Protein kinase CK2 is a central regulator of topoisomerase I hyperphosphorylation and camptothecin sensitivity in cancer cell lines.

Topoisomerase I (topo I) is required to unwind DNA during synthesis and provides the unique target for camptothecin-derived chemotherapeutic agents, including Irinotecan and Topotecan. While these agents are highly effective anticancer agents, some tumors do not respond due to intrinsic or acquired resistance, a process that remains poorly understood. Because of treatment toxicity, there is interest in identifying cellular factors that regulate tumor sensitivity and might serve as predictive biomarkers of therapy sensitivity. Here we identify the serine kinase, protein kinase CK2, as a central regulator of topo I hyperphosphorylation and activity and cellular sensitivity to camptothecin. In nine cancer cell lines and three normal tissue-derived cell lines we observe a consistent correlation between CK2 levels and camptothecin responsiveness. Two other topo I-targeted serine kinases, protein kinase C and cyclin-dependent kinase 1, do not show this correlation. Camptothecin-sensitive cancer cell lines display high CK2 activity, hyperphosphorylation of topo I, elevated topo I activity, and elevated phosphorylation-dependent complex formation between topo I and p14ARF, a topo I activator. Camptothecin-resistant cancer cell lines and normal cell lines display lower CK2 activity, lower topo I phosphorylation, lower topo I activity, and undetectable topo I/p14ARF complex formation. Experimental inhibition or activation of CK2 demonstrates that CK2 is necessary and sufficient for regulating these topo I properties and altering cellular responses to camptothecin. The results establish a cause and effect relationship between CK2 activity and camptothecin sensitivity and suggest that CK2, topo I phosphorylation, or topo I/p14ARF complex formation could provide biomarkers of therapy-responsive tumors.

Progressive silencing of p14ARF in oesophageal adenocarcinoma.

The frequency of oesophageal adenocarcinoma is increasing in Western countries for unknown reasons, and correlates with a corresponding increase in the pre-malignant condition, Barrett's Oesophagus, which raises the risk of adenocarcinoma by some 40- to 125-fold. We have examined how disease progression correlates with changes in expression of the p14ARF (ARF) tumour suppressor, a key regulator of the p53 tumour suppressor pathway that is silenced in some 30% of cancers overall, but for which a role in oesophageal cancer is unclear. We have used quantitative PCR, RT-PCR, methylation-specific PCR and chromatin-immunoprecipitation to examine the regulation and function of ARF in oesophageal adenocarcinoma tissue specimens and cell lines. We find highly significant reductions (P< 0.001) in ARF expression during disease progression from normal oesophageal epithelium to Barrett's Oesophagus to adenocarcinoma, with 57/76 (75%) adenocarcinomas displaying undetectable levels of ARF expression. Retention of ARF expression in adenocarcinoma is a highly significant indicator of increased survival (P< 0.001) and outperforms all clinical variables in a multivariate model. CpG methylation as well as histone H3 methylation of lysines 9 and 27 contribute independently to ARF gene silencing in adenocarcinoma cell lines and can be reversed by 5-aza-2'-deoxycytidine. The results suggest that silencing of ARF is involved in the pathogenesis of oesophageal adenocarcinoma and show that either DNA or histone methylation can provide the primary mechanism for ARF gene silencing. Silencing of ARF could provide a useful marker for increased risk of progression and poor prognosis.

Mesenchymal stem cell-mediated delivery of therapeutic adenoviral vectors to prostate cancer.

