Reciprocal regulation of CIP2A and AR expression in prostate cancer cells

Reciprocal regulation of CIP2A and AR expression in prostate cancer cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Reciprocal regulation of CIP2A and AR expression in prostate cancer cells Hao-Wen Chuang, Jian-Hua Pan, Yi-Xuan Cai, Darius Rupa, Ting-Syuan Huang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1856958/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein overexpressed in human malignancies, including prostate cancer (PCa). In this study, we aimed to explore the oncogenic function of CIP2A in PCa cells and its underlying mechanism. We showed that 63.3% (38/60 cases) of PCa tissues exhibited a high CIP2A immunostaining, compared to 25% (3/12 cases) of BPH samples. Furthermore, CIP2A expression was positively correlated with patients’ short survival time and nuclear AR levels in PCa tissues. Compared to PZ-HPV-7, an immortalized prostate cell line, androgen-sensitive LNCaP C-33, androgen-independent LNCaP C-81, or 22Rv1 cells exhibited a high CIP2A expression, associated with high AR expression and phosphorylation. While AR expression and activity modulated CIP2A expression, manipulating CIP2A expression in PCa cells regulated their AR expression and proliferation. The reduction of CIP2A expression also enhanced the sensitivity of PCa cells toward Enzalutamide treatment. Our data further showed that depletion of polo-kinase 1 (PLK1) expression or activity in C-81 or 22Rv1 cells caused reduced expression of c-Myc and AR. Notably, inhibition of PLK1 activity could abolish CIP2A-promoted expressions in c-Myc, AR, and prostate-specific antigen (PSA) in C-33 cells under an androgen-deprived condition, suggesting the role of PLK1 activity in CIP2A-promoted AR expression. In summary, our data showed the existence of a novel regulation between CIP2A and AR expression, which is critical for promoting PCa malignancy. Thus, CIP2A could serve as a therapeutic target for PCa. Prostate cancer CRPC CIP2A AR PLK1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Prostate cancer (PCa) is the most commonly diagnosed cancer among men in the developed world. Like normal prostate cells, most PCa cells require androgens for their growth and survival. Thus, androgen deprivation therapy (ADT) which aims to reduce androgen levels or block the activity of androgen receptor (AR) becomes the standard treatment for locally advanced or metastatic PCa. Most tumors initially respond to ADT but later are refractory to hormonal therapies. Within 2–3 years, the diseases eventually develop the castration-resistant PCa (CRPC), resulting in tumor relapse and cancer-related death [ 1 ]. Until recently, therapeutic options for CRPC are limited and provide a minimal increase in overall survival. Thus, it is urgently needed to develop suitable therapies for patients with CRPC. The AR is a steroid hormone receptor that functions as a ligand-regulated transcription factor. Upon activation by androgens, AR dimerizes as a homodimer and subsequently binds to androgen-responsive elements (ARE) on target genes, activating gene expression for regulating the growth and survival of prostate epithelial cells. However, deregulated AR expression or activity plays a key role in the development of CRPC. Accumulated evidence demonstrates that the majority of CRPC is still dependent on the AR signaling and exhibits a reactivated AR even under castration. Numerous mechanisms have been identified to drive the progression from androgen-dependent to CRPC [ 2 ]. Interestingly, many studies have shown that progression of CRPC is associated with AR overexpression. However, only 10–20% of CRPC exhibits AR gene amplification [ 3 ]. It indicates that increased AR expression in CRPC may be due to the mechanism other than gene amplification, such as activated transcription factors. Besides, multiple signaling pathways that activate different protein kinases promote AR transcriptional activity by serine/threonine phosphorylation [ 4 ]. CIP2A, encoded by the KIAA1524 gene, is an oncoprotein overexpressing approximately 39–90% of malignant tissues. Its expression is correlated with aggressive disease and the poor survival rate [ 5 ]. Knockdown of CIP2A expression in various cancer cells inhibits the growth of xenografted tumors [6; 7]. Accumulated evidence indicates that CIP2A executes its oncogenic function by suppressing the phosphatase activity of protein phosphatase 2A (PP2A). CIP2A could directly bind to PP2A and inhibition of CIP2A leads to enhanced PP2A activity [ 6 ]. Thus, CIP2A promotes the activities of various kinases and transcription factors by keeping them away from PP2A-mediated dephosphorylation and degradation. It is well-characterized that CIP2A interacts with c-Myc to prevent PP2A activity toward c-Myc serine 62, stabilizing c-Myc against degradation [ 6 ]. Furthermore, CIP2A regulates polo-like kinase 1 (PLK1) stability and activity, facilitating cell-cycle progression and tumor development [ 8 ]. In PCa cells, CIP2A is highly expressed in patient specimens of hormone-naïve PCa and CRPC [9; 10]. Knockdown of CIP2A expression in LNCaP cells led to reduced cell viability and colony formation [ 9 ]. However, the role of CIP2A in facilitating castration resistance in PCa cells and its underlying molecular mechanism is mostly unknown. In this study, we investigated the novel regulatory mechanism between CIP2A and AR in PCa cells. We showed that positive correlation exhibited between CIP2A and AR expression in PCa tumors. We further provided evidence that AR signaling could transcriptionally regulate CIP2A expression. Importantly, CIP2A modulated AR expression required the activity of PLK1. Our data demonstrated the oncogenic role of CIP2A in promoting AR expression, which potentially serves as a therapeutic target for CRPC treatment. Materials And Methods Antibodies and chemicals Antibodies against CIP2A, PP2Ac, PSA, or β-actin and horseradish peroxidase (HRP)-conjugated secondary antibodies were from Santa Cruz Biotechnology (Dallas, Texas, USA). Antibodies against PLK1, phospho-c-Myc (Ser62), or c-Myc were purchased from Cell Signaling (Danvers, MA, USA). Antibodies against phospho-AR (Ser81) or AR were obtained from Millipore (Burlington, MA, USA). Enzalutamide was obtained from MedChemExpress (Monmouth Junction, NJ, USA). LB100 and BI6727 were purchased from BioVision (Milpitas, CA, USA). Okadaic acid, TD52, Bicalutamide, and other chemicals were obtained from Sigma-Aldrich (Burlington, MA, USA). Cell culture Cell culture media, FBS, charcoal/dextran-treated FBS (c-FBS), and supplements were purchased from ThermoFisher Scientific (Waltham, MA, USA). Immortalized prostate cell line PZ-HPV-7 cells, androgen-sensitive LNCaP (passage 28), and androgen-independent 22Rv1 prostate carcinoma cell lines were purchased from the Biosource Collection and Research Center (BCRC, Hsinchu, Taiwan) and cultured as described previously [11]. Cells were fed twice and split once weekly with trypsinization defined as one passage. LNCaP cells with passage numbers less than 33 were designated as LNCaP C-33. The passage numbers of LNCaP cells over 80 were designated as LNCaP C-81 [12], obtained from Dr. Ming-Shyue Lee at National Taiwan University (Taipei, Taiwan). To reduce androgen effects on cell culture, a steroid-reduced (SR), i.e., phenol red-free RPMI-1640 medium supplemented with 5% charcoal/dextran-treated FBS, 1% glutamine, and 0.5% gentamicin, was used. Construction of KIAA1524 expression vector and lentiviral infection For constructing the KIAA1524 expression vector, the full-length human KIAA1524 cDNA flanked by att L sequences and cloned in the pENTR223.1 vector was purchased from MyBioSource (San Diego, CA, USA). Using the Gateway LR Clonase II enzyme mix kit (ThermoFisher Scientific; Waltham, MA, USA), the KIAA1524 cDNA was shuttled from the pENTR223.1 vector to the att R-contained pLX302 lentiviral vector (Academia Sinica; Taipei, Taiwan). For gene overexpression or knockdown experiments, the production or infection of vector-contained lentiviruses was performed as described previously [13]. Real-time PCR Total RNA content was isolated from cells using an RNA purification kit (Geneaid; Taipei, Taiwan), and cDNA was synthesized using a GoScript reverse transcription system (Promega; Madison, WI, USA), following the manufacturer’s protocols. All real-time PCR reactions were conducted by the Bio-Rad CFX96 Real-time PCR Detection System (Hercules, CA, USA). The amplification was conducted using the iQ SYBR Green Supermix (Bio-Rad). The thermal cycling conditions were 30 cycles (94°C for 1 min, 60°C for 1 min, and 72°C for 2 min). The paired primers used for AR and GAPDH were AR (forward): 5’-CCTGGCTTCCGCAACTTACAC-3’; AR (reverse): 5’-GGACTTGTGCATGCGGTACTC-3’; GAPDH (forward): 5’-TGGTATCGTGGAAGGACTCATGAC-3’; and GAPDH (reverse): 5’-TGCCAGTGAGCTTCCCGTTCAGC-3’. These primers were designed according to the database of RTPrimerDB [14]. Cell proliferation assay The virus-infected or inhibitor-treated cells with a density of 0.7-1x10 5 cells/well were seeded onto 24-well plates. After incubation for 48 hours, one set of attached cells was harvested and counted as day 0. The remaining cells were fed with fresh medium, and the cell number was counted on day 3. Cell lysis and immunoblotting The experimental procedure was described previously [13]. Briefly, an aliquot of cell lysates in the SDS-PAGE sample buffer was separated by electrophoresis and then transferred to a nitrocellulose membrane for immunoblotting. The membrane was then blocked with 5% nonfat milk in TBST and incubated with primary antibodies overnight at 4°C. After rinsing, the membrane was incubated with HRP-conjugated secondary antibodies for 1 hour at RT. The specific protein was then detected by an ECL reagent kit (PerkinElmer; Waltham, MA, USA). Patient samples Tissue samples, including 12 benign prostatic hyperplasia (BPH) and 60 PCa, were collected from patients who underwent prostatectomy and whose personal information was de-identified between 2003 and 2006 at Kaohsiung Veterans General Hospital (Kaohsiung, Taiwan). All specimens from the archival files and reviewed by two experienced pathologists. None of the patients had received chemotherapy, hormonotherapy, or radiotherapy before surgery. The study protocol was approved by the Institutional Review Board of Kaohsiung Veterans General Hospital (#KSVGH21-CT5-01) in accordance with the IRB’s guidelines and regulations. TMA construction and immunohistochemistry The tissue microarrays (TMA) were prepared from the formalin-fixed paraffin-embedded tissue blocks using a manual TMA Arrayer MTA-1 (Beecher Instruments; Sun Prairie, WA, USA). Immunohistochemical (IHC) procedures were conducted following the manufacturer’s protocol of the Bond III Autostainer (Leica Biosystems; Wetzlar, Germany). Briefly, tissue sections were deparaffinized and rehydrated. Subsequently, the slides were immersed in ethylenediaminetetraacetic acid buffer (pH 9.0) for 40 min. The sections were then incubated with an anti-CIP2A (clone 2G10; Novus Biologicals, Littleton, CO, USA) at 1:100 dilution or an anti-AR (clone AR27; Leica Biosystems, Wetzlar, Germany) at 1:50 dilution at RT for 30 min. The signal was amplified using a BOND Polymer Refine Detection kit (Leica Biosystems). The expression of CIP2A and nuclear AR in the prostate epithelia was evaluated independently by two experienced pathologists who scored using a semi-quantitative H-score method, which considered both the staining intensity and the percentage of positively stained cells. The intensity of the staining was divided into three grades: negative (0), weak (1+), moderate (2+), and strong (3+). The percentage (0 to 100%) of staining was determined and a total score ranging from 0 to 300 was obtained [15]. Statistical analysis The MedCalc version 19.1.6 (MedCalc Software; Ostend, Belgium) and PRISM version 5.0 (Graphpad Software; San Diego, CA, USA) were used for statistical analyses. A median cut-off immunoscore value of 160 was used to separate cases into low and high CIP2A immunoexpression. A student’s two-tailed t-test or a one-way ANOVA was applied to determine the significance between groups. Fisher’s exact or chi-square test was applied to evaluate associations between categorical variables. Spearman’s correlation was used to assess the correlation between variables. The Kaplan–Meier method was used to analyze overall survival (OS), and the log-rank test was used to compare groups. A p - value of <0.05 was considered significant. Results The expression of CIP2A is correlated with AR expression in PCa tumors We initially examined CIP2A expression in PCa tissue arrays containing 12 BPH and 60 PCa specimens. The representative images showed