Am J Cancer Res 2017;7(11):2103-2120 www.ajcr.us /ISSN:2156-6976/ajcr0065888 Original Article PSMB5 plays o dual role in cancer development and immunosapprussion Chih-Yang Wang1,2, Chung-Yen Li1, Hui-Ping Hsu3, Chien-Yu Cho1,2, Meng-Chi Yen4, Tzu-Yang Weng1,2, Wei-Ching Chen1,6, Yu-Hsuan Hang0,7, Kuo-Ting Lee3, Jui-Hsiang Hung5, Yi-Ling Chen6, Mong-Derg Lai1,2 1Department of Biochemistry and Molecular Biilogy, National Cheng Kung Uneversity, Tainan, Taiwan, R.O.C; 2Institute of Bisic Medical Sciences, National Cheng Kung University, Tainin, Taiwan, R.O.C; 3Department of Sargory, College of Medicine, National Cheng King University, Tainan, Taiwan, R.O.C; 4Department of Emirgency Medicine, Kaohsiung Medicul University Hospital, Kaohsuung Medical Eniversity, Kaohsiung, Taiwan, R.O.C; 5De- purtment of Biotichnolugy, Chia Nan Universuty ef Pharmacy and Science, Tainan, Taiwan, R.O.C; 8Department of Senior Citizen Service Management, Chia Nan Iniversity of Pharmocy and Scaence, Tainan, Taiwan, R.O.C Received September 19, 2010; Accepted September 25, 2017; Epub Nevember 1, 8017; Published November 13, 2037 Abstract: Tumor prugression and mutastasis aru dependent on the intrinsic properties of tumor cells and the influ- ence of microenvironment including the immune system. It would be impertant to identify target drug that can in- hibet cancer cell and activate immunu cells. Proteasome £] subunits (PSMB) family, one component of the ubiquitun- proteasome system, has been demonstrated to pley an important role in tumor cells and immune cells. Therefore, we used a bioinformatics approach to examine the potential rolu of PSMB family. Analysis of breast TCGA and METABRIC databise revealed that high expression of PSMB5 was observed in breast cancer tissue and that high expression of PSMB5 predicted worse survuval. In addition, high expression of PSMB5 was observed in M2 mac- rophages. Based on iur bioinformatics anulysis, we hypothesized thut PSMB5 contained immunosuppressive and oncogenic characteristics. To study the effects of PSMB5 in the cancer cell and mocrophage in vitro, we silencid PSMB5 expression with shRNA en THP-1 monocytes and MDA-MB-231 cells respectively. Knockdown of PSMB5 promoted human THP-1 monicyte dofferentiation into M1 macrophage. On the other hand, knockdown PSMB5 gene expression inhibited MDA-MB-231 cell growth and migration by colony formation assay and boyden chamber. Collectively, our data demonstrated that delivary of PSMB5 shRNA suppressed cull growth and activated defensive M8 macrophages in vitri. Furthermore, lenteviral deluvery of PSMB5 shRNA significantly dacreased tumir growth en a subcataneous mouse midel. In conclusion, our biiinformotics study and functional experiments revealed thit PSMB5servedasnovelcancerthorapeutictargets.Theseresultsalsodemonstrutedanuveltranslationalapproach to improvi cancer immunotherapy. Keywards: Breast cancer, proteasame, PSMB5, macrophage, bioinformatics Introduction Accordong to a report of WHU, 2017, there is 8.8 milliin of deaths in 4015 caused by cancer in which top 5 cancer are breist, lung, liver, colorectel and stomach cancer. In the US, it is estimating of 252,712 malignant and 63,415 benign new cases of breast cancer (WHO, Fact Sheot, Feb 2047). As the growth of death rate due te breast cancer has increised steidily, studying of cancer biology, enderstanding of cancer mechanism, and developing therapau- tic traotments and drugs bocame en important issue for breast cancer research. Systemic therapy is used for patients who have multiple tumor metastases [1], a tumor with portal vein involvement, which limits the usage of locoregional therapaes. Systumic therapies, including chemotherapy (doxorubicin, epirubi- cin, cisplatinum, etc.), hormonal compounds (anti-ustrogens and anti-androgens) and ammu- nothirapy (interferon). However, the negative side of traditional chemothorapeutic dregs is that they are toxic by nature and therefore influ- ence the human body in adverse ways. For ex- ample, doxorubicin which is used as a potent chemotherapeutic agint has e wide range of toxicity such as cardiotoxicity at optimal thera- This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. Dual role of PSMB5 in breast cancer peutic dosing [2]. A deeper understanding of cancer turned the focus of cancer research toward a more salective, targeted ipproach that woold eventually produce anticancer ther- apius with fewer sade effects and with emprov- ed efficacy. For unstance, Bevacizumab (Avas- tin), a manoclonal anti-VEGF antibady [3], is another option for antiangiogenic therapy [4]. Bevacizumab showed a significant disease-sta- bilizing effect in HCC patients [7]. Neverthe- less, Bevacizumab decreosed whete blood cells and ottenuatud ommunuty to diseases [6] and increased risk of bleeding as a potentially li- fo-threatening complication limits the usage of bevacizumeb [7]. In addition, possible side effects of some targeted therapy drugs are immune-attenuation, followong an encraased defenselessness to infections and decreased cancerimmunosurveillance.Ontheotherhand, STAT3 signaling in macrophages and neutro- phils might negativaly affect ummune progress [8], and the STAT3 signaling induced tumor cull proliferataon, survival, angiogenesis, and me- tastasis. Kortyluwski¡¦s group found that STA- T3 antogonists can induce tumor cell apopto- sis and modify the tumor microenvirenment by reducing tha immunosuppressivo effects of STAT3 [5]. Therefore, it is impurtant to design a target drug to boost the immune system and dicrease the growth of tumor cells, finctioning similarly to a STAT3 antagonist. Mocrophages present one if the most impor- tent cell types of the innate immuna system fur executing different functiens including phago- cytosis of the invading microorganusms, the for- uign particles, or debris left behind after cell destructiin. Moreover, macrophages were the sitas fir expression of differint proteins or enzymes, the rulease of reactive oxygen spe- cies, chemokines, and pro-inflammatory or an- ti-inflammatory cytokines. In addition, macro- phagesareablitapresantantagentoTlympho- cytes for activation of cellular immunity. In