Breast Cancer Cells Reprogram the Oncogenic lncRNAs/mRNAs Coexpression Networks in Three-Dimensional Microenvironment

Abstract

Organotypic three-dimensional (3D) cell cultures more accurately mimic the characteristics of solid tumors in vivo in comparison with traditional two-dimensional (2D) monolayer cell models. Currently, studies on the regulation of long non-coding RNAs (lncRNAs) have not been explored in breast cancer cells cultured in 3D microenvironments. In the present research, we studied the expression and potential roles of lncRNAs in estrogen receptor-positive luminal B subtype BT-474 breast cancer cells grown over extracellular matrix proteins-enriched 3D cultures. Global expression profiling using DNA microarrays identifies 290 upregulated and 183 downregulated lncRNAs in 3D cultures relative to 2D condition. Using a co-expression analysis approach of lncRNAs and mRNAs pairs expressed in the same experimental conditions, we identify hundreds of regulatory axes modulating genes involved in cancer hallmarks, such as responses to estrogens, cell proliferation, hypoxia, apical junctions, and resistance to endocrine therapy. In addition, we identified 102 lncRNAs/mRNA correlations in 3D cultures, which were similar to those reported in TCGA datasets obtained from luminal B breast cancer patients. Interestingly, we also found a set of mRNAs transcripts co-expressed with LINC00847 and CTD-2566J3.1 lncRNAs, which were predictors of pathologic complete response and overall survival. Finally, both LINC00847 and CTD -2566J3.1 were co-expressed with essential genes for cancer genetic dependencies, such as FOXA1 y GINS2. Our experimental and predictive findings show that co-expressed lncRNAs/mRNAs pairs exhibit a high degree of similarity with those found in luminal B breast cancer patients, suggesting that they could be adequate pre-clinical tools to identify not only biomarkers related to endocrine therapy response and PCR, but to understand the biological behavior of cancer cells in 3D microenvironments.

Organotypic 3D Cell-Architecture Impacts the Expression Pattern of miRNAs–mRNAs Network in Breast Cancer SKBR3 Cells

“…In two previous reports from our group, using the triple-negative breast cancer (TNBC) cell line Hs578T and the luminal B breast cancer cell line BT-474 cultivated under the same cell culture conditions used here, we also obtained compact and large organoids, but with different morphology [4,27]. In the case of the BT-474 cell line, it has been shown that a 3D microenvironment can reprogram the oncogenic lncRNAs/mRNAs coexpression networks [4]. Similarly, in our case, SKBR3 cells reprogram the oncogenic miRNA/mRNA coexpresion network, which makes it obvious that lncRNA expression is also modified in our HER2+ subtype.…”

“…To obtain SKBR3 organoids, a standard On-Top protocol was used [4,27]; briefly, 24well flat-bottom plates were coated with 150 µL of LDEV-free growth factor-reduced Geltrex (Gibco, USA) and incubated at 37 • C for 30 min to promote its gelation. Then, 4.2 × 10 4 cells were seeded, and DMEM/F-12 supplemented medium was added to incubate for up to 5 days.…”

“…Breast cancer exhibits a high degree of heterogeneity, encompassing distinct genotypic, phenotypic, and anatomical characteristics that exert profound influences on the clinical outcomes of patients [3]. Although the histological and molecular classifications of breast cancer may appear simplistic, they can predict biological behavior and facilitate the development of targeted therapeutic approaches [4]. Approximately 84 breast cancer cell lines have been categorized based on the presence of four major molecular subtypes: human epidermal growth factor receptor 2 (HER2)-enriched, basal-like, luminal A, and luminal B [5].…”

Regulatory Roles of Non-Coding RNAs in Cancer

“…The use of co-expressed lncRNAs/mRNAs pairs as preclinical tools to identify biomarkers related to response to endocrine therapy and to understand the biological behavior of cancer cells in 3D microenvironments could have significant implications in the development of personalized cancer treatment. It is important to note that further studies are needed to confirm the results and assess their clinical utility [12].…”