Chamberlain, F.E., Wilding, C., Jones, R.L. & Huang, P.
(2019). Pazopanib in patients with advanced intermediate-grade or high-grade liposarcoma. Expert opinion on investigational drugs,
Harrison, P.T., Vyse, S. & Huang, P.H.
(2019). Rare epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer. Seminars in cancer biology,
Vyse, S. & Huang, P.H.
(2019). Targeting EGFR exon 20 insertion mutations in non-small cell lung cancer. Signal transduction and targeted therapy,
Lee, A.T., Jones, R.L. & Huang, P.H.
(2019). Pazopanib in advanced soft tissue sarcomas. Signal transduction and targeted therapy,
Lee, A.T., Chew, W., Wilding, C.P., Guljar, N., Smith, M.J., Strauss, D.C., Fisher, C., Hayes, A.J., Judson, I., Thway, K., et al.
(2019). The adequacy of tissue microarrays in the assessment of inter- and intra-tumoural heterogeneity of infiltrating lymphocyte burden in leiomyosarcoma. Scientific reports,
Lee, A.T., Huang, P.H. & Jones, R.L.
(2019). Negative phase III trials announce the need for biomarkers in sarcoma. European journal of cancer,
Martinez, V.G., Pankova, V., Krasny, L., Singh, T., White, I.J., Benjamin, A.C., Dertschnig, S., Horsnell, H.L., Kriston-Vizi, J., Burden, J.J., et al.
(2019). Conduit Integrity is Compromised During Acute Lymph Node Expansion. ,
Vyse, S., McCarthy, F., Broncel, M., Paul, A., Wong, J.P., Bhamra, A. & Huang, P.H.
(2018). Quantitative phosphoproteomic analysis of acquired cancer drug resistance to pazopanib and dasatinib. Journal of proteomics,
Harrison, P. & Huang, P.H.
(2018). Exploiting vulnerabilities in cancer signalling networks to combat targeted therapy resistance. Essays in biochemistry,
Drug resistance remains one of the greatest challenges facing precision oncology today. Despite the vast array of resistance mechanisms that cancer cells employ to subvert the effects of targeted therapy, a deep understanding of cancer signalling networks has led to the development of novel strategies to tackle resistance both in the first-line and salvage
therapy settings. In this review, we provide a brief overview of the major classes of resistance mechanisms to targeted therapy, including signalling reprogramming and tumour evolution; our discussion also focuses on the use of different forms of polytherapies (such as
inhibitor combinations, multi-target kinase inhibitors and HSP90 inhibitors) as a means of combating resistance. The promise and challenges facing each of these polytherapies are elaborated with a perspective on how to effectively deploy such therapies in patients. We
highlight efforts to harness computational approaches to predict effective polytherapies and the emerging view that exceptional responders may hold the key to better understanding drug resistance. This review underscores the importance of polytherapies as an effective
means of targeting resistance signalling networks and achieving durable clinical responses in the era of personalised cancer medicine..
Antoniou, G., Lee, A.T., Huang, P.H. & Jones, R.L.
(2018). Olaratumab in soft tissue sarcoma – Current status and future perspectives. European journal of cancer,
Luczynski, M.T., Harrison, P.T., Lima, N., Krasny, L., Paul, A. & Huang, P.H.
(2018). Spatial localisation of Discoidin Domain Receptor 2 (DDR2) signalling is dependent on its collagen binding and kinase activity. Biochemical and biophysical research communications,
Jenks, A.D., Vyse, S., Wong, J.P., Kostaras, E., Keller, D., Burgoyne, T., Shoemark, A., Tsalikis, A., de la Roche, M., Michaelis, M., et al.
(2018). Primary Cilia Mediate Diverse Kinase Inhibitor Resistance Mechanisms in Cancer. Cell reports,
Lee, A.T., Thway, K., Huang, P.H. & Jones, R.L.
(2018). Clinical and Molecular Spectrum of Liposarcoma. Journal of clinical oncology,
Krasny, L., Bland, P., Kogata, N., Wai, P., Howard, B.A., Natrajan, R.C. & Huang, P.H.
(2018). SWATH mass spectrometry as a tool for quantitative profiling of the matrisome. J proteomics,
Proteomic analysis of extracellular matrix (ECM) and ECM-associated proteins, collectively known as the matrisome, is a challenging task due to the inherent complexity and insolubility of these proteins. Here we present sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH MS) as a tool for the quantitative analysis of matrisomal proteins in both non-enriched and ECM enriched tissue without the need for prior fractionation. Utilising a spectral library containing 201 matrisomal proteins, we compared the performance and reproducibility of SWATH MS over conventional data-dependent analysis mass spectrometry (DDA MS) in unfractionated murine lung and liver. SWATH MS conferred a 15-20% increase in reproducible peptide identification across replicate experiments in both tissue types and identified 54% more matrisomal proteins in the liver versus DDA MS. We further use SWATH MS to evaluate the quantitative changes in matrisome content that accompanies ECM enrichment. Our data shows that ECM enrichment led to a systematic increase in core matrisomal proteins but resulted in significant losses in matrisome-associated proteins including the cathepsins and proteins of the S100 family. Our proof-of-principle study demonstrates the utility of SWATH MS as a versatile tool for in-depth characterisation of the matrisome in unfractionated and non-enriched tissues. SIGNIFICANCE: The matrisome is a complex network of extracellular matrix (ECM) and ECM-associated proteins that provides scaffolding function to tissues and plays important roles in the regulation of fundamental cellular processes. However, due to its inherent complexity and insolubility, proteomic studies of the matrisome typically require the application of enrichment workflows prior to MS analysis. Such enrichment strategies often lead to losses in soluble matrisome-associated components. In this study, we present sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH MS) as a tool for the quantitative analysis of matrisomal proteins. We show that SWATH MS provides a more reproducible coverage of the matrisome compared to data-dependent analysis (DDA) MS. We also demonstrate that SWATH MS is capable of accurate quantification of matrisomal proteins without prior ECM enrichment and fractionation, which may simplify sample handling workflows and avoid losses in matrisome-associated proteins commonly linked to ECM enrichment..
Tan, A.-., Vyse, S. & Huang, P.H.
(2017). Exploiting receptor tyrosine kinase co-activation for cancer therapy. Drug discovery today,
(2017). Targeting SWI/SNF mutant cancers with tyrosine kinase inhibitor therapy. Expert review of anticancer therapy,
Polychronidou, G., Karavasilis, V., Pollack, S.M., Huang, P.H., Lee, A. & Jones, R.L.
(2017). Novel therapeutic approaches in chondrosarcoma. Future oncology,
Lee, A.T., Pollack, S.M., Huang, P. & Jones, R.L.
(2017). Phase III Soft Tissue Sarcoma Trials: Success or Failure?. Current treatment options in oncology,
Vyse, S., Howitt, A. & Huang, P.H.
(2017). Exploiting Synthetic Lethality and Network Biology to Overcome EGFR Inhibitor Resistance in Lung Cancer. Journal of molecular biology,
Vyse, S., Desmond, H. & Huang, P.H.
(2017). Advances in mass spectrometry based strategies to study receptor tyrosine kinases. Iucrj,
Receptor tyrosine kinases (RTKs) are key transmembrane environmental sensors that are capable of transmitting extracellular information into Phenotypic responses, including cell proliferation, survival and metabolism. Advances in mass spectrometry (MS)-based phosphoproteomics have been instrumental in providing the foundations of much of our current understanding of RTK signalling networks and activation dynamics. Furthermore, new insights relating to the deregulation of RTKs in disease, for instance receptor coactivation and kinome reprogramming, have largely been identified using phosphoproteomic-based strategies. This review outlines the current approaches employed in phosphoproteomic workflows, including phosphopeptide enrichment and MS data-acquisition methods. Here, recent advances in the application of MS-based phosphoproteomics to bridge critical gaps in our knowledge of RTK signalling are focused on. The current limitations of the technology are discussed and emerging areas such as computational modelling, high-throughput phosphoproteomic workflows and next-generation single-cell approaches to further our understanding in new areas of RTK biology are highlighted..
Lima, N., Lee, A. & Huang, P.H.
(2017). Progress and impact of clinical phosphoproteomics on precision oncology. Translational cancer research,
Advances in phosphoproteomic technologies have enabled a step-change in the ability to make precise measurements of global protein phosphorylation states within tumours, providing critical information on the activation status of key oncogenic pathways driving disease. As an ever increasing number of molecularly targeted drugs enact their effect through modulation of such pathways, translational phosphoproteomic studies represent an avenue for biomarker discovery that provides insight into the direct functional impact of drug treatment whilst uncovering previously unrecognised mechanisms of drug response and resistance. Array- and mass spectrometry (MS)-based techniques allow for the characterisation of hundreds to thousands of phosphorylation sites that in principle can be readily integrated with genomic and transcriptomic datasets to produce highly detailed molecular portraits of human cancers. However, in practice, the routine application of phosphoproteomics to clinically-derived material has been limited by factors relating to sample integrity, assay resolution and computational challenges. In this perspective, we highlight a number of recent studies that have applied phosphoproteomics to the analysis of tumour specimens and discuss their impact on our knowledge of dynamic pathway biology and response to targeted therapies. We provide examples of pioneering studies that have integrated phosphoproteomic data with genomic methods to demonstrate the utility of these approaches in identifying clinically important biology for applications in personalised medicine. Collectively, these studies suggest that the increasing use of phosphoproteomics in translational precision oncology holds the promise of contributing to practice-changing discoveries over the coming decade..
Payne, L.S. & Huang, P.H.
(2017). Targeted Analysis of Phosphotyrosine Signaling by Multiple Reaction Monitoring Mass Spectrometry. ,
Broncel, M. & Huang, P.H.
(2017). Analysis of Phosphotyrosine Signaling Networks in Lung Cancer Cell Lines. ,
Lee, A., Huang, P., DeMatteo, R.P. & Pollack, S.M.
