Tumor Suppressor Gene Product Research Articles

Immunocytochemical markers of neuronal maturation in human diagnostic neuropathology.

Histological descriptions of morphogenesis in human fetal brain and in malformations and tumours can now be supplemented by the timing and sequence of the maturation of individual neurons. In human neuropathology, this is principally achieved by immunocytochemical reactivities used as maturational markers of neuronal properties denoted by molecules and cell products. Cytological markers can appear early and then regress, often being replaced by more mature molecules, or might not exhibit the onset of immunoreactivity until a certain stage of neuronal differentiation is achieved, some early, others intermediate and some late during the maturational process. Inter-specific differences occur in some structures of the brain. The classification of markers of neuronal maturation can be based, in addition to those mentioned above, on several criteria: cytological localisation, water solubility, biochemical nature of the antigen, specificity and various technical factors. The most useful immunocytochemical markers of neuronal maturation in human neuropathology are NeuN, synaptophysin, calretinin and other calcium-binding molecules, various microtubule-associated proteins and chromogranins. Non-antibody histochemical stains that denote maturational processes include luxol fast blue for myelination, acridine orange fluorochrome for nucleic acids, mitochondrial respiratory chain enzymes and argentophilic impregnations. Neural crest derivatives of the peripheral nervous system, including chromaffin and neuroendocrine cells, have special features that are shared and others that differ greatly between lineages. Other techniques used in human diagnostic neuropathology, particularly as applied to tumours, include chromosomal and genetic analyses, the mTOR signalling pathway, BRAF V600E and other tumour-suppressor gene products, transcription products of developmental genes and the proliferation index of the tumour cells and of mitotic neuroepithelial cells.

P75NTR is highly expressed in vestibular schwannomas and promotes cell survival by activating nuclear transcription factor κB

Vestibular schwannomas (VSs) arise from Schwann cells (SCs) and result from the loss of function of merlin, the protein product of the NF2 tumor suppressor gene. In contrast to non‐neoplastic SCs, VS cells survive long‐term in the absence of axons. We find that p75NTR is overexpressed in VSs compared with normal nerves, both at the transcript and protein level, similar to the response of non‐neoplastic SCs following axotomy. Despite elevated p75NTR expression, VS cells are resistant to apoptosis due to treatment with proNGF, a high affinity ligand for p75NTR. Furthermore, treatment with proNGF protects VS cells from apoptosis due to c‐Jun N‐terminal kinase (JNK) inhibition indicating that p75NTR promotes VS cell survival. Treatment of VS cells with proNGF activated NF‐κB while inhibition of JNK with SP600125 or siRNA‐mediated knockdown reduced NF‐κB activity. Significantly, proNGF also activated NF‐κB in cultures treated with JNK inhibitors. Thus, JNK activity appears to be required for basal levels of NF‐κB activity but not for proNGF‐induced NF‐κB activity. To confirm that the increase in NF‐κB activity contributes to the prosurvival effect of proNGF, we infected VS cultures with Ad.IκB.SerS32/36A virus, which inhibits NF‐κB activation. Compared with control virus, Ad.IκB.SerS32/36A significantly increased apoptosis including in VS cells treated with proNGF. Thus, in contrast to non‐neoplastic SCs, p75NTR signaling provides a prosurvival response in VS cells by activating NF‐κB independent of JNK. Such differences may contribute to the ability of VS cells to survive long‐term in the absence of axons. GLIA 2014;62:1699–1712

BLMP-1/Blimp-1 regulates the spatiotemporal cell migration pattern in C. elegans.

