CK2 Pro-Survival Role in Prostate Cancer Is Mediated via Maintenance and Promotion of Androgen Receptor and NFκB p65 Expression.

The prosurvival protein kinase CK2, androgen receptor (AR), and nuclear factor kappa B (NFκB) interact in the function of prostate cells, and there is evidence of crosstalk between these signals in the pathobiology of prostate cancer (PCa). As CK2 is elevated in PCa, and AR and NFκB are involved in the development and progression of prostate cancer, we investigated their interaction in benign and malignant prostate cells in the presence of altered CK2 expression. Our results show that elevation of CK2 levels caused increased levels of AR and NFκB p65 in prostate cells of different phenotypes. Analysis of TCGA PCa data indicated that AR and CK2α RNA expression are strongly correlated. Small molecule inhibition or molecular down-regulation of CK2 caused reduction in AR mRNA expression and protein levels in PCa cells and in orthotopic xenograft tumors by various pathways. Among these, regulation of AR protein stability plays a unifying role in CK2 maintenance of AR protein levels. Our results show induction of various endoplasmic reticulum stress signals after CK2 inhibition, which may play a role in the PCa cell death response. Of note, CK2 inhibition caused loss of cell viability in both parental and enzalutamide-resistant castrate-resistant PCa cells. The present work elucidates the specific link of CK2 to the pathogenesis of PCa in association with AR and NFκB expression; further, the observation that inhibition of CK2 can exert a growth inhibitory effect on therapy-resistant PCa cells emphasizes the potential utility of CK2 inhibition in patients who are on enzalutamide treatment for advanced cancer.

CDK11 Loss Induces Cell Cycle Dysfunction and Death of BRAF and NRAS Melanoma Cells.

Cyclin dependent kinase 11 (CDK11) is a protein kinase that regulates RNA transcription, pre-mRNA splicing, mitosis, and cell death. Targeting of CDK11 expression levels is effective in the experimental treatment of breast and other cancers, but these data are lacking in melanoma. To understand CDK11 function in melanoma, we evaluated protein and RNA levels of CDK11, Cyclin L1 and Cyclin L2 in benign melanocytes and BRAF- as well as NRAS-mutant melanoma cell lines. We investigated the effectiveness of reducing expression of this survival kinase using RNA interference on viability, clonal survival, and tumorsphere formation in melanoma cell lines. We examined the impact of CDK11 loss in BRAF-mutant melanoma on more than 700 genes important in cancer signaling pathways. Follow-up analysis evaluated how CDK11 loss alters cell cycle function in BRAF- and NRAS-mutant melanoma cells. We present data on CDK11, CCNL1 and CCNL2 mRNA expression in melanoma patients, including prognosis for survival. In sum, we found that CDK11 is necessary for melanoma cell survival, and a major impact of CDK11 loss in melanoma is to cause disruption of the cell cycle distribution with accumulation of G1- and loss of G2/M-phase cancer cells.

Towards quality assurance and quality control in untargeted metabolomics studies.

We describe here the agreed upon first development steps and priority objectives of a community engagement effort to address current challenges in quality assurance (QA) and quality control (QC) in untargeted metabolomic studies. This has included (1) a QA and QC questionnaire responded to by the metabolomics community in 2015 which recommended education of the metabolomics community, development of appropriate standard reference materials and providing incentives for laboratories to apply QA and QC; (2) a 2-day 'Think Tank on Quality Assurance and Quality Control for Untargeted Metabolomic Studies' held at the National Cancer Institute's Shady Grove Campus and (3) establishment of the Metabolomics Quality Assurance and Quality Control Consortium (mQACC) to drive forward developments in a coordinated manner.

Towards precision prevention: Technologies for identifying healthy individuals with high risk of disease.

The rise of advanced technologies for characterizing human populations at the molecular level, from sequence to function, is shifting disease prevention paradigms toward personalized strategies. Because minimization of adverse outcomes is a key driver for treatment decisions for diseased populations, developing personalized therapy strategies represent an important dimension of both precision medicine and personalized prevention. In this commentary, we highlight recently developed enabling technologies in the field of DNA damage, DNA repair, and mutagenesis. We propose that omics approaches and functional assays can be integrated into population studies that fuse basic, translational and clinical research with commercial expertise in order to accelerate personalized prevention and treatment of cancer and other diseases linked to aberrant responses to DNA damage. This collaborative approach is generally applicable to efforts to develop data-driven, individualized prevention and treatment strategies for other diseases. We also recommend strategies for maximizing the use of biological samples for epidemiological studies, and for applying emerging technologies to clinical applications.

CK2 targeted RNAi therapeutic delivered via malignant cell-directed tenfibgen nanocapsule: dose and molecular mechanisms of response in xenograft prostate tumors.

CK2, a protein serine/threonine kinase, promotes cell proliferation and suppresses cell death. This essential-for-survival signal demonstrates elevated expression and activity in all cancers examined, and is considered an attractive target for cancer therapy. Here, we present data on the efficacy of a tenfibgen (TBG) coated nanocapsule which delivers its cargo of siRNA (siCK2) or single stranded RNA/DNA oligomers (RNAi-CK2) simultaneously targeting CK2α and α' catalytic subunits. Intravenous administration of TBG-siCK2 or TBG-RNAi-CK2 resulted in significant xenograft tumor reduction at low doses in PC3-LN4 and 22Rv1 models of prostate cancer. Malignant cell uptake and specificity in vivo was verified by FACS analysis and immunofluorescent detection of nanocapsules and PCR detection of released oligomers. Dose response was concordant with CK2αα' RNA transcript levels and the tumors demonstrated changes in CK2 protein and in markers of proliferation and cell death. Therapeutic response corresponded to expression levels for argonaute and GW proteins, which function in oligomer processing and translational repression. No toxicity was detected in non-tumor tissues or by serum chemistry. Tumor specific delivery of anti-CK2 RNAi via the TBG nanoencapsulation technology warrants further consideration of translational potential.

Harnessing genomics to identify environmental determinants of heritable disease.

Next-generation sequencing technologies can now be used to directly measure heritable de novo DNA sequence mutations in humans. However, these techniques have not been used to examine environmental factors that induce such mutations and their associated diseases. To address this issue, a working group on environmentally induced germline mutation analysis (ENIGMA) met in October 2011 to propose the necessary foundational studies, which include sequencing of parent-offspring trios from highly exposed human populations, and controlled dose-response experiments in animals. These studies will establish background levels of variability in germline mutation rates and identify environmental agents that influence these rates and heritable disease. Guidance for the types of exposures to examine come from rodent studies that have identified agents such as cancer chemotherapeutic drugs, ionizing radiation, cigarette smoke, and air pollution as germ-cell mutagens. Research is urgently needed to establish the health consequences of parental exposures on subsequent generations.

Identification of novel positive-strand RNA viruses by metagenomic analysis of archaea-dominated Yellowstone hot springs.

There are no known RNA viruses that infect Archaea. Filling this gap in our knowledge of viruses will enhance our understanding of the relationships between RNA viruses from the three domains of cellular life and, in particular, could shed light on the origin of the enormous diversity of RNA viruses infecting eukaryotes. We describe here the identification of novel RNA viral genome segments from high-temperature acidic hot springs in Yellowstone National Park in the United States. These hot springs harbor low-complexity cellular communities dominated by several species of hyperthermophilic Archaea. A viral metagenomics approach was taken to assemble segments of these RNA virus genomes from viral populations isolated directly from hot spring samples. Analysis of these RNA metagenomes demonstrated unique gene content that is not generally related to known RNA viruses of Bacteria and Eukarya. However, genes for RNA-dependent RNA polymerase (RdRp), a hallmark of positive-strand RNA viruses, were identified in two contigs. One of these contigs is approximately 5,600 nucleotides in length and encodes a polyprotein that also contains a region homologous to the capsid protein of nodaviruses, tetraviruses, and birnaviruses. Phylogenetic analyses of the RdRps encoded in these contigs indicate that the putative archaeal viruses form a unique group that is distinct from the RdRps of RNA viruses of Eukarya and Bacteria. Collectively, our findings suggest the existence of novel positive-strand RNA viruses that probably replicate in hyperthermophilic archaeal hosts and are highly divergent from RNA viruses that infect eukaryotes and even more distant from known bacterial RNA viruses. These positive-strand RNA viruses might be direct ancestors of RNA viruses of eukaryotes.

Inhibition of fried meat-induced colorectal DNA damage and altered systemic genotoxicity in humans by crucifera, chlorophyllin, and yogurt.

UNLABELLED: Dietary exposures implicated as reducing or causing risk for colorectal cancer may reduce or cause DNA damage in colon tissue; however, no one has assessed this hypothesis directly in humans. Thus, we enrolled 16 healthy volunteers in a 4-week controlled feeding study where 8 subjects were randomly assigned to dietary regimens containing meat cooked at either low (100°C) or high temperature (250°C), each for 2 weeks in a crossover design. The other 8 subjects were randomly assigned to dietary regimens containing the high-temperature meat diet alone or in combination with 3 putative mutagen inhibitors: cruciferous vegetables, yogurt, and chlorophyllin tablets, also in a crossover design. Subjects were nonsmokers, at least 18 years old, and not currently taking prescription drugs or antibiotics. We used the Salmonella assay to analyze the meat, urine, and feces for mutagenicity, and the comet assay to analyze rectal biopsies and peripheral blood lymphocytes for DNA damage. Low-temperature meat had undetectable levels of heterocyclic amines (HCAs) and was not mutagenic, whereas high-temperature meat had high HCA levels and was highly mutagenic. The high-temperature meat diet increased the mutagenicity of hydrolyzed urine and feces compared to the low-temperature meat diet. The mutagenicity of hydrolyzed urine was increased nearly twofold by the inhibitor diet, indicating that the inhibitors enhanced conjugation. Inhibitors decreased significantly the mutagenicity of un-hydrolyzed and hydrolyzed feces. The diets did not alter the levels of DNA damage in non-target white blood cells, but the inhibitor diet decreased nearly twofold the DNA damage in target colorectal cells. To our knowledge, this is the first demonstration that dietary factors can reduce DNA damage in the target tissue of fried-meat associated carcinogenesis. TRIAL REGISTRATION: ClinicalTrials.gov NCT00340743.

