Abstract P4-03-07: Combined genome-scale CRISPR-Cas9 knockout screening with transcriptome sequencing to identify paclitaxel related drivers in triple negative breast cancer
Authors: Lian B , Xin H , Zhimin S 
Published: February 2018

Background. Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancers, for which the only standard therapeutics is chemotherapy containing Taxol. However, quite a number of TNBC patients cannot get the expected drug response after paclitaxel treatment and the resistant mechanism has not been clear yet. Other than the traditional “genotype-to-phenotype” means, the high-throughput functional screening, such as CRISPR-CAS9 library, selects genes with the phenotype of interest. Here, we combine the novel screening model with the drug-resistant genotype to explore the decisive role in paclitaxel effect.

Methods. Breast cancer cell line MDA-MB-231(231WT) was treated by paclitaxel from 1ug/ml to 5ug/ml to establish a paclitaxel-resistant cell type (231PTX) for transcriptome sequencing. Genome-scale CRISPR-Cas9 sgRNA library was made into lentivirus to affect MDA-MB-231 cells expressed Cas9 protein (231cas9). Then 231cas9-sgRNA was treated by low dose of paclitaxel for 14 days and was read by next generation sequencing. RNA sequencing data was processed to TPM values and sgRNA data to gene ranking and p value. The threshold of “231PTX TPM/231WT TPM” was above 2 or below 1/2 and the gene p value was smaller than 0.05. Biological technology applied in this study includes western blot (WB), immunofluorescence (IF), real time PCR and cell proliferation assay. In vivo, 20 balb/c mouse were injected MDA-MB-231 in situ for tumor formation and were treated with paclitaxel/normal saline for six times.

Results. Crosstalk between these two sequencing data had result of 124 genes related to paclitaxel resistance (fold change&gt; 2 and p value&lt;0.05 compared Day 14 treated group to Day 14 untreated group) and 18 genes related to paclitaxel sensitivity (fold change&lt; 1/2 and p value&lt;0.05 compared Day 14 untreated group to Day 14 treated group). Considering clinical prognosis and gene information, six paclitaxel resistant candidates and four paclitaxel sensitive candidates were chosen for further research. Eight (STRA6, BIRC3, MTUS1, HDAC9, ADAM28, S1PR5, TNNC1, ZKSCAN7) of ten candidates displayed consistent phenotypes with sequencing results including mRNA expression and the cellular proliferation in paclitaxel treatment. HDAC9 is a histone deacetylation gene that is likely to be a paclitaxel resistant gene. Knockout HDAC9 (231H9 KO) contributed to nearly 2-fold decrease IC50 value (1.7nM versus 3.7nM, p value&lt;0.01). Confocal microscopy observed the formation of multiple spindle foci in the paclitaxel treated 231H9 KO cells. After treatment with paclitaxel, the mark of polymerized tubulin, acetylation tubulin and the mark of cell cycle G2/M, cyclin B1 were notably increased when HDAC9 knockout in both MD-MB1-231 and BT-100 cell lines. In vivo assays found that HDAC9 knockout induced the declined tumorigenesis and more sensitive breast tumors to paclitaxel.

Conclusions.Combined Genome-scale CRISPR-Cas9 knockout screening with transcriptome sequencing is efficient to investigate potent drug targets. In vitro assays suggest that HDAC9 is conductive to paclitaxel resistance in TNBC cells. In vivo results imply inhibition HDAC9 may beneficial to paclitaxel therapeutic response.

Citation Format: Lian B, Xin H, Zhimin S. Combined genome-scale CRISPR-Cas9 knockout screening with transcriptome sequencing to identify paclitaxel related drivers in triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-03-07.