The elucidation of species-specific receptor pharmacology: a case study using subtype selective para- and meta-carborane estrogen receptor agonists.

Estrogen receptors are essential pharmacological targets for treating hormonal disorders and estrogen-dependent malignancies. Selective activation of estrogen receptor (ER) ß is hypothesized to provide therapeutic benefit with reduced risk of unwanted estrogenic side-effects associated with ERα activity. However, activating ERß without activating α is challenging due to the high sequence and structural homology between the receptor subtypes. We assessed the impact of structural modifications to the parent compound OSU-ERß-12 on receptor subtype binding selectivity using cell-free binding assays. Functional selectivity was evaluated by transactivation in HEK-293 cells overexpressing human or murine estrogen receptors. In vivo selectivity was examined through the uterotrophic effects of the analogs after oral administration in estrogen-naïve female mice. Furthermore, we evaluated the in vivo pharmacokinetics of the analogs following single dose IV and oral administration. Regarding selectivity, a single compound exhibited greater functional selectivity than OSU-ERß-12 for human ERß. However, like others in the meta-carborane series, its poor in vivo pharmacokinetics limit its suitability for further development. Surprisingly, and at odds with their pharmacokinetic and in vitro human activity data, most analogs potently induced uterotrophic effects in estrogen-naïve female mice. Further investigation of activity in HEK293 cells expressing murine estrogen receptors revealed species-specific differences in the ER-subtype selectivity of these analogs. Our findings highlight species-specific receptor pharmacology and the challenges it poses to characterizing developmental therapeutics in preclinical species. Significance Statement This study investigates para- and meta-substituted carborane analogs targeting estrogen receptors, revealing the greater selectivity of carborane analogs for human ERß compared to the mouse homolog. These findings shed light on the intricacies of using preclinical species in drug development to predict human pharmacology. The report also provides insights for the refinement and optimization of carborane analogs as potential therapeutic agents for estrogen-related disease states.

Decoupling FcRn and tumor contributions to elevated immune checkpoint inhibitor clearance in cancer cachexia.

High baseline clearance of immune checkpoint inhibitors (ICIs), independent of dose or systemic exposure, is associated with cachexia and poor outcomes in cancer patients. Mechanisms linking ICI clearance, cachexia and ICI therapy failure are unknown. Here, we evaluate in four murine models and across multiple antibodies whether altered baseline catabolic clearance of administered antibody requires a tumor and/or cachexia and whether medical reversal of cachexia phenotype can alleviate altered clearance. Key findings include mild cachexia phenotype and lack of elevated pembrolizumab clearance in the MC38 tumor-bearing model. We also observed severe cachexia and decreased, instead of increased, baseline pembrolizumab clearance in the tumor-free cisplatin-induced cachexia model. Liver Fcgrt expression correlated with altered baseline catabolic clearance, though elevated clearance was still observed with antibodies having no (human IgA) or reduced (human H310Q IgG1) FcRn binding. We conclude cachexia phenotype coincides with altered antibody clearance, though tumor presence is neither sufficient nor necessary for altered clearance in immunocompetent mice. Magnitude and direction of clearance alteration correlated with hepatic Fcgrt, suggesting changes in FcRn expression and/or recycling function may be partially responsible, though factors beyond FcRn also contribute to altered clearance in cachexia.

Exploration of Verticillins in High-Grade Serous Ovarian Cancer and Evaluation of Multiple Formulations in Preclinical In Vitro and In Vivo Models.

Verticillins are epipolythiodioxopiperazine alkaloids isolated from a fungus with nanomolar anti-tumor activity in high-grade serous ovarian cancer (HGSOC). HGSOC is the fifth leading cause of death in women, and natural products continue to be an inspiration for new drug entities to help tackle chemoresistance. Verticillin D was recently found in a new fungal strain and compared to verticillin A. Both compounds exhibited nanomolar cytotoxic activity against OVCAR4 and OVCAR8 HGSOC cell lines, significantly reduced 2D foci and 3D spheroids, and induced apoptosis. In addition, verticillin A and verticillin D reduced tumor burden in vivo using OVCAR8 xenografts in the peritoneal space as a model. Unfortunately, mice treated with verticillin D displayed signs of liver toxicity. Tolerability studies to optimize verticillin A formulation for in vivo delivery were performed and compared to a semi-synthetic succinate version of verticillin A to monitor bioavailability in athymic nude females. Formulation of verticillins achieved tolerable drug delivery. Thus, formulation studies are effective at improving tolerability and demonstrating efficacy for verticillins.

The Neonatal Fc Receptor Is Elevated in Monocyte-Derived Immune Cells in Pancreatic Cancer.

The neonatal Fc receptor (FcRn) is responsible for recycling of IgG antibodies and albumin throughout the body. This mechanism has been exploited for pharmaceutic delivery across an array of diseases to either enhance or diminish this function. Monoclonal antibodies and albumin-bound nanoparticles are examples of FcRn-dependent anti-cancer therapeutics. Despite its importance in drug delivery, little is known about FcRn expression in circulating immune cells. Through time-of-flight mass cytometry (CyTOF) we were able to characterize FcRn expression in peripheral blood mononuclear cell (PBMC) populations of pancreatic ductal adenocarcinoma (PDAC) patients and non-cancer donors. Furthermore, we were able to replicate these findings in an orthotopic murine model of PDAC. Altogether, we found that in both patients and mice with PDAC, FcRn was elevated in migratory and resident classical dendritic cell type 2 (cDC2) as well as monocytic and granulocytic myeloid-derived suppressor cell (MDSC) populations compared to tumor-free controls. Furthermore, PBMCs from PDAC patients had elevated monocyte, dendritic cells and MDSCs relative to non-cancer donor PBMCs. Future investigations into FcRn activity may further elucidate possible mechanisms of poor efficacy of antibody immunotherapies in patients with PDAC.

Sensitivity of osteosarcoma cells to HDAC inhibitor AR-42 mediated apoptosis.

BACKGROUND: Osteosarcoma (OS) is the most common primary bone tumor in both humans and dogs and is the second leading cause of cancer related deaths in children and young adults. Limb sparing surgery along with chemotherapy has been the mainstay of treatment for OS. Many patients are not cured with current therapies, presenting a real need for developing new treatments. Histone deacetylase (HDAC) inhibitors are a promising new class of anticancer agents. In this study, we investigated the activity of the novel HDAC inhibitor AR-42 in a panel of human and canine OS cell lines. METHODS: The effect of AR-42 and suberoylanilide hydroxamic acid (SAHA) alone or in combination with doxorubicin on OS cell viability was assessed. Induction of histone acetylation after HDAC inhibitor treatment was confirmed by Western blotting. Drug-induced apoptosis was analyzed by FACS. Apoptosis was assessed further by measuring caspase 3/7 enzymatic activity, nucleosome fragmentation, and caspase cleavage. Effects on Akt signaling were demonstrated by assessing phosphorylation of Akt and downstream signaling molecules. RESULTS: AR-42 was a potent inhibitor of cell viability and induced a greater apoptotic response compared to SAHA when used at the same concentrations. Normal osteoblasts were much less sensitive. The combination of AR-42 with doxorubicin resulted in a potent inhibition of cell viability and apparent synergistic effect. Furthermore, we showed that AR-42 and SAHA induced cell death via the activation of the intrinsic mitochondrial pathway through activation of caspase 3/7. This potent apoptotic activity was associated with the greater ability of AR-42 to downregulate survival signaling through Akt. CONCLUSIONS: These results confirm that AR-42 is a potent inhibitor of HDAC activity and demonstrates its ability to significantly inhibit cell survival through its pleiotropic effects in both canine and human OS cells and suggests that spontaneous OS in pet dogs may be a useful large animal model for preclinical evaluation of HDAC inhibitors. HDAC inhibition in combination with standard doxorubicin treatment offers promising potential for chemotherapeutic intervention in both canine and human OS.

Pharmacological strategies to target oncogenic KRAS signaling in pancreatic cancer.

The clear importance of mutated KRAS as a therapeutic target has driven the investigation of multiple approaches to inhibit oncogenic KRAS signaling at different molecular levels. However, no KRAS-targeted therapy has reached the clinic to date, which underlies the intrinsic difficulty in developing effective, direct inhibitors of KRAS. Thus, this article provides an overview of the history and recent progress in the development of pharmacological strategies to target oncogenic KRAS with small molecule agents. Mechanistically, these KRAS-targeted agents can be classified into the following four categories. (1) Small-molecule RAS-binding ligands that prevent RAS activation by binding within or outside the nucleotide-binding motif. (2) Inhibitors of KRAS membrane anchorage. (3) Inhibitors that bind to RAS-binding domains of RAS-effector proteins. (4) Inhibitors of KRAS expression. The advantage and limitation of each type of these anti-KRAS agents are discussed.

Integrin-linked kinase as a novel molecular switch of the IL-6-NF-κB signaling loop in breast cancer.

Substantial evidence has clearly demonstrated the role of the IL-6-NF-κB signaling loop in promoting aggressive phenotypes in breast cancer. However, the exact mechanism by which this inflammatory loop is regulated remains to be defined. Here, we report that integrin-linked kinase (ILK) acts as a molecular switch for this feedback loop. Specifically, we show that IL-6 induces ILK expression via E2F1 upregulation, which, in turn, activates NF-κB signaling to facilitate IL-6 production. shRNA-mediated knockdown or pharmacological inhibition of ILK disrupted this IL-6-NF-κB signaling loop, and blocked IL-6-induced cancer stem cells in vitro and estrogen-independent tumor growth in vivo Together, these findings establish ILK as an intermediary effector of the IL-6-NF-κB feedback loop and a promising therapeutic target for breast cancer.

Extra-prostatic transgene-associated neoplastic lesions in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice.

Male transgenic adenocarcinoma of the mouse prostate (TRAMP) mice are frequently used in prostate cancer research because their prostates consistently develop a series of preneoplastic and neoplastic lesions. Disease progression in TRAMP mouse prostates culminates in metastatic, poorly differentiated carcinomas with neuroendocrine features. The androgen dependence of the rat probasin promoter largely limits transgene expression to the prostatic epithelium. However, extra-prostatic transgene-positive lesions have been described in TRAMP mice, including renal tubuloacinar carcinomas, neuroendocrine carcinomas of the urethra, and phyllodes-like tumors of the seminal vesicle. Here, we describe the histologic and immunohistochemical features of 2 novel extra-prostatic lesions in TRAMP mice: primary anaplastic tumors of uncertain cell origin in the midbrain and poorly differentiated adenocarcinomas of the submandibular salivary gland. These newly characterized tumors apparently result from transgene expression in extra-prostatic locations rather than representing metastatic prostate neoplasms because lesions were identified in both male and female mice and in male TRAMP mice without histologically apparent prostate tumors. In this article, we also calculate the incidences of the urethral carcinomas and renal tubuloacinar carcinomas, further elucidate the biological behavior of the urethral carcinomas, and demonstrate the critical importance of complete necropsies even when evaluating presumably well characterized phenotypes in genetically engineered mice.

Targeting the Warburg effect with a novel glucose transporter inhibitor to overcome gemcitabine resistance in pancreatic cancer cells.

Gemcitabine resistance remains a significant clinical challenge. Here, we used a novel glucose transporter (Glut) inhibitor, CG-5, as a proof-of-concept compound to investigate the therapeutic utility of targeting the Warburg effect to overcome gemcitabine resistance in pancreatic cancer. The effects of gemcitabine and/or CG-5 on viability, survival, glucose uptake and DNA damage were evaluated in gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer cell lines. Mechanistic studies were conducted to determine the molecular basis of gemcitabine resistance and the mechanism of CG-5-induced sensitization to gemcitabine. The effects of CG-5 on gemcitabine sensitivity were investigated in a xenograft tumor model of gemcitabine-resistant pancreatic cancer. In contrast to gemcitabine-sensitive pancreatic cancer cells, the resistant Panc-1 and Panc-1(GemR) cells responded to gemcitabine by increasing the expression of ribonucleotide reductase M2 catalytic subunit (RRM2) through E2F1-mediated transcriptional activation. Acting as a pan-Glut inhibitor, CG-5 abrogated this gemcitabine-induced upregulation of RRM2 through decreased E2F1 expression, thereby enhancing gemcitabine-induced DNA damage and inhibition of cell survival. This CG-5-induced inhibition of E2F1 expression was mediated by the induction of a previously unreported E2F1-targeted microRNA, miR-520f. The addition of oral CG-5 to gemcitabine therapy caused greater suppression of Panc-1(GemR) xenograft tumor growth in vivo than either drug alone. Glut inhibition may be an effective strategy to enhance gemcitabine activity for the treatment of pancreatic cancer.

Prospects on strategies for therapeutically targeting oncogenic regulatory factors by small-molecule agents.

Although the Human Genome Project has raised much hope for the identification of druggable genetic targets for cancer and other diseases, this genetic target-based approach has not improved productivity in drug discovery over the traditional approach. Analyses of known human target proteins of currently marketed drugs reveal that these drugs target only a limited number of proteins as compared to the whole proteome. In contrast to genome-based targets, mechanistic targets are derived from empirical research, at cellular or molecular levels, in disease models and/or in patients, thereby enabling the exploration of a greater number of druggable targets beyond the genome and epigenome. The paradigm shift has made a tremendous headway in developing new therapeutic agents targeting different clinically relevant mechanisms/pathways in cancer cells. In this Prospects article, we provide an overview of potential drug targets related to the following four emerging areas: (1) tumor metabolism (the Warburg effect), (2) dysregulated protein turnover (E3 ubiquitin ligases), (3) protein-protein interactions, and (4) unique DNA high-order structures and protein-DNA interactions. Nonetheless, considering the genetic and phenotypic heterogeneities that characterize cancer cells, the development of drug resistance in cancer cells by adapting signaling circuitry to take advantage of redundant pathways or feedback/crosstalk systems is possible. This "phenotypic adaptation" underlies the rationale of using therapeutic combinations of these targeted agents with cytotoxic drugs.

Sensitization of intracellular Salmonella enterica serovar Typhimurium to aminoglycosides in vitro and in vivo by a host-targeted antimicrobial agent.

Aminoglycosides exhibit relatively poor activity against intracellular Salmonella enterica serovar Typhimurium due to their low permeativity across eukaryotic cell membranes. Previously, we identified the unique ability of AR-12, a celecoxib-derived small-molecule agent, to eradicate intracellular Salmonella Typhimurium in macrophages by facilitating autophagosome formation and suppressing Akt kinase signaling. In light of this unique mode of antibacterial action, we investigated the ability of AR-12 to sensitize intracellular Salmonella to aminoglycosides in macrophages and in an animal model. The antibacterial activities of AR-12 combined with various aminoglycosides, including streptomycin, kanamycin, gentamicin, and amikacin, against intracellular S. Typhimurium in murine RAW264.7 macrophages were assessed. Cells were infected with S. Typhimurium followed by treatment with AR-12 or individual aminoglycosides or with combinations for 24 h. The in vivo efficacies of AR-12, alone or in combination with gentamicin or amikacin, were also assessed by treating S. Typhimurium-infected BALB/c mice daily for 14 consecutive days. Exposure of S. Typhimurium-infected RAW264.7 cells to a combination of AR-12 with individual aminoglycosides led to a reduction in bacterial survival (P < 0.05), both intracellular and extracellular, that was greater than that seen with the aminoglycosides alone. This sensitizing effect, however, was not associated with increased aminoglycoside penetration into bacteria or macrophages. Moreover, daily intraperitoneal injection of AR-12 at 0.1 mg/kg of body weight significantly increased the in vivo efficacy of gentamicin and amikacin in prolonging the survival of S. Typhimurium-infected mice. These findings indicate that the unique ability of AR-12 to enhance the in vivo efficacy of aminoglycosides might have translational potential for efforts to develop novel strategies for the treatment of salmonellosis.

