CDK4/6 inhibitors have been widely used around the world for advanced estrogen receptor positive breast cancer patients in the past several years. However, the benefit offered by CDK4/6 inhibitors is individually different, therefore it is imperative need to identify the biomarker and/or monitoring marker.
We established CDK6 over expression cell lines (MCF7-C6) from MCF-7 by stably transfected CDK6 expression vector. We also established Ribociclib-resistant cell lines (RIBR) after long-term culture under the condition of sufficient doses of Ribociclib from estrogen deprivation-resistant cell lines (EDR) which established from MCF-7 cultured with steroid depleted medium as aromatase inhibitor resistance models. We further established RIBR(-R) cell lines from RIBR by long-term cultured without Ribociclib.
First, we assessed IC50 of Ribociclib in several cell lines. Luminal cell lines exhibited lower Ribociclib IC50 than non-luminal cell lines. Immunoblot analysis of Luminal cell lines showed extremely lower levels of CDK6 compared with others. Then we established MCF7-C6. MCF7-C6 reduced Ribociclib sensitivity equivalent to non-luminal cell lines. Next, we established RIBR to understand the characteristics in acquired resistance. We confirmed RIBR showed higher Ribociclib IC50 than EDR. Surprisingly the expression levels of CDK6 were not reduced in RIBR, indicating that the mechanism of resistance to Ribociclib would be different between MCF-7-C6 and RIBR. Then, we explored the efficacy of other CDK4/6 inhibitors on MCF7-C6 and RIBR. MCF7-C6 and RIBR cells showed cross-resistant not only to Palbociclib but Abemaciclib. The expression levels of p21 were reduced in both cell lines though the mechanisms of resistance to CDK4/6 inhibitors were different. Finally, we established RIBR(-R). RIBR(-R) showed more sensitive to Ribociclib than RIBR. In addition, p21 levels of RIBR(-R) were restored to the same degree as EDR.
Ribociclib sensitivity was proportional to the expression levels of p21, suggesting that p21 levels might be the monitoring marker for the CDK4/6 inhibitors resistance in breast cancer.
Clinical trial identification
Legal entity responsible for the study
Grant-in-aid for scientific research from the Ministry of Education, Science, Sports, Science and Technology of Japan; Grant-in-aid for cancer research from the Ministry of Health, Labour and Welfare of Japan; Program for Promotion of Fundamental Studies in Health Science of the National Institute of Biomedical Innovation (NIBIO); Grant from the Smoking Research Foundation.
S-I. Hayashi: Research grants: Novartis Pharma K.K, AstraZeneca K.K. All other authors have declared no conflicts of interest.