Stefano M. Cirigliano

PKCδ Inhibition Impairs Mammary Cancer Proliferative Capacity But Selects Cancer Stem Cells, Involving Autophagy

Protein kinase C (PKC) is a family of serine/threonine kinases that regulate diverse cellular functions including cell death, proliferation, and survival. Recent studies have reported that PKCδ, are involved in apoptosis or autophagy induction. In the present study we focused on how PKCδ regulates proliferation and cancer stem cell (CSC) properties of the hormone‐independent mammary cancer cell line LM38‐LP, using pharmacological and genetic approaches. We found that pharmacological inhibition of PKCδ, by Rottlerin treatment, impairs in vitro LM38‐LP proliferation through cell cycle arrest, inducing the formation of cytoplasmic‐vacuoles. Using immunofluorescence we confirmed that Rottlerin treatment induced the apparition of LC3 dots in cell cytoplasm, and increased autophagy flux. On the other side, the same treatment increased CSC growth rate and self‐renewal. Furthermore, Rottlerin pre‐treatment induced in CSC the development of a “grape‐like” morphology when they are growing in 3D cultures (Matrigel), usually associated with a malignant phenotype, as well as an increase in the number of experimental lung metastasis when these cells were inoculated in vivo. The PKCδ knockdown, by RNA interference, induced autophagy and increased CSC number, indicating that these effects are indeed exerted through a PKCδ dependent pathway. Finally, the increase in the number of mammospheres could be reversed by a 3MA treatment, suggesting that autophagy mechanism is necessary for the increased of CSC self‐renewal induced by PKCδ inhibition. Here we demonstrated that PKCδ activity exerts a dual role through the autophagy mechanism, decreasing proliferative capacity of mammary tumor cells but also regulating tumor stem cell self‐renewal. J. Cell. Biochem. 117: 730–740, 2016.

CIGB-300, an anti-CK2 peptide, inhibits angiogenesis, tumor cell invasion and metastasis in lung cancer models.

OBJECTIVES Casein kinase 2 (CK2) is overexpressed in several types of cancer. It has more than 300 substrates mainly involved in DNA reparation and replication, chromatin remodeling and cellular growth. In recent years CK2 became an interesting target for anticancer drug development. CIGB-300 is a peptidic inhibitor of CK2 activity, designed to bind to the phospho-acceptor domain of CK2 substrates, impairing the correct phosphorylation by the enzyme. The aim of this work was to explore the antitumor effects of this inhibitor in preclinical lung cancer models. MATERIALS AND METHODS Human H125 and murine 3LL Lewis lung carcinoma cell lines were used to evaluate the effect of CIGB-300 treatment in vitro. For this purpose, adhesion, migration and invasion capabilities of cancer cells were tested. Proteolytic activity of tumor cell-secreted uPA and MMP after CIGB-300 incubation was also analyzed. In vivo anticancer efficacy of the peptide was evaluated using experimental and spontaneous lung colonization assays in C57BL/6 mice. Finally, in order to test the effect of CIGB-300 on tumor cell-induced angiogenesis, a modified Matrigel plug assay was conducted. RESULTS AND CONCLUSION We demonstrate that treatment with low micromolar concentrations of CIGB-300 caused a drastic reduction of adhesion, migration and invasion of lung cancer cells. Reduced invasiveness after CIGB-300 incubation was associated with decreased proteolytic activity of tumor cell-conditioned medium. In vivo, intravenous administration of CIGB-300 (10mg/kg) markly decreased lung colonization and metastasis development of 3LL cells. Interestingly, after 5days of systemic treatment with CIGB-300, tumor cell-driven neovascularization was significantly reduced in comparison to control group. Altogether our data suggest an important role of CK2 in lung tumor development, suggesting a potential use of CIGB-300 as a novel therapeutic agent against lung cancer.

Modulation of pancreatic tumor potential by overexpression of protein kinase C β1.

