Panoply™ Human CDK4 Over-expressing Stable Cell Line

CDK4/6 inhibitors have become a standard treatment option for estrogen receptor-positive (ER+) breast cancer patients; therefore, elucidating the mechanisms of resistance has become a pressing issue. Here, researchers found that elevated CDK6 expression is a key determinant of acquired resistance to palbociclib in ER+ breast cancer cells. During palbociclib treatment, CDK6 expression is crucial for cell survival. The elevated CDK6 expression observed in resistant cells is dependent on miR-432-5p expression-mediated inhibition of the TGF-β pathway. Exosomal miR-432-5p expression mediates the transfer of the resistance phenotype between adjacent cell populations. Levels of miR-432-5p are higher in primary breast cancers resistant to CDK4/6 inhibitors compared to those sensitive to CDK4/6 inhibitors. Analysis of pre-treatment and post-progression biopsy samples from a parotid gland cancer patient treated effectively with ribociclib further confirmed these data and demonstrated the clinical relevance of this mechanism. Finally, both in vitro and in vivo studies showed that the CDK4/6 inhibitor resistance phenotype can be reversed by extending the drug-free interval.

Non-targeting (NT) short hairpin RNA (shRNA) had no effect on the cell cycle distribution of drug-resistant cells (R100–R500). shRNA-mediated CDK6 knockdown had no effect on the cell cycle distribution of parental cells (Figure 1A), but restored sensitivity in all drug-resistant cells (cultured in the presence of palbociclib), as evidenced by a significant increase in the proportion of cells in the G1 phase (Figure 1D). In contrast, CDK4 knockdown failed to restore sensitivity in drug-resistant cells. Treatment of parental T47D cells with 100 or 300 nM palbociclib resulted in sustained G1 phase arrest, characterized by a lower proportion of cells in the S and G2 phases of the cell cycle, and this arrest persisted for 14 days. CDK4-overexpressing cells showed a return to normal cell cycle distribution after 14 days of continuous treatment with 100 nM palbociclib, but remained arrested in the presence of 300 nM palbociclib. In contrast, CDK6-overexpressing cells developed resistance to both 100 and 300 nM palbociclib within 10 days (Figure 1E). Furthermore, overexpression of CDK6 in parental T47D or MCF7 cells not only significantly increased the half-maximal inhibitory concentration (GI50) of palbociclib, but also increased the GI50 of ribociclib and abemaciclib in growth inhibition experiments.

Figure 1. Resistance to CDK4/6 inhibition is mediated by high CDK6 expression. (Cornell L, et al., 2019)