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FBXW7 modulates cellular stress response and metastatic potential through ​HSF1 post-translational modification.

Kourtis N, Moubarak RS, Aranda-Orgilles B, Lui K, Aydin IT, Trimarchi T, Darvishian F, Salvaggio C, Zhong J, Bhatt K, Chen EI, Celebi JT, Lazaris C, Tsirigos A, Osman I, Hernando E, Aifantis I - Nat. Cell Biol. (2015)

Bottom Line: ​Heat-shock factor 1 (​HSF1) orchestrates the heat-shock response in eukaryotes.Although this pathway has evolved to help cells adapt in the presence of challenging conditions, it is co-opted in cancer to support malignancy.Here we show that the ubiquitin ligase ​FBXW7α interacts with ​HSF1 through a conserved motif phosphorylated by ​GSK3β and ​ERK1. ​FBXW7α ubiquitylates ​HSF1 and loss of ​FBXW7α results in impaired degradation of nuclear ​HSF1 and defective heat-shock response attenuation. ​FBXW7α is either mutated or transcriptionally downregulated in melanoma and ​HSF1 nuclear stabilization correlates with increased metastatic potential and disease progression. ​FBXW7α deficiency and subsequent ​HSF1 accumulation activates an invasion-supportive transcriptional program and enhances the metastatic potential of human melanoma cells.

View Article: PubMed Central - PubMed

ABSTRACT
​Heat-shock factor 1 (​HSF1) orchestrates the heat-shock response in eukaryotes. Although this pathway has evolved to help cells adapt in the presence of challenging conditions, it is co-opted in cancer to support malignancy. However, the mechanisms that regulate ​HSF1 and thus cellular stress response are poorly understood. Here we show that the ubiquitin ligase ​FBXW7α interacts with ​HSF1 through a conserved motif phosphorylated by ​GSK3β and ​ERK1. ​FBXW7α ubiquitylates ​HSF1 and loss of ​FBXW7α results in impaired degradation of nuclear ​HSF1 and defective heat-shock response attenuation. ​FBXW7α is either mutated or transcriptionally downregulated in melanoma and ​HSF1 nuclear stabilization correlates with increased metastatic potential and disease progression. ​FBXW7α deficiency and subsequent ​HSF1 accumulation activates an invasion-supportive transcriptional program and enhances the metastatic potential of human melanoma cells. These findings identify a post-translational mechanism of regulation of the ​HSF1 transcriptional program both in the presence of exogenous stress and in cancer.

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FBXW7 regulates nuclear HSF1 levels and invasion ability in human melanoma(a) FBXW7 deficiency stabilizes nuclear HSF1 in melanoma. Nuclear fractions from wild type (COLO829, SKMEL28, SKMEL5, SKMEL24) and deficient for FBXW7 (WC00125, WM3862, WM39) melanoma cell lines were analyzed by immunoblotting as indicated. (b) FBXW7 knockdown results in nuclear HSF1 accumulation. 501mel cells were treated with non-coding (NC) siRNA or siRNA against FBXW7. Nuclear fractions were analyzed by immunoblotting as indicated. (c) FBXW7 knockdown results in increased HSF1 targets expression in melanoma. DNAJB1 and HSP90AB1 mRNA expression in 501mel cells treated with non-coding (NC) siRNA or siRNA against FBXW7 (P<0.001 for NC siRNA versus FBXW7 siRNA; unpaired t-test, and n=3 independent experiments) (d) 451Lu cells were treated with the BRAF inhibitor Vemurafenib (2 μM, 9 h) and MEK inhibitor Trametinib (50 nM, 9 h). Nuclear fractions were analyzed by immunoblotting as indicated. (e) FBXW7 depletion results in increased invasion in melanoma. 451Lu, SKMEL239 and A375 cells were infected with the indicated shRNA-encoding lentiviruses. One week after transduction, trans-well invasion assay was performed (P<0.05 for 451Lu shLUC versus shFBXW7, P<0.001 for SKMEL239 scrambled versus shFBXW7, P>0.05 for A375 shLUC versus shFBXW7; n= 10 fields per biological replicate; 4 biological replicates; unpaired t-test). Nuclear fractions were analyzed by immunoblotting as indicated. (f) Representative microphotographs of 451Lu and A375 cells after 8 days of infection with the indicated shRNA-encoding lentiviruses (scale bar, 200 μm). Error bars indicate mean ± SD.. Uncropped blots are shown in Supplementary Fig. 8.
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Figure 5: FBXW7 regulates nuclear HSF1 levels and invasion ability in human melanoma(a) FBXW7 deficiency stabilizes nuclear HSF1 in melanoma. Nuclear fractions from wild type (COLO829, SKMEL28, SKMEL5, SKMEL24) and deficient for FBXW7 (WC00125, WM3862, WM39) melanoma cell lines were analyzed by immunoblotting as indicated. (b) FBXW7 knockdown results in nuclear HSF1 accumulation. 501mel cells were treated with non-coding (NC) siRNA or siRNA against FBXW7. Nuclear fractions were analyzed by immunoblotting as indicated. (c) FBXW7 knockdown results in increased HSF1 targets expression in melanoma. DNAJB1 and HSP90AB1 mRNA expression in 501mel cells treated with non-coding (NC) siRNA or siRNA against FBXW7 (P<0.001 for NC siRNA versus FBXW7 siRNA; unpaired t-test, and n=3 independent experiments) (d) 451Lu cells were treated with the BRAF inhibitor Vemurafenib (2 μM, 9 h) and MEK inhibitor Trametinib (50 nM, 9 h). Nuclear fractions were analyzed by immunoblotting as indicated. (e) FBXW7 depletion results in increased invasion in melanoma. 451Lu, SKMEL239 and A375 cells were infected with the indicated shRNA-encoding lentiviruses. One week after transduction, trans-well invasion assay was performed (P<0.05 for 451Lu shLUC versus shFBXW7, P<0.001 for SKMEL239 scrambled versus shFBXW7, P>0.05 for A375 shLUC versus shFBXW7; n= 10 fields per biological replicate; 4 biological replicates; unpaired t-test). Nuclear fractions were analyzed by immunoblotting as indicated. (f) Representative microphotographs of 451Lu and A375 cells after 8 days of infection with the indicated shRNA-encoding lentiviruses (scale bar, 200 μm). Error bars indicate mean ± SD.. Uncropped blots are shown in Supplementary Fig. 8.