(2016). Immunotherapy for Soft Tissue Sarcoma: Tomorrow Is Only a Day Away. American society of clinical oncology educational book. american society of clinical oncology. meeting,
Despite the advances taking place for patients with many types of cancer, to date there has been little success in meeting the great need for novel treatments of advanced soft tissue sarcoma with effective immunologic therapies. Here, we review recent clinical and preclinical data that indicate immune responses against sarcomas occur spontaneously and can also be successfully provoked. Efforts to manipulate the sarcoma immune microenvironment have the potential to eradicate disease and may also sensitize tumors to other tumor-targeted immunotherapeutic approaches. Other approaches, including vaccines and genetic engineering of T cells, offer a promising opportunity to actively direct cytotoxic lymphocytes toward antigen-bearing sarcomas. Drawing parallels with recent advances made in other cancer types, we identify ways in which sarcomas can be included in the ongoing immunotherapy revolution..
Krasny, L., Paul, A., Wai, P., Howard, B.A., Natrajan, R.C. & Huang, P.H.
(2016). Comparative proteomic assessment of matrisome enrichment methodologies. Biochemical journal,
The matrisome is a complex and heterogeneous collection of extracellular matrix (ECM) and ECM-associated proteins that play important roles in tissue development and homeostasis. While several strategies for matrisome enrichment have been developed, it is currently unknown how the performance of these different methodologies compares in the proteomic identification of matrisome components across multiple tissue types. In the present study, we perform a comparative proteomic assessment of two widely used decellularisation protocols and two extraction methods to characterise the matrisome in four murine organs (heart, mammary gland, lung and liver). We undertook a systematic evaluation of the performance of the individual methods on protein yield, matrisome enrichment capability and the ability to isolate core matrisome and matrisome-associated components. Our data find that sodium dodecyl sulphate (SDS) decellularisation leads to the highest matrisome enrichment efficiency, while the extraction protocol that comprises chemical and trypsin digestion of the ECM fraction consistently identifies the highest number of matrisomal proteins across all types of tissue examined. Matrisome enrichment had a clear benefit over non-enriched tissue for the comprehensive identification of matrisomal components in murine liver and heart. Strikingly, we find that all four matrisome enrichment methods led to significant losses in the soluble matrisome-associated proteins across all organs. Our findings highlight the multiple factors (including tissue type, matrisome class of interest and desired enrichment purity) that influence the choice of enrichment methodology, and we anticipate that these data will serve as a useful guide for the design of future proteomic studies of the matrisome..
Noujaim, J., Payne, L.S., Judson, I., Jones, R.L. & Huang, P.H.
(2016). Phosphoproteomics in translational research: a sarcoma perspective. Annals of oncology,
Wong, J.P., Todd, J.R., Finetti, M.A., McCarthy, F., Broncel, M., Vyse, S., Luczynski, M.T., Crosier, S., Ryall, K.A., Holmes, K., et al.
(2016). Dual Targeting of PDGFRα and FGFR1 Displays Synergistic Efficacy in Malignant Rhabdoid Tumors. Cell reports,
Maguire, S.L., Peck, B., Wai, P.T., Campbell, J., Barker, H., Gulati, A., Daley, F., Vyse, S., Huang, P., Lord, C.J., et al.
(2016). Three-dimensional modelling identifies novel genetic dependencies associated with breast cancer progression in the isogenic MCF10 model. The journal of pathology,
Lee, A., Huang, P.H., Pollack, S.M. & Jones, R.L.
(2016). Drug Repositioning in Sarcomas and Other Rare Tumors. Ebiomedicine,
Corallino, S., Iwai, L.K., Payne, L.S., Huang, P.H., Sacco, F., Cesareni, G. & Castagnoli, L.
(2016). Alterations in the phosphoproteomic profile of cells expressing a non-functional form of the SHP2 phosphatase. N biotechnol,
(5 Pt A),
The phosphatase SHP-2 plays an essential role in growth factor signaling and mutations in its locus is the cause of congenital and acquired pathologies. Mutations of SHP-2 are known to affect the activation of the RAS pathway. Gain-of-function mutations cause the Noonan syndrome, the most common non-chromosomal congenital disorder. In order to obtain a holistic picture of the intricate regulatory mechanisms underlying SHP-2 physiology and pathology, we set out to characterize perturbations of the cell phosphorylation profile caused by an altered localization of SHP-2. To describe the proteins whose activity may be directly or indirectly modulated by SHP-2 activity, we identified tyrosine peptides that are differentially phosphorylated in wild type SHP-2 cells and isogenic cells expressing a non-functional SHP-2 variant that cannot dephosphorylate the physiological substrates due to a defect in cellular localization upon growth factor stimulation. By an iTRAQ based strategy coupled to mass spectrometry, we have identified 63 phosphorylated tyrosine residues in 53 different proteins whose phosphorylation is affected by SHP-2 activity. Some of these confirm already established regulatory mechanisms while many others suggest new possible signaling routes that may contribute to the modulation of the ERK and p38 pathways by SHP-2. Interestingly many new proteins that we found to be regulated by SHP-2 activity are implicated in the formation and regulation of focal adhesions. .
