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

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.

Chk1 targeting reactivates PP2A tumor suppressor activity in cancer cells.

Checkpoint kinase Chk1 is constitutively active in many cancer cell types and new generation Chk1 inhibitors show marked antitumor activity as single agents. Here we present a hitherto unrecognized mechanism that contributes to the response of cancer cells to Chk1-targeted therapy. Inhibiting chronic Chk1 activity in cancer cells induced the tumor suppressor activity of protein phosphatase protein phosphatase 2A (PP2A), which by dephosphorylating MYC serine 62, inhibited MYC activity and impaired cancer cell survival. Mechanistic investigations revealed that Chk1 inhibition activated PP2A by decreasing the transcription of cancerous inhibitor of PP2A (CIP2A), a chief inhibitor of PP2A activity. Inhibition of cancer cell clonogenicity by Chk1 inhibition could be rescued in vitro either by exogenous expression of CIP2A or by blocking the CIP2A-regulated PP2A complex. Chk1-mediated CIP2A regulation was extended in tumor models dependent on either Chk1 or CIP2A. The clinical relevance of CIP2A as a Chk1 effector protein was validated in several human cancer types, including neuroblastoma, where CIP2A was identified as an NMYC-independent prognostic factor. Because the Chk1-CIP2A-PP2A pathway is driven by DNA-PK activity, functioning regardless of p53 or ATM/ATR status, our results offer explanative power for understanding how Chk1 inhibitors mediate single-agent anticancer efficacy. Furthermore, they define CIP2A-PP2A status in cancer cells as a pharmacodynamic marker for their response to Chk1-targeted therapy.

Detection of smell print differences between nonmalignant and malignant prostate cells with an electronic nose.

AIM: To determine whether an electronic nose can differentiate cultured nonmalignant and malignant prostatic cells from each other and whether the smell print is secreted to the surrounding medium. MATERIALS & METHODS: Prostatic nonmalignant (EP-156T and controls) and malignant (LNCaP) cell lines, as well as conditioned and unconditioned media, were collected. The smell prints of the samples were analyzed by a ChemPro(®) 100 electronic nose device. The data were normalized and dimension reduction was conducted. The samples were classified and misclassification rates were calculated. RESULTS: The electronic nose differentiated the nonmalignant and malignant cell lines from each other, achieving misclassification rates of 2.9-3.6%. Cells did not differ from the conditioned medium but differed from the unconditioned medium (misclassification rates: 0.0-25.6%). CONCLUSION: Malignant and nonmalignant prostatic cell lines have distinct smell prints. Prostatic cancer cells seem to modify the smell print of their medium.

Increased expression of androgen receptor sensitizes prostate cancer cells to low levels of androgens.

Androgen receptor (AR) is known to be overexpressed in castration-resistant prostate cancer. To interrogate the functional significance of the AR level, we established two LNCaP cell sublines expressing in a stable fashion two to four times (LNCaP-ARmo) and four to six times (LNCaP-ARhi) higher level of AR than the parental cell line expressing the empty vector (LNCaP-pcDNA3.1). LNCaP-ARhi cell line grew faster than the control line in low concentrations, especially in 1 nmol/L 5alpha-dihydrotestosterone (DHT). Microarray-based transcript profiling and subsequent unsupervised hierarchical clustering showed that LNCaP-ARhi cells clustered together with VCaP cells, containing endogenous AR gene amplification and overexpression, indicating the central role of AR in the overall regulation of gene expression in prostate cancer cells. Two hundred forty genes showed >2-fold changes on DHT treatment in LNCaP-ARhi at 4 h time point, whereas only 164 and 52 showed changes in LNCaP-ARmo and LNCaP-pcDNA3.1, respectively. Many androgen-regulated genes were upregulated in LNCaP-ARhi at 10-fold lower concentration of DHT than in control cells. DHT (1 nmol/L) increased expression of several cell cycle-associated genes in LNCaP-ARhi cells. ChIP-on-chip assay revealed the presence of chromatin binding sites for AR within +/-200 kb of most of these genes. The growth of LNCaP-ARhi cells was also highly sensitive to cyclin-dependent kinase inhibitor, roscovitine, at 1nmol/L DHT. In conclusion, our results show that overexpression of AR sensitizes castration-resistant prostate cancer cells to the low levels of androgens. The activity of AR signaling pathway is regulated by the levels of both ligand and the receptor.

Androgen regulation of the androgen receptor coregulators.