Objective This study aimed to investigate whether the overexpression of protein kinase C &bgr;1 (PKC&bgr;1) is able to modulate the malignant phenotype displayed by the human ductal pancreatic carcinoma cell line PANC1. Methods PKC&bgr;1 overexpression was achieved using a stable transfection approach. PANC1-PKC&bgr;1 and control cells were analyzed both in vitro and in vivo. Results PANC1-PKC&bgr;1 cells displayed a lower growth capacity associated with the down-regulation of the MEK/ERK pathway and cyclin expression. Furthermore, PKC&bgr;1 overexpression was associated with an enhancement of cell adhesion to fibronectin and with reduced migratory and invasive phenotypes. In agreement with these results, PANC1-PKC&bgr;1 cells showed an impaired ability to secrete proteolytic enzymes. We also found that PKC&bgr;1 overexpressing cells were more resistant to cell death induced by serum deprivation, an event associated with G0/G1 arrest and the modulation of PI3K/Akt and NF-&kgr;B pathways. Most notably, the overexpression of PKC&bgr;1 completely abolished the ability of PANC1 cells to induce tumors in nude mice. Conclusions Our results established an important role for PKC&bgr;1 in PANC1 cells suggesting it would act as a suppressor of tumorigenic behavior in pancreatic cancer.

The synthetic peptide CIGB-300 modulates CK2-dependent signaling pathways affecting the survival and chemoresistance of non-small cell lung cancer cell lines

BackgroundLung cancer is the most frequently diagnosed cancer and the leading cause of cancer-related deaths worldwide. Up to 80% of cancer patients are classified as non-small-cell lung cancer (NSCLC) and cisplatin remains as the gold standard chemotherapy treatment, despite its limited efficacy due to both intrinsic and acquired resistance. The CK2 is a Ser/Thr kinase overexpressed in various types of cancer, including lung cancer. CIGB-300 is an antitumor peptide with a novel mechanism of action, since it binds to CK2 substrates thus preventing the enzyme activity. The aim of this work was to analyze the effects of CIGB-300 treatment targeting CK2-dependent signaling pathways in NSCLC cell lines and whether it may help improve current chemotherapy treatment.MethodsThe human NSCLC cell lines NCI-H125 and NIH-A549 were used. Tumor spheroids were obtained through the hanging-drop method. A cisplatin resistant A549 cell line was obtained by chronic administration of cisplatin. Cell viability, apoptosis, immunoblotting, immunofluorescence and luciferase reporter assays were used to assess CIGB-300 effects. A luminescent assay was used to monitor proteasome activity.ResultsWe demonstrated that CIGB-300 induces an anti-proliferative response both in monolayer- and three-dimensional NSCLC models, presenting rapid and complete peptide uptake. This effect was accompanied by the inhibition of the CK2-dependent canonical NF-κB pathway, evidenced by reduced RelA/p65 nuclear levels and NF-κB protein targets modulation in both lung cancer cell lines, as well as conditionally reduced NF-κB transcriptional activity. In addition, NF-κB modulation was associated with enhanced proteasome activity, possibly through its α7/C8 subunit. Neither the peptide nor a classical CK2 inhibitor affected cytoplasmic β-CATENIN basal levels. Given that NF-κB activation has been linked to cisplatin-induced resistance, we explored whether CIGB-300 could bring additional therapeutic benefits to the standard cisplatin treatment. We established a resistant cell line that showed higher p65 nuclear levels after cisplatin treatment as compared with the parental cell line. Remarkably, the cisplatin-resistant cell line became more sensitive to CIGB-300 treatment.ConclusionsOur data provide new insights into CIGB-300 mechanism of action and suggest clinical potential on current NSCLC therapy.

Abstract 5111: Norcantharidin impairs tumor growthin vivoand inhibits stemness of triple-negative breast cancer cells

