Cyclin D1 extensively reprograms metabolism to support biosynthetic pathways in hepatocytes.

Cell proliferation requires metabolic reprogramming to accommodate biosynthesis of new cell components, and similar alterations occur in cancer cells. However, the mechanisms linking the cell cycle machinery to metabolism are not well defined. Cyclin D1, along with its main partner cyclin-dependent kinase 4 (Cdk4), is a pivotal cell cycle regulator and driver oncogene that is overexpressed in many cancers. Here, we examine hepatocyte proliferation to define novel effects of cyclin D1 on biosynthetic metabolism. Metabolomic studies reveal that cyclin D1 broadly promotes biosynthetic pathways including glycolysis, the pentose phosphate pathway, and the purine and pyrimidine nucleotide synthesis in hepatocytes. Proteomic analyses demonstrate that overexpressed cyclin D1 binds to numerous metabolic enzymes including those involved in glycolysis and pyrimidine synthesis. In the glycolysis pathway, cyclin D1 activates aldolase and GAPDH, and these proteins are phosphorylated by cyclin D1/Cdk4 in vitro. De novo pyrimidine synthesis is particularly dependent on cyclin D1. Cyclin D1/Cdk4 phosphorylates the initial enzyme of this pathway, carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and metabolomic analysis indicates that cyclin D1 depletion markedly reduces the activity of this enzyme. Pharmacologic inhibition of Cdk4 along with the downstream pyrimidine synthesis enzyme dihydroorotate dehydrogenase synergistically inhibits proliferation and survival of hepatocellular carcinoma cells. These studies demonstrate that cyclin D1 promotes a broad network of biosynthetic pathways in hepatocytes, and this model may provide insights into potential metabolic vulnerabilities in cancer cells.

Protein kinase CK2 - diverse roles in cancer cell biology and therapeutic promise.

The association of protein kinase CK2 (formerly casein kinase II or 2) with cell growth and proliferation in cells was apparent at early stages of its investigation. A cancer-specific role for CK2 remained unclear until it was determined that CK2 was also a potent suppressor of cell death (apoptosis); the latter characteristic differentiated its function in normal versus malignant cells because dysregulation of both cell growth and cell death is a universal feature of cancer cells. Over time, it became evident that CK2 exerts its influence on a diverse range of cell functions in normal as well as in transformed cells. As such, CK2 and its substrates are localized in various compartments of the cell. The dysregulation of CK2 is documented in a wide range of malignancies; notably, by increased CK2 protein and activity levels with relatively moderate change in its RNA abundance. High levels of CK2 are associated with poor prognosis in multiple cancer types, and CK2 is a target for active research and testing for cancer therapy. Aspects of CK2 cellular roles and targeting in cancer are discussed in the present review, with focus on nuclear and mitochondrial functions and prostate, breast and head and neck malignancies.

CX-4945 and siRNA-Mediated Knockdown of CK2 Improves Cisplatin Response in HPV(+) and HPV(-) HNSCC Cell Lines.

Head and neck squamous cell carcinoma (HNSCC) can be categorized into human papillomavirus (HPV) positive or negative disease. Elevated protein kinase CK2 level and activity have been historically observed in HNSCC cells. Previous studies on CK2 in HNSCC did not generally include consideration of HPV(+) and HPV(-) status. Here, we investigated the response of HPV(+) and HPV(-) HNSCC cells to CK2 targeting using CX-4945 or siRNA downregulation combined with cisplatin treatment. HNSCC cell lines were examined for CK2 expression levels and activity and response to CX-4945, with and without cisplatin. CK2 levels and NFκB p65-related activity were high in HPV(+) HNSCC cells relative to HPV(-) HNSCC cells. Treatment with CX-4945 decreased viability and cisplatin IC50 in all cell lines. Targeting of CK2 increased tumor suppressor protein levels for p21 and PDCD4 in most instances. Further study is needed to understand the role of CK2 in HPV(+) and HPV(-) HNSCC and to determine how incorporation of the CK2-targeted inhibitor CX-4945 could improve cisplatin response in HNSCC.

Identification of high protein kinase CK2α in HPV(+) oropharyngeal squamous cell carcinoma and correlation with clinical outcomes.

