Regulation of TGF-beta family signaling by E3 ubiquitin ligases

Members of the transforming growth factor-beta (TGF-beta) family, including TGF-beta, activin and bone morphogenetic proteins (BMPs), are multifunctional proteins that regulate a wide variety of cellular responses, such as proliferation, differentiation, migration and apoptosis. Alterations in their downstream signaling pathways are associated with a range of human diseases like cancer. TGF-beta family members transduce signals through membrane serine/threonine kinase receptors and intracellular Smad proteins. The ubiquitin-proteasome pathway, an evolutionarily conserved cascade, tightly regulates TGF-beta family signaling. In this pathway, E3 ubiquitin ligases play a crucial role in the recognition and degradation of target proteins by the 26S proteasomes. Smad degradation regulates TGF-beta family signaling; HECT (homologous to the E6-accessory protein C-terminus)-type E3 ubiquitin ligases, Smad ubiquitin regulatory factor 1 (Smurf1), Smurf2, and a RING-type E3 ubiquitin ligase, ROC1-SCF(Fbw1a) have been implicated in Smad degradation. Smurf1 and Smurf2 bind to TGF-beta family receptors via the inhibitory Smads, Smad6 and Smad7, to induce their ubiquitin-dependent degradation. Arkadia, a RING-type E3 ubiquitin ligase, induces the ubiquitination and degradation of Smad7 and corepressors, c-Ski and SnoN, to enhance TGF-beta family signaling. Abnormalities in E3 ubiquitin ligases that control components of TGF-beta family signaling may lead to the development and progression of various cancers.     

Viral and cellular MARCH ubiquitin ligases and cancer

Covalent conjugation of proteins with ubiquitin is one the most important post-translational modifications because it controls intracellular protein trafficking typically resulting in protein degradation. Frequently ubiquitinated proteins are targeted to the proteasome for degradation in the cytosol. However, ubiquitinated membrane bound proteins can also be targeted for endocytosis and degradation in the lysosome. Ubiquitin-dependent degradation pathways have clear cancer relevance due to their integral involvement in protein quality control, regulation of immune responses, signal transduction, and cell cycle regulation. In spite of its fundamental importance, little is known regarding how proteins are specifically identified for ubiquitin-dependent degradation. In this article we review a newly discovered family of viral and cellular ubiquitin ligases called MARCH proteins. Recent studies of MARCH proteins define new paradigms showing how ubiquitin E3 ligases determine the intracellular location and fate of proteins.     

Targeting E3 ubiquitin ligases for cancer therapy

E3 ubiquitin ligases are a large family of proteins that can be classified into three major structurally distinct types: N-end rule E3s, E3s containing the HECT (Homology to E6AP C-Terminus) domain, and E3s with the RING (Really Interesting New Gene) finger, including its derivatives, the U- Box and the PHD (Plant Homeo-Domain). E3 ubiquitin ligases exist as single polypeptide or multimeric complexes. Together with ubiquitin activating enzyme E1 and ubiquitin conjugating enzyme E2, E3 ubiquitin ligases catalyze the ubiquitination of a variety of protein substrates for targeted degradation via the 26S proteasome. E3 ubiqutin ligases, therefore, play an essential role in regulation of many biological processes. Furthermore, E3s are enzymes that determine the specificity of protein substrates; they represent a class of "drugable" targets for pharmaceutical intervention. In this review, I will mainly focus on E3 ubiquitin ligases as potential cancer targets and discuss three of the most promising E3s, Mdm2/Hdm2, IAPs, and SCF, for their target rationales, target validation, and critical issues associated with them. These E3 ligases or their components are overexpressed in many human cancers and their inhibition leads to growth suppression or apoptosis. In addition, I will evaluate two current methodologies available for the high throughput screening for small molecular weight chemical inhibitors of the E3 ubiquitin ligases. Although targeting E3 ubiquitin ligases is still in its infancy, speedy approval of the general proteasome inhibitor, Velcade (bortezomib) by the FDA for the treatment of relapsed and refractory multiple myeloma suggests the promise of specific E3 inhibitors in anti-cancer therapy. Emerging technologies, such as siRNA, will provide a better validation of many E3s. It is anticipated that E3 ubiquitin ligases will represent an important new target platform for future mechanism-driven drug discovery.     

