Biomolecules & Therapeutics 2024; 32(1): 115-122  https://doi.org/10.4062/biomolther.2023.104
Contribution of HSP90 Cleavage to the Cytotoxic Effect of Suberoylanilide Hydroxamic Acid In Vivo and the Involvement of TXNIP in HSP90 Cleavage
Sangkyu Park1,†, Dongbum Kim2,†, Haiyoung Jung3,4,5, In Pyo Choi4, Hyung-Joo Kwon2,6 and Younghee Lee1,7,*
1Biotechnology Research Institute, Chungbuk National University, Cheongju 28644,
2Institute of Medical Science, College of Medicine, Hallym University, Chuncheon 24252,
3Aging Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141,
4Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141,
5Department of Functional Genomics, Korea University of Science and Technology (UST), Daejeon 34113,
6Department of Microbiology, College of Medicine, Hallym University, Chuncheon 24252,
7Department of Biochemistry, College of Natural Sciences, Chungbuk National University, Cheongju 28644, Republic of Korea
*E-mail: yhl4177@cbnu.ac.kr
Tel: +82-43-261-3387, Fax: +82-43-267-2306
The first two authors contributed equally to this work.
Received: May 30, 2023; Revised: July 11, 2023; Accepted: July 21, 2023; Published online: January 1, 2024.
© The Korean Society of Applied Pharmacology. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Heat shock protein (HSP) 90 is expressed in most living organisms, and several client proteins of HSP90 are necessary for cancer cell survival and growth. Previously, we found that HSP90 was cleaved by histone deacetylase (HDAC) inhibitors and proteasome inhibitors, and the cleavage of HSP90 contributes to their cytotoxicity in K562 leukemia cells. In this study, we first established mouse xenograft models with K562 cells expressing the wild-type or cleavage-resistant mutant HSP90β and found that the suppression of tumor growth by the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) was interrupted by the mutation inhibiting the HSP90 cleavage in vivo. Next, we investigated the possible function of thioredoxin interacting protein (TXNIP) in the HSP90 cleavage induced by SAHA. TXNIP is a negative regulator for thioredoxin, an antioxidant protein. SAHA transcriptionally induced the expression of TXNIP in K562 cells. HSP90 cleavage was induced by SAHA also in the thymocytes of normal mice and suppressed by an anti-oxidant and pan-caspase inhibitor. When the thymocytes from the TXNIP knockout mice and their wild-type littermate control mice were treated with SAHA, the HSP90 cleavage was detected in the thymocytes of the littermate controls but suppressed in those of the TXNIP knockout mice suggesting the requirement of TXNIP for HSP90 cleavage. We additionally found that HSP90 cleavage was induced by actinomycin D, β-mercaptoethanol, and p38 MAPK inhibitor PD169316 suggesting its prevalence. Taken together, we suggest that HSP90 cleavage occurs also in vivo and contributes to the anti-cancer activity of various drugs in a TXNIP-dependent manner.
Keywords: Heat shock protein 90 cleavage, Thioredoxin interacting protein, Histone deacetylase inhibitor, Anti-cancer, Reactive oxigen species, Caspase
INTRODUCTION

Heat shock proteins (HSPs) are expressed in most living organisms, and their expression is increased by exposure to stresses such as heat shock (Welch, 1993). Because HSP90 is a molecular chaperone, it contributes to the stability of client proteins, and many of the client proteins are necessary for cancer cell growth and survival. Thus, most cancer cells highly express HSP90 compared to normal cells, and HSP90 has been investigated as a target for anti-cancer drugs (Modi et al., 2007, 2011; Dickson et al., 2013). HSP90 cleavage induces a decrease in the HSP90 activity; therefore, it may be one of the HSP90 suppression mechanisms (Park et al., 2015, 2017, 2019, 2021). The cleavage of HSP90 occurs upon various stimuli and can be classified as enzymatic cleavage and non-enzymatic cleavage (Shen et al., 2008; Beck et al., 2009; Chen et al., 2009; Karkoulis et al., 2010; Beck et al., 2012; Liu et al., 2014; Park et al., 2015; Fritsch et al., 2016; Wu et al., 2016; Park et al., 2017; Castro et al., 2019; Park et al., 2019). Enzymatic cleavage is executed by caspase 10, and it is activated by the Fas axis, reactive oxygen species (ROS) generation, etc. (Chen et al., 2009; Park et al., 2015, 2017). Non-enzymatic cleavage is generated by chemical degradation by ROS generation (Beck et al., 2009, 2012; Castro et al., 2019). The enzymatically cleaved HSP90 fragment is approximately 55 kDa, and the non-enzymatically cleaved HSP90 fragment is approximately 70 kDa (Beck et al., 2009; Chen et al., 2009; Beck et al., 2012; Park et al., 2015, 2017; Castro et al., 2019; Park et al., 2019). The enzymatic HSP90 cleavage mainly occurs in the HSP90β isotype among cytosolic HSP90, HSP90α and HSP90β (Park et al., 2017), and this phenomenon leads to a reduction in the HSP90β activity and a decrease in cell viability in human cancer cells (Park et al., 2021).