Triple-negative breast cancer (TNBC) is characterized by an abundance of treatment-resistant cancer stem cells (CSC). The absence of a molecular target, coupled with its highly aggressiveness, leads to the lack of an effective therapy for TNBC. Norcantharidin (NCTD) is a synthetic demethylated small-molecule analog of the naturally occurring cantharidin isolated from blister beetles (Mylabris phalerata Pall). Unlike the conventional chemotherapeutics, NCTD toxicity is higher to cancer cells than normal ones, making this small molecule promising for cancer treatment. The aims of this work were: A) To study the effect of NCTD on 4T1 cell line proliferation in vitro. B) To analyze the effect of NCTD on 4T1 derived CSC on self-renewal and clonogenic capacity. C) To evaluate the effect of NCTD on 4T1 tumor growth in vivo. We employed the well-known 4T1 triple-negative breast cancer cell model, which presents a huge proportion of CSC. We observed that NCTD treatment during 96 h significantly reduced 4T1 cell proliferation in vitro. In addition, the IC50 value of NCTD was 27.35 ± 2.83 μM. Related to CSC, NCTD pre-treatment for 96 h impaired CSC self-renewal (Number of secondary mammospheres: Control: 276±39; NCTD: 163±18; p≤0.05) as well as the clonogenic capacity (Number of colonies: Control: 359±38; NCTD: 122±11; p≤0.05). By q-PCR, we observed that NCTD treatment for 48 h significantly induced an increase of Gli-1 and Smooth in CSC, keys member of Sonic Hedgehog pathway. Finally, we performed an in vivo assay, where 4T1 cells were orthotopically inoculated on mammary gland of BALB/c mice, and NCTD was i.p. inoculated twice a week (5mg/kg). We observed that NCTD treatment significantly reduced tumor growth in vivo. Our data suggest that NCTD treatment reduces tumor growth both in vitro and in vivo, possibly through the direct effect on CSC self-renewal and clonogenic capacity, by modulating Sonic Hedgehog pathway. Citation Format: Damian E. Berardi, Guido Cicuttin, Maria A. Taruselli, Stefano M. Cirigliano, Elisa D. Bal de Kier Joffe, Alejandro J. Urtreger, Laura B. Todaro. Norcantharidin impairs tumor growth in vivo and inhibits stemness of triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5111. doi:10.1158/1538-7445.AM2017-5111

Abstract 1289: The synthetic peptide CIGB-300 inhibits nuclear factor κB (NF-κB) affecting the survival and chemoresistance of human lung cancer cells

Lung cancer is the leading cause of cancer deaths worldwide and despite significant progress, current therapies are limited in efficacy. The CK2 Ser/Thr kinase has been historically linked with cancer. It is involved in cell proliferation, survival and apoptosis by modulating diverse signaling pathways, including Wnt and NF-κB among the most relevant. CIGB-300 is an antitumor peptide with a novel mechanism of action, capable of binding to CK2 substrates thus preventing the enzyme activity. Previously, we have determined that CIGB-300 induces apoptosis through caspase-3 activation in different lung cancer cell lines. Moreover, CIGB-300 strongly inhibited RelA/NF-κB (p65) nuclear translocation, even in the presence of a phorbol-ester activating stimulus. NF-κB activation is known to reduce chemotherapy efficiency in different malignancies, including lung cancer. Based on this evidence, we hypothesize that supplementing cisplatin with CIGB-300 would improve the treatment efficiency. Indeed, we observed by Western blot that nuclear p65 levels were highly increased after treating human NCI-H125 cells with cisplatin. Moreover, when cells were treated with cisplatin plus CIGB-300, NF-κB activation was completely abolished. Therefore, the CIGB-300 effect on NF-κB signaling pathway prevails over cisplatin. These promising results on NF-κB inhibition led us to evaluate the combined treatment in chemoresistant setting. For this purpose we developed a cisplatin resistant A549 lung cancer cell line (A549-Rcisp) by the chronic administration of cisplatin during six months. A549-Rcisp viability was 40% higher than parental cells, confirming the cisplatin-acquired resistance. Remarkably, cisplatin resistant cells showed a significant increase in CIGB-300 sensitivity as compared to the parental cell line (p Given that NF-κB dimer stability is regulated by the proteasome-selective proteolysis of its inhibitory proteins, we studied the effect of CIGB-300 on this process. Surprisingly, we observed a significant increase on protease activities associated with the proteasome after 30 minutes of CIGB-300 treatment. Thus, proteasome complex is a newly identified target of CIGB-300 that could be relevant for its mechanism of action and deserves further exploration in order to determine the association with the observed perturbation of different signaling pathways. Citation Format: Stefano M. Cirigliano, Maria Ines Diaz Bessone, Carolina Flumian, Damian E. Berardi, Silvio Perea, Elisa Bal De Kier Joffe, Hernan Farina, Laura Todaro, Alejandro Urtreger. The synthetic peptide CIGB-300 inhibits nuclear factor κB (NF-κB) affecting the survival and chemoresistance of human lung cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1289.

