MYBL1 Knockdown in a Triple Negative Breast Cancer Line: Evidence of Down-Regulation of MYBL2, TCF19 and KIF18b Expression

Abstract

Purpose: The MYBL1 gene is a strong transcriptional activator, associated with cell cycle signaling and differentiation. Data show the gene is overexpressed in triple negative breast cancers. Considering the possibility that MYBL1 might be involved in events associated with the pathogenesis of these cancers, we sought to identify genes associated with MYBL1 expression in triple negative breast cancer. Methods: shRNA lentiviral knockdown was used to down-regulate the MYBL1 gene. Microarray analyses were used to identify genes either directly or indirectly affected by targeting MYBL1 knockdown. Data analyses was performed utilizing Affymetrix TAC 4.0, Chip X transcription factor analyses, Target Scan miRNA analyses, and STRING analyses was used to determine protein: protein interaction and pathway analyses. Web Gestalt and Gene Ontology were used to determine pathway and gene-set enrichments. Publicly available patient and cell line datasets were retrieved and processed using resources available in Gene Expression Omnibus and Oncomine. The polymerase chain reaction and western analyses were used to determine transcript and protein levels, respectively. Results: Knockdown of MYBL1 in a triple negative breast cell line led to down-regulation of MYBL2, TCF19, KIF18b along with an enrichment of cell cycle signaling genes. Gene expression analyses show that MYBL1, MYBL2, TCF19 and KIF18b display a similar pattern of expression in breast cell lines and many of the archival patient datasets examined. Conclusion: TNBC is a heterogeneous subtype, so these data suggest that cancers that over-express MYBL1, express MYBL2, TCF19 and KIF18b. Bioinformatic analyses suggest MYBL1 regulates MYBL2 which leads to regulation of TCF19 and KIF18b.

Characterization of MYBL1 Gene in Triple-Negative Breast Cancers and the Genes’ Relationship to Alterations Identified at the Chromosome 8q Loci

“…In an earlier study, we performed an unsupervised meta-analysis of GEO Affymetrix microarray datasets and identified MYBL1 as differentially expressed in a subpopulation of TNBC. As a follow-up study, we performed a knockdown of MYBL1 expression in MDA-MB-231 cells, followed by microarray analyses to determine genes that either directly or indirectly associate with MYBL1 in the TNBC [ 18 ]. Reprocessing the differentially expressed dataset, we identified genes at chromosome 8q loci that were either upregulated or downregulated following MYBL1 gene knockdown ( Supplemental Table S1 ).…”

“…Data show that the gene is over-expressed in tumor progressions [ 62 ], confers chemoresistance in TNBC samples [ 63 ], and functions via mitogen-activated protein kinase (MAPK) signaling [ 64 ]. In the current study, the BOP1 gene was over-expressed in TNBC cell lines [ 18 ] and patient samples along with MYBL1, VCPIP1 and MYC genes. Patient sample data show a close relationship between MYBL1 and BOP1; however, in the GENE description of BOP1 located at NCBI, 175 genes are defined as interacting with BOP1, one of which is MYC gene based on affinity capture Mass Spectrometry ( , accessed on 1 February 2024); there is no mention of MYBL1.…”

“…MYBL1 is a strong transcriptional regulator. Following MYBL1 knockdown, we identified an enrichment of genes associated with MYBL1 transcriptional regulation [ 18 ], and a number of differentially expressed genes located at chromosome 8q loci affected either directly or indirectly by the knockdown process. Other studies report frequent copy number gains, high-level amplifications and mutations at the 8q loci [ 21 , 41 ].…”

“…The second part of our study involves the validation of MYBL1 and a subset of candidate genes in cell lines and a TNBC patient sample dataset. In an earlier experiment, we knocked down MYBL1 expression in a TNBC cell line and identified genes affected by the knockdown process [ 18 ]. Along with MYBL1, we identified a subset of genes located on chromosomal regions 8q13.1–8q24.3.…”