Non-tumorigenic Epithelial Cell Line MCF-10A Research Articles

Abstract 1125: Secretory pathway calcium ATPase-2 (SPCA2) regulates metastasis by suppressing mesenchymal markers in triple negative breast cancer cell lines

Abstract Introduction: Over 90% of cancer deaths in breast cancer are associated with metastasis. Epithelial-mesenchymal transition (EMT) is the hallmark of metastasis. Dysregulation of the Ca2+ toolkit has profound consequences for tumor growth and metastasis, raising hopes for novel avenues of therapeutic intervention. The Secretory Pathway Ca2+-ATPase Isoform 2 (SPCA2) transports Ca2+ from cytoplasm to the Golgi, and elicits Ca2+ influx by interacting with plasma membrane Ca2+ channels. Previously, we showed that SPCA2 is implicated in breast cancer progression (Feng et al., Cell 2010). Furthermore, low SPCA2 expression is associated with triple negative breast cancers (TNBC), which are highly metastatic. Therefore, we investigated if ectopic expression of SPCA2 modulates EMT in TNBC cell lines. Methods: TCGA invasive breast carcinoma project datasets were accessed through cBioPortal. Gene expression was determined by qPCR and protein expression by Immunoblotting and confocal microscopy. Live cell calcium imaging was performed using Fura-2 AM dye. NSG mice were used for in vivo studies. Results: Low SPCA2 expression was associated with poor survival in TNBC patients (n=255, P < 0.05). TNBC cell lines show low SPCA2 expression compared to receptor positive cell lines, when normalized to non-tumorigenic epithelial cell line MCF-10A. Similarly, significantly low SPCA2 expression (P < 0.001) was observed in TNBC patients compared to receptor positive subtypes. Interestingly, we did not observe these results in SPCA1, the housekeeping SPCA isoform, revealing isoform-specific function of Golgi calcium pumps in breast cancer subtypes. Ectopic expression of SPCA2 in TNBC cell line MDA-MB-231 significantly increased baseline intracellular Ca2+ levels (P < 0.001) and uptake of extracellular Ca2+ (P < 0.001) through store independent calcium entry. Increased SPCA2 expression suppressed mesenchymal gene markers (CDH2, SNAI1, SNAI2, VIM and ZEB1, P < 0.05), and decreased cell migration in vitro (P < 0.05) in TNBC cell lines. Overexpression of SPCA2 in MDA-MB-231-luc-D3H2LN cell line reduced metastasis in vivo (quantified 5-weeks after injection in mammary fat pad, P < 0.01) (n=6) compared to control group. Treatment of metastatic TNBC patients with histone deacetylase (HDAC) inhibitors is currently being evaluated in clinical trials. We found treatment of TNBC cell lines with FDA-approved HDAC inhibitors vorinostat and romidepsin elevated SPCA2 expression in a dose-dependent manner (P < 0.05), simultaneously decreasing mesenchymal gene expression (P < 0.05). Conclusion: These novel findings point to a causal link between low SPCA2 levels, poor prognosis, and the epithelial-mesenchymal transition required for breast cancer metastasis. Restoration of SPCA2 expression in TNBC by HDAC inhibitors may have therapeutic potential. Citation Format: Monish Ram Makena, Donna K. Dang, Myungjun Ko, Manuj Bandral, Rajini Rao. Secretory pathway calcium ATPase-2 (SPCA2) regulates metastasis by suppressing mesenchymal markers in triple negative breast cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1125.

Discovery and Identification of Small Molecules as Methuosis Inducers with in Vivo Antitumor Activities.

