Cytoskeleton-associated Protein 2 Research Articles

Human CKAP2L shows a cell cycle-dependent expression pattern and exhibits microtubule-stabilizing properties.

Cytoskeleton-associated protein 2-like (CKAP2L) is a paralogue of cytoskeleton-associated protein 2 (CKAP2). We characterized the expression pattern, subcellular localization, and microtubule-stabilizing properties of human CKAP2L. The levels of both CKAP2L transcript and protein were cell cycle phase-dependent, peaking during the G2/M phase and relatively high in certain human tissues, including testis, intestine, and spleen. CKAP2L protein was detectable in all human cancer cell lines we tested. CKAP2L localized to the mitotic spindle apparatus during mitosis, as reported previously. During interphase, however, CKAP2L localized mainly to the nucleus. Ectopic overexpression of CKAP2L resulted in 'microtubule bundling', and, consequently, an elevated CKAP2L level led to prolonged mitosis. These findings support the mitotic role of CKAP2L during the human cell cycle.

The spindle protein CKAP2 regulates microtubule dynamics and ensures faithful chromosome segregation

Regulation of microtubule dynamics by microtubule-associated proteins (MAPs) is essential for mitotic spindle assembly and chromosome segregation. Altered microtubule dynamics, particularly increased microtubule growth rates, were found to be a contributing factor for the development of chromosomal instability, which potentiates tumorigenesis. The MAP XMAP215/CKAP5 is the only known microtubule growth factor, and whether other MAPs regulate microtubule growth in cells is unclear. Our recent in vitro reconstitution experiments have demonstrated that Cytoskeleton-Associated Protein 2 (CKAP2) increases microtubule nucleation and growth rates, and here, we find that CKAP2 is also an essential microtubule growth factor in cells. By applying CRISPR-Cas9 knock-in and knock-out (KO) as well as microtubule plus-end tracking live cell imaging, we show that CKAP2 is a mitotic spindle protein that ensures faithful chromosome segregation by regulating microtubule growth. Live cell imaging of endogenously labeled CKAP2 showed that it localizes to the spindle during mitosis and rapidly shifts its localization to the chromatin upon mitotic exit before being degraded. Cells lacking CKAP2 display reduced microtubule growth rates and an increased proportion of chromosome segregation errors and aneuploidy that may be a result of an accumulation of kinetochore-microtubule misattachments. Microtubule growth rates and chromosome segregation fidelity can be rescued upon ectopic CKAP2 expression in KO cells, revealing a direct link between CKAP2 expression and microtubule dynamics. Our results unveil a role of CKAP2 in regulating microtubule growth in cells and provide a mechanistic explanation for the oncogenic potential of CKAP2 misregulation.

ZC3H13 Enhances the Malignancy of Cervical Cancer by Regulating m6A Modification of CKAP2.

Sustained expression of zinc finger CCCH-type containing 13 (ZC3H13) in tumors is essential for cancer cell malignancy; however, our understanding of its clinical effects and mechanisms in cervical cancer (CC) is limited. In this study, we aimed to reveal the effect on CC progression of ZC3H13-mediated N6-methyladenosine (m6A) modification to stabilize cytoskeleton-associated protein 2 (CKAP2) expression. CC tissues and paired adjacent normal tissues were collected from 50 patients. qRT-PCR was used to clarify ZC3H13 and CKAP2 expression levels in the CC tissues. The functional roles of ZC3H13 and CKAP2 in CC were analyzed by detecting the changes in CC cell proliferation, migration, invasion, and tumor growth in vivo. The regulatory relationship between ZC3H13 and CKAP2 was investigated by confirming m6A modification levels and their expression correlation. ZC3H13 and CKAP2 were highly expressed in CC and linked with poor prognosis. We observed that ZC3H13 inhibition decreased CC cell proliferation, invasion, and migration, while its facilitation promoted CC cell malignancy. ZC3H13 mediated m6A modification of CKAP2 to enhance CKAP2 expression in CC cells. Furthermore, CKAP2 overexpression partially restored the malignant phenotypic promotion induced by ZC3H13 overexpression in CC cells. In summary, this study revealed that ZC3H13-mediating m6A modification of CKAP2 promotes CC development. This finding should be conducive to an understanding of the role of ZC3H13-m6A-CKAP2 in CC and should provide an effective therapeutic target for this cancer.

Knockdown of CKAP2 Inhibits Proliferation, Migration, and Aggregate Formation in Aggressive Breast Cancer.

Simple SummaryCancer is a complex disease where cells grow and divide in an uncontrolled manner. It is well established that its development and progression involve major alterations in the activity of mitotic regulators. In order to improve our understanding of the contribution of cell-cycle progression defects to the development of disease, the aim of this study is to identify genes relevant to the proper progression of mitosis that are deregulated in breast cancer. Our findings identified CKAP2 as an important mitotic regulator in BC tumors. Moreover, in vitro experiments showed that gene silencing of CKAP2 blocked cell growth, cell migration, and formation of cell aggregates. These results demonstrated the important role of CKAP2 in breast cancer tumor formation.Loss of mitotic regulation is commonly observed in cancer and is a major cause of whole-chromosome aneuploidy. The identification of genes that play a role in the proper progression of mitosis can help us to understand the development and evolution of this disease. Here, we generated a list of proteins implicated in mitosis that we used to probe a patient-derived breast cancer (BC) continuum gene-expression dataset generated by our group by human transcriptome analysis of breast lesions of varying aggressiveness (from normal to invasive). We identified cytoskeleton-associated protein 2 (CKAP2) as an important mitotic regulator in invasive BC. The results showed that CKAP2 is overexpressed in invasive BC tumors when compared with normal tissues, and highly expressed in all BC subtypes. Higher expression of CKAP2 is also related to a worse prognosis in overall survival and relapse-free survival in estrogen receptor (ER)-positive and human epidermal growth factor receptor type 2 (HER2)-negative BC patients. Knockdown of CKAP2 in SKBR3 cells impaired cell proliferation and cell migration and reduced aggregate formation in a 3D culture. Our results show the important role of CKAP2 in BC tumorigenesis, and its potential utility as a prognostic marker in BC.

CKAP2 overexpression correlates with worse overall survival in patients with lung adenocarcinoma.

Background:Adenocarcinoma is a non–small-cell lung cancer that is common cancer in both genders, and has poor clinical outcomes. We aimed to evaluate the role of cytoskeleton-associated protein 2 (CKAP2), its prognostic significance, and the relationship between CKAP2 expression and lung adenocarcinoma driver genes.Methods:The expression of CKAP2 was studied by immunohistochemical staining of specimens from 88 patients with lung adenocarcinoma. The correlation between clinicopathological features and CKAP2 expression was analyzed. Kaplan-Meier analysis and Cox proportional hazard models were used to examine the prognostic value of CKAP2 in terms of overall survival (OS). The correlation between epidermal growth factor receptor (EGFR) mutation, anaplastic lymphoma kinase (ALK) rearrangement, and CKAP2 expression was analyzed. All histological samples were detected by fluorescence in situ hybridization for EGFR mutations and ALK rearrangements.Results:Eighty-eight patients with positive CKAP2 expression were observed in this study. Patients with high levels of CKAP2 expression were associated with OS (P = .021). Multivariate Cox regression analysis disclosed that positive CKAP2 expression (P = .043) could independently predict unfavorable OS. In addition, CKAP2 expression was not associated with EGFR mutation (P = .219) and ALK rearrangement (P = .389) in lung adenocarcinoma patients.Conclusion:High expression of CKAP2 may serve as a marker of poor prognosis in lung adenocarcinoma.

A chalcone-syringaldehyde hybrid inhibits triple-negative breast cancer cell proliferation and migration by inhibiting CKAP2-mediated FAK and STAT3 phosphorylation

BackgroundAlthough triple-negative breast cancer (TNBC) accounts for only 15% of breast cancer cases, it is associated with a high relapse rate and poor outcome after standard treatment. Currently, the effective drugs and treatment strategies for TNBC remain limited, and thus, developing effective treatments for TNBC is pressing. Several studies have demonstrated that both chalcone and syringaldehyde have anticancer effect, but their potential anti-TNBC bioactivity are still unknown. PurposeThe present study aimed to synthesize a chalcone-syringaldehyde hybrid (CSH1) and explore its potential anti-TNBC effects and the underlying molecular mechanism. MethodsCell cytotoxicity was determined by 3-(4,5-dimethythiazol)-2,5-diphenyltetrazolium bromide (MTT). The activity of cell proliferation was measured by colony formation assay and 5-ethynyl-2’-deoxyuridine (EdU) staining assay. Cell cycle distribution and cell apoptosis were determined by fluorescence-activated cell sorter (FACS). The situation of DNA damage was observed using fluorescence microscopy. The ability of cell-matrix adhesion, migration and invasion was detected using cell adhesion assay and transwell assay. Transcriptome sequencing was performed to find out the changed genes. Levels of various signaling proteins were assessed by western blotting. ResultsCSH1 treatment triggered DNA damage and inhibited DNA replication, cell cycle arrest, and cell apoptosis via suppressing signal transducer and activator of transcription 3 (STAT3) phosphorylation. Whole genome RNA-seq analysis suggested that 4% of changed genes were correlated to DNA damage and repair, and nearly 18% of changed genes were functionally related to cell adhesion and migration. Experimental evidence indicated that CSH1 treatment significantly affected the distribution of focal adhesion kinase (FAK) and its phosphorylation, resulting in cell-matrix-adhesion reduction and migration inhibition of TNBC cells. Further mechanistic studies indicated that CSH1 inhibited TNBC cell proliferation, adhesion, and migration by inhibiting cytoskeleton-associated protein 2 (CKAP2)-mediated FAK and STAT3 phosphorylation signaling. ConclusionThese results suggest that CKAP2-mediated FAK and STAT3 phosphorylation signaling is a valuable target for TNBC treatment, and these findings also reveal the potential of CSH1 as a prospective TNBC drug.

The mitotic spindle protein CKAP2 potently increases formation and stability of microtubules.

Cells increase microtubule dynamics to make large rearrangements to their microtubule cytoskeleton during cell division. Changes in microtubule dynamics are essential for the formation and function of the mitotic spindle, and misregulation can lead to aneuploidy and cancer. Using in vitro reconstitution assays we show that the mitotic spindle protein Cytoskeleton-Associated Protein 2 (CKAP2) has a strong effect on nucleation of microtubules by lowering the critical tubulin concentration 100-fold. CKAP2 increases the apparent rate constant ka of microtubule growth by 50-fold and increases microtubule growth rates. In addition, CKAP2 strongly suppresses catastrophes. Our results identify CKAP2 as the most potent microtubule growth factor to date. These finding help explain CKAP2's role as an important spindle protein, proliferation marker, and oncogene.

Cytoskeleton-associated protein 2 (CKAP2) is regulated by vascular endothelial growth factor and p53 in retinal capillary endothelial cells under high-glucose conditions

PurposeWe previously found the mutation frequency of cytoskeleton-associated protein 2 (CKAP2) was significantly increased in proliferative diabetic retinopathy (PDR) patients through whole exome sequencing. The present study was conducted to explore the expression and possible mechanism of CKAP2 in PDR patients and human retinal capillary endothelial cells (HRCECs) under high-glucose (HG) conditions. MethodsExpression of CKAP2 and p53 in the vitreous fluid and fibrovascular membrane (FVM) of PDR patients and HRCECs under HG conditions was observed. Cell proliferation, migration and tubule formation were assessed. Ranibizumab and siRNA transfection were used in the inhibition assay. ResultsCKAP2 and p53 were significantly increased in the ocular tissues of PDR patients. HG combined with VEGF treatment significantly up-regulated expression of CKAP2 and p53 in HRCECs. Inhibition of CKAP2 attenuated the abilities of cell proliferation, migration and tube formation under HG conditions. Blocking VEGF or p53 significantly decreased CKAP2 expression, whereas inhibition of CKAP2 failed to alter the level of VEGF or p53. ConclusionsCKAP2 is involved in the pathogenesis of PDR and regulated by VEGF and p53 under HG conditions.

