Expression of RIZ1 protein (Retinoblastoma‐interacting zinc‐finger protein 1) in prostate cancer epithelial cells changes with cancer grade progression and is modulated in vitro by DHT and E2

Abstract

The nuclear protein methyl-transferase Retinoblastoma-interacting zinc-finger protein 1 (RIZ1) is considered to be a downstream effector of estrogen action in target tissues. Silencing of RIZ1 expression is common in many tumors. We analyzed RIZ1 expression in normal and malignant prostate tissue and evaluated whether estradiol (E2) or dihydrotestosterone (DHT) treatment modulated RIZ1 in cultured prostate epithelial cells (PEC). Moreover, we studied the possible involvement of RIZ1 in estrogen action on the EPN prostate cell line, constitutively expressing both estrogen receptor (ER)-alpha and beta. RIZ1 protein, found in the nucleus of normal PECs by immunohistochemistry, was progressively lost in cancer tissues as the Gleason score increased and was only detected in the cytoplasmic compartment. RIZ1 transcript levels, as assayed by semi-quantitative RT-PCR in primary PEC cultures, were significantly reduced in cancer cells (P < 0.05). In EPN DHT treatment significantly increased RIZ1 transcript and protein levels (P < 0.05); E2 induced a reduction of S phase without significant changes of RIZ1 expression. In E2-treated EPN cell extracts RIZ co-immunoprecipitated with ERbeta and ERalpha. Our data demonstrate that RIZ1 is expressed in normal PECs and down-regulated in cancer cells, with a switch of its sub-cellular localization from the nucleus to the cytoplasm upon cancer grade progression. RIZ1 expression levels in the PECs were modulated by DHT or E2 treatment in vitro. Furthermore, the E2 effects on ER-expressing prostate cells involve RIZ1, which confirms a possible role for ER-mediated pathways in a non-classic E(2)-target tissue.

Raloxifene induces cell death and inhibits proliferation through multiple signaling pathways in prostate cancer cells expressing different levels of estrogen receptorα and β

“…Stimulation by androgens or estradiol couples classical steroid receptors to cytosolic signaling pathways and cell proliferation through the induction of an ER-AR-src ternary complex in LNCaP (Migliaccio et al, 2000) and EPN (Chieffi et al, 2003). Recently, we demonstrated that E2 treatment inhibited cell proliferation of EPN cells involving retinoblastoma-interacting zinc-finger protein 1 (RIZ1; Rossi et al, 2009), which is a downstream effector of ER activation in target tissues (Medici et al, 1999;Abbondanza et al, 2000;Gazzerro et al, 2006). In present study, we found a rapid transient activation of ERK1/2 after RAL treatment in EPN cells and a more sustained and prolonged ERK1/2 activation in CPEC.…”

“…In this study, we used the EPN prostate cell line, which expresses functional AR and both ERs (Sinisi et al, 2002;Rossi et al, 2009). EPN cells resulted by an apparently spontaneous adaptation to indefinite growth in vitro of primary cultures of epithelial prostate cells derived by normal tissue isolated from a prostate sample collected after radical prostatectomy for cancer.…”

“…These cells did not acquired classical marker of transformation, having anchoragedependent and contact-inhibited growth, and being unable to form tumors in nude mice (Sinisi et al, 2002). Cell line from a prostate cancer specimen (CPEC) was established in culture according to previously described criteria and methods, which did not include any transformation steps (Sinisi et al, 2002;Rossi et al, 2009).…”

“…Hormone treatment of cultured cells, preparation of nuclear extracts, immunoprecipitation, and Western blot analysis were performed as previously described Rossi et al, 2009). The following antibodies were used for the primary immunoreaction: a monoclonal antibody to ERa AER311 (Abbondanza et al, 1993); commercial polyclonal antibodies to ERb (H-150), Casp-3-3-3 (H-277), BCL-2 (DC21), Par-4 (H120) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and to AR (AR441; AbCam, NV); polyclonal anti-phospho-ERK1/2 (Thr202/ Tyr204) antibody (#9101S; New England Biolabs, Beverly, MA); polyclonal anti-Erk1/2 antibody (#sc-94-G; Santa Cruz Biotechnology, Inc.).…”

“…In this study, we evaluated the effects of RAL on prostate cancer epithelial cell growth and apoptosis, using the two following in vitro models: the prostate epithelial cell line EPN, which expresses AR and both ERa and ERb (Sinisi et al, 2002;Rossi et al, 2009); and a stable prostate epithelial cell line derived from a prostate cancer primary culture (CPEC), expressing low levels of ERb and AR, and lacking ERa. Here, we also investigated signaling pathways contributing to cell cycle progression and apoptosis that have been associated with RAL administration in these aforementioned models.…”