Published OnlineFirst December 17, 2019; DOI: 10.1158/1535-7163.MCT-19-0777

MOLECULAR CANCER THERAPEUTICS | SMALL MOLECULE THERAPEUTICS

Aurora B Promotes CHIP-Dependent Degradation of HIF1a in Prostate Cancer Cells Kuntal Biswas1, Sukumar Sarkar1, Neveen Said1, David L. Brautigan2, and James M. Larner1

ABSTRACT ◥ Hypoxia is a major factor in tumor progression and resistance dependent . CHIP-dependent HIF1a degradation also occurs to therapies, which involves elevated levels of the transcription in cells arrested in mitosis by nocodazole instead of taxol. Mitotic factor HIF1a. Here, we report that prostate tumor xenografts kinase Aurora B activity is required for taxol-induced HIF1a express high levels of HIF1a and show greatly enhanced growth degradation. Purified Aurora B directly phosphorylates HIF1a at in response to knockdown of the E3 CHIP (C-terminus of multiple sites, and these modifications enhance its polyubiquitina- Hsp70-interacting protein). In multiple human prostate cancer cell tion by CHIP in a purified reconstituted system. Our results show lines under hypoxia, taxol treatment induces the degradation of how activation of Aurora B promotes CHIP-dependent degradation HIF1a, and this response is abrogated by knockdown of CHIP, but of HIF1a in prostate cancer cells. This new knowledge may affect not by E3 ligase VHL or RACK1. HIF1a degradation is accompa- the use of mitotic kinase inhibitors and open new approaches for nied by loss of function, evidenced by reduced expression of HIF1a- treatment of hypoxic prostate tumors.

Introduction Various studies demonstrate that the oxygen level in tissues can range from approximately 4% to 6% oxygen. The prostate has one of It is now established that hypoxia occurs in most solid tumors and the lowest reported median oxygen levels (4%; ref. 12). Physiologic has a major influence on response to therapies (1–3). Under normal stress responses to low level of oxygen occur between 1% and 3% of oxygen levels, hypoxia-inducible factors (HIF) are constantly hydrox- oxygen. In solid tumors, oxygen levels are typically below 1%, indi- ylated by prolyl hydroxylase (PHD) that use oxygen as a cating severe hypoxic stress. Not surprisingly, HIFs are overexpressed substrate. The hydroxylated HIFs are recognized by the von Hippel– in 70% of human tumors relative to adjacent normal tissue (13), Lindau (VHL) protein, a component of an E3-ubiquitin ligase that making those factors attractive targets for anticancer therapies. Pros- initiates degradation through the proteasome (4). At low levels of tate tumors can be very hypoxic (0.3% oxygen), more than 10 times oxygen (hypoxia), the PHD enzymes have insufficient substrate; lower than oxygen levels found in the normal prostate (12, 14). HIF1a is not hydroxylated and therefore escapes degradation. In Prostate tumor hypoxia has been implicated as a driver of malignant response to hypoxia, nonhydroxylated HIF1a translocates to the progression, genetic instability, endothelial-to-mesenchymal transi- nucleus where it partners with constitutively expressed HIF1b subunit tion (15, 16). Hypoxia provides selective pressure for more resistant to form a dimer and upregulate the expression of sets of response cells with greater invasive potential. The changes in tumors induced by genes (2, 4). HIF-independent prosurvival responses also exist and hypoxia have significant implications for cancer treatment. include mTOR, p38 MAPK, and NF-kB pathways (5–7). Thus, a We previously reported that treatment of prostate cancer cells with complex network of cellular and molecular signaling occurs in cells the naturally occurring estrogen metabolite 2-methoxyestradiol (2- exposed to hypoxic stress. Recent reports demonstrate O /PHD/VHL- 2 ME) activated the E3 ligase CHIP (C-terminus of Hsp70-interacting independent regulation of HIF1a activity through various proteins, protein) and increased proteasomal degradation of the androgen including receptor of activated 1 (RACK1; ref. 8), receptor (AR; ref. 17). CHIP interacts with Hsp70 and Hsp90 while carboxyl terminus of HSP70-interacting protein (CHIP; ref. 9), Cullin- mediating the ubiquitination and degradation of multiple chaperone- 5, B-cell lymphoma 2 (Bcl2), and factor inhibiting HIF1 (FIH-1; associated client proteins, including HIF1a (9). Here, we demonstrate refs. 10, 11). that knockdown of CHIP in prostate cancer cells promotes the growth of xenograft tumors. These tumors had high levels of HIF1a. Taxol is used for therapy with castrate-resistant prostate cancer (18, 19). Therefore, we investigated taxol effects on HIF1a expression in 1Department of Radiation Oncology, University of Virginia School of Medicine, Charlottesville, Virginia. 2Center for Cell Signaling, Department of Microbiology, prostate cancer cells grown under hypoxic conditions. Our data reveal – Immunology and Cancer Biology, University of Virginia School of Medicine, a new taxol-Aurora B HIF1a connection that provides new insights to Charlottesville, Virginia. cellular responses to taxol and hypoxia. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Methods and Materials Current address for Neveen Said: Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, NC. Cell culture and reagents Androgen-dependent human prostate carcinoma, LNCaP, and Corresponding Author: James M. Larner, University of Virginia School of Medicine, PO Box 800383, Charlottesville, VA 22908. Phone: 434-924-5564; androgen-independent human prostate carcinoma C4-2 and Fax: 434-243-9789; E-mail: [email protected] 22Rv1cells (American Type Culture Collection) were maintained in RPMI (Gibco-Life Technologies), and human prostate carcinoma Mol Cancer Ther 2020;XX:XX–XX PC3-M cells (American Type Culture Collection) were maintained doi: 10.1158/1535-7163.MCT-19-0777 in DMEM (Gibco) media supplemented with 10% fetal bovine serum  2019 American Association for Cancer Research. and 1% penicillin/streptomycin. All cell lines were maintained at 37 C