Antibody Drug Clearance: An Underexplored Marker of Outcomes with Checkpoint Inhibitors.

Immune-checkpoint inhibitor (ICI) therapy has dramatically changed the clinical landscape for several cancers, and ICI use continues to expand across many cancer types. Low baseline clearance (CL) and/or a large reduction of CL during treatment correlates with better clinical response and longer survival. Similar phenomena have also been reported with other monoclonal antibodies (mAb) in cancer and other diseases, highlighting a characteristic of mAb clinical pharmacology that is potentially shared among various mAbs and diseases. Though tempting to attribute poor outcomes to low drug exposure and arguably low target engagement due to high CL, such speculation is not supported by the relatively flat exposure-response relationship of most ICIs, where a higher dose or exposure is not likely to provide additional benefit. Instead, an elevated and/or increasing CL could be a surrogate marker of the inherent resistant phenotype that cannot be reversed by maximizing drug exposure. The mechanisms connecting ICI clearance, therapeutic efficacy, and resistance are unclear and likely to be multifactorial. Therefore, to explore the potential of ICI CL as an early marker for efficacy, this review highlights the similarities and differences of CL characteristics and CL-response relationships for all FDA-approved ICIs, and we compare and contrast these to selected non-ICI mAbs. We also discuss underlying mechanisms that potentially link mAb CL with efficacy and highlight existing knowledge gaps and future directions where more clinical and preclinical investigations are warranted to clearly understand the value of baseline and/or time-varying CL in predicting response to ICI-based therapeutics.

Insulin-like growth factor-I receptor is suppressed through transcriptional repression and mRNA destabilization by a novel energy restriction-mimetic agent.

Insulin-like growth factor-I receptor (IGF-IR) represents one of the major targets by which dietary or chemically induced energy restriction mediates chemopreventive effects in animal tumor models. However, the mechanism underlying this cellular response remains unclear. In the course of investigating the suppressive effect of the energy restriction-mimetic agent CG-5 on IGF-IR expression in prostate cancer cells, we identified a novel posttranscriptional mechanism by which the RNA-binding protein human antigen R (HuR) regulates IGF-IR expression through messenger RNA (mRNA) stabilization. Previously, we demonstrated that Sp1 and HuR proteins were concomitantly targeted for ubiquitin-dependent degradation by ß-transducin repeat-containing protein in response to CG-5. Although this loss of Sp1 expression contributed to CG-5-mediated IGF-IR downregulation, enforced specific protein 1 (Sp1) expression could only partially protect cells from the drug effect. The small interfering RNA-mediated silencing of HuR suppressed IGF-IR expression by reducing mRNA stability, whereas ectopic HuR expression increased IGF-IR mRNA stability and protein expression and, when coexpressed with Sp1, blocked CG-5-mediated IGF-IR ablation. RNA pull-down and immunoprecipitation analyses indicated that HuR selectively bound to the distal region of the IGF-IR 3' untranslated region (UTR), whereas no interaction with the 5'UTR was noted. Evaluation of a series of truncated HuR mutants revealed that the RNA recognition motifs (RRM2 and RRM3) were involved in IGF-IR 3'UTR binding and the consequent increase in IGF-IR mRNA stability. Although these data contrast with a previous report that HuR acted as a translation repressor of IGF-IR mRNA through 5'UTR binding, our finding is consistent with the reported oncogenic role of HuR in conferring stability to target mRNAs encoding tumor-promoting proteins.

The mRNA-stabilizing factor HuR protein is targeted by ß-TrCP protein for degradation in response to glycolysis inhibition.

