Proteasome 26S Subunit PSMD1 Regulates Breast Cancer Cell Growth Through P53 Protein Degradation

J. Biochem. 2018;163(1):19–29 doi:10.1093/jb/mvx053

Proteasome 26S subunit PSMD1 regulates breast cancer cell growth through p53 protein degradation

Received June 8, 2017; accepted June 30, 2017; published online August 3, 2017

Toshiyuki Okumura1,2, Kazuhiro Ikeda1, modified Eagle’s medium; EGFR, epidermal growth Takafumi Ujihira1,2, Koji Okamoto3, factor receptor; ERa, oestrogen receptor a; FACS, Downloaded from https://academic.oup.com/jb/article-abstract/163/1/19/4061459 by Juntendo University user on 26 December 2018 Kuniko Horie-Inoue1, Satoru Takeda2 and fluorescence-activated cell sorting; GAPDH, glycer- Satoshi Inoue1,4,* aldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; HER2, human epidermal growth 1Division of Gene Regulation and Signal Transduction, Research factor 2; IGFR, insulin like growth factor 1; mTOR, Center for Genomic Medicine, Saitama Medical University, 1397-1 2 the mechanistic target of rapamycin; NBN, Nibrin; Yamane, Hidaka-shi, Saitama 350-1241, Japan; Department of NF-kB, nuclear factor kappa-light-chain-enhancer of Obstetrics and Gynecology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8431, Japan; activated B cells; Nrf2, nuclear factor elythroid 2- 3Division of Cancer Differentiation, National Cancer Center related factor 2; OHT, 4-hydroxytamoxifen; OHTR, Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, 4-hydroxytamoxifen resistant MCF-7; PI, propidium 4 Japan and Department of Functional Biogerontology, Tokyo iodide; PI3K, phosphatidylinositol-4,5-bisphosphate Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan 3-kinase; PSMD1, proteasome 26S subunit, non- ATPase 1; PTEN, phosphatase and tensin homolog; *Satoshi Inoue, Department of Functional Biogerontology, Tokyo PTTG1, pituitary tumour-transforming 1; qRT-PCR, Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan. Tel: +81-3-3964-3241, Fax: +81-3-3579- quantitative real time polymerase chain reaction; 4776, email: [email protected] RIPA buffer, radioimmunoprecipitation assay buffer; RPL31, ribosomal protein L31; SDS-PAGE, sodium Endocrine therapy using antiestrogens and aromatase dodecyl sulphate polyacrylamide gel electrophoresis; inhibitors is usually efficient to treat patients with hor- shRNA, a short hairpin RNA; siRNA, small inter- mone-sensitive breast cancer. Many patients with endo- fering RNA; SNTB1, syntrophin beta 1; TGFb, crine therapy, however, often acquire resistance. In the transforming growth factor beta; TSPAN12, tetra- present study, we performed functional screening using spanin 12; VEGF, vascular endothelial growth factor; short hairpin RNA library to dissect genes involved in WST-8, (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophe- antiestrogen tamoxifen resistance in MCF-7 breast nyl)-5-(2, 4-disulphophenyl)-2H. cancer cells. We identified seven candidate genes that are associated with poor prognosis of breast cancer patients based on clinical dataset. The expression levels of Breast cancer is the most common cancer among six out of seven genes were higher in 4-hydroxytamox- women worldwide (1). Various therapeutic methods ifen (OHT) resistant MCF-7 (OHTR) cells compared are applied to the management of breast cancer, with parental MCF-7 cells. Among the six selected including surgery, endocrine therapy, radiotherapy, genes, siRNA-mediated knockdown of PSMD1 and chemotherapy, molecular target therapy or a combin- TSPAN12 markedly reduced the proliferation of ation of these treatments (1). It is notable that 70% OHTR cells. Notably, the knockdown of proteasome of breast cancers express estrogen receptor a (ERa) 26S subunit PSMD1 exhibited cell cycle arrest and and such hormone-naı¨ ve breast cancers will primarily the accumulation of p53 protein through inhibiting respond well to endocrine therapy using antiestrogens p53 protein degradation. In accordance with p53 accu- such as tamoxifen or aromatase inhibitors (2). mulation, its target genes p21 and SFN were also upre- Antiestrogens and aromatase inhibitors will inhibit gulated by PSMD1 silencing. Taken together, PSMD1 the ERa-mediated signalling by competing with was identified as a potential gene that plays a role in physiological estrogen ligands and blocking estrogen the development of tamoxifen resistance in breast synthesis, respectively. ERa functions as a ligand-de- cancer cells. These findings will provide a new insight pendent transcription factor that belongs to the canon- for the mechanism underlying endocrine therapy resist- ical steroid hormone receptor family and mediates ance and a prognostic and therapeutic molecular target various estrogen actions. In breast cancer, ERa plays for advanced breast cancer. critical pathophysiological roles in angiogenesis, DNA Keywords: breast cancer; cell cycle; p53; proteasome; repair, cell proliferation, cell-cycle progression and tamoxifen-resistance. apoptosis. Thus, the identification of estrogen-regu- lated genes is the first step towards understanding the Abbreviations: AKT, the serine threonine kinase; mechanism underlying estrogen-dependent transcrip- ATP5A1, ATP synthase, H+ transporting, mito- tional regulation in breast cancer cells. For example, chondrial F1 complex, alpha subunit 1, cardiac it is reported that ERa regulates expressions of insulin muscle; BCL-2, B cell lymphoma 2; CAMP, catheli- like growth factor 1 (IGFR), cyclin D1, B cell lymph- cidin antimicrobial peptide; DMEM, Dulbecco’s oma 2 (BCL-2), vascular endothelial growth factor

