Mesfin Gonit

The ETS Domain Transcription Factor Elk1 Directs a Critical Component of Growth Signaling by the Androgen Receptor in Prostate Cancer Cells

Background: Mechanisms that redirect androgen receptor signaling to primarily support prostate tumor growth are poorly understood. Results: Prostate cancer cells were addicted to ELK1, which tethered AR to activate growth genes in hormone-dependent and castration-recurrent PC without ELK1 phosphorylation. Conclusion: ELK1 directs a critical arm of transcriptional growth signaling by AR that is preserved in CRPC. Significance: The ELK1-AR interaction offers a functionally tumor-selective drug target. 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.

C/EBPα redirects androgen receptor signaling through a unique bimodal interaction

Nuclear expression of CCAAT enhancer binding protein-α (C/EBPα), which supports tissue differentiation through several antiproliferative protein–protein interactions, augurs terminal differentiation of prostate epithelial cells. C/EBPα is also a tumor suppressor, but in many tumors its antiproliferative interactions may be attenuated by de-phosphorylation. C/EBPα acts as a corepressor of the classical androgen response element (ARE)-mediated gene activation by the androgen receptor (AR), but this is paradoxical as the genotropic actions of AR are crucial not only for the growth of the prostate but also for its maintenance and function. We show that DNA-bound C/EPBα recruits AR to activate transcription. C/EBPα-dependent trans-activation by AR also overrode suppression of AREs by C/EBPα elsewhere in a promoter. This mechanism was remarkable in that its androgen dependence was apparently for nuclear translocation of AR; it was otherwise androgen independent, flutamide insensitive and tolerant to disruption of AR dimerization. Gene response profiles and global chromatin associations in situ supported the direct bimodal regulation of AR transcriptional signaling by C/EBPα. This unique mechanism explains the functional coordination between AR and C/EPBα in the prostate and also shows that hormone-refractory AR signaling in prostate cancer could occur through receptor tethering.

Hormone Depletion-Insensitivity of Prostate Cancer Cells Is Supported by the AR Without Binding to Classical Response Elements

A need for androgen response elements (AREs) for androgen receptor (AR)-dependent growth of hormone depletion-insensitive prostate cancer is generally presumed. In such cells, androgen-independent activation by AR of certain genes has been attributed to selective increases in basal associations of AR with putative enhancers. We examined the importance of AR binding to DNA in prostate cancer cells in which proliferation in the absence of hormone was profoundly (∼ 90%) dependent on endogenous AR and where the receptor was not up-regulated or mutated but was predominantly nuclear. Here, ARE-mediated promoter activation and the binding of AR to a known ARE in the chromatin remained entirely androgen dependent, and the cells showed an androgen-responsive gene expression profile with an unaltered sensitivity to androgen dose. In the same cells, a different set of genes primarily enriched for cell division functions was activated by AR independently of hormone and significantly overlapped the signature gene overexpression profile of hormone ablation-insensitive clinical tumors. After knockdown of endogenous AR, hormone depletion-insensitive cell proliferation and AR apoprotein-dependent gene expression were rescued by an AR mutant that was unable to bind to ARE but that could transactivate through a well-established AR tethering protein. Hormone depletion-insensitive AR binding sites in the chromatin were functional, binding, and responding to both the wild-type and the mutant AR and lacked enrichment for canonical or noncanonical ARE half-sites. Therefore, a potentially diverse set of ARE-independent mechanisms of AR interactions with target genes must underlie truly hormone depletion-insensitive gene regulation and proliferation in prostate cancer.

Regulation of Folate Receptor Internalization by Protein Kinase C α

The glycosyl-phosphatidylinositol anchored folate receptor (FR) mediates selective delivery of a broad range of experimental drugs to the receptor-rich tumors, but molecular mechanisms controlling FR internalization have not been adequately studied. FR quantitatively recycles between the cell surface and endocytic compartments via a Cdc42-dependent pinocytic pathway. Protein kinase C (PKC) activators including diacylglycerol and phorbol ester have previously been reported to increase the proportion of FR on the cell surface. Here we identify the alpha-subtype of PKC as the mediator of phorbol ester action on FR recycling and provide evidence that activated PKCalpha is recruited to FR-rich membrane microdomains where, in association with its receptor RACK1, it inhibits FR internalization; the activation state of Cdc42 remains unaltered. We also show that the PKC substrate, annexin II, is required for FR internalization. The studies clarify a molecular mechanism for the regulation of FR recycling through PKC which could potentially be exploited for effective drug delivery.

