CD3Z hypermethylation is associated with severe clinical manifestations in systemic lupus erythematosus and reduces CD3ζ-chain expression in T cells.

Objective: The importance of hypomethylation in SLE is well recognized; however, the significance of hypermethylation has not been well characterized. We screened hypermethylated marks in SLE and investigated their possible implications. Methods: DNA methylation marks were screened in SLE whole-blood DNA by microarray, and two marks ( CD3Z and VHL hypermethylations) were confirmed by a methylation single-base extension method in two independent ethnic cohorts consisting of 207 SLE patients and 151 controls. The correlation with clinical manifestations and the genetic influence on those epigenetic marks were analysed. Results: Two epigenetic marks, CD3Z and VHL hypermethylation, were significantly correlated with SLE: CD3Z hypermethylation (odds ratio = 7.76; P = 1.71 × 10 -13 ) and VHL hypermethylation (odds ratio = 3.77; P = 3.20 × 10 -8 ), and the increased CD3Z methylation was correlated with downregulation of the CD3ζ-chain in SLE T cells. In addition, less genetic influence on CD3Z methylation relative to VHL methylation was found in analyses of longitudinal and twin samples. Furthermore, a higher CD3Z methylation level was significantly correlated with a higher SLE disease activity index and more severe clinical manifestations, such as proteinuria, haemolytic anaemia and thrombocytopenia, whereas VHL hypermethylation was not. Conclusion: CD3Z hypermethylation is an SLE risk factor that can be modified by environmental factors and is associated with more severe SLE clinical manifestations, which are related to deranged T cell function by downregulating the CD3ζ-chain.

Down-regulation of lipid raft-associated onco-proteins via cholesterol-dependent lipid raft internalization in docosahexaenoic acid-induced apoptosis.

Lipid rafts, plasma membrane microdomains, are important for cell survival signaling and cholesterol is a critical lipid component for lipid raft integrity and function. DHA is known to have poor affinity for cholesterol and it influences lipid rafts. Here, we investigated a mechanism underlying the anti-cancer effects of DHA using a human breast cancer cell line, MDA-MB-231. We found that DHA decreased cell surface levels of lipid rafts via their internalization, which was partially reversed by cholesterol addition. With DHA treatment, caveolin-1, a marker for rafts, and EGFR were colocalized with LAMP-1, a lysosomal marker, in a cholesterol-dependent manner, indicating that DHA induces raft fusion with lysosomes. DHA not only displaced several raft-associated onco-proteins, including EGFR, Hsp90, Akt, and Src, from the rafts but also decreased total levels of those proteins via multiple pathways, including the proteasomal and lysosomal pathways, thereby decreasing their activities. Hsp90 overexpression maintained its client proteins, EGFR and Akt, and attenuated DHA-induced cell death. In addition, overexpression of Akt or constitutively active Akt attenuated DHA-induced apoptosis. All these data indicate that the anti-proliferative effect of DHA is mediated by targeting of lipid rafts via decreasing cell surface lipid rafts by their internalization, thereby decreasing raft-associated onco-proteins via proteasomal and lysosomal pathways and decreasing Hsp90 chaperone function.

p53 acetylation enhances Taxol-induced apoptosis in human cancer cells.

Microtubule inhibitors (MTIs) such as Taxol have been used for treating various malignant tumors. Although MTIs have been known to induce cell death through mitotic arrest, other mechanisms can operate in MTI-induced cell death. Especially, the role of p53 in this process has been controversial for a long time. Here we investigated the function of p53 in Taxol-induced apoptosis using p53 wild type and p53 null cancer cell lines. p53 was upregulated upon Taxol treatment in p53 wild type cells and deletion of p53 diminished Taxol-induced apoptosis. p53 target proteins including MDM2, p21, BAX, and ß-isoform of PUMA were also upregulated by Taxol in p53 wild type cells. Conversely, when the wild type p53 was re-introduced into two different p53 null cancer cell lines, Taxol-induced apoptosis was enhanced. Among post-translational modifications that affect p53 stability and function, p53 acetylation, rather than phosphorylation, increased significantly in Taxol-treated cells. When acetylation was enhanced by anti-Sirt1 siRNA or an HDAC inhibitor, Taxol-induced apoptosis was enhanced, which was not observed in p53 null cells. When an acetylation-defective mutant of p53 was re-introduced to p53 null cells, apoptosis was partially reduced compared to the re-introduction of the wild type p53. Thus, p53 plays a pro-apoptotic role in Taxol-induced apoptosis and acetylation of p53 contributes to this pro-apoptotic function in response to Taxol in several human cancer cell lines, suggesting that enhancing acetylation of p53 could have potential implication for increasing the sensitivity of cancer cells to Taxol.