that the PCa tissue exhibited cytoplasmic CIP2A immunopositivity in the epithelial cells with a strong (3+) staining, whereas the BPH sample only showed a weak (1+) immunoreactivity (Fig. 1 A). Figure 1 B showed that CIP2A expression was significantly higher in the PCa tissues than in BPH specimens (p = 0.023). Among 60 PCa tissues, 57 cases (95%) exhibited CIP2A immunopositivity, and 38 cases (63.3%) displayed high CIP2A immunoexpression (Table 1 ). Table 2 summarizes the intensity data of CIP2A immunostaining in arrayed tissues. Despite no significant associations with pT stage, lymph node metastasis, lymphovascular and perineural invasion, or PIN, CIP2A expression was significantly associated with Gleason score in PCa tumors ( p = 0.032). Furthermore, high expression of CIP2A immunoscore was significantly correlated with a short survival time (p = 0.0339, log-rank test) (Fig. 1 C). Similarly, results from the TGCA database showed that the PCa patients with a high expression of the KIAA1524 gene with a low ratio of progression-free survival (PFS) (p = 0.0413) (Fig. 1 D). We further determined the correlation between CIP2A and nuclear AR expression in arrayed PCa tissues. The representative images in Fig. 1 E showed the consistency of CIP2A and AR immunoreactivities in the same tissues, and the scatter plot in Fig. 1 F indicated a positive correlation between CIP2A and nuclear AR expression (r = 0.641, p < 0.0001). Results from the TGCA database ( http://ualcan.path.uab.edu/index.html ) further indicated a positive correlation between the KIAA524 and AR genes in PCa samples with the Pearson correlation coefficient (γ) = 0.6 (Fig. 1 G) [ 16 ]. The data collectively suggested that CIP2A was highly expressed in PCa tissues, and its expression was associated with AR expression in PCa tumors. Table 1 Expression of CIP2A in BPH and PCa tissues n (100%) Low (%) High (%) p value BPH 12 9 (75) 3 (25) 0.023 * PCa 60 22 (36.7) 38 (63.3) * p < 0.05; BPH, benign prostatic hyperplasia; PCa, prostate cancer Table 2 Expression of CIP2A expression and clinicopathological parameters in PCa tissues Characteristics n (100%) Low (%) High (%) p value Gleason score 0.032 * 6 16 2 (12.5) 14 (87.5) >6 44 20 (45.5) 24 (54.5) pT stage 0.285 pT2 31 9 (29.0) 21 (71.0) pT3-4 29 13 (44.8) 16 (55.2) LNMets 1.000 pos 3 1 (33.3) 2 (66.7) neg 57 21 (36.8) 36 (63.2) LVI 0.102 pos 12 7 (58.3) 5 (41.7) neg 48 15 (31.2) 33 (68.7) PNI 0.766 pos 45 16 (35.6) 29 (64.4) neg 15 6 (40.0) 9 (60.0) * p <0.05; LNMets, lymph node metastasis; LVI, lymphovascular invasion; PNI, perineural invasion. The expression and activity of AR modulated CIP2A expression Next, we examined whether AR signaling could regulate CIP2A expression. We analyzed the expression or phosphorylation levels of AR and CIP2A in an immortalized prostate cell line PZ-HPV-7 (PZ), an androgen-sensitive LNCaP C-33 (C-33), and two androgen-independent PCa cell lines, i.e., LNCaP C-81 (C-81) and 22Rv1 [12; 17]. Compared to PZ-HPV-7 cells, C-33, C-81, and 22Rv1 cells exhibited higher expression or phosphorylation of AR and CIP2A. Moreover, the expression or phosphorylation levels of AR and CIP2A in C-81 cells were much higher than those in C-33 cells (Fig. 2 A). Under a steroid-reduced (SR) condition, C-81 cells exhibited higher expression or phosphorylation levels of AR and CIP2A than C-33 cells. Treatment of 10 nM DHT greatly enhanced the expression of AR and CIP2A in C-33 cells but did not affect the level of CIP2A in C-81 cells (Fig. 2 B). The stimulating effects of DHT on the expression of CIP2A and prostate-sepcific antigen (PSA) in C-33 cells can be abolished by bicalutamide, an antiandrogen, following a dose-dependent manner (Fig. 2 C). Results from the quantitative real-time PCR analyses showed that knockdown of AR expression in C-33 cells caused significant decreases in the mRNA levels of AR, CIP2A, and PSA, suggesting the functional role of AR in the transcriptional regulation of CIP2A expression (Fig. 2 D). The knockdown of AR expression in C-81 cells also caused decreases in the protein levels of CIP2A and PSA (Fig. 2 E). Treatment of Enzalutamide, a second-generation nonsteroidal antiandrogen, in C-81 cells resulted in reduced AR expression and phosphorylation, correlating with decreased CIP2A and PSA in a dosage-dependent manner (Fig. 2 F). These results indicated that AR played a critical role in regulating CIP2A expression in PCa cells. The expression of CIP2A regulated AR expression and cell proliferation We next examined whether CIP2A could regulate the AR expression and cell proliferation in PCa cells. As shown in Fig. 3 A, increased expression of CIP2A in PZ-HPV-7 cells led to elevated AR expression and phosphorylation and increased cell proliferation. Knockdown of CIP2A expression in C-33 cells caused significant decreases in AR mRNA levels (Fig. 3 B), suggesting the role of CIP2A in the transcriptional regulation of AR expression. In addition, CIP2A-knockdown C-33 or 22Rv1 cells exhibited diminished AR protein levels and reduced cell proliferation (Fig. 3 C & 3 D). We further treated C-81 cells with different dosages of TD52, a CIP2A inhibitor [ 18 ]. As shown in Fig. 3 E, the treatment of TD52 caused decreased CIP2A expression, correlating with reduced levels of AR and PSA in a dose-dependent manner. We further examined whether reducing CIP2A expression might enhance the sensitization of C-81 or 22Rv1 cells to Enzalutamide. As shown in Fig. 3 F and 3 G, the treatment of Enzalutamide alone did not affect cell proliferation. However, the co-treatment of Enzalutamide and TD52 led to significant decreases in cell proliferation. These data suggested that CIP2A could modulate PCa cells in AR expression, cell proliferation, and sensitivity toward antiandrogen treatment. Decreased PP2A activity or expression caused reduced expression of c-Myc and AR Because CIP2A has been shown to inhibit PP2A, thus promoting the stability and activity of c-Myc [ 6 ], a transcription factor involved in modulating AR gene expression [ 19 ], we speculated that CIP2A-promoted AR expression was mediated by suppressing PP2A activity. Surprisingly, the inhibition of PP2A activity in C-33 cells by treating cells with LB100, a specific PP2A inhibitor [ 20 ], or okadaic acid (OA), a nonspecific PP1 and PP2A inhibitor [ 21 ], was unable to enhance the expression or phosphorylation of c-Myc and AR. In contrast, it caused decreased expression and phosphorylation of c-Myc and AR, following a dose-dependent manner (Fig. 4 A). We further knocked down the expression of PP2A in C-33 or 22Rv1 cells. As shown in Fig. 4 B and 4 C, knockdown of PP2Ac, the catalytic subunit of PP2A, caused reduced expression or phosphorylation of c-Myc and AR. Our data clearly showed that inhibition of PP2A activity or expression failed to promote c-Myc or AR phosphorylation and expression. Thus, CIP2A-promoted AR expression was independent of PP2A inhibition. PLK1 was involved in CIP2A-promoted AR expression We next examined the role of polo-like kinase 1 (PLK1), a serine/threonine kinase, in mediating CIP2A-promoted AR expression. As shown in Fig. 5 A, the knockdown of CIP2A expression in C-81 cells led to a decrease in PLK1 expression, suggesting the role of CIP2A in regulating PLK1 expression. We further examined whether PLK1 could regulate c-Myc or AR expression in PCa cells. As shown in Fig. 5 B, knockdown of PLK1 expression in 22Rv1 cells resulted in reduced phosphorylation or expression of c-Myc and AR. Moreover, treatment of BI6727 (volasertib), a PLK1 inhibitor [ 22 ], led to decreases in the expression and phosphorylation of c-Myc and AR, following a dose-dependent manner (Fig. 5 C). We next examined whether PLK1 is required for CIP2A-modulated AR expression. Results from Fig. 5 D showed that overexpression of CIP2A in C-33 cells caused increases in the expression and phosphorylation of c-Myc, AR, or PSA under an androgen-depleted condition. Notably, CIP2A-promoted effects were abolished by BI6727 treatment following a dose-dependent manner. These results indicated that CIP2A could modulate PLK1 expression or activity, which was required for CIP2A-promoted AR expression. Discussion CIP2A is a well-known oncoprotein in various cancers. Its oncogenic function in modulating castration resistance in PCa cells has not been studied. In this study, we showed a positive correlation between CIP2A and AR expression in PCa tumors. While AR signaling could modulate CIP2A expression, CIP2A reciprocally regulated AR expression in mRNA and protein levels. Importantly, our data showed the CIP2A-modulated AR expression required the activity of PLK1. To our knowledge, this is the first report showing the novel regulation between CIP2A and AR expression in PCa cells, which may contribute to the castration resistance of PCa cells. Based on the TGCA database, no difference is found in the expression of KIAA1524 gene between normal and PCa samples. However, lines of evidence indicated high CIP2A expression in PCa tumors, correlating with high Gleason scores [10; 23]. In concordance with the findings, our data showed the high expression of CIP2A in PCa tumors compared to BPH tissues, which was significantly associated with high Gleason scores. Importantly, our results showed that CIP2A expression was positively correlated with AR expression level in PCa tumors. The cell-line study supported this notion that CIP2A expression was higher in PCa cells than immortalized prostate cells, correlating with their AR expression levels. Interestingly, the expression level of CIP2A in LNCaP C-81 cells and 22Rv1, two androgen-independent cell lines, is even higher than that in androgen-sensitive C-33 cells, similar to the observation in clinical specimens [ 9 ]. The high expression of CIP2A in advanced PCa might be due to activated AR signaling. Indeed, Khanna and his colleagues [ 9 ] first showed that increased AR expression and activity enhance CIP2A expression in LNCaP cells. In contrast, knockdown of CIP2A expression in LNCaP or VCaP cells leads to reduced CIP2A expression. Our data further revealed that AR expression was critical for the mRNA expression of CIP2A. Although the detailed mechanism of how AR regulates CIP2A requires further investigation, overexpressed or activated AR in CRPC cells indirectly activates the function of transcription factors, such as ETS1 [24; 25], ELK1 [ 24 ], and E2F1 [ 26 ], to interact with the CIP2A promoter region and enhanced its expression. Alternatively, overexpressed or activated AR could directly bind to CIP2A intronic region and regulate CIP2A expression [ 9 ]. AR gene amplification or protein overexpression is commonly found in CRPC patients [ 27 ]. Increased AR expression in hormone-sensitive cells enhances their resistance to the antiandrogens [ 28 ]. Our data showed that CIP2A could regulate AR expression at mRNA and protein levels. In addition, changes in CIP2A expression affected the response of PCa cells toward androgens or antiandrogens. These results might be caused by the fact that CIP2A-promoted AR expression sensitized the PCa cells to respond to a lower concentration of androgens [ 29 ]. However, the critical question is how CIP2A regulates AR expression? Although studies have suggested that PP2A negatively regulates AR activity or expression [ 30 ], our data clearly showed that depletion of PP2A activity or expression caused reduced AR expression instead of enhancing its expression. This notion is supported by the observation that treatment of okadaic acid in LNCaP and 22Rv1 caused decreases in their AR levels [ 31 ]. Thus, CIP2A-promoted AR expression in PCa cells was mediated by a PP2A-independent mechanism. Our results clearly showed that CIP2A could modulate PLK1 expression in immortalized prostate and PCa cells. The functional linkage between CIP2A and PLK1 was further supported by the observation that CIP2A interacts with the polo-box domain of PLK1, which maintains PLK1 stability and enhances its kinase activity [ 8 ]. PLK1 is highly expressed in PCa tissues and is linked to higher-grade tumors [ 32 ]. Treatment of PLK1 inhibitor caused reduced AR expression and inhibited tumor growth in LNCaP CRPC xenografts [ 33 ]. In vitro kinase assays further showed that PLK1 binds to c-Myc and phosphorylates it at serine 62, promoting c-Myc protein stability [ 34 ]. In agreement with these findings, our data showed that PLK1 silencing or inhibitor treatment in C-81 or 22Rv1 cells caused reduced expression or phosphorylation of c-Myc and AR. Furthermore, CIP2A-promoted expressions in c-Myc, AR, and PSA were abolished by BI6727 treatment under an androgen-deprived condition, suggesting that PLK1 was required for CIP2A-promoted AR expression. Since CIP2A could regulate AR at the transcriptional level, it is possible that CIP2A promoted PLK1 stability and activity, which in turn enhanced c-Myc transcriptional activity and caused increased AR gene transcription. Alternatively, CIP2A-activated PLK1 may enhance AKT activity and leads to AKT-mediated Twist1 phosphorylation and nuclear accumulation. The activated Twist1 further increases AR expression through binding to E-boxes in the AR promoter [ 35 ]. In summary, we have demonstrated that CIP2A was highly expressed in PCa tumors, associated with AR expression. While AR signaling could upregulate CIP2A expression in an AR-dependent manner, CIP2A inversely modulated AR expression. The underlying mechanism was not mediated by PP2A inhibition but was associated with PLK1 activity. Thus, our data supported the oncogenic role of CIP2A in promoting AR expression and castration-resistant growth in PCa cells, potentially serving as a therapeutic target for CRPC treatment. Abbreviations PCa, prostate cancer; BPH, benign prostatic hyperplasia; CRPC, castration-resistant prostate cancer; AR, androgen receptor; CIP2A, cancerous inhibitor of protein phosphatase 2A; PP2A, protein phosphatase 2A; PLK1, polo-like kinase 1; RT, room temperature. Declarations Funding This work was supported by a grant from the Ministry of Science and Technology in Taiwan (NSC-103-2314-B-259-001). Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions Chuang HW and Yuan TC designed the study, analyzed the results, and wrote the paper. Chuang HW, Lin CC, and Lee HS executed tissue sample collection, IHC staining, and clinicopathological analyses. Pan JH, Cai YX, Rupa D, Huang TS, Kuo TC, and Lin CW conducted the in vitro studies. Chen CW helped to analyze the bioinformatic dataset Data Availability The data sets used and/or analysed during the current study are available from the corresponding author upon reasonable request. Ethics statement This study protocol was approved by the Institutional Review Board of Kaohsiung Veterans General Hospital (#KSVGH21-CT5-01) in accordance with the IRB’s guidelines and regulations. References Imamura Y, Sadar MD. (2016) Androgen receptor targeted therapies in castration-resistant prostate cancer: Bench to clinic. Int J Urol 23:654 – 65. https://doi.org/10.1111/iju.13137 . Huang Y, Jiang X, Liang X, et al. Molecular and cellular mechanisms of castration resistant prostate cancer. Oncol Lett. 2018;15:6063–76. https://doi.org/10.3892/ol.2018.8123 . Shiota M, Yokomizo A, Naito S. Increased androgen receptor transcription: a cause of castration-resistant prostate cancer and a possible therapeutic target. J Mol Endocrinol. 2011;47:R25–41. https://doi.org/10.1530/JME-11-0018 . Koryakina Y, Ta HQ, Gioeli D. Androgen receptor phosphorylation: biological context and functional consequences. Endocr Relat Cancer. 2014;21:T131-45. https://doi.org/10.1530/ERC-13-0472 . Khanna A, Pimanda JE. Clinical significance of cancerous inhibitor of protein phosphatase 2A in human cancers. Int J Cancer. 2016;138:525–32. https://doi.org/10.1002/ijc.29431 . Junttila MR, Puustinen P, Niemela M, et al. CIP2A inhibits PP2A in human malignancies. Cell. 2007;130:51–62. https://doi.org/10.1016/j.cell.2007.04.044 . Come C, Laine A, Chanrion M, et al. CIP2A is associated with human breast cancer aggressivity. Clin Cancer Res. 2009;15:5092–100. https://doi.org/10.1158/1078-0432.CCR-08-3283 . Kim JS, Kim EJ, Oh JS, et al. CIP2A modulates cell-cycle progression in human cancer cells by regulating the stability and activity of Plk1. Cancer Res. 2013;73:6667–78. https://doi.org/10.1158/0008-5472.CAN-13-0888 . Khanna A, Rane JK, Kivinummi KK, et al. CIP2A is a candidate therapeutic target in clinically challenging prostate cancer cell populations. Oncotarget. 2015;6:19661–70. https://doi.org/10.18632/oncotarget.3875 . Vaarala MH, Vaisanen MR, Ristimaki A. CIP2A expression is increased in prostate cancer. J Exp Clin Cancer Res. 2010;29:136. https://doi.org/10.1186/1756-9966-29-136 . Chang YL, Hsu YK, Wu TF, et al. Regulation of estrogen receptor alpha function in oral squamous cell carcinoma cells by FAK signaling. Endocr Relat Cancer. 2014;21:555–65. https://doi.org/10.1530/ERC-14-0102 . Igawa T, Lin FF, Lee MS, et al. Establishment and characterization of androgen-independent human prostate cancer LNCaP cell model. Prostate. 2002;50:222–35. https://doi.org/10.1002/pros.10054 . Hsiao YH, Huang YT, Hung CY, et al. PYK2 via S6K1 regulates the function of androgen receptors and the growth of prostate cancer cells. Endocr Relat Cancer. 2016;23:651–63. https://doi.org/10.1530/ERC-16-0122 . Pattyn F, Speleman F, De Paepe A, et al. RTPrimerDB: the real-time PCR primer and probe database. Nucleic Acids Res. 2003;31:122–3. https://doi.org/10.1093/nar/gkg011 . Miyamoto KK, McSherry SA, Dent GA, et al. Immunohistochemistry of the Androgen Receptor in Human Benign and Malignant Prostate Tissue. J Urol. 1993;149:1015–9. https://doi.org/10.1016/s0022-5347(17)36284-5 . Chandrashekar DS, Bashel B, Balasubramanya SAH, et al. UALCAN: A Portal for Facilitating Tumor Subgroup Gene Expression and Survival Analyses. Neoplasia. 2017;19:649–58. https://doi.org/10.1016/j.neo.2017.05.002 . Sramkoski RM, Pretlow TG 2nd, Giaconia JM, et al. A new human prostate carcinoma cell line, 22Rv1. Vitro Cell Dev Biol Anim. 1999;35:403–9. https://doi.org/10.1007/s11626-999-0115-4 . Yu HC, Hung MH, Chen YL, et al. Erlotinib derivative inhibits hepatocellular carcinoma by targeting CIP2A to reactivate protein phosphatase 2A. Cell Death Dis. 2014;5:e1359. https://doi.org/10.1038/cddis.2014.325 . Grad JM, Dai JL, Wu S, et al (1999) Multiple androgen response elements and a Myc consensus site in the androgen receptor (AR) coding region are involved in androgen-mediated up-regulation of AR messenger RNA. Mol Endocrinol 13:1896 – 911. https://doi.org/10.1210/mend.13.11.0369 ,Lee JG, Zheng R, McCafferty-Cepero JM, et al. (2009) Endothelin-1 enhances the expression of the androgen receptor via activation of the c-myc pathway in prostate cancer cells. Mol Carcinog 48:141-9. https://doi.org/10.1002/mc.20462. Wei D, Parsels LA, Karnak D, et al. Inhibition of protein phosphatase 2A radiosensitizes pancreatic cancers by modulating CDC25C/CDK1 and homologous recombination repair. Clin Cancer Res. 2013;19:4422–32. https://doi.org/10.1158/1078-0432.CCR-13-0788 . Dounay AB, Forsyth CJ. Okadaic acid: the archetypal serine/threonine protein phosphatase inhibitor. Curr Med Chem. 2002;9:1939–80. https://doi.org/10.2174/0929867023368791 . Yim H. Current clinical trials with polo-like kinase 1 inhibitors in solid tumors. Anticancer Drugs. 2013;24:999–1006. https://doi.org/10.1097/CAD.0000000000000007 . Celikden SG, Baspinar S, Ozturk SA, et al. CIP2A expression in high grade prostatic intraepithelial neoplasia and prostate adenocarcinoma: a tissue microarray study. Malays J Pathol. 2020;42:227–36. Pallai R, Bhaskar A, Sodi V, et al. Ets1 and Elk1 transcription factors regulate cancerous inhibitor of protein phosphatase 2A expression in cervical and endometrial carcinoma cells. Transcription. 2012;3:323–35. https://doi.org/10.4161/trns.22518 . Khanna A, Okkeri J, Bilgen T, et al. ETS1 mediates MEK1/2-dependent overexpression of cancerous inhibitor of protein phosphatase 2A (CIP2A) in human cancer cells. PLoS ONE. 2011;6:e17979. https://doi.org/10.1371/journal.pone.0017979 . Laine A, Sihto H, Come C, et al. (2013) Senescence sensitivity of breast cancer cells is defined by positive feedback loop between CIP2A and E2F1. Cancer Discov 3:182 – 97. https://doi.org/10.1158/2159-8290.CD-12-0292 . Hu R, Denmeade SR, Luo J. Molecular processes leading to aberrant androgen receptor signaling and castration resistance in prostate cancer. Expert Rev Endocrinol Metab. 2010;5:753–64. https://doi.org/10.1586/eem.10.49 . Chen CD, Welsbie DS, Tran C, et al. Molecular determinants of resistance to antiandrogen therapy. Nat Med. 2004;10:33–9. https://doi.org/10.1038/nm972 . Waltering KK, Helenius MA, Sahu B, et al. Increased expression of androgen receptor sensitizes prostate cancer cells to low levels of androgens. Cancer Res. 2009;69:8141–9. https://doi.org/10.1158/0008-5472.CAN-09-0919 . Bhardwaj A, Singh S, Srivastava SK, et al (2011) Modulation of protein phosphatase 2A activity alters androgen-independent growth of prostate cancer cells: therapeutic implications. Mol Cancer Ther 10:720 – 31. https://doi.org/10.1158/1535-7163 .MCT-10-1096,McClinch Avelar K, Callejas RA. D et al. (2018) Small-Molecule Activators of Protein Phosphatase 2A for the Treatment of Castration-Resistant Prostate Cancer. Cancer Res 78:2065-80. https://doi.org/10.1158/0008-5472.CAN-17-0123. Chen S, Kesler CT, Paschal BM, et al. Androgen receptor phosphorylation and activity are regulated by an association with protein phosphatase 1. J Biol Chem. 2009;284:25576–84. https://doi.org/10.1074/jbc.M109.043133 . Weichert W, Schmidt M, Gekeler V, et al. Polo-like kinase 1 is overexpressed in prostate cancer and linked to higher tumor grades. Prostate. 2004;60:240–5. https://doi.org/10.1002/pros.20050 . Zhang Z, Chen L, Wang H, et al (2015) Inhibition of Plk1 represses androgen signaling pathway in castration-resistant prostate cancer. Cell Cycle 14:2142-8. https://doi.org/10.1080/15384101.2015.1041689 ,Shin SB, Woo SU, Yim H. (2019) Cotargeting Plk1 and androgen receptor enhances the therapeutic sensitivity of paclitaxel-resistant prostate cancer. Ther Adv Med Oncol 11:1758835919846375. https://doi.org/10.1177/1758835919846375. Ren Y, Bi C, Zhao X, et al. PLK1 stabilizes a MYC-dependent kinase network in aggressive B cell lymphomas. J Clin Invest. 2018;128:5517–30. https://doi.org/10.1172/JCI122533 . Zhang Z, Hou X, Shao C, et al. Plk1 inhibition enhances the efficacy of androgen signaling blockade in castration-resistant prostate cancer. Cancer Res. 2014;74:6635–47. https://doi.org/10.1158/0008-5472.CAN-14-1916 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 11 Aug, 2022 Reviews received at journal 29 Jul, 2022 Reviewers agreed at journal 29 Jul, 2022 Reviewers agreed at journal 20 Jul, 2022 Reviewers invited by journal 18 Jul, 2022 Editor assigned by journal 18 Jul, 2022 Editor invited by journal 17 Jul, 2022 Submission checks completed at journal 17 Jul, 2022 First submitted to journal 14 Jul, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1856958","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":121783201,"identity":"14f31b96-68fa-468b-9db6-386f2da589f1","order_by":0,"name":"Hao-Wen Chuang","email":"","orcid":"","institution":"Kaohsiung Veterans General Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hao-Wen","middleName":"","lastName":"Chuang","suffix":""},{"id":121783202,"identity":"c0a58618-222a-460b-8646-96ff6defb16b","order_by":1,"name":"Jian-Hua Pan","email":"","orcid":"","institution":"National Dong Hwa University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jian-Hua","middleName":"","lastName":"Pan","suffix":""},{"id":121783203,"identity":"00fb8f61-9756-4c4a-afe0-9cd1d89f4d9f","order_by":2,"name":"Yi-Xuan Cai","email":"","orcid":"","institution":"National Dong Hwa University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yi-Xuan","middleName":"","lastName":"Cai","suffix":""},{"id":121783204,"identity":"ac8c9d95-b7f9-4a06-a870-2e408aeedda7","order_by":3,"name":"Darius Rupa","email":"","orcid":"","institution":"National Dong Hwa University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Darius","middleName":"","lastName":"Rupa","suffix":""},{"id":121783205,"identity":"0136cdfd-e10c-486d-93fc-49e5a56c5bb1","order_by":4,"name":"Ting-Syuan Huang","email":"","orcid":"","institution":"National Dong Hwa University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ting-Syuan","middleName":"","lastName":"Huang","suffix":""},{"id":121783206,"identity":"e37dceb6-dbdf-4665-a6a4-3af0e37d96bb","order_by":5,"name":"Tzu-Chien Kuo","email":"","orcid":"","institution":"National Dong Hwa University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tzu-Chien","middleName":"","lastName":"Kuo","suffix":""},{"id":121783207,"identity":"729f73c0-60d4-4a7a-bc49-bf1babb39e20","order_by":6,"name":"Chiao-Wen Lin","email":"","orcid":"","institution":"National Dong Hwa University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chiao-Wen","middleName":"","lastName":"Lin","suffix":""},{"id":121783208,"identity":"1713fe13-0384-4dd3-bfe0-21d096558cb5","order_by":7,"name":"Chi-Wei Chen","email":"","orcid":"","institution":"National Dong Hwa University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chi-Wei","middleName":"","lastName":"Chen","suffix":""},{"id":121783209,"identity":"2301f59f-8c44-4172-925f-919feb838620","order_by":8,"name":"Chia-Chin Lin","email":"","orcid":"","institution":"Kaohsiung Veterans General Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chia-Chin","middleName":"","lastName":"Lin","suffix":""},{"id":121783210,"identity":"e43bcef8-ea79-41f4-8770-91cca32fabb4","order_by":9,"name":"Herng-Sheng Lee","email":"","orcid":"","institution":"Kaohsiung Veterans General Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Herng-Sheng","middleName":"","lastName":"Lee","suffix":""},{"id":121783211,"identity":"5c01e4a6-5b1c-46c9-a1c1-c9afa757578a","order_by":10,"name":"Ta-Chun Yuan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYLACCYN/chAW2wEQaUBYi0XBAWM0LQkEtFR8OJDYQLQWgxvJzx7cMLiTPj/s+APmgrI7iQ3szdskGH8cxqMlzdxwhsGz3I23cwyYZ5x7ltjAc6xMgiEBn5YEM2kJA+bcjbNzGJh52w4nNkjkmAG13MajJf2b9B8D5nTD2ekPIFrk3xDSAjRTwuBwgrx0ggHUFh78WiTPvCkDakkz3CCdY3CY59xh4zaetGKLhLT/OLXwHU/fJiHxx0Zefnb6w8c8ZYdl+9kPb7zxwSYNpxaFAzAXAhlgNhuISMCpgYFBvgGdMQpGwSgYBaMAHQAAn2BZliix1q0AAAAASUVORK5CYII=","orcid":"","institution":"National Dong Hwa University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Ta-Chun","middleName":"","lastName":"Yuan","suffix":""}],"badges":[],"createdAt":"2022-07-14 07:59:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1856958/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1856958/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":24299904,"identity":"0511a05d-2593-42ee-870b-6cec590fae50","added_by":"auto","created_at":"2022-07-25 17:07:15","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":247007,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssociation between CIP2A and AR expression in PCa tumors. (A)\u003c/strong\u003e Hematoxylin-eosin (H\u0026amp;E) and cytoplasmic CIP2A immunostaining in the BPH (1+) or PCa (3+) specimen. Scale bar, 250 μm. \u003cstrong\u003e(B) \u003c/strong\u003eDifferential expression of CIP2A in BPH (n=12) and PCa tissues (n=60) (p=0.0113).