HSP70 Inhibition Synergistically Enhances the Effects of Magnetic Fluid Hyperthermia in Ovarian Cancer Karem A

Published OnlineFirst February 21, 2017; DOI: 10.1158/1535-7163.MCT-16-0519

Models and Technologies Molecular Cancer Therapeutics HSP70 Inhibition Synergistically Enhances the Effects of Magnetic Fluid Hyperthermia in Ovarian Cancer Karem A. Court1, Hiroto Hatakeyama2,3, Sherry Y. Wu2, Mangala S. Lingegowda2, Cristian Rodríguez-Aguayo4, Gabriel Lopez-Berestein 4,5, Lee Ju-Seog6, Carlos Rinaldi7,8, Eduardo J. Juan9, Anil K. Sood2,5, and Madeline Torres-Lugo1

Abstract

Hyperthermia has been investigated as a potential treatment for Hsp70-related genes, siRNA and Hsp70 protein function inhibi- cancer. However, specificity in hyperthermia application remains tion by 2-phenylethyenesulfonamide (PES). Both strategies a significant challenge. Magnetic fluid hyperthermia (MFH) may resulted in decreased cell viability following exposure to MFH. be an alternative to surpass such a challenge, but implications of Combination index was calculated for PES treatment reporting a MFH at the cellular level are not well understood. Therefore, the synergistic effect. In vivo efficacy experiments with HSPA6 siRNA present work focused on the examination of gene expression after and MFH were performed using the A2780cp20 and HeyA8 MFH treatment and using such information to identify target ovarian cancer mouse models. A significantly reduction in tumor genes that when inhibited could produce an enhanced therapeutic growth rate was observed with combination therapy. PES and outcome after MFH. Genomic analyzes were performed using MFH efficacy were also evaluated in the HeyA8 intraperitoneal ovarian cancer cells exposed to MFH for 30 minutes at 43C, tumor model, and resulted in robust antitumor effects. This work which revealed that heat shock protein (HSP) genes, including demonstrated that HSP70 inhibition combination with MFH HSPA6, were upregulated. HSPA6 encodes the Hsp70, and its generate a synergistic effect and could be a promising target expression was confirmed by PCR in HeyA8 and A2780cp20 to enhance MFH therapeutic outcomes in ovarian cancer. ovarian cancer cells. Two strategies were investigated to inhibit Mol Cancer Ther; 16(5); 966–76. 2017 AACR.

Introduction dose, patient discomfort, and hot spots limit the use of this therapy (2, 4). The use of nanotechnology could help overcome Hyperthermia is the application of heat to tissues as a thera- such challenges. Magnetic fluid hyperthermia (MFH) is an attrac- peutic tool using temperatures between 41C and 47C. Hyper- tive alternative as it can deliver heat to the desired area using thermia has been successfully employed as an adjuvant for the magnetic nanoparticles under the influence of a magnetic field treatment of several cancers and is known to enhance the effects of (1, 2, 5). chemotherapy and radiotherapy (1–3). Clinically, heat can be There is evidence that MFH can deliver heat more efficiently applied locally, regionally or in the whole-body, but current than conventional hyperthermia (6–12). Aspects such as thermal modalities are not site specific (1, 4). Challenges such as temper- chemosensitization, membrane permeabilization, and sensitiza- ature homogeneity during application, achieving an efficient heat tion of drug-resistant cancer cells have been observed as responses to MFH in vitro (2, 7–9). These findings demonstrate that MFH has potential as an adjuvant cancer treatment, but there is still a dearth 1Department of Chemical Engineering, University of Puerto Rico-Mayaguez,€ of knowledge in the field regarding the implications of MFH at the 2 Mayaguez,€ Puerto Rico. Department of Gynecologic Oncology, MD Anderson molecular level and how these can be exploited to enhance the 3 Cancer Center, Houston, Texas. Faculty of Pharmaceutical Sciences, Chiba effects of MFH in cancer treatment. University, Chiba, Japan. 4Department of Experimental Therapeutics, MD Ander- son Cancer Center, Houston, Texas. 5Center for RNA Interference and Non- Previous work with conventional hyperthermia demonstrated Coding RNA, MD Anderson Cancer Center, Houston, Texas. 6Department of that molecular and cellular responses depend on the cell line, Systems Biology, MD Anderson Cancer Center, Houston, Texas. 7J. Crayton thermal dose, and recuperation time (13–16). In the clinic, Pruitt Family Department of Biomedical Engineering, University of Florida, intraperitoneal administration of hyperthermia (HIPEC), in com- 8 Gainesville, Florida. Department of Chemical Engineering, University of Florida, bination with chemotherapeutic drugs, have been investigated for 9 Gainesville, Florida. Department of Electrical and Computer Engineering, more than three decades as an attractive adjuvant to cytoreductive University of Puerto Rico-Mayaguez,€ Mayaguez,€ Puerto Rico. surgery in ovarian cancer (17). Currently, there are more than 30 Note: Supplementary data for this article are available at Molecular Cancer clinical trials that have recently started or concluded using this Therapeutics Online (http://mct.aacrjournals.org/). technology (18). A recent meta-analysis of clinical trials per- Corresponding Author: Madeline Torres-Lugo, University of Puerto Rico- formed by Huo and colleagues (19), demonstrated that the € € Mayaguez, PO Box 9000, Mayaguez, 00680, Puerto Rico. Phone: 787-832- combination of HIPEC, cytoreductive surgery, and chemotherapy 4040, ext. 2585; Fax: 787-834-3655; E-mail: [email protected] showed significant patient survival. Still, a challenge remains: the doi: 10.1158/1535-7163.MCT-16-0519 ability to reach a therapeutic temperature without causing toxicity 2017 American Association for Cancer Research. to healthy tissue and risk complications (20–24). Furthermore,

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intrinsic biological aspects such as the inherent thermo-tolerance al Instruments) with type T thermocouples (Omega Engineering). properties of tumors are a challenging aspect that has been After 48 hours, cell viability was assessed by Trypan Blue (Sigma- neglected to this point. Nanoscale heat generation systems such Aldrich). For PES (EMD Chemicals)-treated cells, the concentra- as iron oxide nanoparticles represent an attractive alternative as tion was 5 mmol/L for A2780cp20 and 10 mmol/L for HeyA8 and one could generate heat within the system, only under high- SKOV3. PES was added to cells 2.5 hours before MFH and not frequency and moderate amplitude magnetic fields that can be removed until the end of the treatment. Cells were incubated for constrained to the tumor region. Therefore, the goal of this 48 hours in the presence of PES. work was to investigate gene expression profiles after MFH in ovarian cancer cell lines to elucidate cellular response and select Suspended cells. Cells were suspended in 2.5 mL of media in a glass in vitro in vivo molecular targets to enhance its effect and .Itwas vial with CMDx-IO nanoparticles (0.5 mgFe/mL). An induction hypothesized that upregulated genes and proteins resulting heater (RDO Induction) was used to generate a magnetic field from MFH treatment, when inhibited, could potentially intensity that ranged between 29 and 35 kA/m. The copper coil enhance MFH outcome. Results indicated the upregulation of had 4 turns and an inner diameter of 2 cm. Cells were exposed to HSPA6 , which encodes for heat shock 70 kDa protein 6 MFH for 30 minutes. After treatment, cells were seeded in a 25 cm3 (Hsp70), several heat shock proteins (HSP), and ubiquitin flask and placed in an incubator. After 48 hours, cell viability was C. Hsp70-related genes and proteins were selected as potential assessed with Trypan Blue (Sigma-Aldrich). targets. Two strategies were investigated to inhibit Hsp70 genes and proteins (i) siRNA-based therapy; and, (ii) HSP70 protein function inhibition by 2-phenylethyenesulfonamide (PES; Microarray analysis Ovarian cancer cells were exposed to MFH for 30 minutes at ref. 25). Both strategies resulted in enhanced cell death in various ovarian cancer cell lines in vitro and in vivo. 43 C. RNA was prepared after treatment using a mirVana RNA isolation labeling kit (Life Technology). Three hundred nano- Materials and Methods grams of RNA were used for labeling and hybridization on a Human HT-12 v4 Beadchip (Illumina) following manufacturer's Cell culture protocols. The bead chip was scanned with an Illumina BeadArray Ovarian cancer cell lines HeyA8, HeyA8 MDR, A2780, A2780 Reader (Illumina). Microarray data were normalized using the cp20, and SKOV3 were grown in RPMI1640 (Sigma-Aldrich), quantile normalization method in the Linear Models for Micro- 15% FBS (Life Technologies), and 0.1% gentamicin sulfate (Sig- array Data (LIMMA) package using R language. The expression ma-Aldrich). Cells were maintained at 37 C, 95% relative humid- level of each gene was determined by the fold change in the ity and 5% CO2. Cell lines were obtained from the institutional untreated control group, and MFH treated group (43 C and 30 Cell Line Core Laboratory MD Anderson Cancer Center in 2012. minutes) transformed into a log2 base for analysis. The original Authentication was performed within 6 months of the current microarray data were uploaded to GEO with accession number work by the short tandem repeat method using the Promega GSE92990. Power Plex 16HS Kit (Promega). Quantitative real-time PCR Magnetic nanoparticles RNA was extracted from cells and tumor tissue using Direct-zol Carboxymethyl dextran coated iron oxide nanoparticles RNA MiniPrep (Zymo Research). cDNA was synthesized from (CMDx-IO) were prepared using the co-precipitation method as 1,000 ng of RNA with the Thermo Verso cDNA Synthesis Kit previously described (8, 26). Briefly, an aqueous solution of ferric (Thermo Fisher). Quantitative Real-Time PCR was performed chloride hexahydrate, ferric chloride tetrahydrate, and ammoni- using SYBR Green (Thermo Fisher) on a 7500 Fast Real-Time um hydroxide were mixed at a pH 8.0 under nitrogen at 80 C. PCR System and primers for HSPA6 and HSPA7 (Integrated DNA Nanoparticles were peptized with tetramethyl ammonium Technologies). Specific primers for HSPA6 F-CGCTGCGAGT- hydroxyl and dried. They were functionalized in dimethylsulf- CATTGAAATA, R-GAGATCTCGTCCATGGTGCT and HSPA7 oxide, 3-aminopropyltriethoxysilane, DI water, and acetic acid. F-CAACCTGCTGGGGCGTTTTGA, R-CCGGCCCTTGTCATTGGT- Then washed with ethanol and dried. Later they were exposed to GATCTT, 18s were used as an endogenous control. carboxymethyl dextran, N,N-(3-dimethylaminopropyl)-N0-ethyl- carbodiimide hydrochloride, and N-hydroxysuccinimide, at pH Western blot analysis – 4.5 5.0. Particles were centrifuged and washed with ethanol and Cells were lysed with RIPA (50 mmol/L Tris-HCl, 150 mmol/L dried. Particles characterization is presented in Supplementary NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) Fig. S1. incorporating the Protease Inhibitor sigmaFAST (Sigma-Aldrich). Protein concentration was determined with Bradford Reagent In vitro MFH (Sigma-Aldrich). Equal amounts of protein were separated with Attached cells. Cells were seeded in 35-mm cell culture dishes. 4% to 20% Mini-PROTEAN TGX Gel (Bio-Rad Laboratories) and New culture media and CMDx-IO nanoparticles (0.5 mgFe/mL) transferred to a nitrocellulose membrane (Bio-Rad Laboratories). were added the following day. The dish was placed inside the The membrane was incubated with HSP70 antibody, 1:1,000 magnetic induction coil of an Easy Heat 8310 LI (Ambrell) (Novus Biologicals) and b-actin rabbit mAb 1:1,000 (Cell Sig- induction heater and exposed to a magnetic field intensity that naling Technology) overnight. Primary antibody was detected ranged between 24 and 36 kA/m (245 kHz) to achieve 41 Cor with anti-rabbit IgG, HRP-linked Antibody (Cell Signaling Tech- 43 C for 30 minutes. The copper coil had five turns and an inner nology) and developed with SuperSignal WestPico Chemilumi- diameter 5 cm. Temperature was measured with an NI-9211 nescent Substrate (Thermo Scientific) in Chemidoc XRS (Bio-Rad thermocouple signal conditioner and acquisition board (Nation- Laboratories).