Role of E3 ubiquitin ligases in lung cancer

E3 ubiquitin ligases are a large family of proteins that catalyze the ubiquitination of many protein substrates for targeted degradation by the 26S proteasome. Therefore, E3 ubiquitin ligases play an essential role in a variety of biological processes including cell cycle regulation, proliferation and apoptosis. E3 ubiquitin ligases are often found overexpressed in human cancers, including lung cancer, and their deregulation has been shown to contribute to cancer development. However, the lack of specific inhibitors in clinical trials is a major issue in targeting E3 ubiquitin ligases with currently only one E3 ubiquitin ligase inhibitor being tested in the clinical setting. In this review, we focus on E3 ubiquitin ligases that have been found deregulated in lung cancer. Furthermore, we discuss the processes in which they are involved and evaluate them as potential anti-cancer targets. By better understanding the mechanisms by which E3 ubiquitin ligases regulate biological processes and their exact role in carcinogenesis, we can improve the development of specific E3 ubiquitin ligase inhibitors and pave the way for novel treatment strategies for cancer patients.     

Degradation of HER2 by ansamycins induces growth arrest and apoptosis in cells with HER2 overexpression via a HER3, phosphatidylinositol 3'-kinase-AKT-dependent pathway

Breast cancers with high expression of HER2 are associated frequently with aggressive, poor prognosis disease and resistance to chemotherapy-induced apoptosis. Geldanamycin and its less toxic analogue, 17- (allylamino)-17-demethoxygeldanamycin (17-AAG) are ansamycin antibiotics that bind to a highly conserved pocket in the hsp 90 chaperone protein and inhibit its function. Hsp 90 is required for the refolding of proteins during environmental stress and the conformational maturation of certain signaling proteins. Among the most sensitive targets of 17-AAG are the HER kinases. Therefore, tumors that are dependent on these kinases may be especially sensitive to 17-AAG either alone or in combination with chemotherapy. In this study we demonstrate that cells that overexpress HER2 are 10-100-fold more sensitive to 17-AAG than cancer cells expressing low levels of HER2. We found that HER2 is degraded in several cell lines, but only cell lines with high levels of HER2 are sensitive to the drug. The effects of 17-AAG on growth and apoptosis are because of inhibition of signaling through HER2-HER3, phosphatidylinositol 3'- kinase. The absence of HER3 and the introduction of constitutively active p110alpha rendered cells with high HER2 expression more resistant to 17-AAG. These findings suggest that 17-AAG may be useful for the treatment of breast cancer cells with high levels of HER2. However, the overexpression of HER2 alone may not be predictive of response, because the coexpression of HER3 and the activation of phosphatidylinositol 3'-kinase may play a crucial role in the response of these cells to 17-AAG and other drugs directed against HER2. These observations have important clinical implications because they may help to identify patients that are most likely to benefit from 17-AAG and may explain resistance to Herceptin as seen in many patients.     

Construction and evaluation of a novel humanized HER2-specific chimeric receptor

Introduction

The human epidermal growth factor receptor 2 (HER2) represents one of the most studied tumor-associated antigens (TAAs) for cancer immunotherapy. The monoclonal antibody (mAb) trastuzumab has improved the outcomes of patients with HER2+ breast cancer. However, a large number of HER2+ tumors are not responsive to, or become resistant to, trastuzumab-based therapy, and thus more effective therapies targeting HER2 are needed.

Methods

HER2-specific T cells were generated by the transfer of genes that encode chimeric antigen receptor (CAR). Using a multistep overlap extension PCR method, we constructed a novel, humanized HER2 CAR-containing, chA21 single-chain variable fragment (scFv) region of antigen-specific mAb and T-cell intracellular signaling chains made up of CD28 and CD3ζ. An interferon γ and interleukin 2 enzyme-linked immunosorbent assay and a chromium-51 release assay were used to evaluate the antitumor immune response of CAR T cells in coculture with tumor cells. Furthermore, SKBR3 tumor–bearing nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were treated with HER2 CAR T cells to evaluate antitumor activity. Human CD3+ T cell accumulation in tumor xenograft was detected by immunohistochemistry.

Results

chA21-28z CAR was successfully constructed, and both CD4+ and CD8+ T cells were transduced. The expanded HER2 CAR T cells expressed a central memory phenotype and specifically reacted against HER2+ tumor cell lines. Furthermore, the SKBR3 tumor xenograft model revealed that HER2 CAR T cells significantly inhibited tumor growth in vivo. Immunohistochemical analysis showed robust accumulation of human CD3+ T cells in regressing SKBR3 lesions.