Spatiotemporal regulation of cell migration is crucial for animal development and organogenesis. Compared to spatial signals, little is known about temporal signals and the mechanisms integrating the two. In the Caenorhabditis elegans hermaphrodite, the stereotyped migration pattern of two somatic distal tip cells (DTCs) is responsible for shaping the gonad. Guidance receptor UNC-5 is necessary for the dorsalward migration of DTCs. We found that BLMP-1, similar to the mammalian zinc finger transcription repressor Blimp-1/PRDI-BF1, prevents precocious dorsalward turning by inhibiting precocious unc-5 transcription and is only expressed in DTCs before they make the dorsalward turn. Constitutive expression of blmp-1 when BLMP-1 would normally disappear delays unc-5 transcription and causes turn retardation, demonstrating the functional significance of blmp-1 down-regulation. Correct timing of BLMP-1 down-regulation is redundantly regulated by heterochronic genes daf-12, lin-29, and dre-1, which regulate the temporal fates of various tissues. DAF-12, a steroid hormone receptor, and LIN-29, a zinc finger transcription factor, repress blmp-1 transcription, while DRE-1, the F-Box protein of an SCF ubiquitin ligase complex, binds to BLMP-1 and promotes its degradation. We have therefore identified a gene circuit that integrates the temporal and spatial signals and coordinates with overall development of the organism to direct cell migration during organogenesis. The tumor suppressor gene product FBXO11 (human DRE-1 ortholog) also binds to PRDI-BF1 in human cell cultures. Our data suggest evolutionary conservation of these interactions and underscore the importance of DRE-1/FBXO11-mediated BLMP-1/PRDI-BF1 degradation in cellular state transitions during metazoan development.

Merlin deficiency predicts FAK inhibitor sensitivity: a synthetic lethal relationship.

The goal of targeted therapy is to match a selective drug with a genetic lesion that predicts for drug sensitivity. In a diverse panel of cancer cell lines, we found that the cells most sensitive to focal adhesion kinase (FAK) inhibition lack expression of the neurofibromatosis type 2 (NF2) tumor suppressor gene product, Merlin. Merlin expression is often lost in malignant pleural mesothelioma (MPM), an asbestos-induced aggressive cancer with limited treatment options. Our data demonstrate that low Merlin expression predicts for increased sensitivity of MPM cells to a FAK inhibitor, VS-4718, in vitro and in tumor xenograft models. Disruption of MPM cell-cell or cell-extracellular matrix (ECM) contacts with blocking antibodies suggests that weak cell-cell adhesions in Merlin-negative MPM cells underlie their greater dependence on cell-ECM-induced FAK signaling. This provides one explanation of why Merlin-negative cells are vulnerable to FAK inhibitor treatment. Furthermore, we validated aldehyde dehydrogenase as a marker of cancer stem cells (CSCs) in MPM, a cell population thought to mediate tumor relapse after chemotherapy. Whereas pemetrexed and cisplatin, standard-of-care agents for MPM, enrich for CSCs, FAK inhibitor treatment preferentially eliminates these cells. These preclinical results provide the rationale for a clinical trial in MPM patients using a FAK inhibitor as a single agent after first-line chemotherapy. With this design, the FAK inhibitor could potentially induce a more durable clinical response through reduction of CSCs along with a strong antitumor effect. Furthermore, our data suggest that patients with Merlin-negative tumors may especially benefit from FAK inhibitor treatment.

MiR-34a is essential for p19Arf-driven cell cycle arrest

The Arf tumor suppressor gene product, p19Arf, regulates cell proliferation in incipient cancer cells and during embryo development. Beyond its commonly accepted p53-dependent actions, p19Arf also acts independently of p53 in both contexts. One such p53-independent effect with in vivo relevance includes its repression of Pdgfrβ, a process that is essential for vision in the mouse. We have utilized cell culture-based and mouse models to define a new role for miR-34a in this process. Ectopic expression of Arf in cultured cells enhanced the expression of several microRNAs predicted to target Pdgfrß synthesis, including the miR-34 family. Because miR-34a has been implicated as a p53-dependent effector, we investigated whether it also contributed to p53-independent effects of p19Arf. Indeed, in mouse embryo fibroblasts (MEFs) lacking p53, Arf-driven repression of Pdgfrβ and its blockade of Pdgf-B stimulated DNA synthesis were both completely interrupted by anti-microRNA against miR-34a. Ectopic miR-34a directly targeted Pdgfrβ and a plasmid reporter containing wild-type Pdgfrβ 3′UTR sequence, but not one in which the miR-34a target sequence was mutated. Although miR-34a expression has been linked to p53—a well-known effector of p19Arf—Arf expression and its knockdown correlated with miR-34a level in MEFs lacking p53. Finally, analysis of the mouse embryonic eye demonstrated that Arf controlled expression of miR-34a, and the related miR-34b and c, in vivo during normal mouse development. Our findings indicate that miR-34a provides an essential link between p19Arf and its p53-independent capacity to block cell proliferation driven by Pdgfrβ. This has ramifications for developmental and tumor suppressor roles of Arf.

