Therapeutic modulation of nemo-like kinase in primary and acquired endocrine-resistant breast cancer

Purpose: To assess if NLK modulates ER target gene expression, we performed transcriptome sequencing following NLK inhibition or tamoxifen treatment in the BT483 or T47D-TamR cells under estrogen-deprived condition. Methods: BT483 and T47D-TamR cells were treated with NLK-specific siRNAs, or VX-702, or Tamoxifen under estrogen-deprived condition to examine the expression profile changes of ER target genes. NovaSeq 6000 S2 Reagent Kit (Illumina) was used for paired-end reads (2×150 bp) sequencing reactions. The expression changes of ER target genes following NLK inhibition are compared to their changes following tamoxifen treatment. Results: Our results show that the ER target gene expression changes following NLK silencing or VX-702 treatment correlate with their expression changes following tamoxifen treatment. For RNA sequencing, total RNAs from the BT483 and T47D-TamR cells treated as below were extracted using the standard procedure of RNAzol (Molecular Research Center). Following estrogen deprived (ED) condition for 48hours (phenol red-free medium with 5% charcoal-dextran-stripped FBS), BT483 and T47D-TamR cells are seeded and treated with siCtrl +/- 0.5uM Tamoxifen, siNLK#1, and siNLK#2 for 72 hours. For siRNA treatment, the cells were seeded following 48h ED and reverse transfected with the respective siRNA (final concentration 10 nM). T47D-TamR cells are also treated with vehicle, 0.5uM Tamoxifen, or 0.5uM VX-702 following ED for 48hours. The NovaSeq 6000 library for DNA sequencing was prepared using TruSeq Stranded mRNA Library Prep Kit (Illumina) following the protocol provided by the manufacturer. The final libraries were normalized by quantification with LightCycler 480 II (Roche Applied Science, Indianapolis, IN, USA) and quantification with Bioanalyzer (Agilent, Palo Alto, CA, USA). Final loading concentration was adjusted to 10 pM following the NovaSeq 6000 loading protocol and NovaSeq 6000 S2 Reagent Kit (Illumina) was used for paired-end reads (2×150 bp) sequencing reactions.