Low p16INK4a Expression in Early Passage Human Prostate Basal Epithelial Cells Enables Immortalization by Telomerase Expression Alone.

BACKGROUND: There are two principal senescence barriers that must be overcome to successfully immortalize primary human epithelial cells in culture, stress-induced senescence, and replicative senescence. The p16INK4a /retinoblastoma protein (p16/Rb) pathway mediates stress-induced senescence, and is generally upregulated by primary epithelial cells in response to the artificial conditions from tissue culture. Replicative senescence is associated with telomere loss. Following each round of cell division, telomeres progressively shorten. Once telomeres shorten to a critical length, the DNA damage response pathway is activated, and the tumor suppressor p53 pathway triggers replicative senescence. Exogenous expression of telomerase in normal human epithelial cells extends the replicative capacity of cells, and in some cases, immortalizes cells. However reliable immortalization of epithelial cells usually requires telomerase activity coupled with inactivation of the p16/Rb pathway. METHODS: A lentiviral vector, pLOX-TERT-iresTK (Addgene #12245), containing a CMV promoter upstream of a bicistronic coding cassette that includes loxP sites flanking the catalytic subunit of human telomerase gene (TERT) and herpes simplex virus type-1 thymidine kinase gene (HSV1-tk) was used to transduce normal prostate basal epithelial cells (PrECs) initiated in cell culture from prostate cancer patients undergoing radical prostatectomies. RESULTS: Transduction of early (i.e., <7) passage PrECs with TERT led to successful immortalization. However, attempts to immortalize late (i.e., >7) passage PrECs were unsuccessful. Late passage PrECs, which acquired elevated p16, were unable to overcome the senescence barrier. Immortalized PrECs (TERT-PrECs) retained a normal male karyotype and low p16 expression. Additionally, TERT-PrECs were non-tumorigenic when inoculated into intact male immunodeficient NSG mice. CONCLUSIONS: The present studies document that early passage human PrECs have sufficiently low p16 to permit immortalization by TERT expression alone. TERT-PrECs developed using this transduction approach provides an appropriate and experimentally facile model for clarifying the molecular mechanism(s) involved in both immortalization of human PrECs, as well as identifying genetic/epigenetic "drivers" for conversion of these immortalized non-tumorigenic cells into fully lethal prostate cancers. Notably, loxP sites flank the exogenous TERT gene in the TERT-PrECs. Cre recombinase can be used to excise TERT, and resolve whether TERT expression is required for these cells to be fully transformed into lethal cancer. Prostate 77: 374-384, 2017. © 2016 Wiley Periodicals, Inc.

Human prostate cancer initiating cells isolated directly from localized cancer do not form prostaspheres in primary culture.

BACKGROUND: Recent experimental studies suggest that hierarchical expansion from a minor population of cancer cells with an unlimited self-renewal capacity, termed cancer initiating cells (CICs), drives both lethality and heterogeneity of prostate cancer. Human prostate CICs have been established from only two primary prostate cancer patients, with the remaining established CIC lines being derived from metastatic sites from <10 patients. This suggests that the established CIC lines are significant "outliers" and may not be representative of the prostate CICs seen clinically. Thus, there is an urgent need to develop new approaches to achieve the "routine" establishment of CIC containing lines, particularly derived from primary prostate cancers. METHODS: In the present studies, we confirmed that in serum free, high Ca(2+) (i.e., DMEN: F12) growth factor defined (GFD) media plus androgen, a large (n = 10) series of established human prostate cancer cell lines derived from both localized and metastatic sites characteristically self-associate in suspension and grow as unattached spheroids, termed prostaspheres which contain CICs based upon their self-renewal in vitro and tumorigenicity in vivo. RESULTS: Unfortunately, however, while dissociated single cells from human primary prostate cancer tissues are viable, contain CICs as documented by their ability to take and proliferate as xenografts, and produce prostaspheres when plated with serum free, high Ca(2+) /GFD-media plus androgen onto standard tissue culture flask, these prostasphere do not contain CICs. CONCLUSION: The development of reproducibly methods to culture CICs isolated directly from localized cancers is still an urgent unmeet need of the prostate cancer research community.

Lack of biological relevance of platelet cyclooxygenase-2 dependent thromboxane A2 production.