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fi in 5% CO2 humidi ed atmosphere. Taxol was purchase from Sigma- HIF1a protein in the nucleus of CHIP knockdown tumor cells Aldrich Chemical, paclitaxel (catalog #T7402). inhibi- (Fig. 1B) compared with controls. In addition, the CHIP knockout tors were purchased from Selleckchem.com, VX-680 (Tozasertib, cat. tumors had significantly higher microvascular density versus con- #S1048) and MLN 8054 (cat. #S1100). trol tumors, as measured by CD31-positive cells. These results showed prostate tumor growth and HIF1a levels were significantly siRNA transfection elevated in tumors as a result of reduction of CHIP. siRNA was transfected into different prostate cancer cell line It is possible that the increase in prostate tumor growth of CHIP (LNCaP, C4-2, and 22RV1) cells using RNAi-MAX (Invitrogen) as knockdown cells was due to elevated AR levels because CHIP is also per the manufacturer's protocol. Briefly, 5 Â 105 cells were seeded in an E3 ligase for AR (17). To explore this possibility, we used PC3 10-cm dish in growth medium. Next day, transfection complex (siRNA cells that lack expression of AR. We prepared control and CHIP and RNAiMax) was added to the cells when cells are in OptiMEM knockdown PC3 cells, injected them subcutaneously into the flanks without serum, the cells were incubated for 4 to 6 hours, transfection of SCID mice, and measured tumors for up to 6 weeks. CHIP complex was removed and washed with PBS, and growth medium was knockdown in PC3 cells significantly enhanced tumor size in SCID added. Drugs were added, and cells were harvested after 24 hours mice (Supplementary Fig. S1) showing AR was not required for unless otherwise mentioned. The siRNA sequences are given in promotion of the growth of prostate cancer cells after knockdown of Supplementary Methods. CHIP.