Todd, J.R., Ryall, K.A., Vyse, S., Wong, J.P., Natrajan, R.C., Yuan, Y., Tan, A.-. & Huang, P.H.
(2016). Systematic analysis of tumour cell-extracellular matrix adhesion identifies independent prognostic factors in breast cancer. Oncotarget,
Tumour cell-extracellular matrix (ECM) interactions are fundamental for discrete steps in breast cancer progression. In particular, cancer cell adhesion to ECM proteins present in the microenvironment is critical for accelerating tumour growth and facilitating metastatic spread. To assess the utility of tumour cell-ECM adhesion as a means for discovering prognostic factors in breast cancer survival, here we perform a systematic phenotypic screen and characterise the adhesion properties of a panel of human HER2 amplified breast cancer cell lines across six ECM proteins commonly deregulated in breast cancer. We determine a gene expression signature that defines a subset of cell lines displaying impaired adhesion to laminin. Cells with impaired laminin adhesion showed an enrichment in genes associated with cell motility and molecular pathways linked to cytokine signalling and inflammation. Evaluation of this gene set in the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort of 1,964 patients identifies the F12 and STC2 genes as independent prognostic factors for overall survival in breast cancer. Our study demonstrates the potential of in vitro cell adhesion screens as a novel approach for identifying prognostic factors for disease outcome..
Tan, A.C., Ryall, K.A. & Huang, P.H.
(2016). Expanding the computational toolbox for interrogating cancer kinomes. Pharmacogenomics,
Huang, P.H., Cook, R., Zoumpoulidou, G., Luczynski, M.T. & Mittnacht, S.
(2015). Retinoblastoma family proteins: new players in DNA repair by non-homologous end-joining. Molecular and cellular oncology,
Loss of Retinoblastoma protein (RB1) function is a major driver in cancer
development. We have recently reported that in addition to its well-documented functions in cell cycle and fate control, RB1 and its paralogs have a novel role in regulating DNA repair by non-homologous end-joining (NHEJ). Here we summarise our findings and present mechanistic hypotheses on how RB1 may support the DNA repair process and the therapeutic implications for patients that harbour RB1
Huang, P.H., Cook, R. & Mittnacht, S.
(2015). RB in DNA repair. Oncotarget,
Cook, R., Zoumpoulidou, G., Luczynski, M.T., Rieger, S., Moquet, J., Spanswick, V.J., Hartley, J.A., Rothkamm, K., Huang, P.H. & Mittnacht, S., et al.
(2015). Direct involvement of retinoblastoma family proteins in DNA repair by non-homologous end-joining. Cell rep,
Deficiencies in DNA double-strand break (DSB) repair lead to genetic instability, a recognized cause of cancer initiation and evolution. We report that the retinoblastoma tumor suppressor protein (RB1) is required for DNA DSB repair by canonical non-homologous end-joining (cNHEJ). Support of cNHEJ involves a mechanism independent of RB1's cell-cycle function and depends on its amino terminal domain with which it binds to NHEJ components XRCC5 and XRCC6. Cells with engineered loss of RB family function as well as cancer-derived cells with mutational RB1 loss show substantially reduced levels of cNHEJ. RB1 variants disabled for the interaction with XRCC5 and XRCC6, including a cancer-associated variant, are unable to support cNHEJ despite being able to confer cell-cycle control. Our data identify RB1 loss as a candidate driver of structural genomic instability and a causative factor for cancer somatic heterogeneity and evolution. .
Payne, L.S. & Huang, P.H.
(2014). Discoidin domain receptor 2 signaling networks and therapy in lung cancer. J thorac oncol,
Discoidin domain receptor 2 (DDR2) is an atypical receptor tyrosine kinase that binds to and is activated by collagen in the extracellular matrix. Recent exon sequencing studies have identified DDR2 to be mutated with a 3% to 4% incidence in squamous cell cancers of the lung. This article summarizes the current state of knowledge of DDR2 biology and signaling in lung squamous cell cancer. It also explores the context-dependent role of this receptor as both an oncogene and a tumor suppressor in cancer cells. Promising therapeutic opportunities based on existing and novel targeted small molecule inhibitors against DDR2 may provide new strategies for treating lung squamous cell cancer patients. .
Iwai, L.K., Luczynski, M.T. & Huang, P.H.
(2014). Discoidin domain receptors: a proteomic portrait. Cell mol life sci,
The discoidin domain receptors (DDRs) are collagen-binding receptor tyrosine kinases that have been implicated in a number of fundamental biological processes ranging from growth and development to immunoregulation. In this review, we examine how recent proteomic technologies have enriched our understanding of DDR signaling mechanisms. We provide an overview on the use of large-scale proteomic profiling and chemical proteomics to reveal novel insights into DDR therapeutics, signaling networks, and receptor crosstalk. A perspective of how proteomics may be harnessed to answer outstanding fundamental questions including the dynamic regulation of receptor activation kinetics is presented. Collectively, these studies present an emerging molecular portrait of these unique receptors and their functional role in health and disease. .