The mRNA-stabilizing protein HuR acts a stress response protein whose function and/or protein stability are modulated by diverse stress stimuli through posttranslational modifications. Here, we report a novel mechanism by which metabolic stress facilitates proteasomal degradation of HuR in cancer cells. In response to the glucose transporter inhibitor CG-5, HuR translocates to the cytoplasm, where it is targeted by the ubiquitin E3 ligase ß-TrCP1 for degradation. The cytoplasmic localization of HuR is facilitated by PKCα-mediated phosphorylation at Ser-318 as the Ser-318 → alanine substitution abolishes the ability of the resulting HuR to bind PKCα and to undergo nuclear export. The mechanistic link between ß-TrCP1 and HuR degradation was supported by the ability of ectopically expressed ß-TrCP1 to mimic CG-5 to promote HuR degradation and by the protective effect of dominant negative inhibition of ß-TrCP1 on HuR ubiquitination and degradation. Substrate targeting of HuR by ß-TrCP1 was further verified by coimmunoprecipitation and in vitro GST pull-down assays and by the identification of a ß-TrCP1 recognition site. Although HuR does not contain a DSG destruction motif, we obtained evidence that ß-TrCP1 recognizes an unconventional motif, (296)EEAMAIAS(304), in the RNA recognition motif 3. Furthermore, mutational analysis indicates that IKKα-dependent phosphorylation at Ser-304 is crucial to the binding of HuR to ß-TrCP1. Mechanistically, this HuR degradation pathway differs from that reported for heat shock and hypoxia, which underlies the complexity in the regulation of HuR turnover under different stress stimuli. The ability of glycolysis inhibitors to target the expression of oncogenic proteins through HuR degradation might foster novel strategies for cancer therapy.

Histone deacetylase inhibitor AR42 regulates telomerase activity in human glioma cells via an Akt-dependent mechanism.

Epigenetic regulation via abnormal activation of histone deacetylases (HDACs) is a mechanism that leads to cancer initiation and promotion. Activation of HDACs results in transcriptional upregulation of human telomerase reverse transcriptase (hTERT) and increases telomerase activity during cellular immortalization and tumorigenesis. However, the effects of HDAC inhibitors on the transcription of hTERT vary in different cancer cells. Here, we studied the effects of a novel HDAC inhibitor, AR42, on telomerase activity in a PTEN-null U87MG glioma cell line. AR42 increased hTERT mRNA in U87MG glioma cells, but suppressed total telomerase activity in a dose-dependent manner. Further analyses suggested that AR42 decreases the phosphorylation of hTERT via an Akt-dependent mechanism. Suppression of Akt phosphorylation and telomerase activity was also observed with PI3K inhibitor LY294002 further supporting the hypothesis that Akt signaling is involved in suppression of AR42-induced inhibition of telomerase activity. Finally, ectopic expression of a constitutive active form of Akt restored telomerase activity in AR42-treated cells. Taken together, our results demonstrate that the novel HDAC inhibitor AR42 can suppress telomerase activity by inhibiting Akt-mediated hTERT phosphorylation, indicating that the PI3K/Akt pathway plays an important role in the regulation of telomerase activity in response to this HDAC inhibitor.

Zeranol induces deleterious effects on the testes and the prostate gland of mature rats.

BACKGROUND: Endocrine disrupters have been shown to affect the male and female reproductive systems and to alter potential fertility. OBJECTIVES: This study was conducted to evaluate the effect of a continuous-release pellet containing 12 mg of zeranol for 30 days on the testes and the prostate gland of mature male rats. RESULTS: Zeranol treatment induced significant decrease of the testes and the prostate gland weights which were associated with a remarkable atrophy of the testicular seminiferous tubules and prominent regression of the glandular compartment of the prostate gland. However, zeranol treatment increased the thickness of the periductal layer of stromal cells of the prostate gland from a thin layer that express intense immunostaining of SM-actin and mild vimentin to a thicker layer of cells that exhibited intense immunostaining for both SM-actin and vimentin. CONCLUSION: These findings suggest that zeranol-induced changes to the prostate gland could result from either a direct effect of zeranol on the prostate gland or an indirect effect by interfering with testosterone production through disruption of testicular function.

Targeting energy metabolic and oncogenic signaling pathways in triple-negative breast cancer by a novel adenosine monophosphate-activated protein kinase (AMPK) activator.

