Therapy-induced tumour secretomes promote resistance and tumour progression.
The tumour-promoting secretome of melanoma cells treated with the kinase inhibitor vemurafenib is driven by downregulation of the transcription factor FRA1.Dual inhibition of RAF and the PI(3)K/AKT/mTOR intracellular signalling pathways blunted the outgrowth of the drug-resistant cell population in BRAF mutant human melanoma, suggesting this combination therapy as a strategy against tumour relapse.Thus, therapeutic inhibition of oncogenic drivers induces vast secretome changes in drug-sensitive cancer cells, paradoxically establishing a tumour microenvironment that supports the expansion of drug-resistant clones, but is susceptible to combination therapy.
Affiliation: Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA.
Drug resistance invariably limits the clinical efficacy of targeted therapy with kinase inhibitors against cancer. Here we show that targeted therapy with BRAF, ALK or EGFR kinase inhibitors induces a complex network of secreted signals in drug-stressed human and mouse melanoma and human lung adenocarcinoma cells. This therapy-induced secretome stimulates the outgrowth, dissemination and metastasis of drug-resistant cancer cell clones and supports the survival of drug-sensitive cancer cells, contributing to incomplete tumour regression. The tumour-promoting secretome of melanoma cells treated with the kinase inhibitor vemurafenib is driven by downregulation of the transcription factor FRA1. In situ transcriptome analysis of drug-resistant melanoma cells responding to the regressing tumour microenvironment revealed hyperactivation of several signalling pathways, most prominently the AKT pathway. Dual inhibition of RAF and the PI(3)K/AKT/mTOR intracellular signalling pathways blunted the outgrowth of the drug-resistant cell population in BRAF mutant human melanoma, suggesting this combination therapy as a strategy against tumour relapse. Thus, therapeutic inhibition of oncogenic drivers induces vast secretome changes in drug-sensitive cancer cells, paradoxically establishing a tumour microenvironment that supports the expansion of drug-resistant clones, but is susceptible to combination therapy.
- Disease Progression*
- Drug Resistance, Neoplasm/drug effects*
- Lung Neoplasms/drug therapy/metabolism/pathology/secretion*
- Melanoma/drug therapy/metabolism/pathology/secretion*
- Metabolome/drug effects*
- Protein Kinase Inhibitors/pharmacology*/therapeutic use*
- Adenocarcinoma/drug therapy/metabolism/pathology/secretion
- Cell Line, Tumor
- Cell Movement/drug effects
- Cell Proliferation/drug effects
- Cell Survival/drug effects
- Clone Cells/drug effects/pathology
- Down-Regulation/drug effects
- Enzyme Activation/drug effects
- Neoplasm Metastasis/drug therapy/pathology
- Proto-Oncogene Proteins B-raf/antagonists & inhibitors
- Proto-Oncogene Proteins c-akt/metabolism
- Proto-Oncogene Proteins c-fos/deficiency
- Receptor Protein-Tyrosine Kinases/antagonists & inhibitors
- Receptor, Epidermal Growth Factor/antagonists & inhibitors
- Signal Transduction/drug effects
- Tumor Microenvironment/drug effects
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Figure 6: The secretome of vemurafenib-treated melanoma and crizotinib- or erlotinib-treated lung adenocarcinoma cells stimulates the proliferation and migration of drug resistant cells in vitro and occurs prior to apoptosis and senescencea, Quantification of the co-culture assay, depicted in Fig. 2a, 7 days after addition of resistant A375R-TGL cells (n = 4 biological replicates). P values were calculated using a Student’s t-test. b, c, Drug sensitive cells were pre-treated with vehicle or drug (crizotinib or erlotinib) for 48h before 5×102 TGL-expressing, drug-resistant cells were added. Growth was monitored by BLI and quantified 7 days after addition of the resistant cell population, (n = 8 biological replicates), P values were calculated using a Student’s t-test. b, Quantification and representative images of TGL-expressing H2030 cells alone or co-cultured with crizotinib sensitive H3122 cells and treated with vehicle or crizotinib c, Quantification and representative images of TGL-expressing A375R cells alone or co-cultured with erlotinib sensitive HCC827 cells and treated with vehicle or erlotinib. d, Relative number of vemurafenib-resistant LOXR cells after 3 days in the presence of CM derived from A375 and UACC62 cells (n = 3 biological replicates). e, Representative IF for Ki67 in drug-resistant YUMM1.7R cells cultured in CM from YUMM1.7 cells. f, Relative number of vemurafenib-resistant melanoma cells with different, clinically relevant resistance mechanisms after 3 days in the presence of CM derived from A375 cells. SKMEL239#3 expressing the p61 BRAFV600E splice variant, A375 expressing NRASQ61K or the constitutively active MEK variant MEK-DD (n = 5 biological replicates). g, Relative cell number of intrinsically vemurafenib resistant lung adenocarcioma cells (H2030, PC9) or crizotinib and erlotinib resistant melanoma cells (A375R) after 3 days cultured in the presence of CM from vemurafenib-treated melanoma or crizotinib- and erlotinib-treated lung adenocarcinoma (n = 6 in all, except for A375R with HCC827-CM, n = 4 biological replicates). h, Representative image of A375R cells migrated towards A375-derived CM-vehicle or CM-vemurafenib. i, Relative migration towards CM from different sources and different resistant test cells as indicated (n = 10 FOV). P values were calculated using a two-tailed Mann-Whitney test (** p<0.01, **** p<0.0001). j, Representative graph and quantification of real-time migration of A375R cells in the presence of CM derived from A375 cells as measured by the xCELLigence System (n = 4 biological replicates). P-value shown was calculated using a two-tailed Mann-Whitney test. k, Monolayer gap closing assay of A375R cells in the presence of CM derived from A375 cells with representative light microscopy images and quantification of gap closure over time. l, Immunoblotting for cleaved caspase-3 and phosphorylated ERK protein levels in vemurafenib-sensitive melanoma cell lines after 72h of vemurafenib treatment. m, β-galactosidase staining of A375 cells treated with vemurafenib for 72h or 8 days. Data are presented as averages, error bars represent s.e.m.
Tumours consist of a complex microenvironment composed of immune, stromal, and cancer cells21. Soluble mediators from this microenvironment can foster cancer growth and therapy resistance13,14,22–24. Considering that drug-sensitive cancer cells are the main population affected by targeted therapy, we hypothesized that signals derived from sensitive cancer cells in response to kinase inhibitors drive the outgrowth of drug-resistant cells. To test this hypothesis, we established an in vitro co-culture system and monitored the growth of TGL-expressing resistant cells (A375R, H2030) in the absence or presence of sensitive cells treated with kinase inhibitors or vehicle (Fig. 2a). Mimicking our in vivo findings, co-culture with vemurafenib-, crizotinib-, or erlotinib-treated sensitive cells significantly enhanced the growth of resistant cancer cells (Fig. 2a, Extended Data Fig. 2a–c).