Tomomi Ueki

Involvement of kinesin family member 2C/mitotic centromere‐associated kinesin overexpression in mammary carcinogenesis

To elucidate the molecular mechanisms of mammary carcinogenesis and discover novel therapeutic targets for breast cancer, we previously carried out genome‐wide expression profile analysis of 81 breast cancer cases by means of cDNA microarray coupled with laser microbeam microdissection of cancer cells. Among the dozens of transactivated genes, in the present study we focused on the functional significance of kinesin family member 2C (KIF2C)/mitotic centromere‐associated kinesin (MCAK) in the growth of breast cancer cells. Northern blot and immunohistochemical analyses confirmed KIF2C/MCAK overexpression in breast cancer cells, and showed that it is expressed at undetectable levels in normal human tissues except the testis, suggesting KIF2C/MCAK to be a cancer–testis antigen. Western blot analysis using breast cancer cell lines revealed a significant increase in the endogenous KIF2C/MCAK protein level and its phosphorylation in G2/M phase. Treatment of breast cancer cells with small interfering RNA against KIF2C/MCAK effectively suppressed KIF2C/MCAK expression and inhibited the growth of the breast cancer cell lines T47D and HBC5. In addition, we found that KIF2C/MCAK expression was significantly suppressed by ectopic introduction of p53. These findings suggest that overexpression of KIF2C/MCAK might be involved in breast carcinogenesis and is a promising therapeutic target for breast cancers. (Cancer Sci 2008; 99: 62–70)

Involvement of elevated expression of multiple cell-cycle regulator, DTL/RAMP (denticleless/RA-regulated nuclear matrix associated protein), in the growth of breast cancer cells.

To investigate the detailed molecular mechanism of mammary carcinogenesis and discover novel therapeutic targets, we previously analysed gene expression profiles of breast cancers. We here report characterization of a significant role of DTL/RAMP (denticleless/RA-regulated nuclear matrix associated protein) in mammary carcinogenesis. Semiquantitative RT–PCR and northern blot analyses confirmed upregulation of DTL/RAMP in the majority of breast cancer cases and all of breast cancer cell lines examined. Immunocytochemical and western blot analyses using anti-DTL/RAMP polyclonal antibody revealed cell-cycle-dependent localization of endogenous DTL/RAMP protein in breast cancer cells; nuclear localization was observed in cells at interphase and the protein was concentrated at the contractile ring in cytokinesis process. The expression level of DTL/RAMP protein became highest at G1/S phases, whereas its phosphorylation level was enhanced during mitotic phase. Treatment of breast cancer cells, T47D and HBC4, with small-interfering RNAs against DTL/RAMP effectively suppressed its expression and caused accumulation of G2/M cells, resulting in growth inhibition of cancer cells. We further demonstrate the in vitro phosphorylation of DTL/RAMP through an interaction with the mitotic kinase, Aurora kinase-B (AURKB). Interestingly, depletion of AURKB expression with siRNA in breast cancer cells reduced the phosphorylation of DTL/RAMP and decreased the stability of DTL/RAMP protein. These findings imply important roles of DTL/RAMP in growth of breast cancer cells and suggest that DTL/RAMP might be a promising molecular target for treatment of breast cancer.

Activation of an estrogen/estrogen receptor signaling by BIG3 through its inhibitory effect on nuclear transport of PHB2/REA in breast cancer

Breast cancer is known to be a hormone‐dependent disease, and estrogens through an interaction with estrogen receptor (ER) enhance the proliferative and metastatic activity of breast tumor cells. Here we show a critical role of transactivation of BIG3, brefeldin A‐inhibited guanine nucleotide‐exchange protein 3, in activation of the estrogen/ER signaling in breast cancer cells. Knocking‐down of BIG3 expression with small‐interfering RNA (siRNA) drastically suppressed the growth of breast cancer cells. Subsequent coimmunoprecipitation and immunoblotting assays revealed an interaction of BIG3 with prohibitin 2/repressor of estrogen receptor activity (PHB2/REA). When BIG3 was absent, stimulation of estradiol caused the translocation of PHB2/REA to the nucleus, enhanced the interaction of PHB2/REA and ERα, and resulted in suppression of the ERα transcriptional activity. On the other hand, when BIG3 was present, BIG3 trapped PHB2/REA in the cytoplasm and inhibited its nuclear translocation, and caused enhancement of ERα transcriptional activity. Our results imply that BIG3 overexpression is one of the important mechanisms causing the activation of the estrogen/ERα signaling pathway in the hormone‐related growth of breast cancer cells. (Cancer Sci 2009)

Ubiquitination and Downregulation of BRCA1 by Ubiquitin-Conjugating Enzyme E2T Overexpression in Human Breast Cancer Cells

Breast cancer is generated through a multistep genetic and epigenetic process including activations of oncogenes and inactivations of tumor suppressor genes. Here, we report a critical role of ubiquitin-conjugating enzyme E2T (UBE2T), an E2 ubiquitin-conjugating enzyme, in mammary carcinogenesis. Immunocytochemical staining and in vitro binding assay revealed that UBE2T interacted and colocalized with the BRCA1/BRCA1-associated RING domain protein (BARD1) complex. Knocking down of UBE2T expression with small interfering RNA drastically suppressed the growth of breast cancer cells. Interestingly, in vivo ubiquitination assay indicated BRCA1 to be polyubiquitinated by incubation with wild-type UBE2T protein, but not with C86A-UBE2T protein, an E2 activity-dead mutant, in which the 86th residue of cysteine was replaced with alanine. Furthermore, knocking down of UBE2T protein induced upregulation of BRCA1 protein in breast cancer cells, whereas its overexpression caused the decrease of the BRCA1 protein. Our data imply a critical role of UBE2T in development and/or progression of breast cancer through the interaction with and the regulation of the BRCA1/BARD1 complex.

Comprehensive review of post-liver resection surgical complications and a new universal classification and grading system

Liver resection is the gold standard treatment for certain liver tumors such as hepatocellular carcinoma and metastatic liver tumors. Some patients with such tumors already have reduced liver function due to chronic hepatitis, liver cirrhosis, or chemotherapy-associated steatohepatitis before surgery. Therefore, complications due to poor liver function are inevitable after liver resection. Although the mortality rate of liver resection has been reduced to a few percent in recent case series, its overall morbidity rate is reported to range from 4.1% to 47.7%. The large degree of variation in the post-liver resection morbidity rates reported in previous studies might be due to the lack of consensus regarding the definitions and classification of post-liver resection complications. The Clavien-Dindo (CD) classification of post-operative complications is widely accepted internationally. However, it is hard to apply to some major post-liver resection complications because the consensus definitions and grading systems for post-hepatectomy liver failure and bile leakage established by the International Study Group of Liver Surgery are incompatible with the CD classification. Therefore, a unified classification of post-liver resection complications has to be established to allow comparisons between academic reports.

Prognostic roles of preoperative α-fetoprotein and des-γ-carboxy prothrombin in hepatocellular carcinoma patients

