MB Knockout Cell Line (HEK293)

The five lysyl-oxidase genes share similar enzymatic activities and contribute to tumor progression. We have knocked out the five lysyl-oxidase genes in MDA-MB-231 breast cancer cells using CRISPR/Cas9 in order to identify genes that are regulated by LOX but not by other lysyl-oxidases and in order to study such genes in more mechanistic detail in the future. Re-expression of the full-length cDNA encoding LOX identified four genes whose expression was downregulated in the knock-out cells and rescued following LOX re-expression but not re-expression of other lysyl-oxidases. These were the AGR2, STOX2, DNAJB11 and DNAJC3 genes. AGR2 and STOX2 were previously identified as promoters of tumor progression. In addition, we identified several genes that were not downregulated in the knock-out cells but were strongly upregulated following LOX or LOXL3 re-expression. Some of these, such as the DERL3 gene, also promote tumor progression. There was very little proteolytic processing of the re-expressed LOX pro-enzyme in the MDA-MB-231 cells, while in the HEK293 cells, the LOX pro-enzyme was efficiently cleaved. We introduced point mutations into the known BMP-1 and ADAMTS2/14 cleavage sites of LOX. The BMP-1 mutant was secreted but not cleaved, while the LOX double mutant dmutLOX was not cleaved or secreted. However, even in the presence of the irreversible LOX inhibitor β-aminoproprionitrile (BAPN), these point-mutated LOX variants induced the expression of these genes, suggesting that the LOX pro-enzyme has hitherto unrecognized biological functions.

BACKGROUND:The local anesthetic lidocaine suppresses some cancer cell lines but the mechanism is unclear. The melastatin-like transient receptor potential 7 (TRPM7) ion channel is aberrantly expressed in some cancers and may play a role in the disease. Hence, we suggested that lidocaine affects the viability and migration of breast cancer cells by regulating TRPM7. METHODS:We measured the effects of lidocaine on TRPM7 function in HEK293 with exogenous TRPM7 expression (HEK-M7) using whole-cell patch-clamp and fura-2AM-based quench assay. We measured the effect of lidocaine on TRPM7 function, cell viability, and migration in TRPM7 expressing human breast cancer cell lines using fura-2AM-based quench, MTT, and wound-healing assays respectively. We compared cell viability and migration of wild type HEK293 cells (WT-HEK) with HEK-M7 and wild type MDA-MB-231 (WT-231) with TRPM7 knockout MDA-MB-231 (KO-231). RESULTS:Lidocaine (1-3 mM) inhibited the viability and migration of all of these breast cancer cell lines. Functional evidence for TRPM7 was confirmed in the MDA-MB-231, AU565, T47D, and MDA-MB-468 cell lines where lidocaine at 0.3-3 mM suppressed the TRPM7 function. Lidocaine preferentially suppressed viability and migration of HEK-M7 over WT-HEK and WT-231 over KO-231. CONCLUSIONS:Lidocaine differentially reduced the viability and migration of human breast cancer cell lines tested. TRPM7 is one of the potential targets for the effects of lidocaine on viability and migration in MDA-MB-231, AU565, T47D, and MDA-MB-468.

The human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase receptor and tumor-associated antigen abnormally expressed in various types of cancer, including breast, ovarian, and gastric cancer. HER2 overexpression is highly correlated with increased tumor aggressiveness, poorer prognosis, and shorter overall survival. Consequently, multiple HER2-targeted therapies have been developed and approved; however, only a subset of patients benefit from these treatments, and relapses are common. More potent and durable HER2-targeted therapies are desperately needed for patients with HER2-positive cancers. In this study, we developed a lipid nanoparticle (LNP)-based therapy formulated with mRNA encoding a novel HER2-CD3-Fc bispecific antibody (bsAb) for HER2-positive cancers. The LNPs efficiently transfected various types of cells, such as HEK293S, SKOV-3, and A1847, leading to robust and sustained secretion of the HER2-CD3-Fc bsAb with high binding affinity to both HER2 and CD3. The bsAb induced potent T-cell-directed cytotoxicity, along with secretion of IFN-λ, TNF-α, and granzyme B, against various types of HER2-positive tumor cells in vitro, including A549, NCI-H460, SKOV-3, A1847, SKBR3, and MDA-MB-231. The bsAb-mediated antitumor effect is highly specific and strictly dependent on its binding to HER2, as evidenced by the gained resistance of A549 and A1847 knockout cells and the acquired sensitivity of mouse 4T1 cells overexpressing the human HER2 extracellular domain (ECD) or epitope-containing subdomain IV to the bsAb-induced T cell cytotoxicity. The bsAb also relies on its binding to CD3 for T-cell recruitment, as ablation of CD3 binding abolished the bsAb's ability to elicit antitumor activity. Importantly, intratumoral injection of the HER2-CD3-Fc mRNA-LNPs triggers a strong antitumor response and completely blocks HER2-positive tumor growth in a mouse xenograft model of human ovarian cancer. These results indicate that the novel HER2-CD3-Fc mRNA-LNP-based therapy has the potential to effectively treat HER2-positive cancer.