BACKGROUND: There is an urgent need for targeted biological therapies for prostate cancer with greater efficacy and less toxicity, particularly for metastatic disease, where current therapies are not curative. Therapeutic adenoviral vectors or oncolytic adenoviruses offer the possibility of a competent, nontoxic therapeutic alternative for prostate cancer. However, free viral particles must be delivered locally, an approach that does not address metastatic disease, and they display poor tumor penetration. To fully exploit the potential of these vectors, we must develop methods that improve intratumoral dissemination and allow for systemic delivery. This study establishes a proof-of-principle rationale for a novel human mesenchymal stem (stromal) cell-based approach to improving vector delivery to tumors. METHODS/RESULTS: We have generated mesenchymal stem cell-derived packaging cells for adenoviruses (E1-modified mesenchymal stem cells) by modifying human mesenchymal stem cells with the adenovirus (type C) E1A/B genes needed for viral replication. Using cell-based assays, we have demonstrated that two adenoviral vectors, replication-defective adenovirus expressing p14 and p53 or conditionally replicating oncolytic adenovirus, packaged by E1A/B-modified mesenchymal stem cells, suppress the growth of prostate cancer cells in culture. Using subcutaneous xenograft models for human prostate cancer in mice, we have shown that E1A/B-modified mesenchymal stem cells display tumor tropism in tumor-bearing nude mice, that E1A/B-modified mesenchymal stem cells disseminate well within tumors, and that replication-defective adenovirus expressing p14 and p53 or conditionally replicating oncolytic adenovirus-loaded E1-modified mesenchymal stem cells suppresses tumor growth in mice. CONCLUSION: The results show that this approach, if optimized, could circumvent the obstacles to efficient gene delivery encountered with current gene delivery approaches and provide an effective, nontoxic therapeutic alternative for metastatic disease.

Macrophage-mediated bystander effect triggered by tumor cell apoptosis.

Restoration of apoptosis is an important therapeutic strategy for cancer, but bystander effects may be crucial to treatment success. We examined the involvement of bystander effects in the outcome of pro-apoptotic treatments and investigated the role of macrophages. Using a murine N202 breast cancer chamber model and intravital microscopy, we observed bystander apoptosis in vivo in mixed spheroids consisting of bystander N202 cells plus modified N202 cells overexpressing the p14ARF N-terminal region, which promotes p53-mediated apoptosis. The effect was not observed in cocultures in vitro, and could not be transferred through conditioned medium from modified N202 cells. However, if macrophages were also included in the N202 co-cultures, bystander apoptosis was restored, and correlated with elevated surface expression of phosphatidyl serine, a macrophage recognition molecule, on the modified N202 cells. Bystander killing was not observed in cocultures of N202 cells plus macrophages plus cisplatin-treated or 5-fluorouracil-treated N202 cells, where apoptosis induction in the target cell population was inefficient, suggesting that specific activation of macrophages by apoptotic tumor cells was required. The results suggest that pro-apoptotic therapies benefit both from the intrinsic vulnerability of cancer cells to apoptosis and from an innate immune response that amplifies the therapeutic effect.

DNA damage disrupts the p14ARF-B23(nucleophosmin) interaction and triggers a transient subnuclear redistribution of p14ARF.

The p14 alternate reading frame (ARF) tumor suppressor plays a central role in cancer by binding to mdm2 (Hdm2 in humans) and enhancing p53-mediated apoptosis following DNA damage and oncogene activation. It is unclear, however, how ARF initiates its involvement in the p53/mdm2 pathway, as p53 and mdm2 are located in the nucleoplasm, whereas ARF is largely nucleolar in tumor cells. We have used immunofluorescence and coimmunoprecipitation to examine how the subnuclear distribution and protein-protein interactions of ARF change immediately after DNA damage and over the time course of the DNA damage response in human tumor cells. We find that DNA damage disrupts the interaction of ARF with the nucleolar protein B23(nucleophosmin) and promotes a transient p53-independent translocation of ARF to the nucleoplasm, resulting in a masking of the ARF NH2 terminus that correlates with the appearance of ARF-Hdm2 complexes. The translocation also results in an unmasking of the ARF COOH terminus, suggesting that redistribution disrupts a nucleolar interaction of ARF involving this region. By 24 hours after irradiation, DNA repair has ceased and the pretreatment immunofluorescence patterns and complexes of ARF have been restored. Although the redistribution of ARF is independent of p53 and likely to be regulated by interactions other than Hdm2, ARF does not promote UV sensitization unless p53 is expressed. The results implicate the nucleolus and nucleolar interactions of the ARF, including potentially novel interactions involving its COOH terminus as sites for early DNA damage and stress-mediated cellular events.

IRES-Mediated Protein Translation Overcomes Suppression by the p14ARF Tumor Suppressor Protein.

Internal ribosome entry sites (IRES elements) have attracted interest in cancer gene therapy because they can be used in the design of gene transfer vectors that provide bicistronic co-expression of two transgene products under the control of a single promoter. Unlike cellular translation of most mRNAs, a process that requires a post-translational 5' modification of the mRNA known as the cap structure, IRES-mediated translation is independent of the cap structure. The cellular conditions that may intervene to modulate IRES-mediated, cap-independent versus cap-dependent translation, however, remain poorly understood, although they could be critical to the choice of gene transfer vectors. Here we have compared the effects of the p14ARF (Alternate Reading Frame) tumor suppressor, a translational suppressor frequently overexpressed in cancer, on cap-dependent translation versus cap-independent translation from the EMCV viral IRES often used in bicistronic gene transfer vectors. We find that ectopic overexpression of p14ARF suppresses endogenous and ectopic cap-dependent protein translation, consistent with other studies. However, p14ARF has little or no effect on transgene translation initiated within an IRES element. This suggests that transgenes placed downstream of an IRES element will retain efficient translation of their gene products in the presence of high levels of ectopic or endogenous p14ARF, a finding that could be particularly relevant to therapeutic gene therapy strategies for cancer.

DNA damage, p14ARF, nucleophosmin (NPM/B23), and cancer.

The p53/p14ARF/mdm2 stress response pathway plays a central role in mediating cellular responses to oncogene activation, genome instability, and therapy-induced DNA damage. Abrogation of the pathway occurs in most if not all cancers, and may be essential for tumor development. The high frequency with which the pathway is disabled in cancer and the fact that the pathway appears to be incompatible with tumor cell growth, has made it an important point of focus in cancer research and therapeutics development. Recently, Nucleophosmin (NPM, B23, NO38 and numatrin), a multifunctional nucleolar protein, has emerged as a p14ARF binding protein and regulator of p53. While complex formation between ARF and NPM retains ARF in the nucleolus and prevents ARF from activating p53, DNA damaging treatments promote a transient subnuclear redistribution of ARF to the nucleoplasm, where it interacts with mdm2 and promotes p53 activation. The results add support to a recently proposed model in which the nucleolus serves as a p53-uspstream sensor of stress, and where ARF links nucleolar stress signals to nucleoplasmic effectors of the stress response. A better understanding of ARF's nucleolar interactions could further elucidate the regulation of the p53 pathway and suggest new therapeutic approaches to restore p53 function.

Enhanced tumor suppression by a p14ARF/p53 bicistronic adenovirus through increased p53 protein translation and stability.

The p53 tumor suppressor controls a cell cycle arrest and apoptosis pathway that is central to tumor suppression and often disrupted in cancer. The accumulation and activity of p53 are positively controlled by the p14/ARF tumor suppressor and full restoration of the pathway in cancer cells may require that both p53 and p14ARF be supplied [corrected]. To address this issue, we have constructed a bicistronic adenoviral vector encoding the two proteins (Adp14/p53) and compared its tumor suppressor activity with that of a single gene vector for p53 (Adp53). We find that tumor cells treated with Adp14/p53 undergo a much sharper decrease in viability with increasing multiplicities of infection than do cells treated with Adp53, even when cells express endogenous p14ARF. Adp14/p53 is also more effective than is a combination of single gene vectors for p14 and p53. The sharper decrease in cell viability after treatment of cells with Adp14/p53 correlates with an increased rate of p53 protein synthesis and a decreased rate of p53 protein turnover, leading to increased steady-state levels of p53 protein and increased levels of p53 downstream targets mdm2, p21waf1, and bax. Adp14/p53 treatment leads to an elevated bax:bcl2 ratio and induction of apoptosis in vitro and in vivo, coupled with a failure of the tumor cells to induce neovascularization in vivo. The results indicate that endogenous p14ARF expression may be insufficient to ensure efficient accumulation of ectopic p53 after gene transfer and demonstrate that for tumor suppression, bicistronic coexpression of p14ARF and p53 is superior to p53 alone. The results show that in this setting, p14ARF promotes p53 accumulation by increasing p53 protein synthesis, in addition to its well-characterized ability to oppose mdm2-mediated degradation of p53.

