Ford, K., Hanley, C., Mellone, M., Szyndralewiez, C., Heitz, F., Wiesel, P., Wood, O., Machado, M., Lopez, M., Ganesan, A., Wang, C., Chakravarthy, A., Fenton, T., King, E., Vijayanand, P., Ottensmeier, C., Al-Shamkhani, A., Savelyeva, N. and Thomas, G. (2020). NOX4 inhibition potentiates immunotherapy by overcoming cancer-associated fibroblast-mediated CD8 T-cell exclusion from tumours. Cancer Research [Online]. Available at: http://dx.doi.org/10.1158/0008-5472.CAN-19-3158.
Determining mechanisms of resistance to PD-1/PD-L1 immune checkpoint immunotherapy is key to developing new treatment strategies. Cancer-associated fibroblasts (CAF) have many tumor-promoting functions and promote immune evasion through multiple mechanisms, but as yet, there are no CAF-specific inhibitors clinically available. Here we generated CAF-rich murine tumor models (TC1, MC38, 4T1) to investigate how CAF influence the immune microenvironment and affect response to different immunotherapy modalities (anti-cancer vaccination; TC1, [HPV E7 DNA vaccine];PD-1, MC38) and found that CAFs broadly suppressed response by specifically excluding CD8+ T-cells from tumors (not CD4+ T-cells or macrophages); CD8+ T-cell exclusion was similarly present in CAF-rich human tumors. RNA sequencing of CD8+ T-cells from CAF-rich murine tumors and immunochemistry analysis of human tumors identified significant upregulation of CTLA-4 in the absence of other exhaustion markers; inhibiting CTLA-4 with a non-depleting antibody overcame the CD8+ T-cell exclusion effect without affecting T-regs. We then examined the potential for CAF targeting, focusing on the ROS-producing enzyme NOX4, which is upregulated by CAF in many human cancers, and compared this to TGF-062;1 inhibition, a key regulator of the CAF phenotype. siRNA knockdown or pharmacological inhibition (GKT137831 [Setanaxib]) of NOX4 'normalized' CAF to a quiescent phenotype and promoted intratumoral CD8+T-cell infiltration, overcoming the exclusion effect; TGF-062;1 inhibition could prevent, but not reverse, CAF differentiation. Finally, NOX4 inhibition restored immunotherapy response in CAF-rich tumors. These findings demonstrate that CAF-mediated immunotherapy resistance can be effectively overcome through NOX4 inhibition, and could improve outcome in a broad range of cancers.
Smith, N. and Fenton, T. (2019). The APOBEC3 genes and their role in cancer: insights from human papillomavirus. Journal of Molecular Endocrinology [Online] 64. Available at: https://doi.org/10.1530/JME-19-0011.
The interaction between human papillomaviruses (HPV) and the apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC)3 (A3) genes has garnered increasing attention in recent years, with considerable efforts focused on understanding their apparent roles in both viral editing and in HPV-driven carcinogenesis. Here we review these developments and highlight several outstanding questions in the field. We consider whether editing of the virus and mutagenesis of the host are linked, or whether both are essentially separate events, coincidentally mediated by a common, or distinct A3 enzymes. We discuss the viral mechanisms and cellular signalling pathways implicated in A3 induction in virally-infected cells, examine which of the A3 enzymes might play the major role in HPV-associated carcinogenesis and in the development of therapeutic resistance. We consider the parallels between A3 induction in HPV-infected cells and what might be causing aberrant A3 activity in HPV-independent cancers such as those arising in the bladder, lung and breast. Finally, we discuss the implications of ongoing A3 activity in tumours under treatment and the therapeutic opportunities that this may present.
Ma, J., Benitez, J., Li, J., Miki, S., Ponte de Alberquerque, C., Galatro, T., Orellana, L., Zanca, C., Reed, R., Boyer, A., Koga, T., Varki, N., Fenton, T., Marie, S., Lindahl, E., Gahman, T., Shiau, A., Zhou, H., deGroot, J., Sulman, E., Cavenee, W., Kolodner, R., Chen, C. and Furnari, F. (2019). Inhibition of nuclear PTEN tyrosine phosphorylation enhances glioma radiation sensitivity through attenuated DNA repair. Cancer Cell [Online]. Available at: https://doi.org/10.1016/j.ccell.2019.01.020.
Ionizing radiation (IR) and chemotherapy are standard of care treatments for glioblastoma
(GBM) patients and both result in DNA damage, however, the clinical efficacy is limited due to
therapeutic resistance. We identified a mechanism of such resistance mediated by
phosphorylation of PTEN on tyrosine 240 (pY240-PTEN) by FGFR2. pY240-PTEN is rapidly
elevated and bound to chromatin through interaction with Ki-67 in response to IR treatment and
facilitates the recruitment of RAD51 to promote DNA repair. Blocking Y240 phosphorylation
confers radiation sensitivity to tumors and extends survival in GBM preclinical models. Y240FPten
knock-in mice showed radiation sensitivity. These results suggest that FGFR-mediated
pY240-PTEN is a key mechanism of radiation resistance and is an actionable target for
improving radiotherapy efficacy.
Fenton, T., Garrett, M., Wass, M. and Michaelis, M. (2018). What really matters - response and resistance in cancer therapy. Cancer Drug Resistance [Online] 2018:200-203. Available at: https://doi.org/10.20517/cdr.2018.19.
Fenton, T., Garrett, M., Michaelis, M. and Wass, M. (2018). Meeting Abstracts of the BACR conference: response and resistance in cancer therapy. Cancer Drug Resistance [Online] 1:266-302. Available at: https://doi.org/10.20517/cdr.2018.18.
Chakravarthy, A., Furness, A., Joshi, K., Ghorani, E., Ford, K., Ward, M., King, E., Lechner, M., Marafioti, T., Quezada, S., Thomas, G., Feber, A. and Fenton, T. (2018). Pan-cancer deconvolution of tumour composition using DNA methylation. Nature Communications [Online] 9. Available at: https://doi.org/10.1038/s41467-018-05570-1.
The nature and extent of immune cell infiltration into solid tumours are key determinants of therapeutic response. Here, using a DNA methylation-based approach to tumour cell fraction deconvolution, we report the integrated analysis of tumour composition and genomics across a wide spectrum of solid cancers. Initially studying head and neck squamous cell carcinoma, we identify two distinct tumour subgroups: ‘immune hot’ and ‘immune cold’, which display differing prognosis, mutation burden, cytokine signalling, cytolytic activity and oncogenic driver events. We demonstrate the existence of such tumour subgroups pan-cancer, link clonal-neoantigen burden to cytotoxic T-lymphocyte infiltration, and show that transcriptional signatures of hot tumours are selectively engaged in immunotherapy responders. We also find that treatment-naive hot tumours are markedly enriched for known immune-resistance genomic alterations, potentially explaining the heterogeneity of immunotherapy response and prognosis seen within this group. Finally, we define a catalogue of mediators of active antitumour immunity, deriving candidate biomarkers and potential targets for precision immunotherapy.
