Inhibiting p90 ribosomal S6 kinase (RSK)/Y-box binding protein-1 (YB-1) signaling is a novel targeted therapeutic strategy with the ability to overcome drug resistance in triple-negative breast cancer

Despite advances in treating breast cancer, disease recurrence rates remain high and secondary tumors are often refractory to chemotherapy. Currently, the treatment for triple-negative breast cancer (TNBC) relies upon conventional chemotherapeutics as no targeted therapies are available. Although these tumors initially respond well, they paradoxically have the highest relapse rates. Y-box binding protein-1 (YB-1) is an oncogenic transcription/translation factor abundantly expressed in TNBC (~70% of patients) and associated with disease relapse. It is activated predominantly by phosphorylation via p90 ribosomal S6 kinase (RSK). Once activated YB-1 up-regulates the tumor-initiating cell (TIC) marker, CD44 and promotes drug resistance. These data suggest that blocking YB-1’s activation via RSK inhibition may suppress growth and attenuate the development of chemoresistance in TNBC. Through an unbiased, functional viability screen comparing breast cancer subtypes, we identified RSK2 as a novel target for TNBC. Pharmacological or siRNA inhibition of RSK2 blocks activation of YB-1, which subsequently decreases growth in TNBC cell lines and delays tumor initiation in immunocompromised mice. Contrary to most conventional chemotherapies, inhibiting RSK/YB-1 signaling eliminates the CD44⁺/CD24‾ cell fraction rather than enriching for it. In an effort to identify novel RSK inhibitors, we screened “off-patent” compounds and identified the flavonoid, luteolin, as a RSK inhibitor. We validated that luteolin inhibits RSK in cell-free assays and further demonstrated it blocks the RSK/YB-1/Notch4 signaling pathway. Luteolin phenotypically mirrored the effects of established RSK inhibitor, BI-D1870, and suppressed growth in TNBC (including CD44⁺/CD24‾-sorted cells) providing further support for the use of RSK inhibitors to treat this subtype. Finally, we demonstrate that cells that survive standard-of-care chemotherapeutics (paclitaxel and epirubicin) exhibit elevated RSK/YB-1 signaling. Inhibiting this pathway sensitizes TNBC to chemotherapy and reduces the residual cell burden. Importantly, RSK inhibition also demonstrates efficacy against a multidrug resistant cell line and primary, drug-refractory TNBC. When taken together, our data identify RSK as a promising target for the treatment of TNBC. RSK inhibition has the unique ability to eliminate CD44⁺/CD24‾cells and overcome broad-spectrum chemoresistance by blocking activation of YB-1 and as such holds potential to reduce relapse in this aggressive subtype.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat