KPNA2 Interaction with CBX8 Contributes to the Development And

Zeng et al. J Transl Med (2021) 19:112 https://doi.org/10.1186/s12967-021-02709-5 Journal of Translational Medicine

RESEARCH Open Access KPNA2 interaction with CBX8 contributes to the development and progression of bladder cancer by mediating the PRDM1/c‑FOS pathway Fanchang Zeng1, Liumei Luo2*, Daoyuan Li1, Juncheng Guo3 and Min Guo4

Abstract Background: Bladder cancer (BCa) is a common malignancy characterized by high heterogeneity, yet the current treatment modalities are limited. The aim of the present investigation was to unravel the functional role of Karyo- pherin alpha 2 (KPNA2), a tumor facilitator identifed in multiple malignancies, in the progression of BCa. Methods: BCa tissues and adjacent normal tissues were surgically resected and analyzed from patients with BCa to determine the expression profle of KPNA2 and Chromobox 8 (CBX8) by RT-qPCR, Western blot analysis and immunohistochemistry. The relationship among KPNA2, CBX8 and PR domain zinc fnger protein 1 (PRDM1) was explored by co-immunoprecipitation and chromatin-immunoprecipitation. The functions of KPNA2, CBX8 and PRDM1 on BCa cell proliferation, migration and invasion were evaluated. Next, a nude mouse model of BCa was established for validating the roles of KPNA2, CBX8 and PRDM1 in vivo. Results: KPNA2 and CBX8 were highly expressed in BCa and are in association with dismal oncologic outcomes of patients with BCa. KPNA2 promoted nuclear import of CBX8. CBX8 downregulated PRDM1 by recruiting BCOR in the promoter region of PRDM1. Overexpression of KPNA2 promoted the malignant behaviors of BCa cells, which was counteracted by silencing of CBX8. Overexpressing PRDM1 attenuated the progression of BCa by inhibiting c-FOS expression. The tumor-promoting efects of KPNA2 via the PRDM1/c-FOS pathway were also validated in vivo. Conclusion: Collectively, our fndings attached great importance to the interplay between KPNA2 and CBX8 in BCa in mediating the development and progression of BCa, thus ofering a promising candidate target for better BCa patient management. Keywords: Karyopherin alpha 2, Chromobox 8, PR domain zinc fnger protein 1, Bladder cancer, Proliferation, Migration, Invasion

Background against BCa include complete resection, chemotherapy, Bladder cancer (BCa) ranks the 10th most frequently and radiotherapy depending on the diferent disease sub- occurring malignancy across the world with approxi- types. Te identifcation of biomarker is of great impor- mately 549,000 new cases diagnosed and 200,000 deaths tant for diferent cancers, including colorectal cancer [2], in 2018 [1]. Currently available treatment modalities gastric cancer [3], breast cancer [4] and chronic myeloid leukemia [5, 6]. A successful outcome of the treatment for BCa largely depends on early diagnosis and the avail- *Correspondence: [email protected] ability of personalized therapy [7]. Understanding better 2 Department of Science and Education, Hainan General Hospital (Hainan the molecular features underlying the pathogenesis of Afliated Hospital of Hainan Medical University), No. 19, Xiuhua Road, Xiuying District, Haikou 570311, People’s Republic of China BCa could thus ofer new opportunities for early diagno- Full list of author information is available at the end of the article sis and novel individualized and targeted therapies [8].

© The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdo- main/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zeng et al. J Transl Med (2021) 19:112 Page 2 of 15

Nuclear-cytoplasmic transport has been indicated to BCa samples) and GSE7476 (n = 13, including 3 normal play an important role in mediating cellular functions samples and 10 BCa samples) obtained from the Gene while the defects in this transport process are implicated Expression Omnibus (GEO) database (https://www.ncbi. in multiple types of cancers [9]. Karyopherin (KPN) nlm.nih.gov/gds) was performed using the “limma” pack- nuclear transport receptors have emerged as critical reg- age of R language (http://www.bioconductor.org/packa ulators in altering the progression and outcome of cancer ges/release/bioc/html/limma.html). Te BCa-related [10]. As one of the KPN family members, KPN alpha 2 data from Te Cancer Genome Atlas (TCGA) data- (KPNA2) has been highlighted to be a biomarker of can- base (https://portal.gdc.cancer.gov/) were analyzed by cer, which participates in the translocation of diverse pro- means of Gene Expression Profling Interactive Analysis teins associated with carcinogenesis [11]. For instance, (GEPIA) (http://gepia.cancer-pku.cn/). Te diferentially KPNA2 has been demonstrated to harbor the oncogenic expressed genes (DEGs) were selected with |logFold- property of promoting malignant phenotypes of breast Change| > 1.8 and p < 0.01 as the threshold. A Venn dia- cancer cells [12]. Te pro-tumor efects of KPNA2 have gram was plotted to screen out the most signifcant DEG also been validated in hepatocellular carcinoma (HCC) through intersection of DEGs from the three databases, where KPNA2 mediates the nuclear import of the tran- followed by survival analysis using GEPIA. Ten, a pro- scriptional factor pleomorphic adenoma gene 1 (PLAG1) tein–protein interaction (PPI) network was established protein [13]. Moreover, there is evidence elucidating the using DEGs that were signifcantly correlated with function of aberrantly expressed KPNA2 in BCa by reg- patient survival condition with the help of String (https:// ulating the nuclear transportation of octamer-binding string-db.org/). Te core degree of DEGs in PPI network transcription factor 4 [14]. was analyzed by Cytoscape (https://cytoscape.org/). Te Besides, KPNA2 has been found to interact with Chro- downstream regulatory pathway with highest core degree mobox 8 (CBX8) in human lung adenocarcinoma can- was predicted and verifed by GEPIA database and hTF- cer cells [15]. Expression of CBX8, which is a member target (http://bioinfo.life.hust.edu.cn/hTFtarget#!/). of the polycomb family of epigenetic transcription fac- tors, correlates with tumorigenesis in HCC and esopha- Clinical sample collection geal squamous cell carcinoma [16–18]. Additionally, the In total, 45 patients diagnosed with BCa in Hainan high expression of CBX8 has been elaborated to fore- General Hospital from January 2013 to June 2015 were shadow unsatisfactory overall survival and disease-free enrolled. Te BCa tissues and adjacent non-cancerous survival (DFS) of patients with muscle invasive BCa tissues (≥ 3 cm from the edge of BCa tissues) were sur- [19]. However, the mechanistic evidence for interplay gically resected, rapidly frozen in liquid nitrogen and between KPNA2 and CBX8 in BCa poorly documented. stored at − 80 °C before use. BCa tissues were identifed To rectify this, we investigated in the current study how by histology and pathology and further classifed accord- KPNA2 can afect the development and progression of ing to criteria of Te 2004 World Health Organization BCa through its regulatory action on the translocation grading system. Te clinical staging classifed based on of CBX8. Tus, we endeavored to explore the functional the International Union Against Cancer (UICC) TNM interaction of KPNA2 with CBX8 in BCa in an attempt staging system [20]. Clinical tumor recurrence was to unravel the underlying molecular mechanism of this recorded when tumor recurrence or metastasis was sug- oncogenic pathway. gested by clinical and imaging examination. Overall Sur- vival (OS) designated the time period between the date Materials and methods of randomization to the date of death due to any reason. Ethics statement Disease-free survival (DFS) designated the time period Te study was conducted with approval of Ethics Com- between the date of operation to the date of clinical mittee of Ethics Committee of Hainan General Hospital. tumor recurrence or death. All clinical data were ana- Each participant signed written informed consents prior lyzed using R language software. Te data of KPNA2 and to the study. Te animal experiments were approved by CBX8 expression were Log2-transformed into a bivari- the Animal Committee of Hainan General Hospital and ate model. According to the median of the expression of conducted in accordance with the relevant regulations KPNA2 and CBX8, patients with BCa were divided into and requirements of the Institutional Animal Care and high expression group and low expression group. Use Committee (IACUC). Lentivirus infection In silico analysis A human bladder epithelial immortalized cell line SV- Te diferential analysis of the microarray dataset HUC-1, Human BCa cell lines (T24 and BIU-87) and GSE3167 (n = 50, including 9 normal samples and 41 HEK293T cells were purchased from American Type Zeng et al. J Transl Med (2021) 19:112 Page 3 of 15