The antitumor activities of the novel adenosine monophosphate-activated protein kinase (AMPK) activator, OSU-53, were assessed in in vitro and in vivo models of triple-negative breast cancer. OSU-53 directly stimulated recombinant AMPK kinase activity (EC(50), 0.3 µM) and inhibited the viability and clonogenic growth of MDA-MB-231 and MDA-MB-468 cells with equal potency (IC(50), 5 and 2 µM, respectively) despite lack of LKB1 expression in MDA-MB-231 cells. Nonmalignant MCF-10A cells, however, were unaffected. Beyond AMPK-mediated effects on mammalian target of rapamycin signaling and lipogenesis, OSU-53 also targeted multiple AMPK downstream pathways. Among these, the protein phosphatase 2A-dependent dephosphorylation of Akt is noteworthy because it circumvents the feedback activation of Akt that results from mammalian target of rapamycin inhibition. OSU-53 also modulated energy homeostasis by suppressing fatty acid biosynthesis and shifting the metabolism to oxidation by up-regulating the expression of key regulators of mitochondrial biogenesis, such as a peroxisome proliferator-activated receptor γ coactivator 1α and the transcription factor nuclear respiratory factor 1. Moreover, OSU-53 suppressed LPS-induced IL-6 production, thereby blocking subsequent Stat3 activation, and inhibited hypoxia-induced epithelial-mesenchymal transition in association with the silencing of hypoxia-inducible factor 1a and the E-cadherin repressor Snail. In MDA-MB-231 tumor-bearing mice, daily oral administration of OSU-53 (50 and 100 mg/kg) suppressed tumor growth by 47-49% and modulated relevant intratumoral biomarkers of drug activity. However, OSU-53 also induced protective autophagy that attenuated its antiproliferative potency. Accordingly, cotreatment with the autophagy inhibitor chloroquine increased the in vivo tumor-suppressive activity of OSU-53. OSU-53 is a potent, orally bioavailable AMPK activator that acts through a broad spectrum of antitumor activities.

Energy restriction-mimetic agents induce apoptosis in prostate cancer cells in part through epigenetic activation of KLF6 tumor suppressor gene expression.

Although energy restriction has been recognized as an important target for cancer prevention, the mechanism by which energy restriction-mimetic agents (ERMAs) mediate apoptosis remains unclear. By using a novel thiazolidinedione-derived ERMA, CG-12 (Wei, S., Kulp, S. K., and Chen, C. S. (2010) J. Biol. Chem. 285, 9780-9791), vis-à-vis 2-deoxyglucose and glucose deprivation, we obtain evidence that epigenetic activation of the tumor suppressor gene Kruppel-like factor 6 (KLF6) plays a role in ERMA-induced apoptosis in LNCaP prostate cancer cells. KLF6 regulates the expression of many proapoptotic genes, and shRNA-mediated KLF6 knockdown abrogated the ability of ERMAs to induce apoptosis. Chromatin immunoprecipitation analysis indicates that this KLF6 transcriptional activation was associated with increased histone H3 acetylation and histone H3 lysine 4 trimethylation occupancy at the promoter region. Several lines of evidence demonstrate that the enhancing effect of ERMAs on these active histone marks was mediated through transcriptional repression of histone deacetylases and H3 lysine 4 demethylases by down-regulating Sp1 expression. First, putative Sp1-binding elements are present in the promoters of the affected histone-modifying enzymes, and luciferase reporter assays indicate that site-directed mutagenesis of these Sp1 binding sites significantly diminished the promoter activities. Second, shRNA-mediated knockdown of Sp1 mimicked the repressive effect of energy restriction on these histone-modifying enzymes. Third, ectopic Sp1 expression protected cells from the repressive effect of CG-12 on these histone-modifying enzymes, thereby abolishing the activation of KLF6 expression. Together, these findings underscore the intricate relationship between energy restriction and epigenetic regulation of tumor suppressor gene expression, which has therapeutic relevance to foster novel strategies for prostate cancer therapy.