BACKGROUND: Oropharyngeal squamous cell carcinoma (OPSCC) incidence is rising worldwide, especially human papillomavirus (HPV)-associated disease. Historically, high levels of protein kinase CK2 were linked with poor outcomes in head and neck squamous cell carcinoma (HNSCC), without consideration of HPV status. This retrospective study examined tumor CK2α protein expression levels and related clinical outcomes in a cohort of Veteran OPSCC patient tumors which were determined to be predominantly HPV(+). METHODS: Patients at the Minneapolis VA Health Care System with newly diagnosed primary OPSCC from January 2005 to December 2015 were identified. A total of 119 OPSCC patient tumors were stained for CK2α, p16 and Ki-67 proteins and E6/E7 RNA. CK2α protein levels in tumors and correlations with HPV status and Ki-67 index were assessed. Overall survival (OS) analysis was performed stratified by CK2α protein score and separately by HPV status, followed by Cox regression controlling for smoking status. To strengthen the limited HPV(-) data, survival analysis for HPV(-) HNSCC patients in the publicly available The Cancer Genome Atlas (TCGA) PanCancer RNA-seq dataset was determined for CSNK2A1. RESULTS: The patients in the study population were all male and had a predominant history of tobacco and alcohol use. This cohort comprised 84 HPV(+) and 35 HPV(-) tumors. CK2α levels were higher in HPV(+) tumors compared to HPV(-) tumors. Higher CK2α scores positively correlated with higher Ki-67 index. OS improved with increasing CK2α score and separately OS was significantly better for those with HPV(+) as opposed to HPV(-) OPSCC. Both remained significant after controlling for smoking status. High CSNK2A1 mRNA levels from TCGA data associated with worse patient survival in HPV(-) HNSCC. CONCLUSIONS: High CK2α protein levels are detected in HPV(+) OPSCC tumors and demonstrate an unexpected association with improved survival in a strongly HPV(+) OPSCC cohort. Worse survival outcomes for high CSNK2A1 mRNA levels in HPV(-) HNSCC are consistent with historical data. Given these surprising findings and the rising incidence of HPV(+) OPSCC, further study is needed to understand the biological roles of CK2 in HPV(+) and HPV(-) HNSCC and the potential utility for therapeutic targeting of CK2 in these two disease states.

A negative reciprocal regulatory axis between cyclin D1 and HNF4α modulates cell cycle progression and metabolism in the liver.

Hepatocyte nuclear factor 4α (HNF4α) is a master regulator of liver function and a tumor suppressor in hepatocellular carcinoma (HCC). In this study, we explore the reciprocal negative regulation of HNF4α and cyclin D1, a key cell cycle protein in the liver. Transcriptomic analysis of cultured hepatocyte and HCC cells found that cyclin D1 knockdown induced the expression of a large network of HNF4α-regulated genes. Chromatin immunoprecipitation-sequencing (ChIP-seq) demonstrated that cyclin D1 inhibits the binding of HNF4α to thousands of targets in the liver, thereby diminishing the expression of associated genes that regulate diverse metabolic activities. Conversely, acute HNF4α deletion in the liver induces cyclin D1 and hepatocyte cell cycle progression; concurrent cyclin D1 ablation blocked this proliferation, suggesting that HNF4α maintains proliferative quiescence in the liver, at least, in part, via repression of cyclin D1. Acute cyclin D1 deletion in the regenerating liver markedly inhibited hepatocyte proliferation after partial hepatectomy, confirming its pivotal role in cell cycle progression in this in vivo model, and enhanced the expression of HNF4α target proteins. Hepatocyte cyclin D1 gene ablation caused markedly increased postprandial liver glycogen levels (in a HNF4α-dependent fashion), indicating that the cyclin D1-HNF4α axis regulates glucose metabolism in response to feeding. In AML12 hepatocytes, cyclin D1 depletion led to increased glucose uptake, which was negated if HNF4α was depleted simultaneously, and markedly elevated glycogen synthesis. To summarize, mutual repression by cyclin D1 and HNF4α coordinately controls the cell cycle machinery and metabolism in the liver.

Protein kinase CK2 impact on intracellular calcium homeostasis in prostate cancer.

