Clin Invest Med 2008; 31 (1): E8-E15. AbstractObjective: Ki-67 is a proliferation-associated nuclear antigen and is expressed in all cycling cells except for resting cells in the G0-phase. PCNA is an acidic nuclear protein and has been recognized as a histologic marker for the G1/ S phase in the cell cycle. Ki-67and PCNA labeling indices are considered to reflect cell proliferation, particularly, growth fraction. The purpose of this study is to investigate the expression levels of Ki-67 and PCNA in prostate cancer (PCa) and benign prostatic hyperplasia (BPH) and their potential on the early diagnosis of PCa. Methods: Human prostate cancer cell lines LNCaP and PC-3, human normal prostate epithelial cell line HuPEC, tissues from patients with PCa (121 cases) and BPH (45) and 36 normal cases were examined for the expression of Ki-67 and PCNA by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Then, the association of Ki-67 and PCNA expression with clinical grading of PCa was analyzed by immunohistochemistry staining. Results: The ratios of PCNA and Ki-67 expression levels in LNCaP and PC-3 were higher (P<0.05, P<0.001) than that in HuPEC. The two markers were differentially expressed in three tissues and showed increased expression in PCa (P<0.05) and BPH (P<0.05), relative to human normal prostate tissues. Compared with BPH, the ratio of Ki-67 and PCNA expressed in tumour tissue was increased (P<0.05). The increase of Ki-67 was greater than that of PCNA. Expression of the two markers increased after different grading of PCa cases Prostate cancer (PCa) is the second most frequent cause of male cancer-related death in the United States of America (USA) and Western Europe. Its incidence is continuously rising, with over 200 000 new cancers and 35 000~40 000 deaths per year, 1 Carcinogenesis and the mechanisms influencing the progression and prognosis of PCa is a multistep process, involving both genetic insults to epithelial cells and changes in epithelial-stromal interactions. 2 Despite extensive research PCa is not understood.There are two proliferative markers-Ki-67 and proliferating cell nuclear antigen (PCNA). Ki-67 antigen is expressed in proliferative cells throughout the G1, S, G2, and M phases, and provides a reliable index of cellular proliferation 3,4 PCNA is an acidic nuclear protein, expressed mainly in phase S of the cellular cycle. It becomes active, in various tissues par-

Oncogene-induced senescence is an important tumour-suppressing mechanism to prevent both premalignant transformation and cancer progression. Overcoming this process is a critical step in early cancer development. The druggable orphan nuclear receptor TLX (NR2E1) is characterized as an important regulator of neural stem cells and is also implicated in the development of some brain tumours. However, its exact functional roles in cancer growth regulation still remain unclear. Here we report that TLX can act as a promoter of tumourigenesis in prostate cancer by suppressing oncogene-induced senescence. We determined that TLX exhibited an increased expression in high-grade prostate cancer tissues and many prostate cancer cell lines. Functional studies revealed that TLX could perform an oncogenic function in prostate cancer cells, as its knockdown triggered cellular senescence and cell growth arrest in vitro and in vivo, whereas its over-expression promoted the malignant growth of prostate cancer cells. Furthermore, enhancement of TLX activity, by either ectopic expression or ligand stimulation, could potently prevent doxorubicin-induced senescence in prostate cancer cells and also allow prostatic epithelial cells to escape oncogene-induced senescence induced either by activated oncogene H-Ras(G12V) or knockdown of tumour suppressor PTEN, via a mechanism of direct but differential transcriptional regulation of two senescence-associated genes, repression of CDKN1A and transactivation of SIRT1. Together, our present study shows, for the first time, that TLX may play an important role in prostate carcinogenesis through its suppression of oncogene-induced senescence, and also suggests that targeting the senescence-regulatory TLX is of potential therapeutic significance in prostate cancer.

The growth adaptation of cancer cells to a hypoxic tumour microenvironment is mostly regulated by hypoxia-induced transcription factor HIF-1. HIF-1 transcriptional activity is strictly controlled by protein levels of the HIF-1α subunit, which is tightly regulated by a well-characterized O2 -dependent ubiquitin ligase-proteasomal degradation pathway. The cold-sensitive Ca(2+) channel protein TRPM8 exhibits increased expression in advanced prostate cancer. However, its exact functional roles in prostate cancer growth regulation are unclear and controversial. In this work, we show that TRPM8 promotes in vitro hypoxic growth capacities, drug resistance, and in vivo tumourigenicity, accompanied with enhanced HIF-1α protein levels. These effects are further potentiated by TRPM8 agonists but suppressed by TRPM8 gene knockdown and blocking with antagonists or TRPM8 antibody. TRPM8-induced suppression of HIF-1α ubiquitination and enhanced HIF-1 transactivation were attenuated by forced RACK1 expression and TRPM8 overexpression reduced phospho-RACK1 levels, thus affecting its dimerization status, and promoted RACK1 binding to HIF-1α and calcineurin. These data indicate that TRPM8-induced increase of HIF-1α protein in hypoxia- or normoxia-exposed prostate cancer cells was mediated through a newly characterized Ca(2+) -dependent but O2 -independent mechanism involving binding of RACK1 to HIF-1α and RACK1-mediated ubiquitination of HIF-1α. Collectively, our study not only provides a mechanistic insight into how TRPM8 promotes the hypoxic growth adaptation of cancer cells via its promotion of RACK1-mediated stabilization of HIF-1α but also suggests a potential therapeutic strategy for prostate cancer by targeting TRPM8.

