Abstract P2-05-04: Low molecular weight cyclin E facilitates replication stress tolerance in breast cancer development

Purpose: Low Molecular Weight Cyclin E (LMW-E) are the oncogenic forms of cyclin E and were originally discovered in breast cancer (BC). LMW-E are generated from N-terminal cleavage of the 50 KDa, full-length cyclin E1 (FL-cycE). Our laboratory has established that LMW-E expression (i) correlates with poorer survival in BC patients; (ii) increases in frequency as BC progress from ductal carcinoma in situ to invasive ductal carcinoma; (iii) drives spontaneous and metastatic BC in murine transgenic models; (iii) causes human mammary epithelial cells (hMECs) to transform in vitro and form tumors in vivo. Elucidating the critical oncogenic functions and essential downstream factors of LMW-E is a current void in the field and may have important therapeutic implications. Experimental Design: Immunohistochemistry (IHC) analysis were performed using tumor specimens (n=725) from breast cancer patients diagnosed with Stage 1 or 2 disease to determine the level of LMW-E. Micro-dissected tumor DNA from these tissues were analyzed for copy number variations (CNVs) using Molecular Inversion Probe (MIP) based arrays. CCNE1(encoding Cyclin E1) knock-out 76NE6 and 76NF2V hMEC lines were generated by CRISPR, followed by transfection of lentivirus vector expressing doxycycline inducible EGFP fused LMW-E or FL-cycE. Time-lapse live cell imaging was performed to monitor the cell growth and phenotypes after induced expression of LWM-E or FL-cycE. DNA replication and replication stress were examined by BrdU labeling and immunofluorescent (IF) assays using antibodies against BrdU and Replication Protein A (RPA). IF for gamma-H2AX and western blotting for phosphor-RPA32, ATR-CHK1 and ATM-CHK2 pathways were performed to determine the DNA damage and responses. Results: Analysis of CNVs for the 725 tumors stratified by the cyclin E phenotypes reveal that the frequency of the CNVs (gains & losses) were the most significant in patients whose tumors expressed LMW-E and were predictive of poor prognosis independent of BC subtypes. DNA damage signals including gamma-H2AX foci and phosphor-CHK1 were similarly enhanced in hMECs induced for FL-cycE or LMW-E over-expression. Different effects of FL-cycE and LMW-E on DNA replication and replication stress were observed. Induced FL-cycE overexpression attenuated increased RPA foci and RPA phosphorylation, inhibits DNA replication and cell growth, and ultimately lead to cell death. LMW-E overexpression facilitated DNA replication and cell proliferation with damaged DNA, resulting in multi-nuclei and micro-nuclei formation in daughter cells. Conclusion: LMW-E expression positively correlates with genomic instability in BC samples. LMW-E facilitates hMECs to by-pass replication stress induced tumor barrier and survive with damaged DNA, resulting in abnormal nuclei formation, an oncogenic phenotype. Citation Format: Mi Li, Kwang Huei Low, Tuyen Bui, Kelly K Hunt, Khandan Keyomarsi. Low molecular weight cyclin E facilitates replication stress tolerance in breast cancer development [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-05-04.