(VEGF), human epidermal growth factor 2 (HER2), primers specific for barcodes of the library plasmid DNA (911). transforming growth factor beta (TGF) and fibro- The PCR products were purified by QIAquick PCR purification Kit (QIAGEN). The extracted DNA fragments (1.5 mg) from vehicle- or blast growth factor receptor epidermal growth factor OHT-treated MCF-7 cells were labelled with Cy3 or Cy5 dye, re- receptor (EGFR) in breast cancer cells (35). spectively, using Genome DNA Enzymatic Labelling Kit (Agilent, Despite the primary efficiency of endocrine therapy Santa Clara, CA, USA) and purified with Ultracell YM-30 on the majority of breast cancers, patients will be often Microcon centrifugal filter device (Millipore, Tokyo, Japan). suffered from acquired resistance during long-term treatment and will be threatened by advanced stages Microarray analysis The Cy3- and Cy5-labeled DNAs were subjected to microarray ana- of the disease with a poor prognosis. Efforts have been lysis using the Oligo cDGH/ChIP-on-ChIP Hybridization Kit made to clarify the mechanisms underlying acquired (Agilent). Microarray images were obtained and normalized using Downloaded from https://academic.oup.com/jb/article-abstract/163/1/19/4061459 by Juntendo University user on 26 December 2018 resistance to endocrine therapy. In terms of the resist- Agilent Feature Extractor software. Before comparing the micro- ance to endocrine therapy, several mechanisms have array results between OHT-treated and vehicle-treated control sam- been reported, including the upregulations of receptor ples, the reproducibility of signal intensities was evaluated based on the calculation of the coefficient of variation (CV = 100ÂSD/mean) of tyrosine kinase (RTKs) such as HER2, EGFR, in the duplicated OHT-treated and control experiments (7). shRNAs fibroblast growth factor receptor (FGFR), IGFR and at 25th percentile of CV values in each treatment group were used EGFR, the inhibition of PI3K-PTEN/AKT/mTOR for further analysis. pathway, and the activations of ER signalling and NF-kB signalling (6). We previously identified tamoxi- siRNA transfection fen resistance-associated miRNAs such as miR-574-3p siRNA duplexes targeting selected genes were synthesized using an algorithm that enables to minimize off-target effects and to improve in breast cancer cells through functional lentiviral target specificity (Supplementary Table S2) (12). A negative control miRNA library-based screening in MCF-7 cells (7). siRNA (siControl) with no homology to known gene targets in shRNA library-based functional screening is another mammalian cells was purchased from RNAi Inc (Tokyo, Japan). useful technique to identify potential targets associated OHTR and parental MCF-7 cells were plated in 6-well plates at a density of 250,000 cells per well and simultaneously transfected with with endocrine hormone therapy, as we identified a siRNA at a final concentration of 10 nM using Lipofectamine ribosomal protein subunit RPL31 as a bicalutamide RNAiMAX (Invitrogen, Carlsbad, CA, USA). Knockdown effi- resistance-associated gene in prostate cancer cells (8). ciency of siRNA was determined by qRT-PCR using RNA prepared High-throughput loss-of-function study combined from the cells 60 h after transfection and normalized to that of with a long-term treatment with tamoxifen will be siControl. useful to define the mechanisms underlying the acqui- Western blot analysis sition of tamoxifen resistance in breast cancer. OHTR cells cultured with 1 mM OHT were transfected with In the present study, we aimed to identify novel can- siPSMD1 #1, siPSMD1 #2 or siControl (10 nM each) for 60 h and didate genes that contribute to tamoxifen resistance total cell protein was extracted by RIPA buffer. Extracts were boiled through functional shRNA library-based screening in at 100 C for 5 min after adding sample buffer and subjected to 10% hormone-sensitive MCF-7 cells. We identified PSMD1, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE), followed by transfer to polyvinylidene difluoride membrane a subunit of 19 proteasome, as a potential critical gene (Millipore). Membranes were probed with antibodies: anti-PSMD1 that may regulate cell proliferation and cell-cycle pro- (H-300, Santa Cruz Biotechnology) and anti-p53 (DO-7; gression through p53 protein degradation. The present LeicaBiosystems, Newcastle, UK). Membranes were incubated study would provide us novel information in regard to with horseradish peroxidase-conjugated anti-mouse IgG (GE tamoxifen resistance and potential therapeutic targets Healthcare, Buckinghamshire, UK) and visualized using enhanced chemiluminescence (GE Healthcare). Membranes were stripped and for breast cancer management. reprobed with anti-b-actin (AC-74; Sigma-Aldrich) as a loading control (13). Materials and Methods Quantitative RT-PCR Cell culture Total RNA was prepared from cells using ISOGEN reagent (Nippon MCF-7 cells were purchased from ATCC (Manassas, VA, USA) and Gene, Toyama, Japan) and utilized to synthesize first-strand cDNA cultured in Dulbecco’s modified Eagle’s medium (DMEM) supple- using SuperScript III reverse transcriptase (Invitrogen) with mented with 10% foetal bovine serum, 50 units/mL penicillin, and oligo(dT)20 primer. qRT-PCR was performed on a StepOnePlus 50 mg/mL streptomycin at 37 C in a humidified atmosphere of 5% instrument (Life Technologies) using FAST SYBER Green Master CO2. The cells resistant to 4-hydroxytamoxifen (OHT) were estab- Mix (Life Technologies) and sets of gene-specific primers lished from MCF-7 cells by long-term (> 3 months) culture with (Supplementary Table S1). Cycling condition was 95 C for 2 min, 1 mM OHT, and one of the survived clones was utilized for experi- followed by 40 cycles at 95 C for 2 s and at 60 C for 30 s. The ments (7). relative differences in PCR product amounts were quantified by the comparative cycle threshold method, using glyceraldehyde-3- Screening of tamoxifen resistance-related genes using a lenti- phosphate dehydrogenase (GAPDH) as an internal control (11, viral shRNA library 13). Experiments were performed in triplicate. Student’s t-test was Decode Pooled Lentiviral shRNA Library (RHS5339) was pur- used for statistical analysis, and a probability value of P50.05 was chased from Thermo Scientific (Huntsville, AL, USA) as previously regarded as statistically significant. described (811). MCF-7 cells were infected with the lentiviral library or non-targeting shRNA control at different multiplicity of Cell proliferation analysis infection in 10-cm culture dishes and the infection rates were eval- Cell proliferation assay was performed using a kit containing WST-8 uated by analyzing GFP expression by FACS. Populations of cells (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)- expressing GFP at a 30% positivity were selected for screening to 2H tetrazolium, monosodium salt; Nacalai Tesque, Kyoto, Japan). avoid multiple infection of virus. Cells were further cultured in MCF-7 and OHTR cells were seeded in 96-well plates at densities of DMEM containing vehicle (0.1% ethanol control) or 1 mM OHT 3,000 and 2,000 cells per well, respectively, in culture medium contain- for a month. Genomic DNA was extracted from each survived popu- ing 1 mM OHT or vehicle, and transfected with siRNA targeting a gene lation using DNeasy Purification Kit (QIAGEN, Tokyo, Japan) and of interest or siControl (10 nM each) using Lipofectamine RNAiMAX. integrated shRNAs in genomic DNA were amplified by PCR using Reagent solution containing WST-8 was added to each well at

20 Role of PSMD1 in breast cancer indicated time points after transfection and cells were incubated for 2 h represent shRNAs dropped out by OHT treatment, at 37 C. Absorbance for each well was measured at 450 nm on a putatively targeting genes involved in tamoxifen resist- MULTISKAN FC ELIZA reader (Thermo Scientiﬁc, Ulm, Germany) (12, 14). For the assessment of sensitivity to proteasome ance. The list of shRNAs dropped out by OHT at a inhibitor MG-132, cells were treated with 2.5, 5, 7.5 or 10 mMMG- threshold less than 0.18-fold is shown in Table I. 132 (Peptide Institute, Osaka, Japan) or vehicle (DMSO) for 3 days and cell proliferation was analyzed. Representative results from > 3 inde- Selection of candidate tamoxifen resistance-related pendent experiments are shown as mean ± SD. Student’s t-tests were used for statistical analysis and P50.05 was regarded as statistically genes signiﬁcant. Because the genes selected by shRNA library screen were assumed as tamoxifen resistance-related genes,

Cell-cycle analysis we next investigated whether these genes could be po- Downloaded from https://academic.oup.com/jb/article-abstract/163/1/19/4061459 by Juntendo University user on 26 December 2018 OHTR cells were transfected with siPSMD1 #1, siPSMD1 #2 or tential predictive biomarkers for prognosis of breast siControl (10 nM each) in culture medium with 1 mM OHT. After cancer patients. Based on clinical datasets for breast transfection cells were fixed with 70% ethanol for 60 h, treated with RNase A for 10 min, and stained with 5 mg/mL propidium iodide cancer patients retrieved from KaplanMeier plotter (PI) (Sigma-Aldrich). Samples were subjected to cell cycle analysis (19), elevated expression levels of the following seven based on DNA content stained by PI using a FACScalibur (Becton genes were well associated with a shorter time of re- Dickinson, Cockeysville, MD, USA), and the results were analyzed lapse-free survival at a hazard ratio >1.3: TSPAN12 by CellQuest software (Becton Dickinson) to determine the percent- (tetraspanin 12), PTTG1 (pituitary tumour-transform- ages of cells in G0/G1, S and G2/M phases (11, 15). ing 1), CAMP (cathelicidin antimicrobial peptide), Analysis of protein degradation PSMD1 (proteasome 26S subunit, non-ATPase 1), OHTR cells cultured with 1 mM OHT were transfected with SNTB1 (syntrophin beta 1), ATP5A1 (ATP synthase, siPSMD1 #1, siPSMD1 #2 or siControl (10 nM each). After 60 h, H+ transporting, mitochondrial F1 complex, alpha cells were treated with 50 mg/mL cycloheximide and collected at the subunit 1, cardiac muscle) and NBN (Nibrin) (Fig. indicated time points for western blot analysis by anti-p53 (16, 17). p53 protein levels were evaluated by densitometry and normalized to 2A and Table I). Intriguingly, six of the seven genes the levels of the corresponding b-actin. also showed correlation between their elevated expression levels and shorter relapse-free survival in tamoxi- Bioinformatics fen treated patients (Fig. 2B). The clinical relevance of KaplanMeier curves of relapse free survival times for breast cancer the seven selected genes was also shown by patients were acquired through the KaplanMeier plotter (http:// ONCOMINE microarray dataset (20) as the upregula- kmplot.com), which is an online database for genome-wide valid- ation of survival-associated biomarkers in microarray data for can- tion of these genes was shown in breast cancer tissues cers including breast cancer. The ONCOMINE database is a cancer compared with normal tissues (Supplementary Fig. microarray database and online data-mining platform aimed at facil- S1). Using MCF-7 cells and its derivative OHT-resist- itating discovery from genome-wide expression analyses. The ant MCF-7 (OHTR) cells that we previously generated ONCOMINE database (https://www.oncomine.org/resource/login. (7), we examined the expression levels of the seven html) was searched for candidate genes that are upregulated in breast cancer versus normal breast tissue by at least 1.5-fold