MEK guards proteome stability and inhibits tumor-suppressive amyloidogenesis via HSF1

Abstract

Signaling through RAS/MAP kinase pathway is central to biology. ERK has long been perceived as the only substrate for MEK. Here, we report that HSF1, the master regulator of the proteotoxic stress response, is a new MEK substrate. Beyond mediating cell-environment interactions, the MEK-HSF1 regulation impacts malignancy. In tumor cells, MEK blockade inactivates HSF1 and thereby provokes proteomic chaos, presented as protein destabilization, aggregation, and, strikingly, amyloidogenesis. Unlike their non-transformed counterparts, tumor cells are particularly susceptible to proteomic perturbation and amyloid induction. Amyloidogenesis is tumor suppressive, reducing in vivo melanoma growth and contributing to the potent anti-neoplastic effects of proteotoxic stressors. Our findings unveil a key biological function of the oncogenic RAS-MEK signaling in guarding proteostasis and suppressing amyloidogenesis. Thus, proteomic instability is an intrinsic feature of malignant state, and disrupting the fragile tumor proteostasis to promote amyloidogenesis may be a feasible therapeutic strategy.

Figure 1. MEK and ERK oppositely regulate the PSR

(A) NIH3T3 cells were treated with HS at 43°C for 30min, 10μM tubastatin A for 5hr, 40μM VER155008 for 1hr, 500nM MG132 for 1hr, 200nM 17-DMAG for 1hr, and 2.5mM azetidine for 15min. (B) The dual ELK1 reporter system, comprising a serum response element (SRE)-driven secreted embryonic alkaline phosphatase (SEAP) plasmid and a CMV-driven Gaussia luciferase (GLuc) plasmid, was transfected into HEK293T cells. After 24 hr, cells were treatments as in (A) and recovered overnight before measuring reporter activities (mean±SD, n=6, ANOVA). (C and D) NIH3T3 cells were treated with 20μM U0126 or 20nM AZD6244 for 3 hr followed by HS and 4-hr recovery. mRNA levels were quantitated by qRT-PCR (mean±SD, n=3, Student's t-test). (E) Immediately after HS, nuclear proteins of NIH3T3 cells treated as in (C) were extracted to measure HSF1-DNA binding by an ELISA-based assay (mean±SD, n=3, ANOVA). (F) HEK293T cells were transfected with dual HSF1 reporter plasmids, a heat-shock element (HSE)-driven SEAP plasmid and a CMV-GLuc plasmid. After 24 hr, cells were treated with 20μM U0126, 20nM AZD6244, 1μM FR180204, or 100nM Sch772984 for 3 hr followed by HS for 30min and overnight recovery (mean±SD, n=6, ANOVA). (G) HEK293T cells were treated with different inhibitors overnight. (H)-(K) A LacZ or MEK isoform plasmid was co-transfected with dual HSF1 reporter plasmids into HEK293T cells transduced with lentiviral shRNAs. After 24 hr, cells were heat shocked at 43°C for 30min followed by overnight recovery (mean±SD, n=3, ANOVA). (L)-(O) HEK293T cells were transfected with siRNAs for 48 hr followed by transfection with dual HSF1 reporter plasmids for 24 hr before HS (mean±SD, n=6, ANOVA). See also Figure S1.

Figure 2. ERK, MEK, and HSF1 form a stress-inducible protein complex

(A and B) After HS at 43°C for 30min, endogenous HSF1 proteins were precipitated from HEK293T cells. WCL: whole cell lysate; HC: heavy chain. (C) Endogenous MEK1-HSF1 interactions were detected by PLA in HeLa cells using a rabbit anti-MEK1 antibody and a mouse anti-HSF1 antibody. Scale bars: 50μm for LM, 10μm for HM. (D and E) Endogenous MEK-HSF1 interactions were detected by IP in HEK293T cells stably expressing shRNAs. (F) Endogenous MEK-HSF1 interactions were detected in HEK293T cells transfected with siRNAs. (G) Endogenous HSF1-MEK and HSF1-GFP-ERK1 interactions were detected in HEK293T cells transfected with indicated plasmids. (H) Schematic depictions of three possible scenarios. P: phosphorylation. (I) Immediately after HS, HSF1-ERK interactions were detected by co-IP. (J) Endogenous ERK-HSF1 interactions were detected in HEK293T cells stably expressing shRNAs. (K and L) Endogenous ERK-MEK and ERK-HSF1 interactions were detected by PLA in HeLa cells. Scale bar: 50μm for LM, 10μm HM. See also Figure S2.

