Loss of tumor suppressor NF1 activates HSF1 to promote carcinogenesis

Abstract

Intrinsic stress response pathways are frequently mobilized within tumor cells. The mediators of these adaptive mechanisms and how they contribute to carcinogenesis remain poorly understood. A striking example is heat shock factor 1 (HSF1), master transcriptional regulator of the heat shock response. Surprisingly, we found that loss of the tumor suppressor gene neurofibromatosis type 1 (Nf1) increased HSF1 levels and triggered its activation in mouse embryonic fibroblasts. As a consequence, Nf1-/- cells acquired tolerance to proteotoxic stress. This activation of HSF1 depended on dysregulated MAPK signaling. HSF1, in turn, supported MAPK signaling. In mice, Hsf1 deficiency impeded NF1-associated carcinogenesis by attenuating oncogenic RAS/MAPK signaling. In cell lines from human malignant peripheral nerve sheath tumors (MPNSTs) driven by NF1 loss, HSF1 was overexpressed and activated, which was required for tumor cell viability. In surgical resections of human MPNSTs, HSF1 was overexpressed, translocated to the nucleus, and phosphorylated. These findings reveal a surprising biological consequence of NF1 deficiency: activation of HSF1 and ensuing addiction to this master regulator of the heat shock response. The loss of NF1 function engages an evolutionarily conserved cellular survival mechanism that ultimately impairs survival of the whole organism by facilitating carcinogenesis.

Figure 1. Genetic compromise of NF1 induces the heat shock response.

(A) Whole cell–based screen of a heat shock–oriented shRNA library. Heat shock reporter NIH3T3 cells arrayed in a 384-well format were infected with lentiviral constructs designed to target 175 candidate genes. 5 days later, reporter activation was measured by luciferase assay. The mean Z score calculated from 2 biological replicates per construct is plotted as a measure of activation relative to the entire population of wells assayed. 2 independent Nf1-targeted shRNAs that strongly activate the heat shock reporter are highlighted in bold. (B) Nf1 knockdown increased HSP72 expression in an immortalized MEF cell line. MEFs were stably transduced with shRNA39 and shRNA42, which target different regions of the Nf1 mRNA, and a scrambled control shRNA (Scr). NF1 and HSP72 protein levels were examined by immunoblotting. (C) Nf1 knockdown transcriptionally activates the heat shock response. Transcript levels of chaperones in Nf1-knockdown MEFs were measured by 2-step real-time quantitative RT-PCR technique. Transcript levels relative to cells transduced with scrambled control shRNA for each gene are expressed as fold changes (mean ± SD, n = 3 or 4). *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test.

Figure 2. NF1 loss activates HSF1 via elevated MAPK signaling.

(A and B) Cytoplasmic and nuclear fractions were prepared from immortalized MEFs after overnight treatment with DMSO or 10 μM U0126. Blotting for cytoplasmic lactate dehydrogenase (LDH) and nuclear lamins A/C confirmed appropriate fractionation. (C and D) HSF1 was activated in primary Nf1-knockout MEFs. (E) MEK inhibition impaired HSF1 transcriptional activity. EGFP or HSF1 plasmids were transfected with pHSE–firefly luciferase reporter plasmid and pCMV–renilla luciferase plasmid into HEK293T cells. After 1 day, cells were treated with DMSO or 20 μM U0126 overnight. Firefly luciferase signals were normalized against renilla luciferase signals (mean ± SD; n = 6). (F) Dominant-negative MEK1 impaired p-Ser326. EGFP or MEK1^AA plasmid was transfected into HEK293T cells; after 3 days, cells were harvested for immunoblotting. (G) Dominant-negative MEK1 impaired HSF1 transcriptional activity. In HEK293T cells, EGFP or MEK1^AA plasmid was transfected with the luciferase reporter plasmids. HSF1 plasmid was further cotransfected with either EGFP or MEK1^AA plasmid. 3 days after transfection, luciferase signals were measured (mean ± SD; n = 4). (H) Proteasomal inhibition caused HSF1 protein accumulation. MEFs were treated with DMSO or MG132 overnight. (I) HSF1 polyubiquitination was suppressed in Nf1-knockdown cells and reestablished after MEK inhibition. MEFs were treated with either DMSO or 20 μM U0126 overnight, and whole cell lysates were immunoprecipitated for HSF1. Normal rat IgG served as the control. Precipitates were immunoblotted for polyubiquitinated conjugates and HSF1. LC, light chain; WLC, whole cell lysates. P values were determined by Student’s t test.

Figure 3. HSF1 activation by Nf1 knockout renders cells resistant to proteotoxic stress.

Primary MEFs of the indicated genotypes were plated at low density (2,000 cells/well) and exposed for 5 days to the indicated concentrations of (A) MG132, (B) radicicol, and (C) withaferin A. Resazurin dye reduction was assayed as a measure of relative viable cell number. The mean of triplicate determinations repeated in 2 separate experiments is presented (mean ± SD). ***P < 0.01, 2-way ANOVA.

Figure 4. Genetic compromise of Hsf1 prolongs survival in a mouse model of NF1.