Fu, H.-., Valiathan, R.R., Payne, L., Kumarasiri, M., Mahasenan, K.V., Mobashery, S., Huang, P. & Fridman, R.
(2014). Glycosylation at Asn211 regulates the activation state of the discoidin domain receptor 1 (DDR1). J biol chem,
Discoidin domain receptor 1 (DDR1) belongs to a unique family of receptor tyrosine kinases that signal in response to collagens. DDR1 undergoes autophosphorylation in response to collagen binding with a slow and sustained kinetics that is unique among members of the receptor tyrosine kinase family. DDR1 dimerization precedes receptor activation suggesting a structural inhibitory mechanism to prevent unwarranted phosphorylation. However, the mechanism(s) that maintains the autoinhibitory state of the DDR1 dimers is unknown. Here, we report that N-glycosylation at the Asn(211) residue plays a unique role in the control of DDR1 dimerization and autophosphorylation. Using site-directed mutagenesis, we found that mutations that disrupt the conserved (211)NDS N-glycosylation motif, but not other N-glycosylation sites (Asn(260), Asn(371), and Asn(394)), result in collagen I-independent constitutive phosphorylation. Mass spectrometry revealed that the N211Q mutant undergoes phosphorylation at Tyr(484), Tyr(520), Tyr(792), and Tyr(797). The N211Q traffics to the cell surface, and its ectodomain displays collagen I binding with an affinity similar to that of the wild-type DDR1 ectodomain. However, unlike the wild-type receptor, the N211Q mutant exhibits enhanced receptor dimerization and sustained activation upon ligand withdrawal. Taken together, these data suggest that N-glycosylation at the highly conserved (211)NDS motif evolved to act as a negative repressor of DDR1 phosphorylation in the absence of ligand. The presence of glycan moieties at that site may help to lock the collagen-binding domain in the inactive state and prevent unwarranted signaling by receptor dimers. These studies provide a novel insight into the structural mechanisms that regulate DDR activation. .
Iwai, L.K., Chang, F. & Huang, P.H.
(2013). Phosphoproteomic analysis identifies insulin enhancement of discoidin domain receptor 2 phosphorylation. Cell adh migr,
The discoidin domain receptors (DDRs) are collagen binding receptor tyrosine kinases that play important roles in cell migration, invasion and adhesion. Crosstalk between growth factor signaling and components of the extracellular matrix are drivers of cellular function but the integrated signaling networks downstream of such crosstalk events have not been extensively characterized. In this report, we have employed mass spectrometry-based quantitative phosphotyrosine analysis to identify crosstalk between DDR2 and the insulin receptor. Our phosphoproteomic analysis reveals a cluster of phosphorylation sites in which collagen and insulin cooperate to enhance phosphotyrosine levels. Importantly, Y740 on the DDR2 catalytic loop was found in this cluster indicating that insulin acts to promote collagen I signaling by increasing the activity of DDR2. Furthermore, we identify two additional migration associated proteins that are candidate substrates downstream of DDR2 activation. Our data suggests that insulin promotes collagen I signaling through the upregulation of DDR2 phosphorylation which may have important consequences in DDR2 function in health and disease..
Payne, L.S. & Huang, P.H.
(2013). The pathobiology of collagens in glioma. Mol cancer res,
Malignant gliomas are characterized by a diffuse infiltration into the surrounding brain parenchyma. Infiltrating glioma cells exist in close proximity with components of the tumor microenvironment, including the extracellular matrix (ECM). Whereas levels of collagens in the normal adult brain are low, in glioma, collagen levels are elevated and play a vital role in driving tumor progression. This article provides a comprehensive overview of the nature of collagens found in gliomas and offers unique insight into the mechanisms by which cancer cells interact with this ECM via cellular factors such as integrins, discoidin domain receptors, and mannose receptors. Also discussed are the major remodeling pathways of brain tumor collagen, mediated primarily by matrix metalloproteinases, and the reciprocal relationship between these enzymes and the collagen receptors. Finally, a concluding perspective is offered on how the biophysical properties of the collagen ECM, in particular, mechanical stiffness and compliance, influence malignant outcome. A better understanding of the complex molecular interactions between glioma cells and the collagen ECM will provide new avenues to combat the rampant tumor progression and chemoresistance in brain cancer patients..
Fu, H.-., Valiathan, R.R., Arkwright, R., Sohail, A., Mihai, C., Kumarasiri, M., Mahasenan, K.V., Mobashery, S., Huang, P., Agarwal, G., et al.
(2013). Discoidin domain receptors: unique receptor tyrosine kinases in collagen-mediated signaling. J biol chem,
The discoidin domain receptors (DDRs) are receptor tyrosine kinases that recognize collagens as their ligands. DDRs display unique structural features and distinctive activation kinetics, which set them apart from other members of the kinase superfamily. DDRs regulate cell-collagen interactions in normal and pathological conditions and thus are emerging as major sensors of collagen matrices and potential novel therapeutic targets. New structural and biological information has shed light on the molecular mechanisms that regulate DDR signaling, turnover, and function. This minireview provides an overview of these areas of DDR research with the goal of fostering further investigation of these intriguing and unique receptors..
