“Simultaneous knockdown of CD320 and LRP2 receptors is selectively toxic to cancer cells but not normal cells”

At the American Association of Cancer Research (AACR) Annual Meeting 2021, April-May, 2021

David J. Elzi, William Bauta, Shao-Chiang Lai, Trisha Das, Shweta Mogare, and Vivienne I. Rebel
Designing effective therapies with selective toxicity in cancer cells is a priority of cancer research. Porphyrins are known to selectively accumulate in cancerous tissue, a feature that has been exploited in cancer diagnostics and therapeutics. The porphyrin TCPP is used in the CyPath® Lung test for the diagnosis of lung cancer. During efforts to elucidate the mechanism by which TCPP selectively accumulates in cancer cells, we discovered that simultaneous knockdown of two LDLR-family receptors, CD320 and LRP2, was selectively cytotoxic to cancer cells and not to normal cells.

CD320 is a receptor that internalizes the complex of cobalamin (Vitamin B12)/transcobalamin II). LRP2 is a receptor strongly expressed in the kidney which also internalizes the cobalamin/transcobalamin II complex, in addition to more than 50 other ligands. Cobalamin is the catalytic cofactor for the enzymes methionine synthase and methylmalonyl CoA mutase, which play direct and indirect roles in folate metabolism, nucleotide biosynthesis, DNA methylation, and metabolism of fatty acids and amino acids, which suggests that cobalamin deprivation might be particularly deleterious to metabolically active cancer cells. We designed siRNAs to effectively silence CD320 and LRP2 protein expression and transfected these siRNAs into normal and cancer cell lines using commercial transfection reagents.

Effective gene knockdown (up to 90%) was confirmed by western blotting. Simultaneous siRNA knockdown of CD320 and LRP2 in normal fibroblasts did not affect their cell proliferation, as measured by the Cell Titer-GLO assay. However, knockdown of CD320 and LRP2 in cancer cell lines derived from diverse tissues (lung, breast, prostate, brain, and skin cancers) inhibited cell proliferation or killed the cells by up to 80%. Interestingly, in some cell lines, when either CD320 or LRP2 were silenced individually, a concurrent increase in protein expression of the other receptor was observed, suggesting that CD320 and LRP2 compensate for each other’s function; hence, silencing both receptors is required for optimal cell killing.