Thioredoxin interacting protein (TXNIP; VDUP-1; TBP-2), a negative regulator of thioredoxin (Trx), was first isolated as a protein whose expression increases in vitamin D treated HL60 cells (Chen and DeLuca, 1994). Trx is one of the major antioxidants regulating the intracellular homeostasis of redox in mammalian cells (Kaimul et al., 2007), and TXNIP inhibits the function of Trx by directly interacting with the catalytic active site or reducing the expression of Trx (Nishiyama et al., 1999; Junn et al., 2000; Chung et al., 2006). Trx expression was increased in cancer cells, and ROS generation was suppressed by the increased Trx. Therefore, overexpressed Trx promotes growth through an anti-apoptotic function in cancer cells and induces resistance to anti-cancer agents (Kaimul et al., 2007). TXNIP is commonly downregulated in cancer cells. Expression of TXNIP was found in a small portion of primary tumor samples, including renal cell carcinoma, thyroid cancer, breast cancer, and ductal pancreatic cancer (Schroder et al., 2020). Knockout of TXNIP increases chemically-induced hepatocarcinogenesis in mice (Kwon et al., 2010), and increasing the expression of TXNIP is considered a novel anti-cancer strategy (Zhou and Chng, 2013).

In our previous studies, we found that the HSP90 cleavage was induced by histone deacetylase (HDAC) inhibitors and proteasome inhibitors in leukemia cells (Park et al., 2015, 2017). The cleavage occurs through enzymatic processing triggered by ROS generation and caspase 10 activation (Park et al., 2019). Furthermore, we found that the major cleavage site of HSP90β was the 294th aspartic acid, and the cleavage of HSP90 contributes to the cytotoxic effect of the inhibitors in cancer cells (Park et al., 2021). We previously suggested that the ROS may be generated by the up-regulation of TXNIP upon proteasome inhibitor treatment (Park et al., 2017); however, the relationship between TXNIP and HSP90 cleavage is still unclear. This study shows that the cleavage of HSP90 contributes to the suppression of tumor growth induced by the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA; vorinostat) in a mouse xenograft model. Furthermore, we directly proved that the cleavage of HSP90 is modulated by TXNIP.

MATERIALS AND METHODS

Cell culture

K562 (chronic myelogenous leukemia) cells were maintained in Iscove’s Modified Dulbecco’s Medium (IMDM, Hyclone, Logan, UT, USA) with 10% fetal bovine serum (FBS, Hyclone), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C under a humidified atmosphere of 5% CO2. The cells were treated with SAHA (Sigma-Aldrich, St. Louis, MO, USA), actinomycin D (ActD, Sigma-Aldrich), PD169316 (Sigma-Aldrich), or beta-mercaptoethanol (2-ME, Sigma-Aldrich) at the indicated concentration for the indicated period. DMSO (0.1%) was used as a vehicle control for SAHA, ActD, and PD169316. Distilled water (DW) was used as a control for 2-ME. N-acetylcysteine (NAC, Sigma-Aldrich), cycloheximide (CHX, Sigma-Aldrich) and Z-VAD-FMK (pan-caspase inhibitor, Sigma-Aldrich) were pretreated for 1 h before the SAHA treatment, if necessary. K562-HSP90β WT and K562-HSP90β D294A cells, which are stable cell lines overexpressing HSP90β wild type and D294A mutant under the control of retroviral promoter, respectively (Park et al., 2021), were used for the xenograft study in mice

Animal experiments

All procedures for the animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals of the National Veterinary Research and Quarantine Service of Korea with the approval of the Institutional Animal Care and Use Committee of Hallym University (Permit Number: 2013-98, 2020-31). Five-week-old female immune deficient NOD/ShiLtJ-Rag2em1AMCIl2rgem1AMC (NRGA) mice (n=8/group) were obtained from JA BIO, Inc. (Suwon, Korea). Eight-week-old BALB/c and C57BL/6 mice were obtained from Nara Biotech, Inc. (Seoul, Korea). TXNIP knockout C57BL/6 and the littermate control mice were previously established by our group (Lee et al., 2005). The mice were maintained under specific pathogen-free conditions (20-25°C, 40-45% humidity, 12 h light/dark cycle and food and water access ad libitum).