\u003cstrong\u003e (C) \u003c/strong\u003eKaplan–Meier survival curve in subgroups of PCa patients with high (solid line) or low (dash line) expression of CIP2A (p=0.0339). \u003cstrong\u003e(D)\u003c/strong\u003e Kaplan–Meier analysis of Progression-free survival (PFS) based on clinical and molecular data for prostate cancer patients (TCGA Prostate Cancer, PRAD). The patients were stratified by the expression levels in their tumors of \u003cem\u003eKIAA1524 \u003c/em\u003e(p=0.0413).\u003cstrong\u003e (E)\u003c/strong\u003e The consistency of CIP2A and nuclear AR immunoreactivities in the same tissues. The representative images of negative (Neg), weak (1+), moderate (2+), or strong (3+) immunostaining for CIP2A and AR. Scale bar, 50 μm. \u003cstrong\u003e(F)\u003c/strong\u003e Positive correlation between CIP2A and AR expression in 60 PCa specimens. Correlation coefficients (r)=0.641, p\u0026lt;0.0001. \u003cstrong\u003e(G) \u003c/strong\u003eData were downloaded from the UALCAN database. Pearson’s correlation analysis showing gene expression of CIP2A and AR gene in TCGA database (Prostate Cancer, PRAD). Pearson’s correlation coefficient (r)=0.6.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1856958/v1/e853d25f8c8c39fe892bef74.jpg"},{"id":24299906,"identity":"a3072c1e-118a-4e15-a43f-7dcae22471ef","added_by":"auto","created_at":"2022-07-25 17:07:15","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":109051,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAR signaling modulated CIP2A expression in PCa cells. (A) \u003c/strong\u003eTotal lysates from PZ-HPV-7 (PZ), LNCaP C-33, C-81, and 22Rv1 cells are used for immunoblotting with antibodies against the phosphorylation level of AR at Ser81 (p-AR) and the expression levels of full-length AR (AR-FL) and CIP2A. \u003cstrong\u003e(B)\u003c/strong\u003e LNCaP C-33 or C-81 cells are cultured in a steroid-reduced (SR) medium for 48 hours and are then fed with fresh media containing 10 nM dihydrotestosterone (DHT). After additional 48 hours of incubation, total lysates are prepared for immunoblotting. \u003cstrong\u003e(C)\u003c/strong\u003e LNCaP C-33 cells are cultured in an SR medium for 48 hours and are then fed with fresh media containing 10 nM DHT without or with 1 μM or 10 μM bicalutamide (Bic). After 48 hours of incubation, total lysates are prepared for immunoblotting. \u003cstrong\u003e(D)\u003c/strong\u003e LNCaP C-33 or \u003cstrong\u003e(E)\u003c/strong\u003e C-81 cells are infected with viruses carrying the \u003cem\u003eLacZ\u003c/em\u003e- or\u003cem\u003e AR\u003c/em\u003e-targeted shRNA (i.e., shCt, shAR-1, or shAR-2). The cell lysates are prepared for real-time PCR or western blot analyses. Data are expressed as the mean ± SD from three independent experiments. Each set of experiments is conducted in duplicate. *p\u0026lt;0.05, **p\u0026lt;0.01 vs. the ratio of the corresponding shCt control cells.\u003cstrong\u003e (F)\u003c/strong\u003e LNCaP C-81 cells are incubated with 0.1 μM, 1 μM, or 5 μM of Enzalutamide (Enz) for 24 hours. Subsequently, the total lysates are prepared for immunoblotting. The expression level of CIP2A is quantified using ImageJ software and the relative expression ratio is normalized to the β-actin level.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1856958/v1/b59162ea8d8abb86e0a92639.jpg"},{"id":24299902,"identity":"310274d9-b61d-4bf8-ad28-5e4a6039893c","added_by":"auto","created_at":"2022-07-25 17:07:15","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":167663,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of CIP2A expression on the AR expression and proliferation in PCa cells. (A)\u003c/strong\u003e The vector- or CIP2A-expressed PZ-HPV-7 (PZ) cells are cultured for western blot analyses (upper panel) and cell proliferation assays (lower panel).\u003cstrong\u003e (B) \u003c/strong\u003eLNCaP C-33 cells are infected with viruses carrying the \u003cem\u003eLacZ\u003c/em\u003e- or \u003cem\u003eKIAA1524\u003c/em\u003e-targeted shRNAs (i.e., shCt, shCIP2A-1, or shCIP2A-2). The total lysates are prepared for real-time PCR. \u003cstrong\u003e(C) \u003c/strong\u003eLNCaP C-33 and \u003cstrong\u003e(D)\u003c/strong\u003e 22Rv1 cells infected with viruses carrying the shCt, shCIP2A-1, or shCIP2A shRNA are prepared for western blot analyses (upper panel) or cell proliferation assays (lower panel). \u003cstrong\u003e(E)\u003c/strong\u003e LNCaP C-81 cells are incubated with 5 μM, 7.5 μM, or 12.5 μM of TD52 for 24 hours. Subsequently, the total lysates are prepared for immunoblotting. The expression level of AR is quantified using ImageJ software and the relative expression ratio is normalized to the β-actin level. \u003cstrong\u003e(F)\u003c/strong\u003e C-81 or \u003cstrong\u003e(G)\u003c/strong\u003e 22Rv1 cells are treated without or with 5 μM Enzalutamide (Enz) in the absence or presence of 5 μM or 7.5 μM of TD52. After 72 hours of incubation, cells are harvested for cell proliferation assays. Data are expressed as the mean ± SD from three independent experiments. Each set of experiments is conducted in duplicate. *p\u0026lt;0.05, **p\u0026lt;0.01 ***p\u0026lt;0.001 vs. the ratio of the corresponding control cells. n.s.: not significant\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1856958/v1/7af1ad635d19ee6dccefa77e.jpg"},{"id":24299905,"identity":"0f8acd1d-4712-458f-bf6b-3b3bc435e924","added_by":"auto","created_at":"2022-07-25 17:07:15","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":89233,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of inhibiting or knocking down PP2Ac on the expression of c-Myc and AR in PCa cells. (A)\u003c/strong\u003e LNCaP C-33 cells are incubated with 5 μM or 10 μM of LB100, or 10 nM or 20 nM of okadaic acid (OA) for 24 hours. Subsequently, the total lysates are prepared for immunoblotting. \u003cstrong\u003e(B)\u003c/strong\u003e C-33 or \u003cstrong\u003e(C)\u003c/strong\u003e 22Rv1 cells infected with viruses carrying the \u003cem\u003eLacZ\u003c/em\u003e-targeted shRNA (shCt) or \u003cem\u003ePPP2CA\u003c/em\u003e-targeted shRNAs (shPP2A-1 or shPP2A-2) are harvested for western blot analyses.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1856958/v1/14d84fc0f9ff70c2f496660e.jpg"},{"id":24299903,"identity":"56ba7bca-d75d-454c-8c54-4086c69abb70","added_by":"auto","created_at":"2022-07-25 17:07:15","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":82129,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of PLK1 expression or activity on the CIP2A-regulated AR expression.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e The total lysates from\u003cstrong\u003e \u003c/strong\u003ethe shCt control and CIP2A-knockdown C-81 cells were prepared for western blot analyses.\u003cstrong\u003e (B)\u003c/strong\u003e 22Rv1 cells infected with viruses carrying the \u003cem\u003eLacZ\u003c/em\u003e-targeted shRNA (shCt) or \u003cem\u003ePLK1\u003c/em\u003e-targeted shRNAs (shPLK1-1 or shPLK1-2) were harvested for western blot analyses. \u003cstrong\u003e(C) \u003c/strong\u003eC-81 cells were treated with 0.5 μM, 1 μM, or 2.5 μM of BI6727. Cells treated with an equal volume of solvent (DMSO) served as the control. After 24 hours of incubation, the cell lysates were prepared for immunoblotting. \u003cstrong\u003e(D)\u003c/strong\u003e The CIP2A-overexpressed C-33 cells were cultured in steroid-reduced (SR) media and treated without or with 1 μM or 2.5 μM of BI6727 for 24 hours. The vector-expressed cells (vec) served as the control. Total cell lysates were then prepared for western blot analyses.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1856958/v1/da56cbdce961fc740bdeb757.jpg"},{"id":24299908,"identity":"c2939c79-7dd0-43ee-9787-8a0df840962c","added_by":"auto","created_at":"2022-07-25 17:07:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":806264,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1856958/v1/3efe820d-3585-4829-b20a-7f81593af355.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Reciprocal regulation of CIP2A and AR expression in prostate cancer cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eProstate cancer (PCa) is the most commonly diagnosed cancer among men in the developed world. Like normal prostate cells, most PCa cells require androgens for their growth and survival. Thus, androgen deprivation therapy (ADT) which aims to reduce androgen levels or block the activity of androgen receptor (AR) becomes the standard treatment for locally advanced or metastatic PCa. Most tumors initially respond to ADT but later are refractory to hormonal therapies. Within 2\u0026ndash;3 years, the diseases eventually develop the castration-resistant PCa (CRPC), resulting in tumor relapse and cancer-related death [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Until recently, therapeutic options for CRPC are limited and provide a minimal increase in overall survival. Thus, it is urgently needed to develop suitable therapies for patients with CRPC.\u003c/p\u003e \u003cp\u003eThe AR is a steroid hormone receptor that functions as a ligand-regulated transcription factor. Upon activation by androgens, AR dimerizes as a homodimer and subsequently binds to androgen-responsive elements (ARE) on target genes, activating gene expression for regulating the growth and survival of prostate epithelial cells. However, deregulated AR expression or activity plays a key role in the development of CRPC. Accumulated evidence demonstrates that the majority of CRPC is still dependent on the AR signaling and exhibits a reactivated AR even under castration. Numerous mechanisms have been identified to drive the progression from androgen-dependent to CRPC [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Interestingly, many studies have shown that progression of CRPC is associated with AR overexpression. However, only 10\u0026ndash;20% of CRPC exhibits \u003cem\u003eAR\u003c/em\u003e gene amplification [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. It indicates that increased AR expression in CRPC may be due to the mechanism other than gene amplification, such as activated transcription factors. Besides, multiple signaling pathways that activate different protein kinases promote AR transcriptional activity by serine/threonine phosphorylation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCIP2A, encoded by the \u003cem\u003eKIAA1524\u003c/em\u003e gene, is an oncoprotein overexpressing approximately 39\u0026ndash;90% of malignant tissues. Its expression is correlated with aggressive disease and the poor survival rate [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Knockdown of CIP2A expression in various cancer cells inhibits the growth of xenografted tumors [6; 7]. Accumulated evidence indicates that CIP2A executes its oncogenic function by suppressing the phosphatase activity of protein phosphatase 2A (PP2A). CIP2A could directly bind to PP2A and inhibition of CIP2A leads to enhanced PP2A activity [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Thus, CIP2A promotes the activities of various kinases and transcription factors by keeping them away from PP2A-mediated dephosphorylation and degradation. It is well-characterized that CIP2A interacts with c-Myc to prevent PP2A activity toward c-Myc serine 62, stabilizing c-Myc against degradation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Furthermore, CIP2A regulates polo-like kinase 1 (PLK1) stability and activity, facilitating cell-cycle progression and tumor development [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In PCa cells, CIP2A is highly expressed in patient specimens of hormone-na\u0026iuml;ve PCa and CRPC [9; 10]. Knockdown of CIP2A expression in LNCaP cells led to reduced cell viability and colony formation [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, the role of CIP2A in facilitating castration resistance in PCa cells and its underlying molecular mechanism is mostly unknown.