Persistent human papillomavirus infection in the etiology of cervical carcinoma: the role of immunological, genetic, viral and cellular factors

The aim of this paper was to present the role of human papillomavirus (HPV) in cervical carcinogenesis from several aspects. By explaining the HPV virus lifecycle and structure, its effect on cervical cell cycle and subversion of immune response can be better understood. Early E region of the viral genome encodes proteins that are directly involved in carcinogenesis. The E6 protein binds to p53 protein (product of tumor-suppressor gene) blocking and degrading it, which in turn prevents cell cycle arrest and apoptosis induction. E6 is also capable of telomerase activation, which leads to cell immortalization; it also reacts with host proto-oncogene c-jun, responsible for transcription, shortens G1 phase and speeds up the transition from G1 to S phase of the cells infected by HPV. E7 forms bonds with retinoblastoma protein (product of tumor-suppressor gene) and inactivates it. It can inactivate cyclin inhibitors p21, p27, and abrogate the mitotic spindle checkpoint with the loss of protective effect of pRB and p53. The immune system cannot initiate early immunological reaction since the virus is non-lytic, while the concentration of viral proteins--antigens is low and has a basal intracellular position. Presentation through Langerhans cells (LC) is weak, because the number of these cells is low due to the effect of HPV. E7 HPV reduces the expression of E-cadherin, which is responsible for LC adhesion to HPV-transformed keratinocytes. Based on these considerations, it may be concluded that the process of cervical carcinogenesis includes viral, genetic, cellular, molecular-biological, endocrine, exocrine and immunological factors.

Antinuclear antibodies defining autoimmunity pathways

Immunofluorescent imaging has been a powerful technique in helping to identify intracellular nuclear and cytoplasmic molecules which are target antigens of autoantibodies in systemic autoimmune disorders. Patterns of staining can be correlated with molecules engaged in specific cellular functions and distributed in distinct cellular domains. Different autoimmune disorders have different profiles of autoantibodies, and immunodiagnostics has become an important adjunct in differential diagnosis. An important finding that has eluded explanation is the presence of autoantibodies to many different antigens, manifested strikingly in systemic lupus erythematosus. In cancer, the occurrence of autoantibodies to tumor-associated antigens is not uncommon and a characteristic feature is also the presence of multiple autoantibodies. The targeted tumor-associated antigens are either oncogene or tumor suppressor gene products or their coactivators, which are altered or mutated and driving the autoimmune response. Most cancer cells have between two and eight mutated genes before oncogenic transformation occurs, initiating a process called synthetic lethality in tumorigenesis pathways. These observations beg the question of whether there are similar mechanisms in systemic lupus erythematosus and other disorders driving autoimmunity pathways. Targeting molecules that are synthetic lethal to each other is in the forefront of the search for anticancer therapy, and this could also be an objective in systemic autoimmune disorders.

BOB.1-positive Classical Hodgkin's Lymphoma Carries Hypermethylation of Its Promoter as Epigenetic Marker of Gene-silencing Memory.

Analysis of archival formalin-fixed, paraffin-embedded (FFPE) pathological specimens of three case of Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) and three cases of classical Hodgkin lymphoma (CHL) revealed that hypermethylation of the BOB.1 gene promoter was exclusively observed in CHL. A discrepancy was observed, however, between the methylation status of the BOB.1 gene promoter and its expression in the EBV-positive mixed cellular CHL (MCCHL). Since MCCHL lacks the typical B-cell phenotype even in the presence of abundant BOB.1 transcription factors, functional activity of BOB.1 may be lost or reduced by a mechanism other than epigenetic gene silencing. When some tumor-suppressor gene products have lost their biological function, impact or significance of derepression of such genes may be little. Therefore, when interpreting immunohistochemical results for diagnostic or research purposes, it must be borne in mind that apparent positive immunostaining can merely be the result of chromatin remodeling and that such transient expression often has little functional significance. Any apparent positive immunohistochemical result needs to be interpreted carefully with the help of the hypermethylation status as a molecular marker of gene silencing memory.

HIF1α deubiquitination by USP8 is essential for ciliogenesis in normoxia

Loss of primary cilia is a key feature of von Hippel-Lindau tumor suppressor (VHL)-associated pathology. Although VHL-deficiency predisposes cells to precipitous cilia disassembly in response to growth factor cues, it does not affect ciliogenesis. Here, using a siRNA-based screen to find genes that are essential for ciliogenesis only in the presence of the VHL tumor suppressor gene product pVHL, we identify ubiquitin-specific protease (USP)8. The pVHL-dependency of USP8 for ciliogenesis is directly linked to its function as a HIF1α deubiquitinating enzyme. By counteracting pVHL-mediated ubiquitination of HIF1α, USP8 maintains a basal expression of HIF1α and HIF transcriptional output in normoxia, including the repression of Rabaptin5, which is essential for endosome trafficking-mediated ciliogenesis.

Abstract PR07: Integrative functional genomics of breast cancer

Abstract Cancer is a disease of the genome. Using genomic approaches alone, it is difficult to ascertain which variants drive pathogenesis because most of these are functionally irrelevant passenger mutations. Highly recurrent events point to drivers; however, the majority of genomic alterations in tumors occur at low frequency. In addition, the products of many oncogenes and tumor suppressor genes are not druggable. However, such abnormalities can cause unanticipated gene/pathway dependencies (synthetic lethality), providing alternate avenues for drug development. Lentiviral-based shRNA libraries enables genome-wide screening of cultured cancer cells in a pooled format, facilitating the identification of genes necessary for cancer cell proliferation and survival in cultured cells. We screened a panel of > 75 breast cancer cell lines using an 80,000 lentiviral shRNA library targeting 16,000 genes and integrated these screen results with gene expression, copy-number variation (CNV), and somatic mutations derived from the same lines. We identified several classes of gene dropouts, which are required for survival or growth of all (or some) cell lines, irrespective of subtype and several subtype-specific genes, whose essentiality is restricted to a defined subtype. These include well-known HER2 subtype-specific genes, ERBB2, ERBB3, and TFAP2C and luminal subtype-specific gene FOXA1, SPDEF, GATA3, and ESR1. In addition, the unprecedented number of lines allows the identification of synthetic lethal interaction with common breast cancer somatic mutations or CNV such as PIK3CA and PTEN or 9p21 deletion, respectively, which encodes the CDKN2A, CDKN2B, and MTAP genes. Finally, integration of gene expression, copy number variation, and functional screening results identified potential biomarkers with common genetic changes and functional drivers. Overall, our study represents an extensive functional genetic survey of breast cancer, reveals complexities between genomic and functional genomic results, and uncovers several unexpected gene dependencies and potential novel therapeutic target for each subtype. This abstract is also presented as Poster A033. Citation Format: Richard Marcotte, Azin Sayad, Cathy Iorio, Maliha Haider, Jason Moffat, Benjamin G. Neel. Integrative functional genomics of breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr PR07.

Merlin Expression in Spiral Ganglion Neurons Is Decreased in Deafened Mice

Objectives: 1) Evaluate the expression and phosphorylation status of merlin, the protein product of the Nf2 tumor suppressor gene, in auditory neural structures before and after deafening. 2) Determine the extent to which merlin influences neurite growth in spiral ganglion neurons (SGNs). 3) Determine whether merlin expression in SGNs or spiral ganglion Schwann cells (SGSCs) inhibits neurite growth. Methods: Immunostaining of cochlear sections before and after deafening and organotypic explants with anti-merlin antibodies were used to assess merlin expression in SGNs and SGSCs. A (Tx)-inducible Cre-mediated recombination system was used to knock out merlin in neonatal SGNs. Cultures were maintained with (+) or without (-) Tx for 24h to induce Cre expression. To evaluate loss of merlin expression in SGNs versus SGSCs, we plated (+)Tx and (-)Tx neurons onto a bed of (+)Tx and (-)Tx SGSCs. Neurite length and branching were determined for each condition. Results: Merlin expression in SGNs raises the possibility that merlin regulates SGN neurite growth. Merlin expression in SGNs decreased in cochlear sections taken from P30 versus P60 control rats and in P30 and P60 deafened versus P30 and P60 control rats respectively. Reduced merlin expression in cultured neurons but not SCs significantly increases both the number and length of SGN neurites in SGNs. Conclusions: The pattern of merlin expression and phosphorylation following deafening in SGNs and SGSCs taken together with results indicating that neuronal expression of merlin inhibits neurite growth raises the possibility that merlin plays a role in auditory neural maintenance, regeneration, or both.

ATM Mediates pRB Function To Control DNMT1 Protein Stability and DNA Methylation

The retinoblastoma tumor suppressor gene (RB) product has been implicated in epigenetic control of gene expression owing to its ability to physically bind to many chromatin modifiers. However, the biological and clinical significance of this activity was not well elucidated. To address this, we performed genetic and epigenetic analyses in an Rb-deficient mouse thyroid C cell tumor model. Here we report that the genetic interaction of Rb and ATM regulates DNMT1 protein stability and hence controls the DNA methylation status in the promoters of at least the Ink4a, Shc2, FoxO6, and Noggin genes. Furthermore, we demonstrate that inactivation of pRB promotes Tip60 (acetyltransferase)-dependent ATM activation; allows activated ATM to physically bind to DNMT1, forming a complex with Tip60 and UHRF1 (E3 ligase); and consequently accelerates DNMT1 ubiquitination driven by Tip60-dependent acetylation. Our results indicate that inactivation of the pRB pathway in coordination with aberration in the DNA damage response deregulates DNMT1 stability, leading to an abnormal DNA methylation pattern and malignant progression.

CD74-dependent Deregulation of the Tumor Suppressor Scribble in Human Epithelial and Breast Cancer Cells

The γ subunit of the major histocompatibility complex (MHC) class II complex, CD74, is overexpressed in a significant proportion of metastatic breast tumors, but the mechanistic foundation and biologic significance of this phenomenon are not fully understood. Here, we show that when CD74 is overexpressed in human cancer and noncancerous epithelial cells, it interacts and interferes with the function of Scribble, a product of a well-known tumor suppressor gene. Furthermore, using epithelial cell lines expressing CD74 under the control of tetracycline-inducible promoter and quantitative high-resolution mass spectrometry, we demonstrate that, as a result of CD74 overexpression, the phosphorylation pattern of the C-terminal part of Scribble undergoes specific changes. This is accompanied with a translocation of the protein from the sites of cell-to-cell contacts at the plasma membrane to the cytoplasm, which is likely to effectively enhance the motility and invasiveness of the cancer cells.

Downregulation of chromatin remodeling factor CHD5 is associated with a poor prognosis in human glioma

Chromodomain helicase DNA-binding protein 5 (CHD5), a member of the CHD family, is involved in key cellular processes including chromatin remodeling, cell cycle regulation, and cellular adhesion. Recent studies have demonstrated that CHD5 is the product of a novel tumor suppressor gene and is implicated in certain tumor types. However, the clinicopathological significance of CHD5 expression in human malignant gliomas remains unclear. To address this problem, CHD5 expression in human gliomas and non-neoplastic brain tissues was measured using real-time quantitative polymerase chain reaction (RT–PCR) assay, Western blot, and immunohistochemistry. The association of CHD5 immunostaining with clinicopathological factors or prognosis of glioma patients was statistically analyzed. Genetic and protein expression of CHD5 were downregulated in glioma tissues compared to corresponding non-neoplastic brain tissues (both p<0.001). Additionally, decreased expression of CHD5 in glioma was significantly associated with pathological grade (p=0.007); high pathological grade was associated with low CHD5 expression. Loss of CHD5 protein expression was also significantly correlated with a low Karnofsky performance scale score (p=0.01). Moreover, overall survival of patients with low CHD5 protein expression was dramatically shorter than those of patients with high CHD5 protein expression (p=0.003). Multivariate Cox regression analysis indicated that CHD5 expression was an independent prognostic factor for patients with gliomas (p=0.01). In conclusion, these data offer convincing evidence for the first time that CHD5 might act as a tumor suppressor in glioma, may act as a regulator of aggressive development, and is a candidate prognostic marker for this malignancy.

Integrated Proteomics Identified Novel Activation of Dynein IC2-GR-COX-1 Signaling in Neurofibromatosis Type I (NF1) Disease Model Cells

Neurofibromatosis type 1 (NF1) tumor suppressor gene product, neurofibromin, functions in part as a Ras-GAP, and though its loss is implicated in the neuronal abnormality of NF1 patients, its precise cellular function remains unclear. To study the molecular mechanism of NF1 pathogenesis, we prepared NF1 gene knockdown (KD) PC12 cells, as a NF1 disease model, and analyzed their molecular (gene and protein) expression profiles with a unique integrated proteomics approach, comprising iTRAQ, 2D-DIGE, and DNA microarrays, using an integrated protein and gene expression analysis chart (iPEACH). In NF1-KD PC12 cells showing abnormal neuronal differentiation after NGF treatment, of 3198 molecules quantitatively identified and listed in iPEACH, 97 molecules continuously up- or down-regulated over time were extracted. Pathway and network analysis further revealed overrepresentation of calcium signaling and transcriptional regulation by glucocorticoid receptor (GR) in the up-regulated protein set, whereas nerve system development was overrepresented in the down-regulated protein set. The novel up-regulated network we discovered, "dynein IC2-GR-COX-1 signaling," was then examined in NF1-KD cells. Validation studies confirmed that NF1 knockdown induces altered splicing and phosphorylation patterns of dynein IC2 isomers, up-regulation and accumulation of nuclear GR, and increased COX-1 expression in NGF-treated cells. Moreover, the neurite retraction phenotype observed in NF1-KD cells was significantly recovered by knockdown of the dynein IC2-C isoform and COX-1. In addition, dynein IC2 siRNA significantly inhibited nuclear translocation and accumulation of GR and up-regulation of COX-1 expression. These results suggest that dynein IC2 up-regulates GR nuclear translocation and accumulation, and subsequently causes increased COX-1 expression, in this NF1 disease model. Our integrated proteomics strategy, which combines multiple approaches, demonstrates that NF1-related neural abnormalities are, in part, caused by up-regulation of dynein IC2-GR-COX-1 signaling, which may be a novel therapeutic target for NF1.

CtBP2 contributes to malignant development of human esophageal squamous cell carcinoma by regulation of p16INK4A

C-terminal binding protein-2 (CtBP2), as a transcriptional co-repressor, has been shown to mediate the repression of p16(INK4A) , a tumor suppressor gene product, in primary human cells. Here we aimed to investigate how the correlation between CtBP2 and p16(INK4A) influenced the development of esophageal squamous cell carcinoma (ESCC). Immunohistochemistry of ESCC tissue sections indicated that the CtBP2 and p16(INK4A) expressions were inversely correlated to each other with a linear regression coefficient of -0.747 (P < 0.05), and Western blot analysis revealed that CtBP2 was higher expressed in tumorous tissues than in adjacent non-tumorous tissues. Either CtBP2 or p16(INK4A) expression was significantly related to histological differentiation (P = 0.016 or 0.001) and to the expression of Ki-67, a proliferating marker (P = 0.006 or 0.02), and patients with higher CtBP2 and lower p16(INK4A) expressions had shorter overall survival. We also observed that CtBP2 modulated the cell proliferation and cell cycle in ECA109 cells, an ESCC cell line, by inhibiting p16(INK4A) . Overexpression or knockdown of CtBP2 in ECA109 cells was found to inhibit or activate the mRNA or protein expression of p16(INK4A) , which in turn altered the cell proliferation and cell cycle in ECA109 cells, as measured by flow cytometry and cell count assay. Additionally, after ECA109 cells silenced for CtBP2 were treated with cisplatin (an anti-ESCC agent), the p16(INK4A) expression was up-regulated, and the cell apoptosis was promoted, thus confirming the repression of p16(INK4A) by CtBP2. Collectively, all results suggested that CtBP2 might contribute to the progression of ESCC through a negative transcriptional regulation of p16(INK4A).

