CK2 is a serine/threonine kinase that is overexpressed in breast cancer and its inhibition is associated to reduced tumor growth and disease progression. CIGB-300 is an antitumor peptide with a novel mechanism of action, since it binds to protein kinase CK2 catalytic subunit alpha and to CK2 substrates thus preventing the enzyme activity. Our aim was to evaluate the potential therapeutic benefits of CIGB-300 on breast cancer disease using experimental models with translational relevance. We demonstrated that CIGB-300 reduces breast cancer cell growth in MDA-MB-231, MCF-7 and F3II cells, exerting a pro-apoptotic action and cell cycle arrest. We also found that CIGB-300 decreased cell adhesion, migration and clonogenic capacity of malignant cells. Effect on experimental breast cancer lung metastasis was evaluated after surgical removal of primary F3II tumors or after tail vein injection of tumor cells, also we evaluated CIGB-300 effect on spontaneous lung metastasis in an orthotopic model. Systemic CIGB-300 treatment inhibited breast cancer colonization of the lung, reducing the size and number of metastatic lesions. The present preclinical study establishes for the first time the efficacy of CIGB-300 on breast cancer. These encouraging results suggest that CIGB-300 could be used for the management of breast cancer as an adjuvant therapy after surgery, limiting tumor metastatic spread and thus protecting the patient from distant recurrence.
The mitogen activated protein kinase (MAPK) p38 is a kinase involved in the response to different external stimuli, extremely diverse such as oxidative stress or growth factors. Its activation triggers different responses which include from promoting cell apoptosis or cell cycle arrest, to cell differentiation and the activation of survival pathways. Although the role of p38 in cancer is a well studied field, there are still multiple unanswered questions, given the large amount of evidence supporting both a tumorigenic but also a tumor suppressor function for p38 in cancer development and progression. In this study, the effects produced by p38 negative modulation using the chemical inhibitor SB203580 in different aspects of tumor biology are analyzed in vitro and in vivo. As a study model, F3II cell line was used, which is an aggressive murine mammary carcinoma. We found that p38 inhibition provokes contradictory effects in our mammary carcinoma model. On one hand, p38 inhibition provoked the reduction of cell viability and colony formation. On the other hand, SB203580 treatment increased cell adhesion and proliferation in matrix coated surfaces. In vivo we found that SB203580 treatment increases tumor aggressiveness. The shortened latency times, higher number of lung metastasis and larger local recurrences are a reflection of the aforementioned effect. Taking together, the results presented in this work position p38 as a tumor suppressor kinase, in the context of cells adapting to a new environment and developing the first stages of growth. P38 inhibition represented an advantage for cells newly inoculated in vivo. We consider that this phenotypic adaptation is probably a consequence of ERK activation and integrin α5 increased expression. Data presented here is of importance to show how determinant is the microenvironment in the responses elicited by the modulation of p38 kinase. We propose a differential role according to the environment conditions, which reflects the complexity of the pathway and the importance of the extracellular stimuli when evaluating the implications of treatment with p38 MAP kinase inhibitors. Citation Format: Carla S. Capobianco, Johanna E. Sidabra, Maria F. Gottardo, Julio A. Aguirre-Ghiso, Daniel F. Alonso, Hernan G. Farina. p38 kinase is a negative regulator of tumorigenesis and recurrence after surgery in a mammary carcinoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 78.
The p38 pathway can be activated by three different MAP kinase kinases: MKK3, MKK4 and MKK6, each one activated by different stimuli, like DNA damage, oxidative stress, proinflammatory cytokines or growth factors. In consequence, the responses mediated by this kinase are also significantly diverse. Its participation in such different processes makes it reasonable for the role of p38 in cancer to be a topic of current debate. Several authors reported a pro-tumorigenic role of p38, through the stimulation of tumor progression, epidermal to mesenchymal transition and migration and chemotherapy resistance, as a consequence of the transduction of pro-survival signals. Due to the above mentioned, the combination of p38 inhibitors and chemotherapy is frequently proposed as an interesting clinical approach for cancer treatment. In contrast, several reports underline the importance of p38 suppression of tumor initiation, metastasis inhibition through dormancy maintenance, and also a decisive role in the action mechanism of antitumor treatments under evaluation. Specifically, an important amount of evidence point p38 as a key apoptosis mediator in cancer cells, as a response to treatment with anticancer drugs included in different clinical and preclinical protocols. Previously we studied the effect of p38 inhibition in a mouse mammary carcinoma model named F3II. We described that in vitro inhibition of p38 provokes an increased in cell adhesion, cell spreading, proliferation in 3D cultures, ERK phosphorylation and Integrin α5 expression. In vivo inhibition also resulted in shortened latency times and increased tumor growth rates. In this work, we studied the effect of p38 inhibition in the development of recurrences after tumor surgery. When SB203580 inhibitor was administered after surgery, mice developed larger recurrences. Apoptotic cells in histological samples were detected by TUNEL analysis, showing that SB203580 treatment decreased the number of apoptotic cells in tumor recurrences. In addition, mitotic cell count in recurrence tissue showed that SB203580 treatment increased the mitotic index in the tumors. In conclusion, in this work we demonstrated that in F3II cell line, p38 inhibition promotes tumor recurrence development after surgery, mainly blocking the apoptotic response, and promoting cell duplication. Results presented in this work position p38 as a tumor suppressor kinase. The evidence presented here highlight the possible risk of the administration of a p38 inhibitor to oncological patients that may present newly disseminated cells, or a residual tumor mass that remains after treatment. From our perspective, candidates to be treated with new p38 inhibitors should be carefully selected according to the tumor type and the stage of the disease, since the response might be extremely variable between the different conditions. Citation Format: Carla S. Capobianco, Maria F. Gottardo, Johanna E. Sidabra, Julio A. Aguirre Ghiso, Daniel F. Alonso, Hernan G. Farina. p38 kinase acts as a negative regulator of recurrence after surgery in a mammary carcinoma model, through apoptosis induction and proliferation inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5983.
