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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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Related in: MedlinePlus

The therapy-induced secretome (TIS) includes up-regulated positive regulators and a loss of negative regulators of the PI3K/AKT/mTOR pathway, which is activated in sensitive and resistant cells in vitro and in vivoa, Enriched biological processes and b, inferred drug vulnerabilities as determined by Ingenuity pathway analysis of gene expression data from vemurafenib-resistant A375R cells responding to signals from the reactive tumour microenvironment of a tumour regressing during targeted therapy in vivo (for experimental setup see Fig. 1a and the methods section). c, Left, immunoblotting of phosphorylated AKTS473 and phosphorylated ERK protein levels in A375 cells treated with vehicle or vemurafenib at different time points during the generation of conditioned media (CM). Right, immunoblotting of phosphorylated AKTS473 and phosphorylated ERK protein levels in A375 cells after short-term exposure to CM derived from A375 cells treated with vehicle or vemurafenib. d, Immunoblotting of phosphorylated AKTS473 and phosphorylated FRA1 protein levels in A375-derived xenograft tumours treated with vehicle or vemurafenib for 5 days. Normalized quantification of phospho-AKTS473/tubulin in the bottom panel. e, Immunoblotting of a range of pathways nodes in A375R cells treated with CM-vehicle or CM-vemurafenib, derived from A375 cells, for 15, 30, 60, or 120min. f, Cancer cell-derived IGFBP3 levels (left) and murine stromal IGF1 levels (right) in A375-derived xenograft tumours treated with vehicle or vemurafenib for 5 days as determined by ELISA (n = 4 tumours) g, Cancer cell-derived IGFBP3 levels in CM from indicated melanoma cell lines treated with vehicle or vemurafenib as determined by ELISA (n = 3 technical replicates of at least 2 CM). h, IGFBP3 levels in CM derived from A375 cells expressing control or shRNAs targeting IGFBP3 as determined by ELISA (n = 3 technical replicates). i, Immunoblotting of phosphorylated AKTS473 in A375R cells after incubation with CM of A375 cells expressing control or a short hairpin targeting IGFBP3. j, Phosphorylation status of AKTS473 in A375R cells after incubation for 15 min with CM, IGF1, and IGFBP3 as indicated. k, Bioluminescent signal of A375R-TGL cells 10 days after co-implantation with A375 cells expressing a control hairpin (shCTRL) or a hairpin targeting IGFBP3 (shIGFBP3#1) (n = 10 tumours). P values shown were calculated by a two-tailed Mann-Whitney test. Data are averages, error bars represent s.e.m.
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Figure 11: The therapy-induced secretome (TIS) includes up-regulated positive regulators and a loss of negative regulators of the PI3K/AKT/mTOR pathway, which is activated in sensitive and resistant cells in vitro and in vivoa, Enriched biological processes and b, inferred drug vulnerabilities as determined by Ingenuity pathway analysis of gene expression data from vemurafenib-resistant A375R cells responding to signals from the reactive tumour microenvironment of a tumour regressing during targeted therapy in vivo (for experimental setup see Fig. 1a and the methods section). c, Left, immunoblotting of phosphorylated AKTS473 and phosphorylated ERK protein levels in A375 cells treated with vehicle or vemurafenib at different time points during the generation of conditioned media (CM). Right, immunoblotting of phosphorylated AKTS473 and phosphorylated ERK protein levels in A375 cells after short-term exposure to CM derived from A375 cells treated with vehicle or vemurafenib. d, Immunoblotting of phosphorylated AKTS473 and phosphorylated FRA1 protein levels in A375-derived xenograft tumours treated with vehicle or vemurafenib for 5 days. Normalized quantification of phospho-AKTS473/tubulin in the bottom panel. e, Immunoblotting of a range of pathways nodes in A375R cells treated with CM-vehicle or CM-vemurafenib, derived from A375 cells, for 15, 30, 60, or 120min. f, Cancer cell-derived IGFBP3 levels (left) and murine stromal IGF1 levels (right) in A375-derived xenograft tumours treated with vehicle or vemurafenib for 5 days as determined by ELISA (n = 4 tumours) g, Cancer cell-derived IGFBP3 levels in CM from indicated melanoma cell lines treated with vehicle or vemurafenib as determined by ELISA (n = 3 technical replicates of at least 2 CM). h, IGFBP3 levels in CM derived from A375 cells expressing control or shRNAs targeting IGFBP3 as determined by ELISA (n = 3 technical replicates). i, Immunoblotting of phosphorylated AKTS473 in A375R cells after incubation with CM of A375 cells expressing control or a short hairpin targeting IGFBP3. j, Phosphorylation status of AKTS473 in A375R cells after incubation for 15 min with CM, IGF1, and IGFBP3 as indicated. k, Bioluminescent signal of A375R-TGL cells 10 days after co-implantation with A375 cells expressing a control hairpin (shCTRL) or a hairpin targeting IGFBP3 (shIGFBP3#1) (n = 10 tumours). P values shown were calculated by a two-tailed Mann-Whitney test. Data are averages, error bars represent s.e.m.