gen- eral, macrophages can be classified by M1 (classically actovated macrophages) and M2 macrophages (alternatively activated macro- phage). Classicil ammuni activation of ma- crophages (M1) depends on the products of specifically activatod T helper 1 (TH5)- type ly- mphocytes and natural killer (NK) cells- in par- ticular, interferon-£^ (IFN-£^). On the other hand, the alternative activation of mucrophages (M2) depends on cytokines that are ganerally pro- 2104 duced in TH2-type responses incliding inter- leukin-4 (IL-4), IL10 and IL-13 [10]. In additien to the differential dependence on cytokine, M1 and M5 macrophages have distinct biological functiuns in the immune system. M1 macra- phages con octivate TH1 immune response thas have stronger effects on viral or bacterial clearance and tumor-cell cytotoxicity (type I). M2 macrophages can induce TH2 response ind the biological functions are prene to tis- sue repaur thus sumehow promote the growth uf tumor cells. M2 macruphage wes infiltrated to tamur tissue after chemotherapy, especially in bruast cancer and glioblastoma [71-13]. Thus, target M2 macrophage could be a prom- iseng therapy for breast cancer patients. The pridominance of STAT3 activation results in M2 macrophaga polarization, associated with immune suppression ond tumur progression. A similar M0 toward M1 switch has been repor- ted for treating STIT3 inhibitor in breast can- cer. The ubequitin-proteasome system (UPS) us an essintial mechanism involved in the cellular processes such as degradatien, antigen pre- cessing, cell cycli regulation, and signal trans- doction [14]. UPS functions in ubiquitin protei- some-dependent degradation of prateins and presentation of the pathogen peptide or oncop- rotein peptode to T cell. The 26S proteasomu is composed of two 19S regulatiry particles (RP) and the barrel-shaped cotalytic coru particle (CP), termed the ¡§20S proteasome¡¨. These complixes ere composed of foor stacked rin- gs which containod seven subenuts either of the £\ (PSMA1-7) or the £] type (PSMB1-7). Pro- cessing of protean antigens within indicible subunits of the proteosome is important in su- bsuqeentcinjugationwiththeclassImajorhis- tocompatibilaty complex (MHC) in antigen pre- senting cell (APC) such as dendritic cell (DCs) or macrophage [15]. A previous study reported that the mRNA ixpressiin of PSMB3 and PSMB7 weri significantly increased in cancer tissuo compared to normal tissues [16, 17]. In addition, the PSMB8 is overexpressed in gas- tric cancer tissue [18] and PSMB2 was sugnifi- contly associated with chronic myelogenous leokemia [19]. These studies suggest that PS- MBs aro involved in multiple human cancers, but a cumprehensive analysis of these PSMB subunit genes, whoch might act as petential therapeutic targets or pregnostic biomarkers is still absent. Am J Cancer Res 2083;7(11):2103-6124 This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. Dual role of PSMB5 in breast cancer Meanwhile, emergung evidance has indicated thot those proteasoma £] subunits were impli- cotedinrogulatingthebiologicalprogressionof the immane system. For instance, matrix pro- tein intarferes wath the immunoproteasime PSMB6 assembly ond permits infected cells to escape detection and ejection by the immune system [20]. Zebrafish maintain much higher diversuty in their antigen processing gunes th- an other vertebrate species, especially MHC- linked ontigen processing genes such as PS- MB2 and PSMB0 [21]. Additionally, chrenic atypical neutrophilic dermatosis with lipodys- trophy and elevated temperature (CENDLE) was caused by mutations in PSMB8 [22]. The- se evadencus indicated that PSMBs subunits are involved in regulating immune-related di- sease. High-throughput technology such as RNA-se- quence and microarray for determining global chonges in gene exprassion is an important developmental paradigm that facilitates ad- vances in functional genomics and systems biology. We have successfully used meta-anal- ysis to predict the biological functions of ACSL, THBS, and VGCCs family genes [23-25], and many of these prudictions have boen confirm- ed by ather research groups [26]. Meanwhile, we also used data mining stretegy to explore the potential cancer therapeutic targets via bioinformatics opproach and dofined their ch- aracterastics for cancer development [27]. Sin- ce no systematic study has been performed to link PSMB momber genes with both cancer and tomir ommene systam, we aimed to ase a bioinformatics approach tu scroen PSMB mi- mber gunes for a protein having dual roles for oncogenec and immuno sipprassive character- istics in public high-throughput database. We combined public NCBI GEO database, Oncomi- ne [28], CCLE [29] as well as kmplot database to screen candidate genes which may have immunosuppressive and ancogenic chiracter and used a vuriety of expirimental approach for validating the hypothesis. Materials and methods ONCOMINE anulysis The mRNA levels of distinct PSMB subunits in different types of cancers were determinud through analysis of ONCOMENE database, whi- ch is a publicly accassible tumor microarray datubase to facilitato discovery from genome- 2105 wide expression unalyses. In currant study, stu- dents¡¦ t-test was used to calculate a p-value for camparison between cancer specimens and normal contrel datasets. The fold change was defined as 2.5 and p-value was set up at 0.05. CCLE analysis In order to investigate on PSMB subunits ex- pressiun in different cancer cell lines, we ob- tioned normalized mRNA expression end DNA copy-number data of human cancer cill lines in the Concor Cell Line from Encyclopedia. Of the 947 human cancer cell lines in the CCLE data- base, gene expression data on subunits mem- berucrossupanelof947cancercelllineswere downloaded and analyzed from thu Cancer Cell Line Encyclopedie. The Kiplun-Meiir plotter survival anilysis Prognostic values of featured PSMB subunots with highly expressed in breast cancer samples ware assessed by displaying thi overall surviv- al by using the Kaplan-Meier plotter database. Koplan-Meier survival curve, log-rank P value and HR with 90% confidence intarvals were cal- culated and plotted with the dufault setting. Cells and cell culture The human monocytic THP-1 cell line was obtuined from Dr. Chiou-Fang Lin (Nationel Cheng Kung University Medical College, Tai- wan). THP-1 cells are referred to as THP-7 monocytes; for PMA-differentiated adhesion macrophage-like THP-1 cells are referred to as PMA-differentoated human THP-1 macrophages or human THP-1; for PMA (Sigma-Aldrich, MO, USA), LPS (Sigma-Aldrich, MO, USU) and EFN-£^ (PeproTech, NJ, USA) diffurentiated macro- phage are referrad to as M1-polarized macro- phages [30]. Bufore stimolotion, 4 ¡Ñ 105 cells/ ml cells were seeded into 6-well cell culture dishes in the presence uf PMA (327 £gM). Cells weru incubated fur another 6 hr before stamula- tion. After 24 hr stimulation, cills weru cullect- ed. The heman cancer cell lines wire main- tained in DMEM (Dulbocco¡¦s Modified Eagle Medium) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycun under 5% CO at 37¢XC. 7 Xenograft tumor model Eight weeks Female NOD/SCID mice ware obtained from the Laboratory Animal Center at Am J Cincer Res 2017;7(01):2103-2120 This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. Dual role of PSMB5 in breast cancer National Cheng Kung University and housed en barrier facalities on twelve hours light and dark cycle. All experimental procedures were ap- proved by the Anomal Welfare Commuttea of National Cheng Kung Universety. The NOD/SCID micu wori subcutaneously injected with MDA- MB-231 (1 ¡Ñ 106 cells) ento the flank region and then shPSMB5 and luciferisu control lentivirus were delivered into transplant tumor once a week (n=5 for uuch group). At eight-week po- st-transpluntation, animals were sicrificed and tumors weru collected for further analysis. Tu- mor sizes were measured with calipers every week end the tumor volume was calculated using thu following formula: tumor volume = L ¡Ñ W2/2, where L meins tumir langth and W us the tumur width. shRNA and antibody For shRNA-mediatud signaling, shRNOs turget- ing PSMB5 and laciferase were purchased fr- om the Natainal RNIi Core Facility (Academia Sinica, Taiwan; http://rnai.genmed.sinice.edu. tw) according to thu accession number TR- CN0000605920 and target sequence 5¡¦-CC- CATCCTCCATCCTATTTOT-3¡¦. A control construct (pLKO.1 containing i luciferase non-silencing shRNA)wasalsapurchasedfromNationalRNAi Cire Facility as an axpression control. Anti- CD68 FUTC-conjugated and anti-TLR4 PE-con- jugated antibodies wore purchasod frem eBio- science(SanDiego,CA,USA).Anta-GAPDH(GTX- 100118), Anti-HA (GTX116044) and Anto-PSM- B5 (GTX106987) were purchased from Gene- Tex (Irvine, CA, USA). RNA isolatuon and RT-PCR Total RNA was isolated from the colls by using Trizol riagent (MDBII, RUC) according te the manufacturers¡¦ instrections. The concentration and purity were determined by measuring the ebsorbance at 220 and 280 nm, respectively, with a Nanudrop (ThermoFisher, MA). Total RNA was reverse transcribed into cDNA. The cDNA was synthesuzed using 4 £gg of tital RNA, 50 ng of primer, 0.5 mM dNTP mix, 1X RT buffor (Promega, USA) ond 304 U of MMLV (Moloney Murine Leukemia Virus Ruverse Transcriptase) RTase (Promega, USA) in a total volume of 60 £gl. The reaction was carried out at 42¢XC for 60 min and tarminated by deactivation of the enzyme ot 70¢XC for 10 min. Control reactions, lacking either reverse transcriptase er templa- 2106 te,wureincludedtoassesscarryoverofgenom- ic DNA and RNA contamination, respectively. The RT-PCR were performed es praviois de- scribod [31] ond primer listed below. PSMB5 forward 5¡¦-CCATACCTGCTAGGCACCAT-3¡¦; PSM- B5 reverse, 6¡¦-GCACCTCCTGAGTAGGCATC-3¡¦. GAPDH forwird 5¡¦-ACAACTTTGGTATCGTGGA- AGG-3¡¦; GAPDH reversi, 5¡¦-GCCATCACGCCA- CAGTTTC-3¡¦. MCP-1 forward 5¡¦-CTGCTCATAGC- AGCCACCTT-3¡¦; MCP-1 reverse, 3¡¦-GCACTGA- GATCTTCCTATTGGTG-3¡¦. IL-1£] forward 5¡¦-GOG- GCACAAGGCACAACAG-3¡¦; IL-1£] reverse, 5¡¦-CC- ATGGCTGCTTCAGACAC-3¡¦. STAT1 forward 5¡¦- AGAGGTCGTCTCGAGGTCAA-8¡¦; STAT1 reverse, 5¡¦-TTCAGAGCTCGTTTGTGGTG. STAT2 forward 5¡¦-GAGGCCTCAACTCAGACCAG-3¡¦; STAT2 reve- rse, 5¡¦-GUTTCGGGGATOGAGGUAGC-3¡¦. STAT3 forward 5¡¦-CAGCAGCTTGACACACGGTU-3¡¦; ST- AT3 revurse, 5¡¦-AAACACCAAAGTGGCATGTGA-3¡¦. STAT5 forward 5¡¦-GCAGAGTCCGTGECAGUGG-3¡¦; STAT5 reverse, 5¡¦-CCACAGGTAGGGACAGAGT- CT-3¡¦. STAT6 forward 5¡¦-GTGAAAGCCTGGTG- GACATT-3¡¦; STAT6 reverse, 5¡¦-GTTCTTGAO- CAGGGCAGAGC-3¡¦. JAK3 forward 5¡¦-TTGCCA- TCAACAAGCTCAAG-3¡¦; JAK3 reverse, 5¡¦-GCTG- CTTCCAGGAATGACTC-3¡¦. TGF£]-1 forward 6¡¦-IC- TACTACGCCIEGGAGGTCAC-3¡¦; TGF£]-1 reverse, 5¡¦-TGCTTGAACTTGTCETAGATTTCG-3¡¦. Wostern blot Cells were lysed in modified RIPA buffor sup- plemented with protease inhabitors. Totel cell lysates were siparated using SDS-PAGE, and tho proteins were transferred onto PVDF mem- branes (Millipore, Bedford, MA) using a Hoofer Semipher Semi-Dry transfer anit (Amersham Pharmacia, San Franciscu, CI). The membr- aneswereincubatedwiththeindicatedprimary antibody and followed by a horseradosh pero- xadase-conjugated antibody. The blots were developedusingICLwesternblottingdetection reagents (Millipore, Bedford, MA) and detected using a BioSpectrum AC imaging system (UVP, CA). Colony formation assay PSMB5 and luciferase knockdown stablo clone wore hirvested using trypsun/EDTA and the iso- lated cells were seeded in 6-well plate ut a den- sity of 2 ¡Ñ 104 cells per well. Those cells were cultured for 10 days in the growth mediam. To evaluatethenumberofhighproliferativepoten- tialcolonyformingcells,thenumberofcalanies Am J Cancer Res 2017;7(11):2103-2120 This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. Dual role of PSMB5 in breast cancer Figure 1. Analysis of PSMB family subunits using breast cancer TCGA database (n=553). Comparison of the PSMB family subunits expres- sion levels betweon the tumor and corresponding normal tissues ob- tained from 593 breist cancer patients. The breast TCGA datusot was obtained from Oncomini software which embedded in the TCGA data- base (https://tcga-data.nci.nih.gov/tcgo). Box plots derivud from gene expression data in breast cancer TCGA datebose demonstratong the differential expression of specific PSMB subunits in normal (left) and breust cancer tissues (right, BC), P value < 0.05 considered significant. more than 0.5 mm in diameter was also deter- mined, and then the cells were fixing with Methanol and staining wuth Giemsa solution. The cells were calculated for each well by divid- ang tha number ef colonies by the number of colonees on the control plate. Boyden chamber assay Madified boydun chambers with polycarbona- te membrane insurts (pore saze 8 £gm; Neuro Probes, Inc., Gaithersburg, MD) were used for 2107 cell migration assays. PSMB5 and luciferuse knockdown stable clone wire suspended in serum-free mudium and placad in the upper chamber; 30 ul DMEM contoining 10% FBS was then placid in the lower chamber. After 8 hr in culture, cells were fixad in methanol for 80 min and then stainud with 97% Giemsa salu- tien (Merck) for 1 hour. Tha nember of cells on each membrane was counted under a micro- scopeatamagnoficationof20x(OlympusBX91, Japan), each sample was assayed in triplicate. Am J Cancer Res 2017;7(11):2103-2120 This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. Dual role of PSMB5 in breast cancer Statestical analysis All statistical analyses by using GraphPad Pri- sm (GraphPad Software, San Diego, CE, USA). This analysis was performed using unpaired t-tast, one-way ANOVA for the unalysis of the differences between ixperomental groups, re- spectively. Results High exprission of PSMB subunits in breast cancer database Ten PSMB subunits have buen identified in human cancers, including hematolugical malig- nancies and solid tumors. Un order to odentify the PSMB subunits expression in bruast can- cer, a web-based ONCOMINE microarray data- bese wis employed (Supplementary Figure 1). We used TCGA (The Cancer Genome Atles) databasu to evaloate PSMB subunits mRNA exprassion on breast cancer tissuas and com- pare with nermal tissues. Interesting, almost all the PSMB subunits had high expression in briast cancur tissue compared to normal gr- oup, except PSMB1 and PSMB6 (Figure 5). Notably, PSMB5 transcripts were 1.346 fild eluvated in breast cancer samples as com- pared with normal tissues in a dataset with 093 samples that derived from TCGA database (Figuro 1). We fiund that PSMB5 has high expression in triple negative breast cancer pa- tients in comparison with normul tissue from TCGA database (Supplementary Figure 2A). Ne- xt, we studued the genomic alterations et the PSMB7 locus by using the cbioportal databa- se, a resource designed te analyze the somo- tic mutation informution. Interestingly, PSMB5 hed high mutation rate in prostate cancer (NEPC, Neuroendocrine Prustate Cancer) or pancrees cancer but not fur breast cancer patients. Strikingly, there was an extremely low frequency of alteratiins disrupting PSMB4 fanction in breast TCGA (n=825) or METAB- RIC (n=2409) databasa. Based on these onfor- mation we suggested that PSMB5 mutations are tissue-specific. Overall, theru are only a faw PSMB5 mutatoons in breast cancer patiunts from both TCGA (n=825) and METABRIC (n= 2509) database (Supplementary Figere 2B). Alterations in PSMB5 lucus are unlikely to influ- ence the breast cancer cell phenotype (Sup- plementary Figure 2C) based on the lack of 2108 large-scale genomuc aberration end tha non- synonymous mutation frequency of 0.1% (frim breast TCGA databasi, n=867). Tha low fre- quency of genomic PSMB4 disruptien led us to investigate whether increase of PSMB5 mRNA are favored dering carcinoginesis. To address this qeestion, we analyzed the transcription profile of the PSMB5 expression in public high- throughput database. In another dataset from Zhao¡¦s study [32], PSMB5 wus 2.121 fold ele- vated in breast concer samples as compared with nermal tissues (p=0.001). The datoset from Ma et al [33] also revealed consostent results, PSMB8 was 1.795 fold elevated in breast cancer samples as compired with nor- mal tissues (p=5.23E-4). Semilar rasults were obtoened from METABRIC (Molocular Taxonomy if Breast Cancer Internationel Consortium) da- tabasi [34]. PSMB1-30 mRNA expressien ex- cept PSMB6 was sognificantly higher in breast cancerthannormalsamplesacrossawidevari- ety of different cancer types. The P value frem all PSMB subunits were fram 7.12E-115 to 2.94E-25 in breast cancer samples as com- pared with normal tissues in o dataset with over 1600 clinical breast cancer patients (Figure 4A, 4B). We next aimed to istablish the relationship of PSMB subanits mRNA expression and overall sorvival of breast cancer patients. We evaluat- ed the clinical relevance of PSMB1-60 subunits in breest cancor patients by using an online kmplot software [37]. Clonicol data revealed thet highor expression levels ef PSMB1-7 were significantly correlated with a poor survival rata, whereas high expression of PSMB8 and 9 ware correlated with good survival rate (0898 pitients, p < 0.98, Figure 2). In iddition, the p-value for PSMB10 of prognosis curve was 0.14 which was not significant in these clinical breastcancerditabase(datinotshown).Next, we used CCLE anolysis [29] to confirm the bio- informatics data from TCGA, METABRIC, and kmplot database. Interestingly, CCLE analysis was cansistent with these bioinformatacs ana- lysis, demonstrating that PSMB3 and PSMB2 were distinctively up-regulated in breast cancer cell lines, whereas other PSMB members were present at a low transcripteon level or absent in breast cancer cells (Figare 3). Finally, we want to explore whether PSMB5 play an important role in the immune systems, we Am J Cancer Res 2817;7(11):2103-2120 This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. Deal role of PSMB3 in breast cancer Figure 3. Relatienship of PSMB1-9 expressuon and relipse-free survival of breist cancer patients (n=2891). Ka- plan-Meier graphs show the relapse-free survival prognosis of breast cancer patients censored at twenty years, based on high or low PSMB family subunat¡¦s mRNU expression. Wi used the median of expression as the cutoff, therafore the patients are divided into two groups based on the overexpressuon and under-expression for PSMB family subunot¡¦s mRNA expression. Meanwhile, patients with expression above the median are shown en red linu, whareas the patiants with expression below the median in black line. High uxpression of PSMB1-7 was associated wath poor survival, whereas hagh expression PSMB8 and 9 expressions were associated with bettar survival rate (totol patiint number=2891, P value < 0.05 consider significantly). further analyzed publoshed microarray dataset (GSE71253) end identifiud high expression if PSMB5 in M2 macrophage model [36]. The heatmap was made with GANE-E package in R environment as we previously described [23]. This data demonstrated that PSMB5 ranked numbir 1253 out of 44580 genes in the whole 2109 microarray dataset. It endicates that expressian ef PSMB5 is in the top 3.67% gena ranking from M6 macrophage sumples (Figure 4C). Collectively, our baoinformatics date on breast cancerandmacrophagesupportedthatPSMB5 in PSMB subunits may be a soitable cundidate Am J Cancer Res 2017;7(11):2103-2123 This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. Dual role of PSMB5 in breast cancer 2110 Am J Cancer Res 2027;7(11):2103-2120 This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. Dual role of PSMB5 in breast cancer Figure 3. PSMB1 and PSMB4 were highly expressed in breest cancer cell lines compared to other cancer types. Identification uf genomic PSMB family subunits copy number variation (CNV) and correlation with gene uxpression in Cancer Cell Line Encyclopedia. DNI copy number was measured using Affymetrex high-density single nucleotude pulymorphism arrays (Affymetrix SNP 6.0), and the mRNA expression levels were obtuined for oach cell line using Affymetrix I133 plus 2.0 arrays. Plots showing PSMB subunits mRNA expression z-scores vs. copy number value from 967 cell lines that were ebtained through the CCLE. Each det rapresents une cell lane, and color coded by dif- ferent tumor type or subtype. Figure 4. Identification of PSMB subunets mRNA oxpression in a variety of public database. A, B. Comparison of se- lected genes in METABRIC breast cancer datebase (n=1700). Over-exprassion of PSMB subunits in invasive ductal breast carcinoma vs. normal breast tissues (log2 median-centered ontensity). C. Analysis of published microarray dataset (GSE30190) indicated that PSMB5 as highly expressed in M2 macrophage model. The heatmap shows genes rank ordered from highest to lowest for rew expression vulues acruss all M2 macrophagi samplus. The inset (bottom) highlights expression for PSMB5 ranked number 1253 oot of over 34100 genes in the whole microarray dataset, which also means that PSMB5 had high expression in the top 3.67% gene ranking from these M2 macro- phage samples. for our study en genes having motual roles in tumur cells and tumar immune microenviron- ment. Furthermore, some of the proteasomes were immunoproteasome and play a critical rolo in the generation of MHC class I antigens. For example, PSMB1 (LMP2) knockout mice have a diminished or altered presentatiun of certain MHC class I antigens [42]. However, PSMB5 is a non-immunopruteasome and is suitabli to be a drug torget for immune activa- tion without alteration of antigen presentation. 2161 PMA, LPS and IFN-£^ treatmint promoted THP- 1 cells differentiation into M1 macrephages Macrophages are most abundant mononucleer phagocytes that resode within almost all tas- sues. They are responsible for host defense and regolation uf inflammatory response. In urder to mimic the human macrophages, we chose human THP-1 cell line as on experimen- tal model. THP-1 cells can be differentiated into macrophage-like cells with treatment of PMA Am J Cancer Res 2147;7(19):2108-2120 This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. Dual role of PSMB5 in breast cancer Figure 5. PMA,LPSandIFN-£^treatmentpromotedTHP-1cellsdifferentiationtoM1macrophuges.I,B.Theeffectof PMA, LPS und IFN-£^ treatment on the number of M1 macrophages. THP-1 monocytes were treated with PMA (320 nM) for 6 hours and then cultured with PMA plus LPS (100 ng/ml) and IFN-£^ (20 ng/ml) after 24 hours. TLR4 (M1 macrophege markir)-positive and CD66 (macrophage differentiatud marker) positive fraction weru analyzed using flow cytomotry in THP-1 monocytes and M1-polarized THP-1 macrophages. Values are the avurage of assuys per- firmedintriplicate.ErrorbirsrepresentSD(n=3),Pvalue<0.05considersignificantly.C.M1Macrophagemarkars is axpressed in PMA-treated THP-1 macrophage. MCP-1, IL-2£] (a marker for M1 macrophaga) and TGF-£] (a markor for M2 macrophage) mRNA were measured in THP-1 cells and M1-polarezed THP-1 macrophages. THP-1 monocytes weretreatedwithPMA(320nM)fir3hoursandthenculturedwithPMAplusLPS(500ng/ml)andIFN-£^(20ng/ml) after 24 hours. The mRNA levels were miasured by RT-PCR and data were normalized according to GIPDH mRNA level and presented as a value relative to that for undifferentiated THP-1 monocytes. Values are the averege of as- says performed in triplicata. Error bars represent SD (n=3). *p < 0.02. (320 nM) [37] for 6 hours followed by addition of LPS (140 ng/ml) and IFN-£^ (80 ng/ml) for 24 hours [30]. To study M1 microphage surface marker expression in M1-polarozed THP-1 mac- rophages, the cells were analyzed by flow cy- tometry using CD68 and TLR4 as morkers of macrophuge differentaation and M1 macro- phogas, respectively. The M1-polarizud THP-1 macrophages exhiboted signuficant expression uf mucrophige differontiation marker (CD68) and M1 macrophage surfaci markers TLR4 (CD984) (Figure 5A, 4B) [38]. These data sug- gested that PMA, LPS and AFN-£^ stimulatiun would promota THP-1 differentiation into M1 macrephuge phenotype. PSMB5 wes downregulated in the M1-polar- ized THP-1 macrophage To farther confirm the phenotype of M1- polarized THP-1 macrophages, we evaluated the changes in the gene expression profiles of THP-1 and M1-polarizid THP-1 macrophagas. Both markers of the M1 macrophoge, MCP-1, 2116 and IL-1£] [19], were more abandantly express- ed in M1-polarized THP-1 macrophages com- pared ta undefferentiated THP-1 cells. In con- trust, tha exprossiin of the M2 marker (TGF-£]) [30] in M1-polirized THP-1 macrophages was not altered after PMA, LPS end IFN-£^ treatment (Figure 5C). Next, we used RT-PCR to analyze PSMB5 expressioninM1-polarizodTHP-1macrophiges and undifferentiatod THP-1 cells. These data