Beyond Cytokinesis: the Emerging Roles of CEP55 in Tumorigenesis

REVIEW Beyond cytokinesis: the emerging roles of CEP55 in tumorigenesis

J Jeffery1, D Sinha1,2, S Srihari3, M Kalimutho1 and KK Khanna1

CEP55 was initially identified as a pivotal component of abscission, the final stage of cytokinesis, serving to regulate the physical separation of two daughter cells. Over the past 10 years, several studies have illuminated additional roles for CEP55 including regulating the PI3K/AKT pathway and midbody fate. Concurrently, CEP55 has been studied in the context of cancers including those of the breast, lung, colon and liver. CEP55 overexpression has been found to significantly correlate with tumor stage, aggressiveness, metastasis and poor prognosis across multiple tumor types and therefore has been included as part of several prognostic ‘gene signatures’ for cancer. Here by discussing in depth the functions of CEP55 across different effector pathways, and also its roles as a biomarker and driver of tumorigenesis, we assemble an exhaustive review, thus commemorating a decade of research on CEP55.

Oncogene (2016) 35, 683–690; doi:10.1038/onc.2015.128; published online 27 April 2015

INTRODUCTION CG-NAP and Pericentrin B.8 Like many other centrosomal A decade ago, we identified and characterized CEP55 (also proteins, CEP55 is a highly coiled-coil protein (Figures 1a).1 The known as c10orf3 and FLJ10540) as a centrosome-and midbody- N-terminus contains a homodimerization domain, while a hinge associated protein and a key regulator of cytokinesis.1 Subsequent region in the coiled coil has been termed the ESCRT and ALIX- studies described how CEP55 might cooperate with members of binding region (EABR) and is critical to its role in cytokinesis endosomal sorting complex required for transport (ESCRT) (Figures 1a and b).4,9 The C-terminus of the protein targets it to 2–4 machinery to constrict intracellular bridge to allow abscission. both the centrosome and the midbody.1 CEP55 not only localizes More recently, we and others have deciphered additional roles for primarily to the pericentriolar matrix but also is associated with CEP55 in regulating the PI3K/AKT pathway and stemness as well the mother centriole. It does not require the microtubules for its as in promoting tumorigenesis. Together, these studies have centrosomal localization. CEP55 interacts with the pericentriolar fi unveiled critical functions of CEP55 outside cytokinesis, rede ning matrix proteins CG-NAP and pericentrin B but is not required the paradigm of CEP55 action in cells. Herein, the pleiotropic roles for microtubule nucleation in interphase cells or for the of CEP55 including its function in cytokinesis and the implications localization of CG-NAP or pericentrin B to the centrosome.1,9 for human tumorigenesis are comprehensively discussed. CEP55 remains on the centrosome throughout the cell cycle and is recruited to the mitotic spindle during mitosis.9 Consistent fi CEP55 IN CYTOKINESIS with this, CEP55 has been identi ed in the human mitotic spindle proteome.10 CEP55 also localizes to the central spindle during fi Cytokinesis is the nal stage of cell division that divides the anaphase and the midbody during cytokinesis.1,11 CEP55 is cytoplasm equally amongst two daughter cells. After the DNA is reported to have microtubule-bundling activity in vitro,11 separated at opposite ends of the cell, the plasma membrane fi 5 although the signi canceofthisiscurrentlyunclear. begins to ingress in the middle where the midbody forms. During The localization of CEP55 to the central spindle and midbody is cytokinesis there is a sequential recruitment of protein complexes mediated through an interaction with the centralspindlin to the midbody, resulting in a cytokinetic bridge constricting on complex (comprised of MKLP1 and MgcRacGAP) by direct either side of the midbody and the severing of microtubules contact with MKLP1.11 The importance of this association was contained within the bridge. During abscission, the final stage of cytokinesis, the cytokinetic bridge tears at the constriction site, highlighted in MKLP1-depleted cells, where CEP55 could not thus providing physical separation between the daughter cells.6 localize to the midbody. The breast and ovarian cancer We initially described CEP55 as a centrosome- and midbody- susceptibility gene, BRCA2, was also recently shown to modulate 12 associated protein of ~ 55 kDa in size, which spans 464 amino recruitment of CEP55 to the midbody. Syntaxin16, a protein acids (Figure 1a).1 The centrosome is comprised of two centrioles involved in membrane fusion, has been shown to regulate surrounded by a cloud of electron-dense material known as the cytokinesis through its indispensable role in the recruitment of pericentriolar matrix.7 The pericentriolar matrix is comprised of recycling endosomes, exocyst components and CEP55 to the a number of coiled-coil proteins including key structural proteins midbody.13

1Signal Transduction Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia; 2School of Natural Sciences, Grifﬁth University, Brisbane, Queensland, Australia and 3Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia. Correspondence: Professor KK Khanna, Signal Transduction Laboratory, QIMR Berghofer Medical Research Institute, 300 Herston Road, Brisbane, Queensland, Australia. E-mail: [email protected] Received 25 February 2015; revised 16 March 2015; accepted 16 March 2015; published online 27 April 2015 CEP55 and cancer J Jeffery et al 684

