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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 deficiency results in nuclear HSF1 accumulation and prolonged heat-shock response upon exposure to exogenous stress(a) Loss of FBXW7 results in accumulation of nuclear HSF1 during recovery from heat shock. HCT116 WT and FBXW7 KO cells were heat shocked (42°C for 1 h) following recovery for the indicated time. Nuclear fractions were analyzed by immunoblotting as indicated. (b) Loss of FBXW7 results in accumulation of nuclear HSF1 during recovery from proteotoxic stress. HCT116 WT and FBXW7 KO cells were treated with MG132 (1 μM for 10 h) following recovery for 3 h. Nuclear fractions were analyzed by immunoblotting as indicated. (c) FBXW7 KO cells show defective attenuation of the heat-shock response pathway. Heat maps showing fold changes in expression of HSF1 targets comparing recovery (37°C for 2 h) to heat shock (42°C for 1 h) as determined by high-throughput RNA-Seq. The common targets of HSF1 in HCT116 WT and FBXW7 KO cells after heat shock, as revealed by ChIP-Seq analysis, are displayed on the heat map. Well-characterized genes, positively regulated by HSF1, are indicated. Each column represents a biological replicate. (d) Overlap of genes bound by HSF1 in HCT116 WT and FBXW7 KO cells, under basal conditions. (e) Representative ChIP-Seq tracks for common gene loci between WT and KO (HSPD1/E1, HSP90AB1) and unique for KO gene loci (EIF4A2, CCT6A). The scale corresponds to RPM. (f) Read density profile around TSS's of common HSF1 targets in WT and FBXW7 KO cells. (g) HSPD1 and HSP90AB1 mRNA expression in HCT116 WT and FBXW7 KO cells, under basal conditions (P<0.001 for WT versus KO; unpaired t-test). Error bars indicate mean ± SD, and n=3 independent experiments. Uncropped blots are shown in Supplementary Fig. 8.
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Figure 3: FBXW7 deficiency results in nuclear HSF1 accumulation and prolonged heat-shock response upon exposure to exogenous stress(a) Loss of FBXW7 results in accumulation of nuclear HSF1 during recovery from heat shock. HCT116 WT and FBXW7 KO cells were heat shocked (42°C for 1 h) following recovery for the indicated time. Nuclear fractions were analyzed by immunoblotting as indicated. (b) Loss of FBXW7 results in accumulation of nuclear HSF1 during recovery from proteotoxic stress. HCT116 WT and FBXW7 KO cells were treated with MG132 (1 μM for 10 h) following recovery for 3 h. Nuclear fractions were analyzed by immunoblotting as indicated. (c) FBXW7 KO cells show defective attenuation of the heat-shock response pathway. Heat maps showing fold changes in expression of HSF1 targets comparing recovery (37°C for 2 h) to heat shock (42°C for 1 h) as determined by high-throughput RNA-Seq. The common targets of HSF1 in HCT116 WT and FBXW7 KO cells after heat shock, as revealed by ChIP-Seq analysis, are displayed on the heat map. Well-characterized genes, positively regulated by HSF1, are indicated. Each column represents a biological replicate. (d) Overlap of genes bound by HSF1 in HCT116 WT and FBXW7 KO cells, under basal conditions. (e) Representative ChIP-Seq tracks for common gene loci between WT and KO (HSPD1/E1, HSP90AB1) and unique for KO gene loci (EIF4A2, CCT6A). The scale corresponds to RPM. (f) Read density profile around TSS's of common HSF1 targets in WT and FBXW7 KO cells. (g) HSPD1 and HSP90AB1 mRNA expression in HCT116 WT and FBXW7 KO cells, under basal conditions (P<0.001 for WT versus KO; unpaired t-test). Error bars indicate mean ± SD, and n=3 independent experiments. Uncropped blots are shown in Supplementary Fig. 8.

Mentions: To determine whether FBXW7 affects HSF1 protein turnover, we utilized the human colorectal cancer cell line HCT116, an isogenic cell line containing a homozygous deletion (KO) of the FBXW7 gene and its wild type (WT), FBXW7-expressing counterpart38. HSF1 shuttles between the cytoplasm and the nucleus, but upon stress it accumulates in the nucleus19, 39. Subcellular fractionation analysis of HSF1 revealed identical cytoplasmic HSF1 protein levels in WT and FBXW7 KO cells under basal conditions (37°C; Supplementary Fig. 2a). Strikingly, increased baseline HSF1 nuclear levels were observed in FBXW7 KO cells, compared to WT cells (Fig. 3a), in agreement with the nuclear localization of FBXW7α protein. Next, we exposed both cell lines to heat shock and noticed a marked increase of nuclear HSF1 levels. We also observed a rapid reduction of nuclear HSF1 during the recovery period, in WT cells (Fig. 3a). In contrast, we noted a prolonged stabilization of nuclear HSF1 in FBXW7 KO cells (Fig. 3a). HSF1 mRNA levels did not change during heat shock or recovery (Supplementary Fig. 2b). Subsequently, we asked whether loss of FBXW7 affects the clearance of nuclear HSF1, accumulated upon exposure to MG132, a known proteotoxic stressor. We observed reduced degradation of HSF1 in FBXW7 KO cells, compared to WT cells, upon MG132 removal (Fig. 3b). However, we could not detect any significant difference in the cytoplasmic fraction (Supplementary Fig. 2c). Thus, FBXW7 deficiency results in defective clearance of nuclear HSF1 upon exogenous stress removal.