Iwai, L.K., Payne, L.S., Luczynski, M.T., Chang, F., Xu, H., Clinton, R.W., Paul, A., Esposito, E.A., Gridley, S., Leitinger, B., et al.
(2013). Phosphoproteomics of collagen receptor networks reveals SHP-2 phosphorylation downstream of wild-type DDR2 and its lung cancer mutants. Biochem j,
Collagen is an important extracellular matrix component that directs many fundamental cellular processes including differentiation, proliferation and motility. The signalling networks driving these processes are propagated by collagen receptors such as the β1 integrins and the DDRs (discoidin domain receptors). To gain an insight into the molecular mechanisms of collagen receptor signalling, we have performed a quantitative analysis of the phosphorylation networks downstream of collagen activation of integrins and DDR2. Temporal analysis over seven time points identified 424 phosphorylated proteins. Distinct DDR2 tyrosine phosphorylation sites displayed unique temporal activation profiles in agreement with in vitro kinase data. Multiple clustering analysis of the phosphoproteomic data revealed several DDR2 candidate downstream signalling nodes, including SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), NCK1 (non-catalytic region of tyrosine kinase adaptor protein 1), LYN, SHIP-2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 2], PIK3C2A (phosphatidylinositol-4-phosphate 3-kinase, catalytic subunit type 2α) and PLCL2 (phospholipase C-like 2). Biochemical validation showed that SHP-2 tyrosine phosphorylation is dependent on DDR2 kinase activity. Targeted proteomic profiling of a panel of lung SCC (squamous cell carcinoma) DDR2 mutants demonstrated that SHP-2 is tyrosine-phosphorylated by the L63V and G505S mutants. In contrast, the I638F kinase domain mutant exhibited diminished DDR2 and SHP-2 tyrosine phosphorylation levels which have an inverse relationship with clonogenic potential. Taken together, the results of the present study indicate that SHP-2 is a key signalling node downstream of the DDR2 receptor which may have therapeutic implications in a subset of DDR2 mutations recently uncovered in genome-wide lung SCC sequencing screens..
(2012). Phosphoproteomic studies of receptor tyrosine kinases: future perspectives. Mol biosyst,
In the last decade, large-scale mass spectrometry-based phosphoproteomic studies of receptor tyrosine kinases (RTKs) have generated a compendium of signalling networks that are activated downstream of these receptors. In this article, a brief summary of previous phosphoproteomic studies on epidermal growth factor receptor (EGFR) signalling will be presented together with a perspective on the importance for the field to keep pace with new advances in RTK biology. Using examples drawn primarily from studies on the EGFR, c-Met and Flt3 receptors, areas in RTK biology which will greatly benefit from the power of phosphoproteomics will be discussed, including (a) validating oncogenic RTK mutants identified in cancer genome sequencing efforts, (b) spatial RTK signalling networks and (c) understanding crosstalk and co-activation between members of the RTK superfamily..
Hedgethorne, K. & Huang, P.H.
(2012). Dacomitinib Pan-ErbB inhibitor, Oncolytic. Drugs of the future,
Xu, H., Bihan, D., Chang, F., Huang, P.H., Farndale, R.W. & Leitinger, B.
(2012). Discoidin domain receptors promote α1β1- and α2β1-integrin mediated cell adhesion to collagen by enhancing integrin activation. Plos one,
The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that bind to and are activated by collagens. Similar to collagen-binding β1 integrins, the DDRs bind to specific motifs within the collagen triple helix. However, these two types of collagen receptors recognize distinct collagen sequences. While GVMGFO (O is hydroxyproline) functions as a major DDR binding motif in fibrillar collagens, integrins bind to sequences containing Gxx'GEx". The DDRs are thought to regulate cell adhesion, but their roles have hitherto only been studied indirectly. In this study we used synthetic triple-helical collagen-derived peptides that incorporate either the DDR-selective GVMGFO motif or integrin-selective motifs, such as GxOGER and GLOGEN, in order to selectively target either type of receptor and resolve their contributions to cell adhesion. Our data using HEK293 cells show that while cell adhesion to collagen I was completely inhibited by anti-integrin blocking antibodies, the DDRs could mediate cell attachment to the GVMGFO motif in an integrin-independent manner. Cell binding to GVMGFO was independent of DDR receptor signalling and occurred with limited cell spreading, indicating that the DDRs do not mediate firm adhesion. However, blocking the interaction of DDR-expressing cells with collagen I via the GVMGFO site diminished cell adhesion, suggesting that the DDRs positively modulate integrin-mediated cell adhesion. Indeed, overexpression of the DDRs or activation of the DDRs by the GVMGFO ligand promoted α1β1 and α2β1 integrin-mediated cell adhesion to medium- and low-affinity integrin ligands without regulating the cell surface expression levels of α1β1 or α2β1. Our data thus demonstrate an adhesion-promoting role of the DDRs, whereby overexpression and/or activation of the DDRs leads to enhanced integrin-mediated cell adhesion as a result of higher integrin activation state..
