The Amino-terminal Domain of the Androgen Receptor Co-opts Extracellular Signal-regulated Kinase (ERK) Docking Sites in ELK1 Protein to Induce Sustained Gene Activation That Supports Prostate Cancer Cell Growth.

The ETS domain transcription factor ELK1 is in a repressive association with growth genes and is transiently activated through phosphorylation by ERK1/2. In prostate cancer (PCa) cells the androgen receptor (AR) is recruited by ELK1, via its amino-terminal domain (A/B), as a transcriptional co-activator, without ELK1 hyper-phosphorylation. Here we elucidate the structural basis of the interaction of AR with ELK1. The ELK1 polypeptide motifs required for co-activation by AR versus those required for activation of ELK1 by ERK were systematically mapped using a mammalian two-hybrid system and confirmed using a co-immunoprecipitation assay. The mapping precisely identified the two ERK-docking sites in ELK1, the D-box and the DEF (docking site for ERK, FXFP) motif, as the essential motifs for its cooperation with AR(A/B) or WTAR. In contrast, the transactivation domain in ELK1 was only required for activation by ERK. ELK1-mediated transcriptional activity of AR(A/B) was optimal in the absence of ELK1 binding partners, ERK1/2 and serum-response factor. Purified ELK1 and AR bound with a dissociation constant of 1.9 × 10-8 m A purified mutant ELK1 in which the D-box and DEF motifs were disrupted did not bind AR. An ELK1 mutant with deletion of the D-box region had a dominant-negative effect on androgen-dependent growth of PCa cells that were insensitive to MEK inhibition. This novel mechanism in which a nuclear receptor impinges on a signaling pathway by co-opting protein kinase docking sites to constitutively activate growth genes could enable rational design of a new class of targeted drug interventions.

Hybrid Enzalutamide Derivatives with Histone Deacetylase Inhibitor Activity Decrease Heat Shock Protein 90 and Androgen Receptor Levels and Inhibit Viability in Enzalutamide-Resistant C4-2 Prostate Cancer Cells.

Histone deacetylase inhibitors (HDACIs) can disrupt the viability of prostate cancer (PCa) cells through modulation of the cytosolic androgen receptor (AR) chaperone protein heat shock protein 90 (HSP90). However, toxicities associated with their pleiotropic effects could contribute to the ineffectiveness of HDACIs in PCa treatment. We designed hybrid molecules containing partial chemical scaffolds of enzalutamide and suberoylanilide hydroxamic acid (SAHA), with weakened intrinsic pan-HDACI activities, to target HSP90 and AR in enzalutamide-resistant PCa cells. The potency of the new molecules, compounds 2-75 [4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-2-fluoro-N-(7-(hydroxyamino)-7-oxoheptyl)benzamide] and 1005 [(E)-3-(4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-2-fluorophenyl)-N-hydroxyacrylamide], as inhibitors of nuclear and cytosolic histone deacetylases was substantially lower than that of SAHA in cell-free and in situ assays. Compounds 2-75 and 1005 antagonized gene activation by androgen without inducing chromatin association of AR. Enzalutamide had no effect on the levels of AR or HSP90, whereas the hybrid compounds induced degradation of both AR and HSP90, similar to (compound 1005) or more potently than (compound 2-75) SAHA. Similar to SAHA, compounds 2-75 and 1005 decreased the level of HSP90 and induced acetylation in a predicted approximately 55 kDa HSP90 fragment. Compared with SAHA, compound 2-75 induced greater hyperacetylation of the HDAC6 substrate α-tubulin. In contrast with SAHA, neither hybrid molecule caused substantial hyperacetylation of histones H3 and H4. Compounds 2-75 and 1005 induced p21 and caused loss of viability in the enzalutamide-resistant C4-2 cells, with efficacies that were comparable to or better than SAHA. The results suggest the potential of the new compounds as prototype antitumor drugs that would downregulate HSP90 and AR in enzalutamide-resistant PCa cells with weakened effects on nuclear HDACI targets.

Restoration of the cellular secretory milieu overrides androgen dependence of in vivo generated castration resistant prostate cancer cells overexpressing the androgen receptor.

It is believed that growth of castration resistant prostate cancer (CRPC) cells is enabled by sensitization to minimal residual post-castrate androgen due to overexpression of the androgen receptor (AR). Evidence is derived from androgen-induced colony formation in the absence of cell-secreted factors or from studies involving forced AR overexpression in hormone-dependent cells. On the other hand, standard cell line models established from CRPC patient tumors (e.g., LNCaP and VCaP) are hormone-dependent and require selection pressure in castrated mice to re-emerge as CRPC cells and the resulting tumors then tend to be insensitive to the androgen antagonist enzalutamide. Therefore, we examined established CRPC model cells produced by castration of mice bearing hormone-dependent cell line xenografts including CRPC cells overexpressing full-length AR (C4-2) or co-expressing wtAR and splice-variant AR-V7 that is incapable of ligand binding (22Rv1). In standard colony formation assays, C4-2 cells were shown to be androgen-dependent and sensitive to enzalutamide whereas 22Rv1 cells were incapable of colony formation under identical conditions. However, both C4-2 and 22Rv1 cells formed colonies in conditioned media derived from the same cells or from HEK293 fibroblasts that were proven to lack androgenic activity. This effect was (i) not enhanced by androgen, (ii) insensitive to enzalutamide, (iii) dependent on AR (in C4-2) and on AR-V7 and wtAR (in 22Rv1) and (iv) sensitive to inhibitors of several signaling pathways, similar to androgen-stimulation. Therefore, during progression to CRPC in vivo, coordinate cellular changes accompanying overexpression of AR may enable cooperation between hormone-independent activity of AR and actions of cellular secretory factors to completely override androgen-dependence and sensitivity to drugs targeting hormonal factors.

Differential effects of estrogen-dependent transactivation vs. transrepression by the estrogen receptor on invasiveness of HER2 overexpressing breast cancer cells.

Estrogen (E2) supports breast cancer cell growth but suppresses invasiveness and both actions are antagonized by anti-estrogens. As a consequence, anti-estrogen treatment may increase the invasive potential of estrogen receptor (ER)+ tumor cell sub-populations that are endocrine resistant due to HER2 amplification. Either transactivation or transrepression by E2/ER could lead to both up- and down-regulation of many genes. Inhibition of the transactivation function of ER is adequate to inhibit E2-dependent growth. However, the impact of inhibiting E2-dependent transactivation vs. transrepression by ER on regulation of invasiveness by E2 is less clear. Here we dissect the roles of ER-mediated transactivation and transrepression in the regulation of invasiveness of ER+/HER2+ breast cancer cells by E2. Knocking down the general ER co-activators CBP and p300 prevented activation by E2 of its classical target genes but did not interfere with the ability of E2 to repress its direct target genes known to support invasiveness and tumor progression; there was also no effect on invasiveness or the ability of E2 to regulate invasiveness. On the other hand, overexpression of a co-repressor binding site mutant of ER (L372R) prevented E2-dependent transrepression but not transactivation. The mutant ER abrogated the ability of E2 to suppress invasiveness. E2 can partially down-regulate HER2 but knocking down HER2 below E2-regulated levels did not affect invasiveness or the ability of E2 to regulate invasiveness, although it did inhibit growth. Therefore, in ER+/HER2+ cells, the E2-dependent transrepression by ER rather than its transactivation function is critical for regulation of invasiveness and this is independent of HER2 regulation by E2. The findings suggest that selective inhibitors of transactivation by ER may be more beneficial in reducing tumor progression than conventional anti-estrogens that also antagonize E2-dependent transrepression.

The ETS domain transcription factor ELK1 directs a critical component of growth signaling by the androgen receptor in prostate cancer cells.

