The role of NMDA receptors in the aggressive phenotype of inflammatory breast cancer (IBC).

Abstract

Inflammatory Breast Cancer (IBC) is the most aggressive and lethal type of breast cancer. In most cases, the presence of a solid tumor is replaced by swelling, redness, and skin changes, which results in the misdiagnosis of an infection during the invasion and migration phase. This type of cancer blocks blood and lymphatic vessels under the breast's skin, causing local inflammation and rapid metastasis. Furthermore, IBC has a 73% incidence of brain metastasis as compared to other cancer types like a adenocarcinomas of breast, lung 40%, and kidney 24% cancers (Bos et al., 2009; EMELINE et al., 2012) Previous research shows how tumor cells from different breast cancer subtypes (HR- / HER2-) and (HR- / HER2+) have a high incidence of crossing the Blood- Brain Barrier (BBB) due to its high expression of glutamate receptors such as N-methyl-D- aspartate receptors specifically (NMDAR1 and NMDR2) subunits. However, little is known of the role that these glutamatergic ionotropic receptors have in the development and/or progression of IBC cell lines (SUM149PT and SUM190PT). Therefore, this research has several objectives, the first of which is to provide a new understanding of the cellular and molecular action that NMDA receptors (NMDAR) have on IBC. The second goal is to detect, characterize, and quantify NMDAR expression in IBC cell lines employing Western blot analysis, qRT-PCR, and immunofluorescence to identify their presence in IBC and be able to associate it with phenotypes such as migration and proliferation in quite aggressive cell lines. This study will also examine the effects of inhibition of NMDAR in IBC cell models dose-response curves of the experimental drugs (memantine and dizocilpine) in 2D cultures. In order to achieve this, we conducted several experiments using Dose-response curves and generated 2D cultures to find the drug concentration that inhibits cell viability by 50% (IC50) for each of the inhibitors (memantine and dizocilpine). Functional assays (Wound Healing Assay and 3D colony formation) were performed to measure cell migration and proliferation following each drug treatment. Findings on our first set of experiments revealed the preponderance and quantification of each of the cell lines under study (Non-IBC and IBC) through relative mRNA and protein abundance expression. The NMDAR sub- units were found in areas internal to the cell (endoplasmic reticulum, Golgi apparatus, perinuclear membrane), thus establishing new knowledge at the cellular and molecular level of NMDAR in cancer lines, especially in IBC. During this first discovery, the presence of NMDAR subunits gave IBC (Triple-negative and HER2-enriched) cell lines the ability to survive in a brain microenvironment after breast metastasis. On one hand, our second data set showed a significant decrease during migration and proliferation in IBC cell lines after dizocilpine treatment, on the other hand, memantine treatment had no effect during dose-response tests. We also found that the absence of the NMDAR’s ligand glutamate and the presence of dizocilpine affects cell migration. The present findings demonstrate that the presence of NMDAR is essential for the pro-oncogenic characteristics of IBC cell lines (SUM149PT and SUM190PT) and could emerge as a new study for alternative treatment routes for patients with inflammatory breast cancer.