Stable cell lines Taxol treatment reduces HIF1a in a dose- and time-dependent Prostate cancer cell lines (LNCaP, C4-2b, and PC3) were stably manner in human prostate cancer cells depleted of CHIP using MISSION TRC shRNA lentiviral particles Prostate tumors tend to be hypoxic and taxol is frequently used in from Sigma according to the manufacturer's protocol. Briefly, cells prostate cancer treatment; therefore, we tested for the effects of were seeded in 12-well plates, infected with 30 mL of virus particle taxol on HIF1a levels in prostate cancer cells. We treated LNCaP, solution in 1 mL of complete growth medium containing polybrene C42, and 22RV1 human prostate cancer cells with increasing (4 mg/mL). Cells were selected by puromycin treatment, and CHIP concentrations of taxol (0–5 mmol/L) for 16 hours under hypoxic depletion was confirmed by Western blot analysis. conditions (<1% oxygen). As expected, HIF1a protein was detected by immunoblotting in prostate cancer cells under hypoxia, relative Western blotting and antibodies to normoxia (Fig. 2A–C;lane1vs.2).Underhypoxia,theaddition Cells were lysed in modifiedRIPAlysisbuffer(50mmol/LTris– of 1 nmol/L to 5 mmol/L taxol reduced HIF1a levels in a dose- HCl, 150 mmol/L NaCl, 5 mmol/L EDTA, 5 mmol/L EGTA, 0.5% dependent manner in LNCaP (Fig. 2A), C4-2 (Fig. 2B), and 22RV1 NP-40, 0.1% SDS, 50 mmol/L NaF, 2 mmol/L sodium orthovana- cells (Fig. 2C).ItisimportanttopointoutthatHIF1b levels were date) supplemented with a protease inhibitor mix (Thermo Scien- unchanged under these conditions in all three cell lines, revealing tific). Unless otherwise described, 30 mgofproteinwasresolvedby the specificity of taxol treatment for HIF1a.WetreatedLNCaP SDS–polyacrylamide gel electrophoresis (PAGE), transferred, and (Fig. 2D), C4-2 (Fig. 2E), and 22RV1 cells (Fig. 2F)with0.1mmol/ immunoblotted with various antibodies. The antibodies used were L taxol under hypoxic condition and harvested after 6 hours, anti-GAPDH (sc-32233), anti-RACK1 (sc-7754), anti-TPX-2(sc- 12 hours, and 24 hours. By immunoblotting, we observed a 32863) from Santa Cruz Biotechnology (Santa Cruz); anti-CHIP time-dependent reduction of HIF1a starting at 6 hours, increasing (C3B6), anti-VHL(68437), anti-P-AuroraA/B(D13A11), anti-Aur- at 12 hours, and becoming nearly complete at 24 hours. These oraA(83092), anti AuroraB(3094), anti-H3(9715), anti-p-H3(ser10; results revealed a dose- and time-dependent reduction in HIF1a 3377), anti-HIF1b(5537), and anti-cyclinB1(4138) from Cell Sig- levels in response to adding taxol to human prostate cancer cell naling Technology; and anti-HIF1a (610958) from BD Transduc- lines. tion Laboratories. Taxol treatment of cells severely decreased the formation of colonies in soft agar (Fig. 2G). This suggests that reduction of HIF1a levels reduces the fitness of prostate cancer cells in the assay. The response to Results taxol was not seen with CHIP knockdown cells, supporting the idea CHIP knockdown promotes prostate cancer in xenograft model that without CHIP the cells are resistant to taxol treatment. We tested The E3 ligase CHIP (C-terminus of Hsp70-interacting protein) is both taxol (paclitaxel) and taxotere (docetaxel) and observed degra- implicated in support of tumorigenesis and invasion in several dation of HIF1a. malignancies (20–23). The human protein atlas shows an inverse relationship between the level of CHIP and stage of prostate HIF1a protein is degraded by the proteasome after taxol cancer (24) but to date the role of CHIP in prostate cancer has treatment not been explored. To experimentally test whether CHIP affects What was the mechanism for reduction of HIF1a protein levels in prostate cancer growth, we isolated C42-b cells expressing shRNA response to taxol in prostate cancer cells? To address this, we treated targeted for CHIP and cells control with a scrambled sequence 22RV1 prostate cancer cells with or without the proteasome inhibitor shRNA. Both CHIP knockdown and control cells were injected MG132 in the presence or absence of taxol, under both normoxic and subcutaneously in the flanks of SCID mice and we measured the hypoxic conditions (Supplementary Fig. S2A). Under normoxia when tumors over 8 weeks. The results (Fig. 1A) clearly showed a HIF1a levels are barely detectable (lanes 1, 3), the addition of MG132 statistically significant increase in tumor growth of human prostate blocked proteasome degradation and HIF1a was accumulated in the cancer cells with knockdown of CHIP. There are several reports cells (lanes 2, 4). Under hypoxia, HIF1a was detected (lane 5), and supporting hypoxia in prostate tumors and high levels of HIF1a in addition of MG132 further increased the levels (lane 6). Addition of prostate cancer as compared with benign prostate hyperplasia and taxol under hypoxia provoked the degradation of HIF:1a (lane 7 vs. 5), normal tissue (13, 25). IHC revealed dramatically higher levels of and this was blocked by the addition of MG132 (lane 8 vs. 7). Based on

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Aurora B Kinase Promotes HIF1a Degradation through CHIP

A N 2,000 C4-2b_NTsh ( = 6/10*)

C4-2b_shCHIP (N = 10/10)

) 3 1,500

NTsh shCHIP 1,000

CHIP sh-CHIP GAPDH

500 Tumor volume (mm volume Tumor

0 sh-NT 0 2 4 6 8 Weeks B C4-2b NTsh C4-2b shCHIP 400 P < 0.001 300

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/HPF HIF1 Nuclear a

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20 P < 0.001 15

CD31 10

5

0 Mean vascular density (MVD) density vascular Mean C4-2b_NTsh C4-2b_shCHIP

Figure 1. Knockdown of CHIP enhances growth of prostate cancer xenograft tumors. A, Growth of subcutaneous tumors of human C4-2b cell xenografts in SCID mice. C42b cells were stably knocked down for CHIP by lentiviral transduction with short hairpin RNA targeting CHIP (shCHIP) and cells expressing non-targeted RNA (NTsh) used as controls. Tumor growth was significantly increased with P < 0.0001 by two-way ANOVA. Photomicrographs of representative excised tumors are shown on the right. B, Immunostaining of tumors showing significant increase in both nuclear HIF1a and CD31 immunostaining in C4-2b_shCHIP compared with C4-2b_NTsh. Right: top box plots representing the number of nuclear staining of HIF1a cells counted in 4 tumor sections, 6 high power fields/section. Significance at P < 0.001 by two-tailed Student t test; bottom box plots represent the mean vascular density of tumor xenografts calculated from CD31 immunostaining in 4 tumor sections, 6 high power fields/section. Significance of P < 0.001 by two-tailed Student t test. Ã<0.05; ÃÃ<0.01. the effects of MG132, we concluded that taxol was stimulating the is considered the primary E3 ligase for HIF1a degradation. To test proteasome degradation of HIF1a. We confirmed that the effects of whether VHL and/or CHIP mediate taxol-induced HIF1a degra- taxol were on HIF1a protein levels, not mRNA. Quantitative reverse- dation, we knocked down CHIP or VHL individually using siRNA transcription PCR (qRT-PCR) was performed and HIF1a mRNA in two human prostate cancer cell lines. After siRNA transfection levels were unchanged in taxol treated versus untreated controls in cells were exposed to low oxygen in hypoxia chamber for 16 hours, both LNCaP and C4-2 cells (Supplementary Fig. S2C). Likewise, in the presence or absence of taxol. A parallel set of transfected cells HIF1a-mRNA levels were unchanged in taxol-treated versus untreat- waskeptatambientoxygenlevels.InbothLNCaP(Fig. 3A)and ed controls in 22RV1 cells based on qRT-PCR (Supplementary C42 cells (Fig. 3B), knockdown of CHIP limited degradation of Fig. S2B). Apparently, taxol was not interfering with HIF1a transcrip- HIF1a when compared with control siRNA-treated samples, under tion, splicing, or RNA stability because the levels of mRNA were normoxic conditions (lane 1 vs. lane 3). Surprisingly, knockdown unchanged. of VHL did not elevate HIF1a levels in prostate cell lines tested under normoxia (lanes 5 and 6). Knockdown of CHIP but not VHL Taxol-induced HIF1a degradation is CHIP dependent prevented the taxol-induced HIF1a degradation in either cell line We suspected that CHIP might be the E3 responsible for HIF1a (lane 10 vs. 12). Another protein implicated in degradation of ubiquitination and degradation under hypoxic conditions. Recent- HIF1a is the receptor for activated C-kinase 1 (RACK1; ref. 8), ly, it has been reported that CHIP is required for degradation of originally identified as an anchoring protein for activated protein HIF1a through autophagy activated by nutrient deprivation (26). kinase C (PKC). However, knockdown of RACK1 failed to prevent Under normoxic conditions, the von Hippel–Lindau (VHL) factor taxol-induced HIF1a degradation in C4-2 (Supplementary

