A novel RING-type ubiquitin ligase breast cancer-associated gene 2 correlates with outcome in invasive breast cancer

The RING finger family of proteins possess ubiquitin ligase activity and play pivotal roles in protein degradation and receptor-mediated endocytosis. In this study, we examined whether the breast cancer-associated gene 2 (BCA2), a novel RING domain protein, has E3 ubiquitin ligase activity and investigated its expression status in breast tumors. The full-length BCA2 gene was cloned from the human breast cancer cell line MDA-MB-468. It encodes an open reading frame of 304 amino acids and contains a RING-H2 domain. BCA2 maps to chromosome 1q21.1, a region known to harbor cytogenetic aberrations in breast cancers. We found that the BCA2 protein has an intrinsic autoubiquitination activity, the hallmark of E3 ligases, whereas mutant RING protein is not autoubiquitinated. This indicates that the BCA2 ubiquitin ligase activity is dependent on the RING-H2 domain. Using tissue microarrays and immunohistochemistry, we found strong to intermediate BCA2 staining in 56% of 945 invasive breast cancers cases, which was significantly correlated with positive estrogen receptor status [odds ratio (OR), 1.51; P = 0.004], negative lymph node status (OR, 0.73; P = 0.02), and an increase in disease-free survival for regional recurrence (OR, 0.45; P = 0.03). Overexpression of BCA2 increased proliferation and small interfering RNA inhibited growth of T47D human breast cancer cells and NIH3T3 mouse cells. The autoubiquitination activity of BCA2 indicates that it is a novel RING-type E3 ligase. Its association with clinical measures and its effects on cell growth indicate that BCA2 may be important for the ubiquitin modification of proteins crucial to breast carcinogenesis and growth.     

A portrayal of E3 ubiquitin ligases and deubiquitylases in cancer

E3 ubiquitin ligases and deubiquitylating enzymes (DUBs) are the key components of ubiquitin proteasome system which plays a critical role in cellular protein homeostasis. Any shortcoming in their biological roles can lead to various diseases including cancer. The dynamic interplay between ubiquitylation and deubiquitylation determines the level and activity of several proteins including p53, which is crucial for cellular stress response and tumor suppression pathways. In this review, we describe the different types of E3 ubiquitin ligases including those targeting tumor suppressor p53, SCF ligases and RING type ligases and accentuate on biological functions of few important E3 ligases in the cellular regulatory networks. Tumor suppressor p53 level is tightly regulated by multiple E3 ligases including Mdm2, COP1, Pirh2, etc. SCF ubiquitin ligase complexes are key regulators of cell cycle and signal transduction. BRCA1 and VHL RING type ligases function as tumor suppressors and play an important role in DNA repair and hypoxia response respectively. Further, we discuss the biological consequences of deregulation of the E3 ligases and the implications for cancer development. We also describe deubiquitylases which reverse the process of ubiquitylation and regulate diverse cellular pathways including metabolism, cell cycle control and chromatin remodelling. As the E3 ubiquitin ligases and DUBs work in a substrate specific manner, an improved understanding of them can lead to better therapeutics for cancer.     

RNF115/BCA2 E3 Ubiquitin Ligase Promotes Breast Cancer Cell Proliferation through Targeting p21Waf1/Cip1 for Ubiquitin-Mediated Degradation

The E3 ubiquitin ligase RING finger protein 115 (RNF115), also known as breast cancer-associated gene 2 (BCA2), has previously been reported to be overexpressed in estrogen receptor α (ERα)-positive breast tumors and to promote breast cell proliferation; however, its mechanism is unknown. In this study, we demonstrated that silencing of BCA2 by small interfering RNAs (siRNAs) in two ERα-positive breast cancer cell lines, MCF-7 and T47D, decreases cell proliferation and increases the protein levels of the cyclin-dependent kinase inhibitor p21^Waf/Cip1. The protein stability of p21 was negatively regulated by BCA2. BCA2 directly interacts with p21 and promotes p21 ubiquitination and proteasomal degradation. Knockdown of p21 partially rescues the cell growth arrest induced by the BCA2 siRNA. These results suggest that BCA2 promotes ERα-positive breast cancer cell proliferation at least partially through downregulating the expression of p21.     