C/EBPγ Is a Critical Regulator of Cellular Stress Response Networks through Heterodimerization with ATF4

ABSTRACT The integrated stress response (ISR) controls cellular adaptations to nutrient deprivation, redox imbalances, and endoplasmic reticulum (ER) stress. ISR genes are upregulated in stressed cells, primarily by the bZIP transcription factor ATF4 through its recruitment to cis-regulatory C/EBP:ATF response elements (CAREs) together with a dimeric partner of uncertain identity. Here, we show that C/EBPγ:ATF4 heterodimers, but not C/EBPβ:ATF4 dimers, are the predominant CARE-binding species in stressed cells. C/EBPγ and ATF4 associate with genomic CAREs in a mutually dependent manner and coregulate many ISR genes. In contrast, the C/EBP family members C/EBPβ and C/EBP homologous protein (CHOP) were largely dispensable for induction of stress genes. Cebpg−/− mouse embryonic fibroblasts (MEFs) proliferate poorly and exhibit oxidative stress due to reduced glutathione levels and impaired expression of several glutathione biosynthesis pathway genes. Cebpg−/− mice (C57BL/6 background) display reduced body size and microphthalmia, similar to ATF4-null animals. In addition, C/EBPγ-deficient newborns die from atelectasis and respiratory failure, which can be mitigated by in utero exposure to the antioxidant, N-acetyl-cysteine. Cebpg−/− mice on a mixed strain background showed improved viability but, upon aging, developed significantly fewer malignant solid tumors than WT animals. Our findings identify C/EBPγ as a novel antioxidant regulator and an obligatory ATF4 partner that controls redox homeostasis in normal and cancerous cells.

Expression and sub‐cellular localization of the CCAAT/enhancer binding protein α in relation to postnatal development and malignancy of the prostate

C/EBPα is a critical mediator of terminal differentiation and a tumor suppressor through its strong antiproliferative actions on cell cycle regulatory proteins. C/EBPα also appears to regulate androgen receptor (AR) AR signaling. There, is a paucity of information on the expression and sub‐cellular localization of C/EBPα in normal mouse and human prostate and in prostate cancer.

Androgen activation of the folate receptor α gene through partial tethering of the androgen receptor by C/EBPα

The folate receptor α (FRα) is critical for normal embryonic and fetal development. The receptor has a relatively narrow tissue specificity which includes the visceral endoderm and the placenta and mediates delivery of folate, inadequacy of which results in termination of pregnancy or developmental defects. We have previously reported that the FRα gene is negatively and directly regulated by estrogen and positively but indirectly by progesterone and glucocorticoid. To further investigate hormonal control of this gene and in view of the growing evidence for the importance of the androgen receptor (AR) in endometrial and placental functions, we examined the response of the FRα gene to androgen. Here we demonstrate that the FRα gene is directly activated by androgen. The P4 promoter of the FRα gene is the target of hormone-dependent activation by the androgen receptor (AR) in a manner that is co-activator-dependent. The site of functional association of AR in the FRα gene maps to a 35bp region occurring ∼1500bp upstream of the target promoter. The functional elements within this region are an androgen response element (ARE) half-site and a non-canonical C/EBP element that cooperate to recruit AR in a manner that is dependent on the DNA-bound C/EBPα. Since the placenta is rich in C/EBPα, the findings underscore the multiplicity of mechanisms by which the FRα gene is under the exquisite control of steroid hormones.

Mechanisms of ARE-Independent Gene Activation by the Androgen Receptor in Prostate Cancer Cells: Potential Targets for Better Intervention Strategies

Molecular mechanisms that redirect androgen or AR action to primarily support growth in prostate cancer (PC) cells are not adequately understood. In PC cells in which AR supports robust cell growth in the absence of hormone, AR is localized in the nucleus independent of hormone; still, in these cells androgen is required for activation of its classical target genes that involves AR binding to canonical or noncanonical androgen response elements (AREs). However, following either hormone-dependent or -independent nuclear translocation, AR activates a distinct set of critical growth genes in a ligand-insensitive manner through putative tethered associations of AR with chromatin. Consistent with these observations, splice variants of AR that lack the ligand binding domain support PC growth by activating a transcriptional program distinct from that induced by androgen plus full length AR. Indeed, several studies suggest that specific AR tethering proteins help to redirect AR toward targeting gene sets appropriate to the physiological context. These proteins may also simultaneously suppress the activation of other genes by AR. This review describes how transcriptional signaling by AR is directed by other chromatin bound transcription factors, comprising the AR “tetherome,” that could work either in concert with AREs or completely independent of them. The potential utility of specific tether-dependent growth signaling mechanisms of AR as tumor-selective drug targets in both early stage and advanced prostate cancer is discussed.