Mentions: We next monitored a panel of melanoma cell lines for nuclear HSF1 levels. Notably, melanoma cells deficient for FBXW7 (WM39, WM3862, WC00125) had significantly higher levels of nuclear HSF1, compared to WT cell lines (SKMEL24, SKMEL5, SKMEL28, COLO829; Fig. 5a). Furthermore, FBXW7 siRNA-mediated knockdown resulted in nuclear HSF1 accumulation in 501mel melanoma cell line (Fig. 5b). Interestingly, FBXW7 knockdown did not have any effect on cytoplasmic HSF1 levels (Supplementary Fig. 5a). We reasoned that the increased HSF1 nuclear accumulation upon FBXW7 knockdown might translate to elevated expression of HSF1 gene targets. Indeed, we were able to show increased expression of classical-HSF1 targets upon FBXW7 knockdown (Fig. 5c; Supplementary Fig. 5b).


FBXW7 modulates cellular stress response and metastatic potential through ​HSF1 post-translational modification.

Kourtis N, Moubarak RS, Aranda-Orgilles B, Lui K, Aydin IT, Trimarchi T, Darvishian F, Salvaggio C, Zhong J, Bhatt K, Chen EI, Celebi JT, Lazaris C, Tsirigos A, Osman I, Hernando E, Aifantis I - Nat. Cell Biol. (2015)

