Breast cancer (BC)/mammary gland carcinoma (MGC) is the most frequently diagnosed and leading cause of cancer-related mortality in both women and canines. To better understand both canine MGC and human BC-specific genes, we sequenced RNAs obtained from eight pairs of carcinomas and adjacent normal tissues in dogs. By comprehensive transcriptome analysis, 351 differentially expressed genes (DEGs) were identified in overall canine MGCs. Based on the DEGs, comparative analysis revealed correlation existing among the three histological subtypes of canine MGC (ductal, simple, and complex) and four molecular subtypes of human BC (HER2+, ER+, ER&HER2+, and TNBC). Eight DEGs shared by all three subtypes of canine MGCs had been previously reported as cancer-associated genes in human studies. Gene ontology and pathway analyses using the identified DEGs revealed that the biological processes of cell proliferation, adhesion, and inflammatory responses are enriched in up-regulated MGC DEGs. In contrast, fatty acid homeostasis and transcription regulation involved in cell fate commitment were down-regulated in MGC DEGs. Moreover, correlations are demonstrated between upstream promoter transcripts and DEGs. Canine MGC- and subtype-enriched gene expression allows us to better understand both human BC and canine MGC, yielding new insight into the development of biomarkers and targets for both diseases.

Giant axonal neuropathy (GAN) is caused by mutations in the GAN gene encoding for gigaxonin (GIG), which functions as an adaptor of the CUL3-RBX1-GIG (CRL3 ^GIG ) E3 ubiquitin ligase complex. The pathological hallmark of GAN is characterized by the accumulation of densely packed neurofilaments (NFs) in the axons. However, there are fundamental knowledge gaps in our understanding of the molecular mechanisms by which the ubiquitin–proteasome system controls the homeostasis of NF proteins. Recently, the deubiquitylating enzyme USP15 was reported to play a crucial role in regulating ubiquitylation and proteasomal degradation of CRL4 ^CRBN substrate proteins. Here, we report that the CRL3 ^GIG –USP15 pathway governs the destruction of NF proteins NEFL and INA. We identified a specific degron called NEFL ^L12 degron for CRL3 ^GIG . Notably, mutations in the C-terminal Kelch domain of GIG, represented by L309R, R545C, and C570Y, disrupted the binding of GIG to NEFL and INA, leading to the accumulation of these NF proteins. This accounts for the loss-of-function mutations in GAN patients. In addition to regulating NFs, CRL3 ^GIG also controls actin filaments by directly targeting actin-filament-binding regulatory proteins TPM1, TPM2, TAGLN, and CNN2 for proteasomal degradation. Thus, our findings broadly impact the field by providing fundamental mechanistic insights into regulating extremely long-lived NF proteins NEFL and INA by the CRL3 ^GIG –USP15 pathway and offering previously unexplored therapeutic opportunities to treat GAN patients and other neurodegenerative diseases by explicitly targeting downstream substrates of CRL3 ^GIG .

SUMMARY: 5-Aza-2′-deoxycytidine, also known as decitabine, is a DNA-methyltransferase inhibitor (DNMTi) used to treat acute myeloid leukemia (AML). Decitabine activates the transcription of endogenous retroviruses (ERV), which can induce an immune response by acting as cellular double-stranded RNAs (dsRNAs). Here, we employ an image-based screening platform to identify dsRNA-binding factors that mediate the downstream effect of ERV induction. We find that Staufen1 (Stau1) knockdown decreases the interferon signature and rescues decitabine-mediated cell death. Moreover, Stau1 directly binds to ERV RNAs and stabilizes them, together with a long non-coding RNA TINCR. We further show that TINCR enhances the interaction between Stau1 and ERV RNAs. Analysis of a clinical patient cohort reveals that AML patients with low Stau1 and TINCR expressions exhibits inferior treatment outcomes to the DNMTi therapy. Our study reveals that decitabine-mediated cell death is a consequence of complex interactions among different dsRNA-binding proteins for access to their common dsRNA targets. HIGHLIHGTS: Image-based RNAi screening reveals multiple dsRBPs regulate response to decitabine Stau1 binds to ERV RNAs and affects their stability and subcellular localization TINCR binds to Stau1 and enhances Stau1-ERV interactions AML/MDS patients with low Stau1 and TINCR expressions show poor response to DNMTis

Triple‐negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by high metastatic potential, poor prognosis, and limited effective therapeutic options. In this study, we investigated the molecular mechanisms underlying the anticancer effects of the nucleolin (NCL)‐targeting DNA aptamer AS1411 using label‐free quantitative proteomic profiling. AS1411 treatment significantly reduced TNBC cell viability and migration. To uncover the underlying mechanisms, we performed global proteomic analysis of AS1411‐treated TNBC cells. Bioinformatic analysis of differentially expressed proteins (DEPs) revealed enrichment of tumor‐associated signaling pathways and protein–protein interaction networks regulated by AS1411. Among the DEPs, ATPase H ⁺ ‐transporting accessory protein 1 (ATP6AP1) was markedly downregulated in AS1411‐treated TNBC cells. Functional studies demonstrated that ATP6AP1 knockdown suppressed TNBC cell proliferation and migration, whereas its overexpression enhanced tumorigenic phenotypes. Importantly, modulation of ATP6AP1 expression showed minimal effects on normal breast epithelial cells. Collectively, these findings identify ATP6AP1 as a key downstream mediator of AS1411 and support its potential as a therapeutic target in TNBC.