Cystatin M loss is associated with the losses of estrogen receptor, progesterone receptor, and HER4 in invasive breast cancer

Ko, Eunkyung,Park, Seong-Eun,Cho, Eun Yoon,Kim, Yujin,Hwang, Jung-Ah,Lee, Yeon-Su,Nam, Seok Jin,Bang, Saik,Park, Joobae,Kim, Duk-Hwan BioMed Central 2010 Breast cancer research Vol.12 No.6

<P><B>Introduction</B></P><P>This study was aimed at understanding the clinicopathological significance of cystatin M loss, and investigating possible factors responsible for cystatin M loss in breast cancer.</P><P><B>Methods</B></P><P>The expression of estrogen receptor (ER), progesterone receptor (PR), HER2, HER4, and cystatin M was retrospectively analyzed using immunohistochemistry in 117 patients with ductal carcinoma <I>in situ </I>(DCIS) and in 175 patients with invasive breast cancer (IBC). The methylation status of <I>CST6 </I>gene encoding cystatin M was evaluated using methylation-specific polymerase chain reaction (PCR) in formalin-fixed paraffin-embedded tissues from 292 participants and using pyrosequencing in fresh-frozen tumor and matched normal tissues from 51 IBC patients.</P><P><B>Results</B></P><P>Cystatin M loss was found in 9 (8%) of 117 patients with DCIS and in 99 (57%) of 175 with invasive breast cancer (IBC) (<I>P </I>< 0.0001). Cystatin M loss was found in 58 (57%) of 101 HER2-negative IBCs and in 41 (55%) of 74 HER2-positive IBCs, and this difference was not statistically significant (<I>P </I>= 0.97). However, cystatin M loss was significantly associated with the loss of ER (<I>P </I>= 0.01), PR (<I>P </I>= 0.002), and HER4 (<I>P </I>= 0.003) in IBCs. Cystatin M loss occurred in 34 (76%) of the 45 HER4-negative IBCs and in 65 (50%) of the 130 HER4-positive IBCs. Multivariate analysis showed that cystatin M loss occurred at a 3.57 times (95% CI = 1.28 to 9.98; <I>P </I>= 0.01) higher prevalence in the triple-negative IBCs of ER, PR, and HER4 than in other subtypes, after adjusting for age. The quantity of <I>CST6 </I>methylation was associated with ER loss (<I>P </I>= 0.0002) in IBCs but not with the loss of PR (<I>P </I>= 0.64) or HER4 (<I>P </I>= 0.87).</P><P><B>Conclusions</B></P><P>The present study suggests that cystatin M loss may be associated with the losses of ER, PR, and HER4 in IBC.</P>

Functional Importance of the Anaphase-Promoting Complex-Cdh1-Mediated Degradation of TMAP/CKAP2 in Regulation of Spindle Function and Cytokinesis

Hong, Kyung Uk,Park, Young Soo,Seong, Yeon-Sun,Kang, Dongmin,Bae, Chang-Dae,Park, Joobae American Society for Microbiology 2007 Molecular and cellular biology Vol.27 No.10

<B>ABSTRACT</B><P>Cytoskeleton-associated protein 2 (CKAP2), also known as tumor-associated microtubule-associated protein (TMAP), is a novel microtubule-associated protein that is frequently upregulated in various malignances. However, its cellular functions remain unknown. A previous study has shown that its protein level begins to increase during G1/S and peaks at G2/M, after which it decreases abruptly. Ectopic overexpression of TMAP/CKAP2 induced microtubule bundling related to increased microtubule stability. TMAP/CKAP2 overexpression also resulted in cell cycle arrest during mitosis due to a defect in centrosome separation and subsequent formation of a monopolar spindle. We also show that degradation of TMAP/CKAP2 during mitotic exit is mediated by the anaphase-promoting complex bound to Cdh1 and that the KEN box motif near the N terminus is necessary for its destruction. Compared to the wild type, expression of a nondegradable mutant of TMAP/CKAP2 significantly increased the occurrence of spindle defects and cytokinesis failure. These results suggest that TMAP/CKAP2 plays a role in the assembly and maintenance of mitotic spindles, presumably by regulating microtubule dynamics, and its destruction during mitotic exit serves an important role in the completion of cytokinesis and in the maintenance of spindle bipolarity in the next mitosis.</P>

TMAP/CKAP2 is essential for proper chromosome segregation.