Topoisomerase-I PS506 as a Dual Function Cancer Biomarker.

Novel biomarkers for cancer diagnosis and therapy selection are urgently needed to facilitate early detection and improve therapy outcomes. We have previously identified a novel phosphorylation site at serine 506 (PS506) on topoisomerase-I (topo-I) and have shown that it is widely expressed in cell lines derived from several cancers, including lung cancer, but is low in cell lines derived from non-cancerous tissues. Here we have investigated how PS506 expression in lung tissue specimens correlates with their malignant status. We find that PS506 expression is significantly elevated in malignant tumors of non-small cell lung cancer (NSCLC) compared to adjacent, non-cancerous lung tissue and benign lung tumors. PS506 expression was up to 6-fold higher in malignant specimens than in paired non-malignant tissue. Using the well-characterized NIH/NCI 60-cell line panel, we correlate the most elevated expression levels of PS506 in lung, ovarian, and colon cancer cells lines with increased sensitivity to camptothecin, a plant alkaloid that targets topo-I. This is consistent with our earlier studies in a smaller sampling of cell lines and with our finding that PS506 increases topo-I DNA binding. Two widely used chemotherapeutic drugs for ovarian and colon cancer, topotecan and irinotecan, respectively, are derived from camptothecin. Irinotecan has also displayed efficacy in clinical trials of NSCLC. Our results suggest that elevated PS506 expression may correlate with clinical chemosensitivity to these agents in ovarian, colon, and NSCLC. PS506 may therefore serve as a biomarker for diagnosis or therapy selection.

Growth suppression by a p14(ARF) exon 1beta adenovirus in human tumor cell lines of varying p53 and Rb status.

We have analyzed the ability of an adenoviral vector encoding the exon 1beta region of the p14(ARF) tumor suppressor (ARF) to suppress the growth and viability of an array of tumor cell lines of various origins and varying p53 and Rb status, in order to establish the clinical potential of ARF. An important activity of ARF is regulation of p53 stability and function through binding to the mdm2 protein. By sequestering mdm2, ARF may promote growth suppression through the Rb pathway as well because mdm2 can bind to Rb and attenuate its function. Whereas the high frequency of ARF gene deletion in human cancers, accounting for some 40% of cancers overall, suggests that ARF would be a strong candidate for therapeutic application, the possible dependence of ARF activity on p53 and Rb function presents a potential limitation to its application, as these functions are often impaired in cancer. We show here that a replication-defective adenovirus, Ad1beta, encoding the exon 1beta region of ARF is most effective in tumor cells expressing endogenous wild-type p53. Nevertheless, Ad1beta suppresses tumor cell growth and viability in vitro and in vivo, inducing G1 or G2 cell cycle arrest and cell death even in tumor cells lacking both functional Rb and p53 pathways, and independently of induction of the p53 downstream targets, p21, bax, and mdm2. These results point to an activity of ARF in human tumor cells that is independent of Rb or p53, and suggest that therapeutic applications based on ARF would have a broad clinical application in cancer.

The p14ARF alternate reading frame protein enhances DNA binding of topoisomerase I by interacting with the serine 506-phosphorylated core domain.

In addition to its well-characterized function as a tumor suppressor, p14ARF (ARF) is a positive regulator of topoisomerase I (topo I), a central enzyme in DNA metabolism and a target for cancer therapy. We previously showed that topo I hyperphosphorylation, a cancer-associated event mediated by elevated levels of the protein kinase CK2, increases topo I activity and the cellular sensitivity to topo I-targeted drugs. Topo I hyperphosphorylation also increases its interaction with ARF. Because the ARF-topo I interaction could be highly relevant to DNA metabolism and cancer treatment, we identified the regions of topo I involved in ARF binding and characterized the effects of ARF binding on topo I function. Using a series of topo I deletion constructs, we found that ARF interacted with the topo I core domain, which encompasses most of the catalytic and DNA-interacting residues. ARF binding increased the DNA relaxation activity of hyperphosphorylated topo I by enhancing its association with DNA, but did not affect the topo I catalytic rate. In cells, ARF promoted the chromatin association of hyperphosphorylated, but not basal phosphorylated, topo I, and increased topo I-mediated DNA nicking under conditions of oxidative stress. The aberrant nicking was found to correlate with increased formation of DNA double-strand breaks, which are precursors of many genome destabilizing events. The results suggest that the convergent actions of oxidative stress and elevated CK2 and ARF levels, which are common features of cancer cells, lead to a dysregulation of topo I that may contribute both to the cellular response to topo I-targeted drugs and to genome instability.

CK2-mediated hyperphosphorylation of topoisomerase I targets serine 506, enhances topoisomerase I-DNA binding, and increases cellular camptothecin sensitivity.

Topoisomerase I is the target for a potent class of chemotherapeutic drugs derived from the plant alkaloid camptothecin that includes irinotecan and topotecan. In this study we have identified a novel site of CK2-mediated topoisomerase I (topo I) phosphorylation at serine 506 (PS506) that is relevant to topo I function and to cellular responses to these topo I-targeted drugs. CK2 treatment induced hyperphosphorylation of recombinant topo I and expression of the PS506 epitope, and resulted in increased binding of topo I to supercoiled plasmid DNA. Hyperphosphorylated topo I was approximately three times more effective than the basal phosphorylated enzyme at relaxing plasmid supercoils but had similar DNA cleavage activity once bound to DNA. The PS506 epitope was expressed in cancer cell lines with elevated CK2 activity, hyperphosphorylated topo I, and increased sensitivity to camptothecin. In contrast, PS506 was not detected in normal cells or cancer cell lines with lower levels of CK2 activity. By experimentally manipulating CK2 activity in cancer cell lines, we demonstrate a cause and effect relationship between CK2 activity, PS506 expression, camptothecin-induced cellular DNA damage, and cellular camptothecin sensitivity. Our results show that the PS506 epitope is an indicator of dysregulated, hyperphosphorylated topo I in cancer cells, and may thus serve as a diagnostic or prognostic biomarker and predict tumor responsiveness to widely used topo I-targeted therapies.

Phosphorylation of CRN2 by CK2 regulates F-actin and Arp2/3 interaction and inhibits cell migration.

CRN2 (synonyms: coronin 1C, coronin 3) functions in the re-organization of the actin network and is implicated in cellular processes like protrusion formation, secretion, migration and invasion. We demonstrate that CRN2 is a binding partner and substrate of protein kinase CK2, which phosphorylates CRN2 at S463 in its C-terminal coiled coil domain. Phosphomimetic S463D CRN2 loses the wild-type CRN2 ability to inhibit actin polymerization, to bundle F-actin, and to bind to the Arp2/3 complex. As a consequence, S463D mutant CRN2 changes the morphology of the F-actin network in the front of lamellipodia. Our data imply that CK2-dependent phosphorylation of CRN2 is involved in the modulation of the local morphology of complex actin structures and thereby inhibits cell migration.

Spermidinyl-CoA-based HAT inhibitors block DNA repair and provide cancer-specific chemo- and radiosensitization.

Acetyl group turnover on specific lysine epsilon-amino groups of the core chromosomal histones regulates DNA accessibility function, and the acetylating and deacetylating enzymes that govern the turnover provide important targets for the development of anti-cancer drugs. Histone deacetylase (HDAC) inhibitors have been developed and evaluated extensively in clinical trials, while the development of inhibitors of histone acetyltransferase (HAT) has proceeded more slowly. Here we have examined the cellular effects of an S-substituted coenzyme A (CoA) inhibitor of histone acetylation, consisting of spermidine (Spd) linked to the S-terminus of CoA through a thioglycolic acid linkage (adduct abbreviated as Spd-CoA), as well as the effects of a truncated Spd-CoA derivative lacking the negatively charged portion of the CoA moiety. While exposure of cancer cells to Spd-CoA has little effect on cell viability, it causes a rapid inhibition of histone acetylation that correlates with a transient arrest of DNA synthesis, a transient delay in S-phase progression, and an inhibition of nucleotide excision repair and DNA double strand break repair. These effects correlate with increased cellular sensitivity to the DNA-targeted chemotherapeutic drugs, cisplatin (Platinol()) and 5-fluorouracil, to the DNA damaging drug, camptothecin, and to UV-C irradiation. The sensitization effects of Spd-CoA are not observed in normal cells due to a barrier to uptake. The truncated Spd-CoA derivative displays similar but enhanced chemosensitization effects, suggesting that further modifications of the Spd-CoA structure could further improve potency. The results demonstrate that Spd-CoA and its truncated version are efficiently and selectively internalized into cancer cells, and suggest that the resulting inhibition of acetylation-dependent DNA repair enhances cellular sensitivity to DNA damage. These and related inhibitors of histone acetylation could therefore constitute a novel class of potent therapy sensitizers applicable to a broad range of conventional cancer treatments.

Serine phosphorylation-dependent coregulation of topoisomerase I by the p14ARF tumor suppressor.

p14ARF (ARF) and topoisomerase I play central roles in cancer and have recently been shown to interact. The interaction activates topoisomerase I, an important target for camptothecin-like chemotherapeutic drugs, but the regulation of the interaction is poorly understood. We have used the H358 and H23 lung cancer cell lines and purified recombinant human topoisomerase I to demonstrate that the ARF/topoisomerase I interaction is regulated by topoisomerase I serine phosphorylation, a modification that regulates topoisomerase I activity. Both cell lines express wild-type ARF and topoisomerase I proteins at equivalent levels, but H23 topoisomerase I, unlike that of H358 cells, is largely devoid of serine phosphorylation, has low activity, and complexes poorly with ARF. The ability of H23 topoisomerase I to complex with ARF can be restored by treatment with the serine kinase, casein kinase II. Consistent with these observations, we show that the response of H23 cells to camptothecin treatment is unaffected by changes in intracellular levels of ARF. However, in H358 and PC-3 cells, which express a serine phosphorylated topoisomerase I that complexes with ARF, ectopic overexpression of ARF causes sensitization to camptothecin, and siRNA-mediated down-regulation of endogenous ARF causes desensitization to camptothecin. These biological responses correlate with increased and decreased levels, respectively, of ARF/topoisomerase I complex and DNA-bound topoisomerase I. Thus, ARF is a serine phosphorylation-dependent coregulator of topoisomerase I in vivo, and it regulates cellular sensitivity to camptothecin by interacting with topoisomerase I. Certain cancer associated defects affecting ARF/topoisomerase I complex formation could contribute to cellular resistance to camptothecin.

Regulation of p14ARF through subnuclear compartmentalization.

The p53-mediated pathway cell cycle arrest and apoptosis is central to cancer and an important point of focus for therapeutics development. The p14ARF ("ARF") tumor suppressor induces the p53 pathway in response to oncogene activation or DNA damage. However, ARF is predominantly nucleolar in localization and engages in several interactions with nucleolar proteins, whereas p53 is nucleoplasmic. This raises the question as to how ARF initiates its involvement in the p53 pathway. We have found that UV irradiation of cells disrupts the interaction of ARF with two of its nucleolar binding partners, B23 (NPM, nucleophosmin, NO38, numatrin) and topoisomerase I, and promotes an immediate and transient subnuclear redistribution of ARF to the nucleoplasm, where it can engage the p53 pathway (Lee et al, Cancer Res 65:9834-42; 2005). The results support a model in which the nucleolus serves as a p53 upstream sensor of cellular stress, and add to a growing body of evidence that nucleolar sequestration of ARF prevents activation of p53. The results also have therapeutic implications for therapies based on exploiting p53 and other cellular stress response pathways to suppress cancer.