Abstract 5203: Differential response to retinoid treatment in mouse and human mammary tumor cell lines with alterations in protein kinase C (PKC) expression

PKC is a serine-threonine kinase family that controls malignant transformation and metastatic dissemination. ATRA is the main active metabolite of vitamin A. Some evidences indicate that PKCδ may regulate the expression of some retinoid acid (RA) dependent genes, and others indicate that retinoids could alter PKCα intracellular localization; both processes would lead to cell differentiation. In this work we have developed human (MDA-MB 231) and murine (LM3) cell models overexpressing PKCα or PKCδ, in order to determine whether PKC expression alters the sensitivity to retinoids treatment (ATRA). The effect of ATRA was studied in vitro, analyzing biological responses related to tumor growth. In LM3 cells, only PKCα overexpression was able to reduce in vitro population doubling time (PDT) as compared to control (PDT: 14,8±2,3 h vs 21,2±3,1 h for LM3-PKCα y LM3-Vector respectively). Moreover, these cells also responded to retinoid treatment with a significant delay in cell proliferation (PDT: 24,3±4,2 h vs. 14,8±2,3 h for LM3-PKCα ATRA treated or not respectively. In MDA-MB 231 derived cell lines, PKC overexpression as well as ATRA treatment have no effect on proliferative potential. No differences were observed on migratory and invasive capabilities either. Interestingly, in LM3, PKCδ overexpression induced an important increase in proteolytic enzymes secretion, which correlates with its major invasiveness, but this increase had no impact on in vivo metastatic dissemination. Only PKCα overexpression increased this parameter (lung nodes, median (range): 55 (20-75) vs 0 (0-10) for LM3-PKCα y LM3-Vector respectively). Contrary to LM3-PKCδ in MDA-PKCδ cells we could detect a decrease on proteolytic enzymes production and invasive capability. Moreover, colonies growing in Matrigel as 3D cultures showed a small and branched structures. Finally we studied whether the overexpression of α and δ PKC isoforms is able to alter the activity of retinoic acid responsive elements (RARE) through a reporter gene assay. On LM3 model, the constitutive expression of PKCδ highly increased RARE dependent activity. Surprisingly, in MDA-MB231 derived sublines, we could detect a significant increase on RARE activity when cells were treated with ATRA, Altogether, these results suggest that PKCα overexpression confers a more aggressive phenotype in LM3 model, but also makes these cells sensitive to ATRA effects. We could hypothesize, that the absence of response to ATRA treatment displayed by MDA-MB 231 sublines can be explained by their lack of RARβ, which is implied in AP-1 transrepression in response to ATRA. Among others phenomena, AP-1 is involved in cell proliferation and proteolytic enzymes production. Regarding the differences of response to PKCδ overexpression of both models, it has been reported that this PKC isoform has a differential role, pro or anti tumorigenic, depending on cellular context. Citation Format: Maria I. Diaz Bessone, D. E. Berardi, S. M. Cirigliano, C. Flumian, E. D. Bal de Kier Joffe, L. B. Todaro, A. J. Urtreger. Differential response to retinoid treatment in mouse and human mammary tumor cell lines with alterations in protein kinase C (PKC) expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5203. doi:10.1158/1538-7445.AM2015-5203

Abstract 5204: Parameters associated with metastatic dissemination are differentially modulated by specific isotypes of the retinoic acid receptor