LncRNA DARS-AS1 aggravates the growth and metastasis of hepatocellular carcinoma via regulating the miR-3200-5p-Cytoskeleton associated protein 2 (CKAP2) axis

ABSTRACT Accumulating signs have found that long noncoding RNAs (lncRNAs) contribute to hepatocellular carcinoma (HCC). Here, we probed the effect and mechanism of lncRNA DARS-AS1 in HCC. The profiles of DARS-AS1 and Cytoskeleton associated protein 2 (CKAP2) in 50 HCC tissues and non-tumor tissues were examined by real-time quantitative polymerase chain reaction (RT-qPCR). DARS-AS1 and CKAP2 overexpression and/or knockdown cell models were established. The proliferation, apoptosis, invasion and epithelial-mesenchymal transition (EMT) were determined. CKAP2, and focal adhesion kinase (FAK)-extracellular signal-regulated kinase (ERK) was tested by Western blot (WB). The relationship between DARS-AS1 and CKAP2 was predicted by Bioinformatics, and the dual-luciferase reporter assay was applied to verify the targeting association between miR-3200-5p and DARS-AS1 and CKAP2. DARS-AS1 was overexpressed in HCC tissues (vs. that in non-tumor tissues) and was closely correlated with the patients’ tumor stage. DARS-AS1 facilitated HCC cell proliferation and hampered apoptosis. HCC cell migration and EMT were enhanced by DARS-AS1. DARS-AS1 up-regulated CKAP2, which aggravated HCC. Further investigation illustrated that either DARS-AS1 or CKAP2 activated FAK-ERK pathway, and miR-3200-5p was competitively restrained by DARS-AS1. miR-3200-5p exerted tumor-suppressive effects in HCC and inactivated CKAP2 and FAK-ERK pathway. All in all, this study corroborates that DARS-AS1 facilitates HCC proliferation and metastasis by regulating miR-3200-5p-mediated CKAP2, which provides a potential target for HCC diagnosis and treatment. Abbreviations: CCK-8: cell counting kit-8; CKAP2: Cytoskeleton associated protein 2; cDNA:complementary DNA; DAPI: 4ʹ,6-diamidino-2-phenylindole; DARS-AS1: DARS1 antisense RNA 1; DEPC: diethyl pyrocarbonate; DMEM-F12: Dulbecco’s minimal essential medium/Ham’s-F12; EMT: epithelial-mesenchymal transition; ERK: extracellular signal-regulated kinase; FAK: focal adhesion kinase; FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HCC: hepatocellular carcinoma; HE: hematoxylin-eosin; IHC: Immunohistochemistry; LIHC: Liver hepatocellular carcinoma; lncRNAs: long noncoding RNAs; MIAT: lncRNA myocardial infarction-related transcripts; MT: Mutant; NC: negative control; PBS: phosphate-buffered saline; PMSF: Phenylmethylsulfonyl fluoride; PVDF: polyvinylidene difluoride; RT: room temperature; RT-qPCR: real-time quantitative polymerase chain reaction; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SPF: specific pathogen-free; TMAP: tumor-associated microtubule-associated protein; TUNEL: TdT-mediated dUTP nick end labeling; V: volume; WT: wild type.

Silence of cytoskeleton-associated protein 2 represses cell proliferation and migration and promotes apoptosis in liver cancer cell lines.

To investigate the roles of cytoskeleton-associated protein 2 (CKAP2) in proliferation, apoptosis, and migration in liver cancer cells and the potential mechanisms. Human normal hepatocyte L02 and liver cancer cell lines HepG2, Huh7, and SMMC-7721 were cultured. The CKAP2 expression was detected by real-time PCR and Western blotting. HepG2 cells were randomly divided into a control group, a negative control (NC) group, and a CKAP2 silencing (siCKAP2) group. CCK-8 and BrdU assays were used to evaluate cell viability and proliferation, respectively. Transwell assay was employed to determine cell migration and invasion. The protein levels of cleaved-caspase 3, Bax, E-cadherin, N-cadherin, Vimentin, phosphorylated Janus kinase 2 (p-JAK2), and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) were determined by Western blotting. Compared with normal hepatocyte L02, CKAP2 was highly expressed in liver cancer cell lines HepG2, Huh7, and SMMC-7721 (all P<0.05). Compared with the NC group, cell viability and proliferation rate of the siCKAP2 group were decreased (both P<0.05). The apoptotic rate, protein expression of cleaved-caspase 3 and Bax in the siCKAP2 group were significantly higher than those in the NC group (all P<0.05). Compared with the NC group, cell migration and invasion rates of the siCKAP2 group were significantly attenuated (both P<0.05). Compared with the NC group, E-cadherin protein expression in siCKAP2 group was increased, while protein expression levels of Vimentin, N-cadherin, p-JAK2, and p-STAT3 were decreased (all P<0.05). CKAP2 gene silence inhibits proliferation, migration, and invasion, and promotes apoptosis in liver cancer cells, while JAK2/STAT3 signaling pathway may be involved in these processes.

Silencing of cytoskeleton-associated protein 2 represses cell proliferation and induces cell cycle arrest and cell apoptosis in osteosarcoma cells

Osteosarcoma is the most common primary bone malignancy, mainly occurring in children and adolescents. Cytoskeleton-associated protein 2 (CKAP2), which plays important roles in cell proliferation, has been reported to be overexpressed in diverse human cancers. In the present study, we aimed at exploring the expression and functions of CKAP2 in osteosarcoma. The mRNA and protein expression of CKAP2 was analyzed on collected osteosarcoma and control bone cyst tissues. The results indicated that CKAP2 expression was remarkably elevated in osteosarcoma tissues compared with bone cysts tissues. The expression level of CKAP2 in osteosarcoma was associated with overall survival, tumor size and tumor stage. In addition, down-regulation of CKAP2 by RNA interference in osteosarcoma cell lines, MG63 and SW1353, caused a remarkable inhibition in cell proliferation in vitro and xenograft growth in nude mice. Silencing of CKAP2 also significantly induced G0/G1 arrest and cell apoptosis of osteosarcoma cells. Furthermore, phosphorylation levels of Janus kinase 2 (JAK2) and Signal transducers and activators of transcription 3 (STAT3) were significantly reduced in CKAP2 knockdown cells. The expression of downstream targets of JAK2/STAT3 signaling, Cyclin D1, Bcl-2 and survivin, was also decreased in CKAP2 knockdown cells. Such aberrations can be rescued by re-expression of RNAi-resistant CKAP2. Collectively, the present study indicates that CKAP2 is a potential oncogene by targeting JAK2/STAT3 signaling, and that CKAP2 may serve as a novel target for osteosarcoma therapy.

CKAP2 Promotes Ovarian Cancer Proliferation and Tumorigenesis Through the FAK-ERK Pathway.

Upregulation of cytoskeleton-associated protein 2 (CKAP2) has been observed in ovarian cancer. This study aimed to determine the effects of overexpression of CKAP2 on cell proliferation and tumorigenesis and to explore the potential signal pathway in the oncogenic process. Expression of CKAP2 in tissue of ovarian cancer patients was detected. Human cancer cell line SKOV3 was overexpressed with CKAP2 and measured for cell growth and mobility. Moreover, CKAP2 effect was estimated using mouse tumor xenografts. mRNA expression levels of related genes were assessed. Expression levels of CKAP2 and p-ERK2 in SKOV3 cells were detected before and after treatment of focal adhesion kinase (FAK) inhibitor. mRNA and protein levels of CKAP2 were significantly elevated in human ovarian tumor tissues. Overexpression of CKAP2 in SKOV3 cells promoted cell proliferation and migration. Implantation of overexpressed SKOV3 cells showed significant tumorigenesis ability. mRNA expression levels of proliferating cell nuclear antigen (PCNA), matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), and SNAI1 were elevated in overexpressed SKOV3 cells. Cells with CKAP2 overexpression also had significantly increased p-ERK2 level. Moreover, FAK inhibitor treatment significantly decreased CKAP2 expression levels as well as the phosphorylation level of ERK2 in SKOV3 cells. This study suggests that overexpression of CKAP2 enhanced proliferation and mobility of carcinoma cells through the FAK-ERK2 signaling pathway.

CKAP2 (cytoskeleton-associated protein2) is a new prognostic marker in HER2-negative luminal type breast cancer.

BackgroundRecently, we reported cytoskeleton-associated protein2 (CKAP2) as a possible new prognostic breast cancer marker. However, it has not yet been applied in clinic. Therefore, clinical significance of CKAP2 was evaluated in comparison with that of Ki-67 in a cohort of breast cancer patients, and the expression difference was analyzed in cell cycle-arrested cancer and fibroblast cells.MethodsA total of 579 early breast cancer patients who underwent surgery at the National Cancer Center Hospital in Korea between 2001 and 2005 were accrued. CKAP2-positive cell count (CPCC) and Ki-67 labeling index (Ki-67LI) were evaluated by immunohistochemcal staining. The immunocytochemical staining patterns of CKAP2 and Ki-67 were analyzed in HeLa and human fibroblast cells after synchronization by double thymidine block.ResultsAlthough there was a significant correlation (R = 0.754, P < 0.001) between CPCC and Ki-67LI, only CPCC was correlated with DFS in overall population (HR, 2.029; 95% CI, 1.012–4.068; P = 0.046) and HER2-negative luminal subgroup (HR, 3.984; 95% CI, 1.350–11.762; P = 0.012) by multivariate analysis. In immunocytochemical staining, more than 50% of serum-starved or non-mitotic cell phase HeLa cells were positive for Ki-67, in comparison to the low CKAP2-positivity, which might explain the prognostic difference between CPCC and Ki-67LI.ConclusionsThe current study showed that CPCC but not Ki-67LI is an independent prognostic indicator in early breast cancer, more specifically in HER2-negative luminal breast cancer. The difference between two markers may be related to the lower background expression of CKAP2 in cancer cells.

Involvement of FAK-ERK2 signaling pathway in CKAP2-induced proliferation and motility in cervical carcinoma cell lines

Cervical carcinoma is the fourth most common cause of death in woman, caused by human papillomavirus (HPV) infections and arising from the cervix. Cytoskeleton-associated protein 2 (CKAP2), also known as tumor-associated microtubule-associated protein, has been linked to tumorigenic effects. In the present study, we screened CKAP2 as a new candidate gene which promotes development of cervical carcinoma, in two independent datasets (TCGA and GSE27678). Results showed that CKAP2 expression was significantly up-regulated in cervical cancerous tissues compared with normal counterparts. Gene set enrichment analysis (GSEA) showed that metastasis, cell cycle and FAK pathways were related with elevated CKAP2 expression. Knockdown of CKAP2 expression significantly inhibited cell proliferation, migration and invasion both in HeLa and C-33A cells. And depletion of CKAP2 down-regulated the expression of metastasis and cell cycle related proteins as well as the phosphorylation of ERK2 (p-ERK2), except E-cadherin. In vivo experiment revealed that knockdown of CKAP2 inhibited C-33A cells proliferation. However, FAK inhibitor PF-562271 and ERK2 inhibitor VX-11e treatment significantly inhibited CKAP2 overexpression-induced cell proliferation, migration and invasion in SiHa cells. In conclusion, our study suggests that CKAP2 acts as a functional oncogene in cervical carcinoma development and may exert its function by targeting FAK-ERK2 signaling pathway.

