Satoshi Nakayama

High expression of ubiquitin carboxy-terminal hydrolase-L1 and -L3 mRNA predicts early recurrence in patients with invasive breast cancer

The present study investigated the mRNA expression level of ubiquitin c‐terminal hydrolase (UCH)‐L1 and ‐L3 in breast cancer tissue and aimed to elucidate its association with tumor characteristics and patient prognosis. UCH‐L1 and UCH‐L3 mRNA levels in invasive breast cancer (n = 100) were determined by a real‐time polymerase chain reaction (PCR) assay and their relationship with various clinicopathological characteristics of breast tumors as well as patient prognosis were studied. UCH‐L3 mRNA level was significantly upregulated in breast cancer tissue compared to adjacent normal breast tissue (P < 0.005), and UHC‐L1 mRNA level also showed a non‐significant increase in tumor tissue compared to adjacent normal breast tissue. Both UCH‐L1 and UCH‐L3 mRNA levels were significantly higher in high histological grade tumors than in low histological grade tumors (P < 0.001 and P < 0.005, respectively). High UCH‐L1 mRNA level was significantly associated with negative estrogen receptor status (P < 0.05) and negative progesterone receptor status (P < 0.05). Patients with both UCH‐L1 and UCH‐L3 mRNA high tumors showed a significantly poorer prognosis than those in the UCH‐L1 or UCH‐L3 mRNA low group (P < 0.005). These observations that UCH‐L3 mRNA level is upregulated in breast cancer tissue, and breast tumors with both UCH‐L1 and UCH‐L3 mRNA high expression are associated with a poor prognosis, suggest the possible involvement of UCH‐L1 and UCH‐L3 in the pathogenesis and progression of breast cancer. (Cancer Sci 2006; 97: 523–u2003529)

Growth-inhibitory effect of adiponectin via adiponectin receptor 1 on human breast cancer cells through inhibition of S-phase entry without inducing apoptosis.

Adiponectin is one of the most important adipocytokines secreted from adipose tissue. In addition to its effects on glucose and fatty acid metabolism, it has been reported that adiponectin has a direct growth-inhibitory effect on breast cancer cells. However, it still remains to be established how adiponectin affects cell cycle and apoptosis and whether or not its inhibitory effect is mediated through adiponectin receptors. Here, we demonstrated that adiponectin treatment resulted in a significant dose-dependent growth inhibition of both MDA-MB-231 and T47D cells. In both cell lines, the G0/G1 population significantly increased after adiponectin treatment, but apoptosis was not induced. High expression of mRNA and protein of adiponectin receptor 1 was observed, but expression of adiponectin receptor 2 was very low in both cell lines. Treatment with small interference RNA against adiponectin receptor 1 significantly reduced the growth inhibition induced by adiponectin in both cell lines. Taken together, adiponectin decreases breast cancer cell proliferation by inhibiting the entry into S-phase without inducing apoptosis, and this inhibitory effect is mediated through adiponectin receptor 1.

Prediction of paclitaxel sensitivity by CDK1 and CDK2 activity in human breast cancer cells

IntroductionPaclitaxel is used widely in the treatment of breast cancer. Not all tumors respond to this drug, however, and the characteristics that distinguish resistant tumors from sensitive tumors are not well defined. Activation of the spindle assembly checkpoint is required for paclitaxel-induced cell death. We hypothesized that cyclin-dependent kinase (CDK) 1 activity and CDK2 activity in cancer cells, which reflect the activation state of the spindle assembly checkpoint and the growth state, respectively, predict sensitivity to paclitaxel.MethodsCell viability assays and DNA and chromatin morphology analyses were performed in human breast cancer cell lines to evaluate sensitivity to paclitaxel and the cell cycle response to paclitaxel. We then examined the specific activities of CDK1 and CDK2 in these cell lines and in xenograft models of human breast cancer before and after paclitaxel treatment. Protein expression and kinase activity of CDKs and cyclins were analyzed using a newly developed assay system.ResultsIn the cell lines, biological response to paclitaxel in vitro did not accurately predict sensitivity to paclitaxel in vivo. Among the breast cancer xenograft tumors, however, tumors with significantly increased CDK1 specific activity after paclitaxel treatment were sensitive to paclitaxel in vivo, whereas tumors without such an increase were resistant to paclitaxel in vivo. Baseline CDK2 specific activity was higher in tumors that were sensitive to paclitaxel than in tumors that were resistant to paclitaxel.ConclusionsThe change in CDK1 specific activity of xenograft tumors after paclitaxel treatment and the CDK2 specific activity before paclitaxel treatment are both associated with the drug sensitivity in vivo. Analysis of cyclin-dependent kinase activity in the clinical setting could be a powerful approach for predicting paclitaxel sensitivity.