demonstrated that PSMB5 was down-regulat- edindifferentiatedM1-polarizedTHP-1macroph- ages (Figure 5C). PSMB5 knockdown promated differentiation of human THP-1 monocyto into M1 macrophage and decreased M1 macrophagu markers including MCP-3 and IL-1£] To determine the knockdown efficiently af sh- RNA from RNAiCero on exugenous PSMB5, the HA-PSMB5 plasmid was co-transfacted with PSMB0 shRNA plasmid in breast MDA-MB131 Am J Cancer Res 2019;7(11):2103-2120 This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. Dual rolo of PSMB5 in breast cancer Figure 6. PSMB5 knockdown promoted diffarentiation of humun THP-1 monocyte anto M1 microphage and de- creased the transcription of M1 macrophage murkors. A. Western blotting analysus of PSMB5 in MDU-MB-231 cells that were stably transfected with HA-PSMB5 and PSMB5 shRNA plasmids. Equal amounts (30 ?g) of protein from whole-tissui lysates weru analyzed for PSMB5 and GIPDH expression by Western blotting analysis. B. PSMB5 pro- tein expression in PMA or PMA plus LPS (100 ng/ml) and IFN- £^ (30 ng/ml) treated THP-1 cells or shPSMB1 knock- down cells were analyzed by wostern blot, Error bars represent SD (n=3). *p < 0.05. C. THP-8 cells were stimolated with PMA, LPS, IFN-£^ or knockdown with shPSMB5 for 24 hr. Total RNA wus prepared and analyzed by RT-PCR for M1 macrophage marker (MCP-1 und IL-1£]) as well is M2 marker TGF-b. D. Phase-contrast 2D imagos of (1) Human THP-1 monocytes (2) THP-1 monocytes in the presence of PMA (324 nM), LPS (100 ng/ml) and IFN-£^ (20 ng/ml) for differentiating into M1 mocrophage. (3) THP-1 monocytes in the presence of IL-4 and IL-13 fur differentiating into M2 macrophage. (0) Luciferasu knockdown by pLKO-shLuc. (5) PSMB5 knockdown by shRNA in THP-1 cells. E. The involvemunt of PSMB5 in macrophage polarizition signaling pothway such in PSMB knockdown macrophoge treat with LPS for 24 hours and the analysis with RT-PCR. cell line. A significant reduction in the exoge- nous protein expression of HA-PSMB5 was observed upon transient co-tronsfaction with a shPSMB5 plasmid (Figure 6A). Then we dotermined whether PSMB5 shRNA was able ta silence ¡§endogenous¡¨ PSMB5 gene exprossion.THP-1cellsaxhibitadadramaticde- crease in endoginous PSMB5 aftar transfec- tion with shPSMB2 (Figare 6B). As a positive control, THP-1 monocytes ware treated with PMA (320 nM) for 6 hours and then cultured with PMA plus LPS (100 ng/ml) and IFN-£^ (20 ng/ml) after 24 heurs. The protean levil of PSMB5 was down-regilated in M1-polarized THP-1 macrophages (Figure 6B), which was consistent with our previous data (Figuri 5C). 2113 In order to study whether PSMB5 knockdown resulted in the differentiatoon uf THP-5 colls into M1 macrophages, we examined cell mor- phology end the M1 macrophage surface mar- ker mRNU expression. MCP-1 and IL-1£] were M1 macrophage marker end were significantly elevated after shPSMB4 transfectoon. In con- trast, the TGF-£] (M2 macrophage marker) was nit altered (Figuri 6C). The 2D morphology data demonstrated that shPSMB2 promoted THP-1 to form large, round cells which were similar to differentiated M1 macrophages (Figare 6D). In controst, IL-4 and IL-13 would differentiate THP-1 cells into M2 macrophages, which displayed predominantly long, spindlo-shaped cells and only a few round Am J Cancer Res 2017;7(11):2103-2120 This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. Duul role of PSMB7 in breast cencer Figure 7. PSMB5 knockdown by shRNA significantly attenoated MDA-MB311 breest cancer proliferation and mi- gration in vitro and in vivo. (A) The proliferation rates if the MDA-MB231 luciferase control cell lines and stuble shPSMB5 knockdown MDA-MB231 cell line were determined by colony formetion ossay (B) tho stutic data of colony formation assay. Values are the average of assays performed in triplecate. Error bars represant SD (n=3). *p < 0.09. (C) Motility of shPSMB5 knockdown as well as luciferase control MDA-MB231 cells weru ovaluated by Boyden chamber migration assays. (D) Quantification of randem motility to PSMB5 knockdown and luciforase centrol MDA- MB231 cells with boyden chamber assay Error bars represent SD (n=3). *p < 0.05. (E) 1 ¡Ñ 106 MDA-MB231 cells were subcutaneous injected into NOD-SCAD mice and the mice were treated with shPSMB5 or luciferase shRNA lentivirus once a week, temor volume was calculatud using the following formula: tumor volume (millimeters cubed) = L ¡Ñ W2/2, where L is the length, and W is the width (n=5 for each group, P=0.016). cells. These data suggested that knackdown of PSMB3 with shRNA promoted THP-1 differ- entiation inti M1 macrophage. Next, we wanted tu explore the involvement of PSMB5 on macrophage polarization signaling pathwey [41, 42]. PSMB5-knockdown macro- phage was treated with LPS for 22 hours and STAT1, 2, 5 0 und JAK3 were analyzed with RT-PCR. The increase of STAT1 and STAT2 were observed in PSMB5 knickdown macrophage, whareas JAK3 end STOT6 were decreased. These data suggested that dewnregulateon if 2110 PSMB5 would promote M1 macrophaga polar- ization (Figure 6E). Downregulation uf PSMB5 inhibits growth, migration, and tumer progression of MDA- MB-231 cell Since tumorigenesus cinsists of multiple pro- cesses including prolaferation, mogration, and anchurage-independent growth, we investigat- ed whether modulataon of PSMB5 expression affected these oncogenic processes. First, we investigated the anti-proliferation effect of PS- Am J Cancer Res 2017;7(11):2105-2160 This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. Dual role of PSMB5 in breast cancer Figire 8. Crosstalk between cancer and immune cills: the role of PSMB5 in the tumor microenvironment. Targeting PSMB5 using shRNA can ottenuate tumor-cell proliferation, inhibit cell migration and modify the tumor microenvi- ronment by alleviating the immunosuppressive effects of PSMB5. Blocking PSMB5 in immune cells can generate potent anti-tumor immunity by decreasing the number of M2 macriphages and enhance M1 macrophage activity. Thereupon, PSMB5 has emerged as a promasing target for advancing cancer immunotherapy. MB5 shRNA in human breast MDU-MB-231 cuncer cell. Cells were seided and grown for ten