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 deficiency results in nuclear HSF1 accumulation and prolonged heat-shock response upon exposure to exogenous stress(a) Loss of FBXW7 results in accumulation of nuclear HSF1 during recovery from heat shock. HCT116 WT and FBXW7 KO cells were heat shocked (42°C for 1 h) following recovery for the indicated time. Nuclear fractions were analyzed by immunoblotting as indicated. (b) Loss of FBXW7 results in accumulation of nuclear HSF1 during recovery from proteotoxic stress. HCT116 WT and FBXW7 KO cells were treated with MG132 (1 μM for 10 h) following recovery for 3 h. Nuclear fractions were analyzed by immunoblotting as indicated. (c) FBXW7 KO cells show defective attenuation of the heat-shock response pathway. Heat maps showing fold changes in expression of HSF1 targets comparing recovery (37°C for 2 h) to heat shock (42°C for 1 h) as determined by high-throughput RNA-Seq. The common targets of HSF1 in HCT116 WT and FBXW7 KO cells after heat shock, as revealed by ChIP-Seq analysis, are displayed on the heat map. Well-characterized genes, positively regulated by HSF1, are indicated. Each column represents a biological replicate. (d) Overlap of genes bound by HSF1 in HCT116 WT and FBXW7 KO cells, under basal conditions. (e) Representative ChIP-Seq tracks for common gene loci between WT and KO (HSPD1/E1, HSP90AB1) and unique for KO gene loci (EIF4A2, CCT6A). The scale corresponds to RPM. (f) Read density profile around TSS's of common HSF1 targets in WT and FBXW7 KO cells. (g) HSPD1 and HSP90AB1 mRNA expression in HCT116 WT and FBXW7 KO cells, under basal conditions (P<0.001 for WT versus KO; unpaired t-test). Error bars indicate mean ± SD, and n=3 independent experiments. Uncropped blots are shown in Supplementary Fig. 8.
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Figure 3: FBXW7 deficiency results in nuclear HSF1 accumulation and prolonged heat-shock response upon exposure to exogenous stress(a) Loss of FBXW7 results in accumulation of nuclear HSF1 during recovery from heat shock. HCT116 WT and FBXW7 KO cells were heat shocked (42°C for 1 h) following recovery for the indicated time. Nuclear fractions were analyzed by immunoblotting as indicated. (b) Loss of FBXW7 results in accumulation of nuclear HSF1 during recovery from proteotoxic stress. HCT116 WT and FBXW7 KO cells were treated with MG132 (1 μM for 10 h) following recovery for 3 h. Nuclear fractions were analyzed by immunoblotting as indicated. (c) FBXW7 KO cells show defective attenuation of the heat-shock response pathway. Heat maps showing fold changes in expression of HSF1 targets comparing recovery (37°C for 2 h) to heat shock (42°C for 1 h) as determined by high-throughput RNA-Seq. The common targets of HSF1 in HCT116 WT and FBXW7 KO cells after heat shock, as revealed by ChIP-Seq analysis, are displayed on the heat map. Well-characterized genes, positively regulated by HSF1, are indicated. Each column represents a biological replicate. (d) Overlap of genes bound by HSF1 in HCT116 WT and FBXW7 KO cells, under basal conditions. (e) Representative ChIP-Seq tracks for common gene loci between WT and KO (HSPD1/E1, HSP90AB1) and unique for KO gene loci (EIF4A2, CCT6A). The scale corresponds to RPM. (f) Read density profile around TSS's of common HSF1 targets in WT and FBXW7 KO cells. (g) HSPD1 and HSP90AB1 mRNA expression in HCT116 WT and FBXW7 KO cells, under basal conditions (P<0.001 for WT versus KO; unpaired t-test). Error bars indicate mean ± SD, and n=3 independent experiments. Uncropped blots are shown in Supplementary Fig. 8.
Mentions: To determine whether FBXW7 affects HSF1 protein turnover, we utilized the human colorectal cancer cell line HCT116, an isogenic cell line containing a homozygous deletion (KO) of the FBXW7 gene and its wild type (WT), FBXW7-expressing counterpart38. HSF1 shuttles between the cytoplasm and the nucleus, but upon stress it accumulates in the nucleus19, 39. Subcellular fractionation analysis of HSF1 revealed identical cytoplasmic HSF1 protein levels in WT and FBXW7 KO cells under basal conditions (37°C; Supplementary Fig. 2a). Strikingly, increased baseline HSF1 nuclear levels were observed in FBXW7 KO cells, compared to WT cells (Fig. 3a), in agreement with the nuclear localization of FBXW7α protein. Next, we exposed both cell lines to heat shock and noticed a marked increase of nuclear HSF1 levels. We also observed a rapid reduction of nuclear HSF1 during the recovery period, in WT cells (Fig. 3a). In contrast, we noted a prolonged stabilization of nuclear HSF1 in FBXW7 KO cells (Fig. 3a). HSF1 mRNA levels did not change during heat shock or recovery (Supplementary Fig. 2b). Subsequently, we asked whether loss of FBXW7 affects the clearance of nuclear HSF1, accumulated upon exposure to MG132, a known proteotoxic stressor. We observed reduced degradation of HSF1 in FBXW7 KO cells, compared to WT cells, upon MG132 removal (Fig. 3b). However, we could not detect any significant difference in the cytoplasmic fraction (Supplementary Fig. 2c). Thus, FBXW7 deficiency results in defective clearance of nuclear HSF1 upon exogenous stress removal.

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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Related in: MedlinePlus