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PES cytotoxicity in ovarian cancer cells magnetic field intensity of 23 kA/m. The tumor was positioned in Cells were seeded in 96-well plates in 200 mL of media and the center of the coil. The copper coil consisted of 7.5 turns and an allowed to attach overnight. Cells were exposed to a concentration inner diameter of 3.16 cm. Warm water was circulated through a of PES between 5 and 30 mmol/L for 24 and 48 hours. Cell viability plastic tubing to maintain the environmental temperature inside was measured with EZU4 assay (ALPCO) following the manu- the coil at 37C. Body temperature was measured in the anus to facturer's protocol. guarantee mice safety. The external temperature of the tumor and the surrounding wall was also measured with an NI-9211 ther- Determination of synergism mocouple signal conditioner and acquisition board (National Combination index (CI) was evaluated using the proposed Instruments) and type T thermocouples (Omega Engineering). model of Chou–Talay with the CompuSyn software (27). Com- puSyn uses the median–drug effect analysis. Concentration–effect Intraperitoneal tumor model and MFH curves were assumed to be sigmoidal for the single drug and the The intraperitoneal tumor was generated in an athymic nude combination treatment. Calculation of doses was performed by mouse model. Studies were performed according to the protocol the median–effect equation. The parameters used for the calcu- approved by the M.D. Anderson Cancer Center Institutional lation were the fraction affected and PES and MFH exposure time. Animal Care and Use Committee. 2.5 10 5 HeyA8 cells sus- Equations are described in the Supplementary Material. pended in 200 mL of Hank's balance salt solution were injected directly into the peritoneal cavity (IP) of mice. Treatment started 2 In vitro siRNA delivery and MFH weeks later after cells IP injection. PES was injected a 10 mg/Kg 2 Cells were transfected with Lipofectamine RNAiMax (Invitro- days before the first MFH. CMDx-IO nanoparticles were injected m m HSPA6 gen) at 2.5 L of the reagent: 1 g siRNA. siRNA from intraperitoneally at 2 mg Fe/mouse in 200 mL of PBS the night Rosetta Prediction Human (Sigma-Aldrich) was selected as target before treatment. Next day mice were anesthetized with ketamine HSPA7 gene, sequence 1: SASI_Hs01_00244829, sequence 2: 1 and MFH applied for 30 minutes, with the same coil configuration HSPA7 and 2, sequence 3: SASI_Hs01_00244824 and sequence 4: previously described. The temperature was also measured in the SASI_Hs01_00244827 and SASI_Hs01_00244829 and control mouse belly. siRNA was MISSION siRNA Universal Control #1 (Sigma- Aldrich) at a concentration 60.2 or 100 nmol/L. A total 36 hours Histology of transfection was performed. MFH was applied, and cell viability Tumors were embedded in paraffin and cut (8-mm sections). was measured following 48 hours of incubation as previously Slides were deparaffinized, and rehydrated. The Prussian Blue, described. Iron Stain Kit (Sigma) was used following the manufacturer's protocol. Liposomal nanoparticle preparation (siRNA-DOPC) HSPA6 in vivo and control SiRNAs for delivery were incor- Statistical analysis porated into 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine Statistical analyzes were performed using the Student t test (DOPC) nanoliposomes as previously described (28). DOPC (two-tailed distributions, two samples with unequal variables). and siRNA were mixed in the presence of excess tertiary butanol For values that were not normally distributed, the Mann–Whitney w w at a ratio of 1:10 ( / ) siRNA/DOPC. Tween 20 was added to the rank sum test was used. A P value of <0.05 was considered mixture in a ratio of 1:19 Tween 20:siRNA/DOPC. The mixture statistically significant. was vortexed, frozen in an acetone/dry ice bath and lyophilized. in vivo Before administration, this preparation was hydrated with Results PBS. MFH in ovarian cancer cell lines Subcutaneous MFH and HSPA6 siRNA delivery Four ovarian cancer cell lines, A2780 and HeyA8, and their corresponding drug resistant sub-lines were exposed to MFH at Subcutaneous tumors were generated in an athymic nude mouse model. Studies were performed according to the protocol various temperatures (41 C, 43 C, and 45 C) and exposure times approved by the MD Anderson Cancer Center Institutional Ani- (30 or 60 minutes). Cell viability was assessed after 48 hours of recovery (Fig. 1A). No significant change in cell viability was mal Care and Use Committee. HeyA8 or A2780cp20 cells (1 6 m observed at 41 C for all cell lines. However, at 43 C, a significant 10 ) suspended in 50 L of Hank's balance salt solution were injected subcutaneously in the mice right flank on day 0, to obtain decrease in cell viability was observed for most cell lines. At 45 C, subcutaneous tumors on day 7. Tumor volumes were 35–113 further reduction in cell viability was observed for all cell lines. mm3. siRNA liposomes injection started on day 7, 5 mgofHSPA6 The thermal dose for each case was calculated using cumulative siRNA or control siRNA liposomes were injected intraperitone- equivalent minutes (CEM) as established by Dewey and collea- ally. Three MFH treatments and siRNA deliveries were performed. gues (30). Supplementary Table S1 presents the CEM values for all The first MFH treatment was applied on day 9. Tumor volume was conditions. Temperature and exposure time affected CEM values calculated as (largest diameter) (smallest diameter)2 p/6. and cell viability. Higher CEM values resulted in enhanced cell MFH started with iron oxide nanoparticles injected directly into death. the tumor (5 mgFe/cm3 tumor volume). Nanoparticles were injected for 30 seconds. The tip needle was removed from the Gene expression in MFH in ovarian cancer cell lines tumor after 5 minutes. MFH was applied 4 hours after nanopar- A gene array analyses was carried out to identify genomic ticle injection to allow particle distribution (29). Mice were changes resulting from exposure of ovarian cancer cells to MFH. anesthetized and placed inside the magnetic induction coil of an A control group of untreated HeyA8 cells and an experimental EasyHeat 8310 LI (Ambrell) induction heater for 30 minutes at a group consisting of cells exposed to MFH for 30 minutes at 43C