CKAP2 is necessary to ensure the faithful spindle bipolarity in a dividing diploid hepatocyte

Spindle bipolarity is crucial for segregating chromosome during somatic cell division. Previous studies have suggested that cytoskeleton associated protein 2 (CKAP2) is involved in spindle assembly and chromosome segregation. In this study, we show that CKAP2-depleted primary hepatocytes exhibit over-duplicated centrosomes with disjoined chromosomes from metaphase plate. These cells proceed to apoptosis or multipolar cell division and subsequent apoptotic cell death. In addition, a mouse liver regeneration experiment showed a marked decrease in efficiency of hepatic regeneration in CKAP2-depleted liver. These data suggest a physiological role of CKAP2 in the formation of spindle bipolarity, which is necessary for maintaining chromosomal stability.

Abstract P5-08-25: CKAP2 (cytoskeleton associated protein 2) is a new prognostic marker in HER2-negative luminal breast cancer

Abstract Background: Ki-67 has been increasingly used as a prognostic marker in spite of debates on the evaluation methods and inconsistent results on its clinical values. CKAP2 is a microtubule-associated protein which plays key roles in microtubule assembly and disassembly. In the present study, the clinical significance of CKAP2-positive cells was evaluated and compared with the results of Ki-67 positive cells. Methods: A total of 579 early breast cancer patients who underwent surgery at the National Cancer Center Hospital between 2001 and 2005 were accrued. The proliferation activity was measured by CKAP2-positive cell count (CPCC) and Ki-67 labeling index (Ki-67 LI) using CKAP2 and Ki-67 antibodies, respectively, by immunohistochemcial staining on FFPE tumor tissue. The correlation of CPCC or Ki-67 LI with recurrence free survival (RFS) was analyzed. The immunofluorescent staining was performed on HeLa cells after synchronization by double thymidine block to compare the patterns between CKAP2 and Ki-67. Results: The CPCC (median, 8 with the range of 0- 170) and Ki-67 LI (median, 10.2 with the range of 0%- 91.7%) were highly correlated (R = 0.754, P &amp;lt; 0.001). While CPCC was marginally significant in multivariate analysis for RFS in all cases, it was a significant variable for RFS in the subset analysis with HER2-negative luminal breast cancer patients (HR, 3.154; 95% CI, 1.154-10.693; P = 0.027). On the contrary, Ki-67 LI failed to show any correlation with RFS in all or any subgroups. In the analysis on HeLa cells, CKAP2 staining was more specific to cells in metaphase than Ki-67 staining. Conclusions: CPCC can be an independent prognostic factor specifically in a HER2-negative luminal type of breast cancer. In addition, CPCC appears to be superior to Ki-67 LI as a survival indicator which may be related to the restricted expression pattern of CKAP2 in metaphase cells. Further study is warranted. Citation Format: Sim SH, Bae C-D, Kwon Y, Park IH, Lee KS, Jung S-Y, Lee S, Kang H-S, Lee ES, Kim H-S, Hong K-M, Ro J. CKAP2 (cytoskeleton associated protein 2) is a new prognostic marker in HER2-negative luminal breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-08-25.

Clinical implications of proliferation activity in T1 or T2 male gastric cancer patients.

Proliferation activity has already been established as a prognostic marker or as a marker for anticancer drug sensitivity. In gastric cancer, however, the prognostic significance of proliferation activity is still being debated. Several studies evaluating proliferation activity using Ki-67 have shown controversial results in terms of the relationship between proliferation activity and overall survival (OS) or drug sensitivity in gastric cancer patients. Because cytoskeleton-associated protein 2 (CKAP2) staining has recently been introduced as a marker of proliferation activity, we analyzed 437 gastric cancer tissues through CKAP2 immunohistochemistry, and we evaluated the chromatin CKAP2-positive cell count (CPCC) for proliferation activity. Although the CPCC did not show any significant correlation with OS in the male, female or total number of cases, it did show a significant correlation in the T1 or T2 male patient subgroup, according to log-rank tests (P=0.001) and univariate analysis (P=0.045). Additionally, multivariate analysis with the Cox proportional hazard regression model showed a significant correlation between the CPCC and OS (P=0.039) for the co-variables of age, gender, T stage, N stage, histology, tumor location, tumor size and adjuvant chemotherapy. In male gastric cancer cell lines, faster-growing cancer cells showed higher sensitivity to cisplatin than slow-growing cells. Thus our study indicates that CPCC-measured proliferation activity demonstrates a significantly worse prognosis in T1 or T2 male gastric cancer patients. The CPCC will help to more precisely classify gastric cancer patients and to select excellent candidates for adjuvant chemotherapy, which in turn will facilitate further clinical chemotherapeutic trials.