Culture Collection (https://www.atcc.org/; Manas- Table 1 Primer sequences for RT-qPCR sas, VA, USA) and cultured in Dulbecco’s Modifed Gene Primer sequences Eagle Medium (DMEM, Termo Fisher Scientifc Inc., Waltham, MA, USA) containing 10% fetal bovine serum KPNA2 (human) F: 5′-CTGGGACATCAGAACAAACCAAG-3′ (FBS) (10100147, Gibco BRL, Invitrogen, CA, USA) at R: 5′-ACACTGAGCCATCACCTGCAAT-3′ CBX8 (human) F: 5′-CCTTCGAAACATGGGTTTGT-3′ 37 °C with 5% CO2. Cells at the logarithmic growth phase were detached by R: 5′-CTGGGCTTGTCATCCACTCT-3′ 4 PRDM1 (human) F: 5′-CCCTCATCGGTGAAGTCTA-3′ trypsin and dispersed into single cell suspension (5 × 10 cells/mL), which were then seeded in a 6-well plate R: 5′-ACGTAGCGCATCCAGTTG-3′ (2 mL/well), cultured at 37 °C overnight and infected c-FOS (human) F: 5′-AGAATCCGAAGGGAAAGGAA-3′ with diferent lentivirus. Te lentivirus plasmids har- R: 5′-CTTCTCCTTCAGCAGGTTGG-3′ boring overexpressed genes (oe-KPNA2, oe-CBX8, oe- U6 F: 5′-CGGCGGTCGTGAAGCGTTCCAT-3′ PRDM1 and oe-c-FOS) were constructed on LV5-GFP R: 5′-CCAGTGCAGGGTCCGAGGTAT-3′ and lentivirus plasmids harboring short hairpin RNA GAPDH F: 5′-CACCCACTCCTCCACCTTTG-3′ (shRNA) (sh-KPNA2-1, sh-KPNA2-2, sh-CBX8-1, sh- R: 5′-CCACCACCCTGTTGCTGTAG-3′ CBX8-2, sh-PRDM1-1 and sh-PRDM1-2) were con- RT-qPCR: reverse transcription quantitative polymerase chain reaction; F: structed on pSIH1-H1-copGFP. Te lentivirus plasmids forward; R: reverse; KPNA2: karyopherin α2; CBX8: Chromobox 8; PRDM1: PR domain zinc fnger protein 1; c-FOS: Cellular oncogene fos; GAPDH: harboring shRNA were synthesized by Gene Pharma Co., glyceraldehyde-3-phosphate dehydrogenase Ltd., (Shanghai, China). Te supernatant was harvested, followed by centrifugation to remove virus particles and enable determination of virus titer determination. At 48 h after infection, the cells were collected and the Billerica, MA, USA) in a wet manner. Te membrane expression levels of diferent genes were measured by was blocked in 5% bovine serum albumin at room tem- reverse transcription quantitative polymerase chain reac- perature for 1 h, and then probed with the following tion (RT-qPCR). diluted primary antibodies: rabbit polyclonal antibodies to KPNA2 (ab84440, 1:1000, Abcam Inc., Cambridge, RT‑qPCR UK), CBX8 (ab70796, 1:500, Abcam), PRDM1 (ab119401, Total RNA was extracted from cells and tissues using Tri- 1:1000, Abcam), c-FOS (ab190289, 1:2000, Abcam), and zol (15596026, Invitrogen) and reversely transcribed to GAPDH (ab181602, 1:2000) at 4 °C overnight, and then cDNA using a RT kit (RR047A, Takara, Kusatsu, Shiga, re-probed with horseradish peroxidase-conjugated Japan). qPCR was performed with SYBR Premix EX goat anti-rabbit immunoglobulin G (ab205718, 1:10000, Taq kit (RR420A, Takara) in a real-time fuorescence Abcam) at room temperature for 1 h. Te protein bands qPCR instrument (ABI7500, ABI, Foster City, CA, USA). were visualized by VILBER FUSION FX5 (Vilber Lour- Te relative expression level was analyzed by 2 −ΔΔCt mat, Paris, France) and analyzed by ImageJ 1.48u soft- method with glyceraldehyde-3-phosphate dehydrogenase ware (National Institutes of Health, Bethesda, Maryland, (GAPDH) serving as the loading control. Primers for USA). Te relative expression was calculated as the ratio KPNA2, CBX8, PRDM1 and c-FOS were synthesized by of gray value of target band to that of the internal refer- Shanghai Sangon Biotech Co., Ltd., (Shanghai, China) as ence (GAPDH). shown in Table 1. Immunohistochemistry (IHC) Western blot analysis BCa tissue blocks were fxed in 4% paraformalde- Total protein was extracted from cells and tissues by hyde-phosphate bufer for 12 h and cut into slices of high-efective Radio Immunoprecipitation Assay Lysis 4 μm-thickness. Te tissue slides were dewaxed with Bufer (R0010, Solarbio Life Sciences Co., Ltd., Beijing, xylene, hydrated by gradient ethanol, boiled in 0.01 M China) in strict accordance with the manual. Nucleopro- citric acid bufer solution for 15–20 min and cooled tein and cytoplasmic protein were prepared using cyto- down to room temperature. Te tissue slides were incu- plasm and nucleus protein extraction kits (Termo Fisher bated with 50 μL primary rabbit antibodies to KPNA2 Scientifc). Te protein concentration was determined (ab84440, 1:200, Abcam) and CBX8 (ab182627, 1:50, using a bicinchonininc acid protein assay kit (20201ES76, Abcam) at room temperature for 1 h and with second- Yeasen Biotech Co., Ltd., Shanghai, China). Te pro- ary antibody goat anti-rabbit immunoglobulin G (IgG; teins were separated by 10% sodium dodecylsulphate- ab6728, 1:1000, Abcam) at room temperature for another polyacrylamide gel electrophoresis and wet-transferred 1 h. Ten the slides were incubated with streptavidin per- onto the polyvinylidene fuoride membrane (Millipore, oxidase at 37 °C for 30 min. Color was developed using Zeng et al. J Transl Med (2021) 19:112 Page 4 of 15

3,3-N-diaminobenzidine (DAB) for 5 min and reaction (ab150075, 1:400, Abcam) for 1 h at 37 °C in the dark. Te was terminated by washing under running water for nucleus was stained by 4’,6-diamidino-2-phenylindole 10 min. Hematoxylin was applied for 2-min counterstain- (BioDee Biotechnology Co., Ltd., Beijing, China) in the ing, followed by hydrochloric acid–ethanol for diferen- dark for 5 min. Te cells were fnally sealed by fuorescent tiation. Te sample was dehydrated, cleared, sealed and mounting media and images were acquired under a fuo- observed under an inverted microscope. rescence microscope (IX73, Olympus, Tokyo, Japan).

Co‑immunoprecipitation (Co‑IP) Cell counting kit‑8 (CCK‑8) assay T24 cells were collected and lysed. Te polyclonal anti- Te cells were seeded in a 96-well plate at a density of 3 bodies to KPNA2 (ab70160, 1:100, Abcam) and CBX8 2 × 10 cells/well. After 24 h-infection, cell proliferation (ab182627, 1:100, Abcam) were used for Co-IP with ref- was evaluated with a CCK-8 kit (Dojindo Laboratories, erence to the instructions of the Pierce Classic IP Kit Kumamoto, Japan). Te optical density value at 450 nm (Termo Fisher Scientifc). In brief, protein extracts was measured by a microplate reader (Bio-Rad, Hercules, were incubated with specifc antibody or IgG (negative CA, USA). control) at 4 °C overnight. Te immune complex was collected after incubated with protein A agarose for 2 h 5‑ethynyl‑2′‑deoxyuridine (EdU) assay at 4 °C. Following 3 washes using protein lysis bufer Te cells at the logarithmic growth phase were seeded 3 4 (0.7 mL), the precipitate was boiled, cooled, and ana- in a 96-well plate at a density of 2 × 10 − 4 × 10 cells/ lyzed by 10–12% SDS-PAGE, followed by Western blot well and cultured overnight. 48 h after infection, cells analysis. were labeled with EdU by incubated with EdU (100 μL/ well) for 2-h. Cells were fxed with cell fxative (100 μL/ Chromatin immunoprecipitation (ChIP) well) for 30-min at room temperature and incubated with BCa cells were fxed with formaldehyde for 10 min to 2 mg/mL glycine for another 5 min. Te cells were fur- generate DNA–protein crosslink and then disrupted by ther incubated for 10 min with phosphate bufer saline Ultrasonic Cell Disruptor (UP-250, Scientz, Ningbo, Zhe- (PBS) containing 0.5% TritonX-100 (100 μL/well), stained jiang, China), 10 s each at an interval of 10 s, 15 cycles in with 1 × Apollo for 30 min and with 1 × Hoechst 33342 total. Te supernatant was collected through centrifuga- for 30 min (100 μL/well) at room temperature in the tion at 4 °C for 10 min at 12,000 rpm and sub-packed into dark. Ten, fuorescent mounting media (100 μL/well) 2 tubes. One tube was added with rabbit antibody to IgG was used for sealing. Te number of EdU-labeled cells in (ab109489, 1: 300, Abcam) as NC and the other tube was 3 randomly-selected felds was counted under the fuo- added with antibody to CBX8 (ab182627, 1: 50, Abcam) rescence microscope. EdU labeling rate (%) = the number for incubation at 4 °C overnight. Te DNA–protein com- of positive cells/(the number of positive cells + the num- plex was precipitated by Protein Agarose/Sepharose. ber of negative cells) × 100%. Te supernatant was removed through centrifugation Terminal deoxynucleotidyl transferase dUTP nick end at 12,000×g for 5 min. Te non-specifc complex was washed, followed by de-crosslinking at 65 °C overnight. labeling (TUNEL) staining DNA fragments were purifed and retrieved by phenol/ BCa tissues were fxed in 4% paraformaldehyde over- chloroform. Te binding between CBX8 and PRDM1 was night, embedded with parafn and sliced into 5-mm detected by RT-qPCR using PRDM1 specifc primers. sections. Five sections were dewaxed, hydrated, and incubated with 50 μL of 1% proteinase K dilution at Immunofuorescence 37 °C for 30 min. Te activity of endogenous peroxidase Te cells were fxed in 4% paraformaldehyde for 15 min, (POD) was blocked by incubated with methanol solu- penetrated by 0.5% Triton X-100 (Sangon) at room tem- tion supplemented with 0.3% H2O2 at 37 °C for 30 min. perature for 20 min, and blocked in 5% normal goat TUNEL reaction solution was added and incubated serum (Solarbio Science & Technology Co., Ltd., Beijing, with sections in a humidifed box at 37 °C for 1 h in the China) at room temperature for 30 min. Ten the cells dark. Ten, 50 μL Converter-POD was added and incu- were incubated with rabbit anti-KPNA2 (ab70160, 1:100, bated with the sections in a wet box at 37 °C for another Abcam) and CBX8 (ab182627, 1:100, Abcam) in a humid- 30 min. Color was visualized by incubation with 2% ifed box at 4 °C overnight. Ten the cells were incubated DAB for 15-min at room temperature. Te reaction was with Alexa Fluor 647-labeled donkey anti-rabbit IgG terminated by adding distilled water. Te sections were Zeng et al. J Transl Med (2021) 19:112 Page 5 of 15

then counterstained by hematoxylin, followed by dehy- migration was observed under an inverted microscope dration using ethanol of ascending concentrations and (CKX53, OLYMPUS, Japan), photographed and counted. xylene clearing. After sealed with neutral gum, sections Each experiment was repeated three times. were observed under a microscope (40×) in 10 randomly-selected felds. Te brown nuclei were indicative Scratch test of apoptotic positive cells and blue nuclei were indica- Te transfected cells were seeded in a 6-well plate at tive of normal cells. Te apoptosis rate the number of 5 = 5 × 10 cells/well. When the cell growth and confu- apoptotic cells/the number of normal cells. ence reached about 90%, the axis of the hole was gently scratched using a sterile pipette tip. After washing with Transwell assay PBS to remove foating cells, the cells were added with Invasion assay was performed. Te extracellular matrix serum-free medium and continued to culture for 0.5–1 h (ECM) gel (E1270-1ML, Sigma-Aldrich Chemical Com- to recover the cells. Te cells were photographed at 0 h pany, St Louis, MO, USA) was diluted to 1 mg/mL with and 24 h, respectively, and the migration distance of the serum-free medium. Te polycarbonate membrane cells was measured with Image-Pro Plus Analysis soft- in the apical chambers was supplemented with 40 μL ware (Media Cybernetics, Silver Spring, MD, USA), and ECM gel and incubated for 5 h at 37 °C with 5% CO 2 for the value was averaged. polymerization, which was then incubated with DMEM (70 µL/chamber) for 0.5 h at 37 °C for rehydration. BCa Xenograft tumor in nude mice cells were starved for 24 h in serum free DMEM and In total, 36 BALA/C nude mice (age: 4–5 weeks, weight: re-suspended in serum free DMEM to make the fnal 16–18 g) were purchased from Hunan SJA Laboratory 5 cell concentration 2.5 × 10 cells/mL. Ten, 0.2 mL cell Animal Co., Ltd., (Changsha, Hunan, China) (http:// suspension was added into apical Transwell chambers www.hnsja.com/ ), and raised under specifc patho- with hydrated basement membrane while the basolat- gen free environment. Every 12 mice were inoculated eral chambers were added with 700 µL pre-cold DMEM 3 with BCa cells (5 × 10 cells/mouse) harboring oe-NC, containing 10% FBS, followed by incubation under sat- oe-KPNA2 or oe-KPNA2 + oe-PRDM1. Te volume of urated humidity at 37 °C with 5% CO2 for 24 h. With xenograft tumors was measured on the 10th day after the removal of chambers, the cells on the chambers inoculation and calculated using the formula that V and basement membrane were wiped out using wet 3 2 (mm ) = (A × B )/2, where A is the long diameter and B cotton swab, fxed with methanol for 30 min, stained is the short diameter. Te curve displaying the average with 0.1% crystal violet for 20 min and aired dried. An volume at each time point was plotted. inverted microscope (CKX53, OLYMPUS) was used for observation at 200 × within 5 randomly selected felds Statistical analysis (200×). Te number of cells that crossed through the Data analysis was performed using SPSS 21.0 software membrane was counted. (IBM, Armonk, NY, USA). Measurement data were Migration assay was conducted. Briefy, the cells were expressed as the mean ± standard deviation unless transfected with plasmid DNA for 24 h and then digested otherwise indicated. Data in normal distribution and into a single cell suspension. Cell concentration was 6 homogeneity of variance were compared by paired adjusted to 1 × 10 cells/mL in medium containing 1% t-test within the group, by independent sample t-test FBS. A 24-well plate was added with 10% FBS-containing between two groups and by one-way analysis of vari- complete medium at 600 μL/well. Te Transwell cham- ance (ANOVA) among multiple groups, followed by ber was placed on the plate and added with the adjusted Tukey’s post hoc test. Data comparison at diferent time amount of cell suspension at 100 μL per chamber, fol- points was analyzed by repeated measures ANOVA, lowed by incubation at 37 °C for 24 h. Te chamber was followed by Tukey’s post hoc test. Kaplan–Meier was removed and the medium and non-migrated cells in introduced to plot a survival curve, while Log-rank the chamber were wiped of using a cotton swab, then test was used for diferential analysis. Cox propor- fxed with a fxative with a ratio of methanol and acetic tional hazard regression model was used to evaluate acid of 3: 1 for 15–30 min, and air-dried. Te chamber the individual variables infuencing the survival risk of was stained with 1% crystal violet for 20 min and cell BCa. Wald’s test was used to determine the statistical Zeng et al. J Transl Med (2021) 19:112 Page 6 of 15

abOverall Survival Overall Survival Overall Survival Overall Survival .0 .0 .0 GSE3167 GSE7476 Low FHL1 Group Low KPNA2 Group Low PCP4 Group 1. 0 Low SYNM Group High FHL1 Group High KPNA2 Group High PCP4 Group High SYNM Group Logrank p=0.042 Logrank p=0.005 Logrank p=0.033 Logrank p=0.039 HR(high)=1.4 HR(high)=1.4 HR(high)=1.4 HR(high)=1.6 .8 0. 81 0. 81 0. 81 p(HR)=0.034 p(HR)=0.04 30 2 143 p(HR)=0.043 p(HR)=0.0053 n(high)=299 n(high)=301 al n(high)=300 n(high)=301 al al al iv iv n(low)=301 n(low)=301 iv iv n(low)=301 n(low)=301 .6 .6 .6 19 0. 60