Lechner, M., Chakravarthy, A., Walter, V., Masterson, L., Feber, A., Jay, A., Weinberger, P., McIndoe, R., Forde, C., Chester, K., Kalavrezos, N., O’Flynn, P., Forster, M., Jones, T., Vaz, F., Hayes, D. and Fenton, T. (2018). Frequent HPV-independent p16/INK4A overexpression in head and neck cancer. Oral Oncology [Online] 83:32-37. Available at: https://doi.org/10.1016/j.oraloncology.2018.06.006.
p16INK4A (p16) is the most widely used clinical biomarker for Human Papillomavirus (HPV) in head and neck squamous cell cancer (HNSCC). HPV is a favourable prognostic marker in HNSCC and is used for patient stratification. While p16 is a relatively accurate marker for HPV within the oropharynx, recent reports suggest it may be unsuitable for use in other HNSCC subsites, where a smaller proportion of tumors are HPV-driven.
Materials and methods
We integrated reverse phase protein array (RPPA) data for p16 with HPV status based on detection of viral transcripts by RNA-seq in a set of 210 HNSCCs profiled by The Cancer Genome Atlas project. Samples were queried for alterations in CDKN2A, and other pathway genes to investigate possible drivers of p16 expression.
While p16 levels as measured by RPPA were significantly different by HPV status, there were multiple HPV (?) samples with similar expression levels of p16 to HPV (+) samples, particularly at non-oropharyngeal subsites. In many cases, p16 overexpression in HPV (?) tumors could not be explained by mutation or amplification of CDKN2A or by RB1 mutation. Instead, we observed enrichment for inactivating mutations in the histone H3 lysine 36 methyltransferase, NSD1 in HPV (?)/p16-high tumors.
RPPA data suggest high p16 protein expression in many HPV (?) non-oropharyngeal HNSCCs, limiting its potential utility as an HPV biomarker outside of the oropharynx. HPV-independent overexpression of wild-type p16 in non-oropharyngeal HNSCC may be linked to global deregulation of chromatin state by inactivating mutations in NSD1.
Lechner, M., Vassie, C., Kavasogullari, C., Jones, O., Howard, J., Masterson, L., Fenton, T., Yarbrough, W., Waller, J. and Gilson, R. (2018). A cross-sectional survey of awareness of human papillomavirus-associated oropharyngeal cancers among general practitioners in the UK. BMJ Open [Online] 8. Available at: https://doi.org/10.1136/bmjopen-2018-023339.
Objectives: To examine the level of awareness of the link between human papillomavirus (HPV) and oropharyngeal cancer (OPC) and epidemiological trends in HPV-related OPC among general practitioners (GPs) in the UK.
Design: Cross-sectional survey.
Participants: 384 GPs from England, Scotland, Wales and Northern Ireland.
Setting: The survey was administered at GP training courses and via email to lists of training course attendees.
Primary and secondary outcome measures: Proportion of respondents aware of the link between HPV and OPC; respondents’ self-rated knowledge of OPC; proportion of participants aware of the epidemiological trends in HPV-associated OPC.
Results: 384 questionnaires were completed with an overall response rate of 72.9%. 74.0% of participants recognised HPV as a risk factor for OPC, which was lower than knowledge about the role of smoking, chewing tobacco and alcohol consumption (all >90%?recognition). Overall, 19.4% rated their knowledge of OPC as very good or good, 62.7% as average and 17.7% as poor or very poor. The majority (71.9%) were aware that rates of HPV-associated OPC have increased over the last two decades. Fewer than half (41.5%) of the participants correctly identified being male as a risk factor of HPV-associated OPC, while 58.8% were aware that patients with HPV-associated OPC tend to be younger than those with non-HPV-associated disease.
Conclusions: The association of HPV infection with OPC is a relatively recent discovery. Although the level of awareness of HPV and OPC among GPs was high, the characteristics of HPV-associated OPC were less well recognised, indicating the need for further education.
Holme, H., Gulati, A., Brough, R., Fleuren, E., Bajrami, I., Campbell, J., Chong, I., Costa-Cabral, S., Elliott, R., Fenton, T., Frankum, J., Jones, S., Menon, M., Miller, R., Pemberton, H., Postel-Vinay, S., Rafiq, R., Selfe, J., von Kriegsheim, A., Munoz, A., Rodriguez, J., Shipley, J., van der Graaf, W., Williamson, C., Ryan, C., Pettitt, S., Ashworth, A., Strauss, S. and Lord, C. (2018). Chemosensitivity profiling of osteosarcoma tumour cell lines identifies a model of BRCAness. Scientific Reports [Online] 8. Available at: https://doi.org/10.1038/s41598-018-29043-z.
Osteosarcoma (OS) is an aggressive sarcoma, where novel treatment approaches are required. Genomic studies suggest that a subset of OS, including OS tumour cell lines (TCLs), exhibit genomic loss of heterozygosity (LOH) patterns reminiscent of BRCA1 or BRCA2 mutant tumours. This raises the possibility that PARP inhibitors (PARPi), used to treat BRCA1/2 mutant cancers, could be used to target OS. Using high-throughput drug sensitivity screening we generated chemosensitivity profiles for 79 small molecule inhibitors, including three clinical PARPi. Drug screening was performed in 88 tumour cell lines, including 18 OS TCLs. This identified known sensitivity effects in OS TCLs, such as sensitivity to FGFR inhibitors. When compared to BRCA1/2 mutant TCLs, OS TCLs, with the exception of LM7, were PARPi resistant, including those with previously determined BRCAness LoH profiles. Post-screen validation experiments confirmed PARPi sensitivity in LM7 cells as well as a defect in the ability to form nuclear RAD51 foci in response to DNA damage. LM7 provides one OS model for the study of PARPi sensitivity through a potential defect in RAD51-mediated DNA repair. The drug sensitivity dataset we generated in 88 TCLs could also serve as a resource for the study of drug sensitivity effects in OS.
Periyasamy, M., Singh, A., Gemma, C., Kranjec, C., Farzan, R., Leach, D., Navaratnam, N., Pálinkás, H., Vértessy, B., Fenton, T., Doorbar, J., Fuller-Pace, F., Meek, D., Coombes, R., Buluwela, L. and Ali, S. (2017). p53 controls expression of the DNA deaminase APOBEC3B to limit its potential mutagenic activity in cancer cells. Nucleic Acids Research [Online] 45:11056-11069. Available at: http://dx.doi.org/10.1093/nar/gkx721.
Cancer genome sequencing has implicated the cytosine deaminase activity of apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) genes as an important source of mutations in diverse cancers, with APOBEC3B (A3B) expression especially correlated with such cancer mutations. To better understand the processes directing A3B over-expression in cancer, and possible therapeutic avenues for targeting A3B, we have investigated the regulation of A3B gene expression. Here, we show that A3B expression is inversely related to p53 status in different cancer types and demonstrate that this is due to a direct and pivotal role for p53 in repressing A3B expression. This occurs through the induction of p21 (CDKN1A) and the recruitment of the repressive DREAM complex to the A3B gene promoter, such that loss of p53 through mutation, or human papilloma virus-mediated inhibition, prevents recruitment of the complex, thereby causing elevated A3B expression and cytosine deaminase activity in cancer cells. As p53 is frequently mutated in cancer, our findings provide a mechanism by which p53 loss can promote cancer mutagenesis.
Chakravarthy, A., Henderson, S., Thirdborough, S., Ottensmeier, C., Su, X., Lechner, M., Feber, A., Thomas, G. and Fenton, T. (2016). Human Papillomavirus Drives Tumor Development Throughout the Head and Neck: Improved Prognosis Is Associated With an Immune Response Largely Restricted to the Oropharynx. Journal Of Clinical Oncology [Online] 34:4132-4141. Available at: http://dx.doi.org/10.1200/JCO.2016.68.2955.
