Takahito Nakagawa

Autoinhibitory Regulation of p73 by ΔNp73 To Modulate Cell Survival and Death through a p73-Specific Target Element within the ΔNp73 Promoter

ABSTRACT p73 is a p53-related tumor suppressor but is also induced by oncogene products such as E2F-1, raising a question as to whether p73 is a tumor suppressor gene or oncogene. Unlike p53, p73 has several variants, including ΔNp73, which lacks the NH2-terminal transactivation domain. Although, in developing neurons, ΔNp73 is expressed abundantly and seems to inhibit the proapoptotic function of p53, the role of p73 and ΔNp73 and their regulatory mechanism in cell growth and differentiation are poorly understood. Here we report that p73, but not p53, directly activates the transcription of endogenous ΔNp73 by binding to the p73-specific target element located at positions −76 to −57 within the ΔNp73 promoter region. The activation of ΔNp73 promoter by p63 was marginal. ΔNp73 was associated with p73α, p73β, and p53, as demonstrated by immunoprecipitation assays, and inhibited their transactivation activities when we used reporters of Mdm2, Bax, or ΔNp73 itself in SAOS-2 cells. Furthermore, induction or overexpression of ΔNp73 promoted cell survival by competing with p53 and p73 itself. Thus, our results suggest that the negative feedback regulation of p73 by its target ΔNp73 is a novel autoregulatory system for modulating cell survival and death.

Number of lymph node metastases is a significant prognostic factor in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (IHCC) is a rare primary hepatic tumor. Outcomes after resection and the use of lymph node dissection have not been well described. From a prospective database, we identified 53 patients with IHCC who underwent exploration between April 1983 and March 2004. Hepatic resection was performed in 44 patients, 30 of whom underwent lymph node dissection. Clinicopathological features and outcomes were analyzed. The actuarial 1-year survival was 66.2% in resected patients, compared to 0% in unresectable patients (p < 0.0001), with a 50% overall survival of 21.5 months and 3.1 months, respectively. The actuarial 3-year and 5-year overall survival rates in resected patients were 38.3% and 26.3%, respectively. Univariate analysis revealed that factors associated with poor overall survival included multiple tumors, extrahepatic bile duct involvement, noncurative resection, and involvement of lymph nodes. Multivariate analysis in resected patients revealed that multiple tumors (p < 0.0074) and non-curative resection (p = 0.0068) were significant risk factors for poor overall survival. The survival rate in patients with three or more positive nodes was significantly lower than in those with fewer than three (p < 0.0001). Three patients with solitary tumors and one or two involved lymph nodes have survived beyond 4 years after extended lobectomy with systemic lymphadenectomy. Curative resection, single tumor, and fewer than two lymph node metastases were prognostic factors for good outcome. Curative resection with lymph node dissection improved survival in patients with no more than two positive lymph nodes.

A novel HECT-type E3 ubiquitin ligase, NEDL2, stabilizes p73 and enhances its transcriptional activity.

Expression of p73, a p53 family member regulating cell growth and apoptosis, is maintained at low levels in mammalian cells, and cellular activation of p73 is usually controlled at the protein level. However, the precise molecular mechanisms by which p73 stability is regulated are unclear. During the search for interacting molecules with the COOH-terminal proline-rich region of p73, we identified a novel NEDD4-related protein (termed as NEDL2) which contains a C2 domain at its NH(2)-terminus, two WW domains, and a HECT domain at its COOH-terminus. As expected, NEDL2 catalyzed the ubiquitination of bacterial cellular proteins in vitro. Reciprocal co-immunoprecipitation experiments and in vitro pull-down assays revealed that NEDL2 bound to p73, which carries two putative PY motifs. p73 was efficiently ubiquitinated but stabilized in a NEDL2-dependent manner. Accordingly, p73 decayed at faster rates in the absence of NEDL2 than in its presence. Consistent with the NEDL2-mediated stabilization of p73, NEDL2 enhanced the p73-dependent transcriptional activation. Thus, our results suggest that NEDL2 activates the function of p73 by increasing its stability.

