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CEP55 (also known as c10orf3 and FLJ10540) is a highly coiled coil protein of 55 kDa and was initially identified by our laboratory, as a pivotal component of cell abscission, the final stage of cytokinesis in somatic cells. Various independent studies over the past decade have highlighted the critical role of CEP55 in recruiting ESCRT machinery at the midbody and facilitating equal segregation of cytoplasmic contents between the daughter cells. CEP55 is regulated in a phosphorylation-dependent manner by CDK1, ERK2 and PLK1 for timely recruitment to midbody. Conversely, in germ cells, CEP55 in partnership with TEX14 has also been shown to be an integral component of the intercellular bridge prior to meiosis. Notably, CEP55 overexpression has been linked to tumorigenesis of various major organs including those of the breast, lung, colon and liver. Its expression has been significantly correlated with aggressiveness, tumor stage, metastasis and poor prognosis across multiple tumor types. CEP55 binds to and stabilizes the catalytic subunit, p110 of PIK3CA and increases AKT signaling. Independently, studies have shown the interplay between CEP55 and FOXM1 in the cancer context which is negatively regulated by TP53, although the mechanisms underlying this theory are not well defined. Despite significant in vitro studies, the role of CEP55 in development and promoting tumorigenesis remain incompletely understood. In order to decipher the mechanism by which CEP55 promotes tumorigenesis in vivo, we developed a novel “knock-in” transgenic mouse model that ubiquitously overexpresses Cep55 (Cep55Tg/Tg). Unexpectedly, I found that Cep55Tg/Tg male mice were sterile and suffered severe and progressive defects in spermatogenesis due to spermatogonial stem cell (SSC) dysfunction. Thus, in the first research chapter, we have characterized this male-specific phenotype and shown that Cep55 overexpression results in hyper-activation of PI3K/Akt signaling in testis. In line with this, we observed increased phosphorylation of Foxo1, and suppression of its nuclear retention. Independently, I observed that Cep55 amplification favored upregulation Plzf, Ret and Gfra1, factors required for SSC maintenance. Consistent with this data, I also observed selective down-regulation of genes associated with germ cell differentiation in Cep55 overexpressing testes, including Erg4 and Sohlh1. Thus, Cep55 amplification leads to a shift towards the initial maintenance of SSC stemness while blocking SSC differentiation and entry into meiosis. However, in the long term, it results in progressive germ cell loss. Collectively, in this chapter, we have shown that Cep55 overexpression inactivates Foxo1 resulting in degeneration of germ cells and the manifestation of a Sertoli cell only (SCO)-like phenotype similar to that seen in many azoospermic men. In the second research chapter, I demonstrated that the Cep55Tg/Tg mice were susceptible to a wide spectrum of neoplasias arising at approximately 15 months post birth. The tumor spectrum varied from lung adenoma and carcinoma, papillary adenocarcinoma, B-cell and T-cell lymphoma, myelogenous leukemia, haemangiosarcoma and lipoma suggesting Cep55 to be a broad-spectrum oncogene. This is consistent with the overexpression of CEP55 observed in a wide-variety of human cancers. I have observed that Cep55Tg/Tg mice were prone to a higher incidence of lymphomas and sarcomas mimicking Trp53-/- phenotype. Notably, we observed reduced tumor latency in bi-transgenic Cep55wt/Tg Trp53wt/- mice compared to Cep55wt/wt Trp53wt/- mice, indicating loss or suppression of Trp53 function might be an important event in de novo tumorigenesis observed in Cep55Tg/Tg mice. In addition, I have observed that Cep55 amplification in vivo caused hyper-proliferation due to upregulation of the PI3K/Akt pathway and also the Foxm1-Plk1 pathway. Further, I have also demonstrated that Cep55 overexpression promotes genomic instability in vivo and protects polyploid cells during perturbed mitosis. Collectively, I have characterized the oncogenic potential of Cep55 in vivo and showed that Cep55 amplification in mice leads to de novo tumorigenesis through acquisition of genomic instability. In the third research chapter, I have used Breast Cancer (BC) as a model to study the role of CEP55 in regulating the fate of aneuploid cell populations during perturbed mitosis. I have shown that high CEP55 mRNA expression associates with aggressive breast cancer subtypes with poor clinical outcomes. Moreover, I have demonstrated that depletion of CEP55 impacts cell proliferation, anchorage-independent growth and tumor forming capacity in vivo. I have also illustrated that CEP55 overexpression promotes aneuploid cell survival during perturbed mitosis by evading apoptosis. Collectively, these findings highlight the clinical implications of deregulated CEP55 and how this can be exploited for therapy development. In the fourth and final research chapter, I have demonstrated that CEP55 is transcriptionally controlled by an ERK1/2-MYC axis in BC. Notably, I have shown that inhibition of MEK1/2 using a small molecule inhibitor can mimic depletion of CEP55 in vivo and CEP55-depleted cells cannot tolerate mitotic stress caused by anti-mitotic drugs such as PLK1 inhibitors. Here, I explore the rationale of synergistically targeting the CEP55-dependent PLK1/ERK2 pathways using small the molecule inhibitors AZD6244157 (MEK1/2 inhibitor) and BI2536158 (PLK1 inhibitor) against TNBC. This combination of MEK1/2-PLK1 blockade resulted in selective tumor cell growth inhibition and apoptosis in vitro as well as significant growth retardation and regression in multiple in vivo basal-like breast cancer xenografts models. Mechanistically, I have demonstrated that the dual combination resulted in unscheduled CDK1/Cyclin B activation and favored CDK1-Caspase 3-dependent mitotic catastrophe. Therefore, I have provided preclinical evidence of a novel therapeutic strategy of a MEK1/2-PLK1 dual combination for selectively targeting CEP55 over-expressing BC in the clinic. In summary, these findings illustrate the physiological and oncogenic role of CEP55 in vivo and broaden our understanding of CEP55 function with respect to spermatogenesis and genomic instability. The findings also demonstrate that precise regulation of CEP55 levels are necessary for regular homeostasis, and highlight the therapeutic potential of targeting this protein in aggressive breast cancer.

The centrosomal protein, CEP55, is a key regulator of cytokinesis, and its overexpression is linked to genomic instability, a hallmark of cancer. However, the mechanism by which it mediates genomic instability remains elusive. Here, we showed that CEP55 overexpression/knockdown impacts survival of aneuploid cells. Loss of CEP55 sensitizes breast cancer cells to anti-mitotic agents through premature CDK1/cyclin B activation and CDK1 caspase-dependent mitotic cell death. Further, we showed that CEP55 is a downstream effector of the MEK1/2-MYC axis. Blocking MEK1/2-PLK1 signaling therefore reduced outgrowth of basal-like syngeneic and human breast tumors in invi v models. In conclusion, high CEP55 levels dictate cell fate during perturbed mitosis. Forced mitotic cell death by blocking MEK1/2-PLK1 represents a potential therapeutic strategy for MYC-CEP55-dependent basal-like, triple-negative breast cancers. Refereed/Peer-reviewed