Protein kinase CK2 plays multiple roles in cell function in normal and disease states. CK2 is elevated in numerous types of cancer cells, and CK2 suppression of apoptosis represents a key link to the cancer cell phenotype. CK2 regulation of cell survival and death involves diverse processes, and our previous work suggested that mitochondrial machinery is a key locus of this function. One of the earliest responses of prostate cells to inhibition of CK2 is a change in mitochondrial membrane potential, possibly associated with Ca2+ signaling. Thus, in the present work, we investigated early impact of CK2 on intracellular Ca2+ dynamics. Three prostate cancer (PCa) cell lines, PC3-LN4, C4-2B, and 22Rv1, were studied. PCa cells were treated with the CK2 small molecule inhibitors 4,5,6,7-tetrabrombenzotriazole and CX-4945 followed by analysis of Ca2+ levels in various cellular compartments over time. The results showed dose-dependent loss in cytosolic Ca2+ levels starting within 2 min and reaching maximal loss within 5-10 min. There was a concomitant increase in Ca2+ in the endoplasmic reticulum (ER) and mitochondrial compartments. The results suggest that inhibition of CK2 activity results in a rapid movement of Ca2+ out of the cytosol and into the ER and mitochondria, which may be among the earliest contributory factors for induction of apoptosis in cells subjected to inhibition of CK2. In cells with death-inducing levels of CK2 inhibition, total cellular Ca2+ levels dropped at 2 h post-treatment. These novel observations represent a potential mechanism underlying regulation of cell survival and death by CK2 activity.

Cyclin-Dependent Kinase and Antioxidant Gene Expression in Cancers with Poor Therapeutic Response.

Pancreatic cancer, hepatocellular carcinoma (HCC), and mesothelioma are treatment-refractory cancers, and patients afflicted with these cancers generally have a very poor prognosis. The genomics of these tumors were analyzed as part of The Cancer Genome Atlas (TCGA) project. However, these analyses are an overview and may miss pathway interactions that could be exploited for therapeutic targeting. In this study, the TCGA Pan-Cancer datasets were queried via cBioPortal for correlations among mRNA expression of key genes in the cell cycle and mitochondrial (mt) antioxidant defense pathways. Here we describe these correlations. The results support further evaluation to develop combination treatment strategies that target these two critical pathways in pancreatic cancer, hepatocellular carcinoma, and mesothelioma.

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.

Immune characteristics correlating with HSV-1 immune control and effect of squaric acid dibutyl ester on immune characteristics of subjects with frequent herpes labialis episodes.

INTRODUCTION: Differences in immune characteristics, including immune gene expression by peripheral blood mononuclear cells (PBMCs), correlating with herpes labialis and good or poor immune control of herpes simplex virus type 1 (HSV-1), and how these characteristics change after dosing with squaric acid dibutyl ester (SADBE), were investigated. METHODS: PBMCs were collected from persons positive for IgG against HSV-1 and having frequent, infrequent, or no herpes labialis outbreaks. The PBMCs were tested for proliferation against HSV-1 and a fungal antigen (Candida) and immune gene expression in the presence of HSV-1 and Candida. On day 1 after blood collection the subjects with frequent outbreaks were dosed topically on the arm once with SADBE, and their PBMCs were collected and tested 8 weeks later. RESULTS: Those with good immune control of their HSV-1 infection (fewer outbreaks) differ from those with poorer immune control in these ways: (1) Greater PBMC proliferation in vitro to HSV-1, HSV-1-infected cell extracts, and Candida considered together (P < 0.01). (2) Higher expression of IFNG and five other immune-related genes (P < 0.05 for each) and lower expression of IL5 and two other immune-related genes (P < 0.05 for each) in PBMCs in vitro stimulated with HSV-1 virus. The subjects with frequent outbreaks were treated once with SADBE, and 56 days later the PBMCs of these subjects differed from PBMCs from the same subjects taken on day 1 before treatment in exactly the same ways listed above as differences between those with good and poor immune control of HSV-1, and at the same levels of significance. CONCLUSIONS: Higher IFNG and lower IL5 expression by PBMCs in the presence of HSV-1 correlate with fewer herpes labialis outbreaks, and a single topical dose of SADBE to the arm of subjects with frequent herpes labialis episodes improves immune response to HSV-1.

Evaluation of protein kinase CK2 as a therapeutic target for squamous cell carcinoma of cats.

