ID4 promotes AR expression and blocks tumorigenicity of PC3 prostate cancer cells.

Deregulation of tumor suppressor genes is associated with tumorigenesis and the development of cancer. In prostate cancer, ID4 is epigenetically silenced and acts as a tumor suppressor. In normal prostate epithelial cells, ID4 collaborates with androgen receptor (AR) and p53 to exert its tumor suppressor activity. Previous studies have shown that ID4 promotes tumor suppressive function of AR whereas loss of ID4 results in tumor promoter activity of AR. Previous study from our lab showed that ectopic ID4 expression in DU145 attenuates proliferation and promotes AR expression suggesting that ID4 dependent AR activity is tumor suppressive. In this study, we examined the effect of ectopic expression of ID4 on highly malignant prostate cancer cell, PC3. Here we show that stable overexpression of ID4 in PC3 cells leads to increased apoptosis and decreased cell proliferation and migration. In addition, in vivo studies showed a decrease in tumor size and volume of ID4 overexpressing PC3 cells, in nude mice. At the molecular level, these changes were associated with increased androgen receptor (AR), p21, and AR dependent FKBP51 expression. At the mechanistic level, ID4 may regulate the expression or function of AR through specific but yet unknown AR co-regulators that may determine the final outcome of AR function.

Chronic exposure to benzyl butyl phthalate (BBP) alters social interaction and fear conditioning in male adult rats: alterations in amygdalar MeCP2, ERK1/2 and ERα.

OBJECTIVES: Benzyl Butyl Phthalate (BBP) is an industrial plasticizer that has an unknown action in the central nervous system. Phthalates have recently been associated with behavioral actions that are linked to their endocrine disrupting properties. The purpose of this study was to investigate the behavioral and molecular effects of BBP treatment in male rats. DESIGN: Male rats were chronically exposed to BBP in the drinking water (5.0 ppm and 10.0 ppm) throughout adolescence and into the adult phase of life. Their behavior was then assessed in a learning and memory task (fear conditioning), open field exploration and a test of sociability. RESULTS: BBP treated rats showed decreased freezing in fear conditioning, no changes in open field exploration, and increased aberrant social behavior. Rats were sacrificed at post natal day 140 and blood and brains were harvested and processed. We found increased hormonally active estrogen, 17-ß estradiol, in the serum of BBP treated rats. BBP treatment also induced changes in amygdalar proteins related to synaptic plasticity including decreased MeCP2 levels that correlated with tests of sociability with no changes in stress related proteins such as nuclear factor kappa B (NFkB). We also found alterations in physiological responses as measured by body weight without changes in food consumption suggesting disruption of metabolism and body homeostasis. CONCLUSIONS: We suggest that BBP administration disrupts normal learning and social behavior, and that these effects could be related to alterations of amygdala function.

The helix-loop-helix transcriptional regulator Id4 is required for terminal differentiation of luminal epithelial cells in the prostate.

Inhibitor of differentiation 4 (Id4), a member of the helix-loop-helix family of transcriptional regulators has emerged as a tumor suppressor in prostate cancer. In this study we investigated the effect of loss of Id4 (Id4-/-) on mouse prostate development. Histological analysis was performed on prostates from 25 days, 3 months and 6 months old Id4-/- mice. Expression of Amacr, Ck8, Ck18, Fkbp51, Fkbp52, androgen receptor, Pten, sca-1 and Nkx3.1 was investigated by immunohistochemistry. Results were compared to the prostates from Nkx3.1-/- mice. Id4-/- mice had smaller prostates with fewer and smaller tubules. Subtle PIN like lesions were observed at 6mo. Decreased Nkx3.1 and Pten and increased stem cell marker sca-1, PIN marker Amacr and basal cell marker p63 was observed at all ages. Persistent Ck8 and Ck18 expression suggested that loss of Id4 results in epithelial commitment but not terminal differentiation in spite of active Ar. Loss of Id4 attenuates normal prostate development and promotes hyperplasia/ dysplasia with PIN like lesions. The results suggest that loss of Id4 maintains stem cell phenotype of "luminal committed basal cells", identifying a unique prostate developmental pathway regulated by Id4.

ID4 regulates transcriptional activity of wild type and mutant p53 via K373 acetylation.

Given that mutated p53 (50% of all human cancers) is over-expressed in many cancers, restoration of mutant p53 to its wild type biological function has been sought after as cancer therapy. The conformational flexibility has allowed to restore the normal biological function of mutant p53 by short peptides and small molecule compounds. Recently, studies have focused on physiological mechanisms such as acetylation of lysine residues to rescue the wild type activity of mutant p53. Using p53 null prostate cancer cell line we show that ID4 dependent acetylation promotes mutant p53 DNA-binding capabilities to its wild type consensus sequence, thus regulating p53-dependent target genes leading to subsequent cell cycle arrest and apoptosis. Specifically, by using wild type, mutant (P223L, V274F, R175H, R273H), acetylation mimics (K320Q and K373Q) and non-acetylation mimics (K320R and K373R) of p53, we identify that ID4 promotes acetylation of K373 and to a lesser extent K320, in turn restoring p53-dependent biological activities. Together, our data provides a molecular understanding of ID4 dependent acetylation that suggests a strategy of enhancing p53 acetylation at sites K373 and K320 that may serve as a viable mechanism of physiological restoration of mutant p53 to its wild type biological function.