INTRODUCTION: There is emerging evidence of a considerable variability of the impact of aspirin on clinical outcome and laboratory findings. Persistent TxA2 production seems to be the most likely reason. Aim of this study was to determine whether the mechanism responsible for TxA2 persistent production is, at least partially, dependent upon aspirin-insensitive platelet COX-2 enzymatic pathway. METHODS AND RESULTS: In 100 consecutive patients, under chronic aspirin anti-platelet treatment (100-160 mg/day) selected on the basis of detectable plasma salicylate levels, serum and Arachidonic Acid (AA)-induced platelet TxA2 production, immunoblot analysis of platelet COX-1/COX-2 expression and COX-2 activity were studied. Immunoblot revealed COX-2 expression in 46% patients, in an amount that was markedly lower than COX-1. In 10 COX-2 positive patients with TxA2 levels over the median, AA-induced TxA2 production performed in vitro in the presence of the COX-2 inhibitor CAY10404 and aspirin demonstrated that COX-2 dependent TxA2 production is less than 2%. CONCLUSION: Our data demonstrate that the inter-individual variability of platelet sensitivity to aspirin is due to a reduced efficacy of aspirin on platelet COX-1 despite ascertained patient compliance. We suggest that serum TxA2 assay might be performed in future clinical studies to improve our knowledge on the residual TxA2 production in aspirin-treated patients.

Distinct phenotypes of human prostate cancer cells associate with different adaptation to hypoxia and pro-inflammatory gene expression.

Hypoxia and inflammation are strictly interconnected both concurring to prostate cancer progression. Numerous reports highlight the role of tumor cells in the synthesis of pro-inflammatory molecules and show that hypoxia can modulate a number of these genes contributing substantially to the increase of cancer aggressiveness. However, little is known about the importance of the tumor phenotype in this process. The present study explores how different features, including differentiation and aggressiveness, of prostate tumor cell lines impact on the hypoxic remodeling of pro-inflammatory gene expression and malignancy. We performed our studies on three cell lines with increasing metastatic potential: the well differentiated androgen-dependent LNCaP and the less differentiated and androgen-independent DU145 and PC3. We analyzed the effect that hypoxic treatment has on modulating pro-inflammatory gene expression and evaluated the role HIF isoforms and NF-kB play in sustaining this process. DU145 and PC3 cells evidenced a higher normoxic expression and a more complete hypoxic induction of pro-inflammatory molecules compared to the well differentiated LNCaP cell line. The role of HIF1α and NF-kB, the master regulators of hypoxia and inflammation respectively, in sustaining the hypoxic pro-inflammatory phenotype was different according to cell type. NF-kB was observed to play a main role in DU145 and PC3 cells in which treatment with the NF-kB inhibitor parthenolide was able to counteract both the hypoxic pro-inflammatory shift and HIF1α activation but not in LNCaP cells. Our data highlight that tumor prostate cell phenotype contributes at a different degree and with different mechanisms to the hypoxic pro-inflammatory gene expression related to tumor progression.

Action of retinoic acid receptor on EGFR gene transactivation and breast cancer cell proliferation: Interplay with the estrogen receptor.

In the present report, we investigated the action of retinoic acid (RA) on the transactivation of the epidermal growth factor receptor (EGFR) gene promoter. In a previous study, we showed that the estrogen receptor (ER) α activated by 17ß-estradiol (E2) increased EGFR expression by enhancing the binding of the transcription factor Sp1 to the EGFR minimal promoter in HeLa cells. Here, we demonstrate that ligand-activated RA receptor (RAR) α inhibited EGFR transactivation by competing with Sp1 for binding to the same promoter fragment in the same cell model. When RARα and ERα were coexpressed, the inhibitory effect of RA on transactivation of the EGFR promoter counteracted the enhancement induced by E2-activated ERα and became more pronounced in the presence of ligand-free ERα. In the MCF7 breast cancer cell line, which endogenously expresses RARα and ERα, RA exerted anti-proliferative effects in the presence of ligand-free ERα. Moreover, interplay between the pathways mediated by the two receptors was observed, as RA counteracted E2-induced cell proliferation. Our results suggest that the interference with the activity of Sp1 on the EGFR promoter could be related to the observed RA-mediated growth suppression of breast cancer cells.