4 282 0. 40 0. 4 rcent su rv rcent su rv 0. 40 0. 40 rcent su rv rcent su rv Pe Pe Pe Pe .2 .2 .2 615 .2

GEPIA 0. 00 0. 00 0. 00 0. 00

050100 150 050100 150 050100 150 050100 150 Months Months Months Months c de fg 9 6 FHL1 * p−value = 1.6e−06 R = 0.24 2 4 8 * * NA CBX8

SYMN KP

3 of 6 of (TPM + 1) (TPM + 1) TPX2 2 CDC5L 2

PCP4 ssion 2 4 re pression

KPNB1 KPNA2 log2(CBX8 TPM) xp ex 2345

ee 1 2 ve ti

NUP153 Expression - log Expression - log

RCC1 12345 Rela

Relativ 0 0

2345678 2345678 Adjacent tissue Cancer tissue Adjacent tissue Cancer tissue BLCA log2(KPNA2 TPM) BLCA (num(T)=404; num(N)=28) (num(T)=404; num(N)=28) h i j k KPNA2 58 kDa Adjacent tissue Adjacent tissue Survival function Survival function Survival function Survival function Bladder cancer tissue 1.0 1.0 Bladder cancer tissue l 1.0 1.0 2.0 60 l l * * l 43 kDa CBX2 * 0.8 0.8 0.8 0.8 )

evel 1.5 (%

nl * 0.6 0.6 GAPDH 36 kDa 40 0.6 0.6 ta otei ra 0.4 0.4 0.4 0.4 e e pr 1.0 u su ve

s ve CBX8 ti KPNA2 expression KPNA2 expression CBX8 High expression t tiss 20 0.2 High expression 0.2 High expression 0.2 High expression 0.2 n er Low expression Probability of overall surviva 0.5 Probability of overall surviva Low expression Low expression Low expression ce Positi High expression-censored

High expression-censored Probability of Disease free surviva High expression-censored High expression-censored Probability of Disease free surviva ja Re lati Low expression-censored canc Low expression-censored Ad 0.0 Low expression-censored 0.0 Low expression-censored 0.0 0.0 dder 0 10 20 30 40 a 0.0 0 0 10 20 30 40 01020 30 40 0 10 20 30 40 Time (months) Time (months) Time (months) Time (months) Bl KPNA2 CBX2 KPNA2CBX8 Fig. 1 High expression of KPNA2 and CBX8 in BCa is associated with dismal oncological outcomes of patients with BCa. a Venn diagram illustrating DEGs in BCa through microarray dataset GSE3167 and GSE7476 and TCGA database by GEPIA. b The survival curves displaying the correlation between the expression of KPNA2, PCP4, SYNM or FHL1 and survival of patients with BCa. c The PPI networks of KPNA2, PCP4, SYNM and FHL1. The deeper red color suggests higher core degree and the deeper blue color suggests lower core degree. d The box plot of KPNA2 expression in BCa samples (left red box) and normal samples (right grey box) according to GEPIA; *p < 0.05 vs. normal samples. e The correlation analysis between expression of KPNA2 and CBX8 in BCa according to GEPIA (p 1.6e 06). f The box plot of CBX8 expression in BCa samples (left red box) and = − normal samples (right grey box) according to GEPIA; *p < 0.05 vs. normal samples. g The expression of KPNA2 and CBX8 in BCa tissues and adjacent non-cancerous tissues determined by RT-qPCR; *p < 0.05 vs. adjacent non-cancerous tissues. h The expression of KPNA2 and CBX8 in BCa tissues and adjacent non-cancerous tissues determined by Western blot analysis; *p < 0.05 vs. adjacent non-cancerous tissues. i The expression of KPNA2 and CBX8 in BCa tissues and adjacent non-cancerous tissues identifed by IHC ( 100); *p < 0.05 vs. adjacent non-cancerous tissues. j The correlation × between KPNA2 expression and survival condition analyzed by Kaplan–Meier. k The correlation between CBX8 expression and survival condition analyzed by Kaplan–Meier. n 45. Data comparison between BCa tissues and adjacent non-cancerous tissues was analyzed by paired t-test = signifcance of univariate and multivariate Cox regres- of patients with BCa showed 4 signifcantly correlated sion models. A p value of < 0.05 was considered statisti- DEGs, KPNA2, PCP4, SYNM and FHL1, among which cally signifcant. KPNA2 had the most signifcant correlation (Fig. 1b). Following establishment of a PPI network for the above Results 4 genes using String, Cytoscape was introduced for image High expression of KPNA2 and CBX8 in BCa was correlated processing, which revealed KPNA2 to be the gene with with poor prognosis highest core degree (Fig. 1c, Table 2). Besides, KPNA2 Trough diferential analysis on GEO microarray dataset was revealed as a signifcantly upregulated gene in BCa GSE3167 and GSE7476 combined with GEPIA on TCGA according to the GEPIA results (Fig. 1d, Table 3). database, 55,446 and 920 DEGs were obtained respec- Further GEPIA results demonstrated a positive correla- tively and 19 DEGs were further revealed in the intersection between the expression of KPNA2 and CBX8 in BCa tion by means of Venn diagram (Fig. 1a). Subsequently, samples (Fig. 1e). CBX8 was confrmed to be another a survival curve displaying the correlation between the upregulated gene in BCa (Fig. 1f), which is highly sug- expression profle of 19 DEGs and survival condition gestive of the promotive efects induced by the interplay between KPNA2 and CBX8 on the progression of BCa. Zeng et al. J Transl Med (2021) 19:112 Page 7 of 15