genes by quantitative RT-PCR. The mRNA levels of (P50.01). six genes including ATP5A1, CAMP, NBN, PSMD1, SNTB1 and TSPAN12 were significantly higher in Results OHTR cells compared with parental MCF-7 cells (Fig. 3A). shRNA screen to identify tamoxifen resistance- related genes in breast cancer cells Knockdown of PSMD1 and TSPAN12 decreased To explore tamoxifen resistance-associated genes in OHTR cell proliferation breast cancer cells, hormone-sensitive MCF-7 cells To investigate the functional roles of the above-men- were infected with lentiviral shRNA library that com- tioned six genes in the proliferation of breast cancer prised 10,000 shRNAs with unique barcodes. Cell cells, we employed loss-of-function studies using spe- populations with relatively low infection efficiency cific siRNAs. At first, knockdown efficiency of siRNAs (30%) were selected in order to prevent multiple in- that we designed was examined in OHTR cells by fection of a single gene and cultured continuously in a qRT-PCR (Fig. 3B). siRNAs targeting ATP5A1, culture medium containing either 1 mM 4-hydroxita- NBN, PSMD1 and TSPAN12 could efficiently inhibit moxifen (OHT) or vehicle for a month to establish the expression of the corresponding genes, whereas OHT-resistant or control breast cancer cells. We pre- siRNAs targeting CAMP and SNTB1 were not so ef- pared biologically duplicated genomic DNA samples ficient (Fig. 3B and data not shown). In cell growth for both vehicle-treated and OHT-treated cells. The assay, siRNAs targeting PSMD1 (siPSMD1 #1) and integrated shRNAs in samples were amplified by TSPAN12 (siTSAPAN12) significantly reduced PCR and their amounts were quantified using a OHTR cell proliferation (Fig. 3C). In addition, an- custom-made microarray (Fig. 1A) (9, 18) . We con- other siRNA targeting PSMD1 (siPSMD1 #2) also firmed that the scatter plot for array signals of two significantly repressed PSMD1 mRNA expression independent control samples was shown linearly with (Fig. 3D) and decreased OHTR cell growth (Fig. a high consistent positive correlation (Fig. 1B). When 3E). In MCF-7 cells, siRNAs targeting ATP5A1, analyzed similarly, the plots of array signals for the NBN and TSPAN12, as well as 2 siRNAs against OHT-treated and control samples were rather scat- PSMD1 (siPSMD1 #1 and #2) decreased the expres- tered (Fig. 1C and D). Among differential signals for sion of their target genes (Supplementary Fig. S2A) OHT-treated versus control, we paid particular atten- and cell growth (Supplementary Fig. S2B and C). In tion to those under a diagonal line because they terms of TSPAN12, the gene silencing has been

21 T. Okumura et al.

A B Microarray signal plots

Infected with lentiviral shRNA library 100000

10000 MCF-7 cells

Vehicle treatment OHT treatment channel) 1000 for 1 month for 1 month 100 Control #2 Control Genomic DNA 10 Downloaded from https://academic.oup.com/jb/article-abstract/163/1/19/4061459 by Juntendo University user on 26 December 2018 (Intensity-green (Intensity-green PCR amplification of shRNA 1 1 10 100 1000 10000 100000 Control #1 Microarray analysis (Intensity-red channel)

C Microarray signal plotsD Microarray signal plots

100000 100000

10000 10000

1000 1000 green channel) - OHT #2 OHT #1

100 100 (Intensity (Intensity-green channel) (Intensity-green

10 10 10 100 1000 10000 100000 10 100 1000 10000 100000 Control #1 Control #2 (Intensity-red channel) (Intensity-red channel) Fig. 1 Screening of shRNAs for tamoxifen resistance-related genes in MCF-7 cells. (A) Schematic representation of shRNA screening. MCF-7 cells were infected with a lentiviral shRNA library and further cultured with 4-hydroxytamoxifen (OHT) or vehicle for 1 month. Amounts of shRNAs integrated into each genomic DNA were quantiﬁed by microarray. (BD) Scatter plots of array signal intensities for individual shRNAs (B, Control sample 1 (Control #1) versus Control sample 2 (Control #2); C, OHT sample 1 (OHT #1) versus Control #1; D, OHT sample 2 (OHT #2) versus Control #2).

Table I. The list of dropped out shRNAs in MCF7 cells after 1-month tamoxifen treatment

Microarray KaplanMeier plotter

Targeting gene Controla OHTb OHT/Control Hazard ratio P-value

THEM4 33,418.0 3,661.7 0.10 0.49 51.0EÀ16 MLF1 46,508.4 5,705.9 0.12 1.09 1.5EÀ1 VAPA 49,953.9 6,589.8 0.13 1.20 2.2EÀ3 SOX7 31,568.6 4,282.9 0.13 0.63 2.1EÀ8 GIF 61,471.8 8,527.4 0.13 0.62 4.0EÀ15 ARPP-21 45,134.7 6,733.9 0.14 0.73 4.0EÀ4 TSPAN12c 51.6 7.7 0.14 1.38 9.7EÀ9 PTTG1c 46,135.3 6,975.8 0.15 1.86 51.0EÀ16 CDC14C 41,345.2 6,323.5 0.15 0.83 2.2EÀ2 SYT16 48,435.0 7,875.2 0.16 0.68 5.1EÀ6 CAMPc 62.3 10.2 0.16 1.33 2.3EÀ6 PSMD1c 63,373.0 10,389.8 0.16 1.37 7.1EÀ8 HSPB7 62,671.2 10,401.1 0.16 0.73 2.8EÀ7 SENP2 35,234.2 5896.2 0.16 0.73 4.0EÀ8 SNTB1c 63,014.4 10,586.8 0.16 1.34 8.7EÀ6 ATP5A1c 40,794.2 6,882.4 0.16 1.61 51.0EÀ16 ESRRB 61733.2 10,395.4 0.16 0.72 8.0EÀ5 PHEX 39,344.4 6,698.6 0.17 0.76 7.3EÀ5 SULT1E1 64,298.6 10,965.7 0.17 0.79 4.1EÀ5 NBNc 56,651.6 9,745.0 0.17 1.41 0.4EÀ9 GTF3C5 34,579.8 5,993.2 0.17 d d KLHL5 29,953.5 5,219.8 0.17 0.4 51.0EÀ16 UBA1 64,981.7 11,570.9 0.17 0.94 3.3EÀ1 POLR3K 33,231.0 5,826.6 0.17 1.12 5.2EÀ2 aAveraged signal intensity of shRNA in the vehicle-treated control cells was quantiﬁed by microarray. The results were shown as mean value. bAveraged signal intensity of shRNA in the OHT-treated cells was quantiﬁed by microarray. The results were shown as mean value. cGenes shown in bold exhibit >1.33 hazard ratio in the analysis using the KaplanMeier plotter were further investigated as candidate tamoxifen resistance-associated genes. dProbes corresponding to the genes were not available in the KaplanMeier plotter database.

22 Role of PSMD1 in breast cancer

A Kaplan – Meier plot

1.0 ATP5A1 1.0 CAMP 1.0 NBN 1.0 PSMD1 y t i l i b

a 0.6 0.6 0.6 0.6 b o r P 0.2 Low p 0.2 Low p = 2.3E-6 0.2 Low p = 4E-9 0.2 Low p = 7.1E-8 High <1.6E-16 High High High 0 0 0 0 0 50 150 250 (M) 0 50 150 250 (M) 0 50 150 250 (M) 0 50 150 250 (M) Downloaded from https://academic.oup.com/jb/article-abstract/163/1/19/4061459 by Juntendo University user on 26 December 2018

1.0 PTTG1 1.0 SNTB1 1.0 TSPAN12

0.6 0.6 0.6

Probability 0.2 Low p 0.2 Low p = 8.7E-6 0.2 Low p = 9.7E-9 High 䠘 1.0E-16 High High 0 0 0 0 50 150 250 (M) 0 50 150 250 (M) 0 50 150 250 (M)

B Kaplan – Meier plot 1.0 ATP5A1 1.0 CAMP 1.0 NBN 1.0 PSMD1 y t i l i

b 0.6 0.6 0.6 0.6 a b o r P 0.2 Low p =0.023 0.2 Low p = 0.0012 0.2 Low p = 0.015 0.2 Low p = 1.3E-4 High High High High 0 0 0 0 0 50 150 250(M) 0 50 150 250 (M) 0 50 150 250 (M)0 50 150 250 (M)

1.0 PTTG1 1.0 SNTB1 1.0 TSPAN12

0.6 0.6 0.6

0.2 Low p = 3.5E-7 0.2 Low p = 0.21 0.2 Low p = 0.007 Probability High High High 0 0 0 0 50 150 250 (M) 0 50 150 250 (M) 0 50 150 250 (M) Fig. 2 Relapse-free survival analysis of candidate gene expression in breast cancer. (A, B) Relapse-free survival curves were generated using online survival analysis tool KaplanMeier plotter with the setting of optimal cut point (http://kmplot.com). All patients (n = 3951, A) and patients only with tamoxifen treatment (n = 809, B) were utilized on the analysis. M: months. reported to decrease primary tumour growth of breast PSMD1 knockdown in parental MCF-7 cells also ex- cancer cells in mouse xenograft model (21). Because hibited a similar cell cycle profile compared with that in PSMD1 and TSPAN12 may play particular roles in OHTR cells (Supplementary Fig. S4A and B). Overall, tamoxifen-resistant breast cancer, we focused our PSMD1 may modulate cell cycle profile in hormone- study on PSMD1. In terms of intrinsic subtypes of sensitive and tamoxifen-resistant breast cancer cells. breast cancer, PSMD1 mRNA is more abundantly expressed in aggressive cancer cell lines including luminal PSMD1 is involved in degradation of p53 protein B (ZR-75), claudin-low (MDA-MB-231), HER2 Since PSMD1 is a gene that encodes a 26S proteasome (MDA-MB-453) and basal (MDA-MB-468) subtypes subunit, we questioned whether the modulation of compared with luminal A type MCF-7 and T47D cells PSMD1 influences the degradation of tumour suppres- (Supplementary Fig. S3). sor proteins such as p53 through the alteration of 26S proteasome activity. In cancers, increased proteasome Knockdown of PSMD1 causes cell-cycle inhibition activity often results in a ubiquitin-dependent and -in- The influence of PSMD1 knockdown on cell-cycle pro- dependent degradation of tumour suppressor proteins gression in OHTR cells was assessed by FACS. (22) and proteasome inhibitors are clinically approved Compared with control siRNA (siControl) treatment, for the treatment of some malignancies (23). Cell pro- PSMD1 knockdown in OHTR cells could modulate liferation assay revealed that OHTR cells were highly cell cycle profile by reducing S phase and increasing resistant to MG-132 compared with MCF-7 cells G2/M phase population, respectively (Fig. 4A and B). (Supplementary Fig. S5). We then examined the effects

