Identification of a Modified HOXB9 mRNA in Breast Cancer

Abstract

First identified as a developmental gene, HOXB9 is also known to be involved in tumor biological processes, and its aberrant expression correlates with poor prognosis of various cancers. In this study, we isolated a homeodomain-less, novel HOXB9 variant (HOXB9v) from human breast cancer cell line-derived mRNA. We confirmed that the novel variant was produced from variationless HOXB9 genomic DNA. RT-PCR of mRNA isolated from clinical samples and reanalysis of publicly available RNA-seq data proved that the new transcript is frequently expressed in human breast cancer. Exogenous HOXB9v expression significantly enhanced the proliferation of breast cancer cells, and gene ontology analysis indicated that apoptotic signaling was suppressed in these cells. Considering that HOXB9v lacks key domains of homeobox proteins, its behavior could be completely different from that of the previously described variationless HOXB9. Because none of the previous studies on HOXB9 have considered the presence of HOXB9v, further research analyzing the two transcripts individually is warranted to re-evaluate the true role of HOXB9 in cancer.

Figure 1

Structure and sequence of HOXB9n and HOXB9v. (a) Sequences of genomic DNA HOXB9, mRNA HOXB9n, and mRNA HOXB9v (black and grey highlighting indicates homology between sequences). (b) Schematic diagram of HOXB9n (upper) and HOXB9v (lower) transcripts showing the exons, splicing regions, and the deleted region. In HOXB9v, a 100 bp deletion in exon 1 leads to a frameshift and a stop codon formation (TAA). (c) Protein structure of HOXB9n and HOXB9v. Transcription from the start codon (ATG) to the stop codon (TAA) results in the translation of a full-length HOXB9n protein (upper). The 100 bp deletion in HOXB9v (shown in black) leads to a frameshift from AA85 (shown in grey) and truncation of protein coding by a stop codon (TAG) at AA167, which results in HOXB9v protein without hexapeptide and homeodomain.^∗Stop codon.

Figure 2

Detection of mRNA and genomic DNA of HOXB9 in cell lines. (a) Both HOXB9n and HOXB9v transcripts were detected in breast cancer cell line mRNA (cDNA). (b) Sequencing confirmation of PCR product of Figure 2(a); SKBR3 (HOXB9n, upper column) and MCF10A (HOXB9v, lower column). (c) No genomic variation was detected in genomic DNA of breast cancer cell line genomic DNA. HOXB9v transcripts are commonly found in human breast cancer specimens.

Figure 3

Detection of HOXB9v in clinical samples. (a) HOXB9n and HOXB9v were detected in breast cancer clinical samples. (b) HOXB9v was not detected in normal mammary gland samples. Hormone receptor status and HER2 status of each cancer sample are shown beneath each sample number. We assigned the same sample number if cancer and normal gland samples were acquired from the same patient.

Figure 4

NGS data indicates the presence of HOXB9v in breast cancer. Showing RNA sequence breast cancer sample data (SRR782677) mapped on HOXB9 exon 1. The blue band on the bottom shows the coding region (the thick part) and the 5′ UTR (the thin part) of exon 1. There is a region where data are sparsely mapped (indicated with the red arrow), which matches the deletion region in HOXBv. HOXB9v overexpressing MCF7 and MDA-MB-468 cells proliferated faster compared with HOXB9n overexpressing cells.

Figure 5

(a–d) Confirmation and proliferation of stable MCF7 cell lines that overexpress HOXB9n or HOXB9v. Levels of HOXB9n and HOXB9v (a) mRNA and (b) protein in stable cell lines. Proliferation of the HOXB9n and HOXB9v stable cell lines using a (c) 3D cell culture model and a (d) 2D cell culture model. (e-f) Confirmation and proliferation of MDA-MB-468 cells with transient overexpression of HOXB9n or HOXB9v. (e) Levels of HOXB9n and HOXB9v mRNA expression. (f) Proliferation of HOXB9n and HOXB9v overexpressing cells by 2D cell culture model.

Figure 6

(a) HOXB9n-MCF7 cells and (b) HOXB9v-MCF7 cells were treated with HXR9 or with a control peptide, CXR9. HXR9 significantly inhibited the proliferation of both cell lines.

Figure 7

Microarray data heatmap of HOXB9n-MCF7 and HOXB9v-MCF7. Genomic heatmap compares expression of 37 genes which are involved in suppression of apoptotic process (GO:0043069), with red and green color intensities indicating high and low expressions, respectively. Programmed cell death is suppressed in HOXB9v-MCF7 cells.