Recent studies have shown that nuclear transcription factor cyclic adenosine monophosphate response element binding protein (CREB) is overexpressed in many different types of cancers. Therefore, CREB has been pursued as a novel cancer therapeutic target. Naphthol AS-E and its closely related derivatives have been shown to inhibit CREB-mediated gene transcription and cancer cell growth. Previously, we identified naphthamide 3a as a different chemotype to inhibit CREB’s transcription activity. In a continuing effort to discover more potent CREB inhibitors, a series of structural congeners of 3a was designed and synthesized. Biological evaluations of these compounds uncovered compound 3i (666-15) as a potent and selective inhibitor of CREB-mediated gene transcription (IC50 = 0.081 ± 0.04 μM). 666-15 also potently inhibited cancer cell growth without harming normal cells. In an in vivo MDA-MB-468 xenograft model, 666-15 completely suppressed the tumor growth without overt toxicity. These results further support the potential of CREB as a valuable cancer drug target.

Background: HIF1␣ is a target of anticancer therapy. Results: Lysines within the HIF1␣ N terminus are targets of HDAC4 deacetylation. HDAC4 inhibition causes the increase of HIF1␣ protein acetylation and decrease of protein stability, which lead to the reduction of HIF-1-mediated target gene expressions and activities in cancer cells. Conclusion: HDAC4 provides a novel HIF1␣ regulatory mechanism. Significance: HIF-1 can be targeted by HDAC4 inhibition.

During the reproductive cycle, fluctuations in circulating estrogens affect multiple homeostatic systems controlled by hypothalamic neurons. Two of these neuronal populations are arcuate proopiomelanocortin and neuropeptide Y neurons, which control energy homeostasis and feeding. Estradiol modulates these neurons either through the classical estrogen receptors (ERs) to control gene transcription or through a G protein-coupled receptor (mER) activating multiple signaling pathways. To differentiate between these two divergent ER-mediated mechanisms and their effects on homeostasis, female guinea pigs were ovariectomized and treated systemically with vehicle, estradiol benzoate (EB) or STX, a selective mER agonist, for 4 wk, starting 7 d after ovariectomy. Individual body weights were measured after each injection day for 28 d, at which time the animals were euthanized, and the arcuate nucleus was microdissected. As predicted, the body weight gain was significantly lower for EB-treated females after d 5 and for STX-treated females after d 12 compared with vehicle-treated females. Total arcuate RNA was extracted from all groups, but only the vehicle and STX-treated samples were prepared for gene microarray analysis using a custom guinea pig gene microarray. In the arcuate nucleus, 241 identified genes were significantly regulated by STX, several of which were confirmed by quantitative real-time PCR and compared with EB-treated groups. The lower weight gain of EB-treated and STX-treated females suggests that estradiol controls energy homeostasis through both ERalpha and mER-mediated mechanisms. Genes regulated by STX indicate that not only does it control neuronal excitability but also alters gene transcription via signal transduction cascades initiated from mER activation.

Background: HIF1␣ and p300 are key components of HIF-1 transcription complex. Results: Lysine 709 of HIF1␣ is acetylated by p300, which increases protein stability and HIF-1 activity. Conclusion: p300 has a novel function in stabilizing HIF1␣ by Lys-709 acetylation. Significance: New insights in how HIF1␣ is post-translationally regulated by its cofactor to ensure HIF-1 activity.

Low voltage-activated (T-type) Ca2ϩ channels are responsible for generating low-threshold spikes (LTS) that facilitate burst firing and transmitter release in neurons. The T-type Ca 2ϩ channels contain a regulatory ␣1 subunit, and several isoforms of the ␣1 subunit (Cav3.1, 3.2, 3.3) have been cloned. The Cav 3.1 ␣1 subunit is abundantly expressed in the hypothalamus. Previously, we found that 17 ␤-estradiol (E2) increased the number of arcuate neurons expressing LTS. Therefore, we used an ovariectomized female guinea pig model to measure the distribution and regulation of Cav3.1 mRNA expression by E2. Guinea pig Cav3.1 ␣1 subunit sequences, which were cloned by PCR, were used in ribonuclease protection (RPA) and in situ hybridization assays to evaluate mRNA expression. Based on a RPA, E2 significantly increased the mRNA expression of Cav3.1 ␣1 subunit in the mediobasal hypothalamus and the pituitary. In situ hybridization analysis revealed that E2 significantly increased Cav 3.1 mRNA expression in medial preoptic nuclei, bed nuclei stria terminalis, and the arcuate nucleus. Whole-cell patch recordings in arcuate neurons revealed that E2 treatment significantly increased the peak T-type Ca 2ϩ current density by twofold without affecting the activation/inactivation characteristics and augmented the rebound excitation by threefold to fourfold. These results suggest that estrogen regulates the mRNA expression of T-type calcium channels, which leads to increased functional expression of the channel. Increased expression of T-type channels could be one mechanism by which estrogen augments burst firing and transmitter release in hypothalamic neurons.

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Kisspeptin is essential for reproductive functions in humans. As a model for the human we have used the female guinea pig, which has a long ovulatory cycle similar to that of primates. Initially, we cloned a guinea pig kisspeptin cDNA sequence and subsequently explored the distribution and 17β-estradiol (E2) regulation of kisspeptin mRNA (Kiss1) and protein (kisspeptin) by using in situ hybridization, real-time PCR and immunocytochemistry. In ovariectomized females, Kiss1 neurons were scattered throughout the preoptic periventricular areas (PV), but the vast majority of Kiss1 neurons were localized in the arcuate nucleus (Arc). An E2 treatment that first inhibits (negative feedback) and then augments (positive feedback) serum luteinizing hormone (LH) increased Kiss1 mRNA density and number of cells expressing Kiss1 in the PV at both time points. Within the Arc, Kiss1 mRNA density was reduced at both time points. Quantitative real-time PCR confirmed the in situ hybridization results during positive feedback. E2 reduced the number of immunoreactive kisspeptin cells in the PV at both time points, perhaps an indication of increased release. Within the Arc, the kisspeptin immunoreactivity was decreased during negative feedback but increased during positive feedback. Therefore, it appears that in guinea pig both the PV and the Arc kisspeptin neurons act cooperatively to excite gonadotropin-releasing hormone (GnRH) neurons during positive feedback. We conclude that E2 regulation of negative and positive feedback may reflect a complex interaction of the kisspeptin circuitry, and both the PV and the Arc respond to hormone signals to encode excitation of GnRH neurons during the ovulatory cycle.

Despite recent advances, the efficacy of androgen/androgen receptor (AR)-targeted therapy remains  limited for many patients with metastatic prostate cancer. This is in part because prostate cancers adaptively switch to the androgen/AR-independent pathway for survival and growth, thereby conferring therapy resistance. Tumor hypoxia is considered as a major cause of treatment resistance. However, the exact mechanism is largely unclear. Here we report that chronic-androgen deprivation therapy (ADT) in the condition of hypoxia induces adaptive androgen/AR-independence, and therefore confers resistance to androgen/AR-targeted therapy, e.g., enzalutamide. Mechanistically, this is mediated by glucose-6-phosphate isomerase (GPI), which is transcriptionally repressed by AR in hypoxia, but restored and increased by AR inhibition. In turn, GPI maintains glucose metabolism and energy homeostasis in hypoxia by redirecting the glucose flux from androgen/AR-dependent pentose phosphate pathway (PPP) to hypoxia-induced glycolysis pathway, thereby reducing the growth inhibitory effect of enzalutamide. Inhibiting GPI overcomes the therapy resistance in hypoxia in vitro and increases enzalutamide efficacy in vivo.