Physical Interaction of p73 with c-Myc and MM1, a c-Myc-binding Protein, and Modulation of the p73 Function

p73 shares high sequence homology with the tumor suppressor p53. Like p53, ectopic overexpression of p73 induces cell cycle arrest and/or apoptosis, and these biological activities are linked to its sequence-specific transactivation function. The COOH-terminal region of p73 is unique and has a function to modulate DNA-binding ability and transactivation activity. To identify and characterize cellular proteins that interact with the COOH-terminal region of p73α and regulate its activity, we employed a yeast-based two-hybrid screen with a human fetal brain cDNA library. We found MM1, a nuclear c-Myc-binding protein, was associated with p73α in both yeast two-hybrid and in vitro pull-down assays. In mammalian cells, MM1 co-immunoprecipitated with p73α, whereas p73β and tumor suppressor p53 did not interact with MM1. Overexpression of MM1 in p53-deficient osteosarcoma SAOS-2 cells enhanced the p73α-dependent transcription from the p53/p73-responsiveBax and PG13 promoters, whereas p73β- and p53-mediated transcriptional activation was unaffected in the presence of MM1. MM1 also stimulated the p73α-mediated growth suppression in SAOS-2 cells. More importantly, we found that c-Myc was physically associated with p73α and significantly impaired the transcriptional activity of p73α on Bax and p21 waf1 promoters. Expression of MM1 strongly reduced the c-Myc-mediated inhibitory activity on p73α. These results suggest that MM1 may act as a molecular partner for p73 to prevent the c-Myc-mediated inhibitory effect on its activity.

Function of p73, not of p53, is inhibited by the physical interaction with RACK1 and its inhibitory effect is counteracted by pRB

The newly identified p53-related gene, p73, encodes a nuclear transcription factor. Unlike p53, p73 has various isoforms with different NH2- and COOH-terminal tails. p73α with the longest COOH-terminal extension is most abundantly expressed in many tissues and cells among those splicing isoforms of p73 and the COOH-terminal region appears to have an autoregulatory function. To isolate and characterize the cellular protein(s) that interacts with the unique COOH-terminal region of p73α, we employed a yeast two-hybrid screen with a human fetal brain and 293 cell cDNA libraries. We identified the receptor for activated C kinase (RACK1) as a new member of p73α-binding proteins. The interaction was confirmed by coimmunoprecipitation experiments, whereas RACK1 did not interact with p53 or p73β. Ectopic overexpression of RACK1 in SAOS-2 cells reduced the p73α-mediated transcription from the p53/p73-responsive promoters, and inhibited the p73α-dependent apoptosis. On the other hand, the p53-dependent transcriptional activation as well as apoptosis was unaffected in the presence of RACK1. Furthermore, we found that pRB physically bound to RACK1, and repressed the RACK1-dependent inhibition of p73α. Taken together, our observations suggest that pRB diminishes the RACK1-mediated inhibition of p73α activity through the interaction with RACK1.

N-glycan alterations are associated with drug resistance in human hepatocellular carcinoma

BackgroundCorrelations of disease phenotypes with glycosylation changes have been analysed intensively in the tumor biology field. Glycoforms potentially associated with carcinogenesis, tumor progression and cancer metastasis have been identified. In cancer therapy, drug resistance is a severe problem, reducing therapeutic effect of drugs and adding to patient suffering. Although multiple mechanisms likely underlie resistance of cancer cells to anticancer drugs, including overexpression of transporters, the relationship of glycans to drug resistance is not well understood.ResultsWe established epirubicin (EPI) – and mitoxantrone (MIT) – resistant cell lines (HLE-EPI and HLE-MIT) from the human hepatocellular carcinoma cell line (HLE). HLE-EPI and HLE-MIT overexpressed transporters MDR1/ABCB1 and BCRP/ABCG2, respectively. Here we compared the glycomics of HLE-EPI and HLE-MIT cells with the parental HLE line. Core fucosylated triantennary oligosaccharides were increased in the two resistant lines. We investigated mRNA levels of glycosyltransferases synthesizing this oligosaccharide, namely, N-acetylglucosaminyltransferase (GnT)-IVa, GnT-IVb and α1,6-fucosyltransferase (α1,6-FucT), and found that α1,6-FucT was particularly overexpressed in HLE-MIT cells. In HLE-EPI cells, GnT-IVa expression was decreased, while GnT-IVb was increased. Both GnT-IVs were downregulated in HLE-MIT cells. HLE-MIT cells also showed decreases in fucosylated tetraantennary oligosaccharide, the product of GnT-V. GnT-V expression was decreased in both lines, but particularly so in HLE-MIT cells. Thus both N-glycan and glycosyltransferase expression was altered as cells acquired tolerance, suggesting novel mechanisms of drug resistance.ConclusionN-glycan and glycosyltransferase expression in HLE-EPI and HLE-MIT were analysed and presented that glycans altered according with acquired tolerance. These results suggested novel mechanisms of drug resistance.

UFD2a mediates the proteasomal turnover of p73 without promoting p73 ubiquitination

p73 protein level is kept extremely low in mammalian cultured cells and its stability may be regulated by not only the ubiquitin/proteasome-dependent proteolysis but also through other unidentified mechanisms. Here, we found for the first time that p73 is physically as well as functionally associated with the U-box-type E3/E4 ubiquitin ligase UFD2a. The immunoprecipitation experiments demonstrated that this interaction is mediated by the COOH-terminal region of p73α containing SAM domain. During the cisplatin-induced apoptosis in SH-SY5Y neuroblastoma cells, p73α accumulated at a protein level, whereas the endogenous UFD2a was significantly reduced in response to cisplatin. Ectopic expression of UFD2a decreased the half-life of p73α in association with a significant inhibition of the p73α-mediated transactivation as well as proapoptotic activity. Downregulation of endogenous UFD2a by antisense strategy resulted in a remarkable accumulation of p73α. Unexpectedly, UFD2a-mediated degradation of p73α was sensitive to the proteasomal inhibitor, however, UFD2a did not affect the ubiquitination levels of p73α. Taken together, our present findings imply that UFD2a might promote the proteasomal turnover of p73 in a ubiquitination-independent manner, and also suggest that UFD2a might play an important role in the regulation of cisplatin-induced apoptosis mediated by p73.

Efficacy of preoperative radiotherapy to portal vein tumor thrombus in the main trunk or first branch in patients with hepatocellular carcinoma

BackgroundThe prognosis of hepatocellular carcinoma (HCC) with portal vein tumor thrombus (PVTT) in the main trunk or the first branch is very poor.MethodsRadiotherapy (RT) to PVTT was followed by hepatectomy within 2 weeks. The dose used was 30–36 Gy, in 10–12 fractions, for 15–20 days. The efficacy of preoperative RT to PVTT in the main trunk or first branch was evaluated by comparing results in patients who underwent hepatectomy (group R; n = 15) with preoperative RT and those without preoperative RT (group N; n = 28).ResultsThe 1-, 3-, and 5-year survival rates in group R were 86.2%, 43.5%, and 34.8%, respectively, while these values in group N were 39.0%, 13.1%, and 13.1%, respectively. The survival curve of group R was significantly better than that of group N (P = 0.0359). In group R, five (83.3%) of six patients whose tumor thrombus was completely necrosed (based on pathological examination) and whose follow-up period was over 2 years survived for more than 2 years. Female sex (P = 0.0066), multiple tumors (P = 0.0369), and absence of preoperative RT (P = 0.0359) were ranked as significant factors for a poor prognosis by univariate analysis. Multivariate analysis revealed absence of preoperative RT and female sex to be significant factors for a poor prognosis.ConclusionPreoperative RT to PVTT in the main trunk or first branch improved the prognosis of patients with HCC with PVTT, and could be a promising new modality in the treatment of these patients.

Preoperative Evaluation of Hepatic Vasculature by Three-Dimensional Computed Tomography in Patients Undergoing Hepatectomy

BackgroundHepatectomy is particularly difficult when the tumor is large, close to the inferior vena cava or the main trunk of the hepatic or portal vein, or in the caudate lobe, as well as when the operation is a re-hepatectomy, because two-dimensional computed tomography (CT) often does not clearly show tumor location relative to blood vessels.Study DesignTo evaluate the efficacy of three-dimensional computed tomography (3D-CT), reconstructed from multidetector-row computed tomography (MD-CT) with contrast, MD-CT was performed in 17 patients before hepatectomy.ResultsThe third-order branches of the hepatic artery and the portal vein were clearly shown in all cases. Both the hepatic vein, which drained the same segment that the portal vein fed, and the portal vein were also clearly shown. These vessels could be visualized from any perspective. In 2 patients who underwent hemihepatectomy, large tumors (23.0 and 17.0 cm) displaced the vasculature, but the positions of tumor and vessels could be precisely evaluated by 3D-CT. In patients who required replacement of the vena cava with synthetic grafts, the distance and direction of pressure to IVC by tumor was accurately estimated by 3D-CT. In patients who were limited to segmentectomy or partial hepatectomy because of prior hepatectomy or tumor position, evaluation of the glissons was facilitated by 3D-CT.ConclusionsThree-dimensional-CT was extremely useful for preoperative simulation because it provided important information that could not be obtained with 2D-CT.

AFP mRNA detected in bone marrow by real-time quantitative RT-PCR analysis predicts survival and recurrence after curative hepatectomy for hepatocellular carcinoma.

Objective:To determine whether detection of hepatocellular carcinoma (HCC) cells by real-time quantitative RT-PCR targeting of alpha-fetoprotein mRNA (AFP mRNA) before or after curative hepatectomy predicts HCC recurrence and patient survival. Summary Background Data:The presence of cancer cells in peripheral blood and/or bone marrow in patients with malignant disease has been reported to correlate with outcome. Methods:Between July 2000 and June 2005, 136 consecutive HCC patients underwent primary curative hepatectomy. Bone marrow aspirated preoperatively, and peripheral blood samples collected before and after operation were subjected to real-time quantitative RT-PCR analysis using AFP mRNA as a target molecule. Median follow-up was 23 months (range, 6–54 months). Patient survival (PS), disease-free survival (DFS), and clinicopathologic features were compared between patients with positive and negative AFP mRNA. Results:Twenty-four patients died (22 from HCC). HCC recurred in 66 patients (hepatic in 37 [56.1%]; hepatic and remote in 17 [25.8%], and remote alone in 12 [18.2%]). Bone marrow was positive for AFP mRNA in 38 patients (27.9%) and negative in 98 (72.1%). One- and 3-year PS was 96.6% and 91.4%, respectively, with negative AFP mRNA versus 86.2% and 55.5%, respectively, with positive AFP mRNA (P < 0.0001). One- and 3-year DFS were 73.2% and 44.8%, respectively, with negative AFP mRNA versus 54.5% and 25.8%, respectively, with positive AFP mRNA (P = 0.0399). Portal vascular invasion, tumor size, multiple tumors, and tumor differentiation correlated with inferior PS and DFS on univariate analysis. On multivariate analysis, positive AFP mRNA was the most important risk factor for PS (P = 0.001) and DFS (P = 0.0165). In addition, positive AFP mRNA in peripheral blood after operation tended to predict reduced DFS. Conclusion:AFP mRNA in the bone marrow and systemic circulation during the perioperative period predicts patient survival and recurrence after curative hepatic resection for HCC.