No abstract

Background: UBR5 is a HECT domain E3 ubiquitin ligase that is frequently amplified in breast, ovarian, and prostate cancers. Heightened UBR5 plays a profound role in promoting tumor growth through immune-dependent mechanisms. However, its mode of action in driving tumor metastasis has not been definitively delineated. Methods: In this study, we used a tetracycline (Tet)-inducible RNAi-mediated expression silencing cell system to investigate how UBR5 enables postsurgical mammary tumor metastatic growth in the lungs of mice without the impact of the primary lesion. The effects of such manipulations on tumor metastasis were measured by wound healing, transwell, and clonogenicity assays in vitro, and by genetic deletion and complementation in vivo in syngeneic and xenograft mouse models. The system-wide impact was examined by RNA-seq analyses. Results: In vitro, Ubr5 knockdown induced morphological and molecular changes characteristic of epithelial-mesenchymal transition(EMT). In vivo, UBR5 promoted lung metastasis in an E3 ubiquitin ligase-dependent manner. Moreover, doxycycline-induced knockdown of UBR5 expression in metastatic cells in the lung resulted in increased apoptosis, decreased proliferation, and prolonged survival, whereas silencing the expression of cell division cycle 73 (CDC73), a tumor suppressor and an E3 ligase substrate of UBR5, reversed these effects. Transcriptome analyses revealed a prominent role for the p53 pathway in dovitinib-induced apoptosis of tumor cells differentially regulated by UBR5 and CDC73. In human triple-negative breast cancer (TNBC) patient specimens, a strong inverse correlation was observed between UBR5 and CDC73 protein levels and reduced CDC73 expression at metastatic sites compared to primary lesions. Furthermore, a xenograft model of human TNBC recapitulated the metastatic properties and characteristics of the unique UBR5-CDC73 functional antagonism. Conclusions: This study unveils the novel and critical roles and relationships of UBR5, CDC73 and p53 in postsurgical breast cancer metastasis and implicates the potential of targeting this pathway in cancer therapy.

OBJECTIVES/GOALS: To determine if decellularized costal cartilage (DCC), which could theoretically be obtained “off the shelf,” would provide similar results to autologous cartilage grafts previously studied in this lab, thereby widening the application of our novel nipple engineering approach to all patients undergoing nipple reconstruction. METHODS/STUDY POPULATION: PLA scaffolds (diameter: 1.0 cm, height: 1.0 cm) were printed using a PRUSA 3D printer and sterilized. Lamb costal cartilage was minced (1 mm3) or zested (<0.2 mm3) and then decellularized. The quality of decellularization was assessed using DNA quantification and histological analysis. DCC was then packed into PLA scaffolds and implanted subcutaneously into immunocompetent Sprague Dawley rats using a CV flap technique. The constructs were explanted and evaluated up to 6 months after implantation. RESULTS/ANTICIPATED RESULTS: All nipple reconstructions showed well-preserved diameter and projection due to persistence of the external scaffolds at 1, 3, and 6 months. Mass and volume of engineered tissue was well-preserved over all timepoints. Compared to implantation values, engineered zested nipples demonstrated a 12% mass increase and a 22% volume increase at 6 months. Minced nipples illustrated a similar mass and volume gain with a 21% increase in mass and a 13% increase in volume at 6 months secondary to infiltration of fibrovascular tissue and growth through scaffold wall pores, respectively. Histologic analysis demonstrated a mild inflammatory infiltrate 1 month after implantation which was replaced by fibrovascular tissue by 3 months that remained stable through 6 months. The processed DCC structure remained unchanged over time. DISCUSSION/SIGNIFICANCE: Using acellular ovine xenograft within bioabsorbable scaffolds, we have engineered neonipples that maintain their volume for at least 6 months. DCC architecture is well-preserved with minimal evidence of immune-mediated degradation. By using DCC, this novel approach to nipple engineering may be applied to any patient requiring reconstruction.

Supplementtreatment. PRP should be considered as a safe and effective treatment option for hair loss and included as part of multimodal therapy.