肾脏嗜酸性细胞瘤的诊断与治疗

目的:探讨肾嗜酸性细胞瘤的诊疗方法,提高肾嗜酸性细胞瘤的诊疗水平.方法:回顾性分析本院12例肾嗜酸性细胞瘤患者的临床资料,并结合文献探讨其诊疗方法及预后.结果:12例患者术前影像学均诊断为肾占位性病变,其中T1aN0M0期5例,T1bN0M0期 7例.术中及术后病理学检查证实为肾嗜酸性细胞瘤.2例行根治性肾切除术,10例行根治性肾部分切除术,术后随访18~36个月无复发转移.结论:肾嗜酸细胞瘤系肾脏的良性倾向肿瘤,预后良好,术前仅靠临床症状和影像学特征难以与肾癌相鉴别,易误诊为肾癌.对于可疑病例,须行术中快速冰冻病理检查.诊断需依据病理组织学、免疫组化及特点综合判断.治疗以保肾手术首选,术后应密切随访.     

RING finger ubiquitin protein ligases: implications for tumorigenesis,metastasis and for molecular targets in cancer

Covalent modification of proteins with ubiquitin regulates almost all aspects of eukaryotic cellular function. Ubiquitin protein ligases (E3s) play central regulatory roles in that they provide substrate specificity to this process and therefore, represent attractive molecular targets for disease therapy. We summarize recent advances in our understanding of RING finger and RING finger-related E3s with emphasis on BRCA1 and the tumor autocrine motility factor receptor (gp78), as well as discuss the potential for components of the ubiquitin pathway for proteasomal degradation as molecular targets.     

Regulation of Metformin Response by Breast Cancer Associated Gene 2

Adenosine monophosphate-activated protein kinase (AMPK), a master regulator of cellular energy homeostasis, has emerged as a promising molecular target in the prevention of breast cancer. Clinical trials using the United States Food and Drug Administration (FDA)-approved, AMPK-activating, antidiabetic drug metformin are promising in this regard, but the question of why metformin is protective for some women but not others still remains. Breast cancer associated gene 2 (BCA2/Rabring7/RNF115), a novel Really Interesting New Gene (RING) finger ubiquitin E3 ligase, is overexpressed in >50% of breast tumors. Herein, we report that BCA2 is an endogenous inhibitor of AMPK activation in breast cancer cells and that BCA2 inhibition increases the efficacy of metformin. BCA2 overexpression inhibited both basal and inducible Thr172 phosphorylation/activation of AMPKα1, while BCA2-specific small interfering RNA (siRNA) enhanced phosphorylated AMPKα1 (pAMPKα1). The AMPK-suppressive function of BCA2 requires its E3 ligase-specific RING domain, suggesting that BCA2 targets some protein controlling (de)phosphorylation of AMPKα1 for degradation. Activation of AMPK by metformin triggered a growth inhibitory signal but also increased BCA2 protein levels, which correlated with AKT activation and could be curbed by an AMPK inhibitor, suggesting a potential feedback mechanism from pAMPKα1 to pAkt to BCA2. Finally, BCA2 siRNA, or inhibition of its upstream stabilizing kinase AKT, increased the growth inhibitory effect of metformin in multiple breast cancer cell lines, supporting the conclusion that BCA2 weakens metformin''s efficacy. Our data suggest that metformin in combination with a BCA2 inhibitor may be a more effective breast cancer treatment strategy than metformin alone.     

The role and therapeutic implications of RING‐finger E3 ubiquitin ligases in hepatocellular carcinoma

Increasing evidence indicates that deregulation of RING‐finger ubiquitin‐protein ligases (E3s) involves in the development of hepatocellular carcinoma (HCC). These RING‐finger E3s serve as oncoproteins or tumor suppressors in HCC under specific conditions. In this review, we summarize current knowledge about abnormal RING‐finger E3s and their clinical significance in the development of HCC, and discuss parts of critical substrates for these RING‐finger E3s in detail. Furthermore, in light of success of Bortezomib in treating hematological malignancies, we describe the preclinical and clinical studies of therapeutic approaches targeting aberrant RING‐finger E3s in HCC.     