Figure 1. The role of CEP55 in cytokinesis. (a) Diagramatic representation of CEP55 and its domains. A coiled coil spans most of the protein, with a hinge region containing the ESCRT and ALIX-binding region (EABR). The N-terminal coiled-coil region (CC1) contains a homodimerization region (white dotted region), while the C-terminus (colored blue) targets CEP55 to the centrosome and midbody. This region is phosphorylated on S425, S428 and S436 during mitosis (red circles). (b) The structure of CEP55 complexes at the midbody. Each CEP55 homodimer can bind one molecule of either ALIX or TSG101 via its EABR domain. ALIX can then recruit ESCRT-III complexes directly, whereas TSG101 recruits ESCRT-III complexes via the recruitment of ESCRT-1. (c) The recruitment of ESCRT-III complexes in a CEP55-dependent manner promotes the formation of 17nm ESCRT-III ﬁlaments (pink), which creates a constriction site. ESCRT-III complexes then recruit Spastin to cleave the microtubules (orange).

CEP55 recruits downstream machinery to the midbody Recently, BRCA2 has been reported to promote the ability of CEP55 is critical for correct midbody assembly and CEP55- CEP55 to recruit TSG101 and ALIX to the midbody independent of 12 depleted cells display an abnormal midbody structure with no its function in DNA repair, although the involvement of BRCA2 in 16 midbody visible under light microscopy.11 CEP55 targets cytokinesis remains controversial. Similarly, it is contentious a number of structural and regulatory proteins required for whether CEP55 has a more universal role in the regulation of completion of cytokinesis to the midbody including Aurora B, cytokinesis in mammalian cells as human embryonic kidney cells 14 MKLP2, PRC1, ECT2, Anillin and Syntaxin 2,11 demonstrating that (HEK293) continue to divide following CEP55 depletion. Addi- CEP55 is indispensable for midbody structure maintenance. tional work is required to discern whether this role is direct CEP55 also orchestrates final stage of abscission. During (by facilitating the actual abscission process) or indirect abscission, CEP55 at midbody recruits proteins that are directly (by regulating the recruitment of proteins involved in vesicular involved in the membrane fission (Tumor-susceptibility gene 101 trafficking). (TSG101) and ALG2-interacting protein X (ALIX)) and fusion events (Endobrevin) (Figures 1b and c).2,14 Centriolin, another centroso- The structure of CEP55/ESCRT complexes at the midbody mal protein, also recruits fission and fusion proteins by a distinct CEP55 complex formation with ALIX and TSG101 is required to 11,15 but parallel pathway. TSG101 is a component of ESCRT-I, while allow recruitment of ESCRT complexes to the midbody, which ALIX is an ESCRT-III associated protein.2 Both TSG101 and ALIX facilitate the membrane fission events necessary to create localize to centrosomes and have well-described functions in separation of the two daughter cells.2,14 Although there are retroviral budding as well as the membrane fission events of multiple ESCRT complexes (known as 0, I, II and III), ESCRT-III is the cytokinesis.2,14 Depletion of TSG101 or ALIX results in defective most critical for cytokinesis, forming long filamentous structures cytokinesis, which is reminiscent of the phenotype observed in which create the constriction site.2,14,17 ALIX is able to recruit CEP55-depleted cells with an increased proportion of multi- ESCRT-III directly, whereas TSG101 recruits ESCRT-I, which in turn nucleated cells and/or cells arrested at midbody stage.2 Notably, recruits ESCRT-III (Figure 1b).2,14 unlike TSG101 and ALIX viral budding is unaffected by depletion CEP55 is a highly coiled-coil protein which homodimerizes.9 of CEP55, indicating that CEP55 might act as an adaptor rather Initially, CEP55 was proposed to comprise of two coiled-coil than a component of the core ESCRT pathway to promote regions joined by a hinge region with the first coiled coil acting as completion of cytokinesis.2 the homodimerization region and both ALIX and TSG101 binding