Table 2 Survival analysis of 19 DEGs KPNA2 promoted nuclear import of CBX8 Gene p (HR) HR Gene p (HR) HR To investigate whether the nuclear translocation of CBX8 in BCa was mediated by KPNA2, KPNA2 anti- KPNA2 0.0053 1.6 ACTG2 0.099 1.3 body was introduced for Co-IP assay. CBX8 was detected PCP4 0.034 1.4 ACTC1 0.1 1.3 in KPNA2 antibody-enriched precipitate of the BCa cell SYNM 0.04 1.4 MYH11 0.11 1.3 lines, BIU-87 and T24 (Fig. 2a). Moreover, KPNA2 anti- FHL1 0.043 1.4 CFD 0.21 1.2 body could enrich more CBX8 in T24 cells. Ten, KPNA2 SRPX 0.051 1.4 MYL9 0.22 1.2 and CBX8 mRNA expression was detected in T24 and TNS1 0.056 1.4 TOP2A 0.35 0.85 BIU-87 cell lines using RT-qPCR (Fig. 2b). Te results SPARCL1 0.073 1.3 LMOD1 0.54 1.1 displayed that KPNA2 and CBX8 mRNA expression was TAGLN 0.083 1.3 CKS2 0.59 1.1 increased in T24 and BIU-87 cells relative to SV-HUC-1 CNN1 0.088 1.3 MYLK 0.62 1.1 cells, and their levels were most signifcantly upregulated DES 0.089 1.3 in T24 cells. Terefore, T24 cells were selected for sub- HR: Hazard Ratio (HR > 1 indicates high risk gene, HR < 1 indicates low risk gene); sequent use. Next, in vitro models were developed when DEG: diferentially expressed genes; KPNA2: karyopherin α2; PCP4: Purkinje cell KPNA2 was overexpressed or knocked down in T24 cells. protein 4; SYNM: Synemin; FHL1: Four and a half LIM domain protein 1; SRPX: sushi repeat-containing protein X; TNS1: tensin-1; SPARCL1: Secreted protein According to Western blot analysis, KPNA2 expression acidic and rich in cysteine like-1; TAGLN: transgelin; CNN1: calponin-1; DES: was upregulated/downregulated in response to overex- desulfhydrase pression/knockdown of KPNA2 (Fig. 2c). Following isola- tion of nucleoproteins and cytoplasmic proteins, Western blot analysis showed that overexpressing KPNA2 could Table 3 The core degree of signifcant risk genes signifcantly increase the expression of KPNA2 and Gene Degree CBX8 in the nucleus but slightly decrease the expression of CBX8 in the cytoplasm while opposite results were FHL1 1 observed after silencing KPNA2 (Fig. 2d, e). Observa- PCP4 0 tion through immunofuorescence microscopy revealed SYNM 0 accumulation of KPNA2 and CBX8 in the nucleus after KPNA2 5 overexpression of KPNA2 (Fig. 2f). To conclude, it was FHL1: Four and a half LIM domain protein 1; PCP4: Purkinje cell protein 4; SYNM: suggested that KPNA2 acted as positive regulator for the Synemin; KPNA2: karyopherin α2 nuclear import of CBX8 through its binding with CBX8.

Subsequently, RT-qPCR (Fig. 1g), Western blot analy- KPNA2 mediated the proliferation, migration, and invasion sis (Fig. 1h) and IHC (Fig. 1i) were performed to deter- of BCa cells through regulating CBX8 mine the expression profle of KPNA2 and CBX8 in Te investigation focus was then shifted to an explora- BCa tissues collected from patients with BCa. We saw tion of the functional signifcance of interaction between signifcantly higher expression of KPNA2 and CBX8 in KPNA2 and CBX8 in BCa. Firstly, KPNA2 was overex- BCa tissues than in adjacent non-cancerous tissues was pressed and CBX8 was silenced in T24 cells. RT-qPCR observed. Te correlation analysis between KPNA2 and showed that sh-CBX8 obviously reduced the expression CBX8 expression and prognosis of BCa patients through of CBX8 in the presence of oe-KPNA2 (Fig. 3a). Ten, follow-up visits showed an inverse relationship, whereby the proliferative capability of tumor cells was measured patients with highly expressed KPNA2 and CBX8 by CCK-8 assays (Fig. 3b, c), migration and invasion were exhibited poor overall survival and DFS (Fig. 1j, k). Te evaluated by Transwell assay and scratch test (Fig. 3d–g). expression of KPNA2 and CBX8 was correlated with the Te proliferation, migration, and invasion of BCa cells pathological grade, clinical stage, metastasis, and recur- were signifcantly retarded after inhibition of KPNA2 but rence of the tumor (p < 0.05), but not correlated with age, accelerated after overexpression of KPNA2 (Fig. 3b, d, e). gender, smoking history, and gross hematuria (p > 0.05) Besides, silencing of CBX8 could signifcantly counter- (Table 4). Taken together, these fndings indicated that acted the efects of KPNA2 overexpression on the prolif- KPNA2 and CBX8 were highly expressed in BCa tissues eration, migration, and invasion of BCa cells (Fig. 3c, f, g). and served as adverse prognostic biomarkers. In summary, the results elucidated that the mediation of Zeng et al. J Transl Med (2021) 19:112 Page 8 of 15

Table 4 Correlation between the expression of KPNA2 and CBX8 in tissues and the characteristics of clinical cases Group Cases KPNA2 expression p CBX8 expression p Downregulate Upregulate Downregulate Upregulate

Age > 60 15 5 10 > 0.05 7 8 > 0.05 60 30 17 13 16 14 ≤ Gender Male 35 19 16 > 0.05 18 17 > 0.05 Female 10 3 7 5 5 TNM staging G1 17 5 12 < 0.05 5 12 < 0.05 G2–G3 28 17 11 18 10 Clinical staging T1 24 4 20 < 0.05 8 16 < 0.05 T2–T3 21 18 3 15 6 Smoking history Yes 28 15 13 > 0.05 13 15 > 0.05 No 17 7 10 10 7 Gross hematuria Yes 30 15 15 > 0.05 16 14 > 0.05 No 15 7 8 7 8 Metastasis Yes 14 11 3 < 0.05 11 3 < 0.05 No 31 11 20 12 19 Recurrence Yes 19 15 4 < 0.05 14 5 < 0.05 No 26 7 19 9 17

KPNA2 in BCa cell proliferation, migration and invasion PRDM1 promoter region was then detected by ChIP was dependent on CBX8. assay, which revealed notably reduced enrichment of CBX8 in the promoter region of PRDM1 in response to CBX8 downregulated PRDM1 by recruiting BCOR CBX8 knockdown (Fig. 4d). For further investigation on in the promoter region of PRDM1 whether CBX8 could bind to BCOR in BCa cells, a Co-IP A previous report had elucidated that CBX8 can bind assay was conducted in T24 cells, which showed that to polycomb repressive complex-BCL6 corepressor BCOR was enriched with CBX8 antibody compared with (BCOR) to suppress the transcription of positive regu- IgG (Fig. 4e). Ten, the binding of BCOR in the PRDM1 latory domain 1 (PRDM1), by which lymphomagenesis promoter region was detected by ChIP, which showed is facilitated [21]. In this part of the study, we intended that BCOR enrichment was dramatically reduced in the to explore whether PRDM1 functioned downstream presence of sh-CBX8-1, thus suggesting that CBX8 was of CBX8 in BCa. An inverse relationship between the necessary for BCOR recruitment in the PRDM1 pro- expression of CBX8 and PRDM1 in BCa was revealed moter (Fig. 4f). Further dual luciferase reporter assay from GEPIA (Fig. 4a) and PRDM1 was found to be poorly demonstrated that silencing CBX8 could enhance the expressed in BCa (Fig. 4b). To determine the regulatory luciferase activity of PRDM1-promoter-WT (Fig. 4g). As mechanism of CBX8 expression in BCa cells, CBX8 was shown in Fig. 4h, i, PRDM1 expression sharply decreased silenced in T24 cells. Western blot analysis confrmed in response to CBX8 overexpression, and increased in that CBX8 expression was remarkably decreased by both response to CBX8 knockdown as measured by RT-qPCR sh-CBX8-1 and sh-CBX8-2, and sh-CBX8-1 was selected and Western blot analysis. Collectively, CBX8 was vali- for subsequent experiments because of its higher silenc- dated to inhibit PRDM1 expression through recruitment ing efciency (Fig. 4c). Te enrichment of CBX8 in the of BCOR to the PRDM1 promoter region. Zeng et al. J Transl Med (2021) 19:112 Page 9 of 15

a T24 BIU-87 b 15 6 2 4 3

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Re 0.0 C 2 NC A2 N 2- - A oe- PN sh N -K PNA2-1 P -K -K oe h s sh Fig. 2 KPNA2 assists nucleus shuttling of CBX8. a The binding between KPNA2 and CBX8 in T24 and BIU-87 cells detected by Co-IP assay. b The PNA2 and CBX8 mRNA expression detected in T24 and BIU-87 cell lines using RT-qPCR. c The expression of KPNA2 and CBX8 in BCa cells in response to overexpressing/silencing KPNA2 determined by Western blot analysis. d The expression of CBX8 in the nucleus of BCa cells in response to overexpressing/silencing KPNA2 determined by Western blot analysis. e The expression of CBX8 in the cytoplasm of BCa cells in response to overexpressing/silencing KPNA2 determined by Western blot analysis. f The nucleus accumulation of KPNA2 and CBX8 in T24 cells by immunofuorescence staining. *p < 0.05 vs. T24 cells treated with oe-NC; #p < 0.05 vs. T24 cells treated with sh-NC