钙结合蛋白S100A1在肾细胞肿瘤中的研究进展

不同类型的肾细胞肿瘤形态学、遗传学特征及预后均有不同,但它们在形态学上常有一定的重叠,有时得出准确的病理诊断十分困难。钙结合家族S100蛋白中的S100A1最近发现在。肾细胞肿瘤中表达,尤其在肾嫌色细胞癌及嗜酸细胞瘤的鉴别诊断中很有意义,且可能与肾细胞癌的预后有关。     

79例非透明性肾细胞癌新旧组织学分类的比较分析

目的 比较2004年世界卫生组织(WHO)肾细胞癌组织学分类标准与旧分类标准的异同,总结容易出现的诊断差异及可能原因,纠正对肾细胞癌传统病理分类的观念.方法 天津医科大学肿瘤医院1998年2月至2005年12月间,根治性肾切除术后病理诊断为非透明性细胞癌79例,按2004年WHO肾癌组织学分类标准进行重新分类,并收集相关临床资料进行分析.结果 重新分类后,透明性肾细胞癌14例,乳头状肾细胞癌23例,嫌色性肾细胞癌34例,肾集合管癌1例,未分类的肾细胞癌1例,混合型肾细胞癌5例(透明及乳头状肾细胞癌混合型2例,透明及嫌色性肾细胞癌混合型2例,乳头状及嫌色性肾细胞癌混合型1例),肾嗜酸细胞瘤1例.结论 以往,部分嫌色性肾细胞癌与乳头状肾细胞癌易被诊断为颗粒细胞癌,主要原因相似的嗜酸性颗粒样胞浆特点以及对嫌色性肾细胞癌及乳头状肾细胞癌的组织学特点认识不清.旧有诊断中颗粒细胞癌的结构特点可见于多种肾细胞癌,但不是一种独立的肾癌类型,应从分类中去除.对于混合两种或以上不同类型肾细胞癌的诊断标准有待定义与完善.     

The role of cullin proteins in gastric cancer

The cullin proteins are a family of scaffolding proteins that associate with RING proteins and ubiquitin E3 ligases and mediate substrate–receptor bindings. Thus, cullin proteins regulate the specificity of ubiquitin targeting in the regulation of proteins involved in various cellular processes, including proliferation, differentiation, and apoptosis. There are seven cullin proteins that have been identified in eukaryotes: CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5, and CUL7/p53-associated parkin-like cytoplasmic protein. All of these proteins contain a conserved cullin homology domain that binds to RING box proteins. Cullin–RING ubiquitin ligase complexes are activated upon post-translational modification by neural precursor cell-expressed, developmentally downregulated protein 8. The aberrant expression of several cullin proteins has been implicated in many cancers though the significance in gastric cancer has been less well investigated. This review provides the first systematic discussion of the associations between all members of the cullin protein family and gastric cancer. Functional and regulatory mechanisms of cullin proteins in gastric carcinoma progression are also summarized along with a discussion concerning future research areas. Accumulating evidence suggests a critical role of cullin proteins in tumorigenesis, and a better understanding of the function of these individual cullin proteins and their targets will help identify potential biomarkers and therapeutic targets.     

Renal oncocytoma: report of two cases

The clinico-radiologic, gross, microscopic and ultrastructural findings in 2 cases of renal oncocytoma are reported. The diagnosis of renal oncocytoma has important clinical and prognostic implications, since the neoplasia is usually benign despite its often large size. The differential diagnosis with other renal neoplasms, in particular renal cell carcinoma, is discussed. The problem of the pre-operatory diagnosis of renal oncocytoma is explored on the basis of clinico-radiologic findings and/or histologic examination of pre- or intraoperatory biopsy. In view of the tumor's benign nature, this diagnosis implies tumorectomy, but its close resemblance to renal cell carcinoma at present counsels nephrectomy. A final diagnosis of oncocytoma may be formulated only after histologic examination of several specimens and ultrastructural confirmation.     