Oncogene (2016) 683 – 690 © 2016 Macmillan Publishers Limited CEP55 and cancer J Jeffery et al 685 the hinge region (Figure 1b).14 More recent work demonstrated that the previously identified hinge region is actually a coiled-coil region and probably a continuation of the N-terminal coiled coil (Figure 1a).4 Furthermore, each CEP55 homodimer binds only copy of either ALIX or TSG101, indicating multiple CEP55 homodimers are required for abscission to occur.4 ALIX and TSG101 compete for binding to the EABR region of CEP55, both of which bind the EABR with similar affinity and stoichiometry.4 The EABR is bound by residues 797 to 809 of ALIX, which contain 2,4 a GPPX3Y motif, and residues 154–166 of TSG101. Notably, mutation of either the CEP55 EABR or the ALIX GPPX3Y motif inhibited both the EABR-ALIX interaction as well as ALIX localization to the midbody.4 CEP55 at the midbody likely acts as a scaffold for the recruitment of multiple ESCRT components. Indeed many ESCRT proteins localize to midbodies including CHMP2A, CHMP4A, CHMP5 and VPS4, and depletion of multiple ESCRTIII components also results in cytokinesis defects.14,18 ESCRT proteins are recruited in a sequential manner that is dependent on CEP55, TSG101 and ALIX.14,18 The sequential recruitment of proteins to the midbody is consistent with the observations of Guizetti et al.,17 where CEP55 and ALIX are restricted to the midbody, while members of the ESCRTIII complex localize to the cortical side the midbody as well as the constriction zone (Figure 1c). ESCRT-III oligomerizes and form filaments, which nucleate outwards from the midbody and create a constriction zone, where abscission occurs. The ESCRT filaments then recruit microtubule-severing protein, Spastin, to cleave any remaining microtubules binding the two daughter cells.

CEP55 phosphorylation CEP55 is phosphorylated on serines 425 and 428 during mitosis by CDK1/ERK2 (Figure 1a).1 These phosphorylation events mediate the association of CEP55 with PIN1.19 The interaction with PIN1 stabilizes CEP55 and promotes its phosphorylation on serine 436 by PLK1, most likely by converting CEP55 to a more favorable and stable conformation.19,20 Phosphorylation of all three residues is required for the successful completion of cytokinesis as expression of unphosphorylatable mutants leads to multinucleation and midbody arrest.1 Whilst PLK1 was initially proposed to cooperate with CEP55 at the midbody,1 a more recent report suggests that PLK1 phosphorylation of CEP55 regulates the timing of CEP55 recruitment to the midbody by inhibiting its interaction with MKLP1 21 (Figure 2b). Notably, S436 phosphorylation prevents premature Figure 2. Additional roles of CEP55. (a) CEP55 directly interacts with recruitment of CEP55 to the midbody. Consistent with this, CEP55 the PI3K catalytic subunit, p110, promoting its stability and therefore is not recruited to the midbody until late anaphase, at which point activation. Increased PI3K activation results in an increased pool of PLK1 is being degraded because of mitotic exit. Inhibition of PLK1 PIP3 and a subsequent increase in AKT activation as marked by S473 or expression of CEP55 mutated at PLK1 phosphorylation site phosphorylation. VEGF-A promotes CEP55 localization to the plasma membrane and increases CEP55 levels, although the mechanism for (S436A) results in premature localization and higher levels of this is unclear. (b) Bi-directional regulation of CEP55, PLK1 and CEP55 at the midbody. This early recruitment affects midbody FOXM1. Both PLK1 and CEP55 are transcriptional targets of FOXM1, structure and results in failed abscission, indicating that the spatio- whilst PLK1 phosphorylates FOXM1, creating a positive feedback temporal recruitment of CEP55 to the midbody is critical for both loop. PLK1 phosphorylates CEP55, thereby promoting its stability. integrity of the midbody structure and successful abscission.21 CEP55 has also been reported to promote FOXM1 expression. Remarkably, CEP55 was recently shown to interact with Expression of all three proteins is suppressed by p53. phosphatases MTMR3 and MTMR4 during early mitosis indepen- dently of their roles in lipid regulation.22 CEP55 binds MTMR3- CEP55 IN GERM CELL FUNCTION MTMR4 heterodimers via a direct interaction with the GPPX3Y In adult tissues CEP55 is most highly expressed in the testes and motif of MTMR4, the same motif used by ALIX and TSG101 to cancer cell lines.1,9 In germ cells (testes and ovaries) CEP55 interact with CEP55. These interactions are proposed to prevent localizes to intracellular bridges, comprised of general cytokinesis the premature recruitment of CEP55 to the midbody, similar to components with additional germ cell-speciﬁc factors including phosphorylation on S436 by PLK1.22 Consistent with this, TEX14.23,24 TEX14 is an inactive kinase that maintains stable depletion of either MTMR3 or MTMR4 results in failed abscission. intracellular bridges during meiosis and inhibits CEP55 from Interestingly, the MTMR3-MTMR4 heterodimers also interact with binding to ALIX and TSG101 by binding the EABR, thus blocking PLK1, so the interaction of these proteins may form part of a wider cytokinesis.25 The intracellular bridge is a feature of differentiating network regulating the temporal recruitment of CEP55 to the germ cells, acting to synchronize the cells through the transfer of midbody and its interactions with ALIX and TSG101. organelles and molecules.23 The importance of intracellular bridges