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Figure 1. Gene expression after MFH in ovarian cancer cells. A, Cell viability when MFH was applied to ovarian cancer cells at 41, 43, 45C for 30 and 60 minutes; (n ¼ 3; error SE). B, Pathway of upregulated genes from IPA after MFH was applied to HeyA8 cell lines for 30 minutes at 43C. C, Top 20 upregulated genes after MFH was applied to HeyA8 cell lines for 30 minutes at 43C. Analysis revealed a significant upregulation of HSP70-related genes (one-way ANOVA, n ¼ 3, P < 0.02). were used. This temperature was chosen since the aforementioned overexpressed in various human cancers, playing different roles as results indicated a substantial decrease in cell viability when the an inhibitor of apoptosis (31). HSPA6 and HSPA7 expression temperature was increased from 41Cto43C. Compared with were confirmed by qRT-PCR, and expression was measured for up controls, 60 genes were upregulated (by 1.5-fold) in treated to 8 hours following heat shock treatment (Fig. 2A). A2780cp20 cells. The top 20 upregulated genes are presented Fig. 1C. Sup- showed significant expression at 1 hour for both genes. In HeyA8 plementary Tables S2 and S3 present descriptions of each of the cells, gene expression was sustained for almost 4 hours at 41C dominant upregulated and downregulated genes. and 8 hours at 43C. Data were further analyzed using the Database for Annotation, Hsp70 protein level was also evaluated according to Western Visualization and Integrated Discovery (DAVID) and enrichment blot analysis. At 41C, the highest expression level was observed of Gene Ontology (GO) terms for significantly expressed genes. after 6 hours of MFH treatment. At 43C, protein expression for From this analysis, three main biological functions were found to A2780cp20 cells increased after 1 hour of recovery time, and the be affected by MFH at 43C, including response to unfolded highest expression was observed at 6 hours after MFH (Fig. 2B). proteins, response to protein stimulus, and protein folding HeyA8 cells showed no significant differences in Hsp70 protein (Table 1). Top genes related to the aforementioned functions expression via Western blot analysis (Supplementary Fig. S2C). affected by MFH were HSPs, Hsp70 (HSPA6/HSPA7, HSPA1A, HSPA6 (Hsp70) was selected as the target gene because it was the HSPA1B, HSPA1L, HSPA4L), Hsp60 (LOC643300), Hsp40 most upregulated (32). Two approaches were performed to (DNBAJ family), Hsp20 (CRYAB) and Hsp27 (SERPINH1), and inhibit Hsp70 including gene silencing by siRNA and protein BAG3 (modulator of Hsp70). Besides HSPs, the gene ubiquitin C function inhibition using PES. (UBC) was also in the top ten upregulated genes after MFH. Figure 1B presents the connection of HSPA6 with upregulated genes HSPA6 siRNA enhanced the effects of MFH from the microarray. A2780cp20 and HeyA8 cell lines were selected to further assess To validate the results from the microarray, qRT-PCR was the efficacy of HSPA6 inhibition along with MFH (Fig. 2). performed for HSPA6 and HSPA7. Hsp70 was selected because A2780cp20 and HeyA8 were selected according their capability it has a critical function in cancer cells and hyperthermia. Hsp70 is to create subcutaneous tumor models. First, several siRNA sequences were investigated to evaluate their capacity to inhibit HSPA6 expression. Two siRNA sequences per cell line were estab- Table 1. Biological functions after MFH treatment of HeyA8 cells (43C and 30 lished for HSPA6 inhibition with 50% to 60% of gene knock minutes) down. For HeyA8 cell line, seq # 1 at 60.2 nmol/L and seq #2 at Biological function Number of genes P 100 nmol/L were able to inhibit HSPA6. In the case of A2780cp20 Response to unfolded proteins 14 9.99E 17 cells, seq #3 at 60.2 nmol/L and seq. #4 at 100 nmol/L (Supple- Response to protein stimulus 15 8.43E16 mentary Fig. S2A) were also successful in inhibiting HSPA6. The Protein folding 14 1.88E11 in vitro siRNA effects were evaluated in each cell line (Fig. 2C and