Figure 3. MEK phosphorylates Ser326 to activate HSF1

(A and B) HSF1 Ser326 phosphorylation was measured by immunoblotting in HEK293T cells stably expressing shRNAs or transfected with MEK1^DD plasmid. (C) GFP or MEK1^DD plasmids were co-transfected with dual HSF1 reporter plasmids into HEK293T cells (mean±SD, n=5, Student's t-test). (D) Control or ERK-targeting siRNAs, A (siERK1_1 and siERK2_1) and B (siERK1_3 and siERK2_2), were transfected into HEK293T cells stably expressing shRNAs. (E) GFP or FLAG-HSF1 plasmids were co-transfected with dual HSF1 reporter plasmids into HEK293T cells stably expressing shRNAs (mean±SD, n=6, ANOVA). (F) FLAG-HSF1 plasmids were transfected into HEK293T cells stably expressing HSF1-targeting shRNAs. HSF1-DNA binding was measured after HS as described in Figure 1E using anti-FLAG antibodies. The results were normalized against nuclear FLAG-HSF1 levels (mean±SD, n=3, Student's t-test). (G) FLAG-HSF1 proteins were detected in HEK293T cells treated with 20μg/ml cycloheximide. Co-expressed GFP proteins served as internal controls. (H) 100ng purified GST-MEK1 proteins were incubated with U0126 at RT for 20min followed by incubation with 400ng purified His-HSF1 proteins at RT for 30min. HSF1 phosphorylation was detected by immunoblotting. (I) ERK complexes precipitated from HEK293T cells were treated with U0126 or FR180204, followed by incubation with 400ng His-HSF1, 400ng GST-ERK1, or 1000ng MBP proteins. (J) Inactive GST-ERK1 proteins were incubated with 100ng GST-MEK1 and 400ng His-HSF1 proteins at RT for 30min. (K) LacZ or GFP-ERK1 plasmid was co-transfected with MEK1^WT or MEK1^T292A,T386A plasmid into HEK293T cells stably expressing MEK-targeting shRNAs. (L) HSF1 Ser326 phosphorylation was detected in HEK293T cells transfected with indicated plasmids. (M) HSF1 activities were measured by the dual reporter system in HEK293T cells transfected with indicated plasmids (mean±SD, n=6, ANOVA). (N) WM115 cells were treated with 20nM AZD6244 or 20μM U0126 overnight. (O) HSF1 ChIP assays were performed using WM115 cells treated with DMSO or 20nM AZD6244 overnight. The results were normalized against the values of IgG controls (mean±SD, n=3, ANOVA). See also Figure S3.