Functional characterization of the recombinant human tumour suppressor 101F6 protein, a cytochrome b561 homologue

Candidate human tumour suppressor gene product, 101F6 protein, is a highly hydrophobic transmembrane protein and a member of cytochrome b(561) family. Purified 101F6 protein expressed in Pichia pastoris cells showed visible absorption spectra similar but distinct from those of cytochrome b(561). Haem content analysis indicated presence of two haems B per molecule. Midpoint potentials of the purified protein were found as +109 and +26 mV for two haems, slightly lower than those for bovine chromaffin granule or plant Zea mays cytochromes b(561). Electron paramagnetic resonance (EPR) spectra in oxidized state at 5 K showed only a highly anisotropic low-spin (HALS) signal at g(z) = 3.75. However, at 15 and 20 K, another HALS-type signal appeared at g(z) = 3.65 being overlapped with that of g(z) = 3.75. The rhombic EPR signal at g(z) = 3.16 previously seen in other cytochromes b(561) was not observed, suggesting distinct haem environments. Absence of the inhibition in the electron transfer from ascorbate by a treatment of 101F6 protein with diethylpyrocarbonate showed a remarkable contrast from those of other cytochromes b(561) where the 'concerted H(+)/e(-) transfer mechanism' at the cytosolic haem centre was blocked by specific Nε-carbethoxylation of haem-coordinating imidazole, suggesting that 101F6 protein might accept electrons via a mechanism distinct from other cytochromes b(561).

P53 Arg72Pro Polymorphism, HPV Status and Initiation, Progression, and Development of Cervical Cancer: A Systematic Review and Meta-Analysis

Cervical cancer develops through progression from normal cervical epithelium through squamous intraepithelial lesions (SIL) to invasive cancer. Cervical cancer is associated with oncogenic human papillomavirus (HPV). The HPV E6 oncoprotein binds to the tumor suppressor gene product p53, promoting its degradation; the Arg allele of p53 Arg72Pro polymorphism binds more ardently with HPV E6 than the Pro variant. Here we evaluate the role of p53 Arg72Pro polymorphism and HPV status on the initiation, progression, and development of cervical cancer. A systematic review and meta-analysis were conducted. Events of interest were the initiation of neoplasia (SIL vs. normal), progression to invasive cancer (cervical cancer vs. SIL), and risk of invasive cancer (cervical cancer vs. normal) by HPV status. OR were extracted from individual studies and pooled using generic inverse variance and random effects modeling. Forty-nine studies were included. In individuals showing HPV positivity, there was a significantly higher odds of progression from SIL to cervical cancer with the p53 Arg allele [OR 1.37; 95% confidence intervals (CI), 1.15-1.62; P < 0.001]. This association was not seen in HPV-negative individuals. p53 Arg72Pro was not associated with the risk of cervical cancer or initiation of SIL in either HPV-positive or HPV-negative patient subsets. The Arg variant of p53 Arg72Pro is associated with progression of SIL to cervical cancer only in the presence of HPV positivity. There were no associations of this variant with overall risk or initiation of cancer in either HPV-positive or HPV-negative patients. Clin Cancer Res; 18(23); 6407-15.

Pathways of vulvar intraepithelial neoplasia and squamous cell carcinoma

Vulvar squamous cell carcinoma (VSCC) accounts for >90% of the malignant tumours of the vulva. Most VSCCs originate in intraepithelial lesions, named vulvar intraepithelial neoplasia (VIN), that precede the development of VSCC by a variable period of time. Strong evidence has accumulated showing that there are two different aetiopathogenic pathways for the development of VSCC and VIN, one associated with infection by human papillomavirus (HPV), and a second independent of HPV infection. These two different types of VSCC have different epidemiological, pathological and clinical characteristics, and should therefore be considered as two separate entities. Histologically, HPV-associated VSCCs are of the basaloid or warty type, and arise from VIN of the usual type. Inactivation of p53 and the retinoblastoma tumour suppressor gene product by the viral gene products E6 and E7 is involved in the process of malignant transformation. HPV-independent VSCCs are histologically keratinizing, are associated with differentiated VIN and lichen sclerosus, and frequently show mutations of p53. p16(INK4a) and p53 immunostaining can be useful for classifying VSCC into HPV-associated or HPV-independent. Although large, multicentre studies are needed to definitively assess the involvement of HPV in the prognosis of VSCC, most studies have not found clear differences in survival between HPV-associated and HPV-independent tumours.