BACKGROUND: The critical role of the androgen receptor (AR) in the development of prostate cancer is well recognized. The transcriptional activity of AR is partly regulated by coregulatory proteins. It has been suggested that these coregulators could also be important in the progression of prostate cancer. The aim of this study was to identify coregulators whose expression is regulated by either the androgens and/or by the expression level of AR. METHODS: We used empty vector and AR cDNA-transfected LNCaP cells (LNCaP-pcDNA3.1, and LNCaP-ARhi, respectively), and grew them for 4 and 24 hours in the presence of dihydrotestosterone (DHT) at various concentrations. The expression of 25 AR coregulators (SRC1, TIF2, PIAS1, PIASx, ARIP4, BRCA1, beta-catenin, AIB3, AIB1, CBP, STAT1, NCoR1, AES, cyclin D1, p300, ARA24, LSD1, BAG1L, gelsolin, prohibitin, JMJD2C, JMJD1A, MAK, PAK6 and MAGE11) was then measured by using real-time quantitative RT-PCR (Q-RT-PCR). RESULTS: Five of the coregulators (AIB1, CBP, MAK, BRCA1 and beta-catenin) showed more than 2-fold induction and 5 others (cyclin D1, gelsolin, prohibitin, JMJD1A, and JMJD2C) less than 2-fold induction. Overexpression of AR did not affect the expression of the coregulators alone. However, overexpression of AR enhanced the DHT-stimulated expression of MAK, BRCA1, AIB1 and CBP and reduced the level of expression of beta-catenin, cyclinD1 and gelsolin. CONCLUSION: In conclusion, we identified 5 coactivators whose expression was induced by androgens suggesting that they could potentiate AR signaling. Overexpression of AR seems to sensitize cells for low levels of androgens.

Overexpression of EIF3S3 promotes cancer cell growth.

BACKGROUND: Amplification and overexpression of EIF3S3 gene has been demonstrated in breast and prostate cancer. Here, our goal was to study the effect of EIF3S3 on cell growth. METHODS: The effect of EIF3S3 on growth of NIH 3T3 murine fibroblasts as well as breast (SK-Br-3 and ZR-75-1) and prostate (PC-3 and LNCaP) cancer cell lines was examined by using transfection with inducible pTet-Off system and siRNAs. RESULTS: NIH 3T3 cells with overexpression of EIF3S3 grew significantly faster than cells transfected with empty vector and survived longer when grown in soft agar. The EIF3S3 overexpression was associated with increased fraction of cells in S-phase and with phosphorylation of retinoblastoma (Rb) protein. siRNA treatment inhibited significantly (P = 0.0022) the growth of all breast and prostate cancer cell lines studied. CONCLUSIONS: The results suggest that EIF3S3 regulates cell growth and viability, and that overexpression of the gene may provide growth advantage to the cancer cells.

Amplification of the urokinase gene and the sensitivity of prostate cancer cells to urokinase inhibitors.

OBJECTIVES: To evaluate the frequency of the urokinase-type plasminogen activator (uPA) gene amplification and the sensitivity of prostate cancer cells to uPA inhibition, as we previously found one hormone-refractory prostate tumour with high-level amplification of the uPA (alias PLAU) gene, and also showed that a uPA inhibitor, amiloride, can effectively reduce the invasion potential of the PC-3 prostate cancer cell line. MATERIALS AND METHODS: Sixty-three locally recurrent hormone-refractory tumours and 78 hormone-refractory metastases from 29 patients who died from prostate cancer were analysed for uPA gene-copy number using fluorescence in situ hybridization. The Matrigel invasion assay was used to study the influence of uPA inhibitors on the invasive potential of prostate cancer cell lines. RESULTS: Of the locally recurrent hormone-refractory tumours, 21% had an increased copy number of uPA, but no high-level amplifications were found; 31% of the metastases had increased copy number and one high-level amplification of the uPA. Matrigel invasion assays with two specific uPA inhibitors, B428 and p-aminobenzamidine, showed that invasion of a prostate cancer cell line containing uPA gene amplification was inhibited by these small-molecule uPA inhibitors, while invasion of prostate cell lines without uPA gene amplification were not. CONCLUSION: These results suggest that selective inhibition of the uPA pathway in individuals whose tumours contain uPA gene amplification may provide therapeutic benefit.

Cloning and characterization of a novel six-transmembrane protein STEAP2, expressed in normal and malignant prostate.

By using subtraction and cDNA array hybridizations, we recently identified an anonymous transcript that was differentially expressed in benign prostate hyperplasia and prostate cancer cell line PC-3. Here, we report the cloning of the full-length cDNA of the gene, designated STEAP2 (six-transmembrane epithelial antigen of the prostate 2). The gene is located at the chromosomal region 7q21 and encodes for a 490-amino acid protein with six predicted transmembrane domains and is predominantly expressed in prostate epithelial cells. Green fluorescent protein fusion construct indicated that the STEAP2 protein is localized mainly in the plasma membrane. Real-time quantitative RT-PCR showed that the gene is expressed at levels more than 10 times higher in normal prostate than in other tissues studied. Of the prostate cancer cell lines, STEAP2 was expressed in significant levels only in androgen-responsive LNCaP. The expression of STEAP2 was significantly higher (p = 0.002) in both untreated primary and hormone-refractory prostate carcinomas than in benign prostate hyperplasias, suggesting that it may be involved in the development of prostate cancer. As a cell-surface antigen, STEAP2 is a potential diagnostic or therapeutic target in prostate cancer.