Hong, Kyung Uk,Kim, Eunhee,Bae, Chang-Dae,Park, Joobae Landes Bioscience 2009 Cell Cycle Vol.8 No.2

<P>Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2), is a novel mitotic spindle-associated protein which is frequently up-regulated in various malignances. However, its cellular functions remain unknown. Previous reports suggested that the cellular functions of TMAP/CKAP2 pertain to regulation of the dynamics and assembly of the mitotic spindle. To investigate its role in mitosis, we studied the effects of siRNA-mediated depletion of TMAP/CKAP2 in cultured mammalian cells. Unexpectedly, TMAP/CKAP2 knockdown did not result in significant alterations of the spindle apparatus. However, TMAP/CKAP2-depleted cells often exhibited abnormal nuclear morphologies, which were accompanied by abnormal organization of the nuclear lamina, and chromatin bridge formation between two daughter cell nuclei. Time lapse video microscopy revealed that the changes in nuclear morphology and chromatin bridge formations observed in TMAP/CKAP2-depleted cells are the result of defects in chromosome segregation. Consistent with this, the spindle checkpoint activity was significantly reduced in TMAP/CKAP2-depleted cells. Moreover, chromosome missegregation induced by depletion of TMAP/CKAP2 ultimately resulted in reduced cell viability and increased chromosomal instability. Our present findings demonstrate that TMAP/CKAP2 is essential for proper chromosome segregation and for maintaining genomic stability.</P>

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

Kim, Hyun-Jun,Kwon, Hye-Rim,Bae, Chang-Dae,Park, Joobae,Hong, Kyung U Landes Bioscience 2010 Cell Cycle Vol.9 No.10

<P>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 ((625)RRSRRL(630)) 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.</P>

Promoter hypermethylation of the p16 gene is associated with poor prognosis in recurrent early-stage hepatocellular carcinoma.

Ko, Eunkyung,Kim, Yujin,Kim, Sung-Joo,Joh, Jae-Won,Song, SangYong,Park, Cheol-Keun,Park, Joobae,Kim, Duk-Hwan American Association for Cancer Research 2008 Cancer epidemiology, biomarkers & prevention Vol.17 No.9

<P>Despite significant advances in the detection and treatment of hepatocellular carcinoma, the prognosis of patients with hepatocellular carcinoma remains very poor, in part due to the high incidence of recurrence. This study was aimed at identifying a prognostic indicator of recurrence in patients with hepatocellular carcinoma. We retrospectively analyzed CpG island hypermethylation of the p14, p15, p16, GSTP1, integrin alpha4, SYK, and CDH1 genes in fresh-frozen tissues from 265 patients with hepatocellular carcinoma using the methylation-specific PCR. The expression levels of p16 and p53 were evaluated by immunohistochemistry. CpG island hypermethylation was detected in 6% for p14, 21% for p15, 67% for p16, 75% for GSTP1, 23% for integrin alpha4, 12% for SYK, and 57% for CDH1. Recurrence was observed in 102 (38%) of the 265 patients. There was no association between the risk for recurrence and hypermethylation of any gene studied. However, p16 methylation was associated with a poor survival after surgery for recurrent stage I to II hepatocellular carcinomas (hazard ratio, 4.05; 95% confidence interval, 1.15-14.20; P = 0.03). In addition, the hazard of failure after recurrence was about 3.80 (95% confidence interval, 1.03-14.20; P = 0.04) times higher in patients with p16 methylation than in those without. Negative expression of p16 at a protein level was also associated with poor survival in recurrent stage I to II hepatocellular carcinomas, but p53 expression did not have a synergistic effect on the poor prognosis. In conclusion, the present study suggests that p16 methylation may be associated with a poor prognosis in recurrent early-stage hepatocellular carcinomas.</P>

Association of Global Levels of Histone Modifications with Recurrence-Free Survival in Stage IIB and III Esophageal Squamous Cell Carcinomas.

I, Hoseok,Ko, Eunkyung,Kim, Yujin,Cho, Eun Yoon,Han, Joungho,Park, Joobae,Kim, Kwhanmien,Kim, Duk-Hwan,Shim, Young Mog American Association for Cancer Research 2010 Cancer Epidemiology, Biomarkers & Prevention Vol.19 No.2

<P>This study was aimed at understanding the effects of histone modifications on recurrence-free survival (RFS) after esophagectomy in esophageal squamous cell carcinoma (ESCC). The acetylation of histone H3 lysine (H3K9Ac), histone H3 lysine 18 (H3K18Ac), and histone H4 lysine 12 (H4K12Ac), and the dimethylation of histone H3 lysine 9 (H3K9diMe) and histone H4 arginine 3 (H4R3diMe) were analyzed by immunohistochemistry in 237 ESCCs. The K-means clustering algorithm was used to identify unique patterns of histone modifications. At a median follow-up of 5.1 years, 109 (46%) of 237 patients had developed recurrence of disease. Mean global levels of H3K9Ac, H3K18Ac, H3K9diMe, H4K12Ac, and H4R3diMe were 81.5%, 65.1%, 80.3%, 45.9%, and 27.4%, respectively. In the analysis of individual histones, a 1% increase in the global level of H3K18Ac in pathologic stage III worsened RFS at 1.009 times [95% confidence interval (CI), 1.001-1.016; P = 0.03], after adjusting for age, sex, and operative method. Cluster analysis also showed significant effects of histone modifications on RFS. For stage IIB cancers, Cox proportional hazards analysis showed that RFS of cluster 1, with high global levels of H3K18Ac and H4R3diMe, was 2.79 times poorer (95% CI, 1.14-6.27; P = 0.008) than that of cluster 2, with low levels. RFS for stage III cancers was also poorer in cluster 1 than cluster 2 (adjusted hazard ratio, 2.42; 95% CI, 1.10-5.34; P = 0.02). In conclusion, the present study suggests that global levels of histone modifications in ESCC may be an independent prognostic factor of RFS. Cancer Epidemiol Biomarkers Prev; 19(2); 566-73.</P>

Synergistic effect of Bcl-2 and cyclin A2 on adverse recurrence-free survival in stage I non-small cell lung cancer.

Ko, Eunkyung,Kim, Yujin,Cho, Eun Yoon,Han, Jungho,Shim, Young Mog,Park, Joobae,Kim, Duk-Hwan Raven Press 2013 Annals of surgical oncology Vol.20 No.3

<P>The prognostic significance of cyclin A2 overexpression in non-small cell lung cancer (NSCLC) is controversial.</P>

Downregulation of regenerating islet-derived 3 alpha (REG3A)in primary human gastric adenocarcinomas

최봉근,Woo-Ho Kim,Laurence Christa,Paris Descartes),Joobae Park,Chang-Dae Bae 생화학분자생물학회 2007 Experimental and molecular medicine Vol.39 No.6

Gastric carcinoma is considered to be one of the most prevalent cancers worldwide. We have performed differential- display polymerase chain reaction (DD-PCR) in order to compare the gene expression profile of gastric carcinoma and that of a normal stomach, in an attempt to identifiy differentially expressed genes associated with primary human gastric cancers. One of the down-regulated genes in gastric cancers was identified as regenerating islet-derived 3 alpha (REG3A), also known as hepatocarcinoma-intestine-pancreas/ pancreatitis-associated protein (HIP/PAP). REG3A exhibited relatively high expression levels in normal gastric mucosa. However, REG3A was found to be down-regulated in 67% (20 out of 30 samples) of primary human gastric cancers, as determined by RT-PCR. In addition, REG3A mRNA expression was not detected in stomach cancer cell lines, SNU cells. Immunohistochemical analysis further confirmed the down-regulation of REG3A expression in primary human gastric cancers. Treatment with the demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC) resulted in the restoration of REG3A mRNA expression in the gastric cancer cell line, indicating that the transcriptional silencing of REG3A in SNU cell lines was caused by DNA methylation. Taken together, these data indicate that REG3A is down-regulated in most primary human gastric cancer cells, and might be useful in the diagnosis of gastric cancer. Further characterization of the differentially expressed gene, REG3A, should lead to a better understanding of the changes occurring at the molecular level during the development and progression of primary human gastric cancer.

Cdk1-cyclin B1-mediated phosphorylation of tumor-associated microtubule-associated protein/cytoskeleton-associated protein 2 in mitosis.

Hong, Kyung Uk,Kim, Hyun-Jun,Kim, Hyo-Sil,Seong, Yeon-Sun,Hong, Kyeong-Man,Bae, Chang-Dae,Park, Joobae American Society for Biochemistry and Molecular Bi 2009 The Journal of biological chemistry Vol.284 No.24

<P>During mitosis, establishment of structurally and functionally sound bipolar spindles is necessary for maintaining the fidelity of chromosome segregation. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a mitotic spindle-associated protein whose level is frequently up-regulated in various malignancies. Previous reports have suggested that TMAP is a potential regulator of mitotic spindle assembly and dynamics and that it is required for chromosome segregation to occur properly. So far, there have been no reports on how its mitosis-related functions are regulated. Here, we report that TMAP is hyper-phosphorylated at the C terminus specifically during mitosis. At least four different residues (Thr-578, Thr-596, Thr-622, and Ser-627) were responsible for the mitosis-specific phosphorylation of TMAP. Among these, Thr-622 was specifically phosphorylated by Cdk1-cyclin B1 both in vitro and in vivo. Interestingly, compared with the wild type, a phosphorylation-deficient mutant form of TMAP, in which Thr-622 had been replaced with an alanine (T622A), induced a significant increase in the frequency of metaphase cells with abnormal bipolar spindles, which often displayed disorganized, asymmetrical, or narrow and elongated morphologies. Formation of these abnormal bipolar spindles subsequently resulted in misalignment of metaphase chromosomes and ultimately caused a delay in the entry into anaphase. Moreover, such defects resulting from the T622A mutation were associated with a decrease in the rate of protein turnover at spindle microtubules. These findings suggest that Cdk1-cyclin B1-mediated phosphorylation of TMAP is important for and contributes to proper regulation of microtubule dynamics and establishment of functional bipolar spindles during mitosis.</P>

Cohypermethylation of <i>p14</i> in combination with <i>CADM1</i> or <i>DCC</i> as a recurrence‐related prognostic indicator in stage I esophageal squamous cell carcinoma

Lee, Eunju,Lee, Bo Bin,Ko, Eunkyung,Kim, Yujin,Han, Jungho,Shim, Young Mog,Park, Joobae,Kim, Duk‐,Hwan Wiley Subscription Services, Inc., A Wiley Company 2013 Cancer Vol.119 No.9

<P><B>Abstract</B></P><P><B>BACKGROUND:</B></P><P>The objective of this study was to discover molecular biomarkers associated with the recurrence of esophageal squamous cell carcinoma (ESCC).</P><P><B>METHODS:</B></P><P>The authors retrospectively analyzed the hypermethylation status of 11 genes using methylation‐specific polymerase chain reaction (PCR) and the expression of epidermal growth factor receptor (EGFR), O‐6 methylguanine‐DNA methyltransferase (MGMT), tumor protein 53 (p53), and transforming growth factor β (TGFβ) using immunohistochemistry in 329 formalin‐fixed, paraffin‐embedded ESCCs.</P><P><B>RESULTS:</B></P><P>Recurrence was identified in 151 of 329 ESCCs (46%) at a median follow‐up of 4.5 years. The recurrence was associated with hypermethylation of the genes cell adhesion molecule 1 (<I>CADM1</I>) (<I>P</I> = .003), deleted in colon carcinoma (<I>DCC</I>) (<I>P</I> = .04), or cyclin‐dependent kinase inhibitor 2A (<I>p14</I>) (<I>P</I> = .02) in patients with stage I ESCC. Thirty‐six of 37 Stage I ESCCs (97%) that had cohypermethylation of at least 2 of the 3 genes had hypermethylation of <I>p14</I> plus either <I>CADM1</I> or <I>DCC</I> or both <I>CADM1</I> and <I>DCC</I>. The 5‐year recurrence‐free survival (RFS) rates were 93% in patients who had stage I disease without hypermethylation of the 3 genes and 56% in those who had cohypermethylation of <I>p14</I> in combination with <I>CADM1</I> and/or <I>DCC</I>. Patients who had stage I ESCC with cohypermethylation of <I>p14</I> in combination with <I>DCC</I> and/or <I>CADM1</I> had 7.13 times (95% confidence interval, 1.61‐31.64 times; <I>P</I> = .009) poorer RFS compared with those who had no hypermethylation of the 3 genes after adjusting confounding factors. Hypermethylation of the other 8 genes and altered expression of 4 proteins were not associated with recurrence across pathologic stages.</P><P><B>CONCLUSIONS:</B></P><P>The current results suggested that cohypermethylation of <I>p14</I> in combination with <I>DCC</I> and/or <I>CADM1</I> may be an independent prognostic factor for recurrence in patients with stage I ESCC. Cancer 2013. © 2013 American Cancer Society.</P>