Migration and adhesion are two critical processes highly related to metastasic dissemination and all trans-retinoic acid (ATRA) is known to diminished migration and metalloprotease (MMP) secretion in breast cancer cell lines. Our objective was to evaluate the effect of activating each retinoic acid receptor (RAR) isotype in adhesion, migration and MMPs secretion using two hormone-independent murine cell lines: LM38-LP y 4T1. First we determined that both lines express the different RAR isotypes with the exception of RARβ in 4T1 cell line. Cells were treated for 96h with AM580 (RARα agonist, 200nM), AC55649 (RARβ agonist, 2μM), BMS961 (RARγ agonist, 50nM) or vehicle (DMSO). The migratory potential was evaluated by a “wound healing” assay. LM38-LP cell line reduced its migratory capacity in response to AM580 and AC55649 (AM580: 108.0±24.3μm, AC55649: 114.1±12.8μm vs. vehicle: 186.9±31.0μm, p The same agonist treatments were used to obtain conditioned media in order to evaluate soluble MMPs activity by zymography. AM580, AC55649 and BMS961 pre-treatments decreased soluble MMP2 activity in LM38-LP cells (0.53±0.03au (arbitrary units), 0.47±0.01 au, and 0.75±0.04 au respectively, fold change vs. control). Conversely, AM580 pre-treatment increased MMP2 and MMP9 in 4T1 cells (1.57±0.10 au and 2.04±0.26 au respectively, fold change vs. control). Regarding adhesion assay, after the above mentioned treatments cells were detached, incubated 2h at 37°C in order to recover cell surface proteins and finally seeded. After 2h incubation, non-adherent cells were washed out with PBS and adherent cells were fixed, stained and quantified by densitometry. We observed that AM580 and AC55649 diminished LM38-LP adhesive capacity (0.51±0.02 au, 0.83±0.00 au respectively, fold change vs. control) while AC 55649 increased this parameter in 4T1 cells (1.28±0.06 au fold change vs. control). Finally we performed an experimental lung metastasis assay using LM38-LP cells. Cells where treated with the different agonist during 144h and then inoculated into the tail vein of syngeneic mice. Lungs were removed 21 days later and number of superficial lung metastasis was determined. AC55649 treatment induced an increase in the metastatic potential (Md [Rg]: 143, [86-314] vs. control Md [Rg]: [21.5, 2-73], p In conclusion, the activation of RARα and RARβ isotypes leads to opposite responses in different cell lines. We hypothesized that the differences in RARβ expression between this two cell lines should be responsible of this effect. Surprisingly, the activation of RARβ increased the metastatic potential of LM38-LP cells in spite of the negative regulation of parameters associated with the metastatic process. Citation Format: Carolina Flumian, Damian E. Berardi, Stefano M. Cirigliano, Maria I. Diaz Bessone, Elisa D. Bal de Kier Joffe, Alejandro J. Urtreger, Laura B. Todaro. Parameters associated with metastatic dissemination are differentially modulated by specific isotypes of the retinoic acid receptor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5204. doi:10.1158/1538-7445.AM2015-5204

Abstract 209: Pharmacological inhibition of protein kinase C alpha (PKCα) and all trans retinoic acid (ATRA) synergize to inhibit the proliferation, migration and cancer stem-like properties of a triple-negative mammary cancer model

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA To study the interaction between PKCα and retinoic acid pathways on the biology of breast cancer, we employed a murine mammary triple negative cell model (LM38-LP, composed by luminal (LEP), myoepithelial (MEP) and cancer stem cells (CSC). We proposed to: A) Study the effect of ATRA (1μM) on the expression of PKCα and Retinoic Acid Receptors (RARs) in LEP, MEP and CSC (mammospheres); B) Evaluate the combined effect of ATRA and a PKCα pharmacological inhibitor (1 uM Go6976) on cell proliferation employing a method to determine the interaction type; C) Analyze the effect of ATRA/PKCα inhibitor on migration and MMP activity on LM38-LP;. D) Analyze the effect of ATRA/PKCα inhibitor on proliferation, self-renewal and differentiation of CSC; E) Study the expression profile of pluripotent genes in CSC and their modulation by treatments. By RT-PCR we found that ATRA (48 h) induced a decrease in PKCα in LEP, MEP and CSC. The same treatment increased RARβ2 and RARγ2 in CSC, and only RARβ2 in LEP. ATRA and PKCα inhibitor interaction results from the proliferation assay were analyzed by Chou-Talalay´s method. We found that the inhibitory effect exerted by ATRA/PKCα inhibitor was synergistic with CI=0.59 (Synergistic with CI<0.7). ATRA/PKCα inhibitor treatment reduced LM38-LP migration more efficiently than each treatment alone, showing a synergic effect (% migration: Control: 80,1±6,9, ATRA: 55,4±5,6, PKCα inhibitor: 37,3±9,5, ATRA/PKCα inhibitor: 20,0±1,7; p≤0.05). Furthermore, MMP2 activity was strongly reduced by the combined treatment. Interestingly, we observed that only the combined treatment induced a decrease of RARγ2 expression in the highly migratory MEP cells. ATRA/PKCα inhibitor treatment synergized to reduce mammospheres growth (Diameter in µm at 96h: Control: 176±8, ATRA: 129±10; PKCα inhibitor: 130±11 ATRA/PKCα inhibitor: 103±5 p≤0.05). Pre-treatment with ATRA/PKCα inhibitor for 96h dramatically affected CSC self-renewal (Number of secondary mammospheres: Control: 313±19; ATRA/PKCα inhibitor: 69±21; p≤0.05). While in a 3D matrigel culture assay ATRA-pretreated CSC formed organized colonies with presence of lumen, the combined treatment led to the formation of small undifferentiated structures and evidence of cell death. By RT-PCR we determined that CSC expressed higher levels of pluripotent genes, such as Nanog, Sox2, Slug and Sox9, than the parental LM38-LP cell line. Besides, only the combined treatment for 96 h induced a decrease in the levels of Nanog and Slug in CSC. Our findings suggest that the pharmacological inhibition of PKCα and ATRA synergize to inhibit proliferation and migration possibly through the decrease of RARγ and the inhibition of MMP2 activity. Furthermore, the blockage of CSC expansion by the combined treatment, possibly occurred through the down regulation of Nanog and Slug. Citation Format: Damian E. Berardi, Maria I. Diaz Bessone, Carolina Flumian, Stefano M. Cirigliano, Elisa D. Bal de Kier Joffe, Alejandro J. Urtreger, Laura B. Todaro. Pharmacological inhibition of protein kinase C alpha (PKCα) and all trans retinoic acid (ATRA) synergize to inhibit the proliferation, migration and cancer stem-like properties of a triple-negative mammary cancer model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 209. doi:10.1158/1538-7445.AM2014-209

