Overexpression Of Oncoproteins Research Articles

Redox Status and Protein Glutathionylation in Binase-Treated HPV16-Positive SiHa Carcinoma Cells

Human papillomavirus type 16 (HPV16) belongs to viruses of the high-risk type and is associated by overexpression of E6 and E7 oncoproteins, which determine the oncogenic properties of the virus, such as immortalization and malignant transformation of proliferating epithelial cells. The biogenesis of redox-sensitive proteins E6 and E7 at the early stages of viral infection leads to blocking of the cell antioxidant defense system and ubiquintin-dependent degradation of p53 and Rb tumor suppressors. Maintaining high rates of tumor cell proliferation contributes to an increase in the level of reactive oxygen species (ROS) and a shift in the redox balance towards oxidative processes. Reduced glutathione (GSH) provides antioxidant protection to tumor cells through S-glutathionylation of thiol groups of redox-sensitive proteins, which leads to the appearance of multidrug-resistant forms of cancer. In this regard, drugs restoring redox balance and increasing susceptibility to antitumor therapy are of particular importance. We have established that, Bacillus pumilus RNase (binase) modulates the redox-dependent regulatory mechanisms that ensure tumor cell resistance to apoptosis in HPV-16-positive SiHa cells of cervical squamous cell carcinoma,. Binase in nontoxic concentrations initiates a number of pre-apoptogenic changes, i.e., decreases ROS and reduced glutathione (GSH) levels, suppresses the expression of the E6 oncoprotein, activates the expression of the p53 tumor suppressor, and reduces the mitochondrial potential of tumor cells. Binase-induced disruption of the integrity of the mitochondrial membrane is a signal for activation of the mitochondrial apoptosis pathway.

The Expressions of p16, HPV16-L1 and HPV18-E6 in Salivary Gland Tumours.

This study aimed to investigate the relationship between human papillomavirus (HPV) infection and the development of salivary gland tumour (SGT) by evaluating the expression of p16 and the oncoproteins HPV16-L1 and HPV18-E6 using immunohistochemical (IHC) staining. Additionally, we assess the agreement between these oncoproteins and p16 in diagnosing HPV-infected SGT. This cross-sectional study included all SGT cases undergoing surgical resection at Hospital Universiti Sains Malaysia (HUSM) and Hospital Raja Perempuan Zainab II (HRPZ) in Malaysia from April 2022 to April 2023. IHC staining was performed on paraffin-embedded tissues at the Pathology Laboratory, HUSM, to evaluate the expression of p16, HPV16-L1, and HPV18-E6 oncoproteins. The clinicopathological data were correlated with the staining results. 49 SGT cases were identified, mainly in middle-aged Malay women, with most tumours originating from the parotid gland. Malignant tumours included mucoepidermoid carcinoma (22.4%), adenoid cystic carcinoma (4.1%), acinic cell carcinoma (4.1%), and adenocarcinoma (2%). Benign tumours primarily consisted of pleomorphic adenoma (49%) and Warthin tumours (16.3%). Positive p16 expression was detected in 67% of cases, while HPV16 and HPV18 were detected in 65% and 90% of tumours, respectively. HPV16-L1 exhibited 75.8% sensitivity and 56.3% specificity, while HPV18-E6 showed 100% sensitivity and 31.2% specificity compared to p16. The study findings suggest a correlation between the presence of high-risk HPV types 16 and 18 and the development of SGT, as evidenced by the overexpression of p16, HPV16-L1, and HPV18-E6 oncoproteins. Both HPV16-L1 and HPV18-E6 tests are acceptable, reliable, and sensitive for detecting high-risk HPV in SGT.

