Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion

Abstract

Drug resistance presents a challenge to the treatment of cancer patients. Many studies have focused on cell-autonomous mechanisms of drug resistance. By contrast, we proposed that the tumour micro-environment confers innate resistance to therapy. Here we developed a co-culture system to systematically assay the ability of 23 stromal cell types to influence the innate resistance of 45 cancer cell lines to 35 anticancer drugs. We found that stroma-mediated resistance is common, particularly to targeted agents. We characterized further the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibitors because most patients with this type of cancer show some degree of innate resistance. Proteomic analysis showed that stromal cell secretion of hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-OH kinase (PI(3)K)-AKT signalling pathways, and immediate resistance to RAF inhibition. Immunohistochemistry experiments confirmed stromal cell expression of HGF in patients with BRAF-mutant melanoma and showed a significant correlation between HGF expression by stromal cells and innate resistance to RAF inhibitor treatment. Dual inhibition of RAF and either HGF or MET resulted in reversal of drug resistance, suggesting RAF plus HGF or MET inhibitory combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines. More generally, this study indicates that the systematic dissection of interactions between tumours and their micro-environment can uncover important mechanisms underlying drug resistance.

Figure 1. Effect of stromal cells on chemoresistance of cancer cell lines

a, 45 GFP labeled cancer cell lines were treated with 35 drugs either alone or in co-culture with a panel of up to 23 stromal cell lines and primary cells. The drug effect was calculated by normalizing the number of cells (GFP) after 7 days of treatment to the number of cells (GFP) in DMSO control wells. X-axis represents drug effect in the absence of stromal cells while Y-axis represents drug effect in the presence of stromal cells. The Y axis was also normalized to the effect that each stromal cell type has on cancer cell proliferation when no drug is present (in order to distinguish true rescue from stromal effects on proliferation). The middle diagonal line represents the expected result when stromal cells do not confer resistance. Upper and lower diagonal lines represent one standard deviation from the mid-diagonal line. b, Hierarchical clustering of stromal cells according to their ability to rescue colorectal (CRC) and pancreatic cancer cell lines from 0.1uM gemcitabine. c, Hierarchical clustering of stromal cells according to their ability to rescue melanoma cancer cell lines with V600E BRAF mutation from 2uM PLX4720. d, Hierarchical clustering of stromal cells according to their ability to rescue HER2 amplified breast cancer cell lines from 2uM lapatinib. See Supplementary Figures 1 to 3 for details.

Figure 2. HGF rescues melanoma cancer cell lines from RAF and MEK inhibitors

a, 3 melanoma cell lines were co-cultured with conditioned media from three fibroblast cell lines or with fresh media and treated with 2uM PLX4720. Proliferation was quantified after 7 days and compared to non-treated cells. Bars represent standard error between replicates (n = 3). b, the HGF secretion level of 18 stromal cell lines measured by a protein cytokine array (Supplementary Table 5) is plotted vs. the ability of each stromal cell line to rescue BRAF V600E melanoma cell lines from PLX4720 (Supplementary Fig. 3). c, Effect of HGF (6.25-50ng/ml) on proliferation of melanoma cell lines under PLX4720 or PD184352 treatment. Bars represent standard error between replicates (n = 3). d, Drug resistance manifests only in the presence of HGF-secreting stromal cells, and is reversed by MET inhibitor. Melanoma cell lines were co-cultured with nine stromal cell lines, representing HGF secreting and non-secreting stromal cells and treated with PLX4720 (2uM) or PD184352 (1um) with or without 0.2uM crizotinib. Proliferation was quantified after 7 days and normalized to non-treated cells. Results were averaged across 4 stromal cell lines that secrete HGF and 5 that do not. Non-averaged results are presented in Supplementary Fig. 11. Bars represent standard error between replicates (n = 3). e, 22 cytokines were added to 6 melanoma cell lines that were then treated with 2uM PLX4720 or 1uM PD184352 or DMSO control. Proliferation quantified after 7 days and normalized to No-Cytokine. Results shown are averaged for all cell lines and both drugs. Bars represent standard error between replicates (n = 3).

Figure 3. HGF is present in the stromal cells of melanoma and correlates with poor response to therapy

a, Pre-treatment melanoma section from patient # 32 was analyzed for HGF expression by immunohistochemistry (IHC). Black arrow: normal epidermis. Red arrow: tumor cells. Blue arrow: HGF-expressing stroma (brown staining). Low magnification image shown on the left (scale bar - 200μm) while high magnification image shown on the right (scale bar - 50μm). b, Melanoma sections from patient # 23 analyzed for HGF expression by IHC. On treatment biopsy was obtained 2 weeks after the initiation of treatment with the BRAF inhibitor vemurafenib (PLX4032) and one month after the pre-treatment biopsy was obtained. Third biopsy was obtained 12 months after the initiation of treatment while the patient was progressing under treatment. Low magnification images are shown on top (scale bar - 100μm) while high magnification images are shown on the bottom (scale bar - 50μm). c, Maximal response to treatment of BRAF V600E melanoma patients with or without stromal HGF as measured by IHC. Patients with stromal HGF had a significantly poorer response to treatment compared to those lacking expression (*P < 0.05 by two-sample t-test assuming equal variance). Median values for each group are depicted above the median line.

Figure 4. Characterizing the molecular mechanism of HGF-induced primary resistance

a, Activation of ERK by cytokines. Levels of phosphorylated ERK (T202/Y204) were assayed by immunoblotting 1 hour after treatment with media (-) or with 22 cytokines in the presence of PLX4720 or DMSO (DM) control. b, The activation of AKT by HGF, IGF-1 (IGF), and Insulin (INS). Levels of phosphorylated AKT (S473) were assayed 1 hour and 24 hours after treatment with HGF, IGF-1, or insulin in the presence of PLX4720 (2uM). c, Effect of HGF (25ng/ml) on melanoma cell lines treated with 2uM PLX4720 or 1uM PD184352. MAPK and PI3K/AKT pathways activation was assessed after 24 hours of treatment by immunoblot analysis of pRAF1, pMEK, pERK, pAKT and pMET.