC. (A,) Circos plot of the association between significantly increased biological processes (P<0.05) (see also Supplemental Table 2) and cancer hallmarks (symbols and color-coded labels are indicated on the right) upon MET overexpression. The widths of the connectors represent the absolute values of the Z scores of the biological processes. Bar graphs on the right illustrate the enrichment of some important biological processes. (B,) The left panel shows a heat map of changes in gene expression associated with high MET expression. The right panel shows a Venn diagram of dataset GSE10927 microarray data from pretreatment tumor biopsy samples of ACC patients; representative genes that were significantly up-regulated or down-regulated upon MET overexpression (P≤0.05, log ratio > 0.1) are indicated.

Figure 4

cMET is associated with enhancement of cell proliferation, negative regulation of apoptosis, and drug resistance in ACC. (A) Gene set enrichment analyses of ACC patient dataset GSE10927 for genes involved in cell proliferation (left panel), negative regulation of apoptosis (middle panel) or metabolism of and resistance to cisplatin, etoposide and doxorubicin (right panel). Each bar corresponds to one gene. Gene enrichment scores of all genes in each gene set are listed in Supplemental Tables 4, 5 and 9. (B) Gene set enrichment analyses of dataset GSE49278 showed that high cMET expression is correlated with increased proliferation (upper panel), negative regulation of apoptosis (middle panel) and metabolism of and resistance to cisplatin, etoposide and doxorubicin (bottom panel). Gene enrichment scores of all genes in each gene set are listed in Supplementary Tables 6, 7 and 10. NES, normalized enrichment score.

Figure 5

Increased HGF/cMET signaling is associated with enhanced proliferation and angiogenesis in tumors from ACC patients. (A) Representative images of immunohistochemistry staining of a tissue microarray of samples from two ACC patients showed that high HGF/cMET signaling was accompanied by elevated biomarkers of cellular proliferation (Ki-67) and angiogenesis (CD34) in ACC tumors. (B,C) Pearson correlation analyses indicated an association of HGF/cMET signaling with markers of tumor proliferation (Ki-67) (b) and angiogenesis (CD34) (c). a.u., arbitrary units.

Figure 6

cMET expression is correlated with increased gene expression of enzymes involved in drug metabolism in ACC. (A) NCI-H295R cells were treated with cisplatin (50ng/ml and 500ng/ml), and cMET protein levels were measured by Western blot analysis after 24 h of treatment. (B) NCI-H295R cells were treated with mitotane (5µM and 10µM), and cMET protein levels were measured by Western blot analysis after 24 h of treatment. (C) NCI-H295R cells were treated with radiation (8Gy), and cMET protein levels were measured by Western blot analysis after 0, 1, 3, 6, 12 and 30 h of treatment. (D) Heat map of the genes related to drug metabolism. (E) Pearson correlation analysis of elevated cMET expression and expression of genes related to drug metabolism.

Figure 7

Increased HGF/cMET signaling is associated with enhanced proliferation, tumor growth and reduced apoptosis in ACC. (A) Cell viability measured by MTT assay of NCI-H295R cells cultured at different concentrations of recombinant human HGF for 7 days. (B) Knockdown of MET expression by lentiviral shRNAs decreases ACC cell proliferation. (C) Knockdown of MET expression by lentiviral shRNAs decreases percentages of ACC cells in G2-M or S phase. (D) Cell cycle progression analysis showing the important role of cMET in ACC cell proliferation. (E) Mean tumor volume in mice at different weeks after xenografting of H295R–GFP (green fluorescent protein)–shRNA or H295R-cMET-shRNA cells (5 mice per group). (F) Mean tumor weights in mice 6 weeks after xenografting of H295R-GFP-shRNA or H295R-cMET-shRNA cells (5 mice per group; left panel) and representative images of xenografted tumors harvested from the mice (right panel; scale bars represent 5mm). (G) Mean volumes of tumors formed from xenografted H295R cells at different weeks after treatment of randomized control and cabozantinib-treated mice (6 mice per group). (H) Mean weights of tumors from randomized control and cabozantinib-treated mice after 6 weeks of treatment (6 mice per group; left panel) and representative images of xenografted tumors harvested from the mice (right panel; scale bars represent 5mm) Statistical significance of data in panels f and h was calculated by one-way analysis of variance. The error bars represent 95% confidence intervals; *** P<0.001, and **** P<0.0001.