Targeted degradation of transcription factors by TRAFTACs: TRAnscription Factor TArgeting Chimeras

Many diseases, including cancer, stem from aberrant activation or overexpression of oncoproteins that are associated with multiple signaling pathways. Although proteins with catalytic activity can be successfully drugged, the majority of other protein families, such as transcription factors, remain intractable due to their lack of ligandable sites. In this study, we report the development of TRAnscription Factor TArgeting Chimeras (TRAFTACs) as a generalizable strategy for targeted transcription factor degradation. We show that TRAFTACs, which consist of a chimeric oligonucleotide that simultaneously binds to the transcription factor of interest (TOI) and to HaloTag-fused dCas9 protein, can induce degradation of the former via the proteasomal pathway. Application of TRAFTACs to two oncogenic TOIs, NF-κB and brachyury, suggests that TRAFTACs can be successfully employed for the targeted degradation of other DNA-binding proteins. Thus, TRAFTAC technology is potentially a generalizable strategy to induce degradation of other transcription factors both invitro and invivo.

Regulation of autophagy by high- and low-risk human papillomaviruses.

While high-risk human papillomavirus (HR-HPV) infection is related to the development of cervical, vulvar, anal, penile and oropharyngeal cancer, low-risk human papillomavirus (LR-HPV) infection is implicated in about 90% of genital warts, which rarely progress to cancer. The carcinogenic role of HR-HPV is due to the overexpression of HPV E5, E6 and E7 oncoproteins which target and modify cellular proteins implicated in cell proliferation, apoptosis and immortalization. LR-HPV proteins also target and modify some of these processes; however, their oncogenic potential is lower than that of HR-HPV. HR-HPVs have substantial differences with LR-HPVs such as viral integration into the cell genome, induction of p53 and retinoblastoma protein degradation, alternative splicing in HR-HPV E6-E7 open reading frames, among others. In addition, LR-HPV can activate the autophagy process in infected cells while HR-HPV infection deactivates it. However, in cancer HR-HPV might reactivate autophagy in advance stages. Autophagy is a catabolic process that maintains cell homoeostasis by lysosomal degradation and recycling of damaged macromolecules and organelles; nevertheless, depending upon cellular context autophagy may also induce cell death. Therefore, autophagy can contribute either as a promotor or as a suppressor of tumours. In this review, we focus on the role of HR-HPV and LR-HPV in autophagy during viral infection and cancer development. Additionally, we review key regulatory molecules such as microRNAs in HPV present during autophagy, and we emphasize the potential use of cancer treatments associated with autophagy in HPV-related cancers.

Human Papillomavirus 16 E7 Promotes EGFR/PI3K/AKT1/NRF2 Signaling Pathway Contributing to PIR/NF-κB Activation in Oral Cancer Cells.

A subset of oral carcinomas is etiologically related to high-risk human papillomavirus (HR-HPV) infection, with HPV16 being the most frequent HR-HPV type found in these carcinomas. The oncogenic role of HR-HPV is strongly dependent on the overexpression of E6 and E7 oncoproteins, which, in turn, induce p53 and pRb degradation, respectively. Additionally, it has been suggested that HR-HPV oncoproteins are involved in the regulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), inducing cancer progression and metastasis. Previously, we reported that HPV16 E7 oncoprotein promotes Pirin upregulation resulting in increased epithelial–mesenchymal transition (EMT) and cell migration, with Pirin being an oxidative stress sensor and activator of NF-κB. In this study, we demonstrate the mechanism by which HPV16 E7-mediated Pirin overexpression occurs by promoting EGFR/PI3K/AKT1/NRF2 signaling, thus causing PIR/NF-κB activation in oral tumor cells. Our results demonstrate a new mechanism by which E7 contributes to oral cancer progression, proposing PIR as a potential new therapeutic target.

Selinexor: a first-in-class SINE compound for treatment of relapsed refractory multiple myeloma.

The progression of multiple myeloma is accompanied by complex cytogenetic and epigenetic alterations that include mutation or functional inactivation of tumor suppressor proteins and overexpression of oncoproteins. Patients whose myeloma is refractory to the three major classes of drugs including immunomodulatory agents, proteasome inhibitors and anti-CD38 monoclonal antibodies have a very poor prognosis. Drugs with novel mechanisms of action that can bypass resistance mechanisms are sorely needed for this group of patients. Selinexor represents a novel, oral agent with an innovative mechanism of action that offers a significant therapeutic advance in this group of heavily treated patients. Moreover, this novel mechanism may provide additional options for patients with less refractory disease.