CDK1 and CDK2 activity is a strong predictor of renal cell carcinoma recurrence

BACKGROUNDnIn renal cell carcinoma (RCC), the prediction of metastasis via tumor prognostic markers remains a major problem. The objective of our study was to evaluate the efficacy of cyclin-dependent kinase (CDK)1 and CDK2 activity as a prognostic marker in human RCC.nnnMETHODSnSurgical specimens were obtained from 125 patients with RCC without metastasis. Protein expression and kinase activity of CDKs were analyzed using a newly developed assay system named C2P (Sysmex, Kobe, Japan). We then examined the specific activities (SAs) of CDK1 and CDK2 and calculated CDK2SA-CDK1SA ratio in RCC. Also, risk score (RS) was examined.nnnRESULTSnA total of 125 cases were tested, though 34 cases were excluded because of low sample quality (25 cases) and assay failure (9 cases). In total, 91 cases were analyzed. They included 68 male and 23 female patients, ranging in age from 19 to 83 years. At a median follow-up of 36 months (1-109M), tumor with low CDK2SA-CDK1SA ratio showed significantly better 5-year recurrence-free survival than those with high CDK2SA-CDK1SA ratio (88.7% vs. 54.7%, P = 0.00141). Also, RS enabled the classification of RCCs into high-risk and low-risk groups, and patients with tumors classified as low RS showed better recurrence-free survival than patients with tumors with high RS (88.7% vs. 54.7%, P = 0.0141).nnnCONCLUSIONnCDK1SA of tumors and the CDK2SA are both associated with recurrence and prognosis.nnnIMPACTnCDK-based risk demonstrated is strongly associated with clinical outcome. CDK-based risk should be an accurate system for predicting recurrence and survival for planning follow-up.

Recurrence risk score based on the specific activity of CDK1 and CDK2 predicts response to neoadjuvant paclitaxel followed by 5-fluorouracil, epirubicin and cyclophosphamide in breast cancers

BACKGROUNDnWe established the cell cycle profiling (C2P) assay for specific activity (SA; activity/expression) of cyclin-dependent kinases (CDKs). C2P risk score (C2P-RS) based on CDK1 and CDK2 SAs was significantly associated with relapse in breast cancer (BC). This study was conducted to investigate the predictive value of C2P-RS for neoadjuvant chemotherapy (NAC).nnnPATIENTS AND METHODSnAmong 124 eligible patients, 122 were treated with weekly paclitaxel followed by 5-fluorouracil, epirubicin and cyclophosphamide (P-FEC) and 2 were treated with paclitaxel monotherapy. C2P-RS was determined via C2P using frozen biopsy samples before NAC.nnnRESULTSnNegative estrogen receptor (ER), negative progesterone receptor (PR), positive human epidermal growth factor receptor 2 (HER2), high Ki-67 expression and intermediate + high C2P-RS were significantly associated with high pathological complete response (pCR) rates compared with positive ER (30% versus 9%), positive PR (25% versus 6%), negative HER2 (34% versus 11%), low Ki-67 expression (24% versus 7%) or low C2P-RS (24% versus 9%), respectively. The combination of C2P-RS and Ki-67 had a stronger impact on pCR than each parameter alone, and a multivariate analysis showed that the combination was an independent predictor of pCR (odds ratio 3.3, 95% confidence interval 1.1-9.5).nnnCONCLUSIONSnC2P-RS was significantly associated with pCR after P-FEC and may be a useful predictor for chemotherapy in BCs.

Novel Functional Assay for Spindle-Assembly Checkpoint by Cyclin-Dependent Kinase Activity to Predict Taxane Chemosensitivity in Breast Tumor Patient

Taxanes are among the drugs most commonly used for preoperative chemotherapy for breast cancer. Taxanes induce mitotic arrest and subsequent apoptosis. The spindle-assembly checkpoint (SAC) is known to be activated during mitosis, along with cyclin-dependent kinase-1 (CDK1), and is required for taxane-induced cell death. We hypothesized that CDK1 activity predicts response to taxane-containing chemotherapy. This study included breast cancer patients who received preoperative chemotherapy— taxane-containing treatment followed by anthracycline-based treatment—and then underwent surgery. Before starting taxane-containing chemotherapy, patients underwent fine-needle aspiration biopsy, and the biopsy samples were incubated in paclitaxel solution to measure CDK activity. Clinical were evaluated after taxane therapy, and pathological resposes were evaluated after completion of all preoperative chemotherapy. Thirty five patients were eligible for analysis of clinical response to taxane-containing therapy. Twenty-six patients had taxane-sensitive and 9 taxane-resistant tumors. Using a cut-off of CDK activity determined by the ROC analysis, patients were classified into SAC function and dysfunction groups. Univariate logistic regression analysis with clinicopathologic parameters showed that only CDK-based SAC functionality was significantly correlated with clinical response (P =0.017). No significant correlation was observed between SAC functionality and pathologic response. CDK-based SAC functionality significantly predicted clinical response (P =.0072, overall agreement = 71.4%), and this is a unique mechanism-based marker for predicting taxane chemosensitivity. Further, large prospective study is needed to determine CDK-based SAC functionality could be developed as a predictive biomarker.

Cyclin-Dependent Kinase Activity Correlates with the Prognosis of Patients Who Have Gastrointestinal Stromal Tumors.

AbstractBackgroundnThe estimation of recurrence risk remains a critical issue in relation to gastrointestinal stromal tumors (GISTs) treated with adjuvant therapy. The accuracy of the commonly used risk stratifications is not always adequate.MethodsFor this study, data were prospectively collected from 68 patients with GISTs who underwent R0 surgery between 2004 and 2009. The results from this analysis cohort were evaluated using the data obtained from an additional 40 patients in the validation cohort. Cyclin-dependent kinase 1 (CDK1)- and CDK2-specific activities were measured using a non-RI kinase assay system.ResultsThe specific activities of CDK1 and CDK2, but not their expression, significantly correlated with recurrence. The specific activities of both CDK1 and CDK2 were independently correlated with mitosis and significantly correlated with recurrence-free survival (RFS). In the multivariate analysis, CDK2-specific activity (Pxa0=xa00.0006), tumor size (Pxa0=xa00.0347), and KIT deletion mutations (Pxa0=xa00.0006) were significantly correlated with RFS in the analysis cohort. In the validation cohort, CDK2-specific activity (Pxa0=xa00.0368) was identified as an independent prognostic factor for tumor recurrences with tumor location (Pxa0=xa00.0442).ConclusionThe results suggest that the specific activities of CDK1 and CDK2 may reflect the proliferative activity of GISTs and that CDK2-specific activity is a good prognostic factor predicting recurrence after macroscopic complete resection of GISTs.

The Cell Cycle Profile Test is a Prognostic Indicator for Breast Cancer Patients Treated With Postoperative 5-Fluorouracil-Based Chemotherapy

OBJECTIVEnThe cell cycle profile test is suggested to be an independent prognostic indicator for breast cancer patients. To further clarify the prognostic value, we applied this to breast cancer patients treated with postoperative 5-fluorouracil-based chemotherapy.nnnMETHODSnA total of 153 breast cancer patients, who were treated with postoperative 5-fluorouracil-based chemotherapies, were randomly selected. Specific activities of cyclin-dependent kinases 1 and 2 in the tumor samples were analyzed. Patients were divided into three categories (low, intermediate or high risk) based on cell cycle profile analysis.nnnRESULTSnThe proportions of the cell cycle profile categories were 39% for low risk, 10% for intermediate risk and 45% for high risk, respectively. Although the cell cycle profile test did not show a significant predictive power for relapse-free survival (high vs. low risk; P = 0.052), the cell cycle profile categories were significant prognostic factors in a subgroup of 98 patients with fewer than three involved nodes (high vs. low risk, P = 0.004). Multivariate analyses also indicated that a cell cycle profile parameter (high vs. low risk) was an independent prognostic indicator from the number of involved nodes and clinical stage in this subgroup (hazard ratio = 2.46, P = 0.01). Interestingly, the prognostic power of the cell cycle profile test was significant in 75 patients treated with oral 5-fluorouracil derivatives alone (hazard ratio = 6.29 for high vs. low risk, P = 0.02).nnnCONCLUSIONSnThese findings suggest that the cell cycle profile test is useful for predicting a higher risk of relapse in patients treated with postoperative 5-fluorouracil-based chemotherapy.

P2-11-08: Clinical Value of Combination Assay for Quantitative Determination of Cancer Biomarkers C2P and uPA/PAI-1 for Disease Recurrence Prediction of Early Breast Cancer Patients.