Conclusions

The results of this study show that novel chA21 scFv-based, HER2-specific CAR T cells not only recognized and killed HER2+ breast and ovarian cancer cells ex vivo but also induced regression of experimental breast cancer in vivo. Our data support further exploration of the HER2 CAR T-cell therapy for HER2-expressing cancers.     

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.     

Degradation of HER2/neu by ANT2 shRNA suppresses migration and invasiveness of breast cancer cells

Background  

In breast cancer, the HER2/neu oncoprotein, which belongs to the epidermal growth factor receptor family, may trigger activation of the phosphoinositide-3 kinase (PI3K)/Akt pathway, which controls cell proliferation, survival, migration, and invasion. In this study, we examined the question of whether or not adenine nucleotide translocase 2 (ANT2) short hairpin RNA (shRNA)-mediated down-regulation of HER2/neu and inhibitory effects on the PI3K/Akt signaling pathway suppressed migration and invasiveness of breast cancer cells.     

Screening analysis of ubiquitin ligases reveals G2E3 as a potential target for chemosensitizing cancer cells

Cisplatin is widely used against various tumors, but resistance is commonly encountered. By inducing DNA crosslinks, cisplatin triggers DNA damage response (DDR) and cell death. However, the molecular determinants of how cells respond to cisplatin are incompletely understood. Since ubiquitination plays a major role in DDR, we performed a high-content siRNA screen targeting 327 human ubiquitin ligases and 92 deubiquitinating enzymes in U2OS cells, interrogating the response to cisplatin. We quantified γH2AX by immunofluorescence and image analysis as a read-out for DNA damage. Among known mediators of DDR, the screen identified the ubiquitin ligase G2E3 as a new player in the response to cisplatin. G2E3 depletion led to decreased γH2AX levels and decreased phosphorylation of the checkpoint kinase 1 (Chk1) upon cisplatin. Moreover, loss of G2E3 triggered apoptosis and decreased proliferation of cancer cells. Treating cells with the nucleoside analogue gemcitabine led to increased accumulation of single-stranded DNA upon G2E3 depletion, pointing to a defect in replication. Furthermore, we show that endogenous G2E3 levels in cancer cells were down-regulated upon chemotherapeutic treatment. Taken together, our results suggest that G2E3 is a molecular determinant of the DDR and cell survival, and that its loss sensitizes tumor cells towards DNA-damaging treatment.     

转染嵌合性T细胞受体基因小鼠T淋巴细胞的体外抗肿瘤作用

目的:研究小鼠T淋巴细胞在转染嵌合性T细胞受体基因后的体外抗肿瘤作用.方法:应用重组DNA技术,将HER2特异性嵌合性T-细胞受体构建入逆转录病毒载体;转入包装细胞后,收集病毒上清,转染小鼠T淋巴细胞,转基因后的小鼠T淋巴细胞分别与HER2阳性(SK-OV-3)或阴性(MCF-7)的肿瘤细胞系共培养,检测其细胞因子γ干扰素释放,51Cr释放法检测CTL评价其抗肿瘤效应.结果:所构建载体经酶切鉴定符合要求,乒乓法转染包装细胞系GP E86,检测病毒滴度为1.2×106,Retronectin结合离心法转染经抗CD3/CD28单抗活化的小鼠T淋巴细胞,转染效率可达50%以上;转染嵌合性T细胞受体基因的T淋巴细胞与HER2阳性或阴性的肿瘤细胞系共培养后可检测到HER2特异性的细胞因子γ干扰素释放,51Cr释放法测CTL可见转染嵌合性T细胞受体基因T淋巴细胞对HER2阳性的肿瘤细胞具显著杀伤效应.结论:转染嵌合性T细胞受体基因的小鼠T淋巴细胞在体外可通过细胞因子释放和CTL效应发挥显著的抗肿瘤作用.     

泛素连接酶Cbl对TRAIL诱导Jurkat T细胞凋亡调节作用的探讨

目的:探讨泛素连接酶Cbl在TRAIL诱导Jurkat T细胞凋亡中的调节作用。方法:台盼蓝拒染法检测细胞增殖能力,流式细胞仪PI染色检测细胞凋亡,蛋白质印迹法检测蛋白的表达。结果:不同浓度的TRAIL作用于Jurkat T细胞24 h,TRAIL能以剂量依赖性的方式抑制JurkatT细胞增殖,并能诱导Jurkat T细胞凋亡。100 ng/mL的TRAIL作用24 h,有35.98%的细胞发生凋亡,而对照组仅有2.52%的细胞发生凋亡,P<0.01。在TRAIL诱导Jur-kat T细胞凋亡过程中伴随有p-Akt表达的一过性上调和泛素连接酶Cbl-b和c-Cbl表达的持续上调。结论:TRAIL上调Cbl蛋白的表达是抑制Jurkat T细胞中PI3K/Akt信号过度活化,进而诱导细胞凋亡的重要机制。     