Identification and In-silico Prediction of Hermetiaillucens Larval Protein that Inhibits the Progression of Cervical Cancer

The aim of the current research is to assess the anticancer activity of the proteins identified from the crude protein extract derived from Hermetiaillucens larvae by computational methods. Bioinformatics methods were used to identify the protein sequence and virtual screening for the prediction of protein structure, their physico-chemical characteristics and functional aspects, that aid in exploring the anti-cancer interaction that is inclined to inhibit the oncoprotein activity. Due to the pathological process transpired by HPV, an array of genetic alterations, including the overexpression of oncoproteins or the inactivation of tumor suppressor proteins like TP53, BCL2, MDM2, ARF and BAX occur. The altered protein signaling pathways convulse to cervical cancer. In this research, we identified proteins, from the crude extract derived from Hermetiaillucens by LC-MS method. Further Computational screening of the selected proteins were employed to assess the functional units which has anticancer activity. We predicted four proteins Metallothionine, Defensin like precursor 1 protein, Heat shock protein 90 and NADH dehydrogenase and eluted the protein sequence from NCBI GenBank database. These sequences were used to predict physicochemical properties and protein structure prediction. Pharmacophore analysis of the peptide sequences as potential targets for cancer treatment was evaluated. Molecular docking of peptide sequences with target protein structure was carried out. To screen the best active potential molecule for cancer treatment the Molecular dynamics of docked protein-peptide structures were administered. The molecular docking of the peptides with onco-proteins has been predicted and virtually screened based on RMSD values. The resultant protein sequence was evaluated further by amino acid sequencing, extensive scrutiny of the proteins, advanced computational imaging, molecular docking examination and assessed the results for clinical interpretation. To summate, bioinformatics assessment enabled us to identify four key proteins derived from Hermetiaillucens larvae that have strong inhibitory function against proteins that cause cervical cancer.

UBXN2A enhances CHIP-mediated proteasomal degradation of oncoprotein mortalin-2 in cancer cells.

Overexpression of oncoproteins is a major cause of treatment failure using current chemotherapeutic drugs. Drug‐induced degradation of oncoproteins is feasible and can improve clinical outcomes in diverse types of cancers. Mortalin‐2 (mot‐2) is a dominant oncoprotein in several tumors, including colorectal cancer (CRC). In addition to inactivating the p53 tumor suppressor protein, mot‐2 enhances tumor cell invasion and migration. Thus, mot‐2 is considered a potential therapeutic target in several cancer types. The current study investigated the biological role of a ubiquitin‐like protein called UBXN2A in the regulation of mot‐2 turnover. An orthogonal ubiquitin transfer technology followed by immunoprecipitation, in vitro ubiquitination, and Magnetic Beads TUBE2 pull‐down experiments revealed that UBXN2A promotes carboxyl terminus of the HSP70‐interacting protein (CHIP)‐dependent ubiquitination of mot‐2. We subsequently showed that UBXN2A increases proteasomal degradation of mot‐2. A subcellular compartmentalization experiment revealed that induced UBXN2A decreases the level of mot‐2 and its chaperone partner, HSP60. Pharmacological upregulation of UBXN2A using a small molecule, veratridine (VTD), decreases the level of mot‐2 in cancer cells. Consistent with the in vitro results, UBXN2A+/− mice exhibited selective elevation of mot‐2 in colon tissues. An in vitro Anti‐K48 TUBE isolation approach showed that recombinant UBXN2A enhances proteasomal degradation of mot‐2 in mouse colon tissues. Finally, we observed enhanced association of CHIP with the UBXN2A‐mot‐2 complex in tumors in an azoxymethane/dextran sulfate sodium‐induced mouse CRC model. The existence of a multiprotein complex containing UBXN2A, CHIP, and mot‐2 suggests a synergistic tumor suppressor activity of UBXN2A and CHIP in mot‐2‐enriched tumors. This finding validates the UBXN2A‐CHIP axis as a novel and potential therapeutic target in CRC.