© 2016 Macmillan Publishers Limited Oncogene (2016) 683 – 690 CEP55 and cancer J Jeffery et al 686 is highlighted by the absence of intracellular bridges and male phosphorylation. The CEP55/PIK3CA interaction may be mediated sterility observed in TEX14 knockout mice.26 Similar to its role in by VEGF-A, which promotes CEP55 localization to the plasma cytokinesis, CEP55 may act as an adaptor protein for the membrane, where the PI3K complex resides.38 Fibulin-5, an recruitment of TEX14 and other proteins required for intracellular extracellular matrix protein, has also been reported to increase bridge formation in germ cells. Further studies on CEP55 function AKT activation in a CEP55-dependent manner.39 It has yet to be in testes and germ cell development as well as its contribution to determined if there is an interplay between VEGF-A and Fibulin-5 the formation, maintenance and/or stability of intracellular bridge in promoting the CEP55-dependent AKT activation. Any such are necessary to elucidate the detailed molecular mechanisms interplay may be context dependent as VEGF-A overexpression is responsible for intracellular bridge formation. oncogenic and promotes angiogenesis, whereas Fibulin-5 overexpression has been reported to be oncogenic in some cancer types and tumor-suppressive in others, and antagonizes CEP55 AS A REGULATOR OF STEMNESS angiogenesis.40,41 Further work is required to determine if and Midbody fate how VEGF-A and Fibulin-5 cooperate in promoting CEP55- CEP55 may directly impact upon stemness through its regulation dependent AKT activation. of midbody remnants (also known as midbody derivatives or In line with this, we recently demonstrated the importance of MBds),27 although this is contentious as discussed below. Upon CEP55 in regulating the PI3K/AKT pathway by describing a cep55 completion of cytokinesis, MBds are inherited in an asymmetric mutant zebrafish line mimicking a Cep55 knockout model.42 The fashion, where they may be degraded by autophagy in a CEP55- mutant zebrafish (designated e48) contained a C115T mutation, dependent manner.27 Specifically, the autophagy receptor NBR1 resulting in a premature stop codon at residue 39. Heterozygotes targets MBds for degradation via an interaction with CEP55. Both were indistinguishable from their wildtype siblings, being both CEP55 overexpression and NBR1 depletion resulted in decreased viable and fertile. In contrast, homozygous mutants exhibited MBd degradation, thus uncoupling receptor-mediated entry into abnormal development and larval lethality largely because of the autophagy pathway. Given the accumulation of MBds massively increased apoptosis through Akt destabilization. Nota- is associated with an increase in stemness, CEP55 overexpression bly, we provided the first phenotypic and molecular evidence that as observed in many cancers might promote stemness by Cep55 is indispensible for embryonic development partly by abrogating the degradation of MBds. mediating Pi3k/Akt signaling.42 Interestingly, the phenotype CEP55 has also been implicated in an alternate midbody fate observed in cep55 homozygous mutants differs from the known as midbody release, where the midbody is released into phenotype observed in human cancer cells. Although CEP55 the extracellular space between the two daughter cells.28 Knock- regulates AKT activation in cancer cells,37 it regulates Akt stability down of CEP55 or its binding partners, ALIX and TSG101, impaired during zebrafish embryonic development.42 Further research is this process. Furthermore, contrary to the above report this study required to understand the mechanisms that mediate the demonstrated that cells with impaired midbody release are more different relationships between CEP55 and AKT protein/activation likely to undergo differentiation.28 Thus it is becoming increasingly levels in zebrafish versus human cells. apparent that CEP55 regulates midbody fate, but the implications Consistent with our zebrafish model, CEP55 has recently of this are unclear at present. been implicated in vertebrate neural function. Using mevinphos injection into the rostral ventrolateral medulla of rats as a model Exosomes for organophosphate poisoning, CEP55 mediated an increase in The exosome is an organelle containing proteins, mRNAs, miRNAs, the activation of the PI3K/AKT pathway, resulting in upregulation of the nitric oxide synthase II pathway, progressive hypotension non-coding RNAs and other molecules encapsulated by a lipid 43 bilayer.29 Unlike intracellular organelles, exosomes are released and a reduction of smooth muscle tension in the blood vessels. into the extracellular space and taken up by a donor cell, thus Overall, this suggests a role for CEP55 in brain stem-mediated allowing the transmission of these materials.30 In a cancer setting, cardiovascular regulation. exosomes have been shown to suppress the immune system and CEP55 is overexpressed in lung adenocarcinoma and hepato- promote tumor invasiveness, angiogenesis, metastasis, stemness cellular carcinoma (HCC), where its overexpression promotes cell 31,32 fi migration and invasion via upregulation of the PI3K/AKT and drug resistance. CEP55 mRNA has been puri ed from 37,38 33 pathway. Interestingly, VEGF-A induces CEP55 expression in exosomes derived from colorectal cancer cell lines. The addition 38 of these exosomes to endothelial cells promoted cell proliferation, a dose-dependent manner in lung cancer cell lines. VEGF-A is although the individual contribution of CEP55 to this phenotype is a potent stimulator of angiogenesis, endothelial cell vascular unknown. permeability, motility and proliferation, with a well-established role in cancer.40 VEGF-A also stimulates the PI3K/AKT pathway in a CEP55-dependent manner (Figure 2a).38 Furthermore, CEP55 CEP55 AS A REGULATOR OF THE PI3K/AKT PATHWAY partially translocates from the cytoplasm to the plasma membrane One of the major prosurvival pathways is the PI3K/AKT pathway. in response to stimulation by VEGF-A. Consistent with this, PI3K phosphorylates phosphatidylinositol 4,5-bisphosphate (PIP2), concomitant overexpression of VEGF-A, CEP55 and phospho-AKT 36 converting it to phosphatidylinositol 3,4,5 trisphosphate (PIP3), is observed in patient samples. Similarly, in gastric carcinoma which stimulates AKT activation.34 This phosphorylation event can CEP55 overexpression causes upregulation of PI3K/AKT pathway be reversed by the phosphatase and tensin homolog. AKT has leading to dysregulated cell cycle via inhibition of p21 WAF1/Cip1 44 diverse roles in cell survival, cell cycle, angiogenesis, protein expression. Taken together these studies demonstrate that synthesis and metabolism.35 This pathway is dysregulated in CEP55 may alter tumor cell behavior through activation of the multiple cancers mainly through mutation of PIK3CA (also known prosurvival PI3K/AKT pathway. as p110), the catalytic subunit of PI3K resulting in constitutive activity or loss of phosphatase and tensin homolog expression ADDITIONAL MECHANISMS REGULATING CEP55 EXPRESSION with consequent increase in the pool of PIP3, leading to AKT overactivity.36 FOXM1 CEP55 has been reported to bind PIK3CA (Figure 2a).37 This CEP55 is a direct transcriptional target of FOXM1 (Figure 2b).45,46 interaction increases the stability of this subunit, resulting in A FOXM1 transcriptional program is activated during G2/M to increased AKT activation as observed by an increase in S473 allow normal progression through mitosis in a timely fashion.47