[Background] C2P is an assay measuring specific activities (SA; kinase activity compensated by its protein expression) of cyclin-dependent kinases CDK1 and CDK2 by examining a small piece of fresh-frozen tumor tissue. We reported previously that the C2P risk score given by CDK1SA and CDK2SA is a potent prognostic factor in node-negative breast cancer patients. Likewise, a uPA/PAI-1 ELISA test (FEMTELLE®, American Diagnostica Inc. Stamford, CT) quantitatively determines uPA (urokinase-type plasminogen activator) and PAI-1 (plasminogen activator inhibitor type-1) antigen levels in tumor tissue extracts, to identify patients with high or low risk of disease recurrence of node-negative breast cancer patients. These cancer biomarkers are recommended by the ASCO at the highest level of evidence (LOE-1) for therapy decision making in node-negative breast cancer patients. From the biological point of view, the above two assays can be placed into two categories: tumor cell proliferation for C2P and tumor cell invasion for uPA/PAI-1. This fact led us to examine the concept of combination of the two assays to better select breast cancer patients at risk. [Results] Fifty-nine cases of frozen primary breast cancer tissues were subjected to the C2P assay in a blinded manner. Twenty-one cases (40%) were categorized into “high risk”, 7 cases (14%) into “intermediate risk”, and 24 cases (46%) into “low risk”. Seven cases were judged as “not informative”. The uPA/PAI-1 results and clinical information (26: recurrent cases, 30: non-recurrent cases, 1: stage IV, 2: unknown) were provided by the TUM tumor bank. uPA/PAI-1 risk categories of 36 cases (61%) were classified “high” and 23 cases (39%) categorized “low”. C2P and uPA/PAI-1 showed statistically significant correlation to histological grading (Pearson correlation coefficient; 0.45 and 0.40, respectively). No significant correlation was observed between C2P and uPA/PAI-1. By Kaplan-Meier analysis for disease-free survival, in cases treated with endocrine therapy only, both C2P and uPA/PAI-1 showed a reproducible trend to the respective claimed performances as prognostic factors. In the combination analysis of the two parameters, where low/low was judged as “low” and the others as “high”, 11 cases (24%) were categorized into “low” and 34 cases (76%) into “high”. The sensitivity and negative predictive value for disease recurrence were 90% (19/20) and 91% (10/11), respectively. Strong statistical significance was observed between the risk categories by the log-rank test; p=0.0089, and also by Cox proportional hazards regression analysis; HR=9.18, p=0.032. By multivariate analysis, also including tumor size and nodal status, the CP2 uPA/PAI-1 combination evolved as a significant, statistically independent parameter (HR: 6.51, p Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-11-08.

Abstract 299: CDK1 and CDK2 activity is a strong predictor of renal cell cancer recurrence

Background: We established original methods enabling simultaneous analysis of protein expressions and kinase activities of the CDK (cyclin-dependent kinase) molecules in lysate of tumor tissue in a clinical setting (C2P technology, Ishihara et al: Biochim Biophys Acta. 1741; 226-233, 2005). The clinical utility of the technology was first evaluated in breast cancer, and combination analysis of CDK1 and CDK2 activity was shown to be a significant prognostic indicator for relapse (Kim et al: Ann Oncol. 19; 68-72, 2009). The objective of our study is to evaluate the efficacy of CDK1 and CDK2 activity as a prognostic marker in human renal cell carcinoma (RCC).Methods: Surgical specimens were obtained from 125 patients with RCC without metastasis. These patients were selected randomly for this study. Protein expression and kinase activity of CDKs and cyclins were analyzed using a newly developed assay system. The system to measure the CDK specific activity (SA) is named C2Ps (Sysmex, Kobe, Japan). We then examined the specific activities of CDK1 and CDK2 and calculated CDK2/CDK1 ratio in RCC. Also, risk score (RS) was examined as described in previous study (JGH van Nes et al: Br J Cancer. 100; 494-500, 2009). Cut off value was calculated by ROC analysis.Results: 125 cases were tested, though 34 cases were excluded of low sample quality (25 cases) and of assay failure (9 cases). 91 cases were analyzed. They included 68 male and 23 female patients, ranging in age from 19 to 83 years. At a median follow up of 36 months (1-109M), tumor with low CDK2/CDK1 ratio showed significantly better 5-year progression free survival (PFS) than those with high CDK2/CDK1 ratio (88.7% vs 54.7%, P=0.00141). Also, RS enabled the classification of RCCs into high-risk and low-risk groups, patients with tumors classified as low RS showed better PFS than patients with tumors with high RS (88.7% vs 54.7%, P=0.0141).Conclusion: CDK1 specific activity of tumors and the CDK2 specific activity are both associated with recurrence and prognosis. Analysis of cyclin-dependent kinase activity in the clinical setting could be a powerful approach for predicting cancer recurrence and prognosis in RCC after surgery and has potential for use as a routine laboratory test. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 299. doi:10.1158/1538-7445.AM2011-299