Role of protein ubiquitylation in regulating endocytosis of receptor tyrosine kinases

Growth factors and their transmembrane receptor tyrosine kinases play pivotal roles in morphogenesis, cell fate determination and pathogenesis, including multiple stages of cancer. The amplitude and kinetics of signaling by growth factor receptors are determined by an endocytic process, which sorts activated, autophosphorylated receptors to degradation in lysosomes. Recent studies uncovered the role of protein ubiquitylation in vesicular trafficking of growth factor receptors. Decoration of ligand-activated receptors by multiple monomeric ubiquitins distinguishes this degradative route from the proteasome-mediated pathway, which involves polymeric chains of ubiquitin. Although receptor ubiquitylation occurs at the cell surface, its major role is to sort internalized receptors to the lumen of the multivesicular body, en route to the lysosome. The ubiquitin ligases that control this late sorting event belong to the Cbl family of RING finger adaptors, which bind specific phosphotyrosine residues in the receptors upon activation by ligand. Another group of E3 ubiquitin ligases, the Nedd4 family, regulates the initial sorting event, which targets receptors to clathrin-coated regions of the plasma membrane. This step entails ubiquitin-dependent assembly of a clathrin-binding complex of adaptors such as epsins, which share ubiquitin-interacting motifs. The concerted action of both ubiquitin-binding adaptors of membrane coats and E3 ligases, as well as their regulation by protein phosphorylation and ubiquitylation, ensure robust endocytosis of growth factor receptors. Genetic defects and virus-mediated manipulations of the endocytic pathway divert receptors to a default recycling pathway, thereby enabling unrestrained signaling characteristic to transformed cells.     

Multitechnical pathological diagnosis in chromophobe renal cell carcinoma

Two new cases of chromophobe renal cell carcinoma were diagnosed on the basis of their morphology and their karyotype complemented by flow cytometry. In one of these cases, however, all these investigations were not sufficient and additional histochemistry investigation had to be used to completely rule out other renal tumors such as oncocytoma, the prognosis of which is totally different.     

线粒体蛋白Bcl-2和Bax在肿瘤发生中作用的初步探讨

目的:研究线粒体蛋白(MAB1273)、Bcl-2、Bax在肾细胞癌(RCC)组织内的表达及相关性分析.方法:采用免疫组织化学SP方法检测9例嗜酸细胞瘤、6例嫌色细胞癌、23例透明细胞癌以及12例正常肾组织中MABl273、Bcl-2和Bax蛋白的表达.结果:MABl273和Bel-2在嗜酸细胞瘤、嫌色细胞癌、透明细胞癌中表达明显高于正常肾组织(P=0.006,P=0.008).Bax在各组间表达无明显差异(P=0.057).通过秩相关分析,MAB1273的表达与Bcl-2的表达呈正相关(r=0.341,P=0.015),而Bcl-2表达与Bax表达呈负相关(r=-0.287,P=0.043).结论:线粒体蛋白及Bcl-2的高表达、Bax低表达可能共同参与了肾嗜酸细胞瘤、嫌色细胞癌及透明细胞癌的发生,提示线粒体蛋白表达异常参与RCC细胞凋亡调控过程.     

Histogenesis of renal cell carcinoma and renal oncocytoma. An immunohistochemical study

The histogenesis of renal cell carcinoma and oncocytoma is controversial. We compared immunohistochemical profiles of normal kidney, nine carcinomas, and six oncocytomas. Carcinomas and oncocytomas expressed the following antigens respectively: proximal tubule--Uro 3 (56 & 67%), alpha-1-antitrypsin (89 & 50%); proximal and distal tubule--Uro 10 (67 & 83%); distal tubule--B2-microglobulin (100 & 100%); distal/medullary tubule--epithelial membrane antigen (89 & 83%), neuron-specific enolase (78 & 100%), glandular cytokeratin (78 & 100%), epidermal keratin (67 & 67%); and medullary tubule--Uro 8 (89 & 83%). All tumors, except one oncocytoma, had at least one positive reaction for each antigen group. Oncocytomas predominantly stained for distal/medullary tubular antigens; none showed a predominance of proximal tubular antigens. Carcinomas also demonstrated largely distal/medullary tubule antigens; 44% showed prominence of proximal tubular antigens as well. Assignment of an exclusive proximal, distal or medullary tubule origin to renal neoplasms does not appear valid. Divergent histogenesis from a precursor stem cell is likely.     