OBJECTIVE To investigate protein kinase CK2 (CK2) expression in squamous cell carcinoma (SCC) of cats and to examine effects of CK2 downregulation on in vitro apoptosis and viability in SCC. SAMPLE Biopsy specimens of oral mucosa and testis and blood samples from clinically normal cats, biopsy specimens of oral SCC from cats, and feline SCC (SCCF1) and mammary gland carcinoma (K12) cell lines. PROCEDURES Immunohistochemical labeling for CK2α was performed on biopsy specimens. Sequences of the CK2α subunit gene and CK2α' subunit gene in feline blood and feline cancer cell lines were determined by use of PCR and reverse-transcription PCR assays followed by direct Sanger sequencing. Specific small interfering RNAs (siRNAs) were developed for feline CK2α and CK2α'. The SCCF1 cells were treated with siRNA and assessed 72 hours later for CK2α and CK2α' expression and markers of apoptosis (via western blot analysis) and for viability (via 3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium assays). RESULTS CK2α was expressed in all feline oral mucosa samples and 7 of 8 oral SCC samples. Expression of CK2α and CK2α' was successfully downregulated in SCCF1 cells by use of siRNAs, which resulted in decreased viability and induction of apoptosis. CONCLUSIONS AND CLINICAL RELEVANCE In this study, CK2 appeared to be a promising therapeutic target for SCCs of cats. A possible treatment strategy for SCCs of cats would be RNA interference that targets CK2.

Therapeutic Targeting of Protein Kinase CK2 Gene Expression in Feline Oral Squamous Cell Carcinoma: A Naturally Occurring Large-Animal Model of Head and Neck Cancer.

Protein kinase CK2 (CK2) is a highly promising target for cancer therapy, and anti-CK2 gene expression therapy has shown effectiveness in rodent models of human head and neck cancer (HNC). To date, there has been no large-animal model of cancer in which to further explore anti-CK2 therapies. Feline oral squamous cell carcinoma (FOSCC) has been proposed as a large-animal model for human HNC, and we have previously shown that CK2 is a rational target in FOSCC. Here we have tested the hypothesis that a novel tenfibgen-coated tumor-specific nanocapsule carrying RNA interference (RNAi) oligonucleotides targeting feline CK2α and CK2α' (TBG-RNAi-fCK2αα') would be safe in cats with FOSCC; assessment of target inhibition and tumor response were secondary aims. Nine cats were enrolled and treated at two dose levels in a 3+3 escalation. Cats received a total of six treatments with TBG-RNAi-fCK2αα'. Pre- and posttreatment, tumor and normal oral mucosa biopsies were collected to assess CK2 expression, using immunohistochemistry (IHC) preparations evaluated by light microscopy. Toxicity and tumor response were assessed on the basis of standard criteria. The most common adverse events were grade 1 or 2 weight loss and anorexia. Grade 3 tissue necrosis was seen in association with tumor response in one cat, asymptomatic grade 4 elevations in aspartate transaminase and creatine phosphokinase in one cat, and asymptomatic grade 3 hypokalemia in one cat. Of six cats with evaluable biopsies, two had a reduction in CK2 IHC score in tumors after treatment. Four cats had progressive disease during the study period, three had stable disease, one had partial response, and response could not be evaluated in one cat. We conclude that the drug appeared safe and that there is some evidence of efficacy in FOSCC. Further investigation regarding dosing, schedule, target modulation, toxicity, and efficacy in a larger group of cats is warranted and may inform future clinical studies in human head and neck cancer.

CK2 Molecular Targeting-Tumor Cell-Specific Delivery of RNAi in Various Models of Cancer.

Protein kinase CK2 demonstrates increased protein expression relative to non-transformed cells in the majority of cancers that have been examined. The elevated levels of CK2 are involved in promoting not only continued proliferation of cancer cells but also their resistance to cell death; thus, CK2 has emerged as a plausible target for cancer therapy. Our focus has been to target CK2 catalytic subunits at the molecular level using RNA interference (RNAi) strategies to achieve their downregulation. The delivery of oligonucleotide therapeutic agents warrants that they are protected and are delivered specifically to cancer cells. The latter is particularly important since CK2 is a ubiquitous signal that is essential for survival. To achieve these goals, we have developed a nanocapsule that has the properties of delivering an anti-CK2 RNAi therapeutic cargo, in a protected manner, specifically to cancer cells. Tenfibgen (TBG) is used as the ligand to target tenascin-C receptors, which are elevated in cancer cells. This strategy is effective for inhibiting growth and inducing death in several types of xenograft tumors, and the nanocapsule elicits no safety concerns in animals. Further investigation of this therapeutic approach for its translation is warranted.

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.

Preclinical evaluation of cyclin dependent kinase 11 and casein kinase 2 survival kinases as RNA interference targets for triple negative breast cancer therapy.

INTRODUCTION: Targeted therapies for aggressive breast cancers like triple negative breast cancer (TNBC) are needed. The use of small interfering RNAs (siRNAs) to disable expression of survival genes provides a tool for killing these cancer cells. Cyclin dependent kinase 11 (CDK11) is a survival protein kinase that regulates RNA transcription, splicing and mitosis. Casein kinase 2 (CK2) is a survival protein kinase that suppresses cancer cell death. Eliminating the expression of these genes has potential therapeutic utility for breast cancer. METHODS: Expression levels of CDK11 and CK2 mRNAs and associated proteins were examined in breast cancer cell lines and tissue arrays. RNA expression levels of CDC2L1, CDC2L2, CCNL1, CCNL2, CSNK2A1, CSNK2A2, and CSNK2B genes in breast cancer subtypes were analyzed. Effects following transfection of siRNAs against CDK11 and CK2 in cultured cells were examined by viability and clonal survival assays and by RNA and protein measures. Uptake of tenfibgen (TBG) nanocapsules by TNBC cells was analyzed by fluorescence-activated cell sorting. TBG nanocapsules delivered siRNAs targeting CDK11 or CK2 in mice carrying TNBC xenograft tumors. Transcript cleavage and response parameters were evaluated. RESULTS: We found strong CDK11 and CK2 mRNA and protein expression in most human breast cancer cells. Immunohistochemical analysis of TNBC patient tissues showed 100% of tumors stained positive for CDK11 with high nuclear intensity compared to normal tissue. The Cancer Genome Atlas analysis comparing basal to other breast cancer subtypes and to normal breast revealed statistically significant differences. Down-regulation of CDK11 and/or CK2 in breast cancer cells caused significant loss of cell viability and clonal survival, reduced relevant mRNA and protein expression, and induced cell death changes. TBG nanocapsules were taken up by TNBC cells both in culture and in xenograft tumors. Treatment with TBG- siRNA to CDK11 or TBG- siRNA to CK2αα' nanocapsules induced appropriate cleavage of CDK11 and CK2α transcripts in TNBC tumors, and caused MDA-MB-231 tumor reduction, loss of proliferation, and decreased expression of targeted genes. CONCLUSIONS: CDK11 and CK2 expression are individually essential for breast cancer cell survival, including TNBC. These genes serve as promising new targets for therapeutic development in breast cancer.

Tenfibgen ligand nanoencapsulation delivers bi-functional anti-CK2 RNAi oligomer to key sites for prostate cancer targeting using human xenograft tumors in mice.

Protected and specific delivery of nucleic acids to malignant cells remains a highly desirable approach for cancer therapy. Here we present data on the physical and chemical characteristics, mechanism of action, and pilot therapeutic efficacy of a tenfibgen (TBG)-shell nanocapsule technology for tumor-directed delivery of single stranded DNA/RNA chimeric oligomers targeting CK2αα' to xenograft tumors in mice. The sub-50 nm size TBG nanocapsule (s50-TBG) is a slightly negatively charged, uniform particle of 15 - 20 nm size which confers protection to the nucleic acid cargo. The DNA/RNA chimeric oligomer (RNAi-CK2) functions to decrease CK2αα' expression levels via both siRNA and antisense mechanisms. Systemic delivery of s50-TBG-RNAi-CK2 specifically targets malignant cells, including tumor cells in bone, and at low doses reduces size and CK2-related signals in orthotopic primary and metastatic xenograft prostate cancer tumors. In conclusion, the s50-TBG nanoencapsulation technology together with the chimeric oligomer targeting CK2αα' offer significant promise for systemic treatment of prostate malignancy.

Protein kinase CK2 inhibition induces cell death via early impact on mitochondrial function.