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the novel regulatory mechanism between CIP2A and AR in PCa cells. We showed that positive correlation exhibited between CIP2A and AR expression in PCa tumors. We further provided evidence that AR signaling could transcriptionally regulate CIP2A expression. Importantly, CIP2A modulated AR expression required the activity of PLK1. Our data demonstrated the oncogenic role of CIP2A in promoting AR expression, which potentially serves as a therapeutic target for CRPC treatment.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eAntibodies and chemicals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAntibodies against CIP2A, PP2Ac, PSA, or \u0026beta;-actin and horseradish peroxidase (HRP)-conjugated secondary antibodies were from Santa Cruz Biotechnology (Dallas, Texas, USA). Antibodies against PLK1, phospho-c-Myc (Ser62), or c-Myc were purchased from Cell Signaling (Danvers, MA, USA). Antibodies against phospho-AR (Ser81) or AR were obtained from Millipore (Burlington, MA, USA). Enzalutamide was obtained from MedChemExpress (Monmouth Junction, NJ, USA). LB100 and BI6727 were purchased from BioVision (Milpitas, CA, USA). Okadaic acid, TD52, Bicalutamide, and other chemicals were obtained from Sigma-Aldrich (Burlington, MA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell culture media, FBS, charcoal/dextran-treated FBS (c-FBS), and supplements were purchased from ThermoFisher Scientific (Waltham, MA, USA). Immortalized prostate cell line PZ-HPV-7 cells, androgen-sensitive LNCaP (passage 28), and androgen-independent 22Rv1 prostate carcinoma cell lines were purchased from the Biosource Collection and Research Center (BCRC, Hsinchu, Taiwan) and cultured as described previously [11]. Cells were fed twice and split once weekly with trypsinization defined as one passage. LNCaP cells with passage numbers less than 33 were designated as LNCaP C-33. The passage numbers of LNCaP cells over 80 were designated as LNCaP C-81 [12], obtained from Dr. Ming-Shyue Lee at National Taiwan University (Taipei, Taiwan). To reduce androgen effects on cell culture, a steroid-reduced (SR), i.e., phenol red-free RPMI-1640 medium supplemented with 5% charcoal/dextran-treated FBS, 1% glutamine, and 0.5% gentamicin, was used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruction of \u003cem\u003eKIAA1524\u003c/em\u003e expression vector and lentiviral infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor constructing the \u003cem\u003eKIAA1524\u003c/em\u003e expression vector, the full-length human \u003cem\u003eKIAA1524\u003c/em\u003e cDNA flanked by \u003cem\u003eatt\u003c/em\u003eL sequences and cloned in the pENTR223.1 vector was purchased from MyBioSource (San Diego, CA, USA). Using the Gateway LR Clonase II enzyme mix kit (ThermoFisher Scientific; Waltham, MA, USA), the \u003cem\u003eKIAA1524\u003c/em\u003e cDNA was shuttled from the pENTR223.1 vector to the \u003cem\u003eatt\u003c/em\u003eR-contained pLX302 lentiviral vector (Academia Sinica; Taipei, Taiwan). For gene overexpression or knockdown experiments, the production or infection of vector-contained lentiviruses was performed as described previously [13].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReal-time PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA content was isolated from cells using an RNA purification kit (Geneaid; Taipei, Taiwan), and cDNA was synthesized using a GoScript reverse transcription system (Promega; Madison, WI, USA), following the manufacturer\u0026rsquo;s protocols. All real-time PCR reactions were conducted by the Bio-Rad CFX96 Real-time PCR Detection System (Hercules, CA, USA). The amplification was conducted using the iQ SYBR Green Supermix (Bio-Rad). The thermal cycling conditions were 30 cycles (94\u0026deg;C for 1 min, 60\u0026deg;C for 1 min, and 72\u0026deg;C for 2 min). The paired primers used for AR and GAPDH were AR (forward): 5\u0026rsquo;-CCTGGCTTCCGCAACTTACAC-3\u0026rsquo;; AR (reverse): 5\u0026rsquo;-GGACTTGTGCATGCGGTACTC-3\u0026rsquo;; GAPDH (forward): 5\u0026rsquo;-TGGTATCGTGGAAGGACTCATGAC-3\u0026rsquo;; and GAPDH (reverse): 5\u0026rsquo;-TGCCAGTGAGCTTCCCGTTCAGC-3\u0026rsquo;. These primers were designed according to the database of RTPrimerDB [14].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell proliferation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe virus-infected or inhibitor-treated cells with a density of 0.7-1x10\u003csup\u003e5\u003c/sup\u003e cells/well were seeded onto 24-well plates. After incubation for 48 hours, one set of attached cells was harvested and counted as day 0. The remaining cells were fed with fresh medium, and the cell number was counted on day 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell lysis and immunoblotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental procedure was described previously [13]. Briefly, an aliquot of cell lysates in the SDS-PAGE sample buffer was separated by electrophoresis and then transferred to a nitrocellulose membrane for immunoblotting. The membrane was then blocked with 5% nonfat milk in TBST and incubated with primary antibodies overnight at 4\u0026deg;C. After rinsing, the membrane was incubated with HRP-conjugated secondary antibodies for 1 hour at RT. The specific protein was then detected by an ECL reagent kit (PerkinElmer; Waltham, MA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissue samples, including 12 benign prostatic hyperplasia (BPH) and 60 PCa, were collected from patients who underwent prostatectomy and whose personal information was de-identified between 2003 and 2006 at Kaohsiung Veterans General Hospital (Kaohsiung, Taiwan). All specimens from the archival files and reviewed by two experienced pathologists. None of the patients had received chemotherapy, hormonotherapy, or radiotherapy before surgery. The study protocol was approved by the Institutional Review Board of Kaohsiung Veterans General Hospital (#KSVGH21-CT5-01) in accordance with the IRB\u0026rsquo;s guidelines and regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTMA construction and immunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tissue microarrays (TMA) were prepared from the formalin-fixed paraffin-embedded tissue blocks using a manual TMA Arrayer MTA-1 (Beecher Instruments; Sun Prairie, WA, USA). Immunohistochemical (IHC) procedures were conducted following the manufacturer\u0026rsquo;s protocol of the Bond III Autostainer (Leica Biosystems; Wetzlar, Germany). Briefly, tissue sections were deparaffinized and rehydrated. Subsequently, the slides were immersed in ethylenediaminetetraacetic acid buffer (pH 9.0) for 40 min. The sections were then incubated with an anti-CIP2A (clone 2G10; Novus Biologicals, Littleton, CO, USA) at 1:100 dilution or an anti-AR (clone AR27; Leica Biosystems, Wetzlar, Germany) at 1:50 dilution at RT for 30 min. The signal was amplified using a BOND Polymer Refine Detection kit (Leica Biosystems). The expression of CIP2A and nuclear AR in the prostate epithelia was evaluated independently by two experienced pathologists who scored using a semi-quantitative H-score method, which considered both the staining intensity and the percentage of positively stained cells. The intensity of the staining was divided into three grades: negative (0), weak (1+), moderate (2+), and strong (3+). The percentage (0 to 100%) of staining was determined and a total score ranging from 0 to 300 was obtained [15].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MedCalc version 19.1.6 (MedCalc Software; Ostend, Belgium) and PRISM version 5.0 (Graphpad Software; San Diego, CA, USA) were used for statistical analyses. A median cut-off immunoscore value of 160 was used to separate cases into low and high CIP2A immunoexpression. A student\u0026rsquo;s two-tailed t-test or a one-way ANOVA was applied to determine the significance between groups. Fisher\u0026rsquo;s exact or chi-square test was applied to evaluate associations between categorical variables. Spearman\u0026rsquo;s correlation was used to assess the correlation between variables. The Kaplan\u0026ndash;Meier method was used to analyze overall survival (OS), and the log-rank test was used to compare groups. A p\u003cem\u003e-\u003c/em\u003evalue of \u0026lt;0.05 was considered significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv class=\"Section2\" id=\"Sec12\"\u003e\n \u003cp\u003e\u003cstrong\u003eThe expression of CIP2A is correlated with AR expression in PCa tumors\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eWe initially examined CIP2A expression in PCa tissue arrays containing 12 BPH and 60 PCa specimens. The representative images showed that the PCa tissue exhibited cytoplasmic CIP2A immunopositivity in the epithelial cells with a strong (3+) staining, whereas the BPH sample only showed a weak (1+) immunoreactivity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB showed that CIP2A expression was significantly higher in the PCa tissues than in BPH specimens (p\u0026thinsp;=\u0026thinsp;0.023). Among 60 PCa tissues, 57 cases (95%) exhibited CIP2A immunopositivity, and 38 cases (63.3%) displayed high CIP2A immunoexpression (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Table\u0026nbsp;2 summarizes the intensity data of CIP2A immunostaining in arrayed tissues. Despite no significant associations with pT stage, lymph node metastasis, lymphovascular and perineural invasion, or PIN, CIP2A expression was significantly associated with Gleason score in PCa tumors (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.032). Furthermore, high expression of CIP2A immunoscore was significantly correlated with a short survival time (p\u0026thinsp;=\u0026thinsp;0.0339, log-rank test) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). Similarly, results from the TGCA database showed that the PCa patients with a high expression of the \u003cem\u003eKIAA1524\u003c/em\u003e gene with a low ratio of progression-free survival (PFS) (p\u0026thinsp;=\u0026thinsp;0.0413) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). We further determined the correlation between CIP2A and nuclear AR expression in arrayed PCa tissues. The representative images in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE showed the consistency of CIP2A and AR immunoreactivities in the same tissues, and the scatter plot in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF indicated a positive correlation between CIP2A and nuclear AR expression (r\u0026thinsp;=\u0026thinsp;0.641, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Results from the TGCA database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://ualcan.path.uab.edu/index.html\u003c/span\u003e\u003c/span\u003e) further indicated a positive correlation between the \u003cem\u003eKIAA524\u003c/em\u003e and \u003cem\u003eAR\u003c/em\u003e genes in PCa samples with the Pearson correlation coefficient (\u0026gamma;)\u0026thinsp;=\u0026thinsp;0.6 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eG) [\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e]. The data collectively suggested that CIP2A was highly expressed in PCa tissues, and its expression was associated with AR expression in PCa tumors.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eExpression of CIP2A in BPH and PCa tissues\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003en (100%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLow (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHigh (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eBPH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9 (75)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3 (25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.023\u003cstrong\u003e*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePCa\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22 (36.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38 (63.