An assessment of the penile squamous cell carcinoma surfaceome for therapeutic target discovery.
Authors: Grass George Daniel , Chahoud Jad , Ercan Dalia , Obermayer Alyssa , Shaw Timothy , Stewart Paul , Johnstone Peter A.S. , Rogatto Silvia Regina , Eschrich Steven A. , Spiess Philippe E. 
Published: March 2023

13 Background: There are limited options for relapsed penile squamous cell carcinoma (PSCC) patients after definitive therapy or chemo-refractory disease. Novel target discovery methods are needed to identify potential treatment options and the cell surface represents an actionable target for molecular and cell-based therapies. We evaluated the cell surface molecular catalogue (e.g., surfaceome) in PSCC to identify underexplored targets. Methods: To evaluate proteins enriched on the surface of PSCC cells, we screened published translatomics data from 5 PSCC cell lines (HPV-negative). Ribosome-bound RNA expression values were then analyzed using a validated surfaceome gene list (n=2,886 proteins) to infer surface presence, which were grouped by consensus protein classes. This was complemented by RNA-Seq (n=37) on resected PSCC tumors (HPV+=16, HPV- = 16, unknown HPV status = 5). We used immunohistochemistry (IHC) to assess protein expression and subcellular localization in PSCC tumors; stain intensity was assessed by a semi-quantitative H-score. Non-parametric statistics compared distributions and Kaplan-Meier analysis estimated overall survival (OS; defined from surgery to death or last follow-up) and groups were compared by log-rank testing. The open-source DRPPM-PATH-SURVEIOR app and R statistical software were used to perform analyses. Results: We identified 604 unique surfaceome proteins which were represented in the top 75% of expression in the PSCC cell line translatome. Half of these proteins have N-glycosylations and 49.5% classified as high-confidence surface targets. The largest proportions of surfaceome proteins included: Receptors (25.6%), Transporters (18.5%), and proteins with unclassified function (32.2%). Of note, 16.2% of this surfaceome linked to at least one compound in the DrugBank database.There was a moderate correlation between cell line and tumor surfaceome RNAs (top 75%; r =0.58, p < 2.2x10-16). We selected targets in the 99% (BSG; CD147; regulates MMPs and lactate transport), 90% (FGFR1; receptor tyrosine kinase) and 75% (SLC16A1; metabolite transporter) of the surfaceome expression. Tumor RNA levels for BSG, FGFR1 and SLC16A1 were elevated, but without differences based on HPV. IHC demonstrated robust expression in tumors with plasma membrane scoring being enriched compared to non-plasma membrane compartments (CD147, p =2.9x10-6; SLC16A1, p = 5.1x10-6; FGFR1, p = 0.007). Of note, patients with elevated BSG RNA (based on median log2 value) had worse OS (p = 0.018), though this lost significance after adjusting for HPV. No differences in OS were seen with FGFR1 or SLC16A1 expression. Conclusions: Our analysis of the surfaceome based on RNA expression was associated with increased protein levels in tumor tissues. Evaluation of the PSCC surfaceome may provide opportunity to investigate novel therapeutic targets, which may be actionable regardless of HPV status.