Validating SCAP as a Therapeutic Target for Colorectal Cancer

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide with an increased prevalence in adults below 50 years old, highlighting the need to develop more therapeutic options. Data mining from genome-wide dropout screens revealed the essentiality of sterol regulatory element binding protein cleavage activating protein (SCAP) on the proliferation of CRC cell lines. In this study, we demonstrate that SCAP knockdown leads to marked antiproliferation, with variability in the phenotypic strength across the CRC cell lines tested. SCAP knockdown reduced mRNA expression of its transcription factor binding partners SREBP1/2, and their transcriptional target genes, primarily impacting lipid homeostasis. SCAP knockdown induced autophagy and multiple cell-specific disruptions in cell cycle progression, which preceded caspase-independent cell death. Global metabolic profiling was performed to further characterize the growth suppressive mechanism and uncovered significant metabolic perturbations in multiple pathways. A profound shift was observed in lipid pathways including cholesterol and monounsaturated fatty acid (FA) synthesis. The decrease in FA synthesis affected downstream derivatives, diverting most of the remaining FAs towards phosphatidylcholine and decreasing acyl-carnitine formation. Furthermore, nicotinamide and polyamine metabolism were greatly affected, resulting in significantly elevated metabolites, notably 1-methylnicotinamide, acetylated spermidine, and spermine.Despite challenges, we successfully expressed and purified the full-length human SCAP and its WDR domain. While cells do not favor overexpressing full-length SCAP, we found that the membrane fraction was the most productive for isolating SCAP with a mild detergent buffer (2% DDM+ 0.4% CHS). For the WDR domain we further optimized our expression constructs to shorten the flexible loops and greatly improve the purification quality and protein solubility. Although crystal screening of the WDR domain has been unsuccessful so far, the protein behavior is promising, and more screening conditions will be explored. Our findings underscore the significance of SCAP in CRC cell proliferation, suggesting that targeting SCAP could offer a promising therapeutic intervention for CRC. Additionally, this work provides the starting ground to determine the crystal structure of SCAP, by establishing a robust and stable recombinant protein for future crystallography efforts, and small molecule screening, which are critical for a future drug discovery effort.

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