In squamous cell carcinomas of the head and neck (HNSCC), the increasing incidence of oropharyngeal squamous cell carcinomas (OPSCCs) is attributable to human papillomavirus (HPV) infection. Despite commonly presenting at late stage, HPV-driven OPSCCs are associated with improved prognosis compared with HPV-negative disease. HPV DNA is also detectable in nonoropharyngeal (non-OPSCC), but its pathogenic role and clinical significance are unclear. The objectives of this study were to determine whether HPV plays a causal role in non-OPSCC and to investigate whether HPV confers a survival benefit in these tumors.
Meta-analysis was used to build a cross-tissue gene-expression signature for HPV-driven cancer. Classifiers trained by machine-learning approaches were used to predict the HPV status of 520 HNSCCs profiled by The Cancer Genome Atlas project. DNA methylation data were similarly used to classify 464 HNSCCs and these analyses were integrated with genomic, histopathology, and survival data to permit a comprehensive comparison of HPV transcript-positive OPSCC and non-OPSCC.
HPV-driven tumors accounted for 4.1% of non-OPSCCs. Regardless of anatomic site, HPV+ HNSCCs shared highly similar gene expression and DNA methylation profiles; nonkeratinizing, basaloid histopathological features; and lack of TP53 or CDKN2A alterations. Improved overall survival, however, was largely restricted to HPV-driven OPSCCs, which were associated with increased levels of tumor-infiltrating lymphocytes compared with HPV-driven non-OPSCCs.
Our analysis identified a causal role for HPV in transcript-positive non-OPSCCs throughout the head and neck. Notably, however, HPV-driven non-OPSCCs display a distinct immune microenvironment and clinical behavior compared with HPV-driven OPSCCs.
Masterson, L., Lechner, M., Loewenbein, S., Mohammed, H., Davies-Husband, C., Fenton, T., Sudhoff, H., Jani, P., Goon, P. and Sterling, J. (2016). CD8+ T cell response to human papillomavirus 16 E7 is able to predict survival outcome in oropharyngeal cancer. European Journal of Cancer [Online] 67:141-151. Available at: http://doi.org/10.1016/j.ejca.2016.08.012.
Introduction: Immunological response to human papillomavirus (HPV) in the development and progression of HPV16+ oropharyngeal squamous cell carcinoma (OPSCC) (accounting for the majority of viral associated cases) is largely unknown and may provide important insights for new therapeutic strategies.
Methods: In this prospective clinical trial (UKCRN11945), we examined cell-mediated immune responses to HPV16 E2, E6 and E7 in peripheral blood using IFN-γ enzyme-linked immunosorbent spot assay. CD56+, CD4+, CD8+ and regulatory T cell frequencies were also discerned by flow cytometry. Fifty-one study participants with oropharyngeal carcinoma were recruited. Control subjects were those undergoing tonsillectomy for benign disease. All patients were treated with curative intent by radiotherapy ± chemotherapy. Disease-specific survival was investigated by multivariate analysis.
Results: HPV16 DNA was detected in 41/51 of the OPSCC participants. T cell responses against HPV16 E6 or E7 peptides were detected in 33/51 evaluable patients, respectively and correlated with HPV status. Matched pre- and post-treatment T cell responses were available for 39/51 OPSCC cases. Within the whole cohort, elevated post-treatment CD8+ response to HPV16 E7 correlated with longer disease free survival (multivariate DFS p < 0.03). Within the HPV + OPSCC cohort, a significant increase in regulatory T cells (p < 0.02) was noted after treatment.
Conclusions: This is the first study to provide survival data in OPSCC stratified by cell-mediated immune response to HPV16 peptides. Within the HPV16+ OPSCC cohort, enhanced immunoreactivity to antigen E7 was linked to improved survival. An increase in regulatory T cell frequencies after treatment may suggest that immunosuppression can contribute to a reduced HPV-specific cell-mediated response.
Feber, A., Worth, D., Chakravarthy, A., de Winter, P., Shah, K., Arya, M., Saqib, M., Nigam, R., Malone, P., Tan, W., Rodney, S., Freeman, A., Jameson, C., Wilson, G., Powles, T., Beck, S., Fenton, T., Sharp, T., Muneer, A. and Kelly, J. (2016). CSN1 Somatic Mutations in Penile Squamous Cell Carcinoma. Cancer Research [Online] 76:4720-4727. Available at: http://dx.doi.org/10.1158/0008-5472.CAN-15-3134.
Other than an association with HPV infection, little is known about the genetic alterations determining the development of penile cancer. Although penile cancer is rare in the developed world, it presents a significant burden in developing countries. Here, we report the findings of whole-exome sequencing (WES) to determine the somatic mutational landscape of penile cancer. WES was performed on penile cancer and matched germline DNA from 27 patients undergoing surgical resection. Targeted resequencing of candidate genes was performed in an independent 70 patient cohort. Mutation data were also integrated with DNA methylation and copy-number information from the same patients. We identified an HPV-associated APOBEC mutation signature and an NpCpG signature in HPV-negative disease. We also identified recurrent mutations in the novel penile cancer tumor suppressor genes CSN1(GPS1) and FAT1. Expression of CSN1 mutants in cells resulted in colocalization with AGO2 in cytoplasmic P-bodies, ultimately leading to the loss of miRNA-mediated gene silencing, which may contribute to disease etiology. Our findings represent the first comprehensive analysis of somatic alterations in penile cancer, highlighting the complex landscape of alterations in this malignancy.
Campbell, J., Ryan, C., Brough, R., Bajrami, I., Pemberton, H., Chong, I., Costa-Cabral, S., Frankum, J., Gulati, A., Holme, H., Miller, R., Postel-Vinay, S., Rafiq, R., Wei, W., Williamson, C., Quigley, D., Tym, J., Al-Lazikani, B., Fenton, T., Natrajan, R., Strauss, S., Ashworth, A. and Lord, C. (2016). Large-Scale Profiling of Kinase Dependencies in Cancer Cell Lines. Cell Reports [Online] 14:2490-2501. Available at: http://dx.doi.org/10.1016/j.celrep.2016.02.023.
One approach to identifying cancer-specific vulnerabilities and therapeutic targets is to profile genetic dependencies in cancer cell lines. Here, we describe data from a series of siRNA screens that identify the kinase genetic dependencies in 117 cancer cell lines from ten cancer types. By integrating the siRNA screen data with molecular profiling data, including exome sequencing data, we show how vulnerabilities/genetic dependencies that are associated with mutations in specific cancer driver genes can be identified. By integrating additional data sets into this analysis, including protein-protein interaction data, we also demonstrate that the genetic dependencies associated with many cancer driver genes form dense connections on functional interaction networks. We demonstrate the utility of this resource by using it to predict the drug sensitivity of genetically or histologically defined subsets of tumor cell lines, including an increased sensitivity of osteosarcoma cell lines to FGFR inhibitors and SMAD4 mutant tumor cells to mitotic inhibitors.
Henderson, S. and Fenton, T. (2015). APOBEC3 genes: retroviral restriction factors to cancer drivers. Trends in Molecular Medicine [Online] 21:274-284. Available at: http://dx.doi.org/10.1016/j.molmed.2015.02.007.
The APOBEC3 cytosine deaminases play key roles in innate immunity through their ability to mutagenize viral DNA and restrict viral replication. Recent advances in cancer genomics, together with biochemical characterization of the APOBEC3 enzymes, have now implicated at least two family members in somatic mutagenesis during tumor development. We review the evidence linking these enzymes to carcinogenesis and highlight key questions, including the potential mechanisms that misdirect APOBEC3 activity to the host genome, the links to viral infection, and the association between a common APOBEC3 polymorphism and cancer risk.