Oncogene (2016) 683 – 690 © 2016 Macmillan Publishers Limited CEP55 and cancer J Jeffery et al 687 The program also induces a range of cell cycle genes including stability and hence completion of cytokinesis.53 Direct evidence Cyclin B1, PLK1, Aurora B, CENP-F, and Nek2. Consistent with this, for this axis has been observed, exemplified by decreased CEP55 CEP55 was identified in a screen for genes that are most highly levels and half-life after induction of p53 as compared with expressed in G2/M and its expression in tumor tissues correlated increased CEP55 levels and half-life upon overexpression of PLK1. with known regulators of cytokinesis including PLK1, Aurora A, Further work is required to demonstrate the role of this axis in Aurora B, MKLP1 and MKLP2.11 tumor development. FOXM1 is overexpressed in many cancers, where it is associated 48,49 with chromosomal instability (CIN). For example, FOXM1 Osteopontin overexpression is frequently observed in head and neck CEP55 expression is also regulated by Osteopontin, a glycosylated squamous cell carcinoma, where it correlates with CEP55 over- sialoprotein and component of the mineralized extracellular expression in lymph node metastasis.45 In contrast, both genes are matrices of the bones.59,60 In nasopharyngeal carcinoma CEP55 expressed at low levels in the basal layer of normal oral mucosa. overexpression contributes to cell proliferation, migration and Furthermore, a recent study implicated CEP55 as a driver of invasion in an osteopontin- and CD44-dependent manner.60 FOXM1 expression in oral cavity squamous cell carcinoma lines Blocking of CD44 receptors using anti-CD44 antibody led to wherein CEP55 upregulates FOXM1 levels in a dose-dependent inhibition of CEP55 expression in a dose-dependent manner. manner, which results in increased transcription of MMP-2 and Taken together, these observations indicate the exact mechan- a consequent increase in MMP-2 levels and activity.50 Other isms regulating CEP55 expression in cancer are not yet well FOXM1 transcriptional targets were not examined, so it is unclear understood. However it is clear that there is a complex interplay if CEP55 specifically upregulated the FOXM1 target, MMP-2 or if between CEP55 and other cell cycle regulators, suggesting that MMP-2 is a part of an increased FOXM1 transcriptional program. cancer cells may select for enhanced CEP55 expression. Notably, CEP55 was shown to promote invasion and metastasis by influencing expression of MMP2.50,51 Concomitant overexpression of CEP55, FOXM1 and MMP-2 was observed in patient samples, CEP55 AS A MARKER FOR MALIGNANCY RISK, PROGNOSIS which may influence response to therapy.50 AND THERAPY OUTCOMES fi Taken together, these ndings suggest that CEP55 exists in CEP55 has been identified in prognostic signatures for multiple a positive feedback loop with FOXM1, whereby CEP55 promotes cancers (Table 1). For example, CEP55 was among the top 70 most FOXM1 expression, which in turn results in increased transcription highly overexpressed genes in tumors with CIN (CIN70 signature) of CEP55 (Figure 2b). Such a loop would not be unprecedented as in an analysis across 12 cancer types including breast, lung, a similar loop exists between FOXM1 and PLK1 during mitosis, but medulloblastoma, glioma, mesothelioma and lymphoma.61 The in this case PLK1 phosphorylates FOXM1, which increases its 52 CIN70 signature and its subset the CIN25 signature correlated transcriptional activity. However, the mechanism by which significantly with clinical outcome and distant metastases. CEP55 might increase FOXM1 levels is currently unclear, possibly Similarly, CEP55 was associated with a CIN signature of 10 genes through reciprocal regulation between the two proteins. where high expression conferred drug resistance, cell proliferation and chromosomal instability.62 For prostate cancer, CEP55 was p53 described as part of a 31-gene cell cycle progression signature CEP55 expression is negatively regulated by p53, a potent tumor that strongly correlated with actively dividing prostate cells.63 This suppressor (Figure 2b).53 p53 is inactivated or mutated in many signature when combined with other clinical parameters including human cancers and negatively regulates multiple transcription prostate-specific antigen levels, tumor stage and margin status led factors such as c-Myc and FOXM1 as well as genes associated with to the development of a ‘combined score’ which was highly cell cycle progression such as PLK1, Cyclin B1 and others by predictive of ten-year fatality risk for these patients.63 various mechanisms.54–58 Hence, p53 may influence CEP55 CEP55 was identified in a set of 23 genes whose overexpression expression indirectly through controlling FOXM1 levels. was predictive of poor prognosis in thyroid cancer.64 CEP55 However, we have shown that PLK1 directly mediates CEP55 overexpression correlated with distant metastasis in renal cancer stability via phosphorylation during mitosis to promote cell as part of a 39-gene signature.65 CEP55 was also identified in a abscission (Figure 2b).20 p53 also negatively regulates PLK1, 100-gene overexpression signature for CIN in breast, ovarian and thereby creating a p53-PLK1-CEP55 axis to directly regulate CEP55 colon cancer.66 Consistent with this, CEP55 was described as part