Hormonal Control of Folate Receptor Genes

The two major human folate receptor (FR) isoforms, α and β, are glycosyl phosphatidylinositol-anchored glycoproteins with limited expression in normal tissues including epithelial cells (FRα) and hematopoietic cells (FRβ). FRα plays a critical role during pregnancy in ensuring an adequate supply of folate to the fetus. FRs are a potential means of delivering a range of therapeutics in cancer and inflammatory diseases in a tissue-targeted manner. The FRα gene is under the exquisite control of steroid hormones which regulate the protein expression by diverse and nonclassical molecular mechanisms. The FRβ gene on the other hand is regulated by retinoid compounds through a nonclassical mechanism. The physiological mechanisms of FR gene regulation could be utilized to selectively enhance the receptor expression and consequently drug delivery to the target tissues. The soluble form of FRα may also be effectively utilized in this manner as a serum marker in the early detection of certain cancers. The in vitro and animal model studies in this field have advanced to a stage that warrants direct clinical trials to validate the proposed treatment modalities.

Abstract PR02: C/EBPG: A critical stress response regulator with a pro-oncogenic role

Stress signals such as amino acid deprivation and redox imbalances activate the integrated stress response (ISR), which allows cells to alleviate the stress or to undergo apoptosis if the stress is unresolved. The ISR is also important for the survival of cancer cells, since they experience stress in the form of nutrient and oxygen deprivation and hence frequently activate stress-response pathways. Cellular stresses trigger up-regulation of the transcription factor ATF4, which subsequently activates stress response genes through recruitment to cis-regulatory sites known as C/EBP:ATF response elements (CAREs). Although the role of ATF4 in regulating stress response is well established, the identity of the C/EBP partner that heterodimerizes with ATF4 to execute this crucial function remains obscure. Here we show that the transcription factor C/EBPG is a critical partner of ATF4 and that C/EBPG:ATF4 heterodimers are the predominant CARE-binding species in stressed cells. Similar to ATF4, C/EBPG is necessary for resistance of MEFs to oxidative stress. MEFs lacking C/EBPG show increased levels of reactive oxygen species (ROS) as a consequence of impaired glutathione biosynthesis, as was also seen in ATF4-deficient MEFs. C/EBPG is required for stress-induced association of ATF4 with CAREs and the subsequent activation of several critical stress-responsive genes, including those with known pro-oncogenic functions. Accordingly, resistance to stress conferred by C/EBPG also facilitates the growth of cancer cells. Depletion of C/EBPG impairs the proliferation of cancer cells in vitro and elevates ROS levels. These effects can be suppressed by addition of the antioxidant, N-acetylcysteine. Mice lacking C/EBPG are smaller in size and show defective eye lens formation, similar to ATF4-deficient mice. The absence of C/EBPG also causes perinatal mortality due to pulmonary atelectasis and respiratory failure. This mortality can be rescued by in utero exposure to N-acetylcysteine. Accordingly, gene expression analysis suggests the presence of increased oxidative stress and impaired expression of stress-responsive genes in the lungs of Cebpg -/- newborn mice. Interestingly, Cebpg -/- mice on a mixed strain background, which do not show perinatal lethality, are resistant to the development of solid malignant tumors. These findings suggest that the role of C/EBPG as a stress response regulator may be important for tumor development/progression. A pro-oncogenic function for C/EBPG is also suggested by the observation that elevated C/EBPG levels are associated with poor patient prognosis in several clinical cancer studies. Activation of stress-responsive genes through upregulation of C/EBPG could be a mechanism deployed by cancer cells to mitigate the high levels of ROS and metabolic stresses that they experience. The importance of C/EBPG and its targets in tumor cells could potentially be exploited to devise novel anti-cancer therapies. Citation Format: Manasi K. Mayekar, Christopher J. Huggins, Nancy Martin, Karen L. Saylor, Mesfin Gonit, Parthav Jailwala, Manjula Kasoji, Diana C. Haines, Octavio A. Quinones, Peter F. Johnson. C/EBPG: A critical stress response regulator with a pro-oncogenic role. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr PR02.