Figure 4. MEK preserves proteostasis

(A) GFP and GFP-GR plasmids were co-transfected into HEK293T cells followed by treatments with 20nM AZD6244, 20μM U0126, or 200nM 17-DMAG for 4 hr. (B) GFP-GR plasmids were co-transfected into HEK293T cells with HA-Ub-K48 plasmids, which encode a mutant ubiquitin that can be conjugated to protein substrates only via lysine 48. Following treatments with 20nM AZD6244 or 200nM 17-DMAG for 4 hr, GFP-GR proteins were precipitated and ubiquitination was detected using anti-HA antibodies. (C) Denatured firefly luciferases were incubated with lysates of A2058 cells treated with DMSO or 20nM AZD6244 (mean±SD, n=4, ANOVA). (D) A2058 cells were treated with 20nM AZD6244, and ubiquitinated proteins were detected in both detergent-soluble and –insoluble fractions using Lys48-specific ubiquitin antibodies. (E) A2058 cells stably expressing LacZ or HSF1^S326D were treated with 20nM AZD6244 for 8 hr. (F) C57BL/6J mice were i.p. injected with DMSO or AZD6244 three times a week for 2 weeks. S: spleen; K: kidney; L: liver. (G) Experimental procedures of MS-based quantitation of ubiquitinated peptides, two technical replicates per treatment. (H) Scatter plot of relative changes in peptide abundance between treated and control conditions. The green and red lines indicate 2.5-fold cutoffs. (I) The classification of the 68 proteins was performed using the PANTHER gene list analysis tool (www.pantherdb.org). (J) The Gene Ontology (GO) biological process enrichment analysis was performed using the web-based Enrichr software application. (K) Interaction network of the 68 proteins. Known and predicted protein interactions were derived from the STRING database (www.string-db.org), and the network was visualized using Cytoscape software. (L and M) V5-TOR1AIP2 or V5-RPL3 plasmids were co-transfected with HA-Ub-K48 plasmids into HEK293T cells. Following 20nM AZD6244 treatment for 8 hr, proteins were precipitated with anti-V5 antibodies. (N) Following AZD6244 treatment, endogenous c-MYC proteins were precipitated from A2058 cells and immunoblotted with anti-ubiquitin antibodies. (O and P) V5-RPL15 and V5-RPL3 plasmids were co-transfected with HA-Ub-K48 plasmids into HEK293T cells stably expressing shRNAs. Cells were treated with 500nM MG132 alone or co-treated with 20nM AZD6244 for 8 hr. (Q) Endogenous RPL15 and RPL3 proteins were detected in A2058 cells treated with 20nM AZD6244 alone or co-treated with 500nM MG132. (R) Endogenous RPL15 and RPL3 proteins were detected in A2058 cells stably expressing LacZ or HSF1^S326D with AZD6244 treatment. See also Figure S4 and Table S1.

Figure 5. MEK and proteasome inhibition provoke protein aggregation and amyloidogenesis

(A) WM115 cells treated with 20nM AZD6244, 100nM Bortezomib, or both for 24 hr were stained with Lys48-specific ubiquitin antibodies. Arrowheads mark ubiquitin-positive aggregates. Scale bar: 10μm. Amounts of aggregates per cell were quantitated using ImageJ (median, n≥100, ANOVA). (B and C) Following transfection with polyQ79 plasmids alone or with both polyQ79 and HSF1^S326D plasmids for one day, HEK293T cells were treated with inhibitors as described in (A). Cells were either analyzed for aggregate size or stained with 10μM ThT. (D) Treated tumor cell lines were stained with 10μM ThT. Geometric means were used to calculate fold changes in ThT fluorescence intensity and the log₂(FC) values were presented as a heat map. (E) HEK293T cells were transfected with LacZ or polyQ79 plasmid. Following treatments, AOs were quantitated by ELISA using A11 antibodies (mean±SD, n=3, ANOVA). (F) Intrinsic AOs were detected in human tumor cell lines (mean±SD, n=3, ANOVA). (G and H) A2058 cells stably expressing LacZ or HSF1^S326D were treated for 24 hr. Amyloids were quantitated by ELISA (mean±SD, n=3, Student's t-test). (I) 20μM synthetic Aβ1-42 peptides were incubated at RT with gentle shaking with 20μg lysates of A2058 cells treated with inhibitors. AF formation was monitored by ThT binding (mean±SD, n=3, ANOVA). (J) For TEM studies (left panel 80,000X, right panel 200,000X), 20μM synthetic Aβ1-42 peptides were incubated with A2058 cell lysates in PBS at 37°C with gentle shaking for 2 days. Scale bars: 100nm. (K) HEK293T cells stably expressing different shRNAs were stained with 10μM ThT. (L and M) After pre-incubation with 10μM ThT for 6hr, A2058 cells were treated for 24 hr. Amyloids were quantitated (mean±SD, n=3, Student's t-test). (N and O) After pre-incubation with 10μM ThT for 6 hr or transfection with 100ng A11 antibodies using JBS-Proteoducin for 16 hr, A2058 cells were treated for 24 hr. Viable cells were quantitated by CellTiter® blue (mean±SD, n=6, Student's t-test). (P) A2058 cells stably expressing LacZ or HSF1^S326D were treated with DMSO or 20nM AZD6244. Viable cells were quantitated (mean±SD, n=6, ANOVA). Relative changes in viable cells after treatment were calculated by normalizing the values of AZD6244-treated cells against the values of DMSO-treated cells at each time point. (Q)-(S) Following treatments with 20nM AZD6244, 100nM Bortezomib, or both for 24 hr, AOs were quantitated in primary MEFs and human cells (mean±SD, n=3, ANOVA). (T and U) Cells were treated with 50μM Q-VD-OPh overnight and AOs were quantitated (mean±SD, n=3, Student's t-test). See also Figure S5.