Table 1. Predictive gene signatures containing CEP55

Malignancy Signature function No. of genes Reference

Breast cancer Progression 16 72 Breast cancer Malignancy risk 109 73 Breast cancer Survival and time to distant metastasis 70 69 Triple negative breast cancer Aggressiveness 206 67 ER+ breast cancer Prognosis 6 83 ER+ breast cancer Distant metastasis free survival 9 70 ER+ breast cancer p53 status, prognosis with or without adjuvant 39 71 tamoxifen therapy and chemotherapy sensitivity Breast, ovarian and colon cancer CIN 100 66 Multiple cancers CIN and prognosis 70 61 Multiple myeloma CIN, drug resistance and relapse 10 62 Prostate cancer Prognosis 31 63 Renal cell cancer Progression and distant metastasis 31 65 Thyroid cancer Prognosis 23 64 Abbreviations: CIN, chromosomal instability; ER, estrogen receptor. A list of gene signatures containing CEP55 in multiple cancer types and predicting various outcomes.

© 2016 Macmillan Publishers Limited Oncogene (2016) 683 – 690 CEP55 and cancer J Jeffery et al 688 of a 206-gene signature for aggressiveness in triple negative Overall, the patient data illustrates the critical importance of breast cancer, which is enriched for genes promoting CIN.67 CEP55 to tumorigenesis. CEP55 expression has been particularly well defined for its CEP55 overexpression has been proposed to be an early event significant correlation with poor survival in breast cancer.66,68–71 in tumorigenesis. Organized growth-arrested human mammary For example, CEP55 was one of 85 genes upregulated in invasive epithelial cells correspond to breast tumors with good prognosis, ductal carcinoma compared with their matched ductal carcinoma whereas disorganized proliferative cells correspond to tumors 82 in situ pairs.68 Higher expression of CEP55 also correlated with with poor prognosis. CEP55 was identified as part of a temporal advanced tumor grade. CEP55 was also identified in a 16-gene pattern of 60 genes observed in the transition from a disorganized proliferative state to an organized growth-arrested state, with signature overexpressed in invasive invasive ductal carcinoma 82 compared with preinvasive ductal carcinoma in situ arising in CEP55 expression being downregulated. Similarly, enhanced 72 expression of CEP55 is observed in premalignant lesions of the young women. 85 CEP55 was reported to be a component of a malignancy risk colon, as well as in colorectal cancer itself. Furthermore, we have signature predicting cancer invasion risk and progression.73 This already discussed above CEP55 as a driving force behind signature contained 109 upregulated genes including CEP55 and malignant potential (migration/invasion) of lung adenocarcinoma, HCC, oral cavity squamous cell carcinoma and nasopharyngeal 31 downregulated genes, and was generated by comparing the carcinoma via upregulation of the PI3K/AKT pathway or FOXM1 expression profiles of invasive ductal carcinoma to normal breast – levels (Table 2).37 39,44,50,60 tissue. The upregulation of CEP55 significantly correlated with cancer relapse, progression and metastasis. Some of these signatures and risk scores have been developed CEP55 as cancer vaccine target into patented prognostic ‘kits’ for malignancy risk, prognosis and CEP55 has been identified both as a cancer testis antigen (CTA) 87 therapy selection in patients.74–79 For example, the CIN70 and a tumor-associated antigen (TAA). Cancer testis antigens are signature61 has been patented as a predictor of treatment proteins normally expressed predominantly in the testes but 88 outcomes in patients with solid tumors including glioma, which become more widely expressed in cancer, as has been