For the mouse xenograft model, K562-HSP90β WT or K562-HSP90β D294A cells (1×106 cells/mouse) in a 50% Matrigel solution (HBSS/Matrigel, 1:1v/v, Corning Inc., Corning, NY, USA) were injected subcutaneously into the NRGA mice in the dorsal right flank (Park et al., 2021). After 10 days, when the tumors reached 50-100 mm3, the mice were injected intraperitoneally with DMSO vehicle control or SAHA (Sigma-Aldrich; 500 μg/mice) every day for 10 days. The body weight and tumor volumes were measured at 2-day intervals starting from the initial SAHA injection. Twenty days after implantation, the mice were sacrificed, and the tumors were dissected and weighed.

Mouse primary cells were isolated from the BALB/c, C57BL/6, TXNIP knockout C57BL/6 and littermate control mice. After the mice were sacrificed, the spleen and thymus were prepared and passed through a 40 μm cell strainer (BD, San Jose, CA, USA). Whole splenocytes and thymocytes were incubated with a red blood cell lysis buffer (Sigma-Aldrich) for 3 min at 37°C to remove the red blood cells and then resuspended in Roswell Park Memorial Institute (RPMI) 1640 with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. After counting, about 1×108 cells were placed on a 10 cm dish and incubated overnight at 37°C under a humidified atmosphere of 5% CO2. The cells were treated with SAHA for 24 h.

Reverse transcription PCR (RT-PCR)

Total RNA was isolated using TRI Reagent® according to the manufacturer’s instructions (MRC, Cincinnati, OH, USA). Two μg of total RNA were reverse-transcribed in first-strand synthesis buffer containing 6 μg/mL oligo(dT) primer, 50 U M-MLV reverse transcriptase, 2 mM dNTP, 10 mM DTT, and 40 U RNaseOUT™ recombinant ribonuclease inhibitor (Invitrogen, Carlsbad, CA, USA). The reaction was done at 37°C for 50 min and heat inactivated at 70°C for 15 min. One μL of synthetic cDNA was subjected to a standard PCR reaction for 20 cycles of denaturation for 40 s at 95°C, annealing for 40 s at 58°C, and extension for 40 s at 72°C. The primer set sequences used were as follows: GAPDH, 5’-TCC ACC ACC CTG TTG CTG TA-3’ (sense) and 5’-ACC ACA GTC CAT GCC ATC AC-3’ (anti-sense) (product size 452 bp); TXNIP, 5’-CAG CCA ACA GGT GAG AAT GA-3’ (sense) and 5’-AGG GGT ATT GAC ATC CAC CA-3’ (anti-sense) (product size 223 bp).

Quantitative RT-PCR (RT-qPCR)

For a quantitative analysis of the mRNA expression, real-time RT-PCR analysis was performed using an iQ™ SYBR® Green Supermix and CFX Connect™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The TXNIP and GAPDH primers and the cDNA synthesis procedure were the same as those for the standard PCR. The Trx primer set sequences used were as follows: Trx, 5’-GAG AGC AAG CAG CGA GTC TT-3’ (sense) and 5’-TTG GCT CCA GAA AAT TCA CC-3’ (anti-sense) (product size 371 bp). The mRNA levels were normalized using GAPDH as an internal control. Then, all normalized values within the dataset were calculated relative to untreated K562 cells with the 2-ΔΔCT method (Livak and Schmittgen, 2001).

Western blotting

Harvested cells were lysed in a lysis buffer (pH 8.0, 20 mM Tris-HCl, 137 mM NaCl, 10% glycerol, 10 mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40, protease inhibitor cocktail, and phosphatase inhibitor). Samples were resolved by SDS-polyacrylamide gel electrophoresis, and electro-transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA). The membranes were blocked with 5% dry milk in phosphate buffered saline-Tween 20 (PBS-T; 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, and 0.05% Tween 20) and probed with the appropriate primary antibodies. The monoclonal antibodies anti-HSP90α/β (#sc-13119) and anti-GAPDH (#sc-32233) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The monoclonal antibody anti-TXNIP (#K0205-3) and anti-Actin (#MAB1501) were from MBL International Corporation (Woburn, MA, USA) and Millipore, respectively. Immunoreactive proteins were detected by horseradish peroxidase-conjugated anti-rabbit (#111-035-003) and anti-mouse (#115-035-003) secondary antibodies (Jackson Immunoresearch, West Grove, PA, USA) and an ECL solution (iNtRon, Seongnam, Korea).

Statistics

The results are shown as the mean ± standard deviation (SD) or standard error of the mean (SEM) from at least three independent experiments. The statistical significance of the differences between two samples was evaluated using the student’s t-test. p<0.05 was considered statistically significant.

RESULTS

The HSP90 cleavage contributes to the anti-cancer effect of SAHA in a mouse xenograft model

Previously, we found that the cleavage of HSP90β was suppressed by the mutation of the 294th aspartic acid residue to alanine (D294A). The cytotoxicity of the HDAC inhibitor or proteasome inhibitor was decreased by the suppression of the HSP90 cleavage in transgenic K562 cells expressing HSP90β D294A compared to the transgenic control cells expressing the wild-type HSP90β (Park et al., 2021). To check whether the HSP90 cleavage in tumors contributes to the cytotoxic effect of SAHA in vivo, we established a mouse xenograft model by implanting K562-HSP90β WT or K562-HSP90β D294A cells into the immune-deficient NRGA mice. After SAHA was intraperitoneally injected into the mice according to the scheme shown in Fig. 1A, the mice were sacrificed, and the tumors were dissected. The tumor growth was significantly suppressed (42% reduction) by the SAHA treatment in the mice implanted with the K562-HSP90β WT cells, but there was no significant effect in the mice implanted with the K562-HSP90β D294A cells (Fig. 1B-1D) in terms of the tumor volumes (Fig. 1B, 1C) and tumor weights (Fig. 1D). On the other hand, the SAHA treatment did not induce significant side effects because there was no difference in the body weights between the SAHA-treated mice and the control mice during the experiment (Fig. 1E). These data suggest that HSP90 cleavage contributes to the cytotoxic effect of SAHA in a mouse xenograft model.

Figure 1. Modulation of the anti-cancer activity of SAHA by HSP90 cleavage in a mouse xenograft model. (A) Experimental schedule. A xenograft mouse model was established by implantation of K562-HSP90β WT or K562-HSP90β D294A cells in the immune deficient NOD/ShiLtJ-Rag2em1AMCIl2rgem1AMC (NRGA) mice. From ten days after the injection, DMSO or SAHA was intraperitoneally injected into the mice every day, and various characteristics such as the tumor volume and mice weight were monitored (n=8/group). (B) Macroscopic appearance of the tumor tissues derived from the K562-HSP90β WT and K562-HSP90β D294A cells. (C) Tumor volumes were calculated as (length x width2)/2 (n=8/group). (D) Tumor weight (n=8/group). (E) Body weight. Values are the mean ± SEM (n=8/group). ***p<0.001 vs. DMSO control. SAHA, suberoylanilide hydroxamic acid.

Up-regulation of the TXNIP gene expression is induced by SAHA in K562 cells

To better understand the regulatory mechanism of the HSP90 cleavage, we investigated the role of TXNIP. Our previous study showed that MG132, a proteasome inhibitor, induced the expression of TXNIP (Park et al., 2017). Therefore, we hypothesized that similar increase in TXNIP expression in response to SAHA triggers caspase 10 activation by ROS generation, leading to HSP90 cleavage and ultimately contributing to the cytotoxic effect of SAHA. To evaluate this possibility, we first examined the HDAC inhibitor-mediated modulation of the TXNIP expression in K562 cells. SAHA increased the expression of TXNIP mRNA at 6 h and further expression of TXNIP was detected at 24 h after the treatment (Fig. 2A). Accordingly, the protein level of TXNIP was increased as well (Fig. 2B). Because TXNIP is also known to reduce the expression of Trx (Nishiyama et al., 1999; Junn et al., 2000; Chung et al., 2006), the expression of Trx mRNA was also investigated and found to be decreased by SAHA at 24 h without any significant decrease at 6 h after the treatment (Fig. 2A). In addition, the increase of the TXNIP protein induced by SAHA was suppressed by pretreatment with cycloheximide (CHX), a translation inhibitor, implying the requirement of new protein synthesis (Fig. 2C). These data support our hypothesis that SAHA induces the up-regulation of TXNIP, and the increased TXNIP may suppress the Trx expression and increase the ROS level.