Figure 6. Combined MEK and proteasome inhibition exerts potent tumor-suppressive effects

(A) After treatments with 20nM AZD6244, 100nM Bortezomib, or both for 24 hr, viable cells were quantitated by CellTiter® Blue (mean±SD, n=6, ANOVA). (B and C) 1×10⁶ A2058 cells were s.c. injected into NOD/SCID mice. After 7 days, mice were treated with DMSO, 5mg/Kg AZD6244, 0.5mg/Kg Bortezomib, or the combination via i.p. injection three times a week. Tumor volumes were measured using a caliper weekly (mean±SEM, ANOVA). Tumor growth curves were fitted to exponential growth models to derive tumor-doubling time (DT). Kaplan-Meier survival curve was plotted for each group (Log-rank test). (D) Proteins were detected by immunoblotting, 3 tumors per group. (E) Tumor lysates were used to quantitate AOs, 5 tumors per group (mean±SD, n=3, ANOVA). (F) Tumor lysates were used to seed Aβ1-42 peptides, 5 tumors per group (mean±SD, n=3, ANOVA). (G) Tumor sections were stained with CR, 5 tumors per group. Ten random fields were taken for each section. Scale bar: 50μm. Total CR fluorescence in each field was quantitated using ImageJ and normalized against total nuclei (median, n=50, ANOVA). (H) Following CR staining, tumor sections were visualized under polarized light microscopy. Scale bar: 50μm. (I) Following staining with AF-specific antibodies (OC), sections of tumors receiving combined treatment were further stained with CR. Scale bar: 50μm. (J) 1×10⁶ A2058 cells stably expressing firefly luciferase transgene were i.v. injected into NOD/SCID mice. Treatments were initiated one day after as described in (B) for 6 weeks. Body weights were monitored weekly (mean±SD, n=10, ANOVA). (K) Detection of metastases by in vivo bioluminescence imaging. (L) Representative micrographs illustrate metastatic melanomas in the lung, skeletal muscle, pelvic adipose tissue, and ovary. T: tumors; L: lung; M: muscle; B: bone; A: adipose tissue; OF: ovarian follicle. Scale bar: 500μm. (M) Combined MEK and proteasome inhibition prevents melanoma metastasis (Barnard's exact test). See also Figure S6 and Table S2.

Figure 7. Amyloidogenesis suppresses tumor growth

(A) Sections of melanomas receiving combined treatment were stained with cleaved caspase 3 antibodies followed by CR staining. Arrowheads and arrows indicate condensed and fragmented nuclei, respectively. Scale bar: 50μm. (B) 1×10⁶ A2058 cells were s.c. injected into NOD/SCID mice. After 7 days, mice were treated with 1mg/30g CR via i.p. injection one day prior to combined treatment. Tumor volumes were measured weekly (mean±SD, ANOVA). (C) Lysates of CR-treated tumors exhibited absorbance at 498nm, 3 tumors per group (mean±SD, n=3). Lysis buffer containing CR served as a positive control. (D) Kaplan-Meier survival curves were compared (Log-rank test). (E and F) Both detergent-soluble and -insoluble fractions of tumor lysates were used to quantitate amyloids, 3 tumors per group (mean±SD, n=3, Student's t-test). (G) Proteins were detected by immunoblotting, 3 tumors per group. (H) Schematic depiction of the interplay among MEK, ERK, and HSF1, and its role in regulating proteome stability. Balanced proteostasis suppresses toxic protein aggregation and amyloidogenesis, thereby facilitating tumorigenesis. See also Figure S7.