Abstract 2635: E3 ubiquitin ligase ARIH1 mechanism and regulation in triple-negative breast cancer

Abstract We have recently identified a novel role for the ubiquitin E3 ligase ARIH1 in Epithelial-to-Mesenchymal transition (EMT) induction, tumor initiation, and metastatic progression of triple-negative breast cancer (TNBC). In TNBC cells, ARIH1 knockdown (KD) leads to a reduction in cell invasion and mammosphere formation, and loss of tumor initiation and metastasis. In this current study, we aim to define the underlying mechanisms controlling ARIH1 expression and contributing to ARIH1 function in TNBC. Quantitative proteomic analysis of control and ARIH1 silenced TNBC cells revealed an enrichment of mitochondrial proteins consistent with ARIH1’s role in mitophagy. Additionally, a striking deregulation of proteins involved in oxidative stress response was observed. This analysis revealed that ARIH1 increases in response to various oxidative stressors, as detected by qPCR and immunoblot. The ARIH1 promotor contains conserved motifs for transcription factors (TFs), including CREB1 and HIF1α sites, linked to oxidative stress. Of note, CREB1 is positively correlated with ARIH1 transcript in breast cancer patient tumors. These findings support the transcriptional upregulation of ARIH1 in response to oxidative stress and may explain why we see an upregulation of this protein during breast cancer progression.As ARIH1 plays a role in both mitochondrial dynamics and EMT, we tested ARIH1 silencing in response to Doxorubicin (Dox), a DNA-damaging agent that induces oxidative stress, and saw improved sensitivity compared to WT MDA-MB-231 cells. Next, we assessed ARIH1 expression in the Dox-resistant cell line of our model, showing ARIH1 is upregulated in these cells at the transcript level, supporting the oxidative stress model. To further understand the crucial players impacting chemoresistance mechanisms in relation to this protein, we will test the contribution of downstream targets in response to chemotherapy. We hypothesize ARIH1’s upregulation in response to oxidative stress and DNA-damaging treatment promotes cancer cell survival and enhances chemoresistance. This study expands on our novel discovery of ARIH1’s role in EMT and cancer progression. The significance of ARIH1 in TNBC is highlighted in patient data, where high ARIH1 expression is correlated with worse patient survival. Citation Format: Casey B. Staby, Vanessa Zapata, Sean Cruz, Kelly Anderson, Abigail Quesnelle, Breege Howley, Philip Howe. E3 ubiquitin ligase ARIH1 mechanism and regulation in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2635.