PRDM1 contributed to the progression of BCa by inhibiting Transwell assay (Fig. 5h) and scratch test (Fig. 5i) revealed c‑FOS expression that overexpressing c-FOS reversed the efects of upregu- Since PRDM1 was poorly expressed in BCa, we overex- lation of PRDM1 on cell proliferation, apoptosis, migra- pressed PRDM1 in T24 cells. Results showed that upreg- tion, and invasion. To conclude, PRDM1 downregulated ulating PRDM1 suppressed cell proliferation, induced c-FOS expression in contribution to the progression of cell apoptosis and inhibited cell migration and invasion. BCa. Collectively, silencing of PRDM1 promotes the As revealed from the hTFtarget database, PRDM1 could expression of C-FOS to promote the development of mediate the expression of c-FOS as a transcription factor BCa. (Fig. 5a). We overexpressed or silenced PRDM1 expression in T24 cells and then the transfection efciency of KPNA2/PRDM1/c‑FOS axis promoted tumor growth of BCa PRDM1 were successfully validated by RT-qPCR (Fig. 5b) in vivo and Western blot analysis (Fig. 5c), among which the sh- Finally, we endeavored to reproduce the efects of the PRDM1-1 with the most silencing efect was selected for KPNA2/PRDM1/c-FOS axis in vivo through subcu- subsequent use. Next, we overexpressed PRDM1 in T24 taneous implantation of T24 cells with overexpressed cells, and performed RT-qPCR (Fig. 5d) and Western KPNA2/PRDM1 into BALA/C nude mice. Results blot analysis (Fig. 5e), which showed that c-FOS expres- revealed that tumor volume (Fig. 6a) and weight (Fig. 6b) sion was signifcantly diminished by oe-PRDM1. Further- were signifcantly enhanced in the presence of oe- more, CCK-8 assay (Fig. 5f), TUNEL staining (Fig. 5g), KPNA2, whereas KPNA2 and c-FOS expression was Zeng et al. J Transl Med (2021) 19:112 Page 10 of 15

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20 gration mi 0 0 0 Fig. 3 CBX8 is required for the mediatory function of KPNA2 in BCa cell proliferation, migration and invasion. a The expression of CBX8 determined by RT-qPCR. *p < 0.05 vs. T24 cells treated with oe-KPNA2 sh-NC. b, c The proliferation of BCa cells detected by CCK-8 assay. d The number of + migratory and invasive BCa cells detected by Transwell assay ( 200). e Migration of BCa cells detected by scratch test ( 200). f The number × × of migratory and invasive BCa cells detected by Transwell assay ( 200). g Migration of BCa cells detected by scratch test ( 200). *p < 0.05 vs. × × T24 cells treated with oe-NC (panels b and d) or oe-NC sh-NC (panels c and e). #p < 0.05 vs. T24 cells treated with sh-NC (panels b and d) or + oe-KPNA2 sh-NC (panels c and e) + upregulated and PRDM1 expression was downregulated Discussion (Fig. 6c, d) accompanied by accelerated cell proliferation In recent years, research attention has been increasingly (Fig. 6e) and suppressed cell apoptosis (Fig. 6f). Further- paid to the diagnostic, prognostic, and therapeutic sig- more, PRDM1 overexpression could counteract the pro- nifcance of nuclear transportation in malignancies. In tumor efects of KPNA2. To sum up, KPNA2 was proved this context, the KPN superfamily stands out as a group to promote tumorigenesis of BCa in vivo through the of soluble transport receptors [22]. During the current PRDM1/c-FOS pathway. investigation, we selected KPNA2 as the research target Zeng et al. J Transl Med (2021) 19:112 Page 11 of 15

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Re oe-NC oe-CBX8sh-NC sh-CBX8-2 oe-NC oe-CBX8sh-NC sh-CBX8-2 Fig. 4 CBX8 recruits PRC1-BCOR to the PRDM1 promoter region to downregulate PRDM1 expression. a The correlation analysis between expression of CBX8 and PRDM1 in BCa according to GEPIA (p 0.00017). b The box plot of PRDM1 expression in BCa samples (left red box) and normal samples = (right grey box) according to GEPIA. *p < 0.05 vs. normal samples analyzed by paired t-test. c The silencing efciency of sh-CBX8 determined by Western blot analysis. d The enrichment of CBX8 in the PRDM1 promoter region detected by ChIP assay. e The interaction between CBX8 and BCOR detected by co-IP. f The enrichment of BCOR in the PRDM1 promoter region detected by ChIP. g The relative luciferase activity determined by dual luciferase reporter assay. h The expression of PRDM1 in response to CBX overexpression/knockdown determined by RT-qPCR. i The expression of PRDM1 in response to CBX overexpression/knockdown determined by Western blot analysis. *p < 0.05 vs. T24 cells treated with sh-NC (panels c, d, f and g) or T24 cells treated with oe-NC (panels h and i); #p < 0.05 vs. T24 cells treated with sh-NC (panels h and i)

to assess its prognostic value in BCa. Collectively, the as a novel biomarker predictive for muscle invasive BCa experimental data demonstrated a detrimental contri- in patients. bution of KPNA2 to BCa in enhancing the proliferation, An initial fnding of our study was that KPNA2 and migration, and invasion of BCa cells in both in vitro and CBX8 were found to be highly expressed in BCa tissues in vivo. Tis detrimental efect was obtained by assist- and cell, both of which serve as independent risk factor ing the nuclear shuttling of CBX8 and therefore regu- for overall survival and DFS of patients with BCa, indi- lating the PRDM1/c-FOS pathway. Results of our study cating the clinical signifcance of these proteins. Largely suggested that high expression of CBX8 played a critical in agreement with our present results, high expression oncogenic role in the aggressiveness of urothelial carci- of KPNA2 has been identifed in patients with BCa as noma cells of the bladder by promoting cancer cell prolif- an independent biomarker for dismal outcomes in BCa eration. Tese results suggested that CBX8 could be used patients, and to be highly suggestive of greater risk for Zeng et al. J Transl Med (2021) 19:112 Page 12 of 15

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g h Migration Invasion i oe-NC oe-NC oe-NC oe-NC oe-PRDM1 oe-PRDM1 oe-PRDM1 oe-PRDM1 sh-NC sh-NC sh-NC sh-NC sh-PRDM1-1 sh-PRDM1-1 sh-PRDM1-1 sh-PRDM1-1 200 & 150 50 oe-PRDM1 + oe-C-FOS oe-PRDM1 + oe-C-FOS # oe-PRDM1 + oe-C-FOS & 30 oe-PRDM1 + oe-C-FOS # ) & * # 160

40 (mm %) er s( 100 20 ll 30 120 b m ce u istance n number ic l l 20 ll 80 e & * nd