Antimutagenicity of cinnamaldehyde and vanillin in human cells: Global gene expression and possible role of DNA damage and repair.

Vanillin (VAN) and cinnamaldehyde (CIN) are dietary flavorings that exhibit antimutagenic activity against mutagen-induced and spontaneous mutations in bacteria. Although these compounds were antimutagenic against chromosomal mutations in mammalian cells, they have not been studied for antimutagenesis against spontaneous gene mutations in mammalian cells. Thus, we initiated studies with VAN and CIN in human mismatch repair-deficient (hMLH1(-)) HCT116 colon cancer cells, which exhibit high spontaneous mutation rates (mutations/cell/generation) at the HPRT locus, permitting analysis of antimutagenic effects of agents against spontaneous mutation. Long-term (1-3 weeks) treatment of HCT116 cells with VAN at minimally toxic concentrations (0.5-2.5mM) reduced the spontaneous HPRT mutant fraction (MF, mutants/10(6) survivors) in a concentration-related manner by 19-73%. A similar treatment with CIN at 2.5-7.5microM yielded a 13-56% reduction of the spontaneous MF. Short-term (4-h) treatments also reduced the spontaneous MF by 64% (VAN) and 31% (CIN). To investigate the mechanisms of antimutagenesis, we evaluated the ability of VAN and CIN to induce DNA damage (comet assay) and to alter global gene expression (Affymetrix GeneChip) after 4-h treatments. Both VAN and CIN induced DNA damage in both mismatch repair-proficient (HCT116+chr3) and deficient (HCT116) cells at concentrations that were antimutagenic in HCT116 cells. There were 64 genes whose expression was changed similarly by both VAN and CIN; these included genes related to DNA damage, stress responses, oxidative damage, apoptosis, and cell growth. RT-PCR results paralleled the Affymetrix results for four selected genes (HMOX1, DDIT4, GCLM, and CLK4). Our results show for the first time that VAN and CIN are antimutagenic against spontaneous mutations in mammalian (human) cells. These and other data lead us to propose that VAN and CIN may induce DNA damage that elicits recombinational DNA repair, which reduces spontaneous mutations.

Inhibition of spontaneous mutagenesis by vanillin and cinnamaldehyde in Escherichia coli: Dependence on recombinational repair.

Vanillin (VAN) and cinnamaldehyde (CIN) are dietary antimutagens that effectively inhibit both induced and spontaneous mutations. We have shown previously that VAN and CIN reduced the spontaneous mutant frequency in Salmonella TA104 (hisG428, rfa, DeltauvrB, pKM101) by approximately 50% and that both compounds significantly reduced mutations at GC sites but not at AT sites. Previous studies have suggested that VAN and CIN may reduce mutations in bacterial model systems by modulating DNA repair pathways, particularly by enhancing recombinational repair. To further explore the basis for inhibition of spontaneous mutation by VAN and CIN, we have determined the effects of these compounds on survival and mutant frequency in five Escherichia coli strains derived from the wild-type strain NR9102 with different DNA repair backgrounds. At nontoxic doses, both VAN and CIN significantly reduced mutant frequency in the wild-type strain NR9102, in the nucleotide excision repair-deficient strain NR11634 (uvrB), and in the recombination-proficient but SOS-deficient strain NR11475 (recA430). In contrast, in the recombination-deficient and SOS-deficient strain NR11317 (recA56), both VAN and CIN not only failed to inhibit the spontaneous mutant frequency but actually increased the mutant frequency. In the mismatch repair-defective strain NR9319 (mutL), only CIN was antimutagenic. Our results show that the antimutagenicity of VAN and CIN against spontaneous mutation required the RecA recombination function but was independent of the SOS and nucleotide excision repair pathways. Thus, we propose the counterintuitive notion that these antimutagens actually produce a type of DNA damage that elicits recombinational repair (but not mismatch, SOS, or nucleotide excision repair), which then repairs not only the damage induced by VAN and CIN but also other DNA damage-resulting in an antimutagenic effect on spontaneous mutation.