Abstract 1775: Scope of cell cycle inhibition in HER2+ breast cancer
Authors: Koirala Nischal , Aske Jennifer , Lin Xiaoqian , Dey Nandini , De Pradip 
Published: June 2022

Background: Proliferative signaling of the HER2- and ER-pathways converge and propagate via cyclin D and CDK4/6, leading to uncontrolled cell cycle progression and resistance to anti- HER2- and ER-targeted therapies.

Purpose: We hypothesize that co-targeting HER2 and CDK4/6 will counter the uncontrolled cell cycle progression, initiate cell cycle arrest, and induce apoptosis in Rb-proficient HER2-amplified breast cancer (BC) cells.

Methods: HER2+ BC cell lines (Rb-WT): BT474 (HR-positive), SKBR3 (HR-negative), and MDA-MB453 (HR-negative, PIK3CA mutated [H1047R]) were used for the study. We tested real-time proliferation, 3D-ON TOP colony formation, cell cycle inhibition, changes in mitochondrial membrane potential, and apoptosis following drug treatment as a single-agent (Ribociclib [R]/Trastuzumab [T]) or combination (R+T). Basal-level mRNA transcripts for CDK4 and CCND1 and changes in Ki67 following treatment were quantified with RT-qPCR. Cyclin D expression was evaluated in HER2+ tissue microarrays (TMA) by immunohistochemistry. The mechanistic evaluation of the critical downstream signaling nodes was performed by Western blot.

Results: Basal-level mRNA transcript expression for CDK4 and CCND1 were substantially higher (relative to GAPDH) in BT474 and MDA-MB453 whereas, moderate-low expression was obtained in SKBR3. Ki67 transcriptional level was attenuated with R, and further attenuation was observed with dual agent treatment except for SKBR3, where a similar trend was conserved but of reduced intensity. Immunohistochemistry showed substantial cyclin D expression in HER2+ TMA signifying opportunities for CDK4/6 inhibitors. BT474 and MDA-MB453 had similar anti-proliferative responses to the combinatorial treatment, whereas SKBR3 showed resistance to growth inhibition. Similar to real-time proliferation, 3D-ON TOP colony formation showed a reduction in proliferative activities with the combination. The cytostatic response was primarily induced by R and did not differ significantly from the combination of treatment. BT474 and MDA-MB453 had &gt;90% G1 phase arrest, while SKBR3 had &gt;70% G1 phase arrest. Changes in the mitochondrial membrane potential and apoptosis were pronounced with the combination in BT474 and MDA-MB453, whereas SKBR3 did not show any significant response. Western blot showed a reduction in Rb phosphorylation (pRb Ser 780 & Ser 807/811) in all cell lines with R treatment, and further attenuation was observed with combinatorial treatment. Changes in the phospho-Akt were more evident with T and R+T combination in BT474 and MDA-MB453 over HER2-amplified/HR-negative SKBR3 cells.

Conclusions: The R+T combination counteracted the proliferative signaling in the HR-positive conditions and sensitized neoplastic cells to elicit an enhanced anti-tumorigenic response irrespective of PIK3CA hotspot mutation.

Citation Format: Nischal Koirala, Jennifer Aske, Xiaoqian Lin, Nandini Dey, Pradip De. Scope of cell cycle inhibition in HER2+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1775.