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LNCaP A D LNCaP Hypoxia Norm- Hypoxia Taxol (mmol/L) oxia 0 0.001 0.01 0.05 0.1 1 5 6 h 12 h 24 h HIF1a Taxol (0.1 mmol/L) - + --++ HIF1a HIF1b

GAPDH GAPDH

B C42 E Norm- Hypoxia C42 Hypoxia Taxol (mmol/L) oxia 0 0.001 0.01 0.05 0.1 1 5 6 h 12 h 24 h a HIF1 Taxol (0.1 mmol/L) --+ ++ - HIF1a HIF1b

GAPDH GAPDH

22RV1 22RV1 C Hypoxia F Norm- Hypoxia Taxol (mmol/L) oxia 0 0.001 0.01 0.05 0.1 1 5 6 h 12 h 24 h m -- - HIF1a Taxol (0.1 mol/L) ++ + HIF1a HIF1b GAPDH GAPDH

Sh-NT Sh-CHIP G Soft-agar colony assay

40 DMSO 35 30 25 20 15 10

5 Taxol No. of colonies of No. 0 DMSO Taxol TaxolDMSO Sh-NT Sh-CHIP

Figure 2. Taxol induces dose- and time-dependent loss of HIF1a in hypoxic cells. LNCaP (A), C42 (B), and 22RV1 (C) cells were treated with different doses of taxol for 16 hours under hypoxic conditions. Cells were harvested and extracts were immunoblotted for HIF1a and HIF1b, plus GAPDH, used as loading control. LNCaP (D), C42 (E), and 22RV1 (F) cells were treated with 0.1 mmol/L taxol and exposed to hypoxia for indicated times. Cell extracts were analyzed for HIF1a by immunoblotting. G, Soft-agar colony formation assay in control and CHIP-depleted cells, treated with or without taxol. The bar graph shows three independent experiments; right, a representative image. No., number.

Fig. S3A) and 22RV1 (Supplementary Fig. S3B) cells in hypoxic (Fig. 3D) was compromised in cells knocked down for CHIP. These conditions. Together, these results support our conclusion that results provide further evidence that CHIP is required for the taxol- CHIP is the primary E3 ligase triggering the degradation of HIF1a induced degradation of HIF1a. To address the possibility that the in response to taxol treatment. effects of siRNA targeting of CHIP were due to off-target The lack of VHL involvement was reinforced by the observation that effects, we knocked out CHIP using CRISPR/Cas9 in C4-2 cells and a VHL-resistant mutant of HIF1a (P402A/P564A) was degraded stably knocked down CHIP using a shRNA (with a different similar to the wild-type (WT) HIF1a in response to taxol treatment sequence from siRNA) in LNCaP cells. Knockout or knock- of cells, under both normoxic and hypoxic conditions (Fig. 3C). We down of CHIP prevented taxol-induced HIF1a degradation in noted that levels of this HIF1a mutant were higher than the WT in cells both C4-2 and LNCaP replicating the response seen with siRNA under normoxia. Both the WT and (P402A/P564A) mutant HIF1a (Fig. 3E). These experiments all support the conclusion that CHIP is were extensively degraded in response to taxol treatment of cells, the primary E3 ligase for HIF1a degradation in cells treated with taxol. indicating that the pathway for taxol stimulation did not depend on Transcription of a number of genes including VEGFA and VHL, consistent with the results in Fig. 3A and B. Moreover, the GLUT1 is dependent on HIF1a (27). We assayed for expression degradation of mutant HIF1a (P402A/P564A) in response to taxol of VEGFA and GLUT1 in cells under normoxic and hypoxic

