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 5: Targeted therapy or oncogene knockdown leads to regression of sensitive melanoma and lung adenocarcinoma tumours but accelerates the proliferation and seeding of residual drug-resistant cells in vivoa, FACS analysis of sensitive A375 and vemurafenib-resistant A375R cells expressing TK-GFP-Luciferase (TGL), at tumour implantation and after two weeks at start of therapy (n = 8 tumours) Plots depict representative images. b, Tumour volume of A375 cells treated with vehicle or vemurafenib over time (vehicle, n = 8; vemurafenib, n = 12 tumours). c, Representative sections of A375/A375R-TGL tumours at 0, 1, 3, and 6 days of vemurafenib treatment analysed with immunofluorescence (IF) against GFP. Arrows indicate emerging clusters of GFP+ resistant cells. Scale bar: 2mm. d, Quantification of BrdU incorporation into vemurafenib-resistant A375R-TGL cells in A375/A375R tumours treated with vehicle or vemurafenib for 6 days (vehicle, n = 13 fields of vision (FOV) of 3 tumours; vemurafenib n = 18 FOV of 4 tumours). e, Fold change of photon flux of TGL-expressing A375R cells in A375 tumours or A375R tumours alone treated with vehicle or dabrafenib for 8 days (A375/A375R: vehicle, n = 15; dabrafenib, n = 14; A375R: vehicle, n = 8; dabrafenib, n = 7 tumours). f, Tumour volume of doxycycline-inducible BRAF knockdown A375-i-shBRAF-derived xenograft tumours treated with vehicle or doxycycline over time (vehicle, n = 5; doxycycline, n = 4 tumours). g, Photon flux of TGL-expressing A375R cells mixed in A375-i-shBRAF tumours treated with vehicle or doxycycline (vehicle, n = 10; doxycycline, n = 11 tumours). h, Fold change of photon flux of TGL-expressing vemurafenib-resistant M249R4 tumours treated with vehicle or vemurafenib (n = 16 tumours). i–k. Co-implantation assay of tumours treated with vehicle or corresponding targeted therapy with BLI quantification after 5–8 days. i, Fold change of photon flux of TGL-expressing vemurafenib-resistant YUMM1.7R cells mixed in unlabelled, vemurafenib-sensitive YUMM1.7 tumours or YUMM1.7R tumours alone (YUMM1.7/YUMM1.7R: n = 24; YUMM1.7R: n = 20 tumours ). j, Fold change of photon flux of TGL-expressing, intrinsically vemurafenib resistant B16 cells mixed in vemurafenib-sensitive YUMM1.1 tumours or B16 tumours alone (YUMM1.1/B16: vehicle, n = 12; vemurafenib, n = 16; B16: n = 20 tumours) k, A375R mixed in crizotinib-sensitive H3122 cells or A375R tumours alone (H3122/A375R: vehicle, n = 14; crizotinib, n = 13; A375R: n = 12 tumours). l, Photon flux of tumours established from intrinsically resistant drug resistant cells alone, treated with vehicle, crizotinib or erlotinib (crizotinib resistant PC9, H2030 or erlotinib resistant A375R) (n (from left to right on the graph, in this order) = 12, 12, 7, 12, 16, 16 tumours, respectively). m, Summary table of the model systems and conditions used in vivo. n, On the left, representative IF images of vemurafenib treated, sensitive tumours 7h or 5d after intracardiac injection with A375R-TGL cells; sections stained for GFP (A375R, green), collagen type IV (blood vessels, red), and DAPI (nuclei, blue). On the right, quantification of A375R single cells and cell clusters (≥2 cells) infiltrating an A375 tumour treated with vehicle or vemurafenib after intracardiac injection of A375R cells (GFP+ cells were scored in at least 10 whole sections of at least 4 tumours). Data in b, e–l, n are presented as averages, error bars represent s.e.m., in f, center line is median, whiskers are min to max. P values shown were calculated by a two-tailed Mann-Whitney test (n.s.=not significant).
In order to model therapeutic targeting of heterogeneous tumour cell populations in vivo, we mixed a small percentage of vemurafenib-resistant A375 human melanoma cells (A375R), labelled with a TK-GFP-Luciferase vector (TGL), together with a majority of non-labelled, vemurafenib-sensitive A375 cells, and injected the admixture (A375/A375R, 99.95/0.05%) subcutaneously in mice (Extended Data Fig. 1a). After the tumours were established, we treated the mice with vemurafenib or vehicle, and monitored the growth of resistant cells by bioluminescent imaging (BLI) in vivo (Fig. 1a). While vemurafenib treatment decreased the volume of sensitive tumours (A375 alone) (Extended Data Fig. 1b), the number of admixed resistant cells in regressing tumours (A375/A375R) significantly increased compared to vehicle-treated controls (Fig. 1b). GFP staining confirmed increased numbers of resistant cells in regressing tumours, and EdU or BrdU staining confirmed their increased proliferation rate compared to the vehicle treated controls (Fig. 1c, Extended Data Fig. 1c, d). Tumours comprised of only resistant cells showed no growth difference when treated with vehicle or vemurafenib (Fig. 1d), indicating that the growth advantage of resistant cells in regressing tumours was not caused by direct effects of vemurafenib on cancer or stromal cells.