Chakravarthy, A., Henderson, S. and Fenton, T. (2014). When defense turns into attack: Antiviral cytidine deaminases linked to somatic mutagenesis in HPV-associated cancer. Molecular & Cellular Oncology [Online] 1:e29914 - e29914. Available at: http://dx.doi.org/10.4161/mco.29914.
The APOBEC3 cytidine deaminases play an important role in innate immunity but have also emerged as mediators of somatic mutations in human cancer. We recently reported a high incidence of APOBEC-mediated driver mutations in human papillomavirus-associated cancer, suggesting a key role for these enzymes in the development of such tumors.
Henderson, S., Chakravarthy, A., Su, X., Boshoff, C. and Fenton, T. (2014). APOBEC-Mediated Cytosine Deamination Links PIK3CA Helical Domain Mutations to Human Papillomavirus-Driven Tumor Development. Cell reports [Online] 7:1833-1841. Available at: http://doi.org/10.1016/j.celrep.2014.05.012.
APOBEC3B cytosine deaminase activity has recently emerged as a significant mutagenic factor in human cancer. APOBEC activity is induced in virally infected cells, and APOBEC signature mutations occur at high frequency in cervical cancers (CESC), over 99% of which are caused by human papillomavirus (HPV). We tested whether APOBEC-mediated mutagenesis is particularly important in HPV-associated tumors by comparing the exomes of HPV+ and HPV- head and neck squamous cell carcinomas (HNSCCs) sequenced by The Cancer Genome Atlas project. As expected, HPV- HNSCC displays a smoking-associated mutational signature, whereas our data suggest that reduced exposure to exogenous carcinogens in HPV+ HNSCC creates a selective pressure that favors emergence of tumors with APOBEC-mediated driver mutations. Finally, we provide evidence that APOBEC activity is responsible for the?generation of helical domain hot spot mutations in the PIK3CA gene across multiple cancers. Our?findings implicate APOBEC activity as a key driver?of PIK3CA mutagenesis and HPV-induced transformation.
Feber, A., Guilhamon, P., Lechner, M., Fenton, T., Wilson, G., Thirlwell, C., Morris, T., Flanagan, A., Teschendorff, A., Kelly, J. and Beck, S. (2014). Using high-density DNA methylation arrays to profile copy number alterations. Genome Biology [Online] 15. Available at: http://dx.doi.org/10.1186/gb-2014-15-2-r30.
The integration of genomic and epigenomic data is an increasingly popular approach for studying the complex mechanisms driving cancer development. We have developed a method for evaluating both methylation and copy number from high-density DNA methylation arrays. Comparing copy number data from Infinium HumanMethylation450 BeadChips and SNP arrays, we demonstrate that Infinium arrays detect copy number alterations with the sensitivity of SNP platforms. These results show that high-density methylation arrays provide a robust and economic platform for detecting copy number and methylation changes in a single experiment. Our method is available in the ChAMP Bioconductor package: http://www.bioconductor.org/packages/2.13/bioc/html/ChAMP.html.
Wilson, G., Lechner, M., and H Carén, A., Butcher, L., Feber, A., Fenton, T., Jay, A., Boshoff, C. and Beck, S. (2013). Integrated virus-host methylome analysis in head and neck squamous cell carcinoma. Epigenetics [Online] 8:953-961. Available at: http://dx.doi.org/10.4161/epi.25614.
One in six cancers worldwide is caused by infection and human papillomavirus (HPV) is one of the main culprits. To better understand the dynamics of HPV integration and its effect on both the viral and host methylomes, we conducted whole-genome DNA methylation analysis using MeDIP-seq of HPV+ and HPV- head and neck squamous cell carcinoma (HNSCC). We determined the viral subtype to be HPV-16 in all cases and show that HPV-16 integrates into the host genome at multiple random sites and that this process predominantly involves the transcriptional repressor gene (E2) in the viral genome. Comparative analysis identified 453 (FDR ? 0.01) differentially methylated regions (DMRs) in the HPV+ host methylome. Bioinformatics characterization of these DMRs confirmed the previously reported cadherin genes to be affected but also revealed new targets for HPV-mediated methylation changes at regions not covered by array-based platforms, including the recently identified super-enhancers.
Lechner, M., Frampton, G., Fenton, T., Feber, A., Palmer, G., Jay, A., Pillay, N., Forster, M., Cronin, M., Lipson, D., Miller, V., Brennan, T., Henderson, S., Vaz, F., Flynn, P., Kalavrezos, N., Yelenski, R., Beck, S., Stephens, P. and Boshoff, C. (2013). Targeted next-generation sequencing of head and neck squamous cell carcinoma identifies novel genetic alterations in HPV+ and HPV- tumors. Genome Medicine [Online] 5. Available at: http://dx.doi.org/10.1186/gm453.
BACKGROUND: Human papillomavirus positive (HPV+) head and neck squamous cell carcinoma (HNSCC) is an emerging disease, representing a distinct clinical and epidemiological entity. Understanding the genetic basis of this specific subtype of cancer could allow therapeutic targeting of affected pathways for a stratified medicine approach. METHODS: Twenty HPV+ and 20 HPV- laser-capture microdissected oropharyngeal carcinomas were used for paired-end sequencing of hybrid-captured DNA, targeting 3,230 exons in 182 genes often mutated in cancer. Copy number alteration (CNA) profiling, Sequenom MassArray sequencing and immunohistochemistry were used to further validate findings. RESULTS: HPV+ and HPV- oropharyngeal carcinomas cluster into two distinct subgroups. TP53 mutations are detected in 100% of HPV negative cases and abrogation of the G1/S checkpoint by CDKN2A/B deletion and/or CCND1 amplification occurs in the majority of HPV- tumors. CONCLUSION: These findings strongly support a causal role for HPV, acting via p53 and RB pathway inhibition, in the pathogenesis of a subset of oropharyngeal cancers and suggest that studies of CDK inhibitors in HPV negative disease may be warranted. Mutation and copy number alteration of PI3 kinase (PI3K) pathway components appears particularly prevalent in HPV+ tumors and assessment of these alterations may aid in the interpretation of current clinical trials of PI3K, AKT and mTOR inhibitors in HNSCC.
Read, R., Fenton, T., Gomez, G., Wykosky, J., Vandenberg, S., Babic, I., Yang, H., Iwanami, A., Cavenee, W., Mischel, P., Furnari, F. and Thomas, J. (2013). A Kinome-Wide RNAi Screen in Drosophila Glia Reveals That the RIO Kinases Mediate Cell Proliferation and Survival through TORC2-Akt Signaling in Glioblastoma. PLoS Genetics [Online] 9:1-19. Available at: https://doi.org/10.1371/journal.pgen.1003253.
