Christopher A. Blair

Histone lysine-specific methyltransferases and demethylases in carcinogenesis: new targets for cancer therapy and prevention.

Aberrant histone lysine methylation that is controlled by histone lysine methyltransferases (KMTs) and demethylases (KDMs) plays significant roles in carcinogenesis. Infections by tumor viruses or parasites and exposures to chemical carcinogens can modify the process of histone lysine methylation. Many KMTs and KDMs contribute to malignant transformation by regulating the expression of human telomerase reverse transcriptase (hTERT), forming a fused gene, interacting with proto-oncogenes or being up-regulated in cancer cells. In addition, histone lysine methylation participates in tumor suppressor gene inactivation during the early stages of carcinogenesis by regulating DNA methylation and/or by other DNA methylation independent mechanisms. Furthermore, recent genetic discoveries of many mutations in KMTs and KDMs in various types of cancers highlight their numerous roles in carcinogenesis and provide rare opportunities for selective and tumor-specific targeting of these enzymes. The study on global histone lysine methylation levels may also offer specific biomarkers for cancer detection, diagnosis and prognosis, as well as for genotoxic and non-genotoxic carcinogenic exposures and risk assessment. This review summarizes the role of histone lysine methylation in the process of cellular transformation and carcinogenesis, genetic alterations of KMTs and KDMs in different cancers and recent progress in discovery of small molecule inhibitors of these enzymes.

Kava Components Down-Regulate Expression of AR and AR Splice Variants and Reduce Growth in Patient-Derived Prostate Cancer Xenografts in Mice

Men living in Fiji and drinking kava have low incidence of prostate cancer (PCa). However, the PCa incidence among Fijian men who had migrated to Australia, increased by 5.1-fold. We therefore examined the potential effects of kava root extracts and its active components (kavalactones and flavokawains) on PCa growth and androgen receptor (AR) expression. PCa cell lines (LNCaP, LAPC-4, 22Rv1, C4-2B, DU145 and PC-3) with different AR expression, and a transformed prostate myofibroblast cell line (WPMY-1), were treated with a commercial kava extract, kavalactones (kawain, 5′6′-dehydrokawain, yangonin, methysticin) and flavokawain B. Expression of AR and its target genes (PSA and TMPRSS2) was examined. Two novel patient-derived PCa xenograft models from high grade PCa specimens were established by implanting the specimens into nude mice and passing tumor pieces through subcutaneous injection in nude mice, and then treated with kava extract and flavokawain B to examine their effects on tumor growth, AR expression and serum PSA levels. The kava extract and flavokawain B effectively down-regulated the expression of both the full-length AR and AR splice variants. The kava extract and kavalactones accelerated AR protein degradation, while flavokawain B inhibited AR mRNA transcription via decreasing Sp1 expression and the binding of Sp1 to the AR promoter. The kava root extract and flavokawain B reduce tumor growth, AR expression in tumor tissues and levels of serum PSA in the patient-derived PCa xenograft models. These results suggest a potential usefulness of a safe kava product or its active components for prevention and treatment of advanced PCa by targeting AR.

Molecular Targeted Approaches to Cancer Therapy and Prevention Using Chalcones

There is an emerging paradigm shift in oncology that seeks to emphasize molecularly targeted approaches for cancer prevention and therapy. Chalcones (1,3-diphenyl-2-propen-1-ones), naturally-occurring compounds with widespread distribution in spices, tea, beer, fruits and vegetables, consist of open-chain flavonoids in which the two aromatic rings are joined by a three-carbon α, β-unsaturated carbonyl system. Due to their structural diversity, relative ease of chemical manipulation and reaction of α, β-unsaturated carbonyl moiety with cysteine residues in proteins, some lead chalcones from both natural products and synthesis have been identified in a variety of screening assays for modulating important pathways or molecular targets in cancers. These pathways and targets that are affected by chalcones include MDM2/p53, tubulin, proteasome, NF-kappa B, TRIAL/death receptors and mitochondria mediated apoptotic pathways, cell cycle, STAT3, AP-1, NRF2, AR, ER, PPAR-γ and β-catenin/Wnt. Compared to current cancer targeted therapeutic drugs, chalcones have the advantages of being inexpensive, easily available and less toxic; the ease of synthesis of chalcones from substituted benzaldehydes and acetophenones also makes them an attractive drug scaffold. Therefore, this review is focused on molecular targets of chalcones and their potential implications in cancer prevention and therapy.

High Sensitivity of an Ha-RAS Transgenic Model of Superficial Bladder Cancer to Metformin Is Associated with ∼240-Fold Higher Drug Concentration in Urine than Serum

While pharmacoepidemiologic and laboratory studies have supported the hypothesis that the antidiabetic drug metformin may be useful in treating or preventing cancer, there is limited evidence to suggest which specific cancer sites may be particularly sensitive. Sensitivity likely is determined both by features of tumor pathophysiology and by pharmacokinetic factors. We used UPII-mutant Ha-ras transgenic mice that develop hyperplasia and low-grade, papillary urothelial cell carcinoma to determine whether metformin has activity in a model of superficial bladder cancer. Metformin significantly improved survival, reduced urinary tract obstruction, reduced bladder weight (a surrogate for tumor volume), and led to clear activation of AMP α kinase and inhibition of mTOR signaling in neoplastic tissue. We investigated the basis of the unusual sensitivity of this model to metformin, and observed that following oral dosing, urothelium is exposed to drug concentrations via the urine that are approximately 240-fold higher than those in the circulation. In addition, we observed that bladder cancer cell lines (RT4, UMUC-3, and J82) with homozygous deletion of either TSC1 or PTEN are more sensitive to metformin than those (TEU2, TCCSUP, and HT1376) with wild-type TSC1 and PTEN genes. Our findings provide a strong rationale for clinical trials of oral metformin in treatment of superficial bladder cancer. Mol Cancer Ther; 15(3); 430–8. ©2016 AACR.

Effect of perineoplasm perinephric adipose tissues on migration of clear cell renal cell carcinoma cells: a potential role of WNT signaling

To investigate the cellular and molecular interactions between clear-cell renal cell carcinoma (ccRCC) and perinephric adipose tissue (PAT), perineoplasm PAT, PAT away from the neoplasm, renal sinus and subcutaneous adipose tissues were collected at the time of renal surgery for renal masses and conditioned medium (CM) was generated from 62 patients. Perineoplasm PAT CMs from 44 out of 62 (about 71%) of patients with ccRCC or benign renal diseases (e.g. oncocytomas, angiomyolipomas, multicystic kidney, interstitial fibrosis, etc.) enhanced the migration of CaKi-2 cells. Perineoplasm PAT CMs from ccRCC significantly increased migration of ACHN and CaKi-2 cells by ~8.2 and ~2.4 folds, respectively, relative to those from benign renal diseases, whereas there is no significant difference in migration between ccRCC and benign renal diseases in CMs collected from culturing PAT away from neoplasm, renal sinus and subcutaneous adipose tissues. High Fuhrman Grade was associated with increased migration of Caki-2 cells by perineoplasm PAT CMs. Perineoplasm PATs from pT3 RCCs overexpressed multiple WNTs and their CMs exhibited higher WNT/ß-catenin activity and increased the migration of Caki-2 cells compared to CMs from benign neoplasms. Addition of secreted WNT inhibitory factor-1 recombinant protein into perineoplasm PAT CMs completely blocked the cell migration. These results indicate that WNT related factors from perineoplasm PAT may promote progression of local ccRCC to locally advanced (pT3) disease by increasing ccRCC cell mobility.

Abstract 256: Delta tocopherol inhibits urothelial tumorigenesis in the UPII mutant Ha-ras transgenic mouse model and induces apoptosis via activation of the ATF4/CHOP-DR5 pathway

Epidemiological studies have reported that Vitamin E intake was inversely related to the risk of multiple cancers including human urinary bladder cancer. Tocopherols (T) are the major forms of vitamin E in the U.S. diet, exist as α-T, β-T, γ-T and δ-T. Compared to α-T, the anti-cancer effect of other Ts and their mechanisms of action remain largely unknown. We have shown that δ-T is the most effective one among the Ts in reducing the viabilities of bladder cancer cell lines with IC50s of under 10 μM, whereas there is no or minimal effect of α-T at a concentration of up to 50 μM on the viability of bladder cancer cell lines. δ-T treatment of bladder cancer cell lines, RT4 and UMUC-3, resulted in apoptosis via marked induction of death receptor-5 (DR5) expression and then activation of caspase 3, 8, and 9 leading to PARP cleavage and apoptotic morphology. siRNA knockdown of DR5 expression significantly attenuated the apoptotic effects of δ-T in bladder cancer cells. Consistent with the above results, δ-T treatment significantly upregulated the expression of ER stress sensors PERK and IRE1α, as well as downstream components GRP78, ATF4, and CHOP. These results suggested that δ-T can induce endoplasmic reticulum stress and the Unfolded Protein Response, triggering its apoptotic effect. Furthermore, homozygous male UPII mutant Ha-ras transgenic mice that mimic the development and progression of human urothelial cell carcinoma (UCC) with H-ras activation were fed with vehicle control diet (n=20) or diet supplemented with δ-T (0.2% in diet) for 150 days (n=18). The δ-T diet significantly decreased the mean bladder weights (serves as a surrogate of tumor weight) of survived Ha-ras mutant mice by 56% (control vs δ-T, 0.259 ± 0.015 gram vs. 0.114 ± 0.012 gram, P Citation Format: Christopher A. Blair, Maggie Wu, Tim Huynh, Hanze Hu, Arman Walia, Chung S. Yang, Xiaolin Zi. Delta tocopherol inhibits urothelial tumorigenesis in the UPII mutant Ha-ras transgenic mouse model and induces apoptosis via activation of the ATF4/CHOP-DR5 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 256. doi:10.1158/1538-7445.AM2017-256

Abstract 2141: Dietary feeding of Kava root extract inhibits prostate carcinogenesis in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model

Kava kava has been traditionally used to prepare beverages and herbal medicines in the Pacific Islands. Men living in Fiji and drinking kava have low incidence of prostate cancer (PCa). However, the PCa incidence among Fijian men who had migrated to Australia, increased by 5.1-fold. To determine the chemopreventive efficacy of kava root extracts, TRAMP mice were fed with commercial kava root extract supplemented food. In the prevention protocol, TRAMP mice were fed with 3 or 6 g (0.3% or 0.6%) kava root extract /kg food starting at 6 weeks of age and ending at 12 weeks of age, resulting in decreased numbers of high-grade prostatic intraepithelial neoplasia and moderately differentiated adenocarcinomas in kava root extract fed mice compared to the mice fed with control food (p 0.9 gram) were also decreased from 86.4% in the control group to 52.2% and 43.5% in 0.3 and 0.6 % kava food groups, respectively. In addition, we showed that kawain, the main component of kava root extracts, inhibited LSD1 enzyme activity, enhanced H3K9 dimethylation and decreased the mRNA expression of androgen receptor target genes, including PSA and TMPRSS2, in LNCaP cells. Taken together, these results suggested that dietary consumption of kava products have the potential to reduce the risk of prostate cancer development. Citation Format: Xuesen Li, Christopher A. Blair, Xiaolin Zi. Dietary feeding of Kava root extract inhibits prostate carcinogenesis in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2141. doi:10.1158/1538-7445.AM2014-2141

Abstract 4867: The metabolomic signature of Rhodiola rosea L. extracts- (SHR-5) treated mouse bladder cancer in the UPII-mutant Ha-ras transgenic model.

Rhodiola rosea has been used for centuries in the traditional medicine for reducing depression, enhancing work performance, and preventing high altitude sickness. We have recently shown that Rhodiola rosea L. extract, SHR-5, markedly inhibited the growth of bladder cancer and the survival of mice in the UP-II-mutant Ha-ras transgenic model via inhibition of the mTOR pathway. The purpose of this study was to further investigate the effects of SHR-5 on bladder tumor metabolism. The global metabolic profiles were compared for bladder tumors from mice that were fed with normal drinking water (n=7), 1.25 (n=7) or 6.25 (n=7) mg/ml SHR-5 in water for about five months. The analysis was conducted at Metabolon Inc. with a combination of high-throughput LC- and GC-based MS method. From a metabolomics library consisting of more than 2,000 standards, a total of 341 named metabolites were detected. Phenolic metabolites, such as phenylpropionylglycine, hydroxycinnamate and catechol sulfate, were increased, while prostaglandin metabolites, such as prostaglandins A2, D2 and E2, were decreased in tumors treated with SHR-5. This observation suggested the potential anti-inflammation effect of SHR-5 in bladder tumors. Neurotransmitters GABA, taurine, NAA and 2-oxoindole-3-acetate was more abundant; whereas kynurenine, histamine and aminoadipate were less abundant with SHR-5 treatment. This result is consistent with mood-improving effects of SHR-5. In addition, SHR-5 treatment resulted in increased levels of glucose and glycogen metabolites (e.g. maltotriose, maltose), as well as two glycolysis intermediates (3-phosphoglycerate and 2-phosphoglycerate) and the citric acid cycle (TCA) intermediates (fumarate and malate) compared to control treatment. These patterns suggest that SHR-5 may increase mobilization of glycogen for energy production in glycolysis and TCA cycle. These effects of SHR-5 may be mediated by differential effects on the mTOR pathway. Taken together, we show that global metabolomic profiling provides a unique and efficient tool for studying mechanisms of complex herb extracts’ action. Citation Format: Zhongbo Liu, Christopher A. Blair, Xiaolin Zi. The metabolomic signature of Rhodiola rosea L. extracts- (SHR-5) treated mouse bladder cancer in the UPII-mutant Ha-ras transgenic model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4867. doi:10.1158/1538-7445.AM2013-4867

