Geminin Overexpression Promotes Imatinib Sensitive Breast Cancer: A Novel Treatment Approach for Aggressive Breast Cancers, Including a Subset of Triple Negative

Including a Subset of Triple Negative. PLoS ONE 9(4): e95663. doi:10.1371/journal.pone.0095663 Geminin Overexpression Promotes Imatinib Sensitive Breast Cancer: A Novel Treatment Approach for Aggressive Breast Cancers, Including a Subset of Triple Negative Zannel Blanchard 0 Nicole Mullins 0 Pavani Ellipeddi 0 Janice M. Lage 0 Shawn McKinney 0 Rana El-Etriby 0 Xu Zhang 0 Raphael Isokpehi 0 Brenda Hernandez 0 Wael M. ElShamy 0 Todd W. Miller, Dartmouth, United States of America 0 1 Cancer Institute, University of Mississippi Medical Center, Jackson, Mississippi, United States of America, 2 Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi, United States of America, 3 Department of Pathology, University of Mississippi Medical Center, Jackson, Mississippi, United States of America, 4 Department of Surgery, University of Mississippi Medical Center, Jackson, Mississippi, United States of America, 5 Center of Biostatistics and Bioinformatics, University of Mississippi Medical Center, Jackson, Mississippi, United States of America, 6 Center for Bioinformatics & Computational Biology, Department of Biology, Jackson State University , Jackson , Mississippi, United States of America, 7 Cancer Research Center of Hawaii, University of Hawaii , Honolulu, Hawaii , United States of America Breast cancer is the second leading cause of cancer-related deaths in women. Triple negative breast cancer (TNBC) is an aggressive subtype that affects 10-25% mostly African American women. TNBC has the poorest prognosis of all subtypes with rapid progression leading to mortality in younger patients. So far, there is no targeted treatment for TNBC. To that end, here we show that c-Abl is one of several tyrosine kinases that phosphorylate and activate geminin's ability to promote TNBC. Analysis of .800 breast tumor samples showed that geminin is overexpressed in ,50% of all tumors. Although c-Abl is overexpressed in ,90% of all tumors, it is only nuclear in geminin overexpressing tumors. In geminin-negative tumors, c-Abl is only cytoplasmic. Inhibiting c-Abl expression or activity (using imatinib or nilotinib) prevented geminin Y150 phosphorylation, inactivated the protein, and most importantly converted overexpressed geminin from an oncogene to an apoptosis inducer. In pre-clinical orthotopic breast tumor models, gemininoverexpressing cells developed aneuploid and invasive tumors, which were suppressed when c-Abl expression was blocked. Moreover, established geminin overexpressing orthotopic tumors regressed when treated with imatinib or nilotinib. Our studies support imatinib/nilotonib as a novel treatment option for patients with aggressive breast cancer (including a subset of TNBCs)-overexpressing geminin and nuclear c-Abl. - Funding: This research was supported by a grant from the American Cancer Society, Grant # RSG-09-275-01. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Licensing at origin of replications (ORI) starts by sequential binding of component of the pre-replication complex (pre-RC); origin recognition complex (ORC), Cdc6 and Cdt1 [1,2] followed by binding of the rate limiting complex; the mini-chromosome maintenance complex (MCM2-7) [1,2]. Geminin, a coiled-coil protein without sequence homology to any known protein [3] inhibits re-replication by binding to Cdt1 at ORI and preventing MCM binding, thus, maintaining chromosomal integrity and number [1,2]. However, other critical roles for geminin have recently been identified. For instance, in Drosophila, geminin regulates neural cell fate determination, and in mouse it regulates cellular differentiation by binding to and antagonizing Six3 or Hox transcriptional activity [47]. More recently, we showed that geminin is recruited to chromatin (also centrosomes, centromeres and midbody) from the nuclear soluble fraction at the end of S-phase after being tyrosine phosphorylated to promote proper cytokinesis and M-toG1 transition [810]. Geminin accomplishes that by binding to and activating, at least two important cytokinesis inducers; namely topoisomerase II alpha (TopoIIa) [9] and Aurora B kinase (AurB) [10]. However, when overexpressed geminin prematurely inhibits chromosomal decatenation by inactivating TopoIIa [9] and condensation/segregation by inactivating AurB, and inhibiting histone H3(S10) phosphorylation (p-H3S10) [10,11], which triggers aneuploidy. Interestingly, previously we showed that overexpression of any single tyrosine mutant geminin (human geminin carries 3 tyrosine residues, at positions 98, 111 and 150) has no effect on H3S10 phosphorylation, chromosome decatenation/condensation/segregation and triggers apoptosis instead of aneuploidy [9,10]. Suggesting that geminin is a genuine oncogene that promotes genomic instability when overexpressed as a wild-type protein. In keeping with that, geminin is overexpressed in many tumor types [1216], and to our knowledge the protein is wild type in these tumors. In fact, we recently analyzed a cohort of 150 DNA samples freshly isolated from patients breast tumors and found that geminin gene carries no mutation or any alterations that can affect the protein in all these tumors (ElShamy WM and Iglehart D, unpublished data). The proto-oncogene c-Abl is a tightly regulated, ubiquitously expressed multifunctional non-receptor protein tyrosine kinase [1721]. c-Abl can be localized to the plasma membrane, cytoplasm and nucleus and affects a variety of cellular functions and activities. For example, cytoplasmic c-Abl plays an important role in cell proliferation, differentiation, and migration [22,23], whereas nuclear c-Abl induces apoptosis, DNA damage repair [22,23] or chromatin dynamic by phosphorylating heterochromatic histones [24]. Unlike chronic myelogenous leukemia [CML] [23], in solid tumors, such as glioblastoma, melanoma, non-small-cell lung cancer, breast and gastric carcinomas there are no c-Abl translocations. Instead, in these solid tumors, c-Abl is overexpressed [2427]. Imatinib mesylate (aka Gleevec or STI571) [28], is a small molecule inhibitor that targets the ATP-binding site in c-Abl kinase domain and is successful in treating CML patients [29,30], as well as gastrointestinal stromal tumors (GIST) expressing mutant c-KIT, or overexpressing a- or b platelet-derived growth factor receptors (PDGFRa or b) [31]. Nilotinib [aka Tasigna] is a novel tyrosine kinase inhibitor that inhibits BCR-ABL [3035], c-KIT and PDGFRa approved to treat CML or GIST patients, especially those showing imatinibresistance or -intolerance [3033]. Here, we expand our previous data and show that c-Abl binds directly or indirectly to geminin and phosphorylates Y150 in G2/ M/early G1 cells. Inhibiting Y150 phosphorylation by suppressing c-Abl expression or activity destabilizes endogenous (...truncated)