On these features, for both cell lines we observed a stronger divergence of KO than KO from w.t. cells are addicted to CK2, relying on its activity more than healthy cells for their life, and exploiting it for developing multiple oncological hallmarks. However, little is known about CK2 contribution to the metabolic rewiring of cancer cells. With this study we aimed at shedding some light on it, especially focusing on the CK2 role in the glycolytic onco-phenotype. By analyzing neuroblastoma and osteosarcoma cell lines depleted of either one () or the other () CK2 catalytic subunit, we also aimed at disclosing possible pro-tumor functions which are specific of a CK2 isoform. Our DO34 results suggest that both CK2 and contribute to cell proliferation, survival and tumorigenicity. The analyzed metabolic features disclosed a role of CK2 in tumor metabolism, and suggest prominent functions for CK2 isoform. Results were also confirmed by CK2 pharmacological inhibition. BBC2 Overall, our study provides new information on the mechanism of cancer cells addiction to CK2 and on its isoform-specific functions, with fundamental implications for improving future therapeutic strategies based on CK2 targeting. 0.05, (**) 0.01, (***) 0.001, (****) 0.0001. 3. Results 3.1. CK2 Profile in w.t. Cell Lines and KO Clones For our study, we used SK-N-BE (neuroblastoma) and U2OS (osteosarcoma) tumor cells. For both lines, different KO clones of each subunit were obtained throughout this investigation (see Section 2), and they were variably used for performing experiments. Unless differently specified, the results shown for a certain KO clone were reproducible with the other clone of the same subunit (while, for quantifications, all data from experiments with different clones were considered). In some analysis, the two KO clones of the same subunit displayed different behaviors (suggesting possible effects due to compensating events, and/or not directly ascribable to the CK2 subunit deletion); in those cases, the results obtained with both clones are shown. SK-N-BE and DO34 U2OS cells express significant amount of both and . The relative percentage of the catalytic isoforms is around 70% and 30% in SKNBE, and 45% and 55% in U2OS, as assessed by using an antibody which recognizes with the same efficacy the two subunits (Figure 1A). Therefore, these two lines provide a model for two different conditions as far as the most abundant DO34 isoform is concerned. Applying the CRISPR-Cas9 technology, we produced cells of both lines that do not express either (KO) or (KO) (Figure 1B). The expression of the regulatory subunit of CK2 was roughly unchanged in KO clones, while it was reduced in KO clones, as already observed in other cell lines . The CK2 cellular activity, measured through the level of the endogenous substrate pSer129 Akt , was reduced in the KO clones compared to w.t. cells, and the effect was more evident in KO for SKNBE, and in KO in U2OS, consistent with the proportion of the catalytic isoform suppressed (Figure 1B). Open in a separate window Figure 1 CK2 expression and activity in SK-N-BE and U2OS cells. (A) Titration of the antibody reactivity towards CK2 catalytic subunits. The indicated amounts of recombinant CK2 catalytic subunits (myc- or ) were loaded on SDS-PAGE, and either blotted for the WB (western blot) analysis or stained by Colloidal Coomassie Blue; (B) CK2 expression and activity in w.t. and KO clones of the cells used for this study. 10 g proteins from cell lysates were analyzed by WB with the indicated antibodies. The last two right lanes belong to an independent experiment. As a reporter of CK2 endogenous activity, pS129 Akt signal has been quantified, normalized to total Akt signal, and reported in the bar graph as % of w.t. cells. At least three independent experiments were performed; representative western blots are shown, while quantification in the bar graphs reports the mean values SEM of all experiments and of the two clones of the same KO. Statistical significance refers to w.t. cells. (*) 0.05, (**) 0.01, (***) 0.001 3.2. Deletion of an Individual CK2 Catalytic Subunit Reduces 3D Growth, Clonogenic Potential, and Motility First we wanted to assess if the knockout of CK2 or affects the proliferation rate of SK-N-BE and U2OS tumor cells grown in monolayer. In SK-N-BE neuroblastoma cells, both and KO displayed a slower proliferation compared to w.t. cells (Figure 2). Results were less clear for the U2OS osteosarcoma model: the.