Degradation of HER2 by Cbl-based chimeric ubiquitin ligases

Targeting disease-causing proteins for ubiquitination and degradation by chimeric molecules represents a promising alternative therapeutic strategy in cancer. Here, several Cbl-based chimeric ubiquitin ligases were recombined to achieve effective down-regulation of HER2. These chimeric molecules consisted of the Cbl NH(2)-terminal tyrosine kinase binding domain, linker, and RING domain, with the Src homology 2 domain replaced with that from growth factor receptor binding protein 2 (Grb2), Grb7, p85, or Src. The chimeric proteins not only interacted with HER2 but also enhanced the down-regulation of endogenous overexpressed HER2. After the chimeric proteins were introduced into HER2-overexpressing breast cancer SK-BR-3 cells or ovarian cancer SK-OV-3 cells, they effectively promoted HER2 ubiquitination and degradation in a RING finger domain-dependent manner. Consequently, expression of these chimeric molecules led to an inhibition of colony formation, increased the proportion of cells in the G(1) cycle, and suppressed tumorigenicity. Collectively, our findings suggest that the Cbl-based chimeric ubiquitin ligases designed in the present study may represent a novel approach for the targeted therapy of HER2-overexpressing cancers.     

Association with cullin partners protects ROC proteins from proteasome-dependent degradation

Cullin 1/CDC53 represents a multigene family and has been linked to the ubiquitin-mediated proteolysis of several different proteins. We recently identified two closely related RING finger proteins, ROC1 and ROC2, that share considerable sequence similarity to an APC subunit, APC11, and demonstrated ROC1 as an essential subunit of CUL1 and CDC53 ubiquitin ligases. We report here that the expression of ROC1, ROC2 and APC11 genes are induced by mitogens and remain constant during the cell cycle. Unlike other subunits of SCF and APC E3 ligases, ectopically expressed ROC family proteins are degraded by a proteasome-inhibitor sensitive pathway and are stabilized by associating with cullins. Mutations at the conserved Phe79 and His80 residues in the RING finger of ROC1 diminish its binding with cullins, resulting in a loss of cullin protection and ubiquitin ligase activity. These results suggest a potential mechanism for regulating the activity of ROC-cullin ligases through complex assembly and ROC/APC11 subunit ubiquitination.     

High levels of S-100a0 (alpha alpha) protein in tumor tissues and in sera of patients with renal cell carcinoma

The alpha alpha form of S-100 protein (S-100a0), which is distributed mainly in the heart and striated muscles, and also in the brain and kidney, was determined in tumor tissues and sera of patients with renal cell carcinoma by employing an enzyme immunoassay system for bovine S-100a0 protein. The average content of S-100a0 in the renal cell carcinoma tissue (n = 10) was about 650 ng/mg protein, 4-fold higher than that in the kidney (n = 6, 160 ng/mg protein). Immunohistochemically, S-100a0 antigen was localized in such epithelial cells as proximal tubules, Bowman's capsules and collecting tubules of normal kidney, and in the cytoplasm, nucleus and occasionally plasma membrane of the tumor cells. The contents of S-100a0 protein in various lung carcinoma tissues were low (less than 10 ng/mg protein). Serum S-100a0 concentrations were less than 0.3 ng/ml in healthy subjects, but they were significantly increased in patients with renal cell carcinoma at diagnosis, showing greater than 0.5 ng/ml in 17/32 cases (53%). Serum S-100a0 levels were also enhanced in some patients with lung cancer (10/33, 30%), breast cancer (4/20, 20%) and other non-neoplastic diseases, indicating that S-100a0 protein in the serum is not a specific biomarker for renal cell carcinoma. However, serum S-100a0 concentrations in patients with renal cell carcinoma changed in parallel with the clinical course during treatment. These results suggest that serum S-100a0 may be a useful biomarker at least for monitoring the clinical course of renal cell carcinoma.