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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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The therapy-induced secretome in melanoma promotes relapse by activating the AKT pathway in resistant cellsa, Schematic diagram showing the isolation of polysome-associated transcripts from resistant cells by translating ribosome affinity profiling (TRAP) from tumours during treatment. b, Ingenuity upstream regulator analysis of gene expression profiles from A375R cells responding to a regressing tumour microenvironment (5 days of treatment; n = 3 tumours) c, Phosphorylation status of AKTS473 in A375R cells, stimulated for 15 min with various CM, as indicated by immunoblotting. d, Phosphorylation status of AKTS473 in A375R cells after stimulation with positive regulators of the AKT pathway, up-regulated in the melanoma-TIS; ANGPTL7 (5ug/ml, 30 min; up-regulated in A375, Colo800, UACC62), PDGFD (10ng/ml, 10 min; up-regulated in Colo800), EGF (10ng/ml, 10 min; up-regulated in A375), and IGF1 (10ng/ml, 10 min; up-regulated in UACC62). e, Mice bearing A375/A375R-TGL tumours were treated with drugs, growth of A375R cells was followed by BLI (vehicle, n = 14; vemurafenib, n = 16; vemurafenib+BEZ235, n = 16; vemurafenib+MK2206, n = 8 tumours). f, Graphical summary of the findings. Data are presented as average; error bars represent s.e.m. P values shown were calculated using a two-tailed Mann-Whitney test.
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Figure 4: The therapy-induced secretome in melanoma promotes relapse by activating the AKT pathway in resistant cellsa, Schematic diagram showing the isolation of polysome-associated transcripts from resistant cells by translating ribosome affinity profiling (TRAP) from tumours during treatment. b, Ingenuity upstream regulator analysis of gene expression profiles from A375R cells responding to a regressing tumour microenvironment (5 days of treatment; n = 3 tumours) c, Phosphorylation status of AKTS473 in A375R cells, stimulated for 15 min with various CM, as indicated by immunoblotting. d, Phosphorylation status of AKTS473 in A375R cells after stimulation with positive regulators of the AKT pathway, up-regulated in the melanoma-TIS; ANGPTL7 (5ug/ml, 30 min; up-regulated in A375, Colo800, UACC62), PDGFD (10ng/ml, 10 min; up-regulated in Colo800), EGF (10ng/ml, 10 min; up-regulated in A375), and IGF1 (10ng/ml, 10 min; up-regulated in UACC62). e, Mice bearing A375/A375R-TGL tumours were treated with drugs, growth of A375R cells was followed by BLI (vehicle, n = 14; vemurafenib, n = 16; vemurafenib+BEZ235, n = 16; vemurafenib+MK2206, n = 8 tumours). f, Graphical summary of the findings. Data are presented as average; error bars represent s.e.m. P values shown were calculated using a two-tailed Mann-Whitney test.

Mentions: To determine the effect of the reactive secretome on the drug-resistant tumour subpopulation in a regressing tumour, we expressed the ribosomal protein L10a fused with green fluorescent protein (EGFP-RPL10a) in A375R cells, allowing the specific retrieval of transcripts from A375R cells by polysome immunoprecipitation for subsequent RNAseq analysis28 (Fig. 4a). In line with the in vivo phenotype of accelerated growth, the gene expression pattern of resistant cells in the regressing microenvironment was enriched for biological processes involved in cell viability, proliferation, and cell movement (Extended Data Fig. 7a). Pathway analysis of the expression data suggested activation of several pathways including PI3K/AKT, BMP-SMAD and NFkB (Fig. 4b). The hyperactivity of the PI3K/AKT pathway in this context also suggested a potential vulnerability of the cells to PI3K/mTOR inhibitors (Extended Data Fig. 7b). The pathway analysis-based prediction of PI3K/AKT activation was also reflected at the protein level in both resistant and sensitive cells in the presence of CM-vemurafenib in vitro and under vemurafenib treatment in vivo (Fig. 4c, Extended Data Fig. 7c, d). Moreover, PI3K/AKT emerged as the dominant TIS responsive pathway in a targeted immunoblot analysis of survival pathways in vitro (Extended Data Fig. 7e).