Abstract 2697: Alterations in the HSP70 interactome are induced by overexpression of MUC1 in pancreatic cancer

Abstract Heat Shock Protein 70 (HSP70) is an evolutionarily well-conserved molecular chaperone involved in several cellular processes such as folding of proteins, modulating protein-protein interactions and transport of proteins across membranes. HSP70 works synergistically with a series of chaperones, co-chaperones, adaptors, and folding enzymes known as the HSP70 “interactome”. Previous work has demonstrated that HSP70 interactome changes can be promoted by overexpression of oncoproteins. An important “client” of HSP70 is the oncoprotein MUC1. MUC1 is overexpressed and promotes drug resistance in pancreatic cancer. We applied quantitative affinity-purification mass spectrometry (AP-MS) to characterize the HSP70 interactome differences between PC3/NEO and PC3/MUC1 cell lines. 439 HSP70-interacting proteins were identified and quantified via LC-MS/MS. Interestingly while 53% of HSP70 interactions remained constant upon MUC1 overexpression, 29% significantly increased and 18% decreased. Many of the altered proteins were key co-chaperones that regulate and maintain HSP70 activity. High levels of MUC1 promoted HSP70 association with 15 co-chaperones (HSPA5, DNAJB1, STIP1, TXNDC5, HSPB1, CCT2, TCP1, P4HB, CCT6A, CCT8, PDIA3 and HSP90B). Conversely, MUC1 overexpression triggered the loss of HSP70 interaction with only 2 co-chaperones (PPIB and DNAJA2). We are currently validating these altered interactions using an array of biochemical techniques. Our hope is that by understanding these HSP70 interactome changes, we may be able to uncover novel ways to inhibit MUC1 function in pancreatic cancer. Citation Format: . Nitika, Donald J. Wolfgeher, Pinku Mukherjee, Andrew W. Truman. Alterations in the HSP70 interactome are induced by overexpression of MUC1 in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2697.

MiRNA and Proteomic Dysregulation in Non-Small Cell Lung Cancer in Response to Cigarette Smoke.

Dysregulation of miRNAs is associated with the development of non-small cell lung cancer (NSCLC). It is imperative to study the dysregulation of miRNAs by cigarette smoke which will affect their targets, either leading to the overexpression of oncoproteins or downregulation of tumor suppressor proteins. In this study, we carried out miRNA sequencing and SILAC-based proteomic analysis of H358 cells chronically exposed to cigarette smoke condensate. Using bioinformatics analysis, we mapped the dysregulated miRNAs to differentially expressed target proteins identified in our data. Gene ontology-based enrichment and pathway analysis was performed using the deregulated targets to study the role of cigarette smoke-mediated miRNA dysregulation in NSCLC cell line. miRNA sequencing resulted in the identification of 208 miRNAs, of which 6 miRNAs were found to be significantly dysregulated (2 fold, Log Base 2; p-value ≤ 0.05) in H358-Smoke cells. Proteomic analysis of the smoke exposed cells compared to the untreated parental cells resulted in the quantification of 2,610 proteins, of which 690 proteins were found to be differentially expressed (fold change ≥ 2). Gene ontology based analysis of target proteins revealed enrichment of proteins driving metabolism and a decrease in expression of proteins associated with immune response in the cells exposed to cigarette smoke. Pathway study using Ingenuity Pathway Analysis (IPA) revealed activation of NRF2-mediated oxidative stress response and actin-cytoskeleton signaling, and repression of protein kinase A signaling in H358-Smoke cells. We also identified 5 novel miRNAs in H358-Smoke cells using unassigned reads of small RNA-Seq dataset. In summary, this study indicates that chronic exposure to cigarette smoke leads to widespread dysregulation of miRNAs and their targets, resulting in signaling aberrations in NSCLC cell line. The miRNAs and their targets identified in the study need to be further investigated to explore their role as potential therapeutic targets and/or molecular markers in NSCLC especially in smokers.

Shp2 deletion in hepatocytes suppresses hepatocarcinogenesis driven by oncogenic β-Catenin, PIK3CA and MET

Shp2 is an SH2-tyrosine phosphatase acting downstream of receptor tyrosine kinases (RTKs). Most recent data demonstrated a liver tumor-suppressing role for Shp2, as ablating Shp2 in hepatocytes aggravated hepatocellular carcinoma (HCC) induced by chemical carcinogens or Pten loss. We further investigated the effect of Shp2 deficiency on liver tumorigenesis driven by classical oncoproteins c-Met (receptor for HGF), β-catenin and PIK3CA. We performed hydrodynamic tail vein injection of two pairs of plasmids expressing c-Met and ΔN90-β-catenin (MET/CAT), or c-Met and PIK3CAH1047R (MET/PIK), into WT and Shp2hep-/- mice. We compared liver tumor loads and investigated the pathogenesis and molecular mechanisms involved using multidisciplinary approaches. Despite the induction of oxidative and metabolic stresses, Shp2 deletion in hepatocytes suppressed hepatocarcinogenesis driven by overexpression of oncoproteins MET/CAT or MET/PIK. Shp2 loss inhibited proliferative signaling from c-Met, Wnt/β-catenin, Ras/Erk and PI3K/Akt pathways, but triggered cell senescence following exogenous expression of the oncogenes. Shp2, acting downstream of RTKs, is positively required for hepatocyte-intrinsic tumorigenic signaling from these oncoproteins, even if Shp2 deficiency induces a tumor-promoting hepatic microenvironment. These data suggest a new and more effective therapeutic strategy for HCCs driven by oncogenic RTKs and other upstream molecules, by inhibiting Shp2 and also suppressing any tumor-enhancing stromal factors produced because of Shp2 inhibition. Primary liver cancer is a malignant disease with poor prognosis, largely because there are limited systemic therapies available. We show here that a cytoplasmic tyrosine phosphatase Shp2 is required for liver tumorigenesis. This tumorigenesis is driven by two oncoproteins that are implicated in human liver cancer. This, together with our previous studies, uncovers the complexity of liver tumorigenesis, by elucidating the pro- and anti-tumor effects of Shp2 in mouse models. This data can be used to guide new therapies.

Overexpression of Human Papillomavirus Type 16 Oncoproteins Enhances Epithelial-Mesenchymal Transition via STAT3 Signaling Pathway in Non-Small Cell Lung Cancer Cells.

The human papillomavirus (HPV) infection may be associated with the development and progression of non-small cell lung cancer (NSCLC). However, the role of HPV-16 oncoproteins in the development and progression of NSCLC is not completely clear. Epithelial–mesenchymal transition (EMT), a crucial step for invasion and metastasis, plays a key role in the development and progression of NSCLC. Here we explored the effect of HPV-16 oncoproteins on EMT and the underlying mechanisms. NSCLC cell lines, A549 and NCI-H460, were transiently transfected with the EGFP-N1-HPV-16 E6 or E7 plasmid. Real-time PCR and Western blot analysis were performed to analyze the expression of EMT markers. A protein microarray was used to screen the involved signaling pathway. Our results showed that overexpression of HPV-16 E6 and E7 oncoproteins in NSCLC cells significantly promoted EMT-like morphologic changes, downregulated the mRNA and protein levels of EMT epithelial markers (E-cadherin and ZO-1), and upregulated the mRNA and protein levels of EMT mesenchymal markers (N-cadherin and vimentin) and transcription factors (ZEB-1 and Snail-1). Furthermore, the HPV-16 E6 oncoprotein promoted STAT3 activation. Moreover, WP1066, a specific signal transducer and activator of transcription 3 (STAT3) inhibitor, reversed the effect of HPV-16 E6 on the expression of ZO-1, vimentin, and ZEB-1 in transfected NSCLC cells. Taken together, our results suggest that overexpression of HPV-16 E6 and E7 oncoproteins enhances EMT, and the STAT3 signaling pathway may be involved in HPV-16 E6-induced EMT in NSCLC cells.