Inactivation of ID4 promotes a CRPC phenotype with constitutive AR activation through FKBP52.

Castration-resistant prostate cancer (CRPC) is the emergence of prostate cancer cells that have adapted to the androgen-depleted environment of the prostate. In recent years, targeting multiple chaperones and co-chaperones (e.g., Hsp27, FKBP52) that promote androgen receptor (AR) signaling and/or novel AR regulatory mechanisms have emerged as promising alternative treatments for CRPC. We have shown that inactivation of inhibitor of differentiation 4 (ID4), a dominant-negative helix loop helix protein, promotes de novo steroidogenesis and CRPC with a gene expression signature that resembles constitutive AR activity in castrated mice. In this study, we investigated the underlying mechanism through which loss of ID4 potentiates AR signaling. Proteomic analysis between prostate cancer cell line LNCaP (L+ns) and LNCaP lacking ID4 (L(-)ID4) revealed elevated levels of Hsp27 and FKBP52, suggesting a role for these AR-associated co-chaperones in promoting constitutively active AR signaling in L(-)ID4 cells. Interestingly, protein interaction studies demonstrated a direct interaction between ID4 and the 52-kDa FK506-binding protein (FKBP52) in vitro, but not with AR. An increase in FKBP52-dependent AR transcriptional activity was observed in L(-)ID4 cells. Moreover, pharmacological inhibition of FKBP52-AR signaling, by treatment with MJC13, attenuated the tumor growth, weight, and volume in L(-)ID4 xenografts. Together, our results demonstrate that ID4 selectively regulates AR activity through direct interaction with FKBP52, and its loss, promotes CRPC through FKBP52-mediated AR signaling.

Intra-tumoral delivery of functional ID4 protein via PCL/maltodextrin nano-particle inhibits prostate cancer growth.

ID4, a helix loop helix transcriptional regulator has emerged as a tumor suppressor in prostate cancer. Epigenetic silencing of ID4 promotes prostate cancer whereas ectopic expression in prostate cancer cell lines blocks cancer phenotype. To directly investigate the anti-tumor property, full length human recombinant ID4 encapsulated in biodegradable Polycaprolactone/Maltodextrin (PCL-MD) nano-carrier was delivered to LNCaP cells in which the native ID4 was stably silenced (LNCaP(-)ID4). The cellular uptake of ID4 resulted in increased apoptosis, decreased proliferation and colony formation. Intratumoral delivery of PCL-MD ID4 into growing LNCaP(-)ID4 tumors in SCID mice significantly reduced the tumor volume compared to the tumors treated with chemotherapeutic Docetaxel. The study supports the feasibility of using nano-carrier encapsulated ID4 protein as a therapeutic. Mechanistically, ID4 may assimilate multiple regulatory pathways for example epigenetic re-programming, integration of multiple AR co-regulators or signaling pathways resulting in tumor suppressor activity of ID4.

Inhibitor of differentiation 4 (ID4) inactivation promotes de novo steroidogenesis and castration-resistant prostate cancer.

Prostate cancer (PCa) is the most commonly diagnosed cancer in men in the Western world. The transition of androgen-dependent PCa to castration-resistant (CRPC) is a major clinical manifestation during disease progression and presents a therapeutic challenge. Our studies have shown that genetic ablation of inhibitor of differentiation 4 (Id4), a dominant-negative helix loop helix protein, in mice results in prostatic intraepithelial neoplasia lesions and decreased Nkx3.1 expression without the loss of androgen receptor (Ar) expression. ID4 is also epigenetically silenced in the majority of PCa. However, the clinical relevance and molecular pathways altered by ID4 inactivation in PCa are not known. This study investigates the effect of loss of ID4 in PCa cell lines on tumorigenicity and addresses the underlying mechanism. Stable silencing of ID4 in LNCaP cells (L-ID4) resulted in increased proliferation, migration, invasion, and anchorage-independent growth. An increase in the rate of tumor growth, weight, and volume was observed in L-ID4 xenografts compared with that in the LNCaP cells transfected with nonspecific short hairpin RNA (L+ns) in noncastrated mice. Interestingly, tumors were also observed in castrated mice, suggesting that loss of ID4 promotes CRPC. RNA sequence analysis revealed a gene signature mimicking that of constitutively active AR in L-ID4, which was consistent with gain of de novo steroidogenesis. Prostate-specific antigen expression as a result of persistent AR activation was observed in L-ID4 cells but not in L+ns cells. The results demonstrate that ID4 acts as a tumor suppressor in PCa, and its loss, frequently observed in PCa, promotes CRPC through constitutive AR activation.