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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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Dual inhibition of RAF and the AKT/mTOR pathway blunts the effects of the regressing tumour environment on the resistant cell populationa, Relative photon flux and representative BLI images of GFP/luciferase expressing A375R cells co-cultured with A375 cells and treated with vehicle, vemurafenib, or the combination of vemurafenib and either MK2206 (AKTi, 2μM) or BEZ235 (PI3K/mTORi, 300nM) for 7 days (n = 2–3 biological replicates). b, Relative number of A375R cells after 3 days in the presence of CM-vehicle or CM-vemurafenib with additional BEZ235 (300nM) (n = 3 biological replicates). c, Mice bearing tumours consisting of A375/A375R cells or A375R cells alone were treated with drugs as indicated. Bioluminescent signal of TGL-expressing A375R cells was determined on day 5 of treatment (n = 16, 16, 12, 12, 12, 16 tumours, respectively). d, Mice bearing tumours consisting of unlabelled A375 cells were pre-treated for 3 days with drugs as indicated and 1×105 TGL-expressing A375R cells were inoculated in the arterial circulation. Drug treatment was continued and seeding of resistant cells to the primary tumour was quantified by BLI. Representative BLI images on the right (vehicle, n = 4; vemurafenib n = 10, vemurafenib+BEZ235, n = 10 tumours). P values were calculated by a two-tailed Mann-Whitney test. Data are averages, error bars represent s.e.m.
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Figure 12: Dual inhibition of RAF and the AKT/mTOR pathway blunts the effects of the regressing tumour environment on the resistant cell populationa, Relative photon flux and representative BLI images of GFP/luciferase expressing A375R cells co-cultured with A375 cells and treated with vehicle, vemurafenib, or the combination of vemurafenib and either MK2206 (AKTi, 2μM) or BEZ235 (PI3K/mTORi, 300nM) for 7 days (n = 2–3 biological replicates). b, Relative number of A375R cells after 3 days in the presence of CM-vehicle or CM-vemurafenib with additional BEZ235 (300nM) (n = 3 biological replicates). c, Mice bearing tumours consisting of A375/A375R cells or A375R cells alone were treated with drugs as indicated. Bioluminescent signal of TGL-expressing A375R cells was determined on day 5 of treatment (n = 16, 16, 12, 12, 12, 16 tumours, respectively). d, Mice bearing tumours consisting of unlabelled A375 cells were pre-treated for 3 days with drugs as indicated and 1×105 TGL-expressing A375R cells were inoculated in the arterial circulation. Drug treatment was continued and seeding of resistant cells to the primary tumour was quantified by BLI. Representative BLI images on the right (vehicle, n = 4; vemurafenib n = 10, vemurafenib+BEZ235, n = 10 tumours). P values were calculated by a two-tailed Mann-Whitney test. Data are averages, error bars represent s.e.m.

Mentions: To test the role of AKT activation as a mediator of TIS-induced tumour proliferation, we combined vemurafenib with AKT/PI3K/mTOR inhibitors. In co-culture and proliferation experiments using CM, dual inhibition of the MAPK and AKT pathway diminished the growth benefit of the TIS (Extended Data Fig. 8a, b). We then treated mice with A375/A375R or A375R tumours with vemurafenib and AKT (MK2206) or PI3K/mTOR inhibitors (BEZ235). The combined inhibition of MAPK and PI3K/AKT/mTOR pathways significantly blunted the outgrowth of vemurafenib-resistant cells in the A375/A375R tumours (Fig. 4h). The growth inhibition was specific for the amplified proliferation in the regressing tumour microenvironment and had no effects on the growth of resistant cells alone (Extended Data Fig. 8c). Furthermore, the outgrowth of resistant A375R cells in tumour seeding assays was significantly reduced when regressing tumours were co-treated with BEZ235 (Extended Data Fig. 8d). Thus, the TIS induced proliferation is susceptible to therapeutic targeting.


