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. 2015 Aug 14;6(23):19661-70.
doi: 10.18632/oncotarget.3875.

CIP2A is a candidate therapeutic target in clinically challenging prostate cancer cell populations

Anchit Khanna  1   2 , Jayant K Rane  3 , Kati K Kivinummi  1   4 , Alfonso Urbanucci  1   5   6 , Merja A Helenius  1 , Teemu T Tolonen  1 , Outi R Saramäki  1 , Leena Latonen  1 , Visa Manni  1 , John E Pimanda  2 , Norman J Maitland  3 , Jukka Westermarck  7   8 , Tapio Visakorpi  1
Affiliations

CIP2A is a candidate therapeutic target in clinically challenging prostate cancer cell populations

Anchit Khanna et al. Oncotarget. .

Abstract

Residual androgen receptor (AR)-signaling and presence of cancer stem-like cells (SCs) are the two emerging paradigms for clinically challenging castration-resistant prostate cancer (CRPC). Therefore, identification of AR-target proteins that are also overexpressed in the cancer SC population would be an attractive therapeutic approach.Our analysis of over three hundred clinical samples and patient-derived prostate epithelial cultures (PPECs), revealed Cancerous inhibitor of protein phosphatase 2A (CIP2A) as one such target. CIP2A is significantly overexpressed in both hormone-naïve prostate cancer (HN-PC) and CRPC patients . CIP2A is also overexpressed, by 3- and 30-fold, in HN-PC and CRPC SCs respectively. In vivo binding of the AR to the intronic region of CIP2A and its functionality in the AR-moderate and AR-high expressing LNCaP cell-model systems is also demonstrated. Further, we show that AR positively regulates CIP2A expression, both at the mRNA and protein level. Finally, CIP2A depletion reduced cell viability and colony forming efficiency of AR-independent PPECs as well as AR-responsive LNCaP cells, in which anchorage-independent growth is also impaired.These findings identify CIP2A as a common denominator for AR-signaling and cancer SC functionality, highlighting its potential therapeutic significance in the most clinically challenging prostate pathology: castration-resistant prostate cancer.

Keywords: CIP2A; androgen receptor; cancer stem-like cells; castration-resistant prostate cancer.

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Conflict of interest statement

CONFLICTS OF INTEREST

None.

Figures

Figure 1
Figure 1. CIP2A is overexpressed in prostate cancer
A. Overexpression of CIP2A mRNA in castration resistant prostate cancer (CRPC; n = 15) hormone-naïve primary prostate cancer (HN-PC; n = 27) compared to benign prostatic hyperplasia (BPH; n = 8). * = n < 0.05, *** = p value < 0.001 using the Kruskal-Wallis test with Dunn's multiple comparison post-test. B. CIP2A protein expression in prostate cancer specimens as detected by immunohistochemistry. A score of 0 (negative), 1 (weak), 2 (moderate) and 3 (high) was given based on the cytoplasmic CIP2A immunopositivity. C. Statistical analysis of CIP2A immunopositivity in both HN-PC and CRPC cases. D. Western blot showing CIP2A protein levels in benign and malignant prostate cancer (PC) cells.
Figure 2
Figure 2. CIP2A is overexpressed and promotes growth and viability of cancer stem-like cell (SC) population from patient-derived prostate epithelial cell (PPECs) cultures
A. Overexpression of CIP2A mRNA in the stem-like cell (SC) population of both primary HN-PC and CRPC patient samples in comparison to BPH samples. Note the y-axis scale change between HN-PC and CRPC panels. B. Western blot showing the effect of two different CIP2A siRNAs on CIP2A protein levels in primary HN-PC-SC cells in comparison to the Scrambled (Scr.) or control transfected cells. C. Effect of CIP2A depletion, using two different siRNAs against CIP2A, on cell viability of HN-PC-SC cells from patients in comparison to the control/Scr. transfected cells at 3 and 6 day time point. (* = p-value < 0.05 using student t-test) D. Effect of CIP2A depletion on colony forming capacity of primary cells from BPH and HN-PC patients (total cell populations) in comparison to the control/Scr. transfected cells expressed as 100% for each. Shown are the effects on both small (less than 32 cells) and big colonies (more than 32 cells ; * = p-value < 0.05 using student t-test).
Figure 3
Figure 3. Androgen receptor (AR) binds to CIP2A and its activity and expression positively regulates CIP2A expression in prostate cancer
A. AR binding on CIP2A intronic region. ChIP-qPCR on LNCaP-pcDNA3.1, -ARmo, -ARhi cells, hormone starved for 4 days followed by treatment with 1nM and 100nM DHT for 2h or Ethanol was performed to assess the AR recruitment on the CIP2A intronic region. B. Effect on CIP2A mRNA expression in LNCaP-pcDNA3.1, -ARhi cells on DHT (1nM, 10nM, 100nM) stimulation at 24h time point. C. Effect on CIP2A protein expression in LNCaP-pcDNA3.1, -ARhi cells on DHT (1nM) stimulation at 24h time point according to Western blotting. D. Effect of two independent AR siRNAs on CIP2A and AR protein expression in LNCaP cells 72h post-transfection and effect of AR and CIP2A siRNAs on CIP2A and AR protein expression in VCaP cells 72h post-transfection.
Figure 4
Figure 4. CIP2A promotes viability, clonogenicity and transformation potential of AR-positive prostate cancer cell lines
A. Effect of two independent CIP2A siRNAs on cell viability of LNCaP-pcDNA3.1, -ARhi cells 3 and 6 days post-transfection. Student's t-test was used to obtain the p values. B. Effect of CIP2A siRNA on clonogenicity of LNCaP cells 6 days post-transfection. Student's t-test was used to obtain the p values. C. Effect of two different CIP2A siRNAs on anchorage-independent growth. Here, LNCaP cells were transfected with CIP2A.1, CIP2A.2 or Scr. siRNA and, at 48 hours post transfection, plated at low density in soft agar medium. Student's t-test was used to obtain the p values. D. Schematic representation of the therapeutic potential of targeting CIP2A. Since CIP2A positivity is common to both AR-dependent and cancer SC populations of prostate cancer, it surfaces as an attractive therapeutic target, especially in clinically challenging castration-resistant prostate cancers.
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