Glioblastoma, the most common primary malignant brain tumor, is incurable with current therapies. Genetic and molecular analyses demonstrate that glioblastomas frequently display mutations that activate receptor tyrosine kinase (RTK) and Pi-3 kinase (PI3K) signaling pathways. In Drosophila melanogaster, activation of RTK and PI3K pathways in glial progenitor cells creates malignant neoplastic glial tumors that display many features of human glioblastoma. In both human and Drosophila, activation of the RTK and PI3K pathways stimulates Akt signaling along with other as-yet-unknown changes that drive oncogenesis. We used this Drosophila glioblastoma model to perform a kinome-wide genetic screen for new genes required for RTK- and PI3K-dependent neoplastic transformation. Human orthologs of novel kinases uncovered by these screens were functionally assessed in mammalian glioblastoma models and human tumors. Our results revealed that the atypical kinases RIOK1 and RIOK2 are overexpressed in glioblastoma cells in an Akt-dependent manner. Moreover, we found that overexpressed RIOK2 formed a complex with RIOK1, mTor, and mTor-complex-2 components, and that overexpressed RIOK2 upregulated Akt signaling and promoted tumorigenesis in murine astrocytes. Conversely, reduced expression of RIOK1 or RIOK2 disrupted Akt signaling and caused cell cycle exit, apoptosis, and chemosensitivity in glioblastoma cells by inducing p53 activity through the RpL11-dependent ribosomal stress checkpoint. These results imply that, in glioblastoma cells, constitutive Akt signaling drives RIO kinase overexpression, which creates a feedforward loop that promotes and maintains oncogenic Akt activity through stimulation of mTor signaling. Further study of the RIO kinases as well as other kinases identified in our Drosophila screen may reveal new insights into defects underlying glioblastoma and related cancers and may reveal new therapeutic opportunities for these cancers.
Lechner, M., Fenton, T., West, J., Wilson, G., Feber, A., Henderson, S., Thirlwell, C., Dibra, H., Jay, A., Butcher, L., Chakravarthy, A., Gratrix, F., Patel, N., Vaz, F., O’Flynn, P., Kalavrezos, N., Teschendorff, A., Boshoff, C. and Beck, S. (2013). Identification and functional validation of HPV-mediated hypermethylation in head and neck squamous cell carcinoma. Genome Medicine [Online] 5. Available at: http://dx.doi.org/10.1186/gm419.
ABSTRACT: BACKGROUND: Human Papillomavirus positive (HPV+) head and neck squamous cell carcinoma (HNSCC) represents a distinct clinical and epidemiological entity compared with HPV negative (HPV-) HNSCC. To test the possible involvement of epigenetic modulation by HPV in HNSCC, we conducted a genome-wide DNA methylation analysis. METHODS: Using laser-capture microdissection of 42 formalin-fixed paraffin-embedded (FFPE) HNSCCs, we generated DNA methylation profiles of 18 HPV+ and 14 HPV- samples, using the Infinium 450k BeadArray technology. Methylation data were validated in two sets of independent HPV+/HPV- HNSCC samples (fresh frozen and cell lines) using two independent methods (Infinium 450k and whole-genome MeDIP-seq). For the functional analysis, an HPV- HNSCC cell line was transduced with lentiviral constructs containing the two HPV oncogenes (E6 and E7) and effects on methylation were assayed using the Infinium 450k technology. RESULTS AND DISCUSSION: Unsupervised clustering over the most methylation variable positions (MVPs) showed that samples segregated according to HPV status, but also that HPV+ tumours are heterogeneous. MVPs were significantly enriched at transcriptional start sites, leading to the identification of a candidate CpG Island Methylator Phenotype in a sub-group of the HPV+ tumours. Supervised analysis revealed a strong preponderance (87%) of MVPs towards hypermethylation in HPV+ HNSCC. Meta-analysis of our HNSCC and publicly available methylation data in cervical and lung cancer confirmed the observed DNA methylation signature to be HPV-specific and tissue-independent. Grouping of MVPs into functionally more significant differentially methylated regions (DMRs) identified 43 hypermethylated promoter DMRs, including for three Cadherins of the Polycomb group target genes. Integration with independent expression data showed strong negative correlation, especially for the Cadherin gene family members. Combinatorial ectopic expression of the two HPV oncogenes (E6 and E7) in an HPV- HNSCC cell line partially phenocopied the hypermethylation signature observed in HPV+ HNSCC tumours and established E6 as the main viral effector gene. CONCLUSIONS: Our data establish archival FFPE tissue to be highly suitable for this type of methylome analysis and suggest that HPV modulates the HNSCC epigenome through hypermethylation of Polycomb repressive complex 2 target genes such as Cadherins which are implicated in tumour progression and metastasis.
Fenton, T., Nathanson, D., de Albuquerque, C., Kuga, D., Iwanami, A., Dang, J., Yang, H., Tanaka, K., Oba-Shinjo, S., Uno, M., Inda, M., Wykosky, J., Bachoo, R., James, C., DePinho, R., Vandenberg, S., Zhou, H., Marie, S., Mischel, P., Cavenee, W. and Furnari, F. (2012). Resistance to EGF receptor inhibitors in glioblastoma mediated by phosphorylation of the PTEN tumor suppressor at tyrosine 240. Proceedings of the National Academy of Sciences [Online] 109:14164 - 14169. Available at: http://dx.doi.org/10.1073/pnas.1211962109.
Glioblastoma multiforme (GBM) is the most aggressive of the astrocytic malignancies and the most common intracranial tumor in adults. Although the epidermal growth factor receptor (EGFR) is overexpressed and/or mutated in at least 50% of GBM cases and is required for tumor maintenance in animal models, EGFR inhibitors have thus far failed to deliver significant responses in GBM patients. One inherent resistance mechanism in GBM is the coactivation of multiple receptor tyrosine kinases, which generates redundancy in activation of phosphoinositide-3'-kinase (PI3K) signaling. Here we demonstrate that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is frequently phosphorylated at a conserved tyrosine residue, Y240, in GBM clinical samples. Phosphorylation of Y240 is associated with shortened overall survival and resistance to EGFR inhibitor therapy in GBM patients and plays an active role in mediating resistance to EGFR inhibition in vitro. Y240 phosphorylation can be mediated by both fibroblast growth factor receptors and SRC family kinases (SFKs) but does not affect the ability of PTEN to antagonize PI3K signaling. These findings show that, in addition to genetic loss and mutation of PTEN, its modulation by tyrosine phosphorylation has important implications for the development and treatment of GBM.
Feng, H., Hu, B., Jarzynka, M., Li, Y., Keezer, S., Johns, T., Tang, C., Hamilton, R., Vuori, K., Nishikawa, R., Sarkaria, J., Fenton, T., Cheng, T., Furnari, F., Cavenee, W. and Cheng, S. (2012). Phosphorylation of dedicator of cytokinesis 1 (Dock180) at tyrosine residue Y722 by Src family kinases mediates EGFRvIII-driven glioblastoma tumorigenesis. Proceedings of the National Academy of Sciences [Online] 109:3018-3023. Available at: http://dx.doi.org/10.1073/pnas.1121457109.
Glioblastoma, the most common primary malignant cancer of the brain, is characterized by rapid tumor growth and infiltration of tumor cells throughout the brain. These traits cause glioblastomas to be highly resistant to current therapies with a resultant poor prognosis. Although aberrant oncogenic signaling driven by signature genetic alterations, such as EGF receptor (EGFR) gene amplification and mutation, plays a major role in glioblastoma pathogenesis, the responsible downstream mechanisms remain less clear. Here, we report that EGFRvIII (also known as \ensuremath?EGFR and de2-7EGFR), a constitutively active EGFR mutant that is frequently co-overexpressed with EGFR in human glioblastoma, promotes tumorigenesis through Src family kinase (SFK)-dependent phosphorylation of Dock180, a guanine nucleotide exchange factor for Rac1. EGFRvIII induces phosphorylation of Dock180 at tyrosine residue 722 (Dock180(Y722)) and stimulates Rac1-signaling, glioblastoma cell survival and migration. Consistent with this being causal, siRNA knockdown of Dock180 or expression of a Dock180(Y722F) mutant inhibits each of these EGFRvIII-stimulated activities. The SFKs, Src, Fyn, and Lyn, induce phosphorylation of Dock180(Y722) and inhibition of these SFKs by pharmacological inhibitors or shRNA depletion markedly attenuates EGFRvIII-induced phosphorylation of Dock180(Y722), Rac1 activity, and glioblastoma cell migration. Finally, phosphorylated Dock180(Y722) is coexpressed with EGFRvIII and phosphorylated Src(Y418) in clinical specimens, and such coexpression correlates with an extremely poor survival in glioblastoma patients. These results suggest that targeting the SFK-p-Dock180(Y722)-Rac1 signaling pathway may offer a novel therapeutic strategy for glioblastomas with EGFRvIII overexpression.