Breast cancer is the first cause of death from female cancer. After treatments against the primary tumor, a few cells can prevail in a dormant state for a long period, becoming minimal residual disease. These cells can resume growth and establish as metastasis, which are responsible for 90% of deaths from cancer. Patients with breast cancer diagnosed at early stage with small tumours have a 25-30% chance of recurrence after 10-15 years after treatment. These new growth often entails chemo and radio resistance and there is not response to conventional treatment. For all these reasons, there is a clear need for the development of novel therapies against these cells in a dormant state. The main limitation on the study of these dormant cells is the lack of relevant in vitro an in vivo models. In this work, we isolated and characterized two novel cell lines F3II-TP and F3II-NM. F3II-TP represents tumours with high proliferation rate whereas F3II-NM represents a dormant cell phenotype. The comparison between these cell lines is expected to yield new molecular targets for the design of new drugs that point dormant cells. The study began with the isolation and establishment of new tumour cell lines. After inoculation of the F3II murine mammary carcinoma cell line, we established cell populations in vitro, one from the primary tumour and another from a spontaneous metastatic nodule, F3II TP and F3II NM cell lines respectively. First, we analysed the expression of EMT markers such as E-cadherin, N-cadherin, Vimentin, Pan-Cytoqueratin and β-catenin by immunofluorescence. In in vitro assays, F3II NM presented major doubling time, major adhesion capacity and lower clonogenic potential. In addition, the expression of the dormancy related genes such as NR2F1 and BHLHE41 was analysed by qPCR, and we found that F3II-NM showed higher levels of transcript than F3II-TP. In addition, F3II NM showed lower chemo sensibility to cisplatin and in an orthotopic model of BALB/c mice, presented a longer latency time and lower tumour growth rate in comparison to F3II-TP. Taking all these together, F3II NM present a dormant phenotype and F3II TP a proliferative one. Finally, analysis and comparison of the cell lines using LFQ-Mass spectrometry revealed 256 differentially expressed proteins between the lines studied. Overexpressed genes of survival paths were identified in F3II-NM, such as DDX42, COX-2, ATGB4 involved in the inhibition of apoptosis, promotion of tumour recurrence via SOX-2 and therapy resistance, respectively. To sum up, we established a new interesting dormant tumour cell model, F3II NM that could help to a better understanding of the behaviour and survival of dormant cells. Moreover, we identified interesting protein targets that encourage the possibility of design new drugs that point and eliminate dormant cells Citation Format: Johanna E. Sidabra, Carla S. Capobianco, Maria F. Gottardo, Daniel F. Alonso, Hernan G. Farina. Isolation and characterization of novel murine mammary cell line with dormant phenotype and identification of new potential targets by mass spectrometry [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2649.
CK2 is a serine-threonine kinase that has been involved in growth, proliferation and cell apoptosis. CK2 has a constitutive expression and it has more than 300 substrates. In recent years, CK2 became an interesting target for anticancer therapies. Inhibition of CK2 showed antitumor activity in different types of cancer. Because breast cancer is one of the main tumor types in which CK2 is overexpressed, we focus on the effect of CK2 inhibition as a modulator of key features of breast cancer cell biology. The aim of the present study was to evaluate the role of inhibition of CK2 by CIGB-300 in murine and human mammary carcinoma cell lines. For this purpose, we evaluated the action of CIGB-300 on viability, apoptosis, cell cycle, clonogenic capacity and migration using three different breast tumor cell lines, MCF7 (positive for estrogen, progesterone and HER2-neu receptors), MDA MB 231 (representative of triple negative tumors) and F3II, a murine mammary carcinoma. As compared the potential of CK2 inhibition in these models we included another chemical inhibitor of the enzyme, CX-4945. Our results showed that CIGB-300 reduced the viability of MDA MB 231 (IC50=120 μM) and MCF-7 (IC50=140 μM) (p<0.05, ANOVA). We observed that in both human and murine cell lines CIGB-300 exerts a pro-apoptotic action (TUNEL assay, p<0.05, χ2) and arrest cell cycle. We also found that the inhibition of CK2 by CIGB-300 decreased the clonogenic capacity and migration of F3II, MCF-7 and MDA-MB-231 (p<0.05, ANOVA). In vivo studies using the syngeneic F3II breast cancer model in BalB/c mice showed a decrease in the number of lung metastases in mice treated systemically with CIGB-300 after primary tumor surgery (Mann Whitney test, p<0.05). However, CK2 inhibition did not affect the growth of local recurrences. Also, intravenous administration of CIGB-300 decreased lung metastasis development of F3II cells. These results demonstrate an antimetastatic effect of CIGB-300 in a systemic inoculation model after surgical removal of primary tumors. In this work we demostrated the antitumor effect of CIGB-300 in breast cancer models such as those reported in myeloid leukemia, lung and cervix and head cancer. Citation Format: Maria F. Gottardo, Carla S. Capibianco, Johanna E. Sidabra, Yasser Perera, Silvio Perea, Daniel F. Alonso, Hernan G. Farina. Antitumor activity of CIGB-300, a peptide CK2 inhibitor, in breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2899.
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