Figure 2. SAHA-induced expression of TXNIP mRNA and protein in K562 cells. (A, B) K562 cells were treated with DMSO or the indicated dose of SAHA. (A) The expression of mRNA was determined by RT-qPCR. Values are the mean ± SD. *p<0.05, **p<0.01 vs. 0 h (n=3). (B) The protein expression was determined by western blot analysis. (C) K562 cells were treated with the indicated dose of cycloheximide for 1 h, followed by treatment with DMSO or 5 μM of SAHA for 24 h. The cell lysates were subjected to western blot analysis using the indicated antibodies. GAPDH was used as a loading control. CHX, cycloheximide. SAHA, suberoylanilide hydroxamic acid.

SAHA induces HSP90 cleavage in mouse thymocytes

To directly investigate the effect of TXNIP on HSP90 cleavage, we used TXNIP knockout mice (Lee et al., 2005). Before this investigation, we first checked whether SAHA-induced HSP90 cleavage occurs in mouse primary cells. Because we observed HSP90 cleavage in human leukemia cells previously (Park et al., 2015, 2017, 2021), mouse thymocytes and splenocytes were isolated from BALB/c mice and treated with SAHA. The HSP90 cleavage was induced by SAHA in the mouse thymocytes but not in the splenocytes (Fig. 3A). Because we used TXNIP knockout C57BL/6 mice for the next experiments, HSP90 cleavage was also investigated in thymocytes isolated from C57BL/6. Considering that the cleavage of HSP90 was induced by ROS generation and caspase 10 activation in human cells (Park et al., 2015, 2017), the thymocytes were pretreated with media control, NAC, an antioxidant, or Z-VAD-FMK, a pan-caspase inhibitor, and then observed their effects on the SAHA-induced HSP90 cleavage. The HSP90 cleavage induced by SAHA also occurred in the thymocytes from C57BL/6, and the cleavage was inhibited when the ROS generation and caspase activation were suppressed (Fig. 3B, 3C). Therefore, we confirmed that the HSP90 cleavage is induced in mouse thymocytes through the same mechanism.

Figure 3. SAHA-induced HSP90 cleavage in mouse thymocytes. (A) The thymocytes and splenocytes were isolated from BALB/c mice and treated with DMSO or indicated dose of SAHA for 24 h. (B, C) The thymocytes were isolated from C57BL/6 mice and treated with 10 mM of NAC (B) or 2.5 μM of Z-VAD-FMK (C) for 1 h, followed by treatment with DMSO or 10 μM of SAHA for 24 h. The cell lysates were subjected to western blot analysis using the indicated antibodies. GAPDH or actin were used as a loading control. NAC, N-acetylcysteine. Z-VAD-FMK, pan-caspase inhibitor. Con, control. SAHA, suberoylanilide hydroxamic acid.

SAHA-induced HSP90 cleavage is mediated by TXNIP

To investigate whether TXNIP is involved in the HSP90 cleavage using TXNIP knockout mice, we first confirmed TXNIP expression in the thymocytes of TXNIP knockout mice and littermate controls (Fig. 4A). When the thymocytes of the mice were treated with SAHA, the HSP90 cleavage was detected in the littermate controls but barely detected in the TXNIP knockout mice (Fig. 4B). Therefore, we conclude that the ROS-mediated enzymatic cleavage of HSP90 is modulated by TXNIP.

Figure 4. Decrease of the SAHA-induced HSP90 cleavage in TXNIP knockout mice. (A, B) Thymocytes were isolated from TXNIP knockout and wild-type littermate control mice. (A) The expression of TXNIP mRNA in the thymocytes was determined by RT-PCR analysis. (B) The thymocytes were treated with DMSO or 10 μM of SAHA for 24 h. The cell lysates were subjected to western blot analysis using the indicated antibodies. Actin was used as a loading control. SAHA, suberoylanilide hydroxamic acid.

Cleavage of HSP90 occurs in response to various compounds

To investigate the molecular mechanism of TXNIP up-regulation induced by SAHA, we pretreated the cells with actinomycin D (ActD), a transcription inhibitor, and investigated the effect on the TXNIP expression. Unexpectedly, we found that ActD also induces HSP90 cleavage in a dose-dependent manner (Fig. 5A, upper panel). ActD increased the activity of various caspases, including caspase 10, in the K562 cells (Fig. 5B), which is in accordance with previous reports showing that ActD induces caspase activation and apoptosis (Kleeff et al., 2000; Wang et al., 2007; Lu et al., 2015). Therefore, we speculate that ActD activates caspase 10 and induces the cleavage of HSP90. Furthermore, we found that PD169316, a p38 MAPK inhibitor, and β-mercaptoethanol (2-ME), an antioxidant, also induce HSP90 cleavage (Fig. 5A, middle and lower panel). PD169316 and 2-ME commonly increased the caspase 10 activity shown in Fig. 5C. These data suggest that the cleavage of HSP90 is induced by various compounds.

Figure 5. HSP90 cleavage was induced by various compounds via caspase 10 activation. (A) K562 cells were treated with the indicated dose of actinomycin D (ActD), PD169316 or β-mercaptoethanol (2-ME) for 24 h. DMSO or distilled water (DW) were used as a control. The cell lysates were subjected to western blot analysis using the indicated antibodies. GAPDH was used as a loading control. (B, C) K562 cells were treated with DMSO or 50 ng/mL of ActD, 50 μM of PD169316 or 20 mM of 2-ME for 24 h. The cell lysates were incubated with each colorimetric caspase substrate (B (n=6)) or caspase 10 substrate (C (n=8)), and the activation of caspase was monitored by measuring the color development at a wavelength of 405 nm. Values are the mean ± SD. ***p<0.001 vs. DMSO control.
DISCUSSION

HSP90 contributes to the survival and growth of cancer cells through the stabilization of client proteins (Park et al., 2019). HDAC inhibitors and proteasome inhibitors are known anti-cancer drugs (Bolden et al., 2006; Dokmanovic et al., 2007; Curran and McKeage, 2009; Crawford et al., 2011; Eckschlager et al., 2017), and we previously found that these inhibitors induce HSP90 cleavage through ROS generation and caspase 10 activation (Chen et al., 2009; Park et al., 2015, 2017). We also confirmed that HSP90 cleavage contributes to the anti-cancer activity of these inhibitors in cancer cells (Park et al., 2021). In this study, we confirmed the contribution of the HSP90 cleavage to the suppression of tumor growth by SAHA in vivo using a human leukemia xenograft mouse model and proved that the cleavage of HSP90 is modulated by TXNIP using thymocytes from TXNIP knockout mice in vitro.

HDAC deacetylates histone proteins and other proteins such as HSP90. HDAC6, a class 2 HDAC, regulates the HSP90 activity by deacetylation of the 294th (HSP90α)/287th (HSP90β) lysine, which is necessary for HSP90 to function as a molecular chaperone (Bali et al., 2005; Scroggins et al., 2007). Therefore, it is generally assumed that HDAC inhibitors suppress HSP90 activity by HDAC6 inhibition. However, the HSP90 cleavage induced by HDAC inhibitors also contributes to the reduced activity of HSP90 resulting in growth suppression of cancer cells (Park et al., 2021). Therefore, we believe that HSP90 cleavage is another mechanism for the anti-cancer effects of HDAC inhibitors and other anti-cancer reagents that induce HSP90 cleavage. In this study, to clarify whether HSP90 cleavage also occurs in vivo, we first analyzed the effect of SAHA on two kinds of mouse primary cells, thymocytes and splenocytes in vitro. For the previous in vitro study, we mainly used K562 cells which are lymphoblast cells isolated from the bone marrow of chronic myelogenous leukemia patients; however, HSP90 cleavage also occurred in THP-1 (acute monocytic leukemia; monocyte), KG1a (acute myelogenous leukemia; promyeloblast, macrophage cell), U937 (histiocytic lymphoma; monocyte) and RPMI8226 (Plasmacytoma; B lymphocyte) cells (Park et al., 2015). Therefore, we believe that the cleavage of HSP90 may occur in various blood cells including thymocytes and splenocytes. However, the cleavage of HSP90 was detected only in the thymocytes and not in the splenocytes. Most of the murine thymus is composed of the cortex (approximately 85%) (Scollay and Shortman, 1983), and CD4/CD8 double-negative immature T cells are distributed in the cortex. On the other hand, B cells constitute a large proportion of the splenocytes (about 74%), and CD4+ or CD8+ T cells account for about 19% (Langeveld et al., 2006). Thymocytes have a higher percentage of T cells and a lower percentage of B cells compared to spleen cells; therefore, this result suggests a cell type specificity: HSP90 cleavage may occur only in T cells (possibly premature T cells) but not in B cells. However, given that the cleavage occurs also in RPMI8226, a cell line derived from B lymphocytes, it may imply a different sensitivity to specific cells as we previously observed in several cell lines (Park et al., 2015, 2021). Recently, we found that the cleavage of HSP90 is induced by HDAC inhibitors and proteasome inhibitors in pluripotent cells such as mouse embryonic cells and induced pluripotent cells. However, the cleaved HSP90 was not found in the cell lysates because the cleaved HSP90 fragment is secreted out of the cells through exosomes (Choi et al., 2022). Therefore, further research is needed to confirm this issue. On the other hand, the cleavage of HSP90 in mouse thymocytes suggests that the inhibition of HSP90 function in normal cells may be one of the side effects induced by anti-cancer drugs causing HSP90 cleavage.