FBXW7 regulates nuclear HSF1 levels and invasion ability in human melanoma(a) FBXW7 deficiency stabilizes nuclear HSF1 in melanoma. Nuclear fractions from wild type (COLO829, SKMEL28, SKMEL5, SKMEL24) and deficient for FBXW7 (WC00125, WM3862, WM39) melanoma cell lines were analyzed by immunoblotting as indicated. (b) FBXW7 knockdown results in nuclear HSF1 accumulation. 501mel cells were treated with non-coding (NC) siRNA or siRNA against FBXW7. Nuclear fractions were analyzed by immunoblotting as indicated. (c) FBXW7 knockdown results in increased HSF1 targets expression in melanoma. DNAJB1 and HSP90AB1 mRNA expression in 501mel cells treated with non-coding (NC) siRNA or siRNA against FBXW7 (P<0.001 for NC siRNA versus FBXW7 siRNA; unpaired t-test, and n=3 independent experiments) (d) 451Lu cells were treated with the BRAF inhibitor Vemurafenib (2 μM, 9 h) and MEK inhibitor Trametinib (50 nM, 9 h). Nuclear fractions were analyzed by immunoblotting as indicated. (e) FBXW7 depletion results in increased invasion in melanoma. 451Lu, SKMEL239 and A375 cells were infected with the indicated shRNA-encoding lentiviruses. One week after transduction, trans-well invasion assay was performed (P<0.05 for 451Lu shLUC versus shFBXW7, P<0.001 for SKMEL239 scrambled versus shFBXW7, P>0.05 for A375 shLUC versus shFBXW7; n= 10 fields per biological replicate; 4 biological replicates; unpaired t-test). Nuclear fractions were analyzed by immunoblotting as indicated. (f) Representative microphotographs of 451Lu and A375 cells after 8 days of infection with the indicated shRNA-encoding lentiviruses (scale bar, 200 μm). Error bars indicate mean ± SD.. Uncropped blots are shown in Supplementary Fig. 8.
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Figure 5: FBXW7 regulates nuclear HSF1 levels and invasion ability in human melanoma(a) FBXW7 deficiency stabilizes nuclear HSF1 in melanoma. Nuclear fractions from wild type (COLO829, SKMEL28, SKMEL5, SKMEL24) and deficient for FBXW7 (WC00125, WM3862, WM39) melanoma cell lines were analyzed by immunoblotting as indicated. (b) FBXW7 knockdown results in nuclear HSF1 accumulation. 501mel cells were treated with non-coding (NC) siRNA or siRNA against FBXW7. Nuclear fractions were analyzed by immunoblotting as indicated. (c) FBXW7 knockdown results in increased HSF1 targets expression in melanoma. DNAJB1 and HSP90AB1 mRNA expression in 501mel cells treated with non-coding (NC) siRNA or siRNA against FBXW7 (P<0.001 for NC siRNA versus FBXW7 siRNA; unpaired t-test, and n=3 independent experiments) (d) 451Lu cells were treated with the BRAF inhibitor Vemurafenib (2 μM, 9 h) and MEK inhibitor Trametinib (50 nM, 9 h). Nuclear fractions were analyzed by immunoblotting as indicated. (e) FBXW7 depletion results in increased invasion in melanoma. 451Lu, SKMEL239 and A375 cells were infected with the indicated shRNA-encoding lentiviruses. One week after transduction, trans-well invasion assay was performed (P<0.05 for 451Lu shLUC versus shFBXW7, P<0.001 for SKMEL239 scrambled versus shFBXW7, P>0.05 for A375 shLUC versus shFBXW7; n= 10 fields per biological replicate; 4 biological replicates; unpaired t-test). Nuclear fractions were analyzed by immunoblotting as indicated. (f) Representative microphotographs of 451Lu and A375 cells after 8 days of infection with the indicated shRNA-encoding lentiviruses (scale bar, 200 μm). Error bars indicate mean ± SD.. Uncropped blots are shown in Supplementary Fig. 8.
Mentions: We next monitored a panel of melanoma cell lines for nuclear HSF1 levels. Notably, melanoma cells deficient for FBXW7 (WM39, WM3862, WC00125) had significantly higher levels of nuclear HSF1, compared to WT cell lines (SKMEL24, SKMEL5, SKMEL28, COLO829; Fig. 5a). Furthermore, FBXW7 siRNA-mediated knockdown resulted in nuclear HSF1 accumulation in 501mel melanoma cell line (Fig. 5b). Interestingly, FBXW7 knockdown did not have any effect on cytoplasmic HSF1 levels (Supplementary Fig. 5a). We reasoned that the increased HSF1 nuclear accumulation upon FBXW7 knockdown might translate to elevated expression of HSF1 gene targets. Indeed, we were able to show increased expression of classical-HSF1 targets upon FBXW7 knockdown (Fig. 5c; Supplementary Fig. 5b).

Bottom Line: ​Heat-shock factor 1 (​HSF1) orchestrates the heat-shock response in eukaryotes.Although this pathway has evolved to help cells adapt in the presence of challenging conditions, it is co-opted in cancer to support malignancy.Here we show that the ubiquitin ligase ​FBXW7α interacts with ​HSF1 through a conserved motif phosphorylated by ​GSK3β and ​ERK1. ​FBXW7α ubiquitylates ​HSF1 and loss of ​FBXW7α results in impaired degradation of nuclear ​HSF1 and defective heat-shock response attenuation. ​FBXW7α is either mutated or transcriptionally downregulated in melanoma and ​HSF1 nuclear stabilization correlates with increased metastatic potential and disease progression. ​FBXW7α deficiency and subsequent ​HSF1 accumulation activates an invasion-supportive transcriptional program and enhances the metastatic potential of human melanoma cells.

View Article: PubMed Central - PubMed

ABSTRACT
​Heat-shock factor 1 (​HSF1) orchestrates the heat-shock response in eukaryotes. Although this pathway has evolved to help cells adapt in the presence of challenging conditions, it is co-opted in cancer to support malignancy. However, the mechanisms that regulate ​HSF1 and thus cellular stress response are poorly understood. Here we show that the ubiquitin ligase ​FBXW7α interacts with ​HSF1 through a conserved motif phosphorylated by ​GSK3β and ​ERK1. ​FBXW7α ubiquitylates ​HSF1 and loss of ​FBXW7α results in impaired degradation of nuclear ​HSF1 and defective heat-shock response attenuation. ​FBXW7α is either mutated or transcriptionally downregulated in melanoma and ​HSF1 nuclear stabilization correlates with increased metastatic potential and disease progression. ​FBXW7α deficiency and subsequent ​HSF1 accumulation activates an invasion-supportive transcriptional program and enhances the metastatic potential of human melanoma cells. These findings identify a post-translational mechanism of regulation of the ​HSF1 transcriptional program both in the presence of exogenous stress and in cancer.

Show MeSH
Related in: MedlinePlus