Intersection of autophagy and vesicle trafficking in Her2-positive breast cancer

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Breast cancer is the most common malignancy among US women and remains a major health threat with high incidence and lethality due to therapeutic resistance and metastasis. Breast cancer is also a heterogenous disease with different subtypes, including HER2+ subtype accounting for about 25% of patients. Despite the remarkable progress in recent years including anti-HER2 targeted therapy, our understanding of the mechanistic basis for breast cancer, particularly metastasis is still very limited. The long-term goal of the proposed studies is to understand the molecular and cellular mechanisms of metastasis and therapeutic resistance of HER2+ and other breast cancers that are ultimately responsible for patient lethality. In the prior funding period, we generated and analyzed mouse models for HER2+ breast cancer with deletion of an essential autophagy gene FIP200 or Fip200-4A mutation specifically blocking its autophagy activity and discovered that blocking FIP200-mediated autophagy abolished mammary tumorigenesis and metastasis through a new mechanism directly regulating the oncogenic driver HER2 itself. We showed that autophagy blockade abolishes mammary tumorigenesis and metastasis by decreasing levels of HER2 on the plasma membrane of mouse and human tumor cells due to aberrant HER2 trafficking from the Golgi to endosomes and multiple vesicular bodies (MVBs) and eventually released from tumor cells in small extracellular vesicles (sEVs). Additionally, employing single-cell RNA sequencing (scRNA-seq) and bioinformatics analysis, we developed a workflow to determine pharmacological interventions that would yield similar effects as FIP200 ablation. We also found FIP200 acetylation at K276 by CBP that regulates its stability. Lastly, we performed an in vivo CRISPR-Cas9 screen of a custom designed library of autophagy regulatory genes using our unique HER2+ mammary tumor cells and identified p47 as a putative suppressor for HER2+ breast cancer metastasis. Previous studies showed a role for p47 in regulating membrane fusion events, autophagy, and NFκB signaling, suggesting potential crosstalk between autophagy with vesicle trafficking in breast cancer metastasis. Based on these strong preliminary data and using our unique mouse and cell models, we propose to 1) determine the mechanism of autophagy regulation of HER2 trafficking in mouse and human breast cancer cells, 2) identify pharmacological agents that can disrupt the functions of FIP200 in mouse and human breast cancer cells as well as patient-derived models, and 3) explore the role and mechanisms of regulation of HER2+ breast cancer metastasis by p47. Together, these studies will provide significant insights into the molecular and cellular mechanisms of breast cancer metastasis that may contribute to novel therapies for this devastating disease.