Cellular Oncoprotein Research Articles

Surface charge of Merkel cell polyomavirus small T antigen determines cell transformation through allosteric FBW7 WD40 domain targeting

Merkel cell polyomavirus (MCV) small T (sT) is the main oncoprotein in Merkel cell carcinoma (MCC) development. A unique domain of sT, LT stabilization domain (LSD), has been reported to bind and inactivate multiple SCF (Skp1-Cullin-F-box) E3 ligases. These interactions impede the turnover of MCV large T (LT) antigen and cellular oncoproteins such as c-Myc and cyclin E, thereby promoting viral replication and cell transformation. However, it is currently unclear how this LSD region contributes to multiple transforming activities of sT. Structural docking simulation of sT and F-box and WD repeat domain-containing 7 (FBW7) revealed a novel allosteric interaction between sT and FBW7 WD40 domain. This model is supported by experimental evidence confirming that charge engineering in the LSD alters sT-WD40 binding. Specifically, loss of net positive charge in the LSD prevents sT-FBW7 binding by abrogating the electrostatic interaction, thus impeding inhibition of FBW7 by sT. Furthermore, positively charged mutations in the LSD significantly restored the sT function and its ability to transform rodent fibroblast cells. We infer that the surface charge of sT is a major determinant for targeting E3 ligases, which leads to sT-induced cell transformation, an observation that could be used to develop targeted therapeutics for MCC.

Proteasomal inhibition triggers viral oncoprotein degradation via autophagy-lysosomal pathway.

Epstein-Barr virus (EBV) nuclear oncoprotein EBNA3C is essential for B-cell transformation and development of several B-cell lymphomas particularly those are generated in an immuno-compromised background. EBNA3C recruits ubiquitin-proteasome machinery for deregulating multiple cellular oncoproteins and tumor suppressor proteins. Although EBNA3C is found to be ubiquitinated at its N-terminal region and interacts with 20S proteasome, the viral protein is surprisingly stable in growing B-lymphocytes. EBNA3C can also circumvent autophagy-lysosomal mediated protein degradation and subsequent antigen presentation for T-cell recognition. Recently, we have shown that EBNA3C enhances autophagy, which serve as a prerequisite for B-cell survival particularly under growth deprivation conditions. We now demonstrate that proteasomal inhibition by MG132 induces EBNA3C degradation both in EBV transformed B-lymphocytes and ectopic-expression systems. Interestingly, MG132 treatment promotes degradation of two EBNA3 family oncoproteins–EBNA3A and EBNA3C, but not the viral tumor suppressor protein EBNA3B. EBNA3C degradation induced by proteasomal inhibition is partially blocked when autophagy-lysosomal pathway is inhibited. In response to proteasomal inhibition, EBNA3C is predominantly K63-linked polyubiquitinated, colocalized with the autophagy-lysosomal fraction in the cytoplasm and participated within p62-LC3B complex, which facilitates autophagy-mediated degradation. We further show that the degradation signal is present at the first 50 residues of the N-terminal region of EBNA3C. Proteasomal inhibition reduces the colony formation ability of this important viral oncoprotein, induces apoptotic cell death and increases transcriptional activation of both latent and lytic gene expression which further promotes viral reactivation from EBV transformed B-lymphocytes. Altogether, this study offers rationale to use proteasome inhibitors as potential therapeutic strategy against multiple EBV associated B-cell lymphomas, where EBNA3C is expressed.

Epidermal growth factor receptor (EGFR) overexpression in endometrial carcinoma: association with histopathologic parameters

BackgroundEpidermal growth factor receptor (EGFR) is a cellular oncoprotein which is overexpressed in many human cancers including a subset of endometrial cancers. Immunohistochemical (IHC) expression of EGFR has been investigated in previous studies; Role of EGFR in endometrial carcinoma as a prognostic biomarker has not been studied in our population; therefore we aimed to evaluate the expression of EGFR in cases of endometrial carcinoma in loco-regional population and its association with histologic variables.MethodsTotal 89 cases of endometrial carcinoma were selected from records of pathology department archives. All patients underwent surgeries at Liaquat National hospital, Karachi from January 2012 till December 2017 over a period of 6 years. Slides of all cases were retrieved and reviewed by two senior histopathologists and pathologic characteristics were evaluated. Moreover, representative tissue blocks of all 89 cases were selected for EGFR immunohistochemistry.Results73% (65 cases) showed no EGFR expression, while 21.3% (19 cases) showed low EGFR expression and 5.6% (5 cases) revealed high EGFR expression. Significant association of EGFR expression was noted with histologic type. Serous carcinoma and carcinosarcoma showed high expression of EGFR. On the other hand, no significant association of EGFR with other histopathologic parameters was found.ConclusionOverall, we found a low EGFR expression in endometrial carcinoma in our population without any significant pathological association except for its high expression in serous carcinoma and carcinosarcoma; however, more large scale studies are warranted to validate these findings.

Activity of antibacterial compounds from Bacillus subtilis against cellular oncoproteins by in silico approach

Abstract The cellular oncoproteins Caspase-3, Caspase-8 and Nuclear Factor-kappa B (NF-κB) are the factors implicated in several cancers. Synthetic and natural compounds were demonstrated to interact with these oncoproteins. Though bacteria are the source of novel compounds including antimicrobial compounds, their anticancer activity remains elusive. Hence in this study, 10 antibacterial compounds of Bacillus subtilis retrieved from the pubchem database and literature were checked for the ability to bind with the chosen oncoproteins in silico. Among those, the compounds Bacilosarcin C, Lipoamicoumacin D and Lipoamicoumacin C showed higher binding GLIDE scores with Caspase-8 (−13.406), Caspase-3 (−9.955) and NF-κB (−8.702) respectively. The strength of hydrogen bond interactions for Bacilosarcin C, Lipoamicoumacin D and Lipoamicoumacin C with the corresponding oncoproteins were 2.62–3.35 A, 2.05 to 3.15 A and 2.59 to 3.21 A respectively. In the binding of these 10 compounds of Bacillus with NF-κB, Thr, Ser and Gly were commonly involved. Similarly, Asn and Phe with Caspase-3 and Arg and Ser with Caspase-8. It is demonstrated that microbial compounds could be used to design novel drugs against oncoproteins to combat cancer.

P53 Degradation by a Coronavirus Papain-like Protease Suppresses Type I Interferon Signaling

Infection by human coronaviruses is usually characterized by rampant viral replication and severe immunopathology in host cells. Recently, the coronavirus papain-like proteases (PLPs) have been identified as suppressors of the innate immune response. However, the molecular mechanism of this inhibition remains unclear. Here, we provide evidence that PLP2, a catalytic domain of the nonstructural protein 3 of human coronavirus NL63 (HCoV-NL63), deubiquitinates and stabilizes the cellular oncoprotein MDM2 and induces the proteasomal degradation of p53. Meanwhile, we identify IRF7 (interferon regulatory factor 7) as a bona fide target gene of p53 to mediate the p53-directed production of type I interferon and the innate immune response. By promoting p53 degradation, PLP2 inhibits the p53-mediated antiviral response and apoptosis to ensure viral growth in infected cells. Thus, our study reveals that coronavirus engages PLPs to escape from the innate antiviral response of the host by inhibiting p53-IRF7-IFNβ signaling.

Exosomal sorting of the viral oncoprotein LMP1 is restrained by TRAF2 association at signalling endosomes

The Epstein–Barr virus (EBV)-encoded oncoprotein latent membrane protein 1 (LMP1) constitutively activates nuclear factor κB (NFκB) from intracellular membranes to promote cell growth and survival. LMP1 associates with CD63 in intracellular membranes and is released via exosomes. Whether tumour necrosis factor (TNF) receptor-associated factors (TRAFs) mediate LMP1 NFκB signalling from endosomes and modulate exosomal sorting is unknown. In this article, we show that LMP1–TRAF2 signalling complexes accumulate at endosomes in a palmitoylation-dependent manner, thereby driving LMP1-dependent oncogenicity. Palmitoylation is a reversible post-translational modification and is considered to function as a membrane anchor for proteins. Mutagenesis studies showed that LMP1–TRAF2 trafficking to endosomes is dependent on one single cysteine residue (C78), a known palmitoylation site of LMP1. Notably, growth assays in soft agar revealed that oncogenic properties of the palmitoylation-deficient LMP1 mutant C78A were diminished compared to wild-type LMP1. Since LMP1 recruitment of TRAF2 and downstream NFκB signalling were not affected by a disturbance in palmitoylation, the specific localization of LMP1 at endosomal membranes appears crucial for its transforming potential. The importance of palmitoylation for trafficking to and signalling from endosomal membranes was not restricted to LMP1, as similar observations were made for the cellular oncoproteins Src and Fyn. Despite abundant LMP1–TRAF2 association at endosomal membranes TRAF2 could not be detected in exosomes by Western blotting or proteomics. Interestingly, point mutations that prevented TRAF binding strongly promoted the sorting and release of LMP1 via exosomes. These observations reveal that LMP1–TRAF2 complexes at endosomes support oncogenic NFκB activation and suggest that LMP1 dissociates from the activated signalling complexes upon sorting into intraluminal vesicles. We propose that “signalling endosomes” in EBV-infected tumour cells can fuse with the plasma membrane, explaining LMP1 release via exosomes.

Berberine alters epigenetic modifications, disrupts microtubule network, and modulates HPV-18 E6–E7 oncoproteins by targeting p53 in cervical cancer cell HeLa: A mechanistic study including molecular docking

Increased evidence of chemo-resistance, toxicity and carcinogenicity necessitates search for alternative approaches for determining next generation cancer therapeutics and targets. We therefore tested the efficacy of plant alkaloid berberine on human papilloma virus (HPV) -18 positive cervical cancer cell HeLa systematically-involving certain cellular, viral and epigenetic factors. We observed disruptions of microtubule network and changes in membrane topology due to berberine influx through confocal and atomic force microscopies (AFM). We examined nuclear uptake, internucleosomal DNA damages, mitochondrial membrane potential (MMP) alterations and cell migration assays to validate possible mode of cell death events. Analytical data on interactions of berberine with pBR322 through fourier transform infrared (FTIR) and gel migration assay strengthen berberine׳s biologically significant DNA binding abilities. We measured cellular uptake, DNA ploidy and DNA strand-breaks through fluorescence activated cell sorting (FACS). To elucidate epigenetic modifications, in support of DNA binding associated processes, if any, we conducted methylation-specific restriction enzyme (RE) assay, methylation specific-PCR (MSP) and expression studies of histone proteins. We also analyzed differential interactions and localization of cellular tumor suppressor p53 and viral oncoproteins HPV-18 E6–E7 through siRNA approach. We further made in-silico approaches to determine possible binding sites of berberine on histone proteins. Overall results indicated cellular uptake of berberine through cell membrane depolarization causing disruption of microtubule networks and its biological DNA binding abilities that probably contributed to epigenetic modifications. Results of modulation in p53 and viral oncoproteins HPV-18 E6–E7 by berberine further proved its potential as a promising chemotherapeutic agent in cervical cancer.

Interaction of Hepatitis C Viral Proteins with Cellular Oncoproteins in the Induction of Liver Cancer

Hepatitis C virus infection is a major health problem all over the world. A large proportion of patients infected by HCV develop liver cirrhosis or cancer. However, the mechanism(s) remain to be elucidated. Since HCV does not carry any known oncogene, it is thought that interaction between virally encoded proteins and host proteins is responsible for carcinogenesis. Many crucial interactions between HCV-encoded proteins and host proteins have been reported. In this review we focus on the interaction of viral proteins with important regulators of cell cycle—oncoproteins YB-1, p53, and cyclin D1—which play a major role in cell proliferation, apoptosis, DNA repair, and genomic stability. Genetic variants of HCV accumulate in patients and alter these interactions of host cell proteins. It is a battle between the virus and host and the final outcome depends on the winner; if the host succeeds in clearing the virus the patient may not develop serious liver diseases. On the other hand, if the virus dominates by evolving quasispecies which code for altered proteins that interact differently with host proteins, or induce mutations in host protooncogenes, then the patient may develop liver cirrhosis and/or liver cancer.

The viral oncoprotein HBx of Hepatitis B virus promotes the growth of hepatocellular carcinoma through cooperating with the cellular oncoprotein RMP.

The smallest gene HBx of Hepatitis B virus (HBV) is recognized as an important viral oncogene (V-oncogene) in the hepatocarcinogenesis. Our previous work demonstrated that RMP is a cellular oncogene (C-oncogene) required for the proliferation of hepatocellular carcinoma (HCC) cells. Here we presented the collaboration between V-oncogene HBx and C-oncogene RMP in the development of HCC. The coexpression of HBx and RMP resulted in the cooperative effect of antiapoptosis and proliferation of HCC cells. In vivo, overexpression of RMP accelerated the growth of HBx-induced xenograft tumors in nude mice and vice versa HBx promoted the growth of RMP-driven xenograft tumors. Although HBx didn't regulate the expression of RMP, HBx and RMP interact with each other and collocalized in the cytoplasm of HCC cells. HBx and RMP collaboratively inhibited the expression of apoptotic factors and promoted the expression of antiapoptotic factors. This finding suggests that HBV may induce, or at least partially contributes to the carcinogenesis of HCC, through its V-oncoprotein HBx interacting with the C-oncoprotein RMP.

Response of Merkel Cell Polyomavirus-Positive Merkel Cell Carcinoma Xenografts to a Survivin Inhibitor

Merkel cell carcinoma (MCC) is a neuroendocrine skin cancer associated with high mortality. Merkel cell polyomavirus (MCV), discovered in 2008, is associated with ~80% of MCC. The MCV large tumor (LT) oncoprotein upregulates the cellular oncoprotein survivin through its conserved retinoblastoma protein-binding motif. We confirm here that YM155, a survivin suppressor, is cytotoxic to MCV-positive MCC cells in vitro at nanomolar levels. Mouse survival was significantly improved for NOD-Scid-Gamma mice treated with YM155 in a dose and duration dependent manner for 3 of 4 MCV-positive MCC xenografts. One MCV-positive MCC xenograft (MS-1) failed to significantly respond to YM155, which corresponds with in vitro dose-response activity. Combination treatment of YM155 with other chemotherapeutics resulted in additive but not synergistic cell killing of MCC cell lines in vitro. These results suggest that survivin targeting is a promising therapeutic approach for most but not all MCV-positive MCCs.

Circulating Tumor Cell Cultures as a Predictive Marker during Salvage Therapy of Refractory Merkel Cell Carcinoma with Chemotherapy and Electron Beam Radiation

Metastatic Merkel Cell carcinoma (MCC) is a highly unusual and aggressive skin cancer that presents as a small, pink to violet skin lesion and metastasizes early in its growth. Metastatic MCC is generally treated with small cell lung cancer chemotherapy regimens, because the tumor consists of neuroendocrine cells, but patients generally do not have durable responses. The pathogenesis of MCC has recently been attributed to the Merkel Cell polyoma virus. This virus activates the cellular retinoblastoma oncoprotein and cell cycle machinery, triggering continual cellular proliferation. A 77-year-old man developed extensive MCC metastases, involving more than one fourth of his scalp and numerous cervical lymph nodes. Following failure of initial chemotherapy and radiation, effective palliation was achieved by using a sequence of electron-beam radiotherapy, low dose gemcitabine, and etoposide, resulting in significant periods of tumor regression and prolonged survival. A novel circulating tumor cell (CTC) culture assay was performed on four separate clinic visits during the treatment period. Tumor colonies were cultured from the patient’s peripheral blood and CTC colony counts were correlated with clinical treatment response. Not only did the patient respond to palliative cell cycle directed chemotherapy and electron beam radiation, but we demonstrated that CTC can be cultured from peripheral blood of MCC patients and serve as a predictive marker to monitor treatment response.

P27kip1 Protein Levels Reflect a Nexus of Oncogenic Signaling during Cell Transformation

SV40 small t-antigen (ST) collaborates with SV40 large T-antigen (LT) and activated rasv12 to promote transformation in a variety of immortalized human cells. A number of oncogenes or the disruption of the general serine-threonine phosphatase protein phosphatase 2A (PP2A) can replace ST in this paradigm. However, the relationship between these oncogenes and PP2A activity is not clear. To address this, we queried the connectivity of these molecules in silico. We found that p27 was connected to each of those oncogenes that could substitute for ST. We further determined that p27 loss can substitute for the expression of ST during transformation of both rodent and human cells. Conversely, knock-in cells expressing the degradation-resistant S10A and T187A mutants of p27 were resistant to the transforming activities of ST. This suggests that p27 is an important target of the tumor-suppressive effects of PP2A and likely an important target of the multitude of cellular oncoproteins that emulate the transforming function of ST.

Survivin Is a Therapeutic Target in Merkel Cell Carcinoma

Merkel cell polyomavirus (MCV) causes ~80% of primary and metastatic Merkel cell carcinomas (MCCs). By comparing digital transcriptome subtraction deep-sequencing profiles, we found that transcripts of the cellular survivin oncoprotein [BIRC5a (baculoviral inhibitor of apoptosis repeat-containing 5)] were up-regulated sevenfold in virus-positive compared to virus-negative MCC tumors. Knockdown of MCV large T antigen in MCV-positive MCC cell lines decreased survivin mRNA and protein expression. Exogenously expressed MCV large T antigen increased survivin protein expression in non-MCC primary cells. This required an intact retinoblastoma protein-targeting domain that activated survivin gene transcription as well as expression of other G(1)-S-phase proteins including E2F1 and cyclin E. Survivin expression is critical to the survival of MCV-positive MCC cells. A small-molecule survivin inhibitor, YM155, potently and selectively initiates irreversible, nonapoptotic, programmed MCV-positive MCC cell death. Of 1360 other chemotherapeutic and pharmacologically active compounds screened in vitro, only bortezomib (Velcade) was found to be similarly potent, but was not selective in killing MCV-positive MCC cells. YM155 halted the growth of MCV-positive MCC xenograft tumors and was nontoxic in mice, whereas bortezomib was not active in vivo and mice displayed serious morbidity. Xenograft tumors resumed growth once YM155 treatment was stopped, suggesting that YM155 may be cytostatic rather than cytotoxic in vivo. Identifying the cellular pathways, such as those involving survivin, that are targeted by tumor viruses can lead to rapid and rational identification of drug candidates for treating virus-induced cancers.

P1-17-08: A Phase II Trial of Ganetespib: Efficacy and Safety in Patients (pts) with Metastatic Breast Cancer (MBC).