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Apoptot 10 40 gratio mi 0 0 0 0 Fig. 5 The interaction between PRDM1 and c-FOS facilitates the malignant behaviors of BCa cells. a The targeting relationship between PRDM1 and c-FOS verifed by hTFtarget. b The expression of PRDM1 determined by RT-qPCR. c The expression of PRDM1 determined by Western blot analysis. d The expression of c-FOS determined by RT-qPCR. e The expression of c-FOS determined by Western blot analysis. f The proliferation of BCa cells detected by CCK-8 assay. g The apoptosis of BCa cells detected by TUNEL staining ( 200). h The number of migratory and invasive BCa cells × detected by Transwell assay ( 200). i migration of BCa cells detected by scratch test ( 200). *p < 0.05 vs. T24 cells treated with oe-NC. #p < 0.05 vs. × × T24 cells treated with sh-NC. &p < 0.05 vs. T24 cells treated with oe-PRDM1

visceral metastasis and bladder recurrence after treat- diferent malignancies, including breast cancer, esopha- ment [23, 24]. In the context of colorectal cancer, non- geal carcinoma, and HCC [28–30]. Moreover, upregu- small-cell lung cancer and HCC, patients with high lated CBX8 expression exerts oncogenic function on the expression of KPNA2 have been reported to usually have aggressiveness of BCa cells by promoting the malignant generally unsatisfactory overall survival condition [25– prototype by repressing the p53 pathway [19] support- 27]. Besides, patients with muscle invasive BCa exhib- ing the generalization of our fndings. On the other hand, ited high expression of CBX8 in association with reduced inhibition of CBX8 in T24 cells could counterbalance the overall survival and DFS [19]. Accumulating evidence has pro-tumor efects of KPNA2, suggesting that the onco- revealed the involvement of CBX8 in regulatory work of genic role of KPNA2 in BCa was dependent on CBX8. Zeng et al. J Transl Med (2021) 19:112 Page 13 of 15

oe-NC oe-NC a oe-NC b c oe-KPNA2 oe-KPNA2 oe-KPNA2 oe-KPNA2 + oe-PRDM1 oe-KPNA2 + oe-PRDM1 oe-KPNA2 + oe-PRDM1 2000 2.0 5 * l *

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# Rel # # * 0 0.0 0 03691215 KPNA2PRDM1 C-FOS Time (days) oe-NC oe-NC oe-NC d oe-KPNA2 e oe-KPNA2 f oe-KPNA2 oe-KPNA2 + oe-PRDM1 oe-KPNA2 + oe-PRDM1 oe-KPNA2 + oe-PRDM1 2.5 40 40

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Pe 10 10 0.5 # Ap o Re la

# Ed Up * 0.0 0 0 KPNA2PRDM1 C-FOS Fig. 6 KPNA2 contributes to the xenograft tumor growth of BCa in vivo by mediating the PRDM1/c-FOS pathway. a The curve displaying tumor volume at various time points. b The representative images of resected tumors and curve displaying tumor weight at various time points. c The expression of KPNA2, PRDM1 and c-FOS in transplanted tumor tissues determined by RT-qPCR. d The expression of KPNA2, PRDM1 and c-FOS in transplanted tumor tissues determined by Western blot analysis. e The tumor cell proliferation detected by EdU assay ( 400). f The tumor cell × apoptosis detected by TUNEL assay ( 200). n 12. * p < 0.05 vs. nude mice bearing T24 cells treated with oe-NC. #p < 0.05 vs. nude mice bearing T24 × = cells treated with oe-KPNA2

Furthermore, silencing of KPNA2 led to suppressed its mediation of the subcellular localization of proteins cell proliferation, migration, and invasion in vitro as associated with DNA damage response in breast cancer well as inhibited carcinogenic potency in vivo. Concord- [33]. ant with our study, KPNA2 is known for its capacity to Additionally, our study showed CBX8 downregu- shuttle cargo proteins related to tumorigenicity between lated the PRDM1 expression by recruiting BCOR to nucleus and cytoplasm [31, 32]. Concordantly, a previ- the PRDM1 promoter region. Likewise, the interplay ous study has confrmed the crosstalk between KPNA2 between CBX8 and BCOR has been documented to and OCT in BCa, showing that nuclear transportation of play a role in lymphomagenesis [21]. Downregulation of OCT can be inhibited by silencing KPNA2 [14]. Similar PRDM1 has been elucidated to correspond to poor prog- to our fndings, the nuclear import of PLAG1 has been nosis and increased malignant phenotypes in lung cancer illustrated to be aided by KPNA2 in hepatocellular carci- and colon cancer [34, 35]. Moreover, silencing of PRDM1 noma (HCC) and PLAG1 seemed to be partly responsible exerted promoting efects on the development of BCa by for the functional role of KPNA2 in HCC [13]. Also, the upregulating c-FOS, which is signifcantly upregulated in signifcance of KPNA2 as a nucleocytoplasmic transport BCa [36–38]. protein in breast cancer has been recognized by virtue of Zeng et al. J Transl Med (2021) 19:112 Page 14 of 15

G contributed to drafting the manuscript. All authors have read and approved the fnal submitted manuscript.

Funding None.

Availability of data and materials The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate The study was conducted with approval of Ethics Committee of Ethics Com- mittee of Hainan General Hospital. Each participant signed written informed consents prior to the study. The animal experiments were approved by the Animal Committee of Hainan General Hospital and conducted in accordance with the relevant regulations and requirements of the Institutional Animal Care and Use Committee (IACUC).

Consent for publication Not applicable.

Competing interests The authors declare that they have no competing interests.

Author details 1 Department of Urology, Hainan General Hospital (Hainan Afliated Hospital of Hainan Medical University), Haikou 570311, People’s Republic of China. 2 Department of Science and Education, Hainan General Hospital (Hainan Afliated Hospital of Hainan Medical University), No. 19, Xiuhua Road, Xiuying District, Haikou 570311, People’s Republic of China. 3 Department of Urol- ogy, Central South University Xiangya School of Medicine Afliated Haikou Hospital, Haikou 570208, People’s Republic of China. 4 Psychological Research Fig. 7 Graphic abstract showing how the KPNA2/CBX8/PRDM1/ Center, Hainan General Hospital (Hainan Afliated Hospital of Hainan Medical c-FOS pathway works in BCa. The interaction of KPNA2 with CBX8 University), Haikou 570311, People’s Republic of China. contributes to the development and progression of BCa by mediating the PRDM1/c-FOS pathway Received: 21 June 2020 Accepted: 20 January 2021

Conclusion Collectively, our results allowed the decipherment of References a functional axis whereby KPNA2 assisted the nuclear 1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global import of CBX8 to activate downstream efectors by cancer statistics 2018: GLOBOCAN estimates of incidence and mor- recruiting BCOR to the promoter region of PRDM1 tality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68:394–424. (see Fig. 7 for details), thus highlighting the tumor-pro- 2. Abdel Ghafar MT, Gharib F, Abdel-Salam S, Elkhouly RA, Elshora A, Shalaby moting properties of KPNA2 in BCa. Our works hints KH, et al. Role of serum Metadherin mRNA expression in the diagnosis at the possibility of using this axis to defne a new ther- and prediction of survival in patients with colorectal cancer. Mol Biol Rep. 2020;47:2509–19. apeutic target to halt the development and progression 3. El-Guindy DM, Wasfy RE, Abdel Ghafar MT, Ali DA, Elkady AM. Oct4 of BCa. expression in gastric carcinoma: association with tumor proliferation, angiogenesis and survival. J Egypt Natl Canc Inst. 2019;31:3. 4. Abdel Ghafar MT, Gharib F, Al-Ashmawy GM, Mariah RA. Serum high- Abbreviations temperature-required protein A2: a potential biomarker for the diagnosis ANOVA: Analysis of variance; BCa: Bladder cancer; CBX8: Chromobox 8; DEGs: of breast cancer. Gene Rep. 2020;20:100706. Diferentially expressed genes; DFS: Disease-free survival; ECM: Extracellular 5. Habib EM, Nosiar NA, Eid MA, Taha AM, Sherief DE, Hassan AE, et al. matrix; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; GEO: Gene Circulating miR-146a expression predicts early treatment response to Expression Omnibus; GEPIA: Gene Expression Profling Interactive Analysis; imatinib in adult chronic myeloid leukemia. J Investig Med. 2020. https:// KPN: karyopherin; KPNA2: KPN alpha 2; RT-qPCR: Reverse transcription quan- doi.org/10.1136/jim-2020-001563. titative polymerase chain reaction; PBS: Phosphate bufer saline; POD: Peroxi- 6. Abdelghafar M, Allam A, Darwish S. Serum HOX transcript antisense RNA dase; PPI: Protein–protein interaction; PRDM1: PR domain zinc fnger protein 1. expression as a diagnostic marker for chronic myeloid leukemia. Egypt J Haematol. 2019;44:91. Acknowledgements 7. Kamat AM, Hahn NM, Efstathiou JA, Lerner SP, Malmstrom PU, Choi W, The authors would like to acknowledge the helpful comments on this paper et al. Bladder cancer. Lancet. 2016;388:2796–810. received from the reviewers. 8. Knowles MA, Hurst CD. Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity. Nat Rev Cancer. 2015;15:25–41. Authors’ contributions 9. Kau TR, Way JC, Silver PA. Nuclear transport and cancer: from mechanism FC Z and LM L designed the study. FC Z, LM L, DY L and JH G collated the data, to intervention. Nat Rev Cancer. 2004;4:106–17. carried out data analyses and produced the initial draft of the manuscript. M Zeng et al. J Transl Med (2021) 19:112 Page 15 of 15

