Autophagy Protects from Trastuzumab-Induced Cytotoxicity in HER2 Overexpressing Breast Tumor Spheroids.

Multicellular tumor spheroids represent a 3D in vitro model that mimics solid tumor essential properties including assembly and development of extracellular matrix and nutrient, oxygen and proliferation gradients. In the present study, we analyze the impact of 3D spatial organization of HER2-overexpressing breast cancer cells on the response to Trastuzumab. We cultured human mammary adenocarcinoma cell lines as spheroids with the hanging drop method and we observed a gradient of proliferating, quiescent, hypoxic, apoptotic and autophagic cells towards the inner core. This 3D organization decreased Trastuzumab sensitivity of HER2 over-expressing cells compared to monolayer cell cultures. We did not observe apoptosis induced by Trastuzumab but found cell arrest in G0/G1 phase. Moreover, the treatment downregulated the basal apoptosis only found in tumor spheroids, by eliciting protective autophagy. We were able to increase sensitivity to Trastuzumab by autophagy inhibition, thus exposing the interaction between apoptosis and autophagy. We confirmed this result by developing a resistant cell line that was more sensitive to autophagy inhibition than the parental BT474 cells. In summary, the development of Trastuzumab resistance relies on the balance between death and survival mechanisms, characteristic of 3D cell organization. We propose the use of spheroids to further improve the understanding of Trastuzumab antitumor activity and overcome resistance

Autophagy in BT474 cells.

<p>(a) Western blot (WB) analysis of LC3 in 2D and 3D cultures after 24 h treatment with Tz. (b) Autophagic flux analyzed by WB in cell monolayers treated with Tz (1μg/ml) and 5 nM Bafilomycin A1 (BAF) (c) Cellular distribution of autophagosomes with LC3 stain. Arrows point to autophagosomes. Images show representative zones of BT474 cell monolayers (200x). (d) Immunofluorescence for LC3 (green) and Propidium iodide (PI, red) in spheroids treated for 15 days with Tz (50 μg/ml) or control IgG. Magnified images correspond to the zones limited by the white squares in the left photographs (600x).</p

Study of spheroid subpopulations.

<p>Spheroids were treated with 50 μg/ml Tz or control IgG for 15 days. (a) H&E (200x); immunofluorescence for pHER2 and HER2, HIF-1α (600x) and cleaved caspase 3 (400x). (b) Immunohistochemistry for Ki67 and quantification of Ki67+ cells (*p<0.05). (c) Confocal analysis of p27 (200x). Filled white arrows correspond to nuclear staining and black filled arrows correspond to cytoplasmatic staining.</p

Cells growth in 3D.

<p>(a) Kinetics of BT474 and MCF7 spheroids growth, n = 6. Upper insert corresponds to representative photographs taken to the same spheroid along time (40X). (b) Hematoxylin-Eosin staining of BT474 spheroid at 7 and 21 days growth showing the necrotic cores and the peripheral rims of viable cells (200X).</p

Apoptosis of cells cultured in 2D and 3D after 6 h treatment with Tz, 3-MA or their combination.

<p>Representative Annexin V/ Propidium iodide graphs are shown. Triplicate cultures were run per group, and the experiment was repeated three times (*p<0.05).</p

Dose-dependent effect of Tz on cells growth.

<p>(a) Western blot analysis for AkT, pAkT and β-actin of BT474 spheroids after 24 h or 5 days of Tz treatment. (b) Viability (MTS assay) of BT474 and MCF7 cells cultured as monolayers treated for 5 days with increasing concentrations of Tz (0.005–50 μg/ml) relative to isotype IgG control. (c) Volume was evaluated in BT474 and MCF7 spheroids chronically treated during 15 days with 50, 10 and 1 μg/ml Tz or human IgG. Each point of the curves expresses the percentage in size change respect to the initial pretreated size (considered 100%), and represents the mean ± SD (n = 6). Inserts (right) correspond to 15 days-treated spheroids (40X). (d) BT474 spheroids chronically treated were analyzed by flow cytometry and the quantification of cell cycle distribution with propidium iodide staining is shown. Triplicate cultures were run per group, and each experiment was repeated twice (*p<0.05). (e) Cells obtained from spheroids chronically treated with Tz (BT474-ETz) or IgG (BT474-EIgG) were cultured as monolayers and treated with increasing concentrations of Tz (0.01–50 μg/ml).</p

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

Background: Lung 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. Methods: The 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. Results: We 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. Conclusions: Our data provide new insights into CIGB-300 mechanism of action and suggest clinical potential on current NSCLC therapy.Fil: Cirigliano, Stéfano Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Díaz Bessone, María Inés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Berardi, Damian Emilio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Flumian, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Bal, Elisa Dora. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Perea, Silvio E.. Centro de Genética Ingeniería y Biotecnología; CubaFil: Farina, Hernán Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Todaro, Laura Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Urtreger, Alejandro Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentin