HOTAIR Modulated Pathways in Early-Stage Breast Cancer Progression

Abstract

The long-non-coding HOX transcript antisense intergenic RNA (HOTAIR) was identified as significantly upregulated in breast ductal carcinoma in situ (DCIS). The aim of this study was to characterize the phenotypic effects and signaling pathways modulated by HOTAIR in early-stage breast cancer progression. We determined that HOTAIR induces premalignant phenotypic changes by increasing cell proliferation, migration, invasion and in vivo growth in normal and DCIS breast cell lines. Transcriptomic studies (RNA-seq) identified the main signaling pathways modulated by HOTAIR which include bioprocesses related to epithelial to mesenchymal transition, cell migration, extracellular matrix remodeling and activation of several signaling pathways (HIF1A, AP1 and FGFR). Similar pathways were identified as activated in primary invasive breast carcinomas with HOTAIR over-expression. We conclude that HOTAIR over-expression behaves as a positive regulator of cell growth and migration both in normal and DCIS breast cells involved with early-stage breast cancer progression.

Keywords: DCIS; HOTAIR; breast cancer; invasion; lncRNA; proliferation.

Figure 1

HOTAIR expression in normal, pre-invasive and invasive breast samples. (A) Top ten most upmodulated lncRNAs in DCIS lesions compared with normal breast samples according to GSE69994 study (2). (B) In silico HOTAIR expression analysis among normal, DCIS and IBC samples obtained from four independent GEO dataset (, –16). HOTAIR expression was significant upregulated in DCIS and IBC samples compared with normal samples (p < 0.01), while non-significant differences were observed between DCIS and IBC cases (p > 0.05). (C) HOTAIR expression analysis among normal and DCIS precursor lesions (early neo.) such as columnar cell lesions and atypical ductal hyperplasia, obtained from GSE47462 dataset (17). (D) HOTAIR expression analysis across DCIS intrinsic subtype obtained from two independent GEO datasets (2, 14). ANOVA or T-test were used to compare the HOTAIR expression among groups. *Statistical significance differences.

Figure 2

Transcriptomic analysis of HOTAIR overexpressing cells. (A) Hierarchical clustering of MCF10A and DCIS.COM stably transduced cells with either vector control (pCDH-empty) or lentivirus expressing HOTAIR (pCDH-HOTAIR) based on RNA-seq profiles. (B) Heat map representation of the differentially expressed genes (DEG) obtained by RNA-seq analysis (FDR<0.05; FC>1.5). Red represents upregulated genes and green downregulated genes. (C, D) Functional enrichment analysis of GO biological processes and pathways identified as affected by expression of HOTAIR in MCF10A cells. (E, F) Functional enrichment analysis of GO biological processes and pathways identified as affected by expression of HOTAIR in DCIS.COM cells. (G) Venn diagram of transcripts commonly modulated among MCF10 and DCIS.COM cells stably transduced with HOTAIR.

Figure 3

Stable overexpression of HOTAIR induces increased cell proliferation, colony growth and invasion in normal breast cells. (A) Levels of HOTAIR expression in stably transduced MCF10A cells based on their RNA-seq profiles (log2 CPM). (B) Stable overexpression of HOTAIR increases cell proliferation in normal breast cells (p < 0.01). Cells were plated 1,000 cells per well on 96 well plates in triplicate and cell proliferation was determined by means of the MTT colorimetric assay and measuring optical density (OD). (C) Cells stably transduced with lentivirus expressing HOTAIR or vector control were plated at clonal density in 6-well plates. Cells were allowed to grow for 9 days, fixed and stained with crystal violet. Bar chart displays increased area occupied by colonies for HOTAIR stably transduced cells compared with vector control. (D) Transwell migration assay of DCIS.COM cells stably transduced with HOTAIR. On the left comparative pictures of cells that migrated through the membrane, on the right bar chart of the relative numbers of cells per membrane for HOTAIR stably transduced cells compared with vector control (p < 0.01). Statistical significance was determined using Mann-Whitney-Wilcoxon test. *Statistical significance differences.

Figure 4

DCIS.COM stably transduced cells with either vector control or lentivirus expressing HOTAIR were compared using the in vivo MIND model. (A) HOTAIR expression levels in stably transduced DCIS.COM cells based on their RNA-seq profiles (log2 CPM). (B) DCIS growth was detected among injected mice. (C) Representative invasive ductal carcinoma (10X H&E staining) induced by DCIS.COM cells stably transduced with HOTAIR. Scale bar: 200 μm.

Figure 5

HOTAIR expression and pathway activity analysis in primary invasive breast carcinomas. (A) Primary breast carcinomas were divided into HOTAIR low or high expression levels based on the StepMiner algorithm using TCGA RNA-seq datasets obtained from the UCSC Xena resource (https://xenabrowser.net/). (B) Percentage of cases with high or low HOTAIR expression among intrinsic subtypes showing a consistent up-regulation in basal-like and HER2 subtypes compared with luminal-like tumors. (C) Heat map with the 68 significantly activated PARADIGM Integrated Pathways Activities (IPAs) among HOTAIR high expression tumors (p-adj.<0.01). (D) Bar plot of the top fifteen activated pathways determined by PARADIGM algorithm in primary invasive breast carcinomas with HOTAIR overexpression.