medulloblastoma and lymphoma.75 Similarly, a mitotic CIN observed with CEP55. As such, CEP55 is also a TAA because of its signature66 has been patented for diagnosis and treatment of aberrant expression in cancer. These properties make CEP55 an breast cancer.80 CEP55 is part of the patented 22-gene ‘3D ideal candidate for cancer vaccine therapies. Initial studies of the signature kit’ to predict the response to chemotherapy in breast effectiveness of CEP55-based immunotherapy vaccines have cancer patients.74 The test predicts patient response to the shown promise in the treatment of chemotherapy-resistant colon cancer stem cells and tumor-initiating cells.89,90 combination chemotherapy, with high expression predicting low pathological complete response. Likewise, CEP55 is also part of a patented 21-gene signature to predict risk of relapse and response CONCLUDING REMARKS 76 to taxane therapy. CEP55 is also part of commercial signatures CEP55 was initially described as having a role in cytokinesis, the from Myriad Genetics Inc for lung cancer prognosis and therapy mechanism of which is becoming well understood. However selection, and for diagnosis and prognosis of prostate, lung, 77,78 the observation that it is dispensable for cytokinesis in normal bladder and brain cancer. Additionally, CEP55 is utilized in the cells together with its expression is being normally limited outside commercially available PAM50 classification for predicting breast the testes raise questions as to the generality of this role. More 81 cancer prognosis. Taken together, this suggests that CEP55 may recent descriptions of CEP55 being involved in the regulation of be clinically useful in predicting malignancy risk, prognosis and the PI3K/AKT pathway, midbody fate and stemness raise the therapeutic response. possibility that CEP55 has a more generalized role, promoting growth and survival at several levels. Hence the evidence of CEP55 being overexpressed in many cancers promoting tumor survival FUNCTIONAL CONSEQUENCES OF CEP55 OVEREXPRESSION IN and progression is not surprising. HUMAN CANCERS Findings discussed herein illustrate that overexpressed CEP55 in CEP55 has been identified as being overexpressed in mRNA cancer has dual functions in activation of PI3K/AKT and FOXM1- – microarray expression profiles of many human cancers.65,68,69,82 85 dependent pathways, although the mechanisms are likely to be Analysis of patient samples has revealed a number of pathways are context dependent. These discoveries also shed light on the – dysregulated in CEP55 overexpressing tumors (Table 2).37 39,44,50,60,86 potential mechanism underlying CEP55-associated tumor pheno- Interestingly, increased multinucleation has not been reported. types. CEP55 has been identified as a marker of poor prognosis,

Table 2. Overexpression of CEP55 in cancer

Malignancy Proliferation Migration p53 AKT FOXM1 Other observations Reference

In vitro In vivo

Gastric carcinoma Yes 68 pairs PS ND ND Yes ND ↓ p21 44 HCC Yes 22 pairs PS ND ND Yes ND ND 37 Lung cancer Yes 56 PS Yes ND Yes ND ↑ VEGF-A 38 Nasopharyngeal carcinoma Yes 63 PS, 10 NT Yes ND ND ND ↑ Osteopontin 60 OCSCC Yes 256 pairs PS Yes ND ND Yes ↑ MMP2 50 OCSCC ND 84 PS, unmatched NM Yes ND Yes ND ↑ Fibulin-5 39 Urinary bladder carcinoma ND 76 PS incl 9 pairs ND ND ND ND ND 86 Abbreviations: HCC, hepatocellular carcinoma; ND, not described; NM, non-malignant tissue; NT, normal tissue; OCSCC, oral cavity squamous cell carcinoma; pairs, matched normal and tumor tissue; PS, patient samples. A list of studies that have analyzed CEP55 expression in cancer and the consequences of overexpression during cancer progression.