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Aurora B Kinase Promotes HIF1a Degradation through CHIP

A LNCaP B Normoxia Hypoxia C42 Normoxia Hypoxia siRNA con conCHIP CHIPVHL VHL Taxol ------+ + ++ + + siRNA con conCHIP VHL VHL CHIP Taxol - - - HIF1a - + - + - + + + + HIF1a CHIP CHIP VHL VHL GAPDH GAPDH 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12 C Normoxia Hypoxia D Normoxia

siRNA con CHIP

Vector

Vector Mutant WT Mutant WT Taxol - + - + Taxol - + - + - + - + - + - + a HIF1a HIF1 (P402A/P564A) GAPDH CHIP

GAPDH LNCaP E C42 1 2 3 4 CRISPR/Cas9 Stable knockdown vec KO Sh-NT Sh-CHIP Taxol ---++++- HIF1a

CHIP

GAPDH 1 2 3 4 5 6 7 8

Figure 3. Taxol-induced HIF1a degradation requires E3 ligase CHIP. LNCaP (A) and C42 (B) cells were knocked down for either CHIP or VHL with specific siRNA and treated with or without taxol in the presence or absence of hypoxia. C, C42 cells transiently transfected with empty vector, wt-HIF1a, or mutant (P402A/P564A) HIF1a were treated with or without taxol and exposed to hypoxia or normoxia. HIF1a protein levels were analyzed by immunoblotting. D, C42 cells stably expressing mutant (P402A/P564A) HIF1a were knocked down for CHIP by specific siRNA and treated with or without taxol for 16 hours under normoxia. Extracts were analyzed by immunoblotting for HIF1a, CHIP, VHL, and GAPDH as internal control. E, CHIP was knocked out of C42 cells by CRISP/Cas9, and LNCaP cells were stably knocked down of CHIP by shRNA, and these were each paired with infected control cells. Cells treated with or without taxol under hypoxia. Cell extracts were immunoblotted for indicated proteins.

conditions, and in the presence or absence of taxol (Fig. 4). normoxia (column 5) and knockdown of CHIP enhanced expres- Expression of both genes was relatively low under normoxia, and sion compared with cells with control siRNA. More important, not much affected by any of the conditions tested (columns 1–4). knockdown of CHIP prevented the reduced expression of both Expression of both genes was elevated under hypoxia relative to genes when cells were treated with taxol (Fig. 4). expression

Figure 4. VEGFA GLUT1 6 HIF1a-regulated genes are dependent 8 on E3 ligase CHIP. RT-PCR of HIF1a 5 regulated genes (VEGFA and GLUT1) in 7 4 6 LNCaP cells with CHIP knockdown by 3 5 siRNA, treated with or without taxol. 4 Normalized expression expressed as 2 3 mean of three independent experi- 1 2 ments Æ SD. 1

0 0

Relative expression Relative

Relative expression Relative

(normalized to GAPDH) to (normalized (normalized to GAPDH) to (normalized

si-Con si-CHIP si-Con si-CHIP si-Con si-CHIP si-Con si-CHIP

si-Con-taxol si-Con-taxol si-Con-taxol si-CHIP-taxol si-CHIP-taxol si-Con-taxolsi-CHIP-taxol si-CHIP-taxol Normoxia Hypoxia Normoxia Hypoxia

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reflected the changes in HIF1a protein levels in response to taxol A Normoxia Hypoxia and CHIP knockdown. DMSO Aph DMSO Aph + ++---- +Taxol (0.1 mmol/L) Taxol-induced HIF1a degradation occurs during mitosis HIF1a Taxol acts as a microtubule stabilizing agent and blocks cells in mitosis. We sought to test whether arrest in mitosis was necessary Cyclin B1 for the CHIP-dependent degradation of HIF1a. We arrested C4-2 p-AurA cells in S phase by treatment for 24 hours with aphidicholin, an p-AurB inhibitor of the replicative DNA polymerase. Cells were then treated with or without taxol for an additional 24 hours. Parallel GAPDH samples were kept in normoxia, as controls, or exposed to low 1 2 3 4 5 6 7 8 oxygen in hypoxia chamber for 6 hours. The taxol-induced deg- radation of HIF1a present in hypoxic cells was completely abro- Hypoxia gated when cells were stalled in S phase by aphidicolin (Fig. 5A). B This suggested to us that degradation of HIF1a was occurring LNCaP PC3 C42 during mitosis. Cyclin B1 levels were analyzed by immunoblotting Nocodazole - + - + - + as a marker for cells in mitosis. Alternatively, we treated LNCaP, C4-2, and PC3M cells (Fig. 5B) with or without the microtubule HIF1a disrupting drug nocodazole for 10 hours, performed mitotic shake off, and put the cells in a hypoxic chamber for 6 hours in the pH3 (ser10) continued presence of nocodazole. We observed that nocodazole H3 treatment induced HIF1a degradation, just like taxol (Fig. 5B). We treated LNCaP cells stably expressing nontargeting and GAPDH shCHIP with nocodazole (Fig. 5C). Both sets of cells were arrested in mitosis, based on the increased phosphorylation of Ser10 in Hypoxia histone H3 (Fig. 5C). However, HIF1a was degraded only in the C control cells (NT, lane 1) but not in the CHIP knockdown cells (lane 2). HIF1a was not degraded in asynchronous cells that were not in mitosis (untreated), even though CHIP was present. These

NT NT-asyn

results show that cells needed to be in mitosis, either by taxol or Sh-CHIP-28 nocodazole treatment, in order for CHIP to promote the degra- a dation of HIF1a. HIF1 CHIP Aurora kinase B is required for HIF1a degradation in response to taxol pH3 (ser10) Aurora are activated during mitosis, so we treated LNCaP (Fig. 6A)andC4-2(Fig. 6B) cells with Aurora A/B inhibitors to test H3 for effects on HIF1a levels. We included cells under normoxic conditions as additional controls, even though HIF1a was not GAPDH detected. Under hypoxic conditions, taxol induced degradation of HIF1a in both cell lines (lane 5 vs. 6). Interestingly, the relatively Figure 5. specific inhibitor MLN8054 (28) had little effect on HIF1a degradation in mitotic cells by E3 ligase CHIP. A, C42 cells were treated taxol-induced HIF1a degradation (Fig. 6A,lane7;Fig. 5B,lane8). with either DMSO or aphidicolin for 24 hours; then cells were treated with or On the other hand, VX680 (29), which inhibits both Aurora A without 0.1 mmol/L taxol for 16 hours and exposed to normoxia or hypoxia for an and Aurora B, prevented taxol-induced HIF1a degradation additional 4 hours. Cell extracts were immunoblotted for HIF1a, pAurora-A, pAurora-B, and GAPDH, as a loading control. B, Prostate cancer cells (LNCaP, (Fig. 6A,lane8; Fig. 6A, lane 7). The effects of the inhibitors PC-3, and C42) were treated with nocodazole for 12 hours; the mitotic cells could be seen by phosphosite immunoblotting for activation of shaken off, collected, and extracts immunoblotted for HIF1a, phospho-Ser10 Aurora A and Aurora B. MLN8054 inhibited Thr288 phosphory- histone H3, total histone H3, and GAPDH, as loading control. C, LNCaP cells lation of Aurora A, but not phosphorylation of Aurora B, while, on stably knockdown of CHIP or cells with nontargeting shRNA were treated with the other hand, VX680 inhibited phosphorylation of both Thr288 in nocodazole for 12 hours; the mitotic cells collected by shake off and extracts P Aurora A and Thr232 in Aurora B. These results indicated that immunoblotted for HIF1a, XHI , phospho-Ser10 histone H3, total histone H3, and GAPDH, as loading control. Aurora B, but not Aurora A, was required for the taxol-induced degradation of HIF1a. The relative involvement of Aurora A, B, and C kinases in taxol- failed to prevent taxol-induced HIF1a degradation. In addition, induced HIF1a degradation was studied by siRNA knockdown in knockdown of TPX2, an activator of Aurora A failed to prevent C4-2 (Fig. 6C) and 22RV1 cells (Fig. 6D). Knockdown of Aurora B HIF1a degradation (Supplementary Fig. S4). Further confirmation increased the basal level of HIF1a in the cells and prevented taxol- of the role of Aurora B was provided by knockdown of INCENP, a induced HIF1a degradation (compare lanes 2 and 6). Knockdown subunit of the active Aurora B complex (30), by either one of two of Aurora B alone or in any combination with Aurora A or C was independent siRNA. Knockdown of INCENP prevented taxol- sufficient to prevent the degradation of HIF1a. In contrast, knock- induced HIF1a degradation. Both the results with chemical inhi- down of either Aurora A or Aurora C, or both together, reduced or bitors and shRNA knockdown strongly support Aurora B as the

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Aurora B Kinase Promotes HIF1a Degradation through CHIP

A C C42 C42 Hypoxia Normoxia Hypoxia siRNA con AurA AurB AurA+B AurC AurB+C AurA+C VX680 - -- + --- + ------MLN8054 ------++ Taxol +++ ++++ Taxol --+ ++ +++ HIF1a HIF1a Aurora A P-Aurora A Aurora B

P-Aurora B CHIP Aurora A Aurora B GAPDH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4 5 6 7 8

LNCaP 22RV1 B Normoxia Hypoxia D Hypoxia

VX680 - - ---+ + - siRNA con AurA AurB AurC AurA+B AurB+C AurA+C MLN8054 - - + ----+ ------Taxol --+ ++ ++ + Taxol +++ ++ ++ HIF1a HIF1a Aurora A P-Aurora A Aurora B P-Aurora B GAPDH Aurora A Aurora B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 GAPDH 1 2 3 4 5 6 7 8

Figure 6. Knockdown of Aurora B abrogates taxol-induced HIF1a degradation. C42 cells (A) and LNCaP cells (B) were treated with Aurora kinase inhibitors MLN8054 or VX680 for 16 hours. Cells were exposed to normoxia or hypoxia for additional 6 hours, and then cell extracts were immunoblotted for HIF1a, phospho-Thr in Aurora kinases, total Aurora A/B, and GAPDH, as loading control. C42 cells (C) and 22RV1 cells (D) were transiently knocked down for Aurora A, B, or C individually or in combination for 24 hours, treated with or without taxol for 16 hours and exposed to hypoxia for additional 4 hours, and then harvested and extracts immunoblotted for indicated proteins. mitotic kinase that is required for the CHIP-mediated degradation ponents, plus HSP70 and CHIP as the E3 ligase. Products were of HIF1a. resolved by SDS-PAGE and immunoblotted for HIF1a,withthe polyubiquitinated HIF1a appearing as slower migrating species Aurora kinase B phosphorylates HIF1a in vitro and enhances (Fig. 7B). Polyubiquitylation of HIF1a by CHIP required complete polyubiquitination reactions and omission of ATP as a control prevented formation of Aurora B could exert effects through direct phosphorylation of ubiquitinated products. The amount of polyubiquitinated HIF1a either HIF1a or CHIP. We performed an in vitro kinase assay using formed was 1.3-fold greater in reactions that included Aurora B bacterial expressed, purified recombinant HIF1a, plus purified recom- (Fig. 7B,lane3)comparedwithHIF1a incubated under identical binant Aurora B kinase and g[32P]ATP (Fig. 7A). The kinase assay conditions, but without added kinase (Fig. 7B,lane4).Theactivity showed time-dependent increase in phosphorylation of HIF1a. The of Aurora B under the assay conditions was confirmed by phos- extent of HIF1a radiolabeling was comparable to histone H3, included phorylation of Ser10 in histone H3 (Fig. 7B, bottom). These results as a known Aurora B substrate (Fig. 7A). Autophosphorylation of demonstrated enhanced polyubiquitination of HIF1a due to phos- Aurora B was evident in all the reactions, including without added phorylation by Aurora B. substrates. Analysis by LC/MS-MS revealed Aurora B phosphorylation of HIF1a at multiple sites, in particular S247, S465, and S657. The S247 and S465 sites both have sequences (RXpS) predicted to be Aurora B Discussion substrates. HIF1a S657 was phosphorylated by Aurora B, even though In this study, we discovered that prostate tumors with RNAi it does not conform to the consensus recognition sequence (TSpSP; knockdown of the E3 ligase CHIP grow significantly larger and express ref. 31). This site was one of two in HIF1a previously reported to be high levels of HIF1a. We show CHIP is the dominant E3 ligase for phosphorylated by PLK3 and mutation of S567A/S657A greatly HIF1a in hypoxic prostate cancer cells and HIF1a degradation is extended the half-life of HIF1a (32). We concluded that Aurora B enhanced by the clinically relevant agent paclitaxel and docetaxel, phosphorylates HIF1a, including at sites that affect its proteasomal which induces mitotic arrest of cells. Nocadazole also blocks cells in degradation. mitosis and induces CHIP-dependent HIF1a degradation. Pharma- To test for effects of Aurora B phosphorylation of HIF1a on cologic inhibition or RNAi knockdown of mitotic Aurora B attenuated reaction with CHIP we performed an in vitro ubiquitination assay. HIF1a degradation. We used in vitro assays with purified proteins to These reactions included purified HIF1a along with E1, E2 com- demonstrate Aurora B directly phosphorylates HIF1a, and this

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A P32-ATP B E1+E2+CHIP+HSP70+ub -ATP +AurB -AurB

1 2 3 4

HIF1 a

AurB

+AurB HIF1 a

+AurB HIF1 a

H3 H3+AurB Ub-HIF1a

HIF1a HIF1a

Aurora B 1.3 1.0 H3 + - ATP H3 pH3 (ser10) 1 2 3 4 5 6 H3

Figure 7. Aurora B phosphorylates HIF1a and enhances polyubiquitination by CHIP. A, Purified recombinant HIF1a and histone H3 were used as substrates in an in vitro kinase assay with commercial purified Aurora B kinase, plus radioactive 32P-ATP. Separate reactions of kinase without added substrate and substrates without added kinase were run in parallel, as controls. Reactions were boiled in SDS sample buffer, proteins resolved by SDS-PAGE, and the gels used to expose X-ray film by autoradiography. The controls show no radiolabeling of either substrate without added kinase, only one band of autophosphorylated Aurora B kinase. Phosphorylation of HIF1a at 30 and 50 minutes of reaction showed an increase in radiolabeling. B, In vitro reconstituted ubiquitination reactions used commercial E1, E2, and ubiquitin, plus HSP70 and CHIP as the E3 ligase. Products were resolved by SDS-PAGE and analyzed by immunoblotting for HIF1a. No reaction occurred without addition of ATP (lane 2), and the formation of polyubiquitinated HIF1a species was greatly enhanced by the addition of Aurora B kinase to the reactions (lane 3 vs. 4). Bottom, active AurB phosphorylates histone H3, as a positive control.