AIM To clarify the utility of using des-γ-carboxy prothrombin (DCP) and α-fetoprotein (AFP) levels to predict the prognosis of hepatocellular carcinoma (HCC) in patients with hepatitis B virus (HBV) and the hepatitis C virus (HCV) infections. METHODS A total of 205 patients with HCC (105 patients with HBV infection 100 patients with HCV infection) who underwent primary hepatectomy between January 2004 and May 2012 were enrolled retrospectively. Preoperative AFP and DCP levels were used to create interactive dot diagrams to predict recurrence within 2 years after hepatectomy, and cutoff levels were calculated. Patients in the HBV and HCV groups were classified into three groups: a group with low AFP and DCP levels (LL group), a group in which one of the two parameters was high and the other was low (HL group), and a group with high AFP and DCP levels (HH group). Liver function parameters, the postoperative recurrence-free survival rate, and postoperative overall survival were compared between groups. The survival curves were compared by log-rank test using the Kaplan-Meier method. Multivariate analysis using a Cox forward stepwise logistic regression model was conducted for a prognosis. RESULTS The preoperative AFP cutoff levels for recurrence within 2 years after hepatectomy in the HBV and HCV groups were 529.8 ng/mL and 60 mAU/mL, respectively; for preoperative DCP levels, the cutoff levels were 21.0 ng/mL in the HBV group and 67 mAU/mL in the HCV group. The HBV group was significantly different from the other groups in terms of vascular invasion, major hepatectomy, volume of intraoperative blood loss, and surgical duration. Significant differences were found between the LL group, the HL group, and the HH group in terms of both mean disease-free survival time (MDFST) and mean overall survival time (MOST): 64.81 ± 7.47 vs 36.63 ± 7.62 vs 18.98 ± 6.17 mo (P = 0.001) and 85.30 ± 6.55 vs 59.44 ± 7.87 vs 46.57 ± 11.20 mo (P = 0.018). In contrast, the HCV group exhibited a significant difference in tumor size, vascular invasion, volume of intraoperative blood loss, and surgical duration; however, no significant difference was observed between the three groups in liver function parameters except for albumin levels. In the LL group, the HL group, and the HH group, the MDFST was 50.09 ± 5.90, 31.01 ± 7.21, and 14.81 ± 3.08 mo (log-rank test, P < 0.001), respectively, and the MOST was 79.45 ± 8.30, 58.82 ± 7.56, and 32.87 ± 6.31 mo (log-rank test, P < 0.001), respectively. CONCLUSION In the HBV group, the prognosis was poor when either AFP or DCP levels were high. In the HCV group, the prognosis was good when either or both levels were low; however, the prognosis was poor when both levels were high. High levels of both AFP and DCP were an independent risk factor associated with tumor recurrence in the HBV and HCV groups. The relationship between tumor marker levels and prognosis was characteristic to the type of viral hepatitis.

The impact of aging on morbidity and mortality after liver resection: a systematic review and meta-analysis

Abstract Surgery involving elderly patients is becoming increasingly common due to the rapid aging of societies all over the world. The objective of this study was to elucidate the prognostic differences between elderly and young patients who undergo liver resection. A systematic review based on the PRISMA flow diagram was conducted. Ovid Medline and PubMed were used to search for relevant literature published between January 2000 and March 2013, and the modified MINORS score was used to assess the methodological quality. In cases of hepatocellular carcinoma and miscellaneous liver tumors, the morbidity and mortality rate did not differ significantly between the elderly and young patients. For patients with colorectal metastatic liver cancer, the mortality of the young patients was 2.7 times lower than that of elderly patients. Our review of high-quality retrospective studies was able to elucidate the clinical risks of age on the outcomes after liver surgery in specific patient populations.

Pancreatic regeneration: basic research and gene regulation

Pancreatic regeneration (PR) is an interesting phenomenon that could provide clues as to how the control of diabetes mellitus might be achieved. Due to the different regenerative abilities of the pancreas and liver, the molecular mechanism responsible for PR is largely unknown. In this review, we describe five representative murine models of PR and thirteen humoral mitogens that stimulate β-cell proliferation. We also describe pancreatic ontogenesis, including the molecular transcriptional differences between α-cells and β-cells. Furthermore, we review 14 murine models which carry defects in genes related to key transcription factors for pancreatic ontogenesis to gain further insight into pancreatic development.

Robot-Assisted Reduced Port TME with Low Colorectal Anastomosis

Advances in minimally invasive laparoscopic surgery have grown to reduced-port surgery (RPS). Single-incision laparoscopic surgery (SILS) is considered the ultimate RPS for improving cosmesis and reducing postoperative pain and the risk of abdominal wall morbidities. Several reports have described both superior short-term and similar long-term results of SILS for colorectal malignancies when compared with standard laparoscopic surgery. However, there are some limitations such as instrument collision and insufficient countertraction. In a recent attempt to overcome the technical issues in SILS, by combining the use of the da Vinci robotic surgical system with the single-incision platform, the recognized challenges are somewhat simplified. Only a few reports of single-port robotic surgery for rectal disease have been conducted; however, the current robotic arm and equipment are still rather bulky and have a limited intrapelvic range of motion. Pure single approach may be not suited for sufficient oncological clearance.