How is Herstatin, a tumor suppressor splice variant of the oncogeneHER2, regulated?

Abstract

The human epidermal growth factor receptor 2 (HER2)/receptor tyrosine-protein kinasebB-2 (ERBB2) is overexpressed in 20-30% of breast tumors leading to faster growing and more aggressive tumors. Alternative splicing generates a functionally distinct HER2 variant called Herstatin, which is produced by the inclusion of intron 8. Herstatin acts as a tumor suppressor by effectively blocking HER2 activity and cell proliferation, while promoting apoptosis. In the present study we investigated HER2 pre-mRNA regulatory sequences and splicing factors which regulate the alternative splicing of Herstatin. A Herstatin minigene, comprising exon 8/intron 8/exon 9 of HER2 was generated and subsequent in vitro splicing assays revealed that RNA secondary structure and somatic mutations did not impact on inclusion of intron 8. However, using RNase-assisted RNA chromatography, followed by mass spectrometry, we identified six RNA-binding proteins (splicing factors) that bind to RNA sequences surrounding exon 8/intron 8 and intron 8/exon 9 boundaries; these included hnRNP I, H1, D, A2/B1 and hnRNPA1 plus the SR protein SRSF1. Specifically, overexpression of hnRNP A1 significantly increased retention of intron 8 resulting in higher levels of Herstatin in SKBR3 breast cancer cells whereas SRSF1 only had a marginal effect in decreasing Herstatin but increased exogenous HER2 levels under these experimental conditions. In conclusion, we have identified the first splicing factors and regulatory sequences that are involved in the production of Herstatin.

HER2-PI9 and HER2-I12: two novel and functionally active splice variants of the oncogene HER2 in breast cancer

“…This finding is made less surprising by the relatively large amounts of protein present at the membrane, perhaps implying it can bind to the cellular membrane or dimerize with other family members with the novel C-terminus. Data suggests that HER2-I12 may behave differently to the two previously identified truncated HER2 splice variants, Herstatin and P100; both block downstream signalling likely by binding to HER2-WT receptors at the cell membrane to halt dimerization (Jackson et al 2013 ; Silipo et al 2017 ).…”

“…Herstatin is a secreted 'auto-inhibitor' with intron 8 retention which causes truncation, producing a HER2 protein with only the extracellular regions and a novel C-terminus (Silipo et al 2017). Herstatin can still bind to a HER2-WT receptor which stops HER2-WT homo-or hetero-dimerization and so blocks downstream signalling (Azios et al 2001;Koletsa et al 2008;Hu et al 2005).…”

The Mammalian Ecdysoneless Protein Interacts with RNA Helicase DDX39A To Regulate Nuclear mRNA Export

“…6G ). Significantly, ECD KD led to an increase in the expression of herstatin, a tumor suppressor splice variant ( 58 – 60 ), but a reduction in the expression of the Δ16HER2 variant, a prooncogenic splice variant ( 57 , 61 , 62 ). Taken together, we demonstrate transcriptional regulation of ErbB2 and its splice variants by ECD.…”

Urinary exosomal expression of long non-coding RNAs as diagnostic marker in bladder cancer

“…Such different pattern of expression of these two variants might imply specific roles for them which should be assessed in future studies. Evidence from protein coding genes has shown that alternative splicing can change oncogenic role of a protein to tumor suppressor role 15,16. However, data regarding the presence of such contrary functions of splice variants of lncRNA are scarce due to the significant difference in splicing dynamics between protein coding and lncRNA genes and lower completed splicing index for lncRNAs in the total chromatin fraction 17.…”