Gretchen M. Unger

Protein kinase CK2 – A key suppressor of apoptosis

Protein kinase CK2 is a ubiquitous and highly conserved protein serine/threonine kinase that is indispensable for cell survival. CK2 has long been implicated in cell growth and proliferation, and studies from several laboratories have suggested that CK2 plays a global role in affecting cell growth related activities. Recently, we documented that CK2, besides its role in cell growth and proliferation, can potently suppress apoptosis. Considering that CK2 has been found to be elevated in all the cancers that have been examined, the ability of CK2 to suppress apoptosis is particularly important in the context of cancer cell pathobiology since these cells exhibit dysregulation of both cell proliferation and cell death. Thus, overexpression of CK2 in cancer cells may impart a survival advantage by its action as a suppressor of apoptotic activity in these cells while promoting cell growth. In experimental studies, we have shown that overexpression of CK2 in cells can potently inhibit apoptosis mediated by a variety of agents including removal of survival factors, chemical and physical agents, and death receptor ligands. On the other hand, inhibition of CK2 by chemical inhibitors or by its molecular downregulation by antisense CK2 ODN or siRNA leads to potent induction of apoptosis. Downregulation of CK2 is associated with apoptosis mediated via effects on several downstream targets, and it appears that CK2 may have a global impact on the apoptotic machinery. While CK2 is present in both the nuclear and cytoplasmic compartments, several of its cell growth and cell death related activities appear to be associated with its signalling to the nuclear structures such as chromatin and nuclear matrix. In general, shuttling of CK2 to these compartments correlates with its role in cell growth and suppression of apoptotic activity whereas loss of CK2 from the nuclear structures is associated with induction of apoptosis and cessation of cell growth. These various observations on the biology of CK2 have led to our original proposal that CK2 is a potentially important target for cancer chemopreventive and therapeutic approaches; this is now being substantiated by recent studies.

Protein Kinase CK2 in Health and Disease

Abstract.Elevated levels of protein kinase CK2 (formerly casein kinase 2 or II) have long been associated with increased cell growth and proliferation both in normal and cancer cells. The ability of CK2 to also act as a potent suppressor of apoptosis offers an important link to its involvement in cancer since deregulation of both cell proliferation and apoptosis are among the key features of cancer cell biology. Dysregulated CK2 may impact both of these processes in cancer cells. All cancers that have been examined show increased CK2 expression, which may also relate to prognosis. The extensive involvement of CK2 in cancer derives from its impact on diverse molecular pathways controlling cell proliferation and cell death. Downregulation of CK2 by various approaches results in induction of apoptosis in cultured cell and xenograft cancer models suggesting its potential as a therapeutic target.

Protein kinase CK2 in health and disease: CK2: a key player in cancer biology.

Abstract.Elevated levels of protein kinase CK2 (formerly casein kinase 2 or II) have long been associated with increased cell growth and proliferation both in normal and cancer cells. The ability of CK2 to also act as a potent suppressor of apoptosis offers an important link to its involvement in cancer since deregulation of both cell proliferation and apoptosis are among the key features of cancer cell biology. Dysregulated CK2 may impact both of these processes in cancer cells. All cancers that have been examined show increased CK2 expression, which may also relate to prognosis. The extensive involvement of CK2 in cancer derives from its impact on diverse molecular pathways controlling cell proliferation and cell death. Downregulation of CK2 by various approaches results in induction of apoptosis in cultured cell and xenograft cancer models suggesting its potential as a therapeutic target.

Targeting CK2 for cancer therapy

Protein kinase CK2 is a highly ubiquitous and conserved protein serine/threonine kinase that has been found to be involved not only in cell growth and proliferation, but also in suppression of apoptosis. CK2 is capable of dynamic intracellular shuttling in response to a variety of signals. It is localized in both the nucleus and cytoplasm in normal cells, but is particularly predominant in the nuclear compartment in cancer cells. CK2 has been found to be uniformly dysregulated in all the cancers that have been examined. Downregulation of CK2 by chemical or molecular methods promotes apoptosis in cells. We have shown that antisense CK2alpha is particularly potent in inducing apoptosis in cancer cells in culture as well as in xenograft models of cancer such as prostate cancer and squamous cell carcinoma of head and neck. The antisense CK2alpha oligodeoxynucleotide (ODN) mediates tumor cell death in a dose- and time-dependent manner such that at an appropriate concentration of the antisense, a complete resolution of the xenograft tumor is observed. Interestingly, normal and benign cells (in culture as well as in vivo) demonstrate a relative resistance to the antisense CK2alpha ODN treatment, which raises the possibility of a significant therapeutic window for this therapy. Further, novel approaches such as the delivery of antisense CK2alpha ODN encapsulated in sub-50-nm tenascin nanocapsules have become available for its targeting specifically in cancer cells. Our studies minimize generally held concerns regarding suitability of CK2 as a target for cancer therapy and provide the first encouraging results for potential future application of this approach for cancer therapy.

Emergence of protein kinase CK2 as a key target in cancer therapy

Protein kinase CK2, a protein serine/threonine kinase, plays a global role in activities related to cell growth, cell death, and cell survival. CK2 has a large number of potential substrates localized in diverse locations in the cell including, for example, NF‐κB as an important downstream target of the kinase. In addition to its involvement in cell growth and proliferation it is also a potent suppressor of apoptosis, raising its key importance in cancer cell phenotype. CK2 interacts with diverse pathways which illustrates the breadth of its impact on the cellular machinery of both cell growth and cell death giving it the status of a “master regulator” in the cell. With respect to cancer, CK2 has been found to be dysregulated in all cancers examined demonstrating increased protein expression levels and nuclear localization in cancer cells compared with their normal counterparts. We originally proposed CK2 as a potentially important target for cancer therapy. Given the ubiquitous and essential for cell survival nature of the kinase, an important consideration would be to target it specifically in cancer cells while sparing normal cells. Towards that end, our design of a tenascin based sub‐50 nm (i.e., less than 50 nm size) nanocapsule in which an anti‐CK2 therapeutic agent can be packaged is highly promising because this formulation can specifically deliver the cargo intracellularly to the cancer cells in vivo. Thus, appropriate strategies to target CK2 especially by molecular approaches may lead to a highly feasible and effective approach to eradication of a given cancer.

Protein Kinase CK2 as Regulator of Cell Survival: Implications for Cancer Therapy

Recent studies have generated sufficient information to warrant a consideration of protein kinase CK2 as a potential target for cancer therapy. CK2 is a ubiquitous and highly conserved protein serine/threonine kinase that has long been considered to play a role in cell growth and proliferation. It is essential for cell survival, and considerable evidence suggests that it can also exert potent suppression of apoptosis in cells. This is important since the cancer phenotype is characterized by deregulation of not only proliferation but also of apoptosis. In normal cells, the level of CK2 appears to be tightly regulated, and cells resist a change in their intrinsic level of CK2. However, in all the cancers that have been examined an elevation of CK2 has been observed. Further, it appears that modest deregulation in the CK2 expression imparts a potent oncogenic potential to the cells. Disruption of CK2 by treatment of cells with antisense CK2 results in induction of apoptosis in a time and dose-dependent manner. Thus, we propose that down-regulation of CK2 by employing specific strategies to deliver antisense CK2 in vivo could have a potential role in cancer therapy.

Downregulation of CK2 induces apoptosis in cancer cells – A potential approach to cancer therapy

We have previously documented that naked antisense CK2α ODN can potently induce apoptosis in cancer cells in culture and in mouse xenograft human prostate cancer. The effects of the antisense CK2α are related to downregulation of CK2α message and rapid loss of the CK2 from the nuclear compartment. Here we demonstrate that downregulation of CK2 elicited by diverse methods leads to inhibition of cell growth and induction of apoptosis. The various approaches to downregulation of CK2 employed were transfection with kinase-inactive plasmid, use of CK2α siRNA, use of inhibitors of CK2 activity, and use of antisense CK2α ODN packaged in sub-50 nm nanocapsules made from tenascin. In all cases, the downregulation of CK2 is associated with loss in cell survival. We have also described preliminary observations on an approach to targeting CK2 in cancer cells. For this, sub-50 nm tenascin-based nanocapsules bearing the antisense CK2α ODN were employed to test that the antisense is delivered to the cancer cells in vivo. The results provide the first preliminary evidence that such an approach may be feasible for targeting CK2 in cancer cells. Together, our results suggest that CK2 is potentially a highly plausible target for cancer therapy.