Oncogene (2016) 683 – 690 © 2016 Macmillan Publishers Limited CEP55 and cancer J Jeffery et al 689 metastasis, risk of relapse, chromosome instability and therapy 12 Mondal G, Rowley M, Guidugli L, Wu J, Pankratz VS, Couch FJ. BRCA2 localization resistance in many cancer types. It has become increasingly clear to the midbody by filamin A regulates cep55 signaling and completion of cyto- the overexpression of cell cycle and proliferation (CCP) genes is kinesis. Dev Cell 2012; 23:137–152. detrimental to survival, as many of these genes are also 13 Neto H, Kaupisch A, Collins LL, Gould GW. Syntaxin 16 is a master recruitment 24 – components of the CEP55-containing signatures. Whether this factor for cytokinesis. Mol Biol Cell 2013; : 3663 3674. 14 Morita E, Sandrin V, Chung HY, Morham SG, Gygi SP, Rodesch CK et al. Human occurs as a result via a single event such as FOXM1 upregulation ESCRT and ALIX proteins interact with proteins of the midbody and function in or through a series of steps contributing to CCP upregulation cytokinesis. Embo J 2007; 26: 4215–4227. remains to be seen. Given these signatures are associated with 15 Gromley A, Yeaman C, Rosa J, Redick S, Chen C-T, Mirabelle S et al. Centriolin such poor outcomes, new ways of treating such tumors need to anchoring of exocyst and snare complexes at the midbody is required for be developed. CEP55 is unique among many of the CCP genes in secretory-vesicle-mediated abscission. Cell 2005; 123:75–87. that it is a CTA, presenting one possible method of directly 16 Lekomtsev S, Guizetti J, Pozniakovsky A, Gerlich DW, Petronczki M. Evidence that targeting cancer tissue. Additionally two other CCP genes, Aurora the tumor-suppressor protein BRCA2 does not regulate cytokinesis in A and Cyclin B1, have been identified as TAAs.91,92 Combination human cells. J Cell Sci 2010; 123: 1395–1400. immunotherapy strategies targeting CCP genes could afford new 17 Guizetti J, Schermelleh L, Mantler J, Maar S, Poser I, Leonhardt H et al. Cortical constriction during abscission involves helices of ESCRT-III-dependent filaments. opportunities for precision therapies because CCP genes seem to Science 2011; 331: 1616–1620. affect multiple tumor types. The recent success with immunother- 18 Morita E, Colf LA, Karren MA, Sandrin V, Rodesch CK, Sundquist WI. Human ESCRT- apy as a cancer treatment has revolutionized the field, and now III and VPS4 proteins are required for centrosome and spindle maintenance. Proc sets the stage for the rational tailoring of immunotherapies to Natl Acad Sci USA 2010; 107: 12889–12894. treat specific cancer types. Perhaps some benefit can be derived 19 van der Horst A, Khanna KK. The peptidyl-prolyl isomerase Pin1 regulates cyto- through the use of CEP55-targeted vaccines. kinesis through Cep55. Cancer Res 2009; 69:6651–6659. The continual discovery of novel roles for CEP55 and different 20 van der Horst A, Simmons J, Khanna KK. Cep55 stabilization is required for normal means of regulating its expression in different cancer types execution of cytokinesis. Cell Cycle 2009; 8:3742–3749. suggests that despite recent advances, thorough understanding of 21 Bastos RN, Barr FA. Plk1 negatively regulates Cep55 recruitment to the midbody to ensure orderly abscission. J Cell Biol 2010; 191: 751–760. the physiological and pathological role of CEP55 has yet to be 22 St-Denis N, Gupta GD, Lin ZY, Gonzalez-Badillo B, Pelletier L, Gingras A-C. realized. Additional studies on alterations in its regulation and Myotubularin-related proteins 3 & 4 interact with PLK1 and CEP55 to control function during cancer development and progression are needed CEP55 recruitment to the midbody and ensure proper abscission. Mol Cell for designing therapeutic strategies that might yield a major Proteom 2015; 14: 946–960. impact on a number of human malignancies with CEP55 23 Greenbaum MP, Iwamori T, Buchold GM, Matzuk MM. Germ cell intercellular overexpression. bridges. Cold Spring Harb Perspect Biol 2011; 3: a005850. 24 Chang YC, Chen YJ, Wu CH, Wu YC, Yen TC, Ouyang P. Characterization of centrosomal proteins Cep55 and pericentrin in intercellular bridges of mouse testes. CONFLICT OF INTEREST J Cell Biochem 2010; 109: 1274–1285. 25 Iwamori T, Iwamori N, Ma L, Edson MA, Greenbaum MP, Matzuk MM. TEX14 The authors declare no conflict of interest. interacts with CEP55 to block cell abscission. Mol Cell Biol 2010; 30: 2280–2292. 26 Greenbaum MP, Yan W, Wu M-H, Lin Y-N, Agno JE, Sharma M et al. TEX14 is ACKNOWLEDGEMENTS essential for intercellular bridges and fertility in male mice. Proc Natl Acad Sci USA 2006; 103: 4982–4987. DS is supported by a Griffith University International and Postgraduate Research 27 Kuo TC, Chen CT, Baron D, Onder TT, Loewer S, Almeida S et al. Midbody accu- Scholarship. MK is supported by a project grant from Cancer Council Queensland mulation through evasion of autophagy contributes to cellular reprogramming (ID1087363). This work was supported by a program grant from the National Health & and tumorigenicity. Nat Cell Biol 2011; 13: 1214–1223. Medical Research Council (NHMRC) to KKK (ID 1017028). KKK is a NHMRC Senior 28 Ettinger AW, Wilsch-Brauninger M, Marzesco A-M, Bickle M, Lohmann A, Maliga Z Principal Research Fellow (ID 613638). et al. Proliferating versus differentiating stem and cancer cells exhibit distinct midbody-release behaviour. 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