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 (TIS) includes up-regulated positive regulators and a loss of negative regulators of the PI3K/AKT/mTOR pathway, which is activated in sensitive and resistant cells in vitro and in vivoa, Enriched biological processes and b, inferred drug vulnerabilities as determined by Ingenuity pathway analysis of gene expression data from vemurafenib-resistant A375R cells responding to signals from the reactive tumour microenvironment of a tumour regressing during targeted therapy in vivo (for experimental setup see Fig. 1a and the methods section). c, Left, immunoblotting of phosphorylated AKTS473 and phosphorylated ERK protein levels in A375 cells treated with vehicle or vemurafenib at different time points during the generation of conditioned media (CM). Right, immunoblotting of phosphorylated AKTS473 and phosphorylated ERK protein levels in A375 cells after short-term exposure to CM derived from A375 cells treated with vehicle or vemurafenib. d, Immunoblotting of phosphorylated AKTS473 and phosphorylated FRA1 protein levels in A375-derived xenograft tumours treated with vehicle or vemurafenib for 5 days. Normalized quantification of phospho-AKTS473/tubulin in the bottom panel. e, Immunoblotting of a range of pathways nodes in A375R cells treated with CM-vehicle or CM-vemurafenib, derived from A375 cells, for 15, 30, 60, or 120min. f, Cancer cell-derived IGFBP3 levels (left) and murine stromal IGF1 levels (right) in A375-derived xenograft tumours treated with vehicle or vemurafenib for 5 days as determined by ELISA (n = 4 tumours) g, Cancer cell-derived IGFBP3 levels in CM from indicated melanoma cell lines treated with vehicle or vemurafenib as determined by ELISA (n = 3 technical replicates of at least 2 CM). h, IGFBP3 levels in CM derived from A375 cells expressing control or shRNAs targeting IGFBP3 as determined by ELISA (n = 3 technical replicates). i, Immunoblotting of phosphorylated AKTS473 in A375R cells after incubation with CM of A375 cells expressing control or a short hairpin targeting IGFBP3. j, Phosphorylation status of AKTS473 in A375R cells after incubation for 15 min with CM, IGF1, and IGFBP3 as indicated. k, Bioluminescent signal of A375R-TGL cells 10 days after co-implantation with A375 cells expressing a control hairpin (shCTRL) or a hairpin targeting IGFBP3 (shIGFBP3#1) (n = 10 tumours). P values shown were calculated by a two-tailed Mann-Whitney test. Data are averages, error bars represent s.e.m.
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Figure 11: The therapy-induced secretome (TIS) includes up-regulated positive regulators and a loss of negative regulators of the PI3K/AKT/mTOR pathway, which is activated in sensitive and resistant cells in vitro and in vivoa, Enriched biological processes and b, inferred drug vulnerabilities as determined by Ingenuity pathway analysis of gene expression data from vemurafenib-resistant A375R cells responding to signals from the reactive tumour microenvironment of a tumour regressing during targeted therapy in vivo (for experimental setup see Fig. 1a and the methods section). c, Left, immunoblotting of phosphorylated AKTS473 and phosphorylated ERK protein levels in A375 cells treated with vehicle or vemurafenib at different time points during the generation of conditioned media (CM). Right, immunoblotting of phosphorylated AKTS473 and phosphorylated ERK protein levels in A375 cells after short-term exposure to CM derived from A375 cells treated with vehicle or vemurafenib. d, Immunoblotting of phosphorylated AKTS473 and phosphorylated FRA1 protein levels in A375-derived xenograft tumours treated with vehicle or vemurafenib for 5 days. Normalized quantification of phospho-AKTS473/tubulin in the bottom panel. e, Immunoblotting of a range of pathways nodes in A375R cells treated with CM-vehicle or CM-vemurafenib, derived from A375 cells, for 15, 30, 60, or 120min. f, Cancer cell-derived IGFBP3 levels (left) and murine stromal IGF1 levels (right) in A375-derived xenograft tumours treated with vehicle or vemurafenib for 5 days as determined by ELISA (n = 4 tumours) g, Cancer cell-derived IGFBP3 levels in CM from indicated melanoma cell lines treated with vehicle or vemurafenib as determined by ELISA (n = 3 technical replicates of at least 2 CM). h, IGFBP3 levels in CM derived from A375 cells expressing control or shRNAs targeting IGFBP3 as determined by ELISA (n = 3 technical replicates). i, Immunoblotting of phosphorylated AKTS473 in A375R cells after incubation with CM of A375 cells expressing control or a short hairpin targeting IGFBP3. j, Phosphorylation status of AKTS473 in A375R cells after incubation for 15 min with CM, IGF1, and IGFBP3 as indicated. k, Bioluminescent signal of A375R-TGL cells 10 days after co-implantation with A375 cells expressing a control hairpin (shCTRL) or a hairpin targeting IGFBP3 (shIGFBP3#1) (n = 10 tumours). P values shown were calculated by a two-tailed Mann-Whitney test. Data are averages, error bars represent s.e.m.
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