increased CHIP-dependent polyubiquitination. Mass spectrometry substrates that are critical for prostate cancer is dependent on cells showed Aurora B phosphorylates multiple sites in HIF1a, including being in mitosis. S657, already known to accelerate protein turnover. Thus, arresting Our observations have potential implications for the therapy of cells in mitosis activates Aurora B to phosphorylate HIF1a,making prostate cancer patients. TCGA data established the inverse relation- it a better substrate for the E3 ligase CHIP, thereby enhancing ship between high HIF1a and low CHIP level in prostate cancer, and HIF1a degradation and reducing the expression of HIF1a depen- correlate with poor outcomes for patients. Thus, HIF1a and CHIP dent genes. could be useful biomarkers to track prostate cancer progression and These new results expand our knowledge about how both mitotic segregate patients for different treatments. Docetaxel is the most kinases Aurora A and Aurora B regulate selective protein turnover by commonly used cytotoxic agent in castrate-resistant prostate cancer. the E3 ligase CHIP. We previously reported Aurora A (not Aurora B) The mechanism of action is thought to be blocking cell-cycle pro- phosphorylation of CHIP during mitosis enhances AR degradation in gression by preventing disassembly the mitotic spindle, causing pro- prostate cancer cells (33). On the other hand, our present results longed mitotic arrest. We suggest that taxanes may also inhibit prostate establish that Aurora B (not Aurora A) promotes CHIP-dependent cancer by promoting HIF1a degradation and downregulating HIF1a- degradation of HIF1a by phosphorylation of HIF1a. In one case, dependent genes. The relationship between CHIP, HIF1a, and the Aurora A activates the (CHIP, an E3 ligase), in the other, response to docetaxel is something for further study. Aurora B Aurora B predisposes a particular substrate (HIF1a) to ubiquitination inhibitors are in clinical trials for multiple solid tumors. Our data by CHIP. Aurora A and Aurora B are activated in different stages of suggest that these inhibitors should be used with caution because we mitosis and in distinct intracellular locations, suggesting that timing predict they will increase HIF1a levels in tumors, and this is associated and location are critical parameters in determining specificity for with therapeutic resistance and poor prognosis. substrates and functions (34). Aurora A kinase is known to regulate Finally, hypoxia is one of the hallmarks of solid tumors and HIF1a is mitotic entry, spindle formation, and centrosome maturation and is activated under hypoxic conditions, which promotes angiogenesis, concentrated at centrosomes (35). Experimental overexpression of EMT, and survival of tumor cells (37, 38). Hypoxia in prostate tumor is Aurora A overrides the mitotic spindle checkpoint and induces considered an early event (39), and several reports supporting hypoxia resistance to paclitaxel (36). In contrast, Aurora B kinase is an itself as an independent risk factor for tumor progression, resistance, integral component of the Passenger Complex that is and treatment failure (40, 41). New knowledge from this study could be associated with chromatin during mitosis, and is differentially useful in creating new therapies for prostate cancer. Agents that regulated at chromosome arms and kinetochores to orchestrate directly activate CHIP, or indirectly support its activation by phos- equal chromosome segregation (35). Agents that disrupt microtu- phorylation or limit its inactivation by dephosphorylation, could lead bule dynamics can arrest cells at different stages in mitosis may to reduced levels of HIF1a and thereby limit tumor growth and therefore have markedly different effects on degradation of pro- survival and prevent resistance to chemotherapies. Phosphorylation teins, such as AR and HIF1a, that are involved in prostate cancer of HIF1a promotes ubiquitination and degradation by CHIP, so progression. CHIP is a common factor, and its actions on different activators of Aurora B or inhibitors of the phosphatase and/or

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Aurora B Kinase Promotes HIF1a Degradation through CHIP

regulatory subunit(s) that target S657 in HIF1a could be effective Writing, review, and/or revision of the manuscript: K. Biswas, S. Sarkar, N. Said, therapeutic agents. Another possibility is for drug inhibitors of the D.L. Brautigan, J.M. Larner deubiquitinases that reverse the CHIP-mediated ubiquitin modifica- Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K. Biswas, S. Sarkar tions, which would promote proteasome degradation of HIF1a to limit Study supervision: K. Biswas, S. Sarkar, D.L. Brautigan, J.M. Larner the growth of prostate tumors and render them more susceptible to chemotherapeutic drugs. Acknowledgments fl We thank Dr. Nicolas Sherman of Biomolecular Analysis Facility, University of Disclosure of Potential Con icts of Interest Virginia, for Mass spectrometry analysis of phosphopeptides. This work was sup- No potential conflicts of interest were disclosed. ported by grant NIH CA192669 and the Charles Burnett fund.

Authors’ Contributions The costs of publication of this article were defrayed in part by the payment of page Conception and design: K. Biswas, S. Sarkar, N. Said, D.L. Brautigan, J.M. Larner charges. This article must therefore be hereby marked advertisement in accordance Development of methodology: K. Biswas, S. Sarkar, N. Said, D.L. Brautigan with 18 U.S.C. Section 1734 solely to indicate this fact. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K. Biswas, S. Sarkar, N. Said, J.M. Larner Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Received August 8, 2019; revised September 25, 2019; accepted December 12, 2019; computational analysis): K. Biswas, S. Sarkar, N. Said, D.L. Brautigan, J.M. Larner published first December 17, 2019.

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Aurora B Kinase Promotes CHIP-Dependent Degradation of HIF1 α in Prostate Cancer Cells

Kuntal Biswas, Sukumar Sarkar, Neveen Said, et al.

Mol Cancer Ther Published OnlineFirst December 17, 2019.

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