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Dual targeted therapy with p53 siRNA and Epigallocatechingallate in a triple negative breast cancer cell model.

ABSTRACT: Triple-negative breast cancer (TNBC) is a highly aggressive phenotype that is resistant to standard therapy. Thus, the development of alternative therapeutic strategies for TNBC is essential. The purpose of our in vitro study was to evaluate the impact of p53 gene silencing in conjunction with the administration of a natural compound, epigallocatechingallate (EGCG). RT2Profiler PCR Array technology was used to evaluate the impact of dual treatment on the main genes involved in apoptosis in the Hs578T cell culture model of TNBC. Gene expression analysis revealed 28 genes were significantly altered (16 upregulated and 12 downregulated) in response to combined p53 siRNA and EGCG treatment. Further analysis revealed that p53 siRNA and EGCG dual therapy leads to the activation of pro-apoptotic genes and the inhibition of pro-survival genes, autophagy, and cell network formation. These results indicate that this dual therapy targets both the apoptotic and angiogenic pathways, which may improve treatment effectiveness for tumors resistant to conventional treatment.

SUBMITTER: Braicu C

PROVIDER: S-EPMC4388814 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Dual targeted therapy with p53 siRNA and Epigallocatechingallate in a triple negative breast cancer cell model.

Braicu Cornelia C Pileczki Valentina V Pop Laura L Petric Roxana Cojocneanu RC Chira Sergiu S Pointiere Eve E Achimas-Cadariu Patriciu P Berindan-Neagoe Ioana I

PloS one 20150407 4

Triple-negative breast cancer (TNBC) is a highly aggressive phenotype that is resistant to standard therapy. Thus, the development of alternative therapeutic strategies for TNBC is essential. The purpose of our in vitro study was to evaluate the impact of p53 gene silencing in conjunction with the administration of a natural compound, epigallocatechingallate (EGCG). RT2Profiler PCR Array technology was used to evaluate the impact of dual treatment on the main genes involved in apoptosis in the H ...[more]

PMID: 25849487

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Project description:BackgroundTriple-negative breast cancer (TNBC) is a uniquely aggressive cancer with high rates of relapse due to resistance to chemotherapy. TNBC expresses higher levels of programmed cell death-ligand 1 (PD-L1) compared to other breast cancers, providing the rationale for the recently approved immunotherapy with anti-PD-L1 monoclonal antibodies (mAbs). A huge effort is dedicated to identify actionable biomarkers allowing for combination therapies with immune-checkpoint blockade. Platelet-derived growth factor receptor β (PDGFRβ) is highly expressed in invasive TNBC, both on tumor cells and tumor microenvironment. We recently proved that tumor growth and lung metastases are impaired in mouse models of human TNBC by a high efficacious PDGFRβ aptamer. Hence, we aimed at investigating the effectiveness of a novel combination treatment with the PDGFRβ aptamer and anti-PD-L1 mAbs in TNBC.MethodsThe targeting ability of the anti-human PDGFRβ aptamer toward the murine receptor was verified by streptavidin-biotin assays and confocal microscopy, and its inhibitory function by transwell migration assays. The anti-proliferative effects of the PDGFRβ aptamer/anti-PD-L1 mAbs combination was assessed in human MDA-MB-231 and murine 4 T1 TNBC cells, both grown as monolayer or co-cultured with lymphocytes. Tumor cell lysis and cytokines secretion by lymphocytes were analyzed by LDH quantification and ELISA, respectively. Orthotopic 4 T1 xenografts in syngeneic mice were used for dissecting the effect of aptamer/mAb combination on tumor growth, metastasis and lymphocytes infiltration. Ex vivo analyses through immunohistochemistry, RT-qPCR and immunoblotting were performed.ResultsWe show that the PDGFRβ aptamer potentiates the anti-proliferative activity of anti-PD-L1 mAbs on both human and murine TNBC cells, according to its human/mouse cross-reactivity. Further, by binding to activated human and mouse lymphocytes, the aptamer enhances the anti-PD-L1 mAb-induced cytotoxicity of lymphocytes against tumor cells. Importantly, the aptamer heightens the antibody efficacy in inhibiting tumor growth and lung metastases in mice. It acts on both tumor cells, inhibiting Akt and ERK1/2 signaling pathways, and immune populations, increasing intratumoral CD8 + T cells and reducing FOXP3 + Treg cells.ConclusionCo-treatment of PDGFRβ aptamer with anti-PD-L1 mAbs is a viable strategy, thus providing for the first time an evidence of the efficacy of PDGFRβ/PD-L1 co-targeting combination therapy in TNBC.

Project description:BackgroundThe effective treatment of triple-negative breast cancer (TNBC) remains a profound clinical challenge. Despite frequent epidermal growth factor receptor (EGFR) overexpression and reliance on downstream signalling pathways in TNBC, resistance to EGFR-tyrosine kinase inhibitors (TKIs) remains endemic. Therefore, the identification of targeted agents, which synergise with current therapeutic options, is paramount.MethodsCompound-based, high-throughput, proliferation screening was used to profile the response of TNBC cell lines to EGFR-TKIs, western blotting and siRNA transfection being used to examine the effect of inhibitors on EGFR-mediated signal transduction and cellular dependence on such pathways, respectively. A kinase inhibitor combination screen was used to identify compounds that synergised with EGFR-TKIs in TNBC, utilising sulphorhodamine B (SRB) assay as read-out for proliferation. The impact of drug combinations on cell cycle arrest, apoptosis and signal transduction was assessed using flow cytometry, automated live-cell imaging and western blotting, respectively. RNA sequencing was employed to unravel transcriptomic changes elicited by this synergistic combination and to permit identification of the signalling networks most sensitive to co-inhibition.ResultsWe demonstrate that a dual cdc7/CDK9 inhibitor, PHA-767491, synergises with multiple EGFR-TKIs (lapatinib, erlotinib and gefitinib) to overcome resistance to EGFR-targeted therapy in various TNBC cell lines. Combined inhibition of EGFR and cdc7/CDK9 resulted in reduced cell proliferation, accompanied by induction of apoptosis, G2-M cell cycle arrest, inhibition of DNA replication and abrogation of CDK9-mediated transcriptional elongation, in contrast to mono-inhibition. Moreover, high expression of cdc7 and RNA polymerase II Subunit A (POLR2A), the direct target of CDK9, is significantly correlated with poor metastasis-free survival in a cohort of breast cancer patients. RNA sequencing revealed marked downregulation of pathways governing proliferation, transcription and cell survival in TNBC cells treated with the combination of an EGFR-TKI and a dual cdc7/CDK9 inhibitor. A number of genes enriched in these downregulated pathways are associated with poor metastasis-free survival in TNBC.ConclusionsOur results highlight that dual inhibition of cdc7 and CDK9 by PHA-767491 is a potential strategy for targeting TNBC resistant to EGFR-TKIs.

Dataset Information

Dual targeted therapy with p53 siRNA and Epigallocatechingallate in a triple negative breast cancer cell model.

Publications

Dual targeted therapy with p53 siRNA and Epigallocatechingallate in a triple negative breast cancer cell model.

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