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)

Dual inhibition of RAF and the AKT/mTOR pathway blunts the effects of the regressing tumour environment on the resistant cell populationa, Relative photon flux and representative BLI images of GFP/luciferase expressing A375R cells co-cultured with A375 cells and treated with vehicle, vemurafenib, or the combination of vemurafenib and either MK2206 (AKTi, 2μM) or BEZ235 (PI3K/mTORi, 300nM) for 7 days (n = 2–3 biological replicates). b, Relative number of A375R cells after 3 days in the presence of CM-vehicle or CM-vemurafenib with additional BEZ235 (300nM) (n = 3 biological replicates). c, Mice bearing tumours consisting of A375/A375R cells or A375R cells alone were treated with drugs as indicated. Bioluminescent signal of TGL-expressing A375R cells was determined on day 5 of treatment (n = 16, 16, 12, 12, 12, 16 tumours, respectively). d, Mice bearing tumours consisting of unlabelled A375 cells were pre-treated for 3 days with drugs as indicated and 1×105 TGL-expressing A375R cells were inoculated in the arterial circulation. Drug treatment was continued and seeding of resistant cells to the primary tumour was quantified by BLI. Representative BLI images on the right (vehicle, n = 4; vemurafenib n = 10, vemurafenib+BEZ235, n = 10 tumours). P values were calculated by a two-tailed Mann-Whitney test. Data are averages, error bars represent s.e.m.
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Figure 12: Dual inhibition of RAF and the AKT/mTOR pathway blunts the effects of the regressing tumour environment on the resistant cell populationa, Relative photon flux and representative BLI images of GFP/luciferase expressing A375R cells co-cultured with A375 cells and treated with vehicle, vemurafenib, or the combination of vemurafenib and either MK2206 (AKTi, 2μM) or BEZ235 (PI3K/mTORi, 300nM) for 7 days (n = 2–3 biological replicates). b, Relative number of A375R cells after 3 days in the presence of CM-vehicle or CM-vemurafenib with additional BEZ235 (300nM) (n = 3 biological replicates). c, Mice bearing tumours consisting of A375/A375R cells or A375R cells alone were treated with drugs as indicated. Bioluminescent signal of TGL-expressing A375R cells was determined on day 5 of treatment (n = 16, 16, 12, 12, 12, 16 tumours, respectively). d, Mice bearing tumours consisting of unlabelled A375 cells were pre-treated for 3 days with drugs as indicated and 1×105 TGL-expressing A375R cells were inoculated in the arterial circulation. Drug treatment was continued and seeding of resistant cells to the primary tumour was quantified by BLI. Representative BLI images on the right (vehicle, n = 4; vemurafenib n = 10, vemurafenib+BEZ235, n = 10 tumours). P values were calculated by a two-tailed Mann-Whitney test. Data are averages, error bars represent s.e.m.
Mentions: To test the role of AKT activation as a mediator of TIS-induced tumour proliferation, we combined vemurafenib with AKT/PI3K/mTOR inhibitors. In co-culture and proliferation experiments using CM, dual inhibition of the MAPK and AKT pathway diminished the growth benefit of the TIS (Extended Data Fig. 8a, b). We then treated mice with A375/A375R or A375R tumours with vemurafenib and AKT (MK2206) or PI3K/mTOR inhibitors (BEZ235). The combined inhibition of MAPK and PI3K/AKT/mTOR pathways significantly blunted the outgrowth of vemurafenib-resistant cells in the A375/A375R tumours (Fig. 4h). The growth inhibition was specific for the amplified proliferation in the regressing tumour microenvironment and had no effects on the growth of resistant cells alone (Extended Data Fig. 8c). Furthermore, the outgrowth of resistant A375R cells in tumour seeding assays was significantly reduced when regressing tumours were co-treated with BEZ235 (Extended Data Fig. 8d). Thus, the TIS induced proliferation is susceptible to therapeutic targeting.

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