Cytoskeleton-associated protein 2 is a potential predictive marker for risk of early and extensive recurrence of hepatocellular carcinoma after operative resection

De principe transplantation is an attractive strategy for the treatment of patients with hepatocellular carcinoma (HCC). The most important issue for this strategy is how to predict the risk of early and extensive recurrence. The present study aimed to identify a molecule associated with early and extensive recurrence of HCC after resection. Differentially expressed genes were screened by DNA microarray analysis with the use of 12 HCC samples from patients who had different clinical courses based on the timing and extent of recurrence after operative resection. Furthermore, the obtained results were validated in 60 independent samples by quantitative real-time reverse transcription-polymerase chain reaction. Immunohistochemistry was performed to assess gene expression at the protein level. Microarray analysis and quantitative reverse transcription-polymerase chain reaction revealed cytoskeleton-associated protein 2 (CKAP2) as a candidate gene associated with early and extensive recurrence of HCC after resection. This observation was confirmed through examination of independent set samples, in which patients with greater-level CKAP2 mRNA expression exhibited shorter recurrence-free survival. Immunohistochemistry showed CKAP2 protein expression was associated with early (≤3years) and extensive recurrence (beyond Milan criteria) after operative resection. Immunohistochemical CKAP2 expression might be a potential biologic marker for identifying HCC patients at risk of early and extensive recurrence after operative resection.

Condensed chromatin staining of CKAP2 as surrogate marker for mitotic figures

Proliferation activity has long been known to be one of the strongest prognostic factors in many different cancers. Nevertheless, microscopic evaluation of mitotic figures remains time-consuming and, furthermore, is relatively subjective. As the expression of cytoskeleton-associated protein 2 (CKAP2) is closely related to the mitotic phase, CKAP2 was evaluated as a surrogate mitotic figure (MF) marker. A monoclonal antibody specific to human CKAP2 was produced, and immunohistochemistry was performed on normal tissue array sections and 30 breast cancer tissues. The expression of CKAP2 in the normal human tissues was limited to well-known cell proliferation zones. Strong, readily visible, condensed chromatin staining of CKAP2 was observed specifically in mitotic cells, and the number of these cells was tightly correlated with the MF count in breast cancer tissues (P<0.001, ρ=0.743), suggesting its usefulness as a surrogate marker for MF counting. Immunohistochemical staining with CKAP2 monoclonal antibody can be considered to be a new, effective approach to the assessment of proliferation activity in cancer tissues.

Specific primary sequence requirements for Aurora B kinase-mediated phosphorylation and subcellular localization of TMAP during mitosis

During mitosis, regulation of protein structures and functions by phosphorylation plays critical roles in orchestrating a series of complex events essential for the cell division process. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a novel player in spindle assembly and chromosome segregation. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis. However, the mechanisms and functional importance of phosphorylation at most of the sites identified are currently unknown. Here, we report that TMAP is a novel substrate of the Aurora B kinase. Ser627 of TMAP was specifically phosphorylated by Aurora B both in vitro and in vivo. Ser627 and neighboring conserved residues were strictly required for efficient phosphorylation of TMAP by Aurora B, as even minor amino acid substitutions of the phosphorylation motif significantly diminished the efficiency of the substrate phosphorylation. Nearly all mutations at the phosphorylation motif had dramatic effects on the subcellular localization of TMAP. Instead of being localized to the chromosome region during late mitosis, the mutants remained associated with microtubules and centrosomes throughout mitosis. However, the changes in the subcellular localization of these mutants could not be completely explained by the phosphorylation status on Ser627. Our findings suggest that the motif surrounding Ser627 (625RRSRRL630) is a critical part of a functionally important sequence motif which not only governs the kinase-substrate recognition, but also regulates the subcellular localization of TMAP during mitosis.