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)

The therapy-induced secretome in melanoma promotes relapse by activating the AKT pathway in resistant cellsa, Schematic diagram showing the isolation of polysome-associated transcripts from resistant cells by translating ribosome affinity profiling (TRAP) from tumours during treatment. b, Ingenuity upstream regulator analysis of gene expression profiles from A375R cells responding to a regressing tumour microenvironment (5 days of treatment; n = 3 tumours) c, Phosphorylation status of AKTS473 in A375R cells, stimulated for 15 min with various CM, as indicated by immunoblotting. d, Phosphorylation status of AKTS473 in A375R cells after stimulation with positive regulators of the AKT pathway, up-regulated in the melanoma-TIS; ANGPTL7 (5ug/ml, 30 min; up-regulated in A375, Colo800, UACC62), PDGFD (10ng/ml, 10 min; up-regulated in Colo800), EGF (10ng/ml, 10 min; up-regulated in A375), and IGF1 (10ng/ml, 10 min; up-regulated in UACC62). e, Mice bearing A375/A375R-TGL tumours were treated with drugs, growth of A375R cells was followed by BLI (vehicle, n = 14; vemurafenib, n = 16; vemurafenib+BEZ235, n = 16; vemurafenib+MK2206, n = 8 tumours). f, Graphical summary of the findings. Data are presented as average; error bars represent s.e.m. P values shown were calculated using a two-tailed Mann-Whitney test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4507807&req=5

Figure 4: The therapy-induced secretome in melanoma promotes relapse by activating the AKT pathway in resistant cellsa, Schematic diagram showing the isolation of polysome-associated transcripts from resistant cells by translating ribosome affinity profiling (TRAP) from tumours during treatment. b, Ingenuity upstream regulator analysis of gene expression profiles from A375R cells responding to a regressing tumour microenvironment (5 days of treatment; n = 3 tumours) c, Phosphorylation status of AKTS473 in A375R cells, stimulated for 15 min with various CM, as indicated by immunoblotting. d, Phosphorylation status of AKTS473 in A375R cells after stimulation with positive regulators of the AKT pathway, up-regulated in the melanoma-TIS; ANGPTL7 (5ug/ml, 30 min; up-regulated in A375, Colo800, UACC62), PDGFD (10ng/ml, 10 min; up-regulated in Colo800), EGF (10ng/ml, 10 min; up-regulated in A375), and IGF1 (10ng/ml, 10 min; up-regulated in UACC62). e, Mice bearing A375/A375R-TGL tumours were treated with drugs, growth of A375R cells was followed by BLI (vehicle, n = 14; vemurafenib, n = 16; vemurafenib+BEZ235, n = 16; vemurafenib+MK2206, n = 8 tumours). f, Graphical summary of the findings. Data are presented as average; error bars represent s.e.m. P values shown were calculated using a two-tailed Mann-Whitney test.
Mentions: To determine the effect of the reactive secretome on the drug-resistant tumour subpopulation in a regressing tumour, we expressed the ribosomal protein L10a fused with green fluorescent protein (EGFP-RPL10a) in A375R cells, allowing the specific retrieval of transcripts from A375R cells by polysome immunoprecipitation for subsequent RNAseq analysis28 (Fig. 4a). In line with the in vivo phenotype of accelerated growth, the gene expression pattern of resistant cells in the regressing microenvironment was enriched for biological processes involved in cell viability, proliferation, and cell movement (Extended Data Fig. 7a). Pathway analysis of the expression data suggested activation of several pathways including PI3K/AKT, BMP-SMAD and NFkB (Fig. 4b). The hyperactivity of the PI3K/AKT pathway in this context also suggested a potential vulnerability of the cells to PI3K/mTOR inhibitors (Extended Data Fig. 7b). The pathway analysis-based prediction of PI3K/AKT activation was also reflected at the protein level in both resistant and sensitive cells in the presence of CM-vemurafenib in vitro and under vemurafenib treatment in vivo (Fig. 4c, Extended Data Fig. 7c, d). Moreover, PI3K/AKT emerged as the dominant TIS responsive pathway in a targeted immunoblot analysis of survival pathways in vitro (Extended Data Fig. 7e).

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