Autophagic Degradation of Hyaluronan Synthase 2: A Novel Mechanism to Regulate Angiogenesis in Breast Cancer
Abstract
An important constituent of the extracellular matrix (ECM), proteoglycans have emerged as dynamic signaling effectors, transducing “outside-in” cues from the extracellular space into surrounding cells. A functional subset of proteoglycans that regulate autophagy and angiogenesis have been shown to have profound therapeutic effects in cancer. Among these, endorepellin, the C-terminal domain V of the heparan sulfate proteoglycan perlecan, evokes a pro-autophagic and anti-angiogenic phenotype in endothelial cells through its selective binding to vascular endothelial growth factor receptor 2 (VEGFR2) and α2β1 integrin. Indeed, endorepellin-evoked angiostasis is contingent upon its ability to induce autophagic flux in endothelial cells, emphasizing a critical link between these two well-established pathways. In this body of work, we discovered that endorepellin induces autophagic degradation of hyaluronan synthase (HAS2), the main producer of pro-angiogenic hyaluronan (HA) in the tissue stroma, thereby identifying HAS2 as the novel link connecting autophagy and angiostasis downstream of endorepellin signaling in endothelial cells. Mechanistically, this catabolic pathway was mediated via VEGFR2 signaling and AMPK activation and mTOR inhibition downstream. We also discovered that HAS2 physically binds to ATG9A, a key multi-pass transmembrane autophagy protein that carries lipid bilayer to the maturing autophagosome, implicating ATG9A as a likely transport protein that shuttles HAS2 to the autophagosomal membrane. Live-cell and super-resolution confocal microscopy in porcine aortic endothelial cells also demonstrated dynamic colocalization between HAS2 and ATG9A within 10 and 2 minutes of endorepellin treatment and nutrient deprivation, respectively.We also demonstrated that autophagic catabolism of HAS2 was a general mechanism as it was also evoked by nutrient deprivation, mTOR inhibition and another pro-autophagic proteoglycan fragment endostatin, and this downregulation was conserved in other cell types and species in vitro. Utilizing fasting and chloroquine (CQ) to induce autophagic flux in vivo, we identified HAS2 as an autophagic substrate in heart and aorta tissue. Functionally, degradation of HAS2 via endorepellin or mTOR inhibition markedly suppressed secreted HA and ex vivo angiogenic sprouting. In summary, we therefore discovered a novel regulatory mechanism in which HAS2 is autophagically degraded, leading to a marked suppression in extracellular HA and robust attenuation of angiogenesis.Shifting to a more translational focus, we then explored the therapeutic ramifications of this novel regulatory HAS2-HA-angiogenesis axis in invasive breast cancer, a disease model that is characterized by an overexpression of HAS2 and a pathological accumulation of HA. We generated a double transgenic inducible Tie2CreERT2;ERKi mouse line that overexpresses recombinant endorepellin directly from the endothelium and utilized a syngeneic orthotopic invasive triple-negative breast cancer allograft model. Inducing recombinant endorepellin expression in the vasculature prior to tumor implantation significantly reduced tumor volume, intratumoral HA and tumor vasculature vis-à-vis uninduced mice. Indeed, inducing intratumoral expression of recombinant endorepellin with adenoviral delivery of Cre in ERKi in all cell types shortly after tumor implantation also inhibited tumor volume, HA and angiogenesis. Overall, our findings thoroughly explore the in vitro and ex vivo mechanisms behind the novel HAS2-HA-angiogenesis axis and further posit endorepellin as an effective protein therapy targeting endothelium-derived HA and angiogenesis in the breast cancer tumor microenvironment in vivo.
Subject Area
Cellular biology|Molecular biology
Recommended Citation
Chen, Carolyn Ge, "Autophagic Degradation of Hyaluronan Synthase 2: A Novel Mechanism to Regulate Angiogenesis in Breast Cancer" (2023). ProQuest ETD Collection - Thomas Jefferson University. AAI28001304.
https://jdc.jefferson.edu/dissertations/AAI28001304
