Systematic identification of signaling pathways with potential to confer anticancer drug resistance

Abstract

Cancer cells can activate diverse signaling pathways to evade the cytotoxic action of drugs. We created and screened a library of barcoded pathway-activating mutant complementary DNAs to identify those that enhanced the survival of cancer cells in the presence of 13 clinically relevant, targeted therapies. We found that activation of the RAS-MAPK (mitogen-activated protein kinase), Notch1, PI3K (phosphoinositide 3-kinase)-mTOR (mechanistic target of rapamycin), and ER (estrogen receptor) signaling pathways often conferred resistance to this selection of drugs. Activation of the Notch1 pathway promoted acquired resistance to tamoxifen (an ER-targeted therapy) in serially passaged breast cancer xenografts in mice, and treating mice with a γ-secretase inhibitor to inhibit Notch signaling restored tamoxifen sensitivity. Markers of Notch1 activity in tumor tissue correlated with resistance to tamoxifen in breast cancer patients. Similarly, activation of Notch1 signaling promoted acquired resistance to MAPK inhibitors in BRAF(V600E) melanoma cells in culture, and the abundance of Notch1 pathway markers was increased in tumors from a subset of melanoma patients. Thus, Notch1 signaling may be a therapeutic target in some drug-resistant breast cancers and melanomas. Additionally, multiple resistance pathways were activated in melanoma cell lines with intrinsic resistance to MAPK inhibitors, and simultaneous inhibition of these pathways synergistically induced drug sensitivity. These data illustrate the potential for systematic identification of the signaling pathways controlling drug resistance that could inform clinical strategies and drug development for multiple types of cancer. This approach may also be used to advance clinical options in other disease contexts.

Fig. 1. Strategy for manipulating oncogenic signaling pathways

Pathway names are indicated in bold and situated in the cellular context in which they function. The engineered cDNA constructs in each pathway are denoted as either wild-type (WT; black), constitutively active mutants (red), or dominant-negative mutants (blue).

Fig. 2. Global analysis of screening results

(A) Results of each screen color coded according to enrichment score (the relative abundance of each construct in the presence of drug normalized to the same value in the absence of drug). (B) Pathway activating constructs scoring in screens involving UACC-62 BRAF^V600E melanoma cells treated with PLX4720 (RAF inhibitor, red/top), AZD6244 (MEK inhibitor, green/middle), or VX-11E (ERK inhibitor, blue/bottom).

Fig. 3. Notch1 pathway activation confers resistance to targeted therapies in breast cancer, including tamoxifen

(A) GI50 values for indicated breast cancer cell lines expressing Notch1 ICD or luciferase and treated with the indicated drugs. Data are means ± SD from three experiments. (B) qRT-PCR analysis of Notch1 target genes and mesenchymal markers in breast cancer cell lines expressing Notch1 ICD (red) or luciferase (gray). Data are means ± SD from three experiments. (C) Expression of indicated genes in parental MCF-7 and TamR cells. Data are means ± SD from three experiments. (D) Growth of TamR xenografts treated with tamoxifen, RO4929097 (γ-secretase inhibitor), or the combination. Data are means ± SEM from 9–13 mice per group. Exponential growth curve and two-way ANOVA analyses were performed using GraphPad Prism 6. (E) Kaplan-Meier plot depicting survival in breast cancer patients treated with tamoxifen as a function of Notch1 pathway gene expression signature level (first and third tertiles). P-value denotes significance between drug treated patients in high and low Notch1 groups. (F) Expression heatmap of indicated genes in Notch1 low and high groups from patients in part (E). * p < 0.1; ** p < 0.05; *** p < 0.01

Fig. 4. Notch1 pathway activation in BRAF-mutant, MAPK pathway inhibitor-resistant melanoma

(A) GI50 values for indicated melanoma cell lines expressing Notch1 ICD or luciferase and treated with the indicated drugs. Data are means ± SD from three experiments. (B) qRT-PCR analysis of Notch1 target genes and differentiation markers in UACC-62 cells expressing Notch1 ICD (red) or luciferase (gray). Data are means ± SD from three experiments. (C) Notch1-dependent resistance in cell lines with evolved resistance to MAPK inhibitors (left) as identified by resensitization in the presence of Notch1 knockdown (examples, right). Data are means ± SD from three experiments. (D) qRT-PCR analysis of Notch1, Notch1 target genes, and NGFR in Colo679 cells with evolved Notch1-dependent resistance to PLX4720. Data are means ± SD from three experiments. (E) Putative mechanisms of resistance in a cohort of 29 relapsed tumors from BRAF^V600 melanoma patients. “Other” indicates AKT1 mutation (one patient) or IGF-1 overexpression (one patient). Inset shows fold changes in expression levels of indicated genes on relapse in the Notch1 activation group. (F) Putative mechanisms of resistance in a second cohort of seven relapsed tumors from BRAF^V600 melanoma patients. Inset shows fold changes in expression levels of indicated genes on relapse in the Notch1 activation group. Data are means ± SD from three experiments. (G) Analysis of Notch1 nuclear localization by immunohistochemistry in matched pretreatment and relapse samples from Patient 24. Examples of nuclear Notch1 are indicated by arrowheads. *p < 0.1; ** p < 0.05; *** p < 0.01.

Fig. 5. Simultaneous co-inhibition of multiple drug resistance pathways converts intrinsically drug resistant melanoma cell lines and short-term cultures to a drug sensitive state

(A) qRT-PCR analysis of Notch1 target genes and differentiation markers in MAPK inhibitor–sensitive or intrinsically resistant melanoma cell lines. Data presented are means ± SD from three experiments. (B) Effect of shRNA-mediated knockdown of Notch1 on the sensitivity of intrinsically resistant melanoma cell lines to VX-11E. Data are means ± SD from three experiments. (C) VX-11E GI50 values measured in BRAF-mutant melanoma cell lines and short-term cultures treated with a cocktail of resistance pathway inhibitors (blue, black) or vehicle (gray, red). Resistance pathway inhibitors are shNotch1, BEZ-235 (200 nM), and fulvestrant (1 μM). Data are means ± SD from three experiments. (D) Resistance pathway dependencies in drug-resistant cell lines inferred from drug sensitizer assays using all combinations of resistance pathway inhibitors. Data for all GI50 curves are means ± SD from three experiments.