Abstract P5-08-02: Inhibition of CDK4/6 induces senescence and autophagy in ER positive breast cancers
Authors: Vijayaraghavan Smruthi , Keyomarsi Khandan 
Published: May 2015

Abstract
Deregulation of the cell cycle machinery is a hallmark of breast cancer, facilitating aberrant proliferation that fuels tumorigenesis and disease progression. This makes the cell cycle, particularly the cyclin dependent kinases (CDKs) an ideal choice for drug targeting in these tumors. Palbociclib or PD0332991, a potent CDK4/6 inhibitor is a known anti-proliferative agent that induces G1 arrest and prevents tumor growth in several cancers including ER+ breast cancer. This drug has shown tremendous success in Phase II clinical trials in ER+ breast cancers and is presently undergoing phase III trials in combination with letrazole. However, little is know about its precise mechanism of action and modes of resistance in ER+ breast cancers. This is critical in understanding the biology of treatment response, drug resistance and combination strategies, and this project aims at addressing these gaps in knowledge.
We have used a series of ER+ breast cancer cell lines to examine the mechanism of action of Palbociclib and identify the nodes that could mediate resistance to this agent. Our results have revealed that continuous treatment of ER+ breast cancer cells with Palbociclib results in a G1 arrest with concomitant downregulation of pRb. Treated cells undergo senescence and autophagy, but not apoptosis. Further, we have shown that the induction of quiescence or senescence and autophagy occurs in a time and dose dependent manner.
Since this agent is known to be a specific CDK4/6 inhibitor, we next downregulated these kinases in our model system and subjected them to Palbociclib treatment. Interestingly, downregulation of CDK4 or CDK6 did not significantly alter the sensitivity of these cells to the anti-tumor effects of Palbociclib. Next we asked if depletion of Rb can render the cells resistant to Palbociclib. Results revealed that the complete depletion of Rb was sufficient to reduce the sensitivity of ER+ cells to Palbociclib by 4-6 fold. However, the very steep pattern of the growth response curves suggest that in the absence of Rb, Palbociclib can effectively inhibit another target, albeit, at a higher drug concentration.
Lastly, to interrogate if deregulation of the G1/S checkpoint can render cells more resistant to this inhibitor, we examined if overexpression of the low molecular weight isoforms of Cyclin-E (LMW-E), which by themselves are sufficient to induce an oncogenic phenotype through constitutive phosphorylation of Rb, can mediate resistance in these cells. These results, which were similar to those from the Rb knockout studies, revealed that LMW-E overexpression can reduce the sensitivity of ER+ breast cancer cells to Palbociclib by 5-7 fold.
Collectively, our studies suggest that inhibition of G1/S transition is not the sole mode of action of Palbociclib and that there is another unidentified target, which is specifically inhibited by this agent, when the G1/S check point is compromised. Since the deregulation of the G1/S checkpoint is likely to occur clinically in patients treated with Palbociclib, identification of this second target will be instrumental in efforts to overcome resistance and identify biomarkers of sensitivity to this agent in breast cancer patients.
Citation Format: Smruthi Vijayaraghavan, Khandan Keyomarsi. Inhibition of CDK4/6 induces senescence and autophagy in ER positive breast cancers [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-08-02.