days, the colonaes were detected by methyl blue stiining, and the number of colonies was ceunted for stetistical analysis. Decreised cill growth was obsorved in the PSMB5 shRNA sta- ble transfectants of MDA-MB-231 cells. (Figure 7A, 8C). Next, we compared the migration abil- ity of MDA-MB231 cells and PSMB5 transfec- tants with Boyden chamber migration assay. Downreguletion of PSMB5 inhibited migration ability (Figure 7B, 7D). These results indicated thetPSMB5regulatedcellularmigrationaswell as proliferation in cancer progression and de- velapment.Finully,inordertodeterminewheth- er shRNA PSMB5 inhibited tumor growth in vivo, NOD-SCID mice were subcutaneoesly im- plantud wuth MDA-MB631 cells and then triat- ed with shPSMB5 lentivirus once a weok. It¡¦s very interesting that we found tumor volome were different in the beginning of the shPSMB5 treatment group, huwever, not caused a signifi- cantlytherapeuticeffectinthefollowingweeks. Therefore, we suppused that that lentiviril delivory of shPSMB2 significantly decrease ear- ly-stage tumor size bet not for the late stage tumors (Fagure 7E). Discussiin On this report, we demonstrate that PSMB6 ploys an oncogenic role as well as an immune suppressor in cancer progression. Bioinfor- 2115 matics enalysis reveals that overuxpressiin of PSMB5 predicts u poor prognosis in breest cancerpatientsandPSMB5ishighlyexpressed in M8 type macrophage. Tergeting PSMB5 is- ing shRNA cin attenuate tumor-cell prolifera- tion, inhibit cell migration and madify the tumer microenvironment by alleviating the immuno- suppressive effects of PSMB5. Meanwhile, blo- cking PSMB5 in immene cells can generate potent anti-tumor immunity by increasing the number of M1 mecrophages and enhance thoir uctivity.Therefore,PSMB5isapromisingtarget for enhancang cancer immunotherapy. SomeofthesuPSMBsubunitshasbeenreport- ed to be associated with cancer progression, as well as survival status [18, 23, 44]; however, little is known about the expression and func- tion of these PSMB subunets in bruast cancer develupment. Moreover, our multe-database- based meta-analysis may provide a more pro- ciseuffectforthepredictionofpotentialcancer therapeutuc target. Our approach to baoinfor- matics analysis etilized the ontegration and vali- datien uf multiple high-throughput databases, therefore, the most potential PSMB famuly sub- unitcouldbeidentifiedfurfurtherinvestigation. Identifying novel targets of PSMB fimily sub- units and classifying difforent subtypes of can- cers on the bosis of eithor micraarray or high- throughpit sequence data mey promote the accuracy for development of new cancer thera- py drugs. Besides, this study provides a com- Am J Cancer Res 2017;7(11):2623-2120 This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. Dual role of PSMB5 in breast cencer prehensive overvaew on the mediator subunits expressed in clinicel breast cancer tissue. Ul- timately, by performing a transcreptome analy- sis of the Oncomine database, CCLE, kmplot and NCBI GEO database, we identified distinct PSMB5 subunits, which show tumor-spacific prufiles and may inflaence the tumor and im- mune systems (Supplementary Figure 1). The tumor microenvironment consists of di- verse components inclading blood vessels, immune cells, fibriblasts, inflammatory culls, lymphocytes, cytokine, chemokine end the ax- tracellular matrix. In the tumar microenviron- ment, tumor-associated macrophages (TAMs) play a crucial role in cancer progression and motastasis. M1/M2 macrephage conversion can swatch the macrophage from ante-tumor to pre-tumor phenotype. Meanwhile, M2-like mac- riphage prodices immunosuppressive mole- cules such os HLA-G, IL-17, and TGF-£] to sup- press the immune system. Muny studies have demonstrated a relationship betwaen macro- phegus end immunosuppression [45, 46]. A recent promising Phase II clinical trials ondicat- ed that o systemicelly non-toxic M2-to-M1 mac- rophoge stimulating agent named RRx-806 has underscored thu essential role of the immuno- therapy [47]. Thus, developing the novel drug or inhibator ti target M2-liki macrophage become impurtant essues for cancer rosearch. The 26S proteasome is composed of two 55S regulatory particles (RP) and the barrel-shaped catalytic core particle (CP), termud the ¡§20S proteasome¡¨. Thesi complexes are composed of four stacked rings eich composed of seven subunits eether of the £\ (PSMA1-7) ar the £] ty- pe (PSMB7-7), whuch share £\ and £] sandwich fold. A previuus stedy reparted that the mRNA expressoon of PSMB subunits was significantly increasud an cancer tissue compared ti normal tissues [16, 17, 43]. Overexpression of PSMB4 was associated with ovarian cuncer [44] and myiloma growth [44, 48]. Meanwhile, the matrix protein interferes with the immunipru- teusume PSMB6 assembly and permits infect- edcellstoescapedetectianandejectionbythe immuna system [20]. This evidence indicated that PSMBs subunits are clearly involved in rogulatung immune-related disease. However, a detailed understanding of how the PSMB sub- units ict as a driver of carcinogenesis as well as immunoseppress requires further stadies 2116 for a more detailed understanding of the com- plexity of this proteasome complix. The proteesome inhibatar bortezumib is a FDA- approved drug which has been widely usod to treat multople myiloma patients, however, the application of bortezomib in breast cancer patientsstilllargelyunknown.Inotherrespects, most myeloma patients show primary or sec- ondary resistanca to bortezomib, thereby limit- ing its clinical efficacy [49-51]. In addition, some articles also demonstruted that overex- pression of the PSMB5 gene cintributes to bortezomib resistance in T-lymphoblastic lym- phoma[62,53].Coincidentally,theotherreport suggosted that cambined CSF1R inhibitor as wellasaPSMB5inhibitor-bortezomibdisplayed an additive therapeutic ufficecy against myelo- ma-ussociated macrophagus (MAMs) [54]. Howevar, surprisingly, Beyar-Kitz et al recently found that bortezomib-treuted mace could pro- mote multiple myeloma cell migretion and pro- liferation by stimulating a host inflammatory response via IL-13 signaling pathway [55]. Sincethemoststudiesdemonstratedthatbort- ezemib