Abstract 2565: Divergentnucleocytoplasmic transport influences escape from HER2-activated DCIS-like state

Abstract HER2/ErbB2 and EGFR/ErbB1 activation incite a phenotype resembling premalignant escape from ductal carcinoma in situ (DCIS) in the 3D multicellular arrangement of breast-epithelial cells. This phenomenon tends to be infrequent, and the exact mechanism for its heterogeneous presentation has been elusive. We sought to investigate the process by which ErbB receptors facilitate a picture of incomplete penetrance in DCIS escape (DE). To identify the transcriptional signatures that prime cells towards DE, we randomly profiled 10-cell transcriptomes of single spheroids after 24 hours of ERBB activation, and frequency matched gene expression to the DE-phenotype. We uncovered a network of nucleocytoplasmic transport (NCT) regulators that alter the DE-phenotype penetrance. Of the regulators, CSE1L (an exportin) synergistically increases the DE-frequency when induced, while its knockdown reduces the phenotype, suggesting its functional role. Furthermore, we evaluated the significance of this result in vivo by perturbing Cse1l in Erbb2-amplified mouse mammary-gland tumors and found that activation of Erbb2 (mimicked by ERBB-inhibitor release) alters the macroscale organization of tumors, reminiscent of DE. Specifically, we found an elevated CSE1L inhibits an accumulation of classical nuclear-localizing cargoes that require binding with importin-α/β (KPNAs, KPNB1) complexes. Using proximity labeling with BirA*-fused with CSE1L, we identified ERBB1/ERBB2/ERBB4 as CSE1L interactors. Mechanistically, hetero-dimerized ERBB receptors interact with importin-α/β complexes and re-localize to the nucleus, corroborating previous observations of receptors’ nuclear translocation. Intriguingly, we observed that Doxycycline-induced knockdown of clathrin heavy chain (CLTC, involved in intracellular trafficking of receptors via endocytosis) synergizes with ERBB activation to increase the DE-phenotype penetrance. Using ChIP-Seq, we found that ERBB1 binds to the locus of microRNA, miR205, which negatively regulates the expression of importin-α, mainly KPNA1. These results indicate that ERBB receptors translocated to the nucleus reduce the DE-phenotype by inhibiting importins via miR205, while CSE1L induction relieves this inhibition by reducing the receptor's nuclear accumulation. Taken together, these results show that nucleocytoplasmic shuttling of receptors in ERBB-active breast epithelia generates tunable cross-inhibitory feedback through nucleocytoplasmic transport that results in long-term, heterogeneous multicellular fates. Citation Format: Lixin Wang, Bishal Paudel, Anthony McKnight, Kevin Janes. Divergentnucleocytoplasmic transport influences escape from HER2-activated DCIS-like state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2565.