Polyubiquitylation of AMF requires cooperation between the gp78 and TRIM25 ubiquitin ligases

gp78 is a ubiquitin ligase that plays a vital role in endoplasmic reticulum (ER)-associated degradation (ERAD). Here we report that autocrine motility factor (AMF), also known as phosphoglucose isomerase (PGI), is a novel substrate of gp78. We show that polyubiquitylation of AMF requires cooperative interaction between gp78 and the ubiquitin ligase TRIM25 (tripartite motif-containing protein 25). While TRIM25 mediates the initial round of ubiquitylation, gp78 catalyzes polyubiquitylation of AMF. The E4-like activity of gp78 was illustrated by an in vitro polyubiquitylation assay using Ub-DHFR as a model substrate. We further demonstrate that TRIM25 ubiquitylates gp78 and that overexpression of TRIM25 accelerates the degradation of gp78. Our data suggest that TRIM25 not only cooperates with gp78 in polyubiquitylation of AMF but also gauges the steady-state level of gp78. This study uncovers a previously unknown functional link between gp78 and TRIM25 and provides mechanistic insight into gp78-mediated protein ubiquitylation.     

The HECT family of E3 ubiquitin ligases: multiple players in cancer development

The involvement of the homologous to E6-AP carboxyl terminus (HECT)-type E3s in crucial signaling pathways implicated in tumorigenesis is presently an area of intense research and extensive scientific interest. This review highlights recent discoveries on the ubiquitin-mediated degradation of crucial tumor suppressor molecules catalyzed by the HECT-type E3s. By providing a portrait of their protein targets, we intend to link the substrate specificity of HECT-type E3s with their contribution to tumorigenesis. Moreover, we discuss the relevance of targeting the HECT E3s, through the development of small-molecule inhibitors, as an anticancer therapeutic strategy.     

Genetically engineered T cells expressing a HER2-specific chimeric receptor mediate antigen-specific tumor regression

In this report, we developed a chimeric receptor (N29gamma chR) involving the single chain Fv (scFv) derived from N29 monoclonal antibody (mAb) specific for p185HER2 and characterized the therapeutic efficacy of primary T cells engineered to express N29gamma chR in two histologically distinct murine tumor models. Murine breast (MT901) and fibrosarcoma (MCA207) cancer cell lines were engineered to express human HER2 as targets. Administration of N29gamma chR-expressing T cells eliminated 3-day pulmonary micrometastases of MT901/HER2 and MCA207/HER2 but not parental tumor cells. A 5 to 8-fold increased dose of N29gamma T cells was required to mediate regression of advanced 8-day macrometastases. Exogenous administration of interleukin-2 (IL-2) after N29gamma T-cell transfer was dispensable for treatment of 3-day micrometastases, but was required for mediating regression of well-established 8-day macrometastases. Moreover, fractionated CD8 T cells expressing N29gamma chR suppressed HER2-positive tumor cell growth after adoptive transfer independent of CD4(+) cells. These data indicate that genetically modified T cells expressing a HER2-targeting chimeric receptor can mediate antigen-specific regression of preestablished metastatic cancers in a cell dose-dependent fashion. Systemic administration of IL-2 augments the therapeutic efficacy of these genetically engineered T cells in advanced diseases. These results are relevant to the implication of genetically redirected T cells in clinical cancer immunotherapy.     

The many faces of beta-TrCP E3 ubiquitin ligases: reflections in the magic mirror of cancer

Beta-transducin repeats-containing proteins (beta-TrCP) serve as the substrate recognition subunits for the SCFbeta-TrCP E3 ubiquitin ligases. These ligases ubiquitinate specifically phosphorylated substrates and play a pivotal role in the regulation of cell division and various signal transduction pathways, which, in turn, are essential for many aspects of tumorigenesis. We review the functions of the SCFbeta-TrCP ligases in the light of their relevance to cell growth, survival and transformation. Mechanisms underlying beta-TrCP regulation and their aberration in human and animal cancer as well as prospective of targeting beta-TrCP as a means of anticancer therapy are also discussed.