Cyclin A regulates a cell-cycle-dependent expression of CKAP2 through phosphorylation of Sp1.

CKAP2 plays crucial roles in proper chromosome segregation and maintaining genomic stability. CKAP2 protein showed cell-cycle-dependent expression, which reached a maximum level at the G2/M phase and disappeared at the onset of G1 phase. To elucidate the mechanisms underlying cell cycle-dependent expression of CKAP2, we cloned and analyzed the human CKAP2 promoter. The upstream 115-bp region from the transcription start site was sufficient for minimal CKAP2 promoter activity. We identified 2 regulatory sequences; a CHR (-110 to -104 bp) and a GC box (-41 to -32 bp). We confirmed Sp1 bound to the GC box using a supershift assay and a ChIP assay. Mutation in the GC box resulted in a near complete loss of CKAP2 promoter activity while mutation in the CHR decreased the promoter activity by 50%. The CHR mutation showed enhanced activity at the G1/S phase, but still retained cyclic activity. The Chromatin IP revealed that the amount of Sp1 bound to the GC box gradually increased and reached a maximum level at the G2/M phase. The amount of Sp1 bound to the GC box was greatly reduced when Cyclin A was depleted, which was restored by adding Cyclin A/Cdk2 complex back into the nuclear extracts. Together, we concluded that the GC box was responsible for the cyclic activity of human CKAP2 promoter through the phosphorylation of Sp1, possibly by Cyclin A/Cdk complex.

Transglutaminase 2 as an independent prognostic marker for survival of patients with non-adenocarcinoma subtype of non-small cell lung cancer.

BACKGROUND: Expression of transglutaminase 2 (TGase 2) is related to invasion and resistance to chemotherapeutic agents in several cancer cells. However, there has been only limited clinical validation of TGase 2 as an independent prognostic marker in cancer. METHODS: The significance of TGase 2 expression as an invasive/migratory factor was addressed by in vitro assays employing down-regulation of TGase 2. TGase 2 expression as a prognostic indicator was assessed in 429 Korean patients with early-stage non-small cell lung cancer (NSCLC) by immunohistochemical staining. RESULTS: TGase 2 expression increased the invasive and migratory properties of NSCLC cells in vitro, which might be related to the induction of MMP-9. In the analysis of the immunohistochemical staining, TGase 2 expression in tumors was significantly correlated with recurrence in NSCLC (p = 0.005) or in the non-adenocarcinoma subtype (p = 0.031). Additionally, a multivariate analysis also showed a significant correlation between strong TGase 2 expression and shorter disease-free survival (DFS) in NSCLC (p = 0.029 and HR = 1.554) and in the non-adenocarcinoma subtype (p = 0.030 and HR = 2.184). However, the correlation in the adenocarcinoma subtype was not significant. CONCLUSIONS: TGase 2 expression was significantly correlated with recurrence and shorter DFS in NSCLC, especially in the non-adenocarcinoma subtype including squamous cell carcinoma.

Reply to Krupenko et al. Comment on "Lee et al. The Combination of Loss of ALDH1L1 Function and Phenformin Treatment Decreases Tumor Growth in KRAS-Driven Lung Cancer Cancers 2020, 12, 1382".

In the Cancers paper, we observed the increase ALDH1L1 protein expression following oncogenesis, as well as a therapeutic effect, by deleting the Aldh1l1 gene in KrasLA2 mice, a model of spontaneous non-small cell lung cancer (NSCLC) [...].

CCL3L3-null status is associated with susceptibility to systemic lupus erythematosus.

The correlation between copy number variation (CNV) and the susceptibility to systemic lupus erythematosus (SLE) has been reported for various immunity-related genes. However, the contribution of CNVs to SLE susceptibility awaits more investigation. To evaluate the copy numbers in immunity-related genes such as TNFAIP3, TNIP1, IL12B, TBX21 (T-bet), TLR7, C4A, C4B, CCL3L1, and CCL3L3, the modified real competitive polymerase chain reaction (mrcPCR) assay was employed, and the association between the copy numbers and SLE susceptibility was analyzed in 334 SLE patients and 338 controls. CCL3L3-null status was significantly associated with SLE susceptibility (OR > 18, P < 0.0001), which remained significant by Bonferroni's correction (corrected P = 0.0007). However, the significant association between C4B low-copy status and SLE susceptibility (OR = 1.6051, P = 0.0331) became non-significant by Bonferroni's correction (corrected P = 0.3938). Except for these results, no other significant association between SLE susceptibility and copy number status in other genes was observed. The CCL3L3-null status may be a significant factor for SLE susceptibility.

Mesenchymal stromal cells inhibit graft-versus-host disease of mice in a dose-dependent manner.

BACKGROUND AIMS: Graft-versus-host disease (GvHD) remains a major complication after allogeneic hematopoietic cell transplantation (HCT). Recent literature demonstrates a potential benefit of human mesenchymal stromal cells (MSC) for the treatment of refractory GvHD; however, the optimal dose remains uncertain. We set out to develop an animal model that can be used to study the effect of MSC on GvHD. METHODS: A GvHD mouse model was established by transplanting C3H/he donor bone marrow (BM) cells and spleen cells into lethally irradiated BALB/c recipient mice. MSC were obtained from C3H/he mice and the C3H/10T1/2 murine MSC line. RESULTS: The mRNA expression of Foxp3 in regional lymph nodes (LN) localized with T cells was markedly increased by the addition of C3H10T1/2 cells in a real-time polymerase chain reaction (PCR). Using a mixed lymphocyte reaction, we determined the optimal splenocyte proliferation inhibition dose (MSC:splenocyte ratios 1:2 and 1:1). Three different C3H10T1/2 cell doses (low, 0.5 x 10(6), intermediate, 1 x 10(6), and high, 2 x 10(6)) with a consistent splenocyte dose (1 x 10(6)) were evaluated for their therapeutic potential in an in vivo GvHD model. The clinical and histologic GvHD score and Kaplan-Meier survival rate were improved after MSC transplantation, and these results demonstrated a dose-dependent inhibition. CONCLUSIONS: We conclude that MSC inhibit GvHD in a dose-dependent manner in this mouse model and this model can be used to study the effects of MSC on GvHD.

Ectopic Overexpression of Coiled-Coil Domain Containing 110 Delays G2/M Entry in U2-OS Cells.

Coiled-coil domain containing 110 (CCDC110, KM-HN-1) is a protein containing C-terminal coiled-coil domain (CCD) which was previously discovered as a member of the human cancer/testis antigen (CTA). In addition, CCDC110 has both nuclear localization signal sequence and the leucine zipper motif. Although the functional role of CCDC110 has yet to be fully identified, the mRNA expression levels of CCDC110 are known to be highly elevated in various cancer types including testis, implying its relevance to cancer pathogenesis. In this study, we first developed several monoclonal antibody (mAb) hybridoma clones targeting CCDC110 and further isolated clone by characterizing for its specificity using immunoblotting and immunoprecipitation approaches with basal parenchymal sperm cells in testis tissue. Next, using these mAbs, we showed that the Tet-inducible overexpression of CCDC110 protein delayed the entry of G2/M phase in U2-OS osteosarcoma cells. Based on these results, we propose that CCDC110 plays a crucial role in cell cycle progression.

Phosphorylation of OCT4 Serine 236 Inhibits Germ Cell Tumor Growth by Inducing Differentiation.

Octamer-binding transcription factor 4 (Oct4) plays an important role in maintaining pluripotency in embryonic stem cells and is closely related to the malignancies of various cancers. Although posttranslational modifications of Oct4 have been widely studied, most of these have not yet been fully characterized, especially in cancer. In this study, we investigated the role of phosphorylation of serine 236 of OCT4 [OCT4 (S236)] in human germ cell tumors (GCTs). OCT4 was phosphorylated at S236 in a cell cycle-dependent manner in a patient sample and GCT cell lines. The substitution of endogenous OCT4 by a mimic of phosphorylated OCT4 with a serine-to-aspartate mutation at S236 (S236D) resulted in tumor cell differentiation, growth retardation, and inhibition of tumor sphere formation. GCT cells expressing OCT4 S236D instead of endogenous OCT4 were similar to cells with OCT4 depletion at the mRNA transcript level as well as in the phenotype. OCT4 S236D also induced tumor cell differentiation and growth retardation in mouse xenograft experiments. Inhibition of protein phosphatase 1 by chemicals or short hairpin RNAs increased phosphorylation at OCT4 (S236) and resulted in the differentiation of GCTs. These results reveal the role of OCT4 (S236) phosphorylation in GCTs and suggest a new strategy for suppressing OCT4 in cancer.

Cancer cells undergoing epigenetic transition show short-term resistance and are transformed into cells with medium-term resistance by drug treatment.

To elucidate the epigenetic mechanisms of drug resistance, epigenetically reprogrammed H460 cancer cells (R-H460) were established by the transient introduction of reprogramming factors. Then, the R-H460 cells were induced to differentiate by the withdrawal of stem cell media for various durations, which resulted in differentiated R-H460 cells (dR-H460). Notably, dR-H460 cells differentiated for 13 days (13dR-H460 cells) formed a significantly greater number of colonies showing drug resistance to both cisplatin and paclitaxel, whereas the dR-H460 cells differentiated for 40 days (40dR-H460 cells) lost drug resistance; this suggests that 13dR-cancer cells present short-term resistance (less than a month). Similarly, increased drug resistance to both cisplatin and paclitaxel was observed in another R-cancer cell model prepared from N87 cells. The resistant phenotype of the cisplatin-resistant (CR) colonies obtained through cisplatin treatment was maintained for 2-3 months after drug treatment, suggesting that drug treatment transforms cells with short-term resistance into cells with medium-term resistance. In single-cell analyses, heterogeneity was not found to increase in 13dR-H460 cells, suggesting that cancer cells with short-term resistance, rather than heterogeneous cells, may confer epigenetically driven drug resistance in our reprogrammed cancer model. The epigenetically driven short-term and medium-term drug resistance mechanisms could provide new cancer-fighting strategies involving the control of cancer cells during epigenetic transition.

False-negative errors in next-generation sequencing contribute substantially to inconsistency of mutation databases.

BACKGROUND: More than 11,000 laboratories and companies developed their own next-generation sequencing (NGS) for screening and diagnosis of various diseases including cancer. Although inconsistencies of mutation calls as high as 43% in databases such as GDSC (Genomics of Drug Sensitivity in Cancer) and CCLE (Cancer Cell Line Encyclopedia) have been reported, not many studies on the reasons for the inconsistencies have been published. Methods: Targeted-NGS analysis of 151 genes in 35 cell lines common to GDSC and CCLE was performed, and the results were compared with those from GDSC and CCLE wherein whole-exome- or highly-multiplex NGS were employed. RESULTS: In the comparison, GDSC and CCLE had a high rate (40-45%) of false-negative (FN) errors which would lead to high rate of inconsistent mutation calls, suggesting that highly-multiplex NGS may have high rate of FN errors. We also posited the possibility that targeted NGS, especially for the detection of low-level cancer cells in cancer tissues might suffer significant FN errors. CONCLUSION: FN errors may be the most important errors in NGS testing for cancer; their evaluation in laboratory-developed NGS tests is needed.

FAK-Copy-Gain Is a Predictive Marker for Sensitivity to FAK Inhibition in Breast Cancer.

BACKGROUND: Cancers with copy-gain drug-target genes are excellent candidates for targeted therapy. In order to search for new predictive marker genes, we investigated the correlation between sensitivity to targeted drugs and the copy gain of candidate target genes in NCI-60 cells. METHODS: For eight candidate genes showing copy gains in NCI-60 cells identified in our previous study, sensitivity to corresponding target drugs was tested on cells showing copy gains of the candidate genes. RESULTS: Breast cancer cells with Focal Adhesion Kinase (FAK)-copy-gain showed a significantly higher sensitivity to the target inhibitor, FAK inhibitor 14 (F14). In addition, treatment of F14 or FAK-knockdown showed a specific apoptotic effect only in breast cancer cells showing FAK-copy-gain. Expression-profiling analyses on inducible FAK shRNA-transfected cells showed that FAK/AKT signaling might be important to the apoptotic effect by target inhibition. An animal experiment employing a mouse xenograft model also showed a significant growth-inhibitory effect of F14 on breast cancer cells showing FAK-copy-gain, but not on those without FAK-copy-gain. CONCLUSION: FAK-copy-gain may be a predictive marker for FAK inhibition therapy in breast cancer.

Monitoring circulating tumor DNA by analyzing personalized cancer-specific rearrangements to detect recurrence in gastric cancer.

Circulating tumor DNA (ctDNA) has emerged as a candidate biomarker for cancer screening. However, studies on the usefulness of ctDNA for postoperative recurrence monitoring are limited. The present study monitored ctDNA in postoperative blood by employing cancer-specific rearrangements. Personalized cancer-specific rearrangements in 25 gastric cancers were analyzed by whole-genome sequencing (WGS) and were employed for ctDNA monitoring with blood up to 12 months after surgery. Personalized cancer-specific rearrangements were identified in 19 samples. The median lead time, which is the median duration between a positive ctDNA detection and recurrence, was 4.05 months. The presence of postoperative ctDNA prior to clinical recurrence was significantly correlated with cancer recurrence within 12 months of surgery (P = 0.029); in contrast, no correlation was found between cancer recurrence and the presence of preoperative ctDNA, suggesting the clinical usefulness of postoperative ctDNA monitoring for cancer recurrence in gastric cancer patients. However, the clinical application of ctDNA can be limited by the presence of ctDNA non-shedders (42.1%, 8/19) and by inconsistent postoperative ctDNA positivity.

Transient phosphorylation of tumor associated microtubule associated protein (TMAP)/cytoskeleton associated protein 2 (CKAP2) at Thr-596 during early phases of mitosis.

Tumor associated microtubule associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2) is a mitotic spindle-associated protein whose expression is cell cycle-regulated and also frequently deregulated in cancer cells. Two monoclonal antibodies (mAbs) against TMAP/CKAP2 were produced: B-1-13 and D-12-3. Interestingly, the reactivity of mAb D-12-3 to TMAP/CKAP2 was markedly decreased specifically in mitotic cell lysate. The epitope mapping study showed that mAb D-12-3 recognizes the amino acid sequence between 569 and 625 and that phosphorylation at T596 completely abolishes the reactivity of the antibody, suggesting that the differential reactivity originates from the phosphorylation status at T596. Immunofluorescence staining showed that mAb D-12-3 fails to detect TMAP/CKAP2 in mitotic cells between prophase and metaphase, but the staining becomes evident again in anaphase, suggesting that phosphorylation at T596 occurs transiently during early phases of mitosis. These results suggest that the cellular functions of TMAP/CKAP2 might be regulated by timely phosphorylation and dephosphorylation during the course of mitosis.

Predisposition of genetic disease by modestly decreased expression of GCH1 mutant allele.

Recently it was shown that single nucleotide polymorphisms (SNPs) can explain individual variation because of the small changes of the gene expression level and that the 50% decreased expression of an allele might even lead to predisposition to cancer. In this study, we found that a decreased expression of an allele might cause predisposition to genetic disease. Dopa responsive dystonia (DRD) is a dominant disease caused by mutations in GCH1 gene. The sequence analysis of the GCH1 in a patient with typical DRD symptoms revealed two novel missense mutations instead of a single dominant mutation. Family members with either of the mutations did not have any symptoms of DRD. The expression level of a R198W mutant allele decreased to about 50%, suggesting that modestly decreased expression caused by an SNP should lead to predisposition of a genetic disease in susceptible individuals.

Determination of Protein Expression Level in Cultured Cells by Immunocytochemistry on Paraffin-embedded Cell Blocks.

Immunofluorescent staining is currently the method of choice for determination of protein expression levels in cell-culture systems when morphological information is also necessary. The protocol of immunocytochemical staining on paraffin-embedded cell blocks, presented herein, is an excellent alternative to immunofluorescent staining on non-paraffin-embedded fixed cells. In this protocol, a paraffin cell block from HeLa cells was prepared using the thromboplastin-plasma method, and immunocytochemistry was performed for the evaluation of two proliferation markers, CKAP2 and Ki-67. The nuclei and cytoplasmic morphology of the HeLa cells were well preserved in the cell-block slides. At the same time, the CKAP2 and Ki-67 staining patterns in the immunocytochemistry were quite similar to those in immunohistochemical staining in paraffin cancer tissues. With modified cell-culture conditions, including pre-incubation of HeLa cells under serum-free conditions, the effect could be evaluated while preserving architectural information. In conclusion, immunocytochemistry on paraffin-embedded cell blocks is an excellent alternative to immunofluorescent staining.

Oncogene-induced senescence mediated by c-Myc requires USP10 dependent deubiquitination and stabilization of p14ARF.

Oncogene-induced senescence (OIS) is a critical tumor-suppressor mechanism, which prevents hyper-proliferation and transformation of cells. c-Myc promotes OIS through the transcriptional activation of p14ARF followed by p53 activation. Although the oncogene-mediated transcriptional regulation of p14ARF has been well addressed, the post-translational modification of p14ARF regulated by oncogenic stress has yet to be investigated. Here, we found that c-Myc increased p14ARF protein stability by inducing the transcription of ubiquitin-specific protease 10 (USP10). USP10, in turn, mediated the deubiquitination of p14ARF, preventing its proteasome-dependent degradation. USP10-null mouse embryonic fibroblasts and human primary cells depleted of USP10 bypassed c-Myc-induced senescence via the destabilization of p14ARF, and these cells displayed accelerated hyper-proliferation and transformation. Clinically the c-Myc-USP10-p14ARF axis was disrupted in non-small cell lung cancer patients, resulting in significantly worse overall survival. Our studies indicate that USP10 induced by c-Myc has a crucial role in OIS by maintaining the stability of key tumor suppressor p14ARF.

Zinc finger protein 143 expression is closely related to tumor malignancy via regulating cell motility in breast cancer.

We previously reported the involvement of zinc-finger protein 143 (ZNF143) on cancer cell motility in colon cancer cells. Here, ZNF143 was further characterized in breast cancer. Immunohistochemistry was used to determine the expression of ZNF143 in normal tissues and in tissues from metastatic breast cancer at various stages. Notably, ZNF143 was selectively expressed in duct and gland epithelium of normal breast tissues, which decreased when the tissue became malignant. To determine the molecular mechanism how ZNF143 affects breast cancer progression, it was knocked down by infecting benign breast cancer cells with shorthairpin (sh) RNA-lentiviral particles against ZNF143 (MCF7 sh-ZNF143). MCF7 sh-ZNF143 cells showed different cell-cell contacts and actin filament (F-actin) structures when compared with MCF7 sh-Control cells. In migration and invasion assays, ZNF143 knockdown induced increased cellular motility in breast carcinoma cells. This was reduced by the recovery of ZNF143 expression. Taken together, these results suggest that ZNF143 expression contributes to breast cancer progression. [BMB Reports 2017; 50(12): 621-627].

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

BACKGROUND: Recently, 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. METHODS: A 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. RESULTS: Although 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. CONCLUSIONS: The 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.