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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 secretome of melanoma cells with FRA1 knockdown stimulates proliferation and migration of A375R cells in vitro and in vivoa, Immunoblotting of phosphorylated and total FRA1 protein levels in A375 cells transduced with control shRNA, with or without additional vemurafenib treatment, or shRNAs targeting FRA1. b, Photon flux and representative BLI images of TGL-expressing A375R cells co-cultured with A375 cells expressing control shRNA (with or without vemurafenib treatment) or FRA1-targeting shRNAs after 7 days (n = 9 biological replicates). c, Relative number of A375R cells after 3 days in the presence of CM derived from A375 cells transduced with control shRNA, with or without additional vemurafenib treatment, or FRA1 shRNAs (n = 3 biological replicates). d, Migration of A375R cells towards CM derived from A375 cells transduced with control shRNA (with or without vemurafenib treatment) or FRA1 shRNAs using a Boyden Chamber Assay (shCTRL, n = 15; all other groups n = 10 FOV) e, Relative mRNA levels of selected secreted factors and TF’s of A375 cells expressing control (shCTRL) or a hairpin targeting FRA1 (shFRA1#1), treated with vehicle or vemurafenib (24h). f, Bioluminescent signal of A375R-TGL cells 8 days after subcutaneous co-implantation with UACC62 cells expressing a control or a short hairpin for FRA1 (shCTRL, n = 12; shFRA1, n = 20 tumours). Data are averages, error bars represent s.e.m. P values were calculated by Student’s t-test, ***p<0.01, ***p<0.001, ****p<0.0001.
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Figure 10: The secretome of melanoma cells with FRA1 knockdown stimulates proliferation and migration of A375R cells in vitro and in vivoa, Immunoblotting of phosphorylated and total FRA1 protein levels in A375 cells transduced with control shRNA, with or without additional vemurafenib treatment, or shRNAs targeting FRA1. b, Photon flux and representative BLI images of TGL-expressing A375R cells co-cultured with A375 cells expressing control shRNA (with or without vemurafenib treatment) or FRA1-targeting shRNAs after 7 days (n = 9 biological replicates). c, Relative number of A375R cells after 3 days in the presence of CM derived from A375 cells transduced with control shRNA, with or without additional vemurafenib treatment, or FRA1 shRNAs (n = 3 biological replicates). d, Migration of A375R cells towards CM derived from A375 cells transduced with control shRNA (with or without vemurafenib treatment) or FRA1 shRNAs using a Boyden Chamber Assay (shCTRL, n = 15; all other groups n = 10 FOV) e, Relative mRNA levels of selected secreted factors and TF’s of A375 cells expressing control (shCTRL) or a hairpin targeting FRA1 (shFRA1#1), treated with vehicle or vemurafenib (24h). f, Bioluminescent signal of A375R-TGL cells 8 days after subcutaneous co-implantation with UACC62 cells expressing a control or a short hairpin for FRA1 (shCTRL, n = 12; shFRA1, n = 20 tumours). Data are averages, error bars represent s.e.m. P values were calculated by Student’s t-test, ***p<0.01, ***p<0.001, ****p<0.0001.

Mentions: In order to test the functional role of FRA1 in modulating the TIS, we used RNAi to inhibit FRA1 expression. Co-culture and conditioned media assays using A375-shFRA1 cells showed similar growth accelerating and chemotactic activity on A375R cells as vemurafenib treatment (Extended Data Fig. 6a–d). In line with these results, FRA1 knockdown in A375 cells induced transcriptional changes similar to those induced by vemurafenib (Extended Data Fig. 6e). A375R cells co-implanted with A375-shFRA1 or UACC62-shFRA1 cells also demonstrated increased growth in vivo (Fig. 3f, Extended Data Fig. 6f). A375-shFRA1 tumours attracted significantly more resistant cells from the circulation than tumours expressing the control vector (Fig. 3g). Thus, FRA1 down-regulation drives the induction of the tumour-promoting secretome of vemurafenib-treated cancer cells.


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 secretome of melanoma cells with FRA1 knockdown stimulates proliferation and migration of A375R cells in vitro and in vivoa, Immunoblotting of phosphorylated and total FRA1 protein levels in A375 cells transduced with control shRNA, with or without additional vemurafenib treatment, or shRNAs targeting FRA1. b, Photon flux and representative BLI images of TGL-expressing A375R cells co-cultured with A375 cells expressing control shRNA (with or without vemurafenib treatment) or FRA1-targeting shRNAs after 7 days (n = 9 biological replicates). c, Relative number of A375R cells after 3 days in the presence of CM derived from A375 cells transduced with control shRNA, with or without additional vemurafenib treatment, or FRA1 shRNAs (n = 3 biological replicates). d, Migration of A375R cells towards CM derived from A375 cells transduced with control shRNA (with or without vemurafenib treatment) or FRA1 shRNAs using a Boyden Chamber Assay (shCTRL, n = 15; all other groups n = 10 FOV) e, Relative mRNA levels of selected secreted factors and TF’s of A375 cells expressing control (shCTRL) or a hairpin targeting FRA1 (shFRA1#1), treated with vehicle or vemurafenib (24h). f, Bioluminescent signal of A375R-TGL cells 8 days after subcutaneous co-implantation with UACC62 cells expressing a control or a short hairpin for FRA1 (shCTRL, n = 12; shFRA1, n = 20 tumours). Data are averages, error bars represent s.e.m. P values were calculated by Student’s t-test, ***p<0.01, ***p<0.001, ****p<0.0001.
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Related In: Results  -  Collection

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Figure 10: The secretome of melanoma cells with FRA1 knockdown stimulates proliferation and migration of A375R cells in vitro and in vivoa, Immunoblotting of phosphorylated and total FRA1 protein levels in A375 cells transduced with control shRNA, with or without additional vemurafenib treatment, or shRNAs targeting FRA1. b, Photon flux and representative BLI images of TGL-expressing A375R cells co-cultured with A375 cells expressing control shRNA (with or without vemurafenib treatment) or FRA1-targeting shRNAs after 7 days (n = 9 biological replicates). c, Relative number of A375R cells after 3 days in the presence of CM derived from A375 cells transduced with control shRNA, with or without additional vemurafenib treatment, or FRA1 shRNAs (n = 3 biological replicates). d, Migration of A375R cells towards CM derived from A375 cells transduced with control shRNA (with or without vemurafenib treatment) or FRA1 shRNAs using a Boyden Chamber Assay (shCTRL, n = 15; all other groups n = 10 FOV) e, Relative mRNA levels of selected secreted factors and TF’s of A375 cells expressing control (shCTRL) or a hairpin targeting FRA1 (shFRA1#1), treated with vehicle or vemurafenib (24h). f, Bioluminescent signal of A375R-TGL cells 8 days after subcutaneous co-implantation with UACC62 cells expressing a control or a short hairpin for FRA1 (shCTRL, n = 12; shFRA1, n = 20 tumours). Data are averages, error bars represent s.e.m. P values were calculated by Student’s t-test, ***p<0.01, ***p<0.001, ****p<0.0001.
Mentions: In order to test the functional role of FRA1 in modulating the TIS, we used RNAi to inhibit FRA1 expression. Co-culture and conditioned media assays using A375-shFRA1 cells showed similar growth accelerating and chemotactic activity on A375R cells as vemurafenib treatment (Extended Data Fig. 6a–d). In line with these results, FRA1 knockdown in A375 cells induced transcriptional changes similar to those induced by vemurafenib (Extended Data Fig. 6e). A375R cells co-implanted with A375-shFRA1 or UACC62-shFRA1 cells also demonstrated increased growth in vivo (Fig. 3f, Extended Data Fig. 6f). A375-shFRA1 tumours attracted significantly more resistant cells from the circulation than tumours expressing the control vector (Fig. 3g). Thus, FRA1 down-regulation drives the induction of the tumour-promoting secretome of vemurafenib-treated cancer cells.

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