Abstract Background: Heat shock protein 90 (Hsp90) is a molecular chaperone that maintains the stability of a number of key cellular oncoproteins (client proteins). When Hsp90 is inhibited, its client proteins undergo ubiquitination and degradation. HER2 is one of the most sensitive client proteins of Hsp90 and we have previously shown that tanespimycin, a geldanamycin-based Hsp90 inhibitor, is active in trastuzumab-refractory HER2+ MBC, with a RR of 22% and clinical benefit rate of 59%. Ganetespib is a synthetic, small molecule, IV administered, non-geldanamycin Hsp90 inhibitor which has broader inhibitory effects on oncoproteins with pre-clinical antitumor activity in more breast cancer (BC) subtypes, including triple negative breast cancer (TNBC). We therefore tested ganetespib in an unselected cohort of patients with advanced BC. Methods: Pts with locally advanced or MBC were treated with single agent ganetespib at 200mg/m2 once weekly for 3 weeks, on a 28 day cycle. The primary endpoint of the trial was overall response rate using RECIST 1.1. Pts with HER2+ BC were required to have received prior therapy with trastuzumab. No more than 3 lines of chemotherapy in the metastatic setting were permitted and there was no limit on prior lines of hormone therapy. Pts were evaluated for response after 2 cycles. The trial used a Simon two-stage design requiring at least 3 responses among the first 22 pts, to allow expansion to a total of 40 pts. Results: A total of 14 pts have been treated thus far with a median age of 45.3 years (36.6 to 59.1) and the following subtypes: 8 ER+/HER2+, 3 ER-/HER2+, 1 ER+/HER2− and 2 TNBC. Pts received a median of 1.84 cycles (0.33 to 4). The most common treatment-related AEs were Grade 1/2 and included the following: diarrhea (64%), fatigue (50%), nausea (35%), vomiting (14%), insomnia (14%) and hypersensitivity reactions (35%). The diarrhea had an early onset (< 24 hrs) and lasted up to 48 hrs post-infusion and was managed with either Imodium or similar anti-diarrheal medication. Pts who developed a hypersensitivity reaction were successfully re-challenged following pre-medication using dexamethasone plus H1/H2 antagonists. Grade 3 AEs were limited to 2 pts: 1 with an asymptomatic and reversible elevation in serum amylase, and the other with abdominal pain and diarrhea requiring a dose reduction to 175mg/m2. Of the 10 pts evaluable for efficacy, there is 1 confirmed partial response (PR), 1 minor response (MR) and 2 pts with stable disease (14%). The pt with MR had TNBC and was taken off-study due to a complicated pneumonia requiring hospitalization (unrelated). The remaining 3 pts had HER2+ BC; 2 of these 3 pts (1 PR, 1 SD) are continuing on study and have completed 4 cycles thus far. Updated toxicity and efficacy data will be presented. Conclusions: These data show activity for single agent ganetespib in different subtypes of breast cancer. Ganetespib was well tolerated, with expected gastrointestinal toxicity that was mild in nature and manageable in all patients. Accrual continues and we will present updated data at the meeting. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-17-08.

Abstract 2114: PTPN13 regulates ephrinB1 reverse signaling, MAP kinase signaling and tumor growth

Abstract Human papillomaviruses (HPV's) are a causative factor in over 90% of cervical and 25% of head and neck squamous cell carcinomas. The C-terminus of high risk HPV 16 E6 associates and degrades PTPN13, a non-receptor protein tyrosine phosphatase. PTPN13 has recently been dubbed a putative tumor suppressor. Moreover, its decreased expression correlates with decreased overall survival in breast cancer. PTPN13 contains 5 PDZ (protein-protein interaction) domains and associates with several proteins. Thus, decreased PTPN13 expression likely results in many cellular consequences, some of which may impact cellular proliferation, tumor growth and metastasis. EphrinB1 belongs to a family of ligands which bind and activate the Eph family of receptor tyrosine kinases. Following activation of Eph receptors, Ephrin ligands too become activated and initiate intracellular signals. Importantly, the Eph/Ephrin system functions in cancer biology. EphrinB1 associates with PDZ4 of PTPN13 and is also a phosphatase substrate. As predicted, shRNA-mediated knock-down of PTPN13 leads to increased phosphorylated EphrinB1. Surprisingly, it resulted in a concomitant increase in phosphorylated Erk1/2. In addition, shRNA-mediated knock-down of EphrinB1 attenuates phosphorylated Erk1/2. These data suggest that activated EphrinB1 crosstalks with the MAP Kinase pathway. We have found that EphrinB1 associates with two cellular oncoproteins, ErbB1 and ErbB2. EphrinB1 co-immunoprecipitates and co-localizes with both ErbB1 and ErbB2. This association of a signaling ligand with known kinase oncoproteins is novel and may significantly increase the complexity of signals mediated both in the context of normal tissue growth and development as well as in cancer. In addition, expression of a constitutively activated erbB2 mutant (mNeuNT) robustly activates EphrinB1 and Erk1/2 signaling. To determine the in vivo significance of EphrinB1 in tumor growth, mouse tonsillar epithelial cells stably expressing HPV16 E6, E7 and Ras (MEERL) or those stably knocked down for PTPBL (murine homolog of PTPN13) and expressing mNeuNT were knocked down for EphrinB1 by shRNA and antibiotic selection (sh-EphrinB1). Stable cells lines over-expressing wildtype EphrinB1 were also generated. Injection of sh-EphrinB1 cells into the hind limb of mice resulted in faster tumor growth and poor survival as compared to controls, while cells over-expressing wildtype EphrinB1 either never grew tumors or grew them very slowly, greatly enhancing survival. These data suggest that the interaction between EphrinB1 and ErbB receptors activate signaling cascades important in tumor growth. PTPN13 modulation of these signals is critical in regulation of tumor growth and survival. 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 2114. doi:10.1158/1538-7445.AM2011-2114

