Epigenetic regulation of the integrin ITGA2 in breast cancer
Creators: name: Verhoeff, Tristan
Publisher: name: University Of Tasmania
Year: 2023

description: Breast cancer (BC) is the most commonly diagnosed and second greatest contributor to cancer deaths in women. Mortality is usually associated with the development of metastases with breast, like prostate cancer, having higher levels of bone metastases relative to other cancers. In fact 70% of terminal BC patients have bone lesions. Integrin ˜í¬±2˜í‚â§1, and specifically the ˜í¬±2 subunit (ITGA2), have altered expression in cancers, including BC, with implications in metastasis, angiogenesis, cell adhesion and survival. The integrin's main function is ligation to collagens and laminin. Despite some regulatory pathways for ITGA2 being described, the contributions of these in BC progression are unclear, as are downstream pathways activated by ligation. ITGA2 has anti-metastatic effects in BC due to its promotion of adhesion in mammary gland epithelia. ITGA2 loss in BC metastases was hypothesised to promote motility while re-expression in bone may promote lesion formation and activate osteolytic/pro-invasive pathways. Furthermore there is emerging, albeit limited, evidence that ITGA2 is epigenetically regulated in cancer and it was hypothesised that this may be important in BC. To examine this hypothesis the expression of the ITGA2 gene and protein, as well as epigenetic modifications and transcription factor expression was investigated in BC cell lines representing different subtypes. ITGA2 expression correlated poorly with subtype; basal/metastatic lines MDA-MB-231 and MDA-MB-453 displayed high and low expression, whereas luminal/non-metastatic T-47D and MCF-7 also displayed comparably high and low expression respectively. Differential expression indicated that regulatory mechanisms affect ITGA2 expression within similar BC cell lines. Notably elevated ITGA2 mRNA and protein expression in MDA-MB-231 cells implied that highly metastatic BC cell lines that can be bone-seeking, do upregulate ITGA2 relative to other metastatic cell lines. This may support implications of ITGA2 in metastatic regulation and bone lesion formation. Protein expression reflected mRNA expression, however with less pronounced differences. This study was the first to examine ITGA2 promoter methylation in BC. Previous studies in prostate cancer (PC) concluded that methylation of the CpG island within the ITGA2 gene promoter was capable of regulating its expression, with hypermethylation reducing ITGA2 expression in PC tumours and hypomethylation elevating ITGA2 expression in normal prostate and bone-seeking PC cell lines. In BC cell lines the promoter was hypomethylated with no correlation to cell line subtype or ITGA2 expression. This novel finding suggests that while BC and PC have some similarities in regards to ITGA2 expression they have fundamentally different means of regulating ITGA2 from an epigenetic standpoint. Interestingly MCF-7 cells with low ITGA2 expression relative to the literature did have a consistently methylated CpG site within a putative NF-˜í‚à´B recognition site. As NF-˜í‚à´B can upregulate ITGA2, this implies that blocking of NF-˜í‚à´B ‚Äö- ITGA2 promoter interaction by specific epigenetic marks, may downregulate ITGA2 in BC. Epigenetic post transcriptional regulation of ITGA2 by micro RNA has also been reported for BC, notably with miR-373-3p downregulating ITGA2 protein expression to promote metastasis. A large number of potential miRNA recognition sequences in the ITGA2 mRNA 3'UTR were identified and preparations for future quantitative analysis of specific miRNAs were made. A brief examination of transcription factors regulating ITGA2 gene expression was also conducted. The TWIST1 gene, associated with EMT, was elevated in luminal BC cell lines, contrary to the literature and suggesting that it has no regulatory effect on ITGA2 expression. By contrast EGR-1 showed an inverse correlation to ITGA2 expression and thus possibly represses ITGA2 expression in BC. In other studies, androgens and transcription factors Sp1, SNAI1, AP-1/2 and EGR-3 have also been shown to regulate ITGA2. Overall this study supported ITGA2 downregulation as a mediator of BC metastasis and subsequent upregulation as a promoter of bone metastases. More importantly for the first time this study showed that ITGA2 is likely not regulated by promoter methylation in BC and its expression may instead be more dependent on regulation by miRNA or differential expression of transcription factors with expression varying greatly regardless of subtype. The data presented also suggest that select methylation may interrupt NF-˜í‚à´B interactions, with the ITGA2 promoter in luminal MCF-7 cells, indicating a novel cooperation of epigenetic regulation and transcription factors in BC. Additionally EGR-1 may downregulate ITGA2 in different basal and luminal BC cell lines. Overall a variety of distinct epigenetic and transcription factor regulatory pathways may contribute to ITGA2 expression changes in BC progression and metastasis and provide insight into both future research directions and BC therapies.