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Figure 2. HSP70 genes are upregulated after MFH and HSPA6 RNA interference and MFH in vitro. A, Levels of HSP70 genes were conﬁrmed by quantitative real- time PCR after MFH treatment for 30 minutes, three temperatures 39C, 41C, 43C and up to 8 hours of recovery time. Higher expression was observed after 1-hour recovery; (, P < 0.05; n ¼ 3; error, SD). B, Western blot analysis of HSP70 protein level in A2780cp20 after 30-minute MFH heat treatment at 41C and 43C, with recovery time of 1, 4, and 6 hours. C, MFH cell viability after HSPA6 gene is knockdown with HSPA6 siRNA sequence 1 at 41Cand 43C and sequence 2 at 41C in HeyA8 cell line. D, A2780cp20 cell viability for sequence 3 and 4. cells treated with MFH at 41C for 30 minutes (, P < 0.05).

D). Combination treatment (HSPA6 siRNA þ MFH) resulted in a increased as the concentration of PES increased as depicted by the higher decrease in cell viability when compared to MFH or HSPA6 higher percentage of pale cells (Supplementary Fig. S3A). Lyso- siRNA applied independently. some permeabilization was also investigated by imaging the release of cathepsin B into the cytosol. Cathepsin B in control Antitumor effects of HSP70 inhibition and MFH in vitro cells was located in lysosomes as small red points (Supplementary The effects of HSP70 inhibition using PES inhibitor were tested. Fig. S3B). In PES-treated cells a diffuse pattern was observed, PES cytotoxicity was measured (Fig. 3A) and results presented a indicating the relocation of cathepsin B into the cytosol conﬁrm- reduction in cell viability as PES concentration was increased. ing lysosome permeabilization (Supplementary Fig. S3B). in vitro þ Supplementary table S4 illustrates the IC50 values for PES when The effect of combination treatment (MFH PES) was exposed to ovarian cancer cell lines. A270cp20 cells showed lower assessed in ovarian cancer cells (Fig. 3B). PES concentrations were IC50 values to PES when compared to HeyA8 and SKOV3 cells. selected as to provide viabilities higher than 90% when used Previous work by Granato and colleagues, revealed PES affected independently (Fig. 3A and Supplementary Fig. S3E), 5 mmol/L membrane permeability since HSP70 supports lysosome mem- for A2780cp20 and 10 mmol/L for HeyA8 and SKOV3. Two MFH brane integrity, especially in cancer cells (33). To conﬁrm such temperatures were selected, 41 C representative of mild hyper- mechanism in ovarian cancer cells lysosomal permeabilization thermia, and 43 C representative of hyperthermia. Combination was measured. Results indicated that lysosome permeabilization treatment decreased cell viability to approximately 40% at 41 C

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Figure 3. PES exposure results in signiﬁcant cell death for ovarian cancer cell lines due to lysosomal damage and combination treatment is synergistic. A, Cytotoxicity for A2780cp20, HeyA8, and SKOV3 at 24 and 48 hours. B, Combination treatment PES and MFH, HeyA8, A2780cp 20, and SKOV3 cell were treated with PES and MFH at 41C (top) and 43C (bottom) for 30 minutes after 48 hours of recovery. C, Combination index of MFH and PES for ovarian cancer cell lines.

compared with MFH alone (Fig. 3B). Results indicated that the min in combination with 5, 10, and 20 mmol/L. Results are combination treatment generated a cytotoxic effect on cells, which illustrated in Supplementary Fig. S3D and the CIs shown in could indicate synergy. For this purpose, combination index Supplementary Table S6 for each experimental condition. Data (CI; Fig. 3C) was calculated using the Chou–Talalay (27) and demonstrated synergism for PES concentrations above 10 mmol/L CompuSyn methods. Cell viabilities and dose response curves and all exposure times. These results confirmed that MFH applied from PES and MFH (Supplementary Fig. S3E) were used to in combination with non-lethal doses of PES produced a syner- compute CI values. The median effect principle of the mass-action gistic effect. law was evaluated using parameters described in Supplementary Table S5. The degree of synergism defined by Chou and Marin was HSPA6 silencing and MFH reduced tumor growth in vivo analyzed (27). According to this analysis, A2780cp20 and HeyA8 To identify the in vivo effect of HSP70 inhibition during MFH, cells at 41 C showed slight synergism with PES treatment. Under HSPA6 siRNA was used to silence HSPA6 in a subcutaneous similar conditions, SKOV3 cells also demonstrated synergism. At ovarian tumor model. siRNA was delivered with DOPC nanoli- a higher temperature (43 C), the effect of all cell lines was posomes. Mice were divided into four groups, control siRNA- synergistic. Further experiments were performed to evaluate syn- DOPC, control siRNA-DOPC and MFH treatment, HSPA6 siRNA- ergy at various PES concentrations and MFH exposure times. For DOPC and combined HSPA6 siRNA-DOPC with MFH treatment. this purpose, HeyA8 cells were exposed to MFH at 30, 45 or 60 The efficacy of siRNA delivery and gene silencing was confirmed

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(Supplementary Fig. S2B). Figure 4A presents the experimental control siRNA-DOPC with MFH presented a 3- to 4.4-fold schedule. Research demonstrated that reduction in tumor growth increase when compared to their initial value whereas HSPA6 can be enhanced by the number of MFH treatments (34, 35). For siRNA-DOPC was only a 1.4-fold. Final tumor weight for HSPA6 this purpose, three MFH treatments were scheduled 48 hours siRNA-DOPC and MFH was 65% lower than control siRNA- apart. CMDx-IO nanoparticles were injected intratumorally, 4 DOPC and MFH (Fig. 4C). The temperature proﬁle is exhibited hours before treatment, to allow them to distribute in the tumor. in Supplementary Fig. S4A. Tumor temperatures of 43C were The siRNA-DOPC was administered according to previous pub- reached. lications, two times per week and injected intraperitoneally (28). A second tumor model was evaluated using the A2780cp20 Tumor growth was slower in groups that were treated with MFH cells under a similar treatment schedule (Fig. 4D and E). Tumor when compared with those not treated with MFH (Fig. 4B and volume and weight were measured. Tumor weight for the com- Supplementary Fig. S4B). Supplementary Table S7 presents the bination treatment, HSPA6 siRNA-DOPC and MFH, was approx- statistical values of the ﬁnal tumor weight. By the end of the imately 65% smaller when compared with MFH and control treatment, relative tumor volume for the control groups and siRNA-DOPC groups. For the control groups and control

Figure 4. HSPA6 RNA interference and MFH decreased subcutaneous HeyA8 and A2780cp20 tumor model growth. A, Timeline for MFH in vivo. B, Relative tumor volume in function of time of HeyA8 tumor model. C, Ex vivo HeyA8 tumor weight. Number of mice 4–5per group. D, Relative tumor volume of A2780cp20 tumor model. E, Ex vivo A2780cp20 tumor weight. F, Prussian Blue staining to determine distribution of iron oxide nanoparticles from collected tumor from A2780cp20 and HeyA8; (, P < 0.05; , P < 0.01).

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ed nanoscale heat delivery with magnetic nanoparticles The inhibition of Hsp70 by RNA interference and the novel Hsp70 inhibitor, PES, enhanced the effect of MFH both in vitro and in vivo in various ovarian cancer cell lines and tumor models (subcutaneous and intraperitoneal). Furthermore, in vitro obser- vations with PES demonstrated, for the first time, that the combined treatment resulted in a synergistic effect at mild hyperthermia temperatures. Findings of this work also indicated that the intraperitoneal injection of iron oxide nanoparticles is a feasible alternative to nanoparticle delivery to peritoneal tumors and that MFH treatment can be successfully administered without dam- aging healthy tissue. To further understand such cellular responses, gene expression and pathway analyses were conducted. An experimental temperature of 43C was selected because an inflection point in cell viability is observed for conventional hyperthermia (1, 39). Such behavior was also observed when MFH was applied as a sole treatment in vitro for various ovarian cancer cell lines as described Figure 5. herein. PES and MFH decrease orthotopic HeyA8 tumor model growth. Timeline Results are supported by prior work demonstrating that HSP- for MFH in vivo (A), ex vivo HeyA8 tumor weight (B). Number of mice related genes, including Hsp70, Hsp40, Hsp105, Hsp110, Hsp47, 3–5 per group. and BAG3 are upregulated when conventional hyperthermia was applied to cancerous and normal human cells (13, 15, 16, 40–44). siRNA-DOPC MFH, an increase in relative tumor volume was However, such experiments have not been previously conducted observed with a fold increase between 2 and 6.6. HSPA6 siRNA- for MFH-treated ovarian cancer cells or any other cancer cell line. DOPC and MFH presented a relative 0.7-fold change in volume. The genetic analysis of the response of ovarian cancer cells HSPA6 inhibition improved the outcome of MFH treatment in exposed to MFH revealed that several HSPs were upregulated as UBC fi subcutaneous models. The biological effect of MFH was assessed well as ubiquitin C ( ). These ndings demonstrated that MFH for apoptosis. Caspase-3 activity was observed in MFH-treated upregulated the expression of HSPs genes in ovarian cancer cell tumors (Supplementary Fig. S4B). The distribution of CMDx-IO lines similar to conventional hyperthermia. Interestingly, MFH nanoparticles was evaluated in both tumors models using Prus- also overexpressed UBC, which has not been previously reported sian Blue (Fig. 4F). Iron oxide nanoparticles remained in the as a top overexpressed gene in microarray for conventional tumor tissue of both subcutaneous tumor models until the tumor hyperthermia in treatments where the range in thermal dose was was collected at the end of the experiment. between 7.5 and 60 cumulative equivalent minutes (CEM; refs. 15, – MFH was also tested in the HeyA8 intraperitoneal tumor model 40 42). At higher CEM ( 300) ubiquitin genes expression have in combination with PES. Treatment started when PES was been reported (45, 46). In this particular work MFH treatments injected IP at day 12 after cells injection (Fig. 5A). PES dose was with a thermal dose of only 30 cumulative equivalent minutes fi selected from previous reports where it was administered every 5 signi cantly upregulated UBC. These results may provide further days (25, 36, 37). The following day, CMDx-IO nanoparticles evidence that the extent of MFH damage in the cell might be more were injected intraperitoneally, the technique allows the inter- extensive than conventional hyperthermia as reported by our group and others (6, 7, 12). nalization of nanoparticles in intraperitoneal tumors as described elsewhere (38). At day 14 mice were exposed to MFH for 30 After studying the gene expression resulting from MFH at 43 C, minutes. A total of three MFH treatment and PES injection were Hsp70 was selected as it was the most upregulated gene of the fi performed. Mice were sacrificed when controls appeared mori- microarray, as con rmed by qPCR and Western blot analysis. In bund. Tumor weight of PES and MFH treated mice (Fig. 5B), was addition, Hsp70 is overexpressed in various cancer models, and 65% smaller when compared with the control MFH group. Mice high levels are associated with poor prognosis (31). Hsp70 plays a fi were monitored and no sign of sickness or pain were observed signi cant role in cancer, suppressing apoptosis and participate in days after MFH. Body weight and feeding habits remained tumor development. Under stress conditions, such as hyperther- unchanged. Results agreed with in vitro outcomes of synergy mia, Hsp70 avoids accumulation of unfolded proteins and refold between PES and MFH. aggregated proteins (31, 47). Several Hsp70 inhibitors have been designed with favorable results in vitro, but have failed efficacy in Discussion pre-clinical or clinical trials, with limitations as toxicity and reduced antitumor effect (11, 31, 48, 49). An alternative of two The present work investigated the molecular and cellular approaches were investigated in this work, inhibition of Hsp70 by responses of ovarian cancer cells to MFH. We and others have RNA interference and, inhibition of protein function using PES. attempted to elucidate the cellular and molecular features of Previous reports have demonstrated that Hsp90 inhibition in MFH; however, there are still aspects at the molecular level that combination with MFH therapy increased cell susceptibility to need to be understood (6–12). Results described herein indicated hyperthermia and in vivo tumor regression (50–52). Hsp90 inhi- that there is overexpression of HSPs, in particular, Hsp70 as a bition is commonly used in hyperthermia because it plays a result of MFH treatment. The resulting genomic analysis provided central role in cancer signaling molecules, but research established the means to design targeted combination therapies using direct- that Hsp90 inhibition also upregulates Hsp70 production

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(50, 51). The goal of this work was to demonstrate that only In summary, this work introduces the use of microarray gene Hsp70 inhibition will improve the antitumor effect of MFH. expression to rationally design targeted combination therapies Hsp70 knockdown with siRNA has been achieved in vitro in using directed nanoscale heat delivery with magnetic nanoparticles. human colon cells, prostate and the ovarian cancer cell line Results demonstrated that MFH gene expression in ovarian cancer A2780 (37, 53, 54). The combination of Hsp70 RNA interfer- was dominated by HSPs and ubiquitin C. Hsp70 was found to be ence with MFH has not been previously reported in vitro or in the most upregulated gene and its combination enhanced the effect vivo. Results demonstrated that HSPA6 inhibition with siRNA in of MFH in vitro and in vivo. In vitro combination treatments dem- combination with MFH enhanced treatment outcomes both in onstrated for the first time the synergistic behavior between MFH vitro and in vivo.Dataconfirmed that Hsp70 inhibition and and PES. The intraperitoneal delivery of iron oxide nanoparticles MFH appear as an effective selective alternative, resulting in revealed that tumors were significantly affected when the combined enhanced MFH therapeutic effects. The second approach was to treatment of MFH and HSP70 inhibition was applied. Therefore, inhibit the Hsp70 protein function. An alternative to this results presented herein suggested that iron oxide magnetic nano- approach was to employ PES because it has been identified as particles under the influence of an alternating magnetic field in a novel Hsp70 inhibitor with direct specificity (25, 36). In this combination with heat response impairment drugs are a significant work, PES was able to reduce cell viability in various ovarian milestone for the translation of nano-based adjuvant therapies for cancer cells by similar mechanisms as those proposed in the ovarian cancer into the clinic. literature (55, 56). The combination of MFH and PES resulted fl in a synergistic effect in ovarian cancer cells. This is the first time Disclosure of Potential Con icts of Interest fl that synergy is demonstrated from the combination treatment No potential con icts of interest were disclosed. between PES and MFH. Sekihara and colleagues (37) demon- Authors' Contributions strated that when PES and conventional hyperthermia were Conception and design: K.A. Court, S.Y. Wu, G. Lopez-Berestein, C. Rinaldi, combined in prostate cancer cells the number of cells in early E.J. Juan, A.K. Sood, M. Torres-Lugo apoptosis and necrosis and late apoptosis were increased, Development of methodology: K.A. Court, S.Y. Wu, C. Rodríguez-Aguayo, concluding that the cell death mechanism of the combination G. Lopez-Berestein, C. Rinaldi, E.J. Juan, A.K. Sood, M. Torres-Lugo treatment partially depended on the caspase pathway. Further- Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K.A. Court, H. Hatakeyama, C. Rodríguez-Aguayo, more, they demonstrated that the combination increased the – L. Ju-Seog, E.J. Juan, A.K. Sood number of cells in the G2 M phase and decreased the expres- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, sion of cell cycle related molecules such as c-Myc and cyclin D1 computational analysis): K.A. Court, H. Hatakeyama, G. Lopez-Berestein, causing cell growth arrest. These findings validated that Hsp70 L. Ju-Seog, C. Rinaldi, E.J. Juan, A.K. Sood, M. Torres-Lugo protein function inhibition is enough to enhance the thera- Writing, review, and/or revision of the manuscript: K.A. Court, H. Hatakeyama, peutic outcome of MFH. S.Y. Wu, M.S. Lingegowda, G. Lopez-Berestein, C. Rinaldi, E.J. Juan, A.K. Sood, M. Torres-Lugo Hsp70 inhibition and MFH were further evaluated in intraper- Administrative, technical, or material support (i.e., reporting or organizing itoneal tumor models with peritoneal administration of nanopar- data, constructing databases): K.A. Court, G. Lopez-Berestein, A.K. Sood ticles. The reason to select this model is that intratumoral injections Study supervision: A.K. Sood, M. Torres-Lugo are not practical in clinical settings. Besides, other methods such as nanoparticle targeting for intravenous administration still face Acknowledgments significant challenges achieving the required levels of nanoparticle The authors are grateful to Dr. Camilo Mora and Dr. Marisel Sanchez for their support in confocal imaging and flow cytometer. accumulation in tumors. Administration in the peritoneal area of antineoplastic agents is currently an alternative for treating small Grant Support tumors located in the area. High drug concentrations and longer This work was supported by grants from the following: HHS (NIH; half-life can be achieved with intraperitoneal injections (57). CA 963000/CA 96297; to M. Torres-Lugo; P50CA083639 and CA016672, Previous work has reported the intraperitoneal administration of A.K. Sood), PR Institute for Functional Nanomaterials (EPS-100241; to Made- iron oxide nanoparticles for MFH treatment in a mouse ovarian line Torres-Lugo), Nanotechnology Center for Biomedical, Environmental and cancer model with peritoneal tumors but did not assess tumor Sustainability Applications (HRD-1345156; to Madeline Torres-Lugo) Ovarian growth, focusing solely on demonstrating cell death through Cancer Research Fund (OCRF; RP101502/RP, to S.Y. Wu), and Cancer Preven- tion and Research Institute of Texas training grants (RP101489, to S.Y. Wu; histological analysis (38). In this work, we administered nanopar- RP110595 and RP120214, to A.K. Sood). ticles intraperitoneally to the HeyA8 intraperitoneal model and The costs of publication of this article were defrayed in part by the payment of applied MFH. Results demonstrated that iron oxide nanoparticles page charges. This article must therefore be hereby marked advertisement in when injected intraperitoneal in combination with MFH on intra- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. peritoneal ovarian tumors showed an antitumor effect. We were also able to enhance MFH therapeutic outcome with the inhibition Received August 10, 2016; revised September 6, 2016; accepted February 4, of HSP70 function using PES. 2017; published OnlineFirst February 21, 2017.

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HSP70 Inhibition Synergistically Enhances the Effects of Magnetic Fluid Hyperthermia in Ovarian Cancer

Karem A. Court, Hiroto Hatakeyama, Sherry Y. Wu, et al.

Mol Cancer Ther 2017;16:966-976. Published OnlineFirst February 21, 2017.

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