Single-chain Fv fragment antibodies selected from an intrabody library as effective mono- or bivalent reagents for in vitro protein detection

In spite of their many potential applications, recombinant antibody molecules selected by phage display are rarely available commercially, one reason being the absence of robust bacterial expression systems that yield sufficient quantities of reagents for routine applications. We previously described the construction and validation of an intrabody library that allows the selection of single-chain Fv (scFv) fragments solubly expressed in the cytoplasm. Here, we show that it is possible to obtain monomeric scFvs binding specifically to human papillomavirus type 16 E6 and cellular gankyrin oncoproteins in quantities higher than 0.5 g/L of shake-flask culture in E. coli cytoplasm after auto-induction. In addition, stable bivalent scFvs of increased avidity were produced by tagging the scFvs with the dimeric glutathione-S-transferase enzyme (GST). These minibody-like molecules were further engineered by fusion with green fluorescent protein (GFPuv), leading to high yield of functional bivalent fluorescent antibody fragments. Our results demonstrate that scFvs selected from an intrabody library can be engineered into cost-effective bivalent reagents suitable for many biomedical and industrial applications.

NEMO stabilizes c-Myc through direct interaction in the nucleus

The transcription factor c-Myc is a cellular oncoprotein generally upregulated in most of human cancers. NF-κB essential modulator (NEMO) caused phosphorylation and stabilization of c-Myc protein in the nucleus through direct interaction. The interaction caused reduced ubiquitination of c-Myc by inhibiting ubiquitinating activity of Fbw7 without blocking the interaction between c-Myc and Fbw7. As a consequence, NEMO enhanced the expression of several selected c-Myc targets. Compared to the classical role as an essential subunit for the activity of IKK complex, stabilization of c-Myc by direct interaction is a unique function of NEMO, representing a new mechanism to regulate c-Myc activity. Structured summary MINT- 8045088: c-Myc (uniprotkb: P01106) and NEMO (uniprotkb: Q9Y6K9) colocalize (MI: 0403) by fluorescence microscopy (MI: 0416) MINT- 8045072, MINT- 8045101: c-Myc (uniprotkb: P01106) physically interacts (MI: 0915) with NEMO (uniprotkb: Q9Y6K9) by anti tag coimmunoprecipitation (MI: 0007)

C-Myc stimulates cell invasion by inhibiting FBX8 function.

c-Myc is a cellular onco-protein and a transcriptional activator important for cell growth, cell division, and tumorigenesis. Despite all that is known of its function, the mechanism of how c-Myc contributes to tumorigenesis is unclear. To gain insight into the mechanism through which c-Myc protein exerts its oncogenic activity, we performed large-scale, tandem repeat affinity purification and identified the F box only protein 8 (FBX8), an F-box- and Sec7 domain-containing protein, as a novel Myc-binding protein. The c-Myc/FBX8 interaction was mediated by the c-Myc box II (MBII) region. We also confirmed that Myc protein overexpression in 293T cells affected FBX8 cellular translocation and led to recovery from FBX8-mediated inhibition of ADP-ribosylation factor 6 (ARF6) function during cell invasion. Together, these results suggest that FBX8 is a novel c-Myc binding protein and that c-Myc induces cell invasive activity through the inhibition of FBX8 effects on ARF6 function during cell invasion.