CK2 (official acronym for casein kinase 2 or II) is a potent suppressor of apoptosis in response to diverse apoptotic stimuli-thus its molecular downregulation or activity inhibition results in potent induction of cell death. CK2 downregulation is known to impact mitochondrial apoptotic circuitry but the underlying mechanism(s) remain unclear. Utilizing prostate cancer cell lines subjected to CK2-specific inhibitors which cause loss of cell viability, we have found that CK2 inhibition in cells causes rapid early decrease in mitochondrial membrane potential (Δψm). Cells treated with the CK2 inhibitors TBB (4,5,6,7-tetrabromobenzotriazole) or TBCA (tetrabromocinnamic acid) demonstrate changes in Δψm which become apparent within 2 h, that is, significantly prior to evidence of activation of other mitochondrial apoptotic signals whose temporal expression ensues subsequent to loss of Δψm. Further, we have demonstrated the presence of CK2 in purified mitochondria and it appears that the effect on Δψm evoked by inhibition of CK2 may involve mitochondrial localized CK2. Results also suggest that alterations in Ca(2+) signaling may be involved in the CK2 mediated regulation of Δψm and mitochondrial permeability. Thus, we propose that a key mechanism of CK2 impact on mitochondrial apoptotic circuitry and cell death involves early loss of Δψm which may be a primary trigger for apoptotic signaling and cell death resulting from CK2 inhibition.

Mechanism and efficacy of sub-50-nm tenfibgen nanocapsules for cancer cell-directed delivery of anti-CK2 RNAi to primary and metastatic squamous cell carcinoma.

Improved survival for patients with head and neck cancers (HNC) with recurrent and metastatic disease warrants that cancer therapy is specific, with protected delivery of the therapeutic agent to primary and metastatic cancer cells. A further objective should be that downregulation of the intracellular therapy target leads to cell death without compensation by an alternate pathway. To address these goals, we report the utilization of a sub-50-nm tenfibgen (s50-TBG) nanocapsule that delivers RNAi oligonucleotides directed against the essential survival signal protein kinase CK2 (RNAi-CK2) in a cancer cell-specific manner. We have evaluated mechanism and efficacy of using s50-TBG-RNAi-CK2 nanocapsules for therapy of primary and metastatic head and neck squamous cell carcinoma (HNSCC). s50-TBG nanocapsules enter cancer cells via the lipid raft/caveolar pathway and deliver their cargo (RNAi-CK2) preferentially to malignant but not normal tissues in mice. Our data suggest that RNAi-CK2, a unique single-stranded oligonucleotide, co-opts the argonaute 2/RNA-induced silencing complex pathway to target the CK2αα' mRNAs. s50-TBG-RNAi-CK2 inhibited cell growth corresponding with reduced CK2 expression in targeted tumor cells. Treatment of three xenograft HNSCC models showed that primary tumors and metastases responded to s50-TBG-RNAi-CK2 therapy, with tumor shrinkage and 6-month host survival that was achieved at relatively low doses of the therapeutic agent without any adverse toxic effect in normal tissues in the mice. We suggest that our nanocapsule technology and anti-CK2 targeting combine into a therapeutic modality with a potential of significant translational promise.

Tenfibgen-DMAT Nanocapsule Delivers CK2 Inhibitor DMAT to Prostate Cancer Xenograft Tumors Causing Inhibition of Cell Proliferation.

CK2 is a master regulator protein kinase which demonstrates heightened expression in diverse cancer types and is considered a promising target for therapy. Given its ubiquitous expression and potent influence on cell survival, cancer cell-directed targeting of the CK2 signal is an important factor for development of an anti-CK2 therapeutic. We previously reported on the malignant cell specificity and effect on CK2 signaling of a tenfibgen (TBG) based nanocapsule for delivery of the CK2 small molecule inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) in cultured prostate cancer cells. Here we tested the ability of TBG-DMAT to affect the growth of prostate xenograft tumors in mice. Our results show that treatment of PC3-LN4 xenograft tumors with TBG-DMAT caused loss of proliferative Ki-67 signal as well as Nuclear Factor-kappa B (NF-κB) expression in the tumors. Further, the TBG-DMAT nanocapsule was detected in tumors and not in liver or testis. In conclusion, TBG-based nanocapsule delivery of anti-CK2 small molecule drugs holds significant promise for treatment of prostate cancer.