Noncanonical open reading frames encode functional proteins essential for cancer cell survival.

Although genomic analyses predict many noncanonical open reading frames (ORFs) in the human genome, it is unclear whether they encode biologically active proteins. Here we experimentally interrogated 553 candidates selected from noncanonical ORF datasets. Of these, 57 induced viability defects when knocked out in human cancer cell lines. Following ectopic expression, 257 showed evidence of protein expression and 401 induced gene expression changes. Clustered regularly interspaced short palindromic repeat (CRISPR) tiling and start codon mutagenesis indicated that their biological effects required translation as opposed to RNA-mediated effects. We found that one of these ORFs, G029442-renamed glycine-rich extracellular protein-1 (GREP1)-encodes a secreted protein highly expressed in breast cancer, and its knockout in 263 cancer cell lines showed preferential essentiality in breast cancer-derived lines. The secretome of GREP1-expressing cells has an increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth-inhibitory effect of GREP1 knockout. Our experiments suggest that noncanonical ORFs can express biologically active proteins that are potential therapeutic targets.

In vivo evaluation of an electrospun and 3D printed cellular delivery device for dermal wound healing.

Burns and chronic wounds are especially challenging wounds to heal. In efforts to heal these wounds, physicians often use autologous skin grafts to help restore mechanical and barrier functionality to the wound area. These grafts are, by nature, limited in availability. In an effort to provide an alternative, we have developed an electrospun wound dressing designed to incorporate into the wound with the option to deliver a cellular payload. Here, a blend of poly(glycolic acid) and poly(ethylene glycol) was electrospun as part of a custom fabrication method that incorporated 3D printed poly(vinyl alcohol) sacrificial elements. This preparation is unique compared to traditional electrospinning as sacrificial elements provide an internal void space for an injectable payload to be delivered to the wound site. When the construct was tested in vivo (full thickness excisional skin wounds), wound closure was slightly delayed by the presence of the scaffold in both normal and challenged wounds. Quality of healing was improved in normal wounds as measured by histomorphometrics when treated with the construct and exhibited increased neovascularization. Our results demonstrate that the extracellular matrix-like scaffold developed in this study is beneficial to healing of full thickness skin defects and may benefit challenged wounds.