Gene Analysis of Epstein-Barr Virus-Associated Lymphomas in Hu-PBL/SCID Chimeras

The mechanisms of Epstein-Barr virus (EBV)-associated tumor development are incompletely understood. The aim of this study was to investigate the gene expression of EBV-associated lymphomas in hu-PBL/SCID mice. Human peripheral blood lymphocytes (hu-PBL) from EBV-seropositive donors were transplanted into severe combined immunodeficiency (SCID) mice. In situ hybridization was used to detect EBV-encoded small RNA- 1 (EBER1) in tumor tissues. Mutation of TP53 exons 5-8 in EBV-induced lymphomas was analyzed by PCR-SSCP. Immunohistochemical staining was used to examine EBV gene products and cellular oncoproteins. Twenty-one of 29 mice developed tumors. EBER1 was positive in the nuclei of almost all tumor cells. Immunohistochemistry showed positive staining of LMP1, EBNA2 and ZEBRA in a small number of tumor cells. Immunohistochemically detectable p53 protein expression was common (85.7%), but TP53 gene mutations were identified in only four cases (19.1%) of EBV-associated lymphomas. Positivity rates of C-myc, Bcl-2 and Bax expression were 100%, 95.2%, and 90.5%, respectively, in the 21 cases of EBV-associated lymphomas. Our preliminary findings suggest that EBV-associated lymphomas in hu-PBL/SCID chimeras show EBV infection, expression of oncogenic viral genes, and overexpression of cellular oncogenes. TP53 gene mutations are rare but p53 protein is commonly expressed in EBV-associated lymphomas.

IL-6 and Tumor Susceptibility Alleles of Strain BALB/C Cause Phenotypic Shift of MYC-Driven Lymphomas in Mice from Diffuse Large B-Cell Lymphoma (DLBCL) to Plasmacytoma (PCT)

Aims: Deregulation of cellular oncoprotein MYC (c-Myc) and the plasma cell growth, differentiation and survival cytokine, interleukin-6 (IL-6), are key pathogenetic factors in human high grade B-cell lymphomas (Burkitt lymphoma and DLBCL) and PCT respectively. Genomic instability, as a consequence of this deregulation, is poorly understood. Transgenic expression of these factors in the B-cell lineage, in mice, generates a pre-clinical model system of great relevance for human B-cell lymphoma counterparts. In this study we evaluated for the incidence of lymphomas in transgenic mouse models, with deregulated CMYC and IL-6. We performed a morphologic classification and correlation of the malignant lymphoid proliferations with the underlying genetic lesions.Materials & Methods: Deregulation of CMYC was observed in gene-targeted C57BL/6 mice that harbor a His6-tagged mouse Myc cDNA gene in themouse immunoglobulin heavy-chain locus (B6.iMyc mice). A double transgenic mouse model, using BALB/c mice, carry the same gene insertion, in addition to a widely expressed human IL-6 transgene (C.iMyc/IL-6 mice). Tumor tissue from the mice was processed by formalin fixation and embedded in paraffin. Sections from the paraffin blocks were stained with hematoxylin and eosin and evaluated for morphology. The morphologic evaluation of the lymphomas was performed blind to the genetic make up of the host, followed by genetic correlation with morphology.Results: B6.iMyc mice developed nodal and extranodal lymphomas. Tumor incidence was 50% (70/140) at 18 months of age. The lymphomas demonstrated sheets of large cells with centroblastic morphology, consistent with diffuse large B-cell lymphoma. The mitotic index was high, with the presence of frequent atypical mitotic figures and apoptotic debris laden macrophages, giving a starry sky pattern. C.iMyc/IL-6 mice developed nodal and extra nodal plasma cell tumors (PCT). Tumor incidence was 100% (20/20) at 4–5 months of age. PCT demonstrated sheets of monotonous and atypical plasma cells with prominent nucleoli. In lymphoid tissue containing germinal centers, the sheets of plasma cells were located in the interfollicular areas. There was no bone marrow involvement.Conclusions: Our findings demonstrate thatB6.iMyc mice with CMYC deregulation develop high grade B-cell lymphomas similar to human DLBCL.The activity of deregulated IL-6 appears to enhance and accelerate the oncogenic potential of deregulated CMYC, in the pathogenesis of lymphoid malignancy.IL-6 and/or tumor susceptibility alleles of strain BALB/c cause a remarkable shift in the phenotype of MYC-driven lymphomas from DLBCL to PCT.