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

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          Abstract

          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.

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          Most cited references 67

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          One-thousand-and-one substrates of protein kinase CK2?

          CK2 (formerly termed "casein kinase 2") is a ubiquitous, highly pleiotropic and constitutively active Ser/Thr protein kinase whose implication in neoplasia, cell survival, and virus infection is supported by an increasing number of arguments. Here an updated inventory of 307 CK2 protein substrates is presented. More than one-third of these are implicated in gene expression and protein synthesis as being either transcriptional factors (60) or effectors of DNA/RNA structure (50) or translational elements. Also numerous are signaling proteins and proteins of viral origin or essential to virus life cycle. In comparison, only a minority of CK2 targets (a dozen or so) are classical metabolic enzymes. An analysis of 308 sites phosphorylated by CK2 highlights the paramount relevance of negatively charged side chains that are (by far) predominant over any other residues at positions n+3 (the most crucial one), n+1, and n+2. Based on this signature, it is predictable that proteins phosphorylated by CK2 are much more numerous than those identified to date, and it is possible that CK2 alone contributes to the generation of the eukaryotic phosphoproteome more so than any other individual protein kinase. The possibility that CK2 phosphosites play some global role, e.g., by destabilizing alpha helices, counteracting caspase cleavage, and generating adhesive motifs, will be discussed.
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            ImmunoRatio: a publicly available web application for quantitative image analysis of estrogen receptor (ER), progesterone receptor (PR), and Ki-67

            Introduction Accurate assessment of estrogen receptor (ER), progesterone receptor (PR), and Ki-67 is essential in the histopathologic diagnostics of breast cancer. Commercially available image analysis systems are usually bundled with dedicated analysis hardware and, to our knowledge, no easily installable, free software for immunostained slide scoring has been described. In this study, we describe a free, Internet-based web application for quantitative image analysis of ER, PR, and Ki-67 immunohistochemistry in breast cancer tissue sections. Methods The application, named ImmunoRatio, calculates the percentage of positively stained nuclear area (labeling index) by using a color deconvolution algorithm for separating the staining components (diaminobenzidine and hematoxylin) and adaptive thresholding for nuclear area segmentation. ImmunoRatio was calibrated using cell counts defined visually as the gold standard (training set, n = 50). Validation was done using a separate set of 50 ER, PR, and Ki-67 stained slides (test set, n = 50). In addition, Ki-67 labeling indexes determined by ImmunoRatio were studied for their prognostic value in a retrospective cohort of 123 breast cancer patients. Results The labeling indexes by calibrated ImmunoRatio analyses correlated well with those defined visually in the test set (correlation coefficient r = 0.98). Using the median Ki-67 labeling index (20%) as a cutoff, a hazard ratio of 2.2 was obtained in the survival analysis (n = 123, P = 0.01). ImmunoRatio was shown to adapt to various staining protocols, microscope setups, digital camera models, and image acquisition settings. The application can be used directly with web browsers running on modern operating systems (e.g., Microsoft Windows, Linux distributions, and Mac OS). No software downloads or installations are required. ImmunoRatio is open source software, and the web application is publicly accessible on our website. Conclusions We anticipate that free web applications, such as ImmunoRatio, will make the quantitative image analysis of ER, PR, and Ki-67 easy and straightforward in the diagnostic assessment of breast cancer specimens.
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              CX-4945, an orally bioavailable selective inhibitor of protein kinase CK2, inhibits prosurvival and angiogenic signaling and exhibits antitumor efficacy.

              Malignant transformation and maintenance of the malignant phenotype depends on oncogenic and non-oncogenic proteins that are essential to mediate oncogene signaling and to support the altered physiologic demands induced by transformation. Protein kinase CK2 supports key prosurvival signaling pathways and represents a prototypical non-oncogene. In this study, we describe CX-4945, a potent and selective orally bioavailable small molecule inhibitor of CK2. The antiproliferative activity of CX-4945 against cancer cells correlated with expression levels of the CK2α catalytic subunit. Attenuation of PI3K/Akt signaling by CX-4945 was evidenced by dephosphorylation of Akt on the CK2-specific S129 site and the canonical S473 and T308 regulatory sites. CX-4945 caused cell-cycle arrest and selectively induced apoptosis in cancer cells relative to normal cells. In models of angiogenesis, CX-4945 inhibited human umbilical vein endothelial cell migration, tube formation, and blocked CK2-dependent hypoxia-induced factor 1 alpha (HIF-1α) transcription in cancer cells. When administered orally in murine xenograft models, CX-4945 was well tolerated and demonstrated robust antitumor activity with concomitant reductions of the mechanism-based biomarker phospho-p21 (T145). The observed antiproliferative and anti-angiogenic responses to CX-4945 in tumor cells and endothelial cells collectively illustrate that this compound exerts its antitumor effects through inhibition of CK2-dependent signaling in multiple pathways. Finally, CX-4945 is the first orally bioavailable small molecule inhibitor of CK2 to advance into human clinical trials, thereby paving the way for an entirely new class of targeted treatment for cancer. ©2010 AACR.
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                Author and article information

                Contributors
                krenx001@umn.edu
                gmu@genesegues.com
                abedin@umn.edu
                isak0023@umn.edu
                chenzler@umn.edu
                ahmedk@umn.edu
                trem0005@umn.edu
                Journal
                Breast Cancer Res
                Breast Cancer Research : BCR
                BioMed Central (London )
                1465-5411
                1465-542X
                11 February 2015
                11 February 2015
                2015
                : 17
                : 1
                Affiliations
                [ ]Research Service (151), Minneapolis VA Health Care System, One Veterans Drive, Minneapolis, MN 55417 USA
                [ ]Department of Laboratory Medicine and Pathology, University of Minnesota, 420 Delaware Street, SE, Minneapolis, MN USA
                [ ]Masonic Cancer Center, University of Minnesota, 717 Delaware Street SE Room 130, Minneapolis, MN 55414 USA
                [ ]GeneSegues Inc, 3180 High Point, Chaska, MN 55318 USA
                [ ]Minnesota Supercomputing Institute, University of Minnesota, 117 Pleasant Street SE, Minneapolis, MN 55455 USA
                [ ]Department of Urology, University of Minnesota, 420 Delaware St. SE, Minneapolis, MN 55455 USA
                Article
                524
                10.1186/s13058-015-0524-0
                4344788
                © Kren et al.; licensee BioMed Central. 2015

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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                Research Article
                Custom metadata
                © The Author(s) 2015

                Oncology & Radiotherapy

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