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Therapy-induced tumour secretomes promote resistance and tumour progression.

Obenauf AC, Zou Y, Ji AL, Vanharanta S, Shu W, Shi H, Kong X, Bosenberg MC, Wiesner T, Rosen N, Lo RS, Massagué J - Nature (2015)

Bottom Line: 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.The tumour-promoting secretome of melanoma cells treated with the kinase inhibitor vemurafenib is driven by downregulation of the transcription factor FRA1.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA.

ABSTRACT
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.

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Characterization of cell lines in response to targeted therapya–h, Relative survival of human melanoma cell lines (A375, Colo800, UACC62) (a,c,e), and the murine melanoma cell line YUMM1.7 (g) and corresponding vemurafenib-resistant derivatives (A375R, Colo800R, UACC62R, YUMM1.7R) under increasing concentrations of vemurafenib. Immunoblotting of phosphorylated ERK protein levels in indicated melanoma cell lines in the presence of increasing concentrations of vemurafenib (b,d,f,h). i, Immunoblotting of protein levels of MET, EGFR, BRAF, PDGFRβ, phosphorylated AKT, and phosphorylated ERK in vemurafenib-sensitive and -resistant pairs of human melanoma cell lines (A375, Colo800, UACC62). j, Immunoblotting of phosphorylated ERK and phosphorylated AKTS473 protein levels in HCC827 lung adenocarcinoma cells in the presence of increasing concentrations of erlotinib. k, Immunoblotting of phosphorylated ERK protein levels in H3122 lung adenocarcinoma cells in the presence of increasing concentrations of crizotinib.
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Figure 13: Characterization of cell lines in response to targeted therapya–h, Relative survival of human melanoma cell lines (A375, Colo800, UACC62) (a,c,e), and the murine melanoma cell line YUMM1.7 (g) and corresponding vemurafenib-resistant derivatives (A375R, Colo800R, UACC62R, YUMM1.7R) under increasing concentrations of vemurafenib. Immunoblotting of phosphorylated ERK protein levels in indicated melanoma cell lines in the presence of increasing concentrations of vemurafenib (b,d,f,h). i, Immunoblotting of protein levels of MET, EGFR, BRAF, PDGFRβ, phosphorylated AKT, and phosphorylated ERK in vemurafenib-sensitive and -resistant pairs of human melanoma cell lines (A375, Colo800, UACC62). j, Immunoblotting of phosphorylated ERK and phosphorylated AKTS473 protein levels in HCC827 lung adenocarcinoma cells in the presence of increasing concentrations of erlotinib. k, Immunoblotting of phosphorylated ERK protein levels in H3122 lung adenocarcinoma cells in the presence of increasing concentrations of crizotinib.

Mentions: To generate vemurafenib-resistant melanoma cell lines, vemurafenib-sensitive cell lines were seeded at low density and exposed to 1–3μM vemurafenib (LC-Labs). After approximately 8 weeks of continuous vemurafenib exposure we derived resistant cell clones that were maintained on vemurafenib (1μM vemurafenib for M249R4, Colo800R, LOXR, UACC62R; 2μM vemurafenib for A375R, YUMM 1.7R). The same protocol was performed to generate a crizotinib resistant cell line from H3122 lung adenocarcinoma cells, which were selected and maintained with 300nM crizotinib. Drug-sensitive and resistant melanoma cell lines from A375, Colo800, UACC62 and YUMM 1.7 and the drug sensitive lung adenocarcionoma cell lines H3122 and HCC827 were exposed to increasing doses of vemurafenib and the number of cells was determined after 3 days and pERK levels after 1h of vemurafenib, crizotinib or erlotinib exposure (Extended Data Fig 9a–j). Receptor status was determined by Western blot and showed an increase in EGFR expression levels in all resistant lines examined as well as an increase in MET receptor expression in A375R and UACC62R cells compared to their parental, drug-sensitive cells (Extended Data Fig. 9k).