Methylation of Human Papillomavirus Type 16 CpG Sites at E2-Binding Site 1 (E2BS1), E2BS2, and the Sp1-Binding Site in Cervical Cancer Samples as Determined by High-Resolution Melting Analysis–PCR

High-risk (HR) human papillomavirus (HPV)-associated carcinogenesis is driven mainly by the overexpression of E7 and E6 oncoproteins following viral DNA integration and the concomitant loss of the E2 open reading frame (ORF). However, the integration of HR-HPV DNA is not systematically observed in cervical cancers. The E2 protein acts as a transcription factor that governs viral oncogene expression. The methylation of CpGs in the E2-binding sites (E2BSs) in the viral long control region abrogates E2 binding, thus impairing the E2-mediated regulation of E7/E6 transcription. Here, high-resolution melting (HRM)-PCR was developed to quantitatively analyze the methylation statuses of E2BS1, E2BS2, and the specificity protein 1 (Sp1)-binding site in 119 HPV16-positive cervical smears. This is a rapid assay that is suitable for the analysis of cervical samples. The proportion of cancer samples with methylated E2BS1, E2BS2, and Sp1-binding site CpGs was 47%, whereas the vast majority of samples diagnosed as being within normal limits, low-grade squamous intraepithelial lesions (LSIL), or high-grade squamous intraepithelial lesions (HSIL) harbored unmethylated CpGs. Methylation levels varied widely, since some cancer samples harbored up to 60% of methylated HPV16 genomes. A pyrosequencing approach was used as a confirmation test and highlighted that quantitative measurement of methylation can be achieved by HRM-PCR. Its prognostic value deserves to be investigated alone or in association with other biomarkers. The reliability of this single-tube assay offers great opportunities for the investigation of HPV16 methylation in other HPV-related cancers, such as head and neck cancers, which are a major public health burden.

Increased expression of MDM2 and NM23 are associated with malignant transformation of pleomorphic adenoma

Abstract This study was designed to correlate the expression of both MDM2 and NM23 in pleomorphic adenoma (PA) to clinical background, histological features, local recurrence, and metastatic potentiality in this tumor. Expression of MDM2 and NM23 was studied immunohistochemically in 23 cases of PA. Our results demonstrated MDM2 and NM23 overexpression in almost all cases of PA. There was a significant difference of the MDM2 and NM23 mean values between benign PA and both carcinoma-ex PA and metastasizing PA. The results of this study show that the overexpression of NM23 and MDM2 oncoproteins could be used as reliable predictors of malignant progression as well as metastatic potentiality of PA.

Overexpression of human papillomavirus (HPV) type 16 oncoproteins promotes angiogenesis via enhancing HIF-1α and VEGF expression in non-small cell lung cancer cells

HPV-16 infection may play an important role in the development of non-small cell lung cancer (NSCLC) among never-smokers. Due to the critical role of angiogenesis in NSCLC development, we describe here the effect of HPV-16 oncoproteins on angiogenesis in NSCLC and the underlying mechanisms. We found that overexpression of HPV-16 E6 and E7 oncoproteins in NSCLC cells significantly promoted angiogenesis both in vitro and in vivo, and correspondingly, an enhanced expression of HIF-1α and VEGF, important pro-angiogenic factors in tumor angiogenesis. Meanwhile, overexpression of HPV-16 oncoproteins also led to HIF-1α-dependent increases in the secretion of several other pro-angiogenic factors, including IL-8. Our findings suggest that HPV-16 oncoproteins contribute to the development of NSCLC possibly by promoting HIF-1α/VEGF-mediated tumor angiogenesis.

Abstract LB-95: Inhibition of CXCR4/Stat3 signaling by AMD3465 exerts major tumor- and metastasis-suppressive effects in syngenic 4T1 breast cancer model