Feng, H., Hu, B., Liu, K., Li, Y., Lu, X., Cheng, T., Yiin, J., Lu, S., Keezer, S., Fenton, T., Furnari, F., Hamilton, R., Vuori, K., Sarkaria, J., Nagane, M., Nishikawa, R., Cavenee, W. and Cheng, S. (2011). Activation of Rac1 by Src-dependent phosphorylation of Dock180(Y1811) mediates PDGFR\ensuremath?-stimulated glioma tumorigenesis in mice and humans. Journal of Clinical Investigation [Online] 121:4670-4684. Available at: https://doi.org/10.1172/JCI58559.
Two hallmarks of glioblastoma multiforme, the most common malignant brain cancer in humans, are aggressive growth and the ability of single glioma cells to disperse throughout the brain. These characteristics render tumors resistant to current therapies and account for the poor prognosis of patients. Although it is known that oncogenic signaling caused by overexpression of genes such as PDGFRA is responsible for robust glioma growth and cell infiltration, the mechanisms underlying glioblastoma malignancy remain largely elusive. Here, we report that PDGFR\ensuremath? signaling in glioblastomas leads to Src-dependent phosphorylation of the guanine nucleotide exchange factor Dock180 at tyrosine 1811 (Dock180(Y1811)) that results in activation of the GTPase Rac1 and subsequent cell growth and invasion. In human glioma cells, knockdown of Dock180 and reversion with an RNAi-resistant Dock180(Y1811F) abrogated, whereas an RNAi-resistant Dock180(WT) rescued, PDGFR\ensuremath?-promoted glioma growth, survival, and invasion. Phosphorylation of Dock180(Y1811) enhanced its association with CrkII and p130(Cas), causing activation of Rac1 and consequent cell motility. Dock180 also associated with PDGFR\ensuremath? to promote cell migration. Finally, phosphorylated Dock180(Y1811) was detected in clinical samples of gliomas and various types of human cancers, and coexpression of phosphorylated Dock180(Y1811), phosphorylated Src(Y418), and PDGFR\ensuremath? was predictive of extremely poor prognosis of patients with gliomas. Taken together, our findings provide insight into PDGFR\ensuremath?-stimulated gliomagenesis and suggest that phosphorylated Dock180(Y1811) contributes to activation of Rac1 in human cancers with PDGFRA amplification.
Wykosky, J., Fenton, T., Furnari, F. and Cavenee, W. (2011). Therapeutic targeting of epidermal growth factor receptor in human cancer: successes and limitations. Chin J Cancer [Online] 30:5-12. Available at: http://dx.doi.org/10.5732/cjc.010.10542.
Epidermal growth factor receptor (EGFR) is one of the most commonly altered genes in human cancer by way of over-expression, amplification, and mutation. Targeted inhibition of EGFR activity suppresses signal transduction pathways which control tumor cell growth, proliferation, and resistance to apoptosis. Small molecule tyrosine kinase inhibitors and monoclonal antibodies are among the most common EGFR-targeting agents and have been used clinically for treating various malignancies. This review discusses the successes and challenges of targeting EGFR in human cancer. The genetic alterations of EGFR tend to occur more often in some solid tumors than others, as do the mechanisms of resistance to targeted inhibition. The clinical and basic science experiences with these agents thus far have important implications for the future of therapeutic targeting of EGFR.
Fenton, T. and Gout, I. (2010). Functions and regulation of the 70 kDa ribosomal S6 kinases. International Journal of Biochemistry and Cell Biology [Online] 43:47-59. Available at: http://dx.doi.org/10.1016/j.biocel.2010.09.018.
The 70 kDa ribosomal protein S6 kinases, S6K1 and S6K2 are two highly homologous serine/threonine kinases that are activated in response to growth factors, cytokines and nutrients. The S6 kinases have been linked to diverse cellular processes, including protein synthesis, mRNA processing, glucose homeostasis, cell growth and survival. Studies in model organisms have highlighted the roles that S6K activity plays in a number of pathologies, including obesity, diabetes, ageing and cancer. The importance of S6K function in human diseases has led to the development of S6K-specific inhibitors by a number of companies, offering the promise of improved tools with which to study these enzymes and potentially the effective targeting of deregulated S6K signalling in patients. Here we review the current literature on the role of S6Ks in the regulation of cell growth, survival and proliferation downstream of various signalling pathways and how their dysregulation contributes to the pathogenesis of human diseases.
Fenton, T., Gwalter, J., Cramer, R. and Gout, I. (2010). S6K1 is acetylated at lysine 516 in response to growth factor stimulation. Biochemical and Biophysical Research Communications [Online] 398:400 - 405. Available at: http://dx.doi.org/10.1016/j.bbrc.2010.06.081.
The 70 kDa ribosomal protein S6 kinase 1 (S6K1) plays important roles in the regulation of protein synthesis, cell growth and metabolism. S6K1 is activated by the phosphorylation of multiple serine and threonine residues in response to stimulation by a variety of growth factors and cytokines. In addition to phosphorylation, we have recently shown that S6K1 is also targeted by lysine acetylation. Here, using tandem mass spectrometry we have mapped acetylation of S6K1 to lysine 516, a site close to the C-terminus of the kinase that is highly conserved amongst vertebrate S6K1 orthologues. Using acetyl-specific K516 antibodies, we show that acetylation of endogenous S6K1 at this site is potently induced upon growth factor stimulation. Although S6K1 acetylation and phosphorylation are both induced by growth factor stimulation, these events appear to be functionally independent. Indeed, experiments using inhibitors of S6K1 activation and exposure of cells to various stresses indicate that S6K1 acetylation can occur in the absence of phosphorylation and vice versa. We propose that K516 acetylation may serve to modulate important kinase-independent functions of S6K1 in response to growth factor signalling. (C) 2010 Elsevier inc. All rights reserved.
Fenton, T., Gwalter, J., Ericsson, J. and Gout, I. (2009). Histone acetyltransferases interact with and acetylate p70 ribosomal S6 kinases in vitro and in vivo. The International Journal of Biochemistry & Cell Biology [Online] 42:359 - 366. Available at: http://dx.doi.org/10.1016/j.biocel.2009.11.022.
The 70 kDa ribosomal protein S6 kinases (S6K1 and 56K2) play important roles in the regulation of protein synthesis, cell growth and survival. S6Ks are activated in response to mitogen stimulation and nutrient sufficiency by the phosphorylation of conserved serine and threonine residues. Here we show for the first time, that in addition to phosphorylation, S6Ks are also targeted by lysine acetylation. Following mitogen stimulation, S6Ks interact with the p300 and p300/CBP-associated factor (PCAF) acetyltransferases. S6Ks can be acetylated by p300 and PCAF in vitro and S6K acetylation is detected in cells expressing p300. Furthermore, it appears that the acetylation sites targeted by p300 lie within the divergent C-terminal regulatory domains of both S6K1 and S6K2. Acetylation of S6K1 and 2 is increased upon the inhibition of class I/II histone deacetylases (HDACs) by trichostatin-A, while the enhancement of S6K1 acetylation by nicotinamide suggests the additional involvement of sirtuin deacetylases in S6K deacetylation. Both expression of p300 and HDAC inhibition cause increases in S6K protein levels, and we have shown that S6K2 is stabilized in cells treated with HDAC inhibitors. The finding that S6Ks are targeted by histone acetyltransferases uncovers a novel mode of crosstalk between mitogenic signalling pathways and the transcriptional machinery and reveals additional complexity in the regulation of S6K function. (C) 2009 Elsevier Ltd. All rights reserved.
Wang, M., Panasyuk, G., Gwalter, J., Nemazanyy, I., Fenton, T., Filonenko, V. and Gout, I. (2008). Regulation of ribosomal protein S6 kinases by ubiquitination. Biochemical and Biophysical Research Communications [Online] 369:382-387. Available at: http://dx.doi.org/10.1016/j.bbrc.2008.02.032.
Ribosomal protein S6 kinase (S6K) is a key player in the regulation of cell growth and energy metabolism via the mTOR and PI3K signalling pathways. The activity and subcellular localization of S6K are regulated by multiple S/T phosphorylations in response to diverse extracellular stimuli. Downregulation of S6K signalling occurs through the action of S/T phosphatases (PP2A and PP1) and tumor suppressors (TSC1/2 and PTEN). We report here that, in addition to phosphorylation, S6Ks are ubiquitinated in cells. The pattern of ubiquitination and the effect of proteasomal inhibitors on the steady-state level of transiently overexpressed and endogenous S6Ks point to proteasome-mediated degradation of ubiquitinated S6Ks. Furthermore, we found that the site(s) of ubiquitination are located in the kinase domain and that the N- and C-terminal regulatory regions modulate the efficiency of S6K ubiquitination. This study suggests that S6K signalling also could be regulated through the proteasome-mediated turnover of S6Ks.
Furnari, F., Fenton, T., Bachoo, R., Mukasa, A., Stommel, J., Stegh, A., Hahn, W., Ligon, K., Louis, D., Brennan, C., Chin, L., DePinho, R. and Cavenee, W. (2007). Malignant astrocytic glioma: genetics, biology, and paths to treatment. Genes Dev [Online] 21:2683-2710. Available at: http://dx.doi.org/10.1101/gad.1596707.
Malignant astrocytic gliomas such as glioblastoma are the most common and lethal intracranial tumors. These cancers exhibit a relentless malignant progression characterized by widespread invasion throughout the brain, resistance to traditional and newer targeted therapeutic approaches, destruction of normal brain tissue, and certain death. The recent confluence of advances in stem cell biology, cell signaling, genome and computational science and genetic model systems have revolutionized our understanding of the mechanisms underlying the genetics, biology and clinical behavior of glioblastoma. This progress is fueling new opportunities for understanding the fundamental basis for development of this devastating disease and also novel therapies that, for the first time, portend meaningful clinical responses.
Carrasco, D., Fenton, T., Sukhdeo, K., Protopopova, M., Enos, M., You, M., Vizio, D., Nogueira, C., Stommel, J., Pinkus, G., Fletcher, C., Hornick, J., Cavenee, W., Furnari, F. and Depinho, R. (2006). The PTEN and INK4A/ARF tumor suppressors maintain myelolymphoid homeostasis and cooperate to constrain histiocytic sarcoma development in humans. Cancer Cell [Online] 9:379-390. Available at: http://dx.doi.org/10.1016/j.ccr.2006.03.028.
Histiocytic sarcoma (HS) is a rare malignant proliferation of histiocytes of uncertain molecular pathogenesis. Here, genetic analysis of coincident loss of Pten and Ink4a/Arf tumor suppressors in the mouse revealed a neoplastic phenotype dominated by a premalignant expansion of biphenotypic myelolymphoid cells followed by the development of HS. Pten protein loss occurred only in the histiocytic portion of tumors, suggesting a stepwise genetic inactivation in the generation of HS. Similarly, human HS showed genetic or epigenetic inactivation of PTEN, p16(INK4A), and p14(ARF), supporting the relevance of this genetically engineered mouse model of HS. These genetic and translational observations establish a cooperative role of Pten and Ink4a/Arf in the development of HS and provide mechanistic insights into the pathogenesis of human HS.
Rebholz, H., Panasyuk, G., Fenton, T., Nemazanyy, I., Valovka, T., Flajolet, M., Ronnstrand, L., Stephens, L., West, A. and Gout, I. (2006). Receptor association and tyrosine phosphorylation of S6 kinases. FEBS J [Online] 273:2023 - 2036. Available at: http://dx.doi.org/10.1111/j.1742-4658.2006.05219.x.
Ribosomal protein S6 kinase (S6K) is activated by an array of mitogenic stimuli and is a key player in the regulation of cell growth. The activation process of S6 kinase involves a complex and sequential series of multiple Ser/Thr phosphorylations and is mainly mediated via phosphatidylinositol 3-kinase (PI3K)-3-phosphoinositide-dependent protein kinase-1 (PDK1) and mTor-dependent pathways. Upstream regulators of S6K, such as PDK1 and protein kinase B (PKB/Akt), are recruited to the membrane via their pleckstrin homology (PH) or protein-protein interaction domains. However, the mechanism of integration of S6K into a multi-enzyme complex around activated receptor tyrosine kinases is not clear. In the present study, we describe a specific interaction between S6K with receptor tyrosine Such as platelet-derived growth factor receptor (PDGFR). The kinases, interaction with PDGFR is mediated via the kinase or the kinase extension domain of S6K. Complex formation is inducible by growth factors and leads to S6K tyrosine phosphorylation. Using PDGFR mutants, we have shown that the phosphorylation is exerted via a PDGFR-src pathway. Furthermore, src kinase phosphorylates and coimmunoprecipitates with S6K in vivo. Inhibitors towards tyrosine kinases, such as genistein and PP1, or src-specific SU6656, but not PI3K and mTor inhibitors, lead to a reduction in tyrosine phosphorylation of S6K. In addition, we mapped the sites of tyrosine phosphorylation in S6K1 and S6K2 to Y39 and Y45, respectively. Mutational and immunofluorescent analysis indicated that phosphorylation of S6Ks at these sites does not affect their activity or subcellular localization. Our data indicate that S6 kinase is recruited into a complex with RTKs and src and becomes phosphorylated on tyrosine/s in response to PDGF or serum.
Zhyvoloup, A., Nemazanyy, I., Panasyuk, G., Valovka, T., Fenton, T., Rebholz, H., Wang, M., Foxon, R., Lyzogubov, V., Usenko, V., Kyyamova, R., Gorbenko, O., Matsuka, G., Filonenko, V. and Gout, I. (2003). Subcellular localization and regulation of coenzyme a synthase. Journal of Biological Chemistry [Online] 278:50316 - 50321. Available at: http://dx.doi.org/10.1074/jbc.M307763200.