Methuosis is a novel nonapoptotic mode of cell death characterized by vacuole accumulation in the cytoplasm. In this article, we describe a series of azaindole-based compounds that cause vacuolization in MDA-MB-231 cells. The most potent vacuole inducer, compound 13 (compound 13), displayed differential cytotoxicities against a broad panel of cancer cell lines, such as MDA-MB-231, A375, HCT116, and MCF-7, but it did not inhibit the growth of the nontumorigenic epithelial cell line MCF-10A. A mechanism study confirmed that the cell death was caused by inducing methuosis. Furthermore, compound 13 exhibited substantial pharmacological efficacy in the suppression of tumor growth in a xenograft mouse model of MDA-MB-231 cells without apparent side effects, which makes this compound the first example of a methuosis inducer with potent in vivo efficacy. These results demonstrate that methuosis inducers might serve as novel therapeutics for the treatment of cancer.

Epstein-Barr virus LMP1 induces focal adhesions and epithelial cell migration through effects on integrin-α5 and N-cadherin.

Epstein–Barr virus (EBV) is a γ-herpesvirus associated with human epithelial and B-cell malignancies. The EBV latent membrane protein (LMP) 1 is expressed in nasopharyngeal carcinoma (NPC) and promotes oncogenic intracellular signaling mechanisms. LMP1 also promotes a pro-migratory phenotype through potential effects on cell surface proteins, as expression of LMP1 induces an epithelial–mesenchymal transition (EMT) in epithelial cell lines. In this study, LMP1 was examined for potential effects on cadherin and integrin surface interactions, and assessed for biological effects on adhesion and motility to fibronectin. Expression of LMP1 in the non-tumorigenic epithelial cell line MCF10a induced an EMT-associated cadherin switch. The induced N-cadherin was ligated and localized to the cell surface as determined by triton-solubility and immunofluorescence assays. In addition, LMP1 induced the assembly of focal adhesions (FAs) with increased production of fibronectin in MCF10a and NP460hTERT-immortalized nasopharyngeal cells. Biochemical enrichment of fibronectin-associated proteins indicated that LMP1 selectively promoted the recruitment of integrin-α5 and Src family kinase proteins to FA complexes. Neutralizing antibodies to N-cadherin and integrin-α5, but not integrin-αV, blocked the adhesion and transwell motility of MCF10a cells to fibronectin induced by LMP1. LMP1-induced transwell motility was also decreased by Src inhibition with the PP2 kinase inhibitor and short hairpin RNAs. These studies reveal that LMP1 has multiple mechanisms to promote the adhesive and migratory properties of epithelial cells through induction of fibronectin and modulation of cell surface interactions involving integrin-α5 and N-cadherin, which may contribute to the metastatic potential of NPC.

Bik subcellular localization in response to oxidative stress induced by chemotherapy, in Two different breast cancer cell lines and a Non-tumorigenic epithelial cell line.

Cancer chemotherapy remains one of the preferred therapeutic modalities against malignancies despite its damaging side effects. An expected outcome while utilizing chemotherapy is apoptosis induction. This is mainly regulated by a group of proteins known as the Bcl-2 family, usually found within the endoplasmic reticulum or the mitochondria. Recently, these proteins have been located in other sites and non-canonic functions have been unraveled. Bik is a pro-apoptotic protein, which becomes deregulated in cancer, and as apoptosis is associated with oxidative stress generation, our objective was to determine the subcellular localization of Bik either after a direct oxidative insult due to H2 O2 , or indirectly by cisplatin, an antineoplastic agent. Experiments were performed in two human transformed mammary gland cell lines MDA-MB-231 and MCF-7, and one non-tumorigenic epithelial cell line MCF-10A. Our results showed that in MCF-7, Bik is localized within the cytosol and that after oxidative stress treatment it translocates into the nucleus. However, in MDA-MB-231, Bik localizes in the nucleus and translocates to the cytosol. In MCF10A Bik did not change its cellular site after either treatment. Interestingly, MCF10A were more resistant to cisplatin than transformed cell lines. This is the first report showing that Bik is located in different cellular compartments depending on the cancer stage, and it has the ability to change its subcellular localization in response to oxidative stress. This is associated with increased sensitivity when exposed to toxic agents, thus rendering novel opportunities to study new therapeutic targets allowing the development of more active and less harmful agents.