The androgen receptor (AR) is essential for diverse aspects of prostate development and function. Molecular mechanisms by which prostate cancer (PC) cells redirect AR signaling to genes that primarily support growth are unclear. A systematic search for critical AR-tethering proteins led to ELK1, an ETS transcription factor of the ternary complex factor subfamily. Although genetically redundant, ELK1 was obligatory for AR-dependent growth and clonogenic survival in both hormone-dependent PC and castration-recurrent PC cells but not for AR-negative cell growth. AR required ELK1 to up-regulate a major subset of its target genes that was strongly and primarily enriched for cell growth functions. AR functioned as a coactivator of ELK1 by association through its A/B domain, bypassing the classical mechanism of ELK1 activation by phosphorylation and without inducing ternary complex target genes. The ELK1-AR synergy per se was ligand-independent, although it required ligand for nuclear localization of AR as targeting the AR A/B domain to the nucleus recapitulated the action of hormone; accordingly, Casodex was a poor antagonist of the synergy. ELK3, the closest substitute for ELK1 in structure/function and genome recognition, did not interact with AR. ELK1 thus directs selective and sustained gene induction that is a substantial and critical component of growth signaling by AR in PC cells. The ELK1-AR interaction offers a functionally tumor-selective drug target.

During hormone depletion or tamoxifen treatment of breast cancer cells the estrogen receptor apoprotein supports cell cycling through the retinoic acid receptor α1 apoprotein.

INTRODUCTION: Current hormonal adjuvant therapies for breast cancer including tamoxifen treatment and estrogen depletion are overall tumoristatic and are severely limited by the frequent recurrence of the tumors. Regardless of the resistance mechanism, development and progression of the resistant tumors requires the persistence of a basal level of cycling cells during the treatment for which the underlying causes are unclear. METHODS: In estrogen-sensitive breast cancer cells the effects of hormone depletion and treatment with estrogen, tamoxifen, all-trans retinoic acid (ATRA), fulvestrant, estrogen receptor α (ER) siRNA or retinoic acid receptor α (RARα) siRNA were studied by examining cell growth and cycling, apoptosis, various mRNA and protein expression levels, mRNA profiles and known chromatin associations of RAR. RARα subtype expression was also examined in breast cancer cell lines and tumors by competitive PCR. RESULTS: Basal proliferation persisted in estrogen-sensitive breast cancer cells grown in hormone depleted conditioned media without or with 4-hydroxytamoxifen (OH-Tam). Downregulating ER using either siRNA or fulvestrant inhibited basal proliferation by promoting cell cycle arrest, without enrichment for ErbB2/3+ overexpressing cells. The basal expression of RARα1, the only RARα isoform that was expressed in breast cancer cell lines and in most breast tumors, was supported by apo-ER but was unaffected by OH-Tam; RAR-ß and -γ were not regulated by apo-ER. Depleting basal RARα1 reproduced the antiproliferative effect of depleting ER whereas its restoration in the ER depleted cells partially rescued the basal cycling. The overlapping tamoxifen-insensitive gene regulation by apo-ER and apo-RARα1 comprised activation of mainly genes promoting cell cycle and mitosis and suppression of genes involved in growth inhibition; these target genes were generally insensitive to ATRA but were enriched in RAR binding sites in associated chromatin regions. CONCLUSIONS: In hormone-sensitive breast cancer, ER can support a basal fraction of S-phase cells (i) without obvious association with ErbB2/3 expression, (ii) by mechanisms unaffected by hormone depletion or OH-Tam and (iii) through maintenance of the basal expression of apo-RARα1 to regulate a set of ATRA-insensitive genes. Since isoform 1 of RARα is genetically redundant, its targeted inactivation or downregulation should be further investigated as a potential means of enhancing hormonal adjuvant therapy.

Chronic p27Kip1 Induction by Dexamethasone Causes Senescence Phenotype and Permanent Cell Cycle Blockade in Lung Adenocarcinoma Cells Over-expressing Glucocorticoid Receptor.

Dexamethasone (Dex), co-administered to lung adenocarcinoma patients with pemetrexed chemotherapy, protects against pemetrexed cytotoxicity by inducing reversible G1 arrest, reflected by the effect of Dex on FLT-PET images of patient tumors. However, perioperative Dex treatment increases survival but the mechanism is unknown. In cells with glucocorticoid receptor-α (GR) expression corresponding to higher clinical tumor levels, Dex-induced growth arrest was followed by marked cell expansion, beta-galactosidase expression and Ki67 negativity, despite variable p53 and K-RAS status. Dex induced a transient early surge in p21Cip1. However, a progressive, irreversible loss of clonogenic growth, whose time of onset was dependent on GR level and Dex dose, was independent of p21Cip1and caused by gradual accumulation of p27Kip1 due to transcriptional activation of p27Kip1 by Dex. This effect was independent of canonical pathways of senescence or p27Kip1 regulation. The in vitro observations were reflected by growth suppression and P27Kip1 induction in GR-overexpressing tumor xenografts compared with isogenic low-GR tumors. Extended Dex treatment induces irreversible cell cycle blockade and a senescence phenotype through chronic activation of the p27Kip1 gene in GR overexpressing lung tumor cell populations and hence could improve outcome of surgery/pemetrexed chemotherapy and sensitize tumors to immunotherapy.

Role of the short isoform of the progesterone receptor in breast cancer cell invasiveness at estrogen and progesterone levels in the pre- and post-menopausal ranges.

Overexpression of the progesterone receptor (PR) isoform A (PR-A) is a negative prognosticator for estrogen receptor (ER)-positive breast cancer but in vitro studies have implicated PR-B in progestin-induced invasiveness. As estrogen is known to suppress invasiveness and tumor progression and as the in vitro studies were conducted in models that either lacked ER or excluded estrogen, we examined the role of PR isoforms in the context of estrogen signaling. Estrogen (< 0.01nM) strongly suppressed invasiveness in various ER+ model cell lines. At low (< 1nM) concentrations, progestins completely abrogated inhibition of invasiveness by estrogen. It was only in a higher (5 nM - 50 nM) concentration range that progestins induced invasiveness in the absence of estrogen. The ability of low dose progestins to rescue invasiveness from estrogen regulation was exclusively mediated by PR-A, whereas PR-B mediated the estrogen-independent component of progestin-induced invasiveness. Overexpression of PR-A lowered the progestin concentration needed to completely rescue invasiveness. Among estrogen-regulated genes, progestin/PR-A counter-regulated a distinctive subset, including breast tumor progression genes (e.g., HES1, PRKCH, ELF5, TM4SF1), leading to invasiveness. In this manner, at relatively low hormone concentrations (corresponding to follicular stage and post-menopausal breast tissue or plasma levels), progesterone influences breast cancer cell invasiveness by rescuing it from estrogen regulation via PR-A, whereas at higher concentrations the hormone also induces invasiveness independent of estrogen signaling, through PR-B. The findings point to a direct functional link between PR-A and progression of luminal breast cancer in the context of the entire range of pre- and post-menopausal plasma and breast tissue hormone levels.

Glucocorticoid receptor status is a principal determinant of variability in the sensitivity of non-small-cell lung cancer cells to pemetrexed.

INTRODUCTION: Pemetrexed is an S-phase targeted drug in front-line or maintenance therapy of advanced nonsquamous non-small-cell lung cancer (NSCLC) but methods are needed for predicting the drug response. Dexamethasone is typically administered the day before, the day of, and the day after pemetrexed. As dexamethasone strongly regulates many genes including p53 through the glucocorticoid receptor (GR), we hypothesized that dexamethasone influences tumor response to pemetrexed. METHODS: Eight nonsquamous NSCLC cell line models with varied p53 and GRα/GRß status were used for gene expression and cell-cycle analyses and for loss- or gain-of-function experiments. RESULTS: In three cell lines dexamethasone profoundly, but reversibly, suppressed the fraction of S-phase cells. Dexamethasone also reversibly repressed expression of thymidylate synthase and dihydrofolate reductase, which are primary targets of pemetrexed but are also quintessential S-phase enzymes as well as the S-phase-dependent expression of thymidine kinase 1. Dexamethasone also decreased expression of the major pemetrexed transporters, the reduced folate carrier and the proton coupled folate transporter. Only cells expressing relatively high GRα showed these dexamethasone effects, regardless of p53 status. In cells expressing low GRα, the dexamethasone response was rescued by ectopic GRα. Further, depletion of p53 did not attenuate the dexamethasone effects. The presence of dexamethasone during pemetrexed treatment protected against pemetrexed cytotoxicity in only the dexamethasone responsive cells. CONCLUSIONS: The results predict that in nonsquamous NSCLC tumors, reversible S-phase suppression by dexamethasone, possibly combined with a reduction in the drug transporters, attenuates responsiveness to pemetrexed and that GR status is a principal determinant of tumor variability of this response.