Therapy-induced tumour secretomes promote resistance and tumour progression.

Obenauf AC, Zou Y, Ji AL, Vanharanta S, Shu W, Shi H, Kong X, Bosenberg MC, Wiesner T, Rosen N, Lo RS, Massagué J - Nature (2015)

Characterization of cell lines in response to targeted therapya–h, Relative survival of human melanoma cell lines (A375, Colo800, UACC62) (a,c,e), and the murine melanoma cell line YUMM1.7 (g) and corresponding vemurafenib-resistant derivatives (A375R, Colo800R, UACC62R, YUMM1.7R) under increasing concentrations of vemurafenib. Immunoblotting of phosphorylated ERK protein levels in indicated melanoma cell lines in the presence of increasing concentrations of vemurafenib (b,d,f,h). i, Immunoblotting of protein levels of MET, EGFR, BRAF, PDGFRβ, phosphorylated AKT, and phosphorylated ERK in vemurafenib-sensitive and -resistant pairs of human melanoma cell lines (A375, Colo800, UACC62). j, Immunoblotting of phosphorylated ERK and phosphorylated AKTS473 protein levels in HCC827 lung adenocarcinoma cells in the presence of increasing concentrations of erlotinib. k, Immunoblotting of phosphorylated ERK protein levels in H3122 lung adenocarcinoma cells in the presence of increasing concentrations of crizotinib.
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Related In: Results  -  Collection

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Figure 13: Characterization of cell lines in response to targeted therapya–h, Relative survival of human melanoma cell lines (A375, Colo800, UACC62) (a,c,e), and the murine melanoma cell line YUMM1.7 (g) and corresponding vemurafenib-resistant derivatives (A375R, Colo800R, UACC62R, YUMM1.7R) under increasing concentrations of vemurafenib. Immunoblotting of phosphorylated ERK protein levels in indicated melanoma cell lines in the presence of increasing concentrations of vemurafenib (b,d,f,h). i, Immunoblotting of protein levels of MET, EGFR, BRAF, PDGFRβ, phosphorylated AKT, and phosphorylated ERK in vemurafenib-sensitive and -resistant pairs of human melanoma cell lines (A375, Colo800, UACC62). j, Immunoblotting of phosphorylated ERK and phosphorylated AKTS473 protein levels in HCC827 lung adenocarcinoma cells in the presence of increasing concentrations of erlotinib. k, Immunoblotting of phosphorylated ERK protein levels in H3122 lung adenocarcinoma cells in the presence of increasing concentrations of crizotinib.
Mentions: To generate vemurafenib-resistant melanoma cell lines, vemurafenib-sensitive cell lines were seeded at low density and exposed to 1–3μM vemurafenib (LC-Labs). After approximately 8 weeks of continuous vemurafenib exposure we derived resistant cell clones that were maintained on vemurafenib (1μM vemurafenib for M249R4, Colo800R, LOXR, UACC62R; 2μM vemurafenib for A375R, YUMM 1.7R). The same protocol was performed to generate a crizotinib resistant cell line from H3122 lung adenocarcinoma cells, which were selected and maintained with 300nM crizotinib. Drug-sensitive and resistant melanoma cell lines from A375, Colo800, UACC62 and YUMM 1.7 and the drug sensitive lung adenocarcionoma cell lines H3122 and HCC827 were exposed to increasing doses of vemurafenib and the number of cells was determined after 3 days and pERK levels after 1h of vemurafenib, crizotinib or erlotinib exposure (Extended Data Fig 9a–j). Receptor status was determined by Western blot and showed an increase in EGFR expression levels in all resistant lines examined as well as an increase in MET receptor expression in A375R and UACC62R cells compared to their parental, drug-sensitive cells (Extended Data Fig. 9k).

Bottom Line: 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.The tumour-promoting secretome of melanoma cells treated with the kinase inhibitor vemurafenib is driven by downregulation of the transcription factor FRA1.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus