Allosteric Hsp70 Modulator YM-1 Induces Degradation of BRD4

Abstract

YM-1, an allosteric modulator of heat-shock 70 kDa protein (Hsp70), inhibits cancer cell growth, but the mechanism is not yet fully understood. Here, we show that YM-1 induces the degradation of bromodomain containing 4 (BRD4), which mediates oncogene expression. Overall, our results indicate that YM-1 promotes the binding of HSP70 to BRD4, and this in turn promotes the ubiquitination of BRD4 by C-terminus of Hsc70-interacting protein (CHIP), an E3 ubiquitin ligase working in concert with Hsp70, leading to proteasomal degradation of BRD4. This YM-1-induced decrease of BRD4 would contribute at least in part to the inhibition of cancer cell growth.

Introduction

Heat shock 70 kDa protein (Hsp70) serves as a molecular chaperone, assisting the proper folding of newly synthesized proteins, as well as misfolded and aggregated proteins.¹⁾ There are 13 members of the Hsp70 family in mammals, including cytosolic heat shock cognate 70 (Hsc70, HSPA8), stress-inducible cytoplasmic heat shock protein 70 (Hsp72, HSPA1A), and mitochondrial Hsp70 (mtHsp70, mot-2, HSPA9).²⁾ Hsp70 interacts with the C-terminus of Hsc70-interacting protein (CHIP), which functions as an E3 ubiquitin ligase.³⁾ Substrates that remain bound to Hsp70 are more likely to be ubiquitylated by CHIP and degraded by proteasome, presumably to prevent unfolded substrate proteins from saturating the chaperone network.⁴⁾

Hsp70 is composed of an N-terminal nucleotide binding domain (NBD), to which ATP/ADP binds, and a C-terminal substrate binding domain (SBD), to which substrate proteins bind.⁴⁾ When Hsp70 hydrolyzes ATP, the ADP-bound protein changes its conformation, resulting in increased affinity for substrate proteins.¹⁾ YM-1 (sometimes referred to as YM-01) and its lead compound MKT-077 (Fig. 1), which are allosteric modulators of Hsp70, bind to the NBD of ADP-bound Hsp70 and promote substrate binding by inhibiting ATP/ADP turnover.^2,5,6) YM-1 and MKT-077 both have cancer-specific growth-inhibitory activity.^7,8) MKT-077 has been reported to inhibit the interaction between the tumor suppressor p53 and mtHsp70, and this action might be partly responsible for its inhibition of cancer cell growth.⁹⁾ However, other factors may also be involved. In this study, we show for the first time that YM-1 promotes the degradation of bromodomain containing 4 (BRD4), and this may also contribute to its cancer cell growth-inhibitory activity.

Fig. 1. Chemical Structures of YM-1, MKT-077, JQ1 and dBET1

BRD4, a member of the bromodomain and extraterminal domain (BET) family, binds to acetylated lysine residues on target proteins, including histones.¹⁰⁾ BRD4 recruits positive transcription elongation factor b (P-TEFb) to the transcription start site, thereby inducing phosphorylation of the C-terminus of RNA polymerase II and activating transcription.¹¹⁾ BRD4 also interacts with the RNA polymerase II complex to facilitate elongation.¹²⁾ Since the expression of the oncogene c-myc is known to be activated by this mechanism, BRD4 has attracted attention as a promising drug target for cancer therapy.^13,14) Indeed, the BET inhibitor JQ1 (Fig. 1) shows anticancer activity.¹⁵⁾ Since the development of JQ1 in 2010, several BET inhibitors have been developed and have entered clinical trials.¹⁶⁾ Furthermore, dBET1 (Fig. 1), a proteolysis targeting chimera (PROTAC) utilizing JQ1, induces degradation of BRD4 and thereby promotes apoptosis of cancer cells more potently than JQ1 itself.¹⁷⁾ Thus, inhibition and/or degradation of BRD4 is a promising approach for cancer treatment. Here, we show that YM-1 promotes BRD4 degradation and we discuss its mode of action.

Results and Discussion

YM-1 Inhibits Proliferation of MCF-7 Cells

First, we synthesized YM-1 and confirmed that it has cancer cell growth-inhibitory activity, as reported.^7,8) We treated human breast cancer cell line MCF-7 with YM-1 for 72 h and quantified viable cells by WST-8 assay. Proliferation was strongly inhibited by YM-1 (Fig. 2).

Fig. 2. Antiproliferative Activity of YM-1

MCF-7 cancer cells were treated with dimethyl sulfoxide (DMSO) or YM-1 at the indicated concentrations for 72 h. Their viability was assessed by WST-8 assay, and the measured absorbance minus the blank value was used for evaluation. Data are the mean (standard deviation (S.D.)) from three independent experiments. Asterisks indicate a significant difference (** p < 0.01) by paired two-sided Student’s t-test in comparison to DMSO-treated cells.

YM-1 Treatment Decreases BRD4 in MCF-7 and HeLa Cells

We found that treatment of MCF-7 cells with YM-1 reduced BRD4 in a concentration-dependent manner (Fig. 3a). To see if YM-1 also has a similar effect in a different cell line, human cervical carcinoma HeLa cells were treated with YM-1. A concentration-dependent decrease in BRD4 was also observed in HeLa cells, though it was less pronounced than in the case of MCF-7 cells (Fig. 3b).

Fig. 3. Decrease of BRD4 Induced by YM-1 Treatment

MCF-7 cells (a) or HeLa cells (b) were treated with YM-1 at the indicated concentrations for 6 h. The level of intercellular BRD4 was determined by Western blotting. α-Tubulin was used as a loading control.

YM-1 Induces Proteasomal Degradation of BRD4

We next examined the mechanism of BRD4 reduction by YM-1. Previous studies have shown that YM-1 promotes proteasomal degradation of androgen receptor having an abnormally extended polyglutamine repeat (polyQ-AR) by stabilizing the ADP-binding state of Hsp70. It has also been shown that YM-1 does not affect the expression level of Hsp70.⁶⁾ We hypothesized that the decrease of BRD4 might involve a similar mechanism. Indeed, the BRD4-reducing activity of YM-1 in MCF-7 cells was blocked by the proteasome inhibitors MG132 and epoxomicin (Fig. 4). This indicates that YM-1 promotes proteasomal degradation of BRD4. Since BRD4 has an intrinsically disordered region (IDR), it may be recognized by Hsp70 as a substrate, and it seems likely that the allosteric Hsp70 modulator YM-1 promotes the recognition of BRD4 by Hsp70, resulting in ubiquitination followed by proteasomal degradation.

Fig. 4. YM-1-Induced Decrease of BRD4 Is Blocked by Proteasome Inhibitors

MCF-7 cells were pretreated with proteasome inhibitor (MG132 or epoxomicin) at the indicated concentrations 1 h before YM-1 treatment (10 µM, 6 h). The levels of BRD4 and vinculin (loading control) were determined by Western blotting. (a) A representative Western blotting image. (b) Quantification of the BRD4/vinculin ratio. Data are the mean (S.D.) of normalized BRD4/vinculin ratio from three independent experiments. Asterisks indicate a significant difference (* p < 0.05 ** p < 0.01) by paired two-sided Student’s t-test (for lane 1 vs. 6) or Dunnett’s test (for lane 6 vs. 8 and 10).

YM-1 is known to bind to several proteins of the Hsp70 family, including Hsc70.²⁾ Since Hsc70 mediates chaperone-mediated autophagy, in which substrate proteins are selectively delivered to lysosomes and degraded,¹⁸⁾ we considered that autophagy may also be involved in the reduction of BRD4 by YM-1. To test this, we treated MCF-7 cells with bafilomycin A1 (bafA1), which inhibits lysosome function, along with YM-1. Although the decrease of BRD4 was slightly reduced, the difference was not statistically significant (Fig. 5), suggesting that the YM-1-induced BRD4 reduction is predominantly due to degradation by the ubiquitin–proteasome system, rather than autophagy.

Fig. 5. Effect of BafilomycinA1 on BRD4 Degradation-Inducing Activity of YM-1

MCF-7 cells were pretreated with bafA1 (1 µM) 1 h before YM-1 treatment (10 µM, 6 h). The levels of BRD4 and vinculin (loading control) were determined by Western blotting. (a) A representative Western blotting image. (b) Quantification of the BRD4/vinculin ratio. Data are the mean (SD) of normalized BRD4/vinculin ratio from three independent experiments. Asterisks indicate a significant difference (** p < 0.01) by paired two-sided Student’s t-test.

Insoluble BRD4 Is Predominantly Reduced by YM-1

Hsp70 binds predominantly to denatured/aggregated proteins, which are present mostly in the insoluble fraction of the lysate. If YM-1 promotes BRD4 degradation via Hsp70, BRD4 in the insoluble fraction should be significantly reduced. Therefore, we analyzed the insoluble fraction of lysate (16710 × g, 5 min) in MCF-7 cells treated with DMSO (control) or YM-1 by Western blotting. Indeed, insoluble BRD4 was greatly reduced by YM-1 treatment (Fig. 6). The reduction of BRD4 in the insoluble fraction was greater than that in the soluble fraction (Fig. 3a), which is consistent with the involvement of Hsp70. This is also in agreement with a previous report that YM-1 treatment resulted in a significant decrease of insoluble polyQ-AR.⁶⁾

Fig. 6. Reduction of Insoluble BRD4

MCF-7 cells were treated with YM-1 (10 µM) for 6 h. The amount of pelleted BRD4 was visualized by Western blotting. Although a loading control in the pellet was not quantified, the protein concentration in the soluble fractions were nearly equal (3.2 µg/µL (DMSO) and 2.8 µg/µL (YM-1)), and the two samples were directly compared.

Based on these results, YM-1 is considered to promote the binding of Hsp70 to BRD4, thereby inducing its ubiquitination by CHIP, followed by proteasomal degradation. The degradation of BRD4 is expected to suppress c-myc expression, and this may play a role in the anticancer activity of YM-1. It has already been reported that JG-98, a derivative of YM-1, induced downregulation of c-myc.¹⁹⁾ Given that Hsp70 recognizes a wide range of proteins as substrates, it is possible that YM-1 also induces degradation of proteins other than BRD4. In this context, derivatives of YM-1, JG-83 and JG-98, are known to decrease Akt.^2,19) In contrast to BRD4-targeting inhibitors and PROTACs, which specifically inactivate BRD4, Hsp70 modulators such as YM-1 may affect a variety of proteins, and thus may exhibit anticancer activity via multiple pathways. Hsp70 is particularly highly expressed in cancer cells compared to normal cells,⁴⁾ which may make Hsp70 a favorite target for cancer therapeutics.

Conclusion

YM-1, an allosteric modulator of Hsp70, decreases the level of the cancer-associated protein BRD4 in MCF-7 and HeLa cells. The decrease is proteasome-dependent, suggesting that YM-1 promotes the binding of Hsp70 to BRD4, resulting in ubiquitination by ubiquitin ligases that cooperate with Hsp70, followed by proteasomal degradation. Given the role of BRD4 in oncogene expression, our results suggest that regulation of Hsp70 has the potential to be a promising therapeutic strategy for cancer.

Experimental

Chemical Synthesis of YM-1

YM-1 was synthesized as described by Wang et al.⁶⁾ Purity 97% (HPLC).

Cell Culture

MCF-7 and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO₂ in air.

Cell Viability Assay

A total of 0.5 × 10⁴ MCF-7 cells were seeded in 96-well plates and incubated for 24 h, then the culture medium was removed, and the cells were cultured in medium containing YM-1 with 0.1% DMSO for 72 h. To avoid the effect of YM-1’s orange color on absorbance measurement, the medium was replaced and 10 µL of Cell Counting Kit-8 (DOJINDO, Kumamoto, Japan) was added. At this time, the same amount of Cell Counting Kit-8 was added to the medium without cells as a blank. After incubation in a CO₂ incubator for 1 h, the absorbance at 450 nm was measured using an Envision 2104 multilabel reader (PerkinElmer, Inc., CT, U.S.A.), and cell viability was evaluated based on the value obtained after subtracting the absorbance of the blank. Relative cell viability was calculated by normalizing the absorbance of each sample by the mean absorbance of the DMSO-treated samples in three experiments.

Western Blotting

5 × Laemmli buffer (0.25% BPB, 0.5 M dithiothreitol (DTT), 50% glycerol, 10% sodium dodecyl sulfate (SDS), 0.25 M Tris–HCl (pH 6.8)) was added to cell lysate and the mixture was heated at 95 °C for 5 min and then cooled on ice. Proteins ware separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) with 7.5% Mini-PROTEAN^® TGX™ Precast Protein Gels, 15-well (4561026, BIO-RAD, CA, U.S.A.) and transferred onto Immun-Blot^® polyvinylidene difluoride (PVDF) membrane (1620177, BIO-RAD). After blocking with Bullet Blocking One for Western blotting (13779-01, Nacalai tesque, Kyoto, Japan) for 15 min, the membrane was probed with antibody as shown in Table 1. The antibodies were diluted with Can Get Signal Immunoreaction Enhancer Solution 1 or 2 (NKB-101, Toyobo, Osaka, Japan). The membrane was rinsed with water (five times) and washed with TBS-T for 5 min, and the immunoblots were visualized by measuring the enhanced chemiluminescence with Chemi-Lumi One L (07880-54, Nacalai tesque) or the fluorescence from fluorescent-dye conjugated antibodies using a ChemiDoc™ MP Imaging System (BIO-RAD). Images were analyzed by Image Lab™ software (BIO-RAD).

Table 1. Antibodies Used for Western Blotting

Primary antibody			Secondary antibody
Antigen	Dilution ratio	Cat. #	Antigen	Dilution ratio	Cat. #
BRD4	1000 : 1	13440, CST	rabbit IgG	2000 : 1	NBP1-75297, NB
α-Tubulin	2000 : 1	3873, CST	mouse IgG	2000 : 1	12-349, Merck
Vinculin	500 : 1	4650, CST	rabbit IgG	2000 : 1	12004162, BIO-RAD

CST: Cell Signaling Technology, NB: Novus Biologicals.

Evaluation of BRD4 Level in Soluble Fraction

MCF-7 cells were seeded at the concentration of 4.0 × 10⁵ cells/mL in 12-well plates and incubated for 24 h, then the culture medium was removed, and the cells were cultured in medium containing YM-1 with 0.1–0.2% DMSO for 6 h. Inhibitors were added 1 h prior to YM-1 treatment. The cells were lysed on ice for 30 min with TNE-T lysis buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, and 5 mM ethylenediaminetetraacetic acid (EDTA)) supplemented with 1% protease inhibitor cocktail (25955-24, Nacalai tesque). The lysates were centrifuged at 16710 × g, 4 °C, 5 min and the supernatants were collected. The protein concentration in the supernatants was determined by BCA protein assay and normalized by the total protein concentration in each lysate. The amount of intercellular proteins was determined by Western blotting.

Evaluation of BRD4 Level in Insoluble Fraction

MCF-7 cells were seeded at the concentration of 4.0 × 10⁵ cells/mL in 12-well plates and incubated for 24 h, then the culture medium was removed, and the cells were cultured in medium containing YM-1 with 0.2% DMSO for 6 h. The cells were lysed in the same manner as above, and the lysates were centrifuged at 16710 × g, 4 °C, 5 min. The pellets were suspended in TNE-T lysis buffer and subjected to Western blotting.

Statistics

Statistical significance was assessed by paired, two-sided Student’s t-test using Excel (Microsoft) or Dunnett’s test using EZR software (Saitama Medical Center, Jichi Medical University).²⁰⁾

Acknowledgments

This work was partially supported by JSPS KAKENHI (Grant Numbers JP23KJ0187 for YM, JP22K15242 for ST, JP22H00436 for MI), AMED-CREST, AMED (JP 21gm1410007 for MI) and MEXT/JSPS WISE Program: Advanced Graduate Program for Future Medicine and Health Care, Tohoku University.

Conflict of Interest

The authors declare no conflict of interest.

References

• 1) Mayer M. P., Bukau B., Cell. Mol. Life Sci., 62, 670–684 (2005).
• 2) Li X., Srinivasan S. R., Connarn J., Ahmad A., Young Z. T., Kabza A. M., Zuiderweg E. R. P., Sun D., Gestwicki J. E., ACS Med. Chem. Lett., 4, 1042–1047 (2013).
• 3) Ballinger C. A., Connell P., Wu Y., Hu Z., Thompson L. J., Yin L.-Y., Patterson C., Mol. Cell. Biol., 19, 4535–4545 (1999).
• 4) Ambrose A. J., Chapman E., J. Med. Chem., 64, 7060–7082 (2021).
• 5) Rousaki A., Miyata Y., Jinwal U. K., Dickey C. A., Gestwicki J. E., Zuiderweg E. R. P., J. Mol. Biol., 411, 614–632 (2011).
• 6) Wang A. M., Miyata Y., Klinedinst S., Peng H.-M., Chua J. P., Komiyama T., Li X., Morishima Y., Merry D. E., Pratt W. B., Osawa Y., Collins C. A., Gestwicki J. E., Lieberman A. P., Nat. Chem. Biol., 9, 112–118 (2013).
• 7) Koya K., Li Y., Wang H., Ukai T., Tatsuta N., Kawakami M., Shishido T., Chen L. B., Cancer Res., 56, 538–543 (1996).
• 8) Koren J. III, Miyata Y., Kiray J., O’Leary J. C. III, Nguyen L., Guo J., Blair L. J., Li X., Jinwal U. K., Cheng J. Q., Gestwicki J. E., Dickey C. A., PLOS ONE, 7, e35566 (2012).
• 9) Wadhwa R., Sugihara T., Yoshida A., Nomura H., Reddel R. R., Simpson R., Maruta H., Kaul S. C., Cancer Res., 60, 6818–6821 (2000).
• 10) Donati B., Lorenzini E., Ciarrocchi A., Mol. Cancer, 17, 164 (2018).
• 11) Yang Z., Yik J. H. N., Chen R., He N., Jang M. K., Ozato K., Zhou Q., Mol. Cell, 19, 535–545 (2005).
• 12) Kanno T., Kanno Y., LeRoy G., Campos E., Sun H.-W., Brooks S. R., Vahedi G., Heightman T. D., Garcia B. A., Reinberg D., Siebenlist U., O’Shea J. J., Ozato K., Nat. Struct. Mol. Biol., 21, 1047–1057 (2014).
• 13) Delmore J. E., Issa G. C., Lemieux M. E., et al., Cell, 146, 904–917 (2011).
• 14) Duan Y., Guan Y., Qin W., Zhai X., Yu B., Liu H., MedChemComm, 9, 1779–1802 (2018).
• 15) Filippakopoulos P., Qi J., Picaud S., et al., Nature (London), 468, 1067–1073 (2010).
• 16) Sarnik J., Popławski T., Tokarz P., Int. J. Mol. Sci., 22, 11102 (2021).
• 17) Winter G. E., Buckley D. L., Paulk J., Roberts J. M., Souza A., Dhe-Paganon S., Bradner J. E., Science, 348, 1376–1381 (2015).
• 18) Kaushik S., Cuervo A. M., Nat. Rev. Mol. Cell Biol., 19, 365–381 (2018).
• 19) Yaglom J. A., Wang Y., Li A., Li Z., Monti S., Alexandrov I., Lu X., Sherman M. Y., Sci. Rep., 8, 3010 (2018).
• 20) Kanda Y., Bone Marrow Transplant., 48, 452–458 (2013).