Protein Arginine Methyltransferase 5 (PRMT5) Over-Expression Is Essential for Epstein-Barr Virus-Driven B-Cell Transformation.

Abstract 2378Epstein-Barr virus (EBV) is a ubiquitous, gamma herpes virus that infects human epithelial cells and B lymphocytes. Over 90% of adults worldwide are infected with EBV and, collectively, this virus is associated with a broad spectrum of benign and malignant disease. EBV is a potent oncogenic virus and is capable of efficiently transforming B cells in both in vitro and in vivo models. While signaling cascades contributing toward B cell immortalization and transformation following EBV infection have been described, epigenetic events that contribute toward the B cell transformation process remain poorly characterized. EBV-transformed lymphoblastoid lines (LCL) and spontaneous B cell lymphomas that arise in the hu-PBL-SCID model of EBV-induced lymphomagenesis show abundant expression of the protein arginine methyltransferase 5 (PRMT5), a type II PRMT enzyme that catalyzes symmetric dimethylation of arginine residues on histones and non histone proteins (P53, CYCLIN D1). PRMT5 partners with multiple co-repressor proteins such as HDAC2, MBD2 and DNMT3a to silence multiple regulatory and tumor suppressor gene products. All EBV-transformed B cell lines and primary tumors showed cytoplasmic and nuclear staining for PRMT5 and its associated epigenetic marks symmetric dimethyl histone 3, arginine 8 (S2Me-H3R8) and S2Me-H4R3. Resting and activated B cells did not demonstrate PRMT5 over expression or associated global epigenetic marks. Infection of primary human B cells with the B95.8 strain (but not mutant P3HR1 or inactivated EBV) led to dysregulated expression of PRMT5 as early as day 4 post infection. By day 8 post EBV infection, PRMT5 location had transitioned to the nucleus and this localization coincided with acquisition of S2Me-H4R3 and S2Me-H3R8 and loss of the asymmetric epigenetic mark 2Me-H4R3,a type I PRMT histone mark. PRMT5 over-expression was dependent on LMP-1-driven NFkB activity and transcriptional silencing of miR96 expression, a micro RNA that targets the PRMT5 3'UTR. To determine if PRMT5 over expression was essential for induction and maintenance of the transformed phenotype, we infected resting B cells with EBV and, at various time points (day 4, 7, 14 or 21), added a novel, highly selective small molecule inhibitor of PRMT5 activity, an inactive small molecule control, shRNA specific for PRMT5 or control shRNA. Absolute CD19+ cell counts and confocal microscopy experiments to monitor PRMT5 and its epigenetic marks were performed and showed that PRMT5 activity was critical for EBV-driven B cell transformation to proceed. PRMT5 inhibition of resting or activated B cells did not result in any loss of viability. Global transcriptome analysis identified several tumor suppressor genes, including the protein tyrosine phosphatase PTPROt, were silenced during EBV-driven B cell transformation. Chromatin immune precipitation (ChIP)-sequencing using monoclonal antibodies specific for PMRT5 and S2Me-H3R8 (or control IgG) confirmed the PTPROt promoter to be directly targeted by PRMT5 and PTPROt transcript was found to become silenced during EBV-driven B cell transformation. Real time PCR and RNA-seq showed PTPROt transcript to become restored with PRMT5 inhibition. PTPROt expression led to dephosphorylation and inhibition of the LYN, SYK, and Bruton's Tyrosine kinase (BTK) kinase proteins, critical proteins involved in regulation of the B cell receptor (BCR). This model provided us with direct evidence that PRMT5 activity is critical to EBV-driven B cell transformation and supports our hypothesis that PRMT5 dysregulation drives epigenetic events that directly contribute to key initiating events during B cell transformation as well as to the maintenance of the malignant phenotype. We believe this is the first example of oncogenic virus driving over expression of an epigenetic modifier that catalyzes placement of global repressive epigenetic marks that silence of regulatory and tumor suppressor genes. This data justifies pursuit of experimental therapeutic strategies focused on selective PRMT5 inhibition in cancer. Disclosures:No relevant conflicts of interest to declare.