Previously, we found that the SAHA-mediated HSP90 cleavage was suppressed by cycloheximide. This implies that certain proteins are required for the SAHA-mediated HSP90 cleavage (Park et al., 2015). Additionally, we found that TXNIP was upregulated at the protein level by MG132 treatment inducing HSP90 cleavage (Park et al., 2017). It was previously reported that over-expression of TXNIP induced an increase of ROS generation in rat cardiomyocytes (Yoshioka et al., 2004), and oxidative stress induced more apoptosis in TXNIP-overexpressed NIH 3T3 cells (Junn et al., 2000). In addition, the intracellular ROS level was decreased in TXNIP knockout mice compared to wild-type mice (Lee et al., 2005). SAHA induces the up-regulation of TXNIP and down-regulation of Trx in LNCaP prostate carcinoma and T24 bladder carcinoma cells (Butler et al., 2002; Park et al., 2017). Here, we confirmed that SAHA induces the up-regulation of TXNIP and down-regulation of Trx in K562 cells. Because ROS generation has an important role in SAHA- and MG132-mediated HSP90 cleavage, we hypothesized that TXNIP up-regulation is involved in the mechanism for the HSP90 cleavage (Park et al., 2015, 2017). Based on the experimental results with the thymocytes from the TXNIP knockout mice and their littermate controls in this study, we proved that TXNIP expression contributes to the HSP90 cleavage induced by SAHA. In addition to the HSP90 cleavage induced by SAHA, ROS is important also for the enzymatic and non-enzymatic cleavage triggered by other factors (Shen et al., 2008; Beck et al., 2009, 2012; Liu et al., 2014; Wu et al., 2016; Castro et al., 2019). Therefore, further experiments are needed to elucidate whether TXNIP upregulation is generally involved in HSP90 cleavage events.

During the basal study to understand the mechanism for the SAHA-mediated TXNIP up-regulation, we investigated the effect of actinomycin D along with cycloheximide to test whether the increase of TXNIP implies an increased transcription or increased stability of mRNA. Unexpectedly, we found that ActD also induces caspase 10 activation and HSP90 cleavage. We also found that other compounds such as 2-ME and PD169316 induce caspase 10 activation and HSP90 cleavage. Therefore, we suggest that HSP90 cleavage may occur more widely than we know in response to various reagents.

In summary, we directly revealed that HSP90 cleavage is regulated by TXNIP using mouse primary thymocytes from TXNIP knockout mice. We also confirmed the contribution of the HSP90 cleavage to the anti-cancer effect of HDAC inhibitors in mice. These results support our notion that HSP90 cleavage is another important mechanism of the anti-cancer effects in vitro and in vivo and that the cleavage of HSP90 and upregulation of TXNIP need to be considered during anti-cancer drug development.

ACKNOWLEDGMENTS

This research was supported by grants from the National Research Foundation (2018R1A2B6002504, NRF-2021R1A2C1006767) funded by the Ministry of Science and ICT in the Republic of Korea.

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