Abstract 3791: The inhibition of protein kinase CK2 activity by the CIGB-300 synthetic peptide impairs the three-dimensional cell growth, Wnt and nuclear factor қB signaling pathways in lung cancer cells

CK2 is a serine/threonine kinase involved in cell growth, survival and apoptosis. The CIGB-300 is a synthetic peptide capable of binding to CK2 substrates thus preventing the enzyme activity. Previously we have determined that CIGB-300 presented an inhibitory concentration 50 dose (IC50) of 119±2.4 µM in NCI-H125 human lung cancer cells. Throughout an Annexin V-FITC assay we could determine that CIGB-300 is a potent apoptosis inductor since the treatment with this drug for 60 minutes induced apoptosis at a level comparable with that observed with etoposide (10 µM). Concomitantly with cell death induction, we could observe caspase-3 activation and the decreased expression of c-myc and cyclin D1 and D2. In this work we analyzed whether CIGB-300 is able to alter the ability of cells to grow in 3D and to modulate signaling pathways involved in tumor progression. First, we developed stable spheroids of NCI-H125 cells, which were able to grow in culture for at least 14 days. After 5 days of treatment, CIGB-300 induced a significant growth inhibition (median volume: 4.07x107±0.70x107 µm3 in control spheroids vs 2.13x107±0.35x107 µm3 in spheroids treated with the IC50 dose p To analyze the effect of CIGB-300 on canonical Wnt signaling, this pathway was initially activated by incubating NCI-H125 cells with a conditioned media containing Wnt3a factor. This incubation led to a significant increase in the levels of cytoplasmic β-catenin. By Western blot we could observe that treatment with CIGB-300 blocked this increase, suggesting its role in the modulation of the Wnt pathway. In order to analyze the nuclear factor κB (NF-κB) dependent pathway, this transcription factor was activated by a phorbol ester treatment (PMA, 15 nM). Short treatment (15 min) with CIGB-300 induced an important reduction in NF-κB nuclear levels. However, this inhibition was reverted 24 h later as determined by Western blot and reporter gene expression assays. Our results indicate that the treatment with CIGB-300 induces a significant anti-proliferative response in a three-dimensional model, which resembles in vivo tumor growth conditions, also affecting key signaling pathways involved in tumor progression. Altogether, our data suggest that this peptide may become a new strategy for the treatment of lung cancer malignancies. Citation Format: Stefano M. Cirigliano, Maria Ines Diaz Bessone, Carolina Flumian, Damian Emilio Berardi, Silvio E. Perea, Elisa Bal De Kier Joffe, Hernan Farina, Laura Todaro, Alejandro Urtreger. The inhibition of protein kinase CK2 activity by the CIGB-300 synthetic peptide impairs the three-dimensional cell growth, Wnt and nuclear factor қB signaling pathways in lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3791. doi:10.1158/1538-7445.AM2014-3791