Background More and more studies have shown that circular RNAs (circRNAs) play a critical regulatory role in many cancers. However, the potential molecular mechanism of circRNAs in prostate cancer (PCa) remains largely unknown. Methods Differentially expressed circRNAs were identified by RNA sequencing. The expression of hsa_circ_0003258 was evaluated using quantitative real-time PCR and RNA in situ hybridization. The impacts of hsa_circ_0003258 on the metastasis of PCa cells were investigated by a series of in vitro and in vivo assays. Lastly, the underlying mechanism of hsa_circ_0003258 was revealed by Western blot, biotin-labeled RNA pulldown, RNA immunoprecipitation, luciferase assays and rescue experiments. Results Increased expression of hsa_circ_0003258 was found in PCa tissues and was associated with advanced TNM stage and ISUP grade. Overexpression of hsa_circ_0003258 promoted PCa cell migration by inducing epithelial mesenchymal transformation (EMT) in vitro as well as tumor metastasis in vivo, while knockdown of hsa_circ_0003258 exerts the opposite effect. Mechanistically, hsa_circ_0003258 could elevate the expression of Rho GTPase activating protein 5 (ARHGAP5) via sponging miR-653-5p. In addition, hsa_circ_0003258 physically binds to insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3) in the cytoplasm and enhanced HDAC4 mRNA stability, in which it activates ERK signalling pathway, then triggers EMT programming and finally accelerates the metastasis of PCa. Conclusions Upregulation of hsa_circ_0003258 drives tumor progression through both hsa_circ_0003258/miR-653-5p/ARHGAP5 axis and hsa_circ_0003258/IGF2BP3 /HDAC4 axis. Hsa_circ_0003258 may act as a promising biomarker for metastasis of PCa and an attractive target for PCa intervention.

BackgroundNon-adherent or ultra-low attachment three-dimensional (3D) culture, also called sphere formation assay, has been widely used to assess the malignant phenotype and stemness potential of transformed or cancer cells. This method is also popularly used to isolate the cancer stem-like cells (CSCs) or tumor-initiating cells based on their unique anchorage-independent growth or anoikis-resistant capacity. Different non-adhesive coating agents, such as poly-2-hydroxyethyl methacrylate (poly-HEMA) and synthetic hydrogels, have been used in this non-adherent 3D culture. However, preparation of non-adherent culture-ware is labor-intensive and technically demanding, and also costs of commercial non-adherent culture-ware prepared with various coating agents are relatively expensive and the culture-ware cannot be used repeatedly.MethodsIn this study, we developed a non-adherent 3D culture method based on agar coating for growing tumor spheres derived from various cancer cell lines and primary prostate cancer tissues under a non-adherent and serum-free condition. The tumor spheres generated by this 3D culture method were analyzed on their expression profiles of CSC-associated markers by reverse transcription quantitative polymerase chain reaction, presence and relative proportion of CSCs by fluorescence-activated cell sorting (CD133+/CD44+ cell sorting) and also a CSC-visualizing reporter system responsive to OCT4 and SOX2 (SORE6), and in vivo tumorigenicity. The repeated use of agar-coated plates for serial passages of tumor spheres was also evaluated.ResultsOur results validated that the multicellular tumor spheres generated by this culture method were enriched of CSCs, as evidenced by their enhanced expression profiles of CSC markers, presence of CD133+/CD44+ or SORE6+ cells, enhanced self-renewal capacity, and in vivo tumorigenicity, indicating its usefulness in isolation and enrichment of CSCs. The agar-coated plates could be used multiple times in serial passages of tumor spheres.ConclusionsThe described agar-based 3D culture method offers several advantages as compared with other methods in isolation of CSCs, including its simplicity and low-cost and repeated use of agar-coated plates for continuous passages of CSC-enriched spheres.Electronic supplementary materialThe online version of this article (10.1186/s13287-018-0987-x) contains supplementary material, which is available to authorized users.