Nutraceuticals in Human Urinary Bladder Cancer Prevention and Treatment

Bladder cancer is a major public health burden. Tumor resection with possible intravesical treatments for superficial disease, and cystectomy or chemotherapy with radiation protocols for invasive bladder cancer have associated limitations and large costs. In addition, exposure to carcinogens contributes to the majority of bladder cancer risk. All of these represent profound opportunities to use nutraceuticals for improvement of current bladder cancer prevention and treatment. We discuss the clinical opportunities for use of nutraceuticals in bladder cancer prevention and treatment, including preventing the first occurrence of bladder cancer in high risk populations, delaying progression of the disease, use in combination with existing intravesical agents, and delaying or preventing radical cystectomy. We review randomized controlled trials of nutraceuticals in bladder cancer, current promising chemopreventive agents under preclinical development for bladder cancer prevention, and future directions of bladder cancer chemoprevention, including the concept of individualized bladder cancer chemoprevention.

Abstract 857: Flavokawains degrade S-phase kinase-associated protein 2 (Skp2) via deneddylation of cullin-1 and inhibit prostate cancer in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The E3 ubiquitin ligase Skp2 was found to be overexpressed in the majority of primary prostate cancer (PCa) and pre-malignant high-grade prostatic intra-epithelial neoplasia (HG-PIN) lesions. In a search for potential Skp2 targeting agents for PCa chemoprevention, we found that flavokawains (including A and B), novel chalcones isolated from the Kava plant, potently decreased protein levels of Skp2 in multiple PCa and other cancer cell lines, including colon, cervical, bladder, melanoma and sarcoma cancer without affecting Skp2 mRNA expression or transcriptional activities. These results indicated that the effects of flavokawains on Skp2 protein expression are not dependent on the genetic backgrounds of these cell lines, suggesting a direct regulation of Skp2 expression by flavokawains. Further studies showed that flavokawains accelerated the degradation of Skp2. Skp2 degradation can be regulated by CDH1 or by autocatalytic degradation. Flavokawains did not increase the expression of CDH1, but decreased cullin-1 neddylation levels in these cell lines. Inhibition of cullin-1 function by expression of a dominant-negative cullin-1 into cells rescued flavokawain B-induced Skp2 degradation, while siRNA knock-down of CSN5 acted synergistically with flavokawain B in degradation of Skp2. In an in vitro neddylation reconstitution assay, flavokawains inhibited the neddylation of both cullin-1 and UBC12, the E2 ligase of neddylation. These results suggest that flavokawains directly inhibit cullin-1 neddylation, resulting in autoubiquition and subsequent degradation of Skp2. To determine the preventive and therapeutic efficacies of flavokawain A, TRAMP mice were fed with flavokawain A supplemented food. In the prevention protocol, TRAMP mice were fed with 3 g flavokawain A /kg food starting at 6 weeks of age and ending at 12 weeks of age, resulting in numbers of HG-PIN and moderately differentiated adenocarcinomas in flavokawain A fed mice which were significantly decreased compared to the mice fed with control food (p<0.01). In the intervention protocol, in which TRAMP mice were fed with 6 g flavokawain A /kg food starting at 12 weeks of age and ending at 24 weeks of age, the average genitourinary weight of flavokawain A fed mice was significantly reduced when compared to control food fed mice (1.45±0.88 gram vs.2.22±0.77 gram, p<0.05, Mann-Whitney U and Kolmogorov-Smirnov test). No toxicity of flavokawain A treatment was observed either in body or organ weight or in histology examination. Western blotting analysis of prostate tissue lystates revealed that the protein levels of Skp2 were markedly reduced in the prostates and tumors of flavokawain A fed mice compared to control. Taken together, Flavokawains are novel Skp2 targeting agents, deserving of further investigation for PCa chemoprevention and therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 857. doi:10.1158/1538-7445.AM2011-857