Suwaki, N., Vanhecke, E., Atkins, K.M., Graf, M., Swabey, K., Huang, P., Schraml, P., Moch, H., Cassidy, A.M., Brewer, D., et al.
(2011). A HIF-regulated VHL-PTP1B-Src signaling axis identifies a therapeutic target in renal cell carcinoma. Sci transl med,
Metastatic renal cell carcinoma (RCC) is a molecularly heterogeneous disease that is intrinsically resistant to chemotherapy and radiotherapy. Although therapies targeted to the molecules vascular endothelial growth factor and mammalian target of rapamycin have shown clinical effectiveness, their effects are variable and short-lived, underscoring the need for improved treatment strategies for RCC. Here, we used quantitative phosphoproteomics and immunohistochemical profiling of 346 RCC specimens and determined that Src kinase signaling is elevated in RCC cells that retain wild-type von Hippel-Lindau (VHL) protein expression. RCC cell lines and xenografts with wild-type VHL exhibited sensitivity to the Src inhibitor dasatinib, in contrast to cell lines that lacked the VHL protein, which were resistant. Forced expression of hypoxia-inducible factor (HIF) in RCC cells with wild-type VHL diminished Src signaling output by repressing transcription of the Src activator protein tyrosine phosphatase 1B (PTP1B), conferring resistance to dasatinib. Our results suggest that a HIF-regulated VHL-PTP1B-Src signaling pathway determines the sensitivity of RCC to Src inhibitors and that stratification of RCC patients with antibody-based profiling may identify patients likely to respond to Src inhibitors in RCC clinical trials..
Huang, P.H., Miraldi, E.R., Xu, A.M., Kundukulam, V.A., Del Rosario, A.M., Flynn, R.A., Cavenee, W.K., Furnari, F.B. & White, F.M.
(2010). Phosphotyrosine signaling analysis of site-specific mutations on EGFRvIII identifies determinants governing glioblastoma cell growth. Mol biosyst,
To evaluate the role of individual EGFR phosphorylation sites in activating components of the cellular signaling network we have performed a mass spectrometry-based analysis of the phosphotyrosine network downstream of site-specific EGFRvIII mutants, enabling quantification of network-level effects of site-specific point mutations. Mutation at Y845, Y1068 or Y1148 resulted in diminished receptor phosphorylation, while mutation at Y1173 led to increased phosphorylation on multiple EGFRvIII residues. Altered phosphorylation at the receptor was recapitulated in downstream signaling network activation levels, with Y1173F mutation leading to increased phosphorylation throughout the network. Computational modeling of GBM cell growth as a function of network phosphorylation levels highlights the Erk pathway as crucial for regulating EGFRvIII-driven U87MG GBM cell behavior, with the unexpected finding that Erk1/2 is negatively correlated to GBM cell growth. Genetic manipulation of this pathway supports the model, demonstrating that EGFRvIII-expressing U87MG GBM cells are sensitive to Erk activation levels. Additionally, we developed a model describing glioblastoma cell growth based on a reduced set of phosphoproteins, which represent potential candidates for future development as therapeutic targets for EGFRvIII-positive glioblastoma patients..
Hedgethorne, K. & Huang, P.H.
(2010). FORETINIB. Drug future,
The c-Met and vascular endothelial growth factor receptor (VEGFR) tyrosine kinases are activated in multiple cancer types and drive a variety of cancer-associated processes, including tumor cell proliferation, invasion, metastasis and angiogenesis. These receptor tyrosine kinases (RTKs) are therefore attractive therapeutic drug targets for a wide range of tumor types. Foretinib is a broadly specific tyrosine kinase inhibitor that targets a spectrum of RTKs, including c-Met and VEGFR-2. It has shown potent preclinical antitumor activity in the laboratory setting, primarily through the reduction of tumor growth and angiogenesis in animal models. Phase I trials have shown foretinib to be well tolerated at clinically active levels when administered once daily on the first 5 days of a 14-day cycle, with the majority of treatment-related adverse responses reversed by either a scheduled break in treatment or a decrease in dose. The recommended dose has been defined as 240 mg and this dose is currently being used in phase II clinical trials in multiple tumor types following the same dosing regimen..
Xu, A.M. & Huang, P.H.
(2010). Receptor tyrosine kinase coactivation networks in cancer. Cancer res,
Cancer cells employ multiple mechanisms to evade tightly regulated cellular processes such as proliferation, apoptosis, and senescence. Systems-wide analyses of tumors have recently identified receptor tyrosine kinase (RTK) coactivation as an important mechanism by which cancer cells achieve chemoresistance. This mini-review discusses our current understanding of the complex and dynamic process of RTK coactivation. We highlight how systems biology and computational modeling have been employed to predict integrated signaling outcomes and cancer phenotypes downstream of RTK coactivation. We conclude by providing an outlook on the feasibility of targeting RTK networks to overcome chemoresistance in cancer..