Abstract Stromal-derived factor-1 α (SDF-1α, also termed CXCL12) and its receptor CXCR4 have been shown to play important roles in hematopoietic stem cell homing, and high expression of CXCR4 has been associated with poor prognosis in hematologic malignancies. The CXCR4/SDF-1α axis is also functional in solid tumors, including breast cancer. It has been postulated that Stat3 may promote metastases in breast cancer through direct interaction with CXCR4, promoting tumor growth and survival. Phosphorylated Stat3 was found to trigger overexpression or activation of several potent oncoproteins. In order to evaluate the role of CXCR4 in metastases, we used the specific CXCR4 inhibitor, AMD3465, to block CXCR4 in the syngeneic 4T1 breast cancer model. Stat3, Akt, Erk1/2, Jak2 and MMP9 activity was found to be almost completely inhibited and expression levels of GSK-3β and cMyc were greatly reduced. Growth of 4T1 cells orthotopically implanted into the mammary fat pad as well as pulmonary and liver metastases were significantly (>95%) reduced in vivo when AMD3465 was given as continuous infusion at a dose of 428.57μg/mouse/day over 14 days. Furthermore, we observed that CXCR4 inhibition by AMD3465 significantly reduced lung metastases in three immunocompetent syngeneic mouse models after surgical resection of the primary breast tumors. This experimental design mimics current clinical treatment strategies. Kozin et al. (Cancer Research 70: 5679–85; 2010) recently suggested that CD11b+ myeloid/monocytic cells (a cell type implicated in supporting angiogenesis) may play a role in tumor re-growth in response to SDF-1 α. In support of this observation, we observed significantly reduced CD11b+ cells in lungs, livers and spleens (P<0.01 for all 3 organs) following treatment with AMD3465, as compared to PBS controls. Hence, our data suggest that pharmacological CXCR4 inhibition in breast cancer-bearing mice following primary tumor resection is well tolerated and potently controls the development of distant metastatic lesions. In conclusion, interruption of the SDF-1/CXCR4/Stat3 signaling axis by the CXCR4-specific inhibitor, AMD3465, effectively inhibits pulmonary and hepatic breast cancer metastasis via disruption of intracellular microenvironment mediated signaling in breast cancer cells and through effects on CD11b+ tumor infiltrating myeloid cells. 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 LB-95. doi:10.1158/1538-7445.AM2011-LB-95

Overexpression of FOXG1 contributes to TGF-β resistance through inhibition of p21WAF1/CIP1 expression in ovarian cancer

Background:Loss of growth inhibitory response to transforming growth factor-β (TGF-β) is a common feature of epithelial cancers. Recent studies have reported that genetic lesions and overexpression of oncoproteins in TGF-β/Smads signalling cascade contribute to the TGF-β resistance. Here, we showed that the overexpressed FOXG1 was involved in attenuating the anti-proliferative control of TGF-β/Smads signalling in ovarian cancer.Methods:FOXG1 and p21WAF1/CIP1 expressions were evaluated by real-time quantitative reverse-transcription polymerase chain reaction (RT–PCR), western blot and immunohistochemical analyses. The effect of FOXG1 on p21WAF1/CIP1 transcriptional activity was examined by luciferase reporter assays. Cell lines stably expressing or short hairpin RNA interference-mediated knockdown FOXG1 were established for studying the gain-or-loss functional effects of FOXG1. XTT cell proliferation assay was used to measure cell growth of ovarian cancer cells.Results:Quantitative RT–PCR and western blot analyses showed that FOXG1 was upregulated and inversely associated with the expression levels of p21WAF1/CIP1 in ovarian cancer. The overexpression of FOXG1 was significantly correlated with high-grade ovarian cancer (P=0.025). Immunohistochemical analysis on ovarian cancer tissue array was further evidenced that FOXG1 was highly expressed and significantly correlated with high-grade ovarian cancer (P=0.048). Functionally, enforced expression of FOXG1 selectively blocked the TGF-β-induced p21WAF1/CIP1 expressions and increased cell proliferation in ovarian cancer cells. Conversely, FOXG1 knockdown resulted in a 20–26% decrease in cell proliferation together with 16–33% increase in p21WAF1/CIP1 expression. Notably, FOXG1 was able to inhibit the p21WAF1/CIP1 promoter activity in a p53-independent manner by transient reporter assays.ConclusionOur results suggest that FOXG1 acts as an oncoprotein inhibiting TGF-β-mediated anti-proliferative responses in ovarian cancer cells through suppressing p21WAF1/CIP1 transcription.