Nanocapsule-delivered Sleeping Beauty mediates therapeutic Factor VIII expression in liver sinusoidal endothelial cells of hemophilia A mice

Liver sinusoidal endothelial cells are a major endogenous source of Factor VIII (FVIII), lack of which causes the human congenital bleeding disorder hemophilia A. Despite extensive efforts, gene therapy using viral vectors has shown little success in clinical hemophilia trials. Here we achieved cell type-specific gene targeting using hyaluronan- and asialoorosomucoid-coated nanocapsules, generated using dispersion atomization, to direct genes to liver sinusoidal endothelial cells and hepatocytes, respectively. To highlight the therapeutic potential of this approach, we encapsulated Sleeping Beauty transposon expressing the B domain-deleted canine FVIII in cis with Sleeping Beauty transposase in hyaluronan nanocapsules and injected them intravenously into hemophilia A mice. The treated mice exhibited activated partial thromboplastin times that were comparable to those of wild-type mice at 5 and 50 weeks and substantially shorter than those of untreated controls at the same time points. Further, plasma FVIII activity in the treated hemophilia A mice was nearly identical to that in wild-type mice through 50 weeks, while untreated hemophilia A mice exhibited no detectable FVIII activity. Thus, Sleeping Beauty transposon targeted to liver sinusoidal endothelial cells provided long-term expression of FVIII, without apparent antibody formation, and improved the phenotype of hemophilia A mice.

CK2 Modulation of NF-κB, TP53, and the Malignant Phenotype in Head and Neck Cancer by Anti-CK2 Oligonucleotides In vitro or In vivo via Sub-50-nm Nanocapsules

Purpose: The aim of this study is to investigate the expression of CK2 subunits and CK2 effects on NF-κB–mediated and TP53-mediated signal activation and gene expression, the malignant phenotype, and chemosensitivity in head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo. Experimental Design: Protein expression of CK2 subunits was investigated by Western blot and immunohistochemistry. CK2 subunits were knocked down by small interfering RNA, and NF-κB activation was examined using DNA binding, Western blot, and luciferase reporter assays. Gene expression was measured by quantitative reverse transcription–PCR. Cell growth, survival, motility, and sensitivity to cisplatin were measured by MTT, flow cytometry, and migration assays. In vivo targeting of CK2α/α′ in HNSCC xenograft models was achieved using anti-CK2α/α′ oligodeoxynucleotide encapsulated in sub–50-nm tenfibgen nanocapsules. Results: CK2 subunit proteins were overexpressed in HNSCC lines and tissues. Knockdown of CK2 subunits differentially inhibited IκBα degradation, NF-κB nuclear localization, phosphorylation, DNA binding, and reporter activity. CK2 subunits modulated gene expression and the malignant phenotype involved in cell cycle and migration, whereas CK2α is critical to promote proliferation, antiapoptosis, and cisplatin resistance in vitro. Furthermore, in vivo delivery of anti-CK2α/α′ oligodeoxynucleotide nanocapsules significantly suppressed tumor growth in HNSCC xenograft models, in association with modulation of CK2 and NF-κB regulated molecules, TP53 family proteins, and induction of apoptosis. Conclusions: Our study reveals a novel role of CK2 in coregulating NF-κB activation, TP53/p63 expression, and downstream gene expression. Downregulation of CK2 in HNSCC models in vitro and in vivo shows antitumor effects as well as sensitization to cisplatin. Clin Cancer Res; 16(8); 2295–307. ©2010 AACR.

Improving Surgical Wound Healing with Basic Fibroblast Growth Factor after Radiation

Objectives/Hypothesis: Delayed wound healing in surgical patients who have received previous irradiation continues to be a significant problem. We investigated whether radiation decreases basic fibroblast growth factor (bFGF) production in skin and whether supplemental bFGF can improve irradiated postsurgical soft tissue healing.