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; BPH, benign prostatic hyperplasia; PCa, prostate cancer\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ccaption\u003e\n \u003cp\u003eTable 2\u003c/p\u003e\n \u003cp\u003eExpression of CIP2A expression and clinicopathological\u0026nbsp;parameters in PCa tissues\u003c/p\u003e\n \u003c/caption\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.904761904761905%\"\u003e\n \u003cp\u003eCharacteristics\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.523809523809526%\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;n (100%)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.333333333333332%\"\u003e\n \u003cp\u003eLow (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"16.38095238095238%\"\u003e\n \u003cp\u003eHigh (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.857142857142858%\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" width=\"43.536121673003805%\"\u003e\n \u003cp\u003e\u003cstrong\u003eGleason score\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.29277566539924%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"16.34980988593156%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.8212927756654%\"\u003e\n \u003cp\u003e0.032\u003cstrong\u003e*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.904761904761905%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.523809523809526%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.333333333333332%\"\u003e\n \u003cp\u003e2 (12.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"16.38095238095238%\"\u003e\n \u003cp\u003e14 (87.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.857142857142858%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.904761904761905%\"\u003e\n \u003cp\u003e\u0026gt;6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.523809523809526%\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.333333333333332%\"\u003e\n \u003cp\u003e20 (45.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"16.38095238095238%\"\u003e\n \u003cp\u003e24 (54.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.857142857142858%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.904761904761905%\"\u003e\n \u003cp\u003e\u003cstrong\u003epT stage\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.523809523809526%\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.333333333333332%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"16.38095238095238%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.857142857142858%\"\u003e\n \u003cp\u003e0.285\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.904761904761905%\"\u003e\n \u003cp\u003epT2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.523809523809526%\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.333333333333332%\"\u003e\n \u003cp\u003e9 (29.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"16.38095238095238%\"\u003e\n \u003cp\u003e21 (71.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.857142857142858%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.904761904761905%\"\u003e\n \u003cp\u003epT3-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.523809523809526%\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.333333333333332%\"\u003e\n \u003cp\u003e13 (44.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"16.38095238095238%\"\u003e\n \u003cp\u003e16 (55.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.857142857142858%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" width=\"43.536121673003805%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLNMets\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.29277566539924%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"16.34980988593156%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.8212927756654%\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.904761904761905%\"\u003e\n \u003cp\u003epos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.523809523809526%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.333333333333332%\"\u003e\n \u003cp\u003e1 (33.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"16.38095238095238%\"\u003e\n \u003cp\u003e2 (66.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.857142857142858%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.904761904761905%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.523809523809526%\"\u003e\n \u003cp\u003e57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.333333333333332%\"\u003e\n \u003cp\u003e21 (36.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"16.38095238095238%\"\u003e\n \u003cp\u003e36 (63.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.857142857142858%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.904761904761905%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLVI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.523809523809526%\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.333333333333332%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"16.38095238095238%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.857142857142858%\"\u003e\n \u003cp\u003e0.102\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.904761904761905%\"\u003e\n \u003cp\u003epos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.523809523809526%\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.333333333333332%\"\u003e\n \u003cp\u003e7 (58.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"16.38095238095238%\"\u003e\n \u003cp\u003e5 (41.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.857142857142858%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.904761904761905%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.523809523809526%\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.333333333333332%\"\u003e\n \u003cp\u003e15 (31.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"16.38095238095238%\"\u003e\n \u003cp\u003e33 (68.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.857142857142858%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.904761904761905%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePNI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.523809523809526%\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.333333333333332%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"16.38095238095238%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.857142857142858%\"\u003e\n \u003cp\u003e0.766\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.904761904761905%\"\u003e\n \u003cp\u003epos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.523809523809526%\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.333333333333332%\"\u003e\n \u003cp\u003e16 (35.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"16.38095238095238%\"\u003e\n \u003cp\u003e29 (64.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.857142857142858%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.904761904761905%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.523809523809526%\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"21.333333333333332%\"\u003e\n \u003cp\u003e6 (40.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"16.38095238095238%\"\u003e\n \u003cp\u003e9 (60.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"18.857142857142858%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; LNMets, lymph node metastasis; LVI, lymphovascular invasion;\u0026nbsp;\u003cp\u003ePNI, perineural invasion.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eThe expression and activity of AR modulated CIP2A expression\u003c/strong\u003e\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003eNext, we examined whether AR signaling could regulate CIP2A expression. We analyzed the expression or phosphorylation levels of AR and CIP2A in an immortalized prostate cell line PZ-HPV-7 (PZ), an androgen-sensitive LNCaP C-33 (C-33), and two androgen-independent PCa cell lines, i.e., LNCaP C-81 (C-81) and 22Rv1 [12; 17]. Compared to PZ-HPV-7 cells, C-33, C-81, and 22Rv1 cells exhibited higher expression or phosphorylation of AR and CIP2A. Moreover, the expression or phosphorylation levels of AR and CIP2A in C-81 cells were much higher than those in C-33 cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Under a steroid-reduced (SR) condition, C-81 cells exhibited higher expression or phosphorylation levels of AR and CIP2A than C-33 cells. Treatment of 10 nM DHT greatly enhanced the expression of AR and CIP2A in C-33 cells but did not affect the level of CIP2A in C-81 cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). The stimulating effects of DHT on the expression of CIP2A and prostate-sepcific antigen (PSA) in C-33 cells can be abolished by bicalutamide, an antiandrogen, following a dose-dependent manner (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). Results from the quantitative real-time PCR analyses showed that knockdown of AR expression in C-33 cells caused significant decreases in the mRNA levels of AR, CIP2A, and PSA, suggesting the functional role of AR in the transcriptional regulation of CIP2A expression (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). The knockdown of AR expression in C-81 cells also caused decreases in the protein levels of CIP2A and PSA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE). Treatment of Enzalutamide, a second-generation nonsteroidal antiandrogen, in C-81 cells resulted in reduced AR expression and phosphorylation, correlating with decreased CIP2A and PSA in a dosage-dependent manner (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF). These results indicated that AR played a critical role in regulating CIP2A expression in PCa cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe expression of CIP2A regulated AR expression and cell proliferation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next examined whether CIP2A could regulate the AR expression and cell proliferation in PCa cells. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, increased expression of CIP2A in PZ-HPV-7 cells led to elevated AR expression and phosphorylation and increased cell proliferation. Knockdown of CIP2A expression in C-33 cells caused significant decreases in AR mRNA levels (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB), suggesting the role of CIP2A in the transcriptional regulation of AR expression. In addition, CIP2A-knockdown C-33 or 22Rv1 cells exhibited diminished AR protein levels and reduced cell proliferation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC \u0026amp; \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD). We further treated C-81 cells with different dosages of TD52, a CIP2A inhibitor [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE, the treatment of TD52 caused decreased CIP2A expression, correlating with reduced levels of AR and PSA in a dose-dependent manner. We further examined whether reducing CIP2A expression might enhance the sensitization of C-81 or 22Rv1 cells to Enzalutamide. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG, the treatment of Enzalutamide alone did not affect cell proliferation. However, the co-treatment of Enzalutamide and TD52 led to significant decreases in cell proliferation. These data suggested that CIP2A could modulate PCa cells in AR expression, cell proliferation, and sensitivity toward antiandrogen treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDecreased PP2A activity or expression caused reduced expression of c-Myc and AR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBecause CIP2A has been shown to inhibit PP2A, thus promoting the stability and activity of c-Myc [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e], a transcription factor involved in modulating AR gene expression [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e], we speculated that CIP2A-promoted AR expression was mediated by suppressing PP2A activity. Surprisingly, the inhibition of PP2A activity in C-33 cells by treating cells with LB100, a specific PP2A inhibitor [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e], or okadaic acid (OA), a nonspecific PP1 and PP2A inhibitor [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e], was unable to enhance the expression or phosphorylation of c-Myc and AR. In contrast, it caused decreased expression and phosphorylation of c-Myc and AR, following a dose-dependent manner (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). We further knocked down the expression of PP2A in C-33 or 22Rv1 cells. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC, knockdown of PP2Ac, the catalytic subunit of PP2A, caused reduced expression or phosphorylation of c-Myc and AR. Our data clearly showed that inhibition of PP2A activity or expression failed to promote c-Myc or AR phosphorylation and expression. Thus, CIP2A-promoted AR expression was independent of PP2A inhibition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePLK1 was involved in CIP2A-promoted AR expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next examined the role of polo-like kinase 1 (PLK1), a serine/threonine kinase, in mediating CIP2A-promoted AR expression. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA, the knockdown of CIP2A expression in C-81 cells led to a decrease in PLK1 expression, suggesting the role of CIP2A in regulating PLK1 expression. We further examined whether PLK1 could regulate c-Myc or AR expression in PCa cells. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB, knockdown of PLK1 expression in 22Rv1 cells resulted in reduced phosphorylation or expression of c-Myc and AR. Moreover, treatment of BI6727 (volasertib), a PLK1 inhibitor [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e], led to decreases in the expression and phosphorylation of c-Myc and AR, following a dose-dependent manner (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). We next examined whether PLK1 is required for CIP2A-modulated AR expression. Results from Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD showed that overexpression of CIP2A in C-33 cells caused increases in the expression and phosphorylation of c-Myc, AR, or PSA under an androgen-depleted condition. Notably, CIP2A-promoted effects were abolished by BI6727 treatment following a dose-dependent manner. These results indicated that CIP2A could modulate PLK1 expression or activity, which was required for CIP2A-promoted AR expression.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCIP2A is a well-known oncoprotein in various cancers. Its oncogenic function in modulating castration resistance in PCa cells has not been studied. In this study, we showed a positive correlation between CIP2A and AR expression in PCa tumors. While AR signaling could modulate CIP2A expression, CIP2A reciprocally regulated AR expression in mRNA and protein levels. Importantly, our data showed the CIP2A-modulated AR expression required the activity of PLK1. To our knowledge, this is the first report showing the novel regulation between CIP2A and AR expression in PCa cells, which may contribute to the castration resistance of PCa cells.\u003c/p\u003e \u003cp\u003eBased on the TGCA database, no difference is found in the expression of \u003cem\u003eKIAA1524\u003c/em\u003e gene between normal and PCa samples. However, lines of evidence indicated high CIP2A expression in PCa tumors, correlating with high Gleason scores [10; 23]. In concordance with the findings, our data showed the high expression of CIP2A in PCa tumors compared to BPH tissues, which was significantly associated with high Gleason scores. Importantly, our results showed that CIP2A expression was positively correlated with AR expression level in PCa tumors. The cell-line study supported this notion that CIP2A expression was higher in PCa cells than immortalized prostate cells, correlating with their AR expression levels. Interestingly, the expression level of CIP2A in LNCaP C-81 cells and 22Rv1, two androgen-independent cell lines, is even higher than that in androgen-sensitive C-33 cells, similar to the observation in clinical specimens [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The high expression of CIP2A in advanced PCa might be due to activated AR signaling. Indeed, Khanna and his colleagues [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] first showed that increased AR expression and activity enhance CIP2A expression in LNCaP cells. In contrast, knockdown of CIP2A expression in LNCaP or VCaP cells leads to reduced CIP2A expression. Our data further revealed that AR expression was critical for the mRNA expression of CIP2A. Although the detailed mechanism of how AR regulates CIP2A requires further investigation, overexpressed or activated AR in CRPC cells indirectly activates the function of transcription factors, such as ETS1 [24; 25], ELK1 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and E2F1 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], to interact with the CIP2A promoter region and enhanced its expression. Alternatively, overexpressed or activated AR could directly bind to CIP2A intronic region and regulate CIP2A expression [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAR gene amplification or protein overexpression is commonly found in CRPC patients [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Increased AR expression in hormone-sensitive cells enhances their resistance to the antiandrogens [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Our data showed that CIP2A could regulate AR expression at mRNA and protein levels. In addition, changes in CIP2A expression affected the response of PCa cells toward androgens or antiandrogens. These results might be caused by the fact that CIP2A-promoted AR expression sensitized the PCa cells to respond to a lower concentration of androgens [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, the critical question is how CIP2A regulates AR expression? Although studies have suggested that PP2A negatively regulates AR activity or expression [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], our data clearly showed that depletion of PP2A activity or expression caused reduced AR expression instead of enhancing its expression. This notion is supported by the observation that treatment of okadaic acid in LNCaP and 22Rv1 caused decreases in their AR levels [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Thus, CIP2A-promoted AR expression in PCa cells was mediated by a PP2A-independent mechanism.\u003c/p\u003e \u003cp\u003eOur results clearly showed that CIP2A could modulate PLK1 expression in immortalized prostate and PCa cells. The functional linkage between CIP2A and PLK1 was further supported by the observation that CIP2A interacts with the polo-box domain of PLK1, which maintains PLK1 stability and enhances its kinase activity [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. PLK1 is highly expressed in PCa tissues and is linked to higher-grade tumors [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Treatment of PLK1 inhibitor caused reduced AR expression and inhibited tumor growth in LNCaP CRPC xenografts [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In vitro kinase assays further showed that PLK1 binds to c-Myc and phosphorylates it at serine 62, promoting c-Myc protein stability [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In agreement with these findings, our data showed that PLK1 silencing or inhibitor treatment in C-81 or 22Rv1 cells caused reduced expression or phosphorylation of c-Myc and AR. Furthermore, CIP2A-promoted expressions in c-Myc, AR, and PSA were abolished by BI6727 treatment under an androgen-deprived condition, suggesting that PLK1 was required for CIP2A-promoted AR expression. Since CIP2A could regulate AR at the transcriptional level, it is possible that CIP2A promoted PLK1 stability and activity, which in turn enhanced c-Myc transcriptional activity and caused increased AR gene transcription. Alternatively, CIP2A-activated PLK1 may enhance AKT activity and leads to AKT-mediated Twist1 phosphorylation and nuclear accumulation. The activated Twist1 further increases AR expression through binding to E-boxes in the AR promoter [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn summary, we have demonstrated that CIP2A was highly expressed in PCa tumors, associated with AR expression. While AR signaling could upregulate CIP2A expression in an AR-dependent manner, CIP2A inversely modulated AR expression. The underlying mechanism was not mediated by PP2A inhibition but was associated with PLK1 activity. Thus, our data supported the oncogenic role of CIP2A in promoting AR expression and castration-resistant growth in PCa cells, potentially serving as a therapeutic target for CRPC treatment.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePCa, prostate cancer; BPH, benign prostatic hyperplasia; CRPC, castration-resistant prostate cancer; AR, androgen receptor; CIP2A, cancerous inhibitor of protein phosphatase 2A; PP2A, protein phosphatase 2A; PLK1, polo-like kinase 1; RT, room temperature.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a grant from the Ministry of Science and Technology in Taiwan (NSC-103-2314-B-259-001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChuang HW and Yuan TC designed the study, analyzed the results, and wrote the paper. Chuang HW, Lin CC, and Lee HS executed tissue sample collection, IHC staining, and clinicopathological analyses. Pan JH, Cai YX, Rupa D, Huang TS, Kuo TC, and Lin CW conducted the in vitro studies. Chen CW helped to analyze the bioinformatic dataset\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data sets used and/or analysed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study protocol was approved by the Institutional Review Board of Kaohsiung Veterans General Hospital (#KSVGH21-CT5-01) in accordance with the IRB\u0026rsquo;s guidelines and regulations.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eImamura Y, Sadar MD. (2016) Androgen receptor targeted therapies in castration-resistant prostate cancer: Bench to clinic. Int J Urol 23:654 \u0026ndash; 65. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/iju.13137\u003c/span\u003e\u003cspan address=\"10.1111/iju.13137\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang Y, Jiang X, Liang X, et al. Molecular and cellular mechanisms of castration resistant prostate cancer. Oncol Lett. 2018;15:6063\u0026ndash;76. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3892/ol.2018.8123\u003c/span\u003e\u003cspan address=\"10.3892/ol.2018.8123\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShiota M, Yokomizo A, Naito S. Increased androgen receptor transcription: a cause of castration-resistant prostate cancer and a possible therapeutic target. J Mol Endocrinol. 2011;47:R25\u0026ndash;41. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1530/JME-11-0018\u003c/span\u003e\u003cspan address=\"10.1530/JME-11-0018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoryakina Y, Ta HQ, Gioeli D. Androgen receptor phosphorylation: biological context and functional consequences. Endocr Relat Cancer. 2014;21:T131-45. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1530/ERC-13-0472\u003c/span\u003e\u003cspan address=\"10.1530/ERC-13-0472\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhanna A, Pimanda JE. Clinical significance of cancerous inhibitor of protein phosphatase 2A in human cancers. Int J Cancer. 2016;138:525\u0026ndash;32. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/ijc.29431\u003c/span\u003e\u003cspan address=\"10.1002/ijc.29431\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJunttila MR, Puustinen P, Niemela M, et al. CIP2A inhibits PP2A in human malignancies. Cell. 2007;130:51\u0026ndash;62. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cell.2007.04.044\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2007.04.044\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCome C, Laine A, Chanrion M, et al. CIP2A is associated with human breast cancer aggressivity. Clin Cancer Res. 2009;15:5092\u0026ndash;100. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1158/1078-0432.CCR-08-3283\u003c/span\u003e\u003cspan address=\"10.1158/1078-0432.CCR-08-3283\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim JS, Kim EJ, Oh JS, et al. CIP2A modulates cell-cycle progression in human cancer cells by regulating the stability and activity of Plk1. Cancer Res. 2013;73:6667\u0026ndash;78. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1158/0008-5472.CAN-13-0888\u003c/span\u003e\u003cspan address=\"10.1158/0008-5472.CAN-13-0888\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhanna A, Rane JK, Kivinummi KK, et al. CIP2A is a candidate therapeutic target in clinically challenging prostate cancer cell populations. Oncotarget. 2015;6:19661\u0026ndash;70. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.18632/oncotarget.3875\u003c/span\u003e\u003cspan address=\"10.18632/oncotarget.3875\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVaarala MH, Vaisanen MR, Ristimaki A. CIP2A expression is increased in prostate cancer. J Exp Clin Cancer Res. 2010;29:136. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1756-9966-29-136\u003c/span\u003e\u003cspan address=\"10.1186/1756-9966-29-136\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChang YL, Hsu YK, Wu TF, et al. Regulation of estrogen receptor alpha function in oral squamous cell carcinoma cells by FAK signaling. Endocr Relat Cancer. 2014;21:555\u0026ndash;65. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1530/ERC-14-0102\u003c/span\u003e\u003cspan address=\"10.1530/ERC-14-0102\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIgawa T, Lin FF, Lee MS, et al. Establishment and characterization of androgen-independent human prostate cancer LNCaP cell model. Prostate. 