HPV-vaccine and screening programs: the new era of global prevention

a vast body of evidence has emerged, in the recent decades, indicating the persistence of high-risk Human Papillomavirus (HPV) infection as the necessary cause, although not sufficient, for the development of cancer of the uterine cervix [1-3]. The biological details that explained the role of HPVs in cervical cancer etiology described some steps (HPV infection and persistence over time, integration into the host genoma and overexpression of E6-E7 oncoproteins, growth of precancer lesions, progression to invasion) that can be distinguished and which provide a background for the secondary prevention of such carcinoma [4]. Cervical cancer is the second most common cancer in women, accounting for at least 250,000 deaths per year worldwide, and overall the disease burden attributable to HPV is significant, with more than 5% of all cancers worldwide attributed to such infections [5]. indeed, it is currently well documented that a significant proportion of vulvo-vaginal, anal and oro-pharyngeal cancers among women (as well as anal, oro-pharyngeal, and penile cancers among men) are etiologically related to the same high-risk HPV types. in terms of prevention the burden of scientific knowledge regarding Papillomavirus infection has translated in two main applications that will provide important health benefits: HPV-Dna testing and HPV-vaccine. in the last years molecular testing for high-risk HPV types become available for clinical use, thus changing the strategy of secondary prevention [6] and management of intraepithelial cervical lesions [7]. Furthermore, an effective screening program will be a prerequisite for the evaluation of vaccines effectiveness, and thus vaccination and cytologic screening will be complementary strategies. although HPV-immunization programs still reveal a number of unanswered questions and some author stressed the necessity of prudence [8, 9], they represents an extraordinary opportunity for primary prevention against cervical cancer and other HPV-related pre-neoplastic/neoplastic diseases. Thus a global prevention program (through HPV immunization integrated into cytological screening) is viewed as an important breakthrough in public health. nevertheless, how to most efficiently carry out such global prevention of cervical cancer is still unclear. This issue will necessitate collaboration among gynecologists, oncologists and experts in preventive medicine, in immunization programs and in sexually transmitted diseases as well.

Human papillomavirus E6 and E7 oncoproteins as risk factors for tumorigenesis

Human papillomavirus (HPV) is small, double-stranded DNA virus that infects mucosal and cutaneous epithelial tissue. HPV is sexually transmitted and the viral DNA replicates extrachromosomally. The virus is non-enveloped and has an icosahedral capsid. There are approximately 118 types of HPV, which are characterized as high-risk or low- risk types. High-risk HPVs cause malignant transformation while the low-risk ones cause benign warts and lesions. The expression of E6 and E7 is normally controlled during the normal viral life cycle when viral DNA replicates extrachromosomally. HPV E6 and E7 oncoproteins are overexpressed when the viral genome integrates into the host DNA.Deregulated overexpression of E6 and E7 oncoproteins can cause several changes in cellular pathways and functions leading to malignant transformation of cells and tumorigenesis. In this review, we focus on several cellular mechanisms and pathways that are altered in the presence of E6 and E7, the target proteins of E6 and E7 inside the host cell and how they contribute to the development of the transformed phenotype.

Regulation of Cell Proliferation and Apoptosis in CHO-K1 Cells by the Coexpression of c-Myc and Bcl-2

Proliferation and cell death are regarded as key targets for the optimization of animal cell culture processes and for the maximization of product yield. Although chemical and physical factors are vitally important, of primary interest is the utilization of genetic engineering to regulate cellular processes. CHO cells were first genetically modified to enhance proliferation rate in both suspension and monolayer cultures. Under the constitutive control of c-myc overexpression the CHO cultures showed an increase in growth rate and maximum cell number accompanied by a similar decrease in specific glucose consumption rate. Although the c-myc transfected cell line exhibited apoptosis at much lower rates than is widely reported and associated with the overexpression of c-Myc, it was nevertheless apparent that c-Myc was responsible for the induction of higher apoptotic rates when compared with the control cell line. Hence, the anti-apoptotic gene bcl-2 was also used to transfect the c-Myc CHO cell line, to reduce cell death. Overexpression of both oncoproteins resulted in a cell line that exhibited higher proliferation rates and maximum cell numbers, with a decrease in apoptosis when compared to the parental cell line. In conclusion, it was shown that Bcl-2 protein overexpression specifically abrogates c-Myc-induced apoptosis without affecting the c-Myc mitogenic function.