10. Cagatay T, Chook YM. Karyopherins in cancer. Curr Opin Cell Biol. 25. Takada T, Tsutsumi S, Takahashi R, Ohsone K, Tatsuki H, Suto T, et al. KPNA2 2018;52:30–42. over-expression is a potential marker of prognosis and therapeutic sensi- 11. Christiansen A, Dyrskjot L. The functional role of the novel biomarker tivity in colorectal cancer patients. J Surg Oncol. 2016;113:213–7. karyopherin alpha 2 (KPNA2) in cancer. Cancer Lett. 2013;331:18–23. 26. Li XL, Jia LL, Shi MM, Li X, Li ZH, Li HF, et al. Downregulation of KPNA2 in 12. Noetzel E, Rose M, Bornemann J, Gajewski M, Knuchel R, Dahl E. Nuclear non-small-cell lung cancer is associated with Oct4 expression. J Transl transport receptor karyopherin-alpha2 promotes malignant breast Med. 2013;11:232. cancer phenotypes in vitro. Oncogene. 2012;31:2101–14. 27. Guo X, Wang Z, Zhang J, Xu Q, Hou G, Yang Y, et al. Upregulated KPNA2 13. Hu ZY, Yuan SX, Yang Y, Zhou WP, Jiang H. Pleomorphic adenoma gene 1 promotes hepatocellular carcinoma progression and indicates prognostic mediates the role of karyopherin alpha 2 and has prognostic signifcance signifcance across human cancer types. Acta Biochim Biophys Sin in hepatocellular carcinoma. J Exp Clin Cancer Res. 2014;33:61. (Shanghai). 2019;51:285–92. 14. Zhou J, Dong D, Cheng R, Wang Y, Jiang S, Zhu Y, et al. Aberrant 28. Lee SH, Um SJ, Kim EJ. CBX8 suppresses Sirtinol-induced premature expression of KPNA2 is associated with a poor prognosis and contrib- senescence in human breast cancer cells via cooperation with SIRT1. utes to OCT4 nuclear transportation in bladder cancer. Oncotarget. Cancer Lett. 2013;335:397–403. 2016;7:72767–76. 29. Xiao W, Ou C, Qin J, Xing F, Sun Y, Li Z, et al. CBX8, a novel DNA repair 15. Wang CI, Wang CL, Wu YC, Feng HP, Liu PJ, Chang YS, et al. Quantitative protein, promotes tumorigenesis in human esophageal carcinoma. Int J proteomics reveals a novel role of karyopherin alpha 2 in cell migration Clin Exp Pathol. 2014;7:4817–26. through the regulation of vimentin-pErk protein complex levels in lung 30. Zhang CZ, Chen SL, Wang CH, He YF, Yang X, Xie D, et al. CBX8 exhibits cancer. J Proteome Res. 2015;14:1739–51. oncogenic activity via AKT/beta-catenin activation in hepatocellular 16. Tang B, Tian Y, Liao Y, Li Z, Yu S, Su H, et al. CBX8 exhibits oncogenic prop- carcinoma. Cancer Res. 2018;78:51–63. erties and serves as a prognostic factor in hepatocellular carcinoma. Cell 31. Yasuhara N, Kumar PK. Aptamers that bind specifcally to human KPNA2 Death Dis. 2019;10:52. (importin-alpha1) and efciently interfere with nuclear transport. J 17. Xiao L, Zhou Z, Li W, Peng J, Sun Q, Zhu H, et al. Chromobox homolog 8 Biochem. 2016;160:259–68. (CBX8) interacts with Y-Box binding protein 1 (YBX1) to promote cellular 32. Han Y, Wang X. The emerging roles of KPNA2 in cancer. Life Sci. proliferation in hepatocellular carcinoma cells. Aging. 2019;11:7123–49. 2020;241:117140. 18. Zhang Y, Chen H, Zhu H, Sun X. CBX8 promotes tumorigenesis and 33. Alshareeda AT, Negm OH, Green AR, Nolan CC, Tighe P, Albarakati N, et al. confers radioresistance in esophageal squamous cell carcinoma cells KPNA2 is a nuclear export protein that contributes to aberrant localisa- through targeting APAF1. Gene. 2019;711:143949. tion of key proteins and poor prognosis of breast cancer. Br J Cancer. 19. Yuan GJ, Chen X, Lu J, Feng ZH, Chen SL, Chen RX, et al. Chromobox 2015;112:1929–37. homolog 8 is a predictor of muscle invasive bladder cancer and 34. Zhu Z, Wang H, Wei Y, Meng F, Liu Z, Zhang Z. Downregulation of PRDM1 promotes cell proliferation by repressing the p53 pathway. Cancer Sci. promotes cellular invasion and lung cancer metastasis. Tumour Biol. 2017;108:2166–75. 2017;39:1010428317695929. 20. Li Y, Zheng F, Xiao X, Xie F, Tao D, Huang C, et al. CircHIPK3 sponges miR- 35. Liu C, Banister CE, Weige CC, Altomare D, Richardson JH, Contreras CM, 558 to suppress heparanase expression in bladder cancer cells. EMBO et al. PRDM1 silences stem cell-related genes and inhibits prolif- Rep. 2017;18:1646–59. eration of human colon tumor organoids. Proc Natl Acad Sci USA. 21. Beguelin W, Teater M, Gearhart MD, Calvo Fernandez MT, Goldstein RL, 2018;115:E5066–75. Cardenas MG, et al. EZH2 and BCL6 cooperate to assemble CBX8-BCOR 36. Li S, Xu X, Xu X, Hu Z, Wu J, Zhu Y, et al. MicroRNA-490-5p inhibits complex to repress bivalent promoters, mediate germinal center forma- proliferation of bladder cancer by targeting c-Fos. Biochem Biophys Res tion and lymphomagenesis. Cancer Cell. 2016;30:197–213. Commun. 2013;441:976–81. 22. Stelma T, Chi A, van der Watt PJ, Verrico A, Lavia P, Leaner VD. Targeting 37. Lan G, Yang L, Xie X, Peng L, Wang Y. MicroRNA-490-5p is a novel tumor nuclear transporters in cancer: diagnostic, prognostic and therapeutic suppressor targeting c-FOS in human bladder cancer. Arch Med Sci. potential. IUBMB Life. 2016;68:268–80. 2015;11:561–9. 23. Jensen JB, Munksgaard PP, Sorensen CM, Fristrup N, Birkenkamp- 38. Long Y, Wu Z, Yang X, Chen L, Han Z, Zhang Y, et al. MicroRNA-101 inhibits Demtroder K, Ulhoi BP, et al. High expression of karyopherin-alpha2 the proliferation and invasion of bladder cancer cells via targeting c-FOS. defnes poor prognosis in non-muscle-invasive bladder cancer and in Mol Med Rep. 2016;14:2651–6. patients with invasive bladder cancer undergoing radical cystectomy. Eur Urol. 2011;59:841–8. 24. Shi B, Su B, Fang D, Tang Y, Xiong G, Guo Z, et al. High expression of Publisher’s Note KPNA2 defnes poor prognosis in patients with upper tract urothelial Springer Nature remains neutral with regard to jurisdictional claims in pub- carcinoma treated with radical nephroureterectomy. BMC Cancer. lished maps and institutional afliations. 2015;15:380.

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