Pines, G., Huang, P.H., Zwang, Y., White, F.M. & Yarden, Y.
(2010). EGFRvIV: a previously uncharacterized oncogenic mutant reveals a kinase autoinhibitory mechanism. Oncogene,
Tumor cells often subvert normal regulatory mechanisms of signal transduction. This study shows this principle by studying yet uncharacterized mutants of the epidermal growth factor receptor (EGFR) previously identified in glioblastoma multiforme, which is the most aggressive brain tumor in adults. Unlike the well-characterized EGFRvIII mutant form, which lacks a portion of the ligand-binding cleft within the extracellular domain, EGFRvIVa and EGFRvIVb lack internal segments distal to the intracellular tyrosine kinase domain. By constructing the mutants and by ectopic expression in naive cells, we show that both mutants confer an oncogenic potential in vitro, as well as tumorigenic growth in animals. The underlying mechanisms entail constitutive receptor dimerization and basal activation of the kinase domain, likely through a mechanism that relieves a restraining molecular fold, along with stabilization due to association with HSP90. Phosphoproteomic analyses delineated the signaling pathways preferentially engaged by EGFRvIVb-identified unique substrates. This information, along with remarkable sensitivities to tyrosine kinase blockers and to a chaperone inhibitor, proposes strategies for pharmacological interception in brain tumors harboring EGFRvIV mutations..
Joughin, B.A., Naegle, K.M., Huang, P.H., Yaffe, M.B., Lauffenburger, D.A. & White, F.M.
(2009). An integrated comparative phosphoproteomic and bioinformatic approach reveals a novel class of MPM-2 motifs upregulated in EGFRvIII-expressing glioblastoma cells. Mol biosyst,
Glioblastoma (GBM, WHO grade IV) is an aggressively proliferative and invasive brain tumor that carries a poor clinical prognosis with a median survival of 9 to 12 months. In a prior phosphoproteomic study performed in the U87MG glioblastoma cell line, we identified tyrosine phosphorylation events that are regulated as a result of titrating EGFRvIII, a constitutively active mutant of the epidermal growth factor receptor (EGFR) associated with poor prognosis in GBM patients. In the present study, we have used the phosphoserine/phosphothreonine-specific antibody MPM-2 (mitotic protein monoclonal #2) to quantify serine/threonine phosphorylation events in the same cell lines. By employing a bioinformatic tool to identify amino acid sequence motifs regulated in response to increasing oncogene levels, a set of previously undescribed MPM-2 epitope sequence motifs orthogonal to the canonical "pS/pT-P" motif was identified. These motifs contain acidic amino acids in combinations of the -5, -2, +1, +3, and +5 positions relative to the phosphorylated amino acid. Phosphopeptides containing these motifs are upregulated in cells expressing EGFRvIII, raising the possibility of a general role for a previously unrecognized acidophilic kinase (e.g. casein kinase II (CK2)) in cell proliferation downstream of EGFR signaling..
Huang, P.H., Xu, A.M. & White, F.M.
(2009). Oncogenic EGFR signaling networks in glioma. Sci signal,
The epidermal growth factor receptor (EGFR) is a primary contributor to glioblastoma (GBM) initiation and progression. Here, we examine how EGFR and key downstream signaling networks contribute to the hallmark characteristics of GBM such as rapid cancer cell proliferation and diffused invasion. Additionally, we discuss current therapeutic options for GBM patients and elaborate on the mechanisms through which EGFR promotes chemoresistance. We conclude by offering a perspective on how the potential of integrative systems biology may be harnessed to develop safe and effective treatment strategies for this disease..
Huang, P.H. & Marais, R.
(2009). Cancer: Melanoma troops massed. Nature,
Huang, P.H. & White, F.M.
(2008). Phosphoproteomics: unraveling the signaling web. Mol cell,
In recent years, phosphoproteomic technologies have increased our understanding of cellular signaling networks. Here, we frame recent phosphoproteomics-based advances in the context of the DNA damage response and ErbB receptor family signaling and offer a perspective on how the molecular insights arising from the integration of such proteomic approaches might be used for clinical applications..
Wilker, E.W., van Vugt, M.A., Artim, S.A., Huang, P.H., Petersen, C.P., Reinhardt, H.C., Feng, Y., Sharp, P.A., Sonenberg, N., White, F.M., et al.
(2007). 14-3-3sigma controls mitotic translation to facilitate cytokinesis. Nature,
14-3-3 proteins are crucial in a wide variety of cellular responses including cell cycle progression, DNA damage checkpoints and apoptosis. One particular 14-3-3 isoform, sigma, is a p53-responsive gene, the function of which is frequently lost in human tumours, including breast and prostate cancers as a result of either hypermethylation of the 14-3-3sigma promoter or induction of an oestrogen-responsive ubiquitin ligase that specifically targets 14-3-3sigma for proteasomal degradation. Loss of 14-3-3sigma protein occurs not only within the tumours themselves but also in the surrounding pre-dysplastic tissue (so-called field cancerization), indicating that 14-3-3sigma might have an important tumour suppressor function that becomes lost early