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. 2016 Nov;18(11):1233-1243.
doi: 10.1038/ncb3427. Epub 2016 Oct 24.

DNAJA1 controls the fate of misfolded mutant p53 through the mevalonate pathway

Alejandro Parrales  1 , Atul Ranjan  1 , Swathi V Iyer  1 , Subhash Padhye  2 , Scott J Weir  3 , Anuradha Roy  4 , Tomoo Iwakuma  1
Affiliations

DNAJA1 controls the fate of misfolded mutant p53 through the mevalonate pathway

Alejandro Parrales et al. Nat Cell Biol. 2016 Nov.

Abstract

Stabilization of mutant p53 (mutp53) in tumours greatly contributes to malignant progression. However, little is known about the underlying mechanisms and therapeutic approaches to destabilize mutp53. Here, through high-throughput screening we identify statins, cholesterol-lowering drugs, as degradation inducers for conformational or misfolded p53 mutants with minimal effects on wild-type p53 (wtp53) and DNA contact mutants. Statins preferentially suppress mutp53-expressing cancer cell growth. Specific reduction of mevalonate-5-phosphate by statins or mevalonate kinase knockdown induces CHIP ubiquitin ligase-mediated nuclear export, ubiquitylation, and degradation of mutp53 by impairing interaction of mutp53 with DNAJA1, a Hsp40 family member. Knockdown of DNAJA1 also induces CHIP-mediated mutp53 degradation, while its overexpression antagonizes statin-induced mutp53 degradation. Our study reveals that DNAJA1 controls the fate of misfolded mutp53, provides insights into potential strategies to deplete mutp53 through the mevalonate pathway-DNAJA1 axis, and highlights the significance of p53 status in impacting statins' efficacy on cancer therapy.

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Figures

Figure 1
Figure 1
Statins reduce levels of conformational or misfolded mutp53. (a) Western blotting (WB) for p53 and β-actin using SK-Br-3 (p53R175H) and U2OS (p53wt) cells treated with the indicated concentrations of DMSO (D) or lovastatin (L) for 24 h. (b) WB for p53 and β-actin or vinculin using SK-Br-3 and U2OS cells treated with the indicated concentrations of DMSO (D) or atorvastatin (A) for 24 h. (c) WB for the indicated proteins using KHOS/NP (p53R156P), BxPC-3 (p53Y220C), HT29 (p53R273H) and MDA-MB-231 (p53R280K) cells treated with D or L for 24 h. (d) Immunoprecipitation (IP) for total p53 (DO1), the folded/native form of p53R248W (PAb1620), and the misfolded/denatured form of p53R248W (PAb240), using MIA PaCa-2 cells treated with D or L, followed by WB for p53 (7F5). Additional results are shown in Supplementary Fig. 1. Unprocessed original scans of blots are shown in Supplementary Fig. 8.
Figure 2
Figure 2
Statins induce mutp53 degradation by CHIP. (a) WB for p53 and vinculin using SK-Br-3 cells treated with cycloheximide (CHX, 50 nM) for the indicated time periods (h) following pre-incubation with DMSO or lovastatin (4 μM) for 12 h. Right: graph showing relative p53R175H levels compared with those without CHX. Error bars, means ± s.d. (n = 3 independent experiments), *P < 0.05; Student's t-test (two-tailed). (b) WB for the indicated proteins using SK-Br-3 cells infected with non-silencing control or MDM2 (M1, 2, 3) shRNA-encoding lentiviral vectors and treated with DMSO (D) or lovastatin (L) at 4 μM for 24 h. (c) WB for the indicated proteins using SK-Br-3 cells infected with lentiviral vectors encoding non-silencing control or CHIP (CH1, 2) shRNAs and treated with D or L (4 μM) for 24 h. (d) Ubiquitin assays for p53R156P and WB for the indicated proteins. KHOS/NP cells with or without CHIP knockdown were transfected with an ubiquitin-encoding plasmid, treated with D or L (4 μM) for 22 h, further incubated with 30 μM of MG132 for 6 h, and harvested with hot SDS lysis buffer. (e) Immunofluorescence for p53R175H in KHOS/NP cells treated with D or L for 18 h. Scale bars, 50 μm. (f) Immunofluorescence (left) and WB (right) for p53R175H using SK-Br-3 cells treated with D or L along with vehicle (ethanol) or leptomycin B (LMB, 50 nM) for 18 h. Scale bars, 50 μm. (g) Immunofluorescence for p53R175H using SK-Br-3 cells infected with control or CHIP shRNA-encoding lentiviral vectors and treated with DMSO or lovastatin. Scale bars, 50 μm. Statistics source data for a are provided in Supplementary Table 1. Additional results are shown in Supplementary Fig. 2. Unprocessed original scans of blots are shown in Supplementary Fig. 8.
Figure 3
Figure 3
Reduced MVP mirrors statins’ effects on mutp53. (a) The mevalonate pathway and its inhibitors. (b) WB for p53R156P and vinculin using KHOS/NP cells treated with DMSO (D) or different mevalonate pathway inhibitors including GGTI-2133 (G, 4 μM), YM-53601 (Y, 4 μM), lovastatin (L, 4 μM), 6-fluoromevalonate (6F, 100 μM) and zoledronic acid (ZA, 25 μM) for 24 h. (c,d) WB for p53 and loading controls (β-actin or vinculin) using SK-Br-3 cells treated with D or L (4 μM) in the presence or absence of mevalonic acid (MVA, 200 μM; c), mevalonate-5-phosphate (MVP, 200 μM, c), or MVA-5PP (100 and 200 μM; d) for 24 h. (e) WB for p53, MVK and loading controls using KHOS/NP, SK-Br-3 and CAL33 (p53R175H) cells infected with lentiviral vectors encoding non-silencing control (C) or MVK shRNAs (K1, K2). (f) Immunofluorescence for p53 using SK-Br-3 infected with lentiviral vectors encoding control (C) or MVK shRNAs. Scale bars, 50 μm. (g) WB for the indicated proteins using SK-Br-3 cells with or without MVK knockdown supplemented with vehicle (water, control) or MVP (200 μM) for 24 h. (h) Ubiquitin assays for p53R156P and WB for the indicated proteins using KHOS/NP cells with or without knockdown of CHIP and/or MVK. (i) WB for the indicated proteins using SK-Br-3 cells with or without knockdown of MVK, along with transfection of control or CHIP siRNAs (50 or 100 nM). (j) WB for the indicated proteins using KHOS/NP and CAL33 cells infected with lentiviral vectors encoding control (C), PMVK (P1, P2) or MVD (D1) shRNAs. Additional results are shown in Supplementary Fig. 3. Unprocessed original scans of blots are shown in Supplementary Fig. 8.
Figure 4
Figure 4
Statins favourably impede mutp53-tumour growth. (a) MTT assays using KHOS/NP and U2OS cells treated with various concentrations of DMSO or lovastatin for 48 h. Error bars, means ± s.d. (n = 3 independent experiments), *P < 0.05; NS, not significant; Student's t-test (two-tailed). (b) Colony formation assays using KHOS/NP and U2OS cells (500) seeded onto 6-well plates and treated with DMSO or lovastatin (4 μM) every other day for 10 days. Representative images (left) and a summarized graph (right). Error bars, means ± s.d. (n = 3 independent experiments), **P <0.01; Student's t-test (two-tailed). (c) Colony formation assays with or without supplementation of vehicle control, mevalonic acid (MVA) or mevalonate-5-pyrophosphate (MVA-5PP). Representative images (left) and summarized graph (right). Error bars, means ± s.d. (n = 3 independent experiments), *P < 0.05, **P < 0.01; Student's t-test (two-tailed). (d) Tumour formation assays in mice subcutaneously injected with KHOS/NP cells (1×106). When tumours reached 3 mm in diameter, mice were intraperitoneally injected with DMSO (D) or atorvastatin (A, 30 mg kg−1) daily. Tumour sizes were measured every 2–3 days. The graph shows tumour growth with representative images of tumours (inset). Error bars, means ± s.d. (n = 5 animals for each group), *P <0.05; Student's t-test (two-tailed). Scale bar, 1 cm. (e) Representative images of IHC for p53, Ki67 and cleaved caspase 3 using KHOS/NP tumours treated with D or A (magnification, ×40). Scale bars, 50 μm. (f) Tumour formation assays in KHOS/NP-injected mice treated with vehicle control (C) or 10 mg kg−1 of rosuvastatin (R) daily. Scale bar, 1 cm. Error bars, means ± s.d. (n = 5 animals for each group), *P < 0.05; Student's t-test (two-tailed). Statistics source data for a–c are provided in Supplementary Table 1. Additional results are shown in Supplementary Fig. 4.
Figure 5
Figure 5
Statins reduce growth of p53R172H MEFs. (a) MTT assays using E1A/H-RasG12V-transformed p53+/+, p53−/−, p53+/R172H, and p53R172H/R172H MEFs treated with various concentrations of lovastatin for 48 h. Error bars, means ± s.d. (n = 3 independent experiments), *P < 0.05; Student's t-test (two-tailed). (b) Tumour formation assays in mice subcutaneously injected with transformed MEFs (1×106) and treated with DMSO (D) or atorvastatin (A) (30 mg kg−1). Scale bars, 1 cm. Error bars, means ± s.d. (n = 5 animals for each group), *P <0.05; NS, not significant; Student's t-test (two-tailed). (c) MTT assays using transformed MEFs treated with various concentrations of doxorubicin together with DMSO or lovastatin (4 μM) for 48 h. Error bars, means ± s.d. (n = 5 independent experiments), **P <0.01; Student's t-test (two-tailed). (d) Quantitative RT–PCR for p21 and BAX using transformed p53+/R172H MEFs pre-treated with DMSO (D) or lovastatin (L) (4 μM) for 24 h and further exposed to vehicle control (C) or doxorubicin (DRB, 0.1 μM near IC50 in p53+/+ MEFs) for 24 h. Error bars, means ± s.d. (n = 3 independent experiments), *P <0.05, **P <0.01; Student's t-test (two-tailed). Statistics source data for a and d are provided in Supplementary Table 1. Additional results are shown in Supplementary Fig. 5.
Figure 6
Figure 6
DNAJA1 knockdown induces mutp53 degradation. (a) WB for p53, Hsc70 and β-actin using CAL33 cells transfected with control or Hsc70 siRNAs and treated with DMSO (D) or lovastatin (L, 4 μM). (b) WB for the indicated proteins using SK-Br-3 cells transfected with siRNAs specific for DNAJA1 (JA1) or DNAJB1 (JB1). (c) WB for the indicated proteins using KHOS/NP cells downregulated for DNAJA1 or DNAJB1 by siRNAs and treated with D or L (4 μM) for 24 h. (d) WB for the indicated proteins using CAL33 cells downregulated for DNAJA1 (JA1) and/or Hsc70. (e) WB for p53, DNAJA1 and vinculin using cell lines with different p53 status transfected with control (C) or DNAJA1 (JA1) siRNAs. (f) WB for the indicted proteins using SK-Br-3 cells downregulated for CHIP and/or DNAJA1. (g) Immunofluorescence for p53R156P using KHOS/NP cells downregulated for DNAJA1 and/or CHIP. Scale bars, 50 μm. (h) Ubiquitin assays for p53R156P and WB for the indicated proteins using KHOS/NP cells downregulated for DNAJA1 and/or CHIP. Additional results are shown in Supplementary Fig. 6. Unprocessed original scans of blots are shown in Supplementary Fig. 8.
Figure 7
Figure 7
DNAJA1 nullifies statins’ effects on mutp53. (a) WB for the indicated proteins using KHOS/NP cells infected with empty (vector) or DNAJA1 cDNA (cDNAJA1)-encoding lentiviral vectors and treated with DMSO (D) or lovastatin (L, 4 μM) for 24 h. (b) Ubiquitin assays for p53R156P and WB for the indicated proteins using KHOS/NP cells with or without overexpression of DNAJA1 (cDNAJA1) and treated with D or L for 24 h in the presence of MG132 for 6 h. (c) Immunofluorescence for p53R156P using KHOS/NP cells with or without overexpression of DNAJA1 and treated with DMSO or lovastatin. Scale bars, 50 μm. (d) Co-immunoprecipitation and WB for DNAJA1 and p53R175H using CAL33 cells treated with D, L or L + mevalonate-5-phosphate (MVP) for 11 h before mutp53 degradation. (e) Co-immunoprecipitation and WB for p53R156P and DNAJA1 using KHOS/NP cells infected with lentiviral vectors encoding control or MVK shRNAs and treated for 18 h with control (water) or MVP (200 μM) along with leptomycin B (LMB, 50 nM). (f) Co-immunoprecipitation and WB for p53R175H and CHIP using CAL33 cells treated with D or L for 11 h. Additional results are shown in Supplementary Fig. 7. Unprocessed original scans of blots are shown in Supplementary Fig. 8.
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References

    1. Lane D, Levine A. p53 research: the past thirty years and the next thirty years. Cold Spring Harb. Perspect. Biol. 2010;2:a000893. - PMC - PubMed
    1. Levav-Cohen Y, et al. The p53-Mdm2 loop: a critical juncture of stress response. Subcell. Biochem. 2014;85:161–186. - PubMed
    1. Rivlin N, Koifman G, Rotter V. p53 orchestrates between normal differentiation and cancer. Semin. Cancer Biol. 2015;32:10–17. - PubMed
    1. Boeckler FM, et al. Targeted rescue of a destabilized mutant of p53 by an in silico screened drug. Proc. Natl Acad. Sci. USA. 2008;105:10360–10365. - PMC - PubMed
    1. Terzian T, et al. The inherent instability of mutant p53 is alleviated by Mdm2 or p16INK4a loss. Genes Dev. 2008;22:1337–1344. - PMC - PubMed

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