(A) Tumor occurrence was suppressed in NPcis mice when Hsf1 was disrupted. Tumor-free survival in relationship to Hsf1 genotype is plotted for NPcis^+/+ and NPcis^+/– mice. Median survival for NPcis mice was as follows: Hsf1^+/+, 22 weeks (n = 24); Hsf1^+/–, 34 weeks (n = 30; P = 0.0002 vs. Hsf1^+/+, log-rank test); Hsf1^–/–, 29 weeks (n = 19; P = 0.028 vs. Hsf1^+/+, log-rank test). (B) Representative micrographs of serial sections from MPNSTs arising in NPcis mice were immunostained for HSF1, showing increased levels in tumor, but not adjacent normal nerve (NV). The inset shows nuclear HSF1 staining in tumors. H&E staining is shown to demonstrate histology and provide orientation. Scale bars: 100 μm; 10 μm (inset). (C) Survival time of NPcis mice that developed MPNSTs only was prolonged by Hsf1 compromise. Median survival was as follows: Hsf1^+/+, 19 weeks; Hsf1^+/–, 23.5 weeks (P = 0.0152 vs. Hsf1^+/+, log-rank test); Hsf1^–/–, 23 weeks (P = 0.0869 vs. Hsf1^+/+, log-rank test).

Figure 5. Hsf1 deficiency attenuates MAPK signaling in NPcis mice.

(A) Immunoblotting demonstrated equivalent levels of total ERK, but less activation-associated p-ERK, in MPNSTs arising in Hsf1^–/– mice. Densitometric quantitation of immunoblots confirmed a statistically significant difference in mean ratio of p-ERK to total ERK (Student’s t test). (B) Basal p-ERK was diminished in NPcis^–/– MEFs derived from Hsf1^+/– and Hsf1^–/– embryos.

Figure 6. HSF1 is overexpressed and activated in human MPNST cell lines lacking neurofibromin.

(A) Immunoblotting of cell lines with complete loss of neurofibromin showed elevated p–C-RAF, p-MEK1/2, total HSF1, p-Ser362, and HSP72 levels compared with lines that maintain full-length neurofibromin expression. (B) IF staining confirmed HSF1 overexpression in cells without neurofibromin. Insets show nuclear HSF1 staining. DAPI counterstaining is presented to visualize cell nuclei. All images were acquired at identical magnification. Scale bars: 25 μm; 5 μm (insets). (C) Immunoblotting of cross-linked cell lysates demonstrated increased activation-associated trimeric (Tri) versus inactive monomeric (Mon) forms of HSF1 in MPNST cells without NF1 compared with lysates from NF1-expressing cell lines. The trimeric/monomeric ratio in lysates, as measured by densitometry, is indicated. (D and E) MEK inhibition reduced p-Ser326 and HSF1 nuclear translocation in human MPNST cells. S462 cells were treated with 20 μM U0126 overnight. Total p-Ser326 and nuclear HSF1 were detected by immunoblotting.

Figure 7. HSF1 knockdown impairs human MPNST cell growth and attenuates MAPK signaling.

(A) Differential efficacy of HSF1-targeting shRNA constructs. S462 cells were transduced with GSP or scrambled control or with hA9 or hA6 HSF1-targeting lentiviral supernatants and harvested for immunoblotting. (B) HSF1 knockdown impaired net growth and survival of MPNST cells. After plating, cells were transduced with viral supernatants as indicated. Relative viable cell number in each well was measured 4 days after viral transduction by resazurin dye reduction assay. Raw fluorescence data were normalized to values obtained in wells that underwent mock transduction (mean ± SD, n = 5). ^#P < 0.001, 2-way ANOVA. (C) Decreased levels of KSR1, p-ERK, HSP90α, and HSP72 after HSF1 knockdown. S462 cells were stably transduced with the indicated viral supernatants and harvested for immunoblotting. (D) Decreased levels of KSR1 and p-ERK after HSP90 inhibition. S462 cells were treated overnight with 1 μM geldanamycin (GA) and harvested for immunoblotting. (E) KSR1 knockdown induced apoptosis in MPNST cells. S462 cells were transfected with a nontargeting siRNA (NC) or 3 independent KSR1-targeting siRNAs at 25 nM final concentration. 2 days after transfection, cells were harvested for immunoblotting. (F) KSR1 knockdown impaired net growth and survival of MPNSTs. S462 cells plated in a 96-well format were transiently transfected without or with 25 nM siRNAs. Relative viable cell number in each well was measured as described in B 3 days after transfection (mean ± SD, n = 5). ***P < 0.001, 1-way ANOVA.

Figure 8. A feed-forward loop of MEK/HSF1.

(A and B) Prolonged MEK inhibition decreased HSF1, HSP90α, and KSR1 protein levels in MPNST cells. S462 cells were treated with 20 μM U0126 for 4 days or 20 μM CI-1040 for 3 days. Whole cell lysates were subjected to immuno­blotting. (C) MEK inhibition enhanced HSF1 polyubiquitination. S462 cells were treated with 20 μM U0126 or CI-1040 overnight. HSF1 polyubiquitination was detected as described in Figure 2I. (D) MEK inhibition impaired net growth and survival of MPNST cells. MPNST cells were plated in a 96-well format (2,000 cells/well) and treated with DMSO or 20 μM CI-1040 for 4 days. Relative viable cell number in each well was measured (mean ± SD, n = 3). (E) HSF1 overexpression increased HSP90α and KSR1 protein levels and enhanced p-ERK. S462 cells were transduced with lentiviral LacZ or HSF1 particles. After stable selection with blasticidin, cells were harvested for immuno­blotting. (F) Proposed feed-forward loop of MEK/HSF1.

Figure 9. HSF1 is overexpressed and activated in NF1 patient tumor resections.

(A) Photomicrographs of dual IF staining for HSF1 (green signal) and neurofilament protein (red signal). DAPI counterstaining was used to visualize cell nuclei (blue signal). Scale bars: 25 μm. (B) Photomicrographs of IHC staining for HSF1 and p-HSF1 in a surgical specimen that shows an MPNST arising from adjacent neurofibroma. Brown signal depicts immunoreactivity. Pale blue counterstaining was provided by Mayer hematoxylin. Scale bars: 250 μm (center); 25 μm (higher magnification); 10 μm (insets).