Discipline: Biological Sciences
Subcategory: Cancer Research
Fayola Levine - The City University of New York, Hunter College
Co-Author(s): Asia N. Matthew-Onabanjo, University of Massachusetts Medical School, Worcester, MA; Leslie M. Shaw, University of Massachusetts Medical School, Worcester, MA
Beclin 1 is an essential autophagy gene that functions in a core complex with vacuolar protein sorting-associated protein 34 (VPS34), the mammalian class III phosphatidylinositol-3 kinase and VPS15/p150. Beclin 1 activates VPS34 to generate phosphatidylinositol-3 phosphate (PI3P), a lipid product that is necessary for the recruitment of FYVE or PX domain containing effector proteins that facilitate membrane fusion and trafficking events. Together this core complex regulates membrane trafficking pathways through two mutually exclusive complexes. Complex I includes ATG14L and regulates autophagy initiation, whereas Complex II includes UVRAG and regulates autophagy independent functions including endocytic receptor trafficking and cytokinesis.
Studies have demonstrated a role for BECN1 in breast cancer. Beclin 1 is a haploinsufficient tumor suppressor that is monoallelically deleted in 40% of human breast cancers. Low beclin 1 expression is common in ER negative (ER-) tumors (HER2 and basal-like/TNBC), and it is associated with poor prognosis in these patients. The role of Beclin 1 in cancer has been attributed to its function in autophagy. However studies from our lab show that Beclin 1 can regulate growth factor receptor signaling, including the Insulin like Growth Factor 1 receptor (IGF-1R). Specifically, loss of Beclin 1 expression enhances and sustains downstream AKT and MAPK signaling. This enhanced signaling is due to the ability of Beclin 1 to regulate endocytic trafficking of the IGF1 receptor. This is relevant for breast cancer, as IGF1R signaling is highly activated in human breast cancers. Using an orthotopic xenograft model, our lab demonstrated that loss of Beclin 1 in vivo leads to enhanced tumor growth that is associated with increased proliferation, but no change in cell death. In this current study, we sought to generate new lentiviral plasmids that could be used for future in vivo rescue experiments because our current Beclin 1 plasmid and Beclin 1 shRNA contained the same selection marker. We employed cloning strategies to subclone the Beclin 1 shRNA into lentiviral plasmids with alternative selection markers. After generating these new constructs, we expressed the shRNA in vitro in breast carcinoma cells to demonstrate its ability to knock down Beclin 1 expression. This work could potentially provide insight into a mechanism, by which Beclin 1 contributes to breast cancer tumorigenesis, by allowing us to restore Beclin 1 with a different selection marker.
Funder Acknowledgement(s): This work was supported by a grant from the National Institution of Health (NIH-CA177167) awarded to Leslie M. Shaw, PhD.
Faculty Advisor: Leslie M. Shaw, email@example.com
Role: I assisted in this project by utilizing cloning strategies to subclone the Beclin 1 shRNA into lentiviral plasmids with Hygromycin and Neomycin as alternative selection markers. Additionally, I assisted with immunoblotting in order to verify that our new plasmids were able to successfully knockdown Beclin 1.