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Functional analysis of N-acetylglucosaminyltransferase-I knockdown in 2D and 3D neuroblastoma cell cultures

ABSTRACT: Tumor development can be promoted/suppressed by certain N-glycans attached to proteins at the cell surface. Here we examined aberrant neuronal properties in 2D and 3D rat neuroblastoma (NB) cell cultures with different N-glycan populations. Lectin binding studies revealed that the engineered N-glycosylation mutant cell line, NB_1(-Mgat1), expressed solely oligomannose N-glycans, and verified that the parental cell line, NB_1, and a previous engineered N-glycosylation mutant, NB_1(-Mgat2), expressed significant levels of higher order N-glycans, complex and hybrid N-glycans, respectively. NB_1 grew faster than mutant cell lines in monolayer and spheroid cell cultures. A 2-fold difference in growth between NB_1 and mutants occurred much sooner in 2D cultures relative to that observed in 3D cultures. Neurites and spheroid cell sizes were reduced in mutant NB cells of 2D and 3D cultures, respectively. Cell invasiveness was highest in 2D cultures of NB_1 cells compared to that of NB_1(-Mgat1). In contrast, NB_1 spheroid cells were much less invasive relative to NB_1(-Mgat1) spheroid cells while they were more invasive than NB_1(-Mgat2). Gelatinase activities supported the ranking of cell invasiveness in various cell lines. Both palladin and HK2 were more abundant in 3D than 2D cultures. Levels of palladin, vimentin and EGFR were modified in a different manner under 2D and 3D cultures. Thus, our results support variations in the N-glycosylation pathway and in cell culturing to more resemble in vivo tumor environments can impact the aberrant cellular properties, particularly cell invasiveness, of NB.

SUBMITTER: Hall M

PROVIDER: S-EPMC8575246 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Invasive lobular carcinoma (ILC) is the second most common subtype of breast cancer following invasive ductal carcinoma (IDC) and characterized by the loss of E-cadherin-mediated adherens junctions. Despite displaying unique histologic and clinical features, ILC still remains a chronically understudied disease, with limited knowledge gleaned from available laboratory research models. Here we report a comprehensive 2D and 3D phenotypic characterization of four estrogen receptor-positive human ILC cell lines: MDA-MB-134, SUM44, MDA-MB-330, and BCK4. Compared with the IDC cell lines MCF7, T47D, and MDA-MB-231, ultra-low attachment culture conditions revealed remarkable anchorage independence unique to ILC cells, a feature not evident in soft-agar gels. Three-dimensional Collagen I and Matrigel culture indicated a generally loose morphology for ILC cell lines, which exhibited differing preferences for adhesion to extracellular matrix proteins in 2D. Furthermore, ILC cells were limited in their ability to migrate and invade in wound-scratch and transwell assays, with the exception of haptotaxis to Collagen I. Transcriptional comparison of these cell lines confirmed the decreased cell proliferation and E-cadherin-mediated intercellular junctions in ILC while uncovering the induction of novel pathways related to cyclic nucleotide phosphodiesterase activity, ion channels, drug metabolism, and alternative cell adhesion molecules such as N-cadherin, some of which were differentially regulated in ILC versus IDC tumors. Altogether, these studies provide an invaluable resource for the breast cancer research community and facilitate further functional discoveries toward understanding ILC, identifying novel drug targets, and ultimately improving the outcome of patients with ILC.Significance: These findings provide the breast cancer research community with a comprehensive assessment of human invasive lobular carcinoma (ILC) cell line signaling and behavior in various culture conditions, aiding future endeavors to develop therapies and to ultimately improve survival in patients with ILC. Cancer Res; 78(21); 6209-22. ©2018 AACR.

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Functional analysis of N-acetylglucosaminyltransferase-I knockdown in 2D and 3D neuroblastoma cell cultures

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