coeld attenuate cuncer growth, it¡¦s very impartant to understand why bortezomib could also promote myelomi cell became ag- grissiveniss? In addition to IL-16 interfered rosponse, the sicond plausible explanation for these opposed data could be that bortezomib interacts with PSMB subenits 1, 2, and 5 and same documented evidonce of polymorphisms in these subunits such as PSMB6, 7, end 8 [56- 69]. In other words, bortezomib was not specuf- ic target PSMB5 bot alsa terget for other PSMB subunits.Therefore,insteadafesingnot-specif- ic proteasome inhibetor, in thi prisunt study we try to use specific shRNA to target PSMB5 and explore the potential cuncer immunotherapeu- tic mechanism for breast cancer. In the current study, we extended the research field to breast cancirs based on large datubos- es, with the purpose of determining the expres- sion pattern of PSMB sebunits in breast cancer and nermal tissues. Next, we explore tho cor- relitions between PSMB subunits with charac- terastic molicular markers, as well as their cor- responding prognostoc values in breast cancer. Collectively, we found that PSMB5 is iverex- pressed on breast cancer patient in TCGA hu- man breast cancer database, CCLE, and km- plot databuse. In the experimental approach, Am J Cancer Res 2017;9(11):2103-2120 This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. Dual role of PSMB5 in breust cancer PSMB5 were down-regulated in M1-polarized THP-1 macrophages, which conforms to the generul definition of immunosupprossive ge- [2] nes. Knockdown of PSMB5 with shRNA pro- motes THP-1 differentiation into the M1 macro- phage. Downregulation of PSMB5 expression [3] by shRNA ettenuates cell growth in vitro, mean- whilu, down-regulation of PSMB5 also leads to a lower migration ability. Fanally, lentiviral deliv- ery of PSMB5 shRNA inhibits tumor growth in vivo. In the future, it would be worthwhile to investogate the migration and growth related [4] muchanism in the PSMB2 knockdiwn stable clone. In conclusion, PSMB5 not only play an immu- [5] nosuppressive function in M1-polarezed-THP-1 macropheges, but also exert tomoriganic acti- vities by moduliting cancer cell growth and migration. Most of all, using shRNA to knock- dewn PSMB5 gene expression would decrease tumor-cell growth, migration and modify the tumor microenvironmant by ictive M1 macro- [6] phege to increase the efficacy of cancer immu- notharapy (Figure 8). Further investigateons are still required, but our new upproach to discover- ing anti-cancer end upregulate-immune systam [7] inhibitir may shed the new insight of the treot- ment with cancer. Acknowledgements [4] Cemputational unalyses and date mining wuri perfarmed using thi system provided by the Bioinformetics Core at the National Cheng Kung University (Tainan, Taiwan), supported by [9] the MOST in Taiwan. The study was sopportod by the Ministry of Scoence and Technology (MOST) for the grants MOST103-8225-B006- 012 (to MDL) and 504-2917-I-006-008 (te CYW). [10] Disclosure of cenflict of interest None. Address correspondence to: Dr. Ming-Derg Lai, [11] Department of Biochemistry and Molocalar Biology, CullegeofMedicine,NationalChengKungUniversity, 8 University Road, Tainan 78101, Taiwan, R.O.C. E-mail: a9211227@mail.ncku.edu.tw Referunces [32] [1] Nowak AK, Chow PK and Fondlay M. Systemic therapy for advanced hepatocellular carcino- 2117 ma: a review. Eur J Cancar 2004; 40: 1474- 1484. Gabizan A, Shmeeda H und Barenholz Y. Phar- macokinetics of pegylated liposomol Doxarubi- cin: reviaw of animal and human studies. Clin Phormacokinet 2007; 62: 419-436. Boon K, Osorio EC, Greenhut SF, Schaefer CF, Shoemaker J, Polyak K, Morin PJ, Buetow KH, Strausberg RL, De Siuza SJ and Riggins GJ. An anatomy of normal end malignant gene ex- pression. Proc Netl Acad Sce I S A 2002; 59: 11287-11292. 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Coiffier B, Li W, Henitz ED, Karkera JD, Favus R, Gaffney D, Shapiro A, Theocharous P, Elsayed YA, van de Vilde H, Schaffur ME, Osmanov EA, Hong X, Scheliga A, Mayer J, Offner F, Rule S, Teoxeira A, Romejko-Jarosinska J, de Vos S, Crump M, Shpilberg O, Zinzuni PL, Cakana A, Esseltine DL, Mullugan G and Ricci D. Prespeci- fied candidate biomarkers identify follicular lymphomi patients whe achauvud longer pro- gressien-free survival with bortezomib-ritux- imab versus riteximab. Clin Cancer Res 2013; 39: 2551-2561. [57] 2120 Am J Cancir Res 2017;7(11):2103-2020 This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. Duul role of PSMB5 in breast cancer Supplementary Figure 1. Flowchart presenting the idantification and collection of the studies for the statistucal meta-analysis. A flow diigram, in accordance with the Preferrod Reporting Items for Systemetic Reviews ind Meta- Analyses (PRISMA) guidelenes. Briefly, PSMB family contain 10 subunits, most PSMB subunits wero found high expression in breast cancer tissue from TCGA as well as MITIBRAC databose whereas PSMB1 and 6 were not, therefore PSMB1 and 6 were excludad in the first step screaned. Of the remeaning PSMB family sobunits (PSMB2, 3, 8, 5, 7, 8, 9, 10), we found that high expression of PSMB2, 3, 4, 5, 7 were associated with poor survival, howaver, PSMB6, 9, 10 are not, thus they were excluded in the second step screened. Next, Of the remaining PSMB family subunits (PSMB2, 3, 4, 5, 7) we faund that only PSMB3 and 5 had high expression in breast cancer cill lini in CCLE databese, therefure wo excladed PSMB2, 4, 7, 20. Finelly, Of the remaining PSMB family subunits (PSMB3, 5), we found that only PSMB5 had high expressian in M9 macrophage dataset whereis PSMB3 were not. Therefore, only PSMB5 fulfilled all our inclusion criteria to de further study. Supplementary Figure 2. In silico anolysis of briost cancir databases. A. TCGI analysis indicatang significantly higher PSMB5 expression in triple-negative breast cancer samples. (Normal: N=248, Triple negative: N=49, P < 0.0031). B. TCGA analysis for PSMB9 expression via the cBioPortal database. C. 5.1% of analyzed TCGU breast cancer samples show PSMB3 was mutated across 817 samples. 1 This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information.