CoA synthase mediates the last two steps in the sequence of enzymatic reactions, leading to CoA biosynthesis. We have recently identified cDNA for CoA synthase and demonstrated that it encodes a bifunctional enzyme possessing 4'-phosphopantetheine adenylyltransferase and dephospho-CoA kinase activities. Molecular cloning of CoA synthase provided us with necessary tools to study subcellular localization and the regulation of this bifunctional enzyme. Transient expression studies and confocal microscopy allowed us to demonstrate that full-length CoA synthase is associated with the mitochondria, whereas the removal of the N-terminal region relocates the enzyme to the cytosol. In addition, we showed that the N-terminal sequence of CoA synthase ( amino acids 1 - 29) exhibits a hydrophobic profile and targets green fluorescent protein exclusively to mitochondria. Further analysis, involving subcellular fractionation and limited proteolysis, indicated that CoA synthase is localized on the mitochondrial outer membrane. Moreover, we demonstrate for the first time that phosphatidylcholine and phosphatidylethanolamine, which are the main components of the mitochondrial outer membrane, are potent activators of both enzymatic activities of CoA synthase in vitro. Taken together, these data provide the evidence that the final stages of CoA biosynthesis take place on mitochondria and the activity of CoA synthase is regulated by phospholipids.
Valovka, T., Verdier, F., Cramer, R., Zhyvoloup, A., Fenton, T., Rebholz, H., Wang, M., Gzhegotsky, M., Lutsyk, A., Matsuka, G., Filonenko, V., Wang, L., Proud, C., Parker, P. and Gout, I. (2003). Protein kinase C phosphorylates ribosomal protein S6 kinase beta II and regulates its subcellular localization. MOL CELL BIOL [Online] 23:852 - 863. Available at: https://mcb.asm.org/content/23/3/852.short.
The ribosomal protein S6 kinase (S6K) belongs to the AGC family of Ser/Thr kinases and is known to be involved in the regulation of protein synthesis and the G(1),/S transition of the cell cycle. There are two forms of S6K, termed S6Kalpha and S6Kbeta, which have cytoplasmic and nuclear splice variants. Nucleocytoplasmic shuttling has been recently proposed for S6Kalpha, based on the use of the nuclear export inhibitor, leptomycin B. However, the molecular mechanisms regulating subcellular localization of S6Ks in response to mitogenic stimuli remain to be elucidated. Here we present data on the in vitro and in vivo phosphorylation of S6Kbeta, but not S6Kalpha, by protein kinase C (PKC). The site of phosphorylation was identified as S486, which is located within the C-terminal nuclear localization signal. Mutational analysis and the use of phosphospecific antibodies provided evidence that PKC-mediated phosphorylation at S486 does not affect S6K activity but eliminates the function of its nuclear localization signal and causes retention of an activated form of the kinase in the cytoplasm. Taken together, this study uncovers a novel mechanism for the regulation of nucleocytoplasmic shuttling of S6KbetaII by PKC-mediated phosphorylation.
Zhyvoloup, A., Nemazanyy, I., Babich, A., Panasyuk, G., Pobigailo, N., Vudmaska, M., Naidenov, V., Kukharenko, O., Palchevskii, S., Savinska, L., Ovcharenko, G., Verdier, F., Valovka, T., Fenton, T., Rebholz, H., Wang, M., Shepherd, P., Matsuka, G., Filonenko, V. and Gout, I. (2002). Molecular cloning of CoA synthase - The missing link in CoA biosynthesis. Journal of Biological Chemistry [Online] 277:22107 - 22110. Available at: http://dx.doi.org/10.1074/jbc.C200195200.
Coenzyme A functions as a carrier of acetyl and acyl groups in living cells and is essential for numerous biosynthetic, energy-yielding, and degradative metabolic pathways. There are five enzymatic steps in CoA biosynthesis. To date, molecular cloning of enzymes involved in the CoA biosynthetic pathway in mammals has been only reported for pantothenate kinase. In this study, we present cDNA cloning and functional characterization of CoA synthase. It has an open reading frame of 563 aa and encodes a protein of similar to60 kDa. Sequence alignments suggested that the protein possesses both phosphopantetheine adenylyltransferase and dephospho-CoA kinase domains. Biochemical assays using wild type recombinant protein confirmed the gene product indeed contained both these enzymatic activities. The presence of intrinsic phosphopantetheine adenylyltransferase activity was further confirmed by site-directed mutagenesis. Therefore, this study describes the first cloning and characterization of a mammalian CoA synthase and confirms this is a bifunctional enzyme containing the last two components of CoA biosynthesis.
Salim, K., Fenton, T., Bacha, J., Urien-Rodriguez, H., Bonnert, T., Skynner, H., Watts, E., Kerby, J., Heald, A., Beer, M., McAllister, G. and Guest, P. (2002). Oligomerization of G-protein-coupled receptors shown by selective co-immunoprecipitation. Journal of Biological Chemistry [Online] 277:15482-15485. Available at: http://dx.doi.org/10.1074/jbc.M201539200.
Recent studies have shown that G-protein-coupled receptors (GPCRs) can assemble as high molecular weight homo- and hetero-oligomeric complexes. This can result in altered receptor-ligand binding, signaling, or intracellular trafficking. We have co-transfected HEK-293 cells with differentially epitope-tagged GPCRs from different subfamilies and determined whether oligomeric complexes were formed by co-immunoprecipitation and immunoblot analysis. This gave the surprising result that the 5HT(1A) receptor was capable of forming hetero-oligomers with all GPCRs tested including the 5HT(1B), 5HT(1D), EDG(1), EDG(3), GPR(26), and GABA(B2) receptors. The testing of other GPCR combinations showed similar results with hetero-oligomer formation occurring for the 5HT(1D) with the 5HT(1B) and EDG(1) receptor. Control studies showed that these complexes were present in co-transfected cells before the time of lysis and that the hetero-oligomers were comprised of GPCRs at discrete stoichiometries. These findings suggest that GPCRs have a natural tendency to form oligomers when co-transfected into cells. Future studies should therefore investigate the presence and physiological role of GPCR hetero-oligomers in cells in which they are endogenously expressed.
Fordham-Skelton, A., Chilley, P., Lumbreras, V., Reignoux, S., Fenton, T., Dahm, C., Pages, M. and Gatehouse, J. (2002). A novel higher plant protein tyrosine phosphatase interacts with SNF1-related protein kinases via a KIS (kinase interaction sequence) domain. The Plant journal : for cell and molecular biology [Online] 29:705-715. Available at: http://dx.doi.org/10.1046/j.1365-313X.2002.01250.x.
A novel protein phosphatase in Arabidopsis thaliana was identified by database searching. This protein, designated AtPTPKIS1, contains a protein tyrosine phosphatase (PTP) catalytic domain and a kinase interaction sequence (KIS) domain. It is predicted to interact with plant SNF1-related kinases (SnRKs), representing central regulators of metabolic and stress responses. AtPTPKIS1 has close homologues in other plant species, both dicots and monocots, but is not found in other kingdoms. The tomato homologue of AtPTPKIS1 was expressed as a recombinant protein and shown to hydrolyse a generic phosphatase substrate, and phosphotyrosine residues in synthetic peptides. The KIS domain of AtPTPKIS1 was shown to interact with the plant SnRK AKIN11 both in vivo in the yeast two-hybrid system, and in vitro in a GST-fusion 'pull down' assay. The genomes of Arabidopsis and other plants contain further predicted proteins related to AtPTPKIS1, which could also interact with SnRKs and act in novel regulatory and signalling pathways.