2002;50:222\u0026ndash;35. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/pros.10054\u003c/span\u003e\u003cspan address=\"10.1002/pros.10054\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHsiao YH, Huang YT, Hung CY, et al. PYK2 via S6K1 regulates the function of androgen receptors and the growth of prostate cancer cells. Endocr Relat Cancer. 2016;23:651\u0026ndash;63. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1530/ERC-16-0122\u003c/span\u003e\u003cspan address=\"10.1530/ERC-16-0122\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePattyn F, Speleman F, De Paepe A, et al. RTPrimerDB: the real-time PCR primer and probe database. Nucleic Acids Res. 2003;31:122\u0026ndash;3. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/gkg011\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkg011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiyamoto KK, McSherry SA, Dent GA, et al. Immunohistochemistry of the Androgen Receptor in Human Benign and Malignant Prostate Tissue. J Urol. 1993;149:1015\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s0022-5347(17)36284-5\u003c/span\u003e\u003cspan address=\"10.1016/s0022-5347(17)36284-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChandrashekar DS, Bashel B, Balasubramanya SAH, et al. UALCAN: A Portal for Facilitating Tumor Subgroup Gene Expression and Survival Analyses. Neoplasia. 2017;19:649\u0026ndash;58. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.neo.2017.05.002\u003c/span\u003e\u003cspan address=\"10.1016/j.neo.2017.05.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSramkoski RM, Pretlow TG 2nd, Giaconia JM, et al. A new human prostate carcinoma cell line, 22Rv1. Vitro Cell Dev Biol Anim. 1999;35:403\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11626-999-0115-4\u003c/span\u003e\u003cspan address=\"10.1007/s11626-999-0115-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu HC, Hung MH, Chen YL, et al. Erlotinib derivative inhibits hepatocellular carcinoma by targeting CIP2A to reactivate protein phosphatase 2A. Cell Death Dis. 2014;5:e1359. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/cddis.2014.325\u003c/span\u003e\u003cspan address=\"10.1038/cddis.2014.325\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrad JM, Dai JL, Wu S, et al (1999) Multiple androgen response elements and a Myc consensus site in the androgen receptor (AR) coding region are involved in androgen-mediated up-regulation of AR messenger RNA. Mol Endocrinol 13:1896 \u0026ndash; 911. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1210/mend.13.11.0369\u003c/span\u003e\u003cspan address=\"10.1210/mend.13.11.0369\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e,Lee JG, Zheng R, McCafferty-Cepero JM, et al. (2009) Endothelin-1 enhances the expression of the androgen receptor via activation of the c-myc pathway in prostate cancer cells. Mol Carcinog 48:141-9. https://doi.org/10.1002/mc.20462.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei D, Parsels LA, Karnak D, et al. Inhibition of protein phosphatase 2A radiosensitizes pancreatic cancers by modulating CDC25C/CDK1 and homologous recombination repair. Clin Cancer Res. 2013;19:4422\u0026ndash;32. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1158/1078-0432.CCR-13-0788\u003c/span\u003e\u003cspan address=\"10.1158/1078-0432.CCR-13-0788\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDounay AB, Forsyth CJ. Okadaic acid: the archetypal serine/threonine protein phosphatase inhibitor. Curr Med Chem. 2002;9:1939\u0026ndash;80. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2174/0929867023368791\u003c/span\u003e\u003cspan address=\"10.2174/0929867023368791\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYim H. Current clinical trials with polo-like kinase 1 inhibitors in solid tumors. Anticancer Drugs. 2013;24:999\u0026ndash;1006. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1097/CAD.0000000000000007\u003c/span\u003e\u003cspan address=\"10.1097/CAD.0000000000000007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCelikden SG, Baspinar S, Ozturk SA, et al. CIP2A expression in high grade prostatic intraepithelial neoplasia and prostate adenocarcinoma: a tissue microarray study. Malays J Pathol. 2020;42:227\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePallai R, Bhaskar A, Sodi V, et al. Ets1 and Elk1 transcription factors regulate cancerous inhibitor of protein phosphatase 2A expression in cervical and endometrial carcinoma cells. Transcription. 2012;3:323\u0026ndash;35. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4161/trns.22518\u003c/span\u003e\u003cspan address=\"10.4161/trns.22518\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhanna A, Okkeri J, Bilgen T, et al. ETS1 mediates MEK1/2-dependent overexpression of cancerous inhibitor of protein phosphatase 2A (CIP2A) in human cancer cells. PLoS ONE. 2011;6:e17979. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0017979\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0017979\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaine A, Sihto H, Come C, et al. (2013) Senescence sensitivity of breast cancer cells is defined by positive feedback loop between CIP2A and E2F1. Cancer Discov 3:182 \u0026ndash; 97. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1158/2159-8290.CD-12-0292\u003c/span\u003e\u003cspan address=\"10.1158/2159-8290.CD-12-0292\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu R, Denmeade SR, Luo J. Molecular processes leading to aberrant androgen receptor signaling and castration resistance in prostate cancer. Expert Rev Endocrinol Metab. 2010;5:753\u0026ndash;64. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1586/eem.10.49\u003c/span\u003e\u003cspan address=\"10.1586/eem.10.49\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen CD, Welsbie DS, Tran C, et al. Molecular determinants of resistance to antiandrogen therapy. Nat Med. 2004;10:33\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nm972\u003c/span\u003e\u003cspan address=\"10.1038/nm972\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWaltering KK, Helenius MA, Sahu B, et al. Increased expression of androgen receptor sensitizes prostate cancer cells to low levels of androgens. Cancer Res. 2009;69:8141\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1158/0008-5472.CAN-09-0919\u003c/span\u003e\u003cspan address=\"10.1158/0008-5472.CAN-09-0919\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhardwaj A, Singh S, Srivastava SK, et al (2011) Modulation of protein phosphatase 2A activity alters androgen-independent growth of prostate cancer cells: therapeutic implications. Mol Cancer Ther 10:720 \u0026ndash; 31. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1158/1535-7163\u003c/span\u003e\u003cspan address=\"10.1158/1535-7163\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.MCT-10-1096,McClinch Avelar K, Callejas RA. D et al. (2018) Small-Molecule Activators of Protein Phosphatase 2A for the Treatment of Castration-Resistant Prostate Cancer. Cancer Res 78:2065-80. https://doi.org/10.1158/0008-5472.CAN-17-0123.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen S, Kesler CT, Paschal BM, et al. Androgen receptor phosphorylation and activity are regulated by an association with protein phosphatase 1. J Biol Chem. 2009;284:25576\u0026ndash;84. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1074/jbc.M109.043133\u003c/span\u003e\u003cspan address=\"10.1074/jbc.M109.043133\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeichert W, Schmidt M, Gekeler V, et al. Polo-like kinase 1 is overexpressed in prostate cancer and linked to higher tumor grades. Prostate. 2004;60:240\u0026ndash;5. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/pros.20050\u003c/span\u003e\u003cspan address=\"10.1002/pros.20050\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Z, Chen L, Wang H, et al (2015) Inhibition of Plk1 represses androgen signaling pathway in castration-resistant prostate cancer. Cell Cycle 14:2142-8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/15384101.2015.1041689\u003c/span\u003e\u003cspan address=\"10.1080/15384101.2015.1041689\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e,Shin SB, Woo SU, Yim H. (2019) Cotargeting Plk1 and androgen receptor enhances the therapeutic sensitivity of paclitaxel-resistant prostate cancer. Ther Adv Med Oncol 11:1758835919846375. https://doi.org/10.1177/1758835919846375.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRen Y, Bi C, Zhao X, et al. PLK1 stabilizes a MYC-dependent kinase network in aggressive B cell lymphomas. J Clin Invest. 2018;128:5517\u0026ndash;30. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1172/JCI122533\u003c/span\u003e\u003cspan address=\"10.1172/JCI122533\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Z, Hou X, Shao C, et al. Plk1 inhibition enhances the efficacy of androgen signaling blockade in castration-resistant prostate cancer. Cancer Res. 2014;74:6635\u0026ndash;47. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1158/0008-5472.CAN-14-1916\u003c/span\u003e\u003cspan address=\"10.1158/0008-5472.CAN-14-1916\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dion","sideBox":"Learn more about [Discover Oncology](https://www.springer.com/12672)","snPcode":"","submissionUrl":"","title":"Discover Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Prostate cancer, CRPC, CIP2A, AR, PLK1","lastPublishedDoi":"10.21203/rs.3.rs-1856958/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1856958/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein overexpressed in human malignancies, including prostate cancer (PCa). In this study, we aimed to explore the oncogenic function of CIP2A in PCa cells and its underlying mechanism. We showed that 63.3% (38/60 cases) of PCa tissues exhibited a high CIP2A immunostaining, compared to 25% (3/12 cases) of BPH samples. Furthermore, CIP2A expression was positively correlated with patients\u0026rsquo; short survival time and nuclear AR levels in PCa tissues. Compared to PZ-HPV-7, an immortalized prostate cell line, androgen-sensitive LNCaP C-33, androgen-independent LNCaP C-81, or 22Rv1 cells exhibited a high CIP2A expression, associated with high AR expression and phosphorylation. While AR expression and activity modulated CIP2A expression, manipulating CIP2A expression in PCa cells regulated their AR expression and proliferation. The reduction of CIP2A expression also enhanced the sensitivity of PCa cells toward Enzalutamide treatment. Our data further showed that depletion of polo-kinase 1 (PLK1) expression or activity in C-81 or 22Rv1 cells caused reduced expression of c-Myc and AR. Notably, inhibition of PLK1 activity could abolish CIP2A-promoted expressions in c-Myc, AR, and prostate-specific antigen (PSA) in C-33 cells under an androgen-deprived condition, suggesting the role of PLK1 activity in CIP2A-promoted AR expression. In summary, our data showed the existence of a novel regulation between CIP2A and AR expression, which is critical for promoting PCa malignancy. Thus, CIP2A could serve as a therapeutic target for PCa.\u003c/p\u003e","manuscriptTitle":"Reciprocal regulation of CIP2A and AR expression in prostate cancer cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-07-25 17:07:13","doi":"10.21203/rs.3.rs-1856958/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-08-11T11:48:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-07-29T20:36:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2531c45a-e4fa-4d89-8f2f-32662a2064e2","date":"2022-07-29T04:20:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1cac94c8-246a-44f0-a905-e800a9623d09","date":"2022-07-20T14:58:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-07-18T22:27:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-07-18T22:18:21+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2022-07-18T01:46:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-07-18T01:43:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Oncology","date":"2022-07-14T07:54:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dion","sideBox":"Learn more about [Discover Oncology](https://www.springer.com/12672)","snPcode":"","submissionUrl":"","title":"Discover Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"11a4ebad-461b-4d81-9323-071035636167","owner":[],"postedDate":"July 25th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-08-30T07:59:11+00:00","versionOfRecord":[],"versionCreatedAt":"2022-07-25 17:07:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1856958","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1856958","identity":"rs-1856958","version":["v1"]},"buildId":"pf3fE39SIOqb-0xH_OWvX","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}