Identification of genes responsible for RelA-dependent proliferation arrest in HMEC conditionally expressing RelA

Immuno-histochemistry for RelA in breast tumors revealed a range of staining intensity and negative correlation between RelA levels and proliferation-index in estrogen receptor-positive tumors. Conditional expression of RelA arrested proliferation in primary mammary and fallopian tube epithelial cells. RelA dependent CDK4 downregulation was responsible for activating the G1/S checkpoint and cell cycle arrest. RelA target genes, including Interferon Response Factors (IRF), were up-regulated in the arrested cells. Among the IRFs, IRF1 expression correlates with RelA expression. Suppressing IRF1 restored CDK4 levels and rescued RelA-dependent proliferation arrest analogous to abrogating the G1/S checkpoint or restoring CDK4 levels. Apart from cyclins, regulation of CDK4 by the RelA-IRF1 transcriptional network controls proliferation of breast tumors and predicts sensitivity to a CDK4/6 inhibitor. Stable HMEC harboring the two plasmid Tetracycline inducible system (Clontech). HRA cells are stable pooled clones of HMEC cells that express RelA in a Doxycycline (Dox) dependent manner. Triplicate samples for gene expression analysis were generated by plating HRA cells at low density and harvested after subjecting them to four conditions 24 hours post plating (time 0 of the experiment): i) Uninduced (No Dox sample), harvested on successive days i.e. 24, 48 and 72 hours after time 0; ii) Induced with Dox for 24 hours (24+ sample) at time 0, 24 and 48, and harvested 24 hours later; iii) Induced with Dox at time 0 and harvested 72 hours later (72+ sample) and iv) Induced with Dox at time 0, Dox withdrawn at time 24 and harvested at 72 hours. Dox+/Dox- media in all plates were changed every 24 hours as required. See supplementary Figure S5 for a schematic representation of the protocol.