METHODS: By utilizing a panel of breast cancer cells and mammospheres culture as cell-based screening platforms, we performed high-throughput chemical library screens to identify agents that are effective against breast CSCs and non-CSCs. The hit molecules were paired with conventional chemotherapy to evaluate the combinatorial treatment effects on breast CSCs and non-CSCs.
RESULTS: We identified a total of 193 inhibitors that effectively targeting both breast CSCs and non-CSCs. We observed that histone deacetylase inhibitors (HDACi) synergized conventional chemotherapeutic agents (i.e., doxorubicin and cisplatin) in targeting breast CSCs and non-CSCs simultaneously. Further analyses revealed that quisinostat, a potent inhibitor for class I and II HDACs, potentiated doxorubicin-induced cytotoxicity in both breast CSCs and non-CSCs derived from the basal-like (MDA-MB-468 and HCC38), mesenchymal-like (MDA-MB-231), and luminal-like breast cancer (MCF-7). It was also observed that the basal-like breast CSCs and non-CSCs were more sensitive to the co-treatment of quisinostat with doxorubicin compared to that of the luminal-like breast cancer subtype.
CONCLUSION: In conclusion, this study demonstrates the potential of HDACi as therapeutic options, either as monotherapy or in combination with chemotherapeutics against refractory breast cancer.
METHODS: Spheroids were generated in suspension spheroidal culture. The ZNF800 mRNA, pluripotency stem cell markers and circZNF800 levels were determined by quantitative RT-PCR. CircZNF800-miRNA interactions were shown in RNA pulldown assays and the miRNA levels determined by stem-loop qRT-PCR. The effects of circZNF800 on cell proliferation were tested by EdU staining followed by flowcytometry. Expression of stem cell markers CD44/CD133, Lgr5 and SOX9 was demonstrated in immunofluorescence microscopy. To manipulate the cellular levels of circZNF800, circZNF800 over-expression was achieved via transfection of in vitro synthesized and circularized circZNF800, and knockdown attained using a CRISPR-Cas13d-circZNF800 vector system. Xenografted nude mice were used to demonstrate effects of circZNF800 over-expression and knockdown on tumor growth in vivo.
RESULTS: CircZNF800 was shown to be over-expressed in late-stage tumor tissues of CRC patients. Data showed that circZNF800 impeded expression of miR-140-3p, miR-382-5p and miR-579-3p while promoted the mRNA levels of ALK/ACVR1C, FZD3 and WNT5A targeted by the miRNAs, as supported by alignments of seed sequences between the circZNF800-miRNA, and miRNA-mRNA paired interactions. Analysis in CRC cells and biopsied tissues showed that circZNF800 positively regulated the expression of intestinal stem cell, pluripotency and cancer stem cell markers, and promoted CRC cell proliferation, spheroid and colony formation in vitro, all of which are cancer stem cell properties. In xenografted mice, circZNF800 over-expression promoted tumor growth, while circZNF800 knockdown via administration of CRISPR Cas13d-circZNF800 viral particles at the CRC tumor sites impeded tumor growth.
CONCLUSIONS: CircZNF800 is an oncogenic factor that regulate cancer stem cell properties to lead colorectal tumorigenesis, and may be used as a predictive marker for tumor progression and the CRISPR Cas13d-circZNF800 knockdown strategy for therapeutic intervention of colorectal cancer.