23 T. Okumura et al.

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B C

Fig. 3 PSMD1 is overexpressed in tamoxifen-resistant breast cancer cells and regulates cell growth. (A) Overexpression of candidate genes in OHTR cells. mRNA expression levels of candidate genes were quantified by qRT-PCR in OHTR cells and parental MCF-7 cells. Data are normalized to GAPDH and presented as mean ± SD. (n = 3; **, P50.01; ***, P50.001) (B) Knockdown efficiency of siRNAs targeting ATP5A1 (siATP5A1), NBN (siNBN), PSMD1 (siPSMD1 #1), and TSPAN12 (siTSPAN12). OHTR cells were transfected with specific siRNAs or control siRNA (siControl) for 60 h, and the expression levels of the genes were analyzed by qRT-PCR. Data are normalized to GAPDH and presented as mean fold change ± SD versus siControl. (n = 3; **, P50.01; ***, P50.001) (C) Growth inhibition of OHTR cells by siATP5A1, siNBN, siPSMD1 #1 and siTSPAN12. WST-8 cell proliferation assay was performed at indicated time points after transfection of each siRNA (10 nM) in OHTR cells cultured with 1 mM OHT. Data are presented as mean ± SD. (n =5;*, P50.05; **, P50.01; ***, P50.001) (D) Knockdown efficiency of siRNAs targeting PSMD1 (siPSMD1 #1 and #2) was examined as in (B). (E) Growth inhibition of OHTR cells by siRNAs targeting PSMD1 (siPSMD1 #1 and #2).

24 Role of PSMD1 in breast cancer

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Fig. 4 Knockdown of PSMD1 induces cell cycle blockade of OHTR cells. (A) OHTR cells were transfected with siPSMD1 #1, siPSMD1 #2 or siControl in culture medium containing 1 mM OHT for 60 h. Cells were stained with propidium iodide and analyzed by FACS. Knockdown of PSMD1 reduced population of cells in S phase whereas increased that in G2/M phases. (B) Percentages of cells in G0/G1, S and G2/M phases were analyzed by using CellQuest software. Data are presented as mean ± SD. (n =3;*, P50.05; **, P50.01). of PSMD1 siRNA on p53 expression and function in cells via functional shRNA library screening. Among OHTR cells. Notably, siRNAs targeting PSMD1 these genes, we selected PSMD1 and TSPAN12 as can- markedly increased the expression levels of p53 protein didate tamoxifen resistance-associated genes based on (Fig. 5A). We next assessed whether PSMD1 silencing clinical data and knockdown experiments. siRNAs tar- influences the degradation of p53 protein in breast geting PSMD1 and TSPAN12 repressed the prolifer- cancer cells. Protein levels of p53 were analyzed by ation of OHTR cells. Knockdown of PSMD1 western blotting in OHTR cells, which were treated increased p53 protein level through the suppression with PSMD1 siRNAs in the presence of protein syn- of protein degradation in OHTR cells. PSMD1 is a thesis inhibitor cycloheximide (Fig. 5B). p53 protein subunit of 19S regulatory complex of 26S proteasome, level at each sample was normalized to b-actin level which plays a crucial role in the degradation of ubiqui- as a loading control (Fig. 5C). p53 protein was more tinated protein that relates to various biological pro- stabilized in PSMD1-silenced OHTR cells than in cesses (25, 26). PSMD1 knockdown also upregulated siControl-treated cells. We examined the influence of p53 target genes including p21 and SFN, and increased PSMD1 silencing on p21 and SFN, both are major the population of OHTR cells in G2/M phase. p53 downstream factors of p53 (24). Knockdown of would exert cell-cycle arrest and represses cell prolifer- PSMD1 upregulated p21 and SFN mRNA levels in ation through the transcriptional activation of p21 and OHTR cells (Fig. 5D). Upregulation of p21 mRNA SFN (27). Intriguingly, it has been reported that level was also observed in MCF-7 cells treated with PSMD1 mRNA is overexpressed in breast cancer to- PSMD1 siRNA (Supplementary Fig. S6). We further gether with PSMD2 and PSMD11, another subunits examined whether p21 substantially contributes to the of 26S proteasome (28). Moreover, it has been re- growth inhibition mediated by PSMD1 silencing. It is ported that MCF-7 and MDA-MB-231 cell lines over- notable that PSMD1 knockdown-mediated repression express 26S proteasome subunits, PSMD5 and of OHTR cell growth was recovered by p21 silencing PSMD3, and show higher proteasome activity than (Fig. 5E). These results indicate that PSMD1 silencing non-tumourigenic mammary epithelial MCF-10A decreased cell proliferation partly through activation cells (29). The report also demonstrates that MCF-7 of p53 pathway. and MDA-MB-231 cells are much more resistant to MG-132 compared with MCF-10A cells. We noted Discussion that OHTR cells were highly resistant to MG-132 compared with MCF-7 cells. Thus, PSMD1 would contrib- In the present study, we identified seven genes that may ute to the enhancement of 26S proteasome activity in be involved in tamoxifen resistance in breast cancer OHTR cells, putatively collaborating with other 26S

25 T. Okumura et al.

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Fig. 5 PSMD1 is involved in the regulation of p53 protein expression. (A) Silencing of PSMD1 increased p53 protein abundance. OHTR cells were transfected with each siRNA in the presence of OHT for 60 h. Cell extracts were subjected to SDS-PAGE and western blot analysis using the indicated antibodies. (B) PSMD1 contributes to the regulation of p53 protein degradation. OHTR cells were transfected with each siRNA for 60 h and treated with 50 mg/mL cycloheximide (CHX) for the indicated time. Cell extracts were analyzed by western blotting. (C) p53 protein levels were quantified by densitometry. Data were normalized to each corresponding level of b-actin and shown as mean ± SD. (n = 5; **, P50.01; ***, P50.001) (D) Knockdown of PSMD1 induces p21 and SFN mRNA expression in OHTR cells. Cells were transfected each siRNA for 60 h and mRNA level was quantified by qRT-PCR. (n =5;*, P50.05; **, P50.01; ***, P50.001) (E) p21 siRNA partially rescues the growth inhibition of OHTR cells induced by PSMD1 siRNA. Cells were transfected with siRNAs at a final concentration of 20 nM: siControl (20 nM siControl), siPSMD1 #1 (10 nM siPSMD1 #1 + 10 nM siControl), sip21 (10 nM sip21 + 10 nM siControl), siPSMD1 #1 + sip21 (10 nM siPSMD1 #1 + 10 nM sip21). Cells were then cultured in the presence of tamoxifen and WST-8 cell proliferation assay was performed at the indicated time points. Data are presented as mean ± SD (n = 5; ***, P50.001).

proteasome subunits. Taken together, the present higher proteasome activity was found with increased study showed that PSMD1 is a gene associated with levels of proteasomal subunit proteins, such as PSMD4 acquired tamoxifen resistance that may contribute to and PSMA5 (34). Mechanisms underlying enhanced the proliferation of breast cancer cells putatively proteasome activity in cancers remain elusive; never- through modulating p53 pathway.p53 protein levels theless, our ﬁndings together with previous literature are strictly controlled at low levels in normal cells by suggest that proteasome genes may contribute to ubiquitination and subsequent proteasomal degrad- cancer pathogenesis and drug resistance. Indeed, ation by 26S proteasome. A major E3 ubiquitin proteasome inhibitors are clinically applied to cancer ligase Mdm2 exerts p53 degradation also through treatment. For example, 20S complex inhibitor borte- 26S proteasome system (30, 31). Activation of prote- zomib (35) has been used as a therapeutic asome system has been shown in various tumours drug against multiple myeloma (36), although the including breast cancer compared with adjacent drug has limitations with its toxicity, relatively low ef- normal tissues (32, 33). In colon cancer, for example, ﬁcacy on solid tumours, and acquired resistance (37).

26 Role of PSMD1 in breast cancer

In triple-negative breast cancers with gain-of-function Acknowledgements p53 mutation, transcription factor Nrf2 interacts with We thank W. Sato and Drs. R. Yamaga, T. Miyazaki, S. Nagai, N. mutated p53 and activates a series of proteasome Kurogi-Ota for their technical assistance. genes, leading to the acquisition of proteasome inhibitor resistance (38). Thus, the development of novel strategy including PSMD1 targeting will enable to Funding manage enhanced proteasome activity in hormone-re- This work was partially supported by Grants of the Cell Innovation fractory breast cancer. It remains to be resolved Program, Development of Innovative Research on Cancer Therapeutics (P-DIRECT), Grants-in-Aid, and Support Project of whether the proteasome pathway specifically contrib- Strategic Research Center in Private Universities from the Ministry utes to tamoxifen resistance. It is also known that the of Education, Culture, Sports, Science, and Technology, Japan; by Downloaded from https://academic.oup.com/jb/article-abstract/163/1/19/4061459 by Juntendo University user on 26 December 2018 inhibition of proteasome function stabilizes wide range the Practical Research for Innovative Cancer Control and the of tumour inhibitory proteins in addition to p53. Project for Cancer Research And Therapeutic Evolution Nevertheless, we noted the correlation between the (P-CREATE) from Japan Agency for Medical Research and development, AMED; by Grants from the Japan Society for the elevated PSMD1 expression levels and shorter re- Promotion of Science, Japan (16K09809, 15K15353, 17H04205 lapse-free survival in tamoxifen-treated breast cancer and 26293223); by the Advanced Research for Medical Products patients. Moreover, our data revealed that the Mining Program of the National Institute of Biomedical p53p21 pathway may be involved in the tamoxifen- Innovation (NIBIO), Japan. resistant growth of OHTR cells. Although other pos- sibilities are not ruled out, proteasomal regulation of Conflict of Interest p53 protein degradation would play a role in tamoxi- None declared. fen resistance in breast cancer. In regard to other genes identified in shRNA library screening, mitochondrial ATP synthase ATP5A1 is References overexpressed in cancers such as glioblastoma, malig- 1. Lozano, R., Naghavi, M., Foreman, K., Lim, S., nant brain tumour (39) and colorectal cancer (40). Shibuya, K., Aboyans, V., Abraham, J., Adair, T., CAMP is one of the cathelicidins that are small, cat- Aggarwal, R., Ahn, S.Y., Alvarado, M., Anderson, ionic, antimicrobial peptides and often plays a role in H.R., Anderson, L.M., Andrews, K.G., Atkinson, C., the migration and invasion in cancer cells (41). In add- Baddour, L.M., Barker-Collo, S., Bartels, D.H., Bell, ition, CAMP is speculated to accelerate with cancer M.L., Benjamin, E.J., Bennett, D., Bhalla, K., Bikbov, proliferation by stimulating the initiation of angiogen- B., Bin Abdulhak, A., Birbeck, G., Blyth, F., Bolliger, I., esis and recruitment of immune cells (42). NBN plays Boufous, S., Bucello, C., Burch, M., Burney, P., an important role in the DNA damage response and Carapetis, J., Chen, H., Chou, D., Chugh, S.S., DNA repair, and its mutation is associated with Coffeng, L.E., Colan, S.D., Colquhoun, S., Colson, K.E., Condon, J., Connor, M.D., Cooper, L.T., Nijmegen breakage syndrome (43). PTTG1 is overex- Corriere, M., Cortinovis, M., de Vaccaro, K.C., pressed in various tumours including breast cancer and Couser, W., Cowie, B.C., Criqui, M.H., Cross, M., may exhibit an oncogenic effect through the modula- Dabhadkar, K.C., Dahodwala, N., De Leo, D., tion of p53 functions (44, 45). Moreover, it has been Degenhardt, L., Delossantos, A., Denenberg, J., Des reported that PTTG1 is overexpressed in ER-positive Jarlais, D.C., Dharmaratne, S.D., Dorsey, E.R., breast cancer samples that relapsed after tamoxifen Driscoll, T., Duber, H., Ebel, B., Erwin, P.J., treatment and associated with shorter relapse-free sur- Espindola, P., Ezzati, M., Feigin, V., Flaxman, A.D., vival (46). In terms of tetraspanin TSPAN12, ablation Forouzanfar, M.H., Fowkes, F.G., Franklin, R., of the protein from MDA-MB-231 cells repressed pri- Fransen, M., Freeman, M.K., Gabriel, S.E., Gakidou, E., Gaspari, F., Gillum, R.F., Gonzalez-Medina, D., mary tumour xenograft growth in mice (21). TSPAN12 Halasa, Y.A., Haring, D., Harrison, J.E., Havmoeller, also induces b-catenin in lung cancer-associated fibro- R., Hay, R.J., Hoen, B., Hotez, P.J., Hoy, D., blasts and promotes the migration and proliferation of Jacobsen, K.H., James, S.L., Jasrasaria, R., adjacent cancer cells (47). These findings suggest that Jayaraman, S., Johns, N., Karthikeyan, G., these five genes may also be involved in the patho- Kassebaum, N., Keren, A., Khoo, J.P., Knowlton, physiology of breast cancer including tamoxifen L.M., Kobusingye, O., Koranteng, A., Krishnamurthi, resistance. R., Lipnick, M., Lipshultz, S.E., Ohno, S.L., In summary, we identified several genes that poten- Mabweijano, J., MacIntyre, M.F., Mallinger, L., tially participate in acquired tamoxifen resistance in March, L., Marks, G.B., Marks, R., Matsumori, A., breast cancer cells by functional shRNA library- Matzopoulos, R., Mayosi, B.M., McAnulty, J.H., McDermott, M.M., McGrath, J., Mensah, G.A., mediated screening. Among the candidate genes, Merriman, T.R., Michaud, C., Miller, M., Miller, T.R., PSMD1 regulates the degradation of p53 protein and Mock, C., Mocumbi, A.O., Mokdad, A.A., Moran, A., cell cycle progression. The present findings would pro- Mulholland, K., Nair, M.N., Naldi, L., Narayan, K.M., vide useful information for alternative diagnostic and Nasseri, K., Norman, P., O’Donnell, M., Omer, S.B., therapeutic options for hormone-refractory or Ortblad, K., Osborne, R., Ozgediz, D., Pahari, B., advanced breast cancer. Pandian, J.D., Rivero, A.P., Padilla, R.P., Perez-Ruiz, F., Perico, N., Phillips, D., Pierce, K., Pope, C.A. 3rd., Porrini, E., Pourmalek, F., Raju, M., Ranganathan, D., Supplementary Data Rehm, J.T., Rein, D.B., Remuzzi, G., Rivara, F.P., Roberts, T., De Leoń, F.R., Rosenfeld, L.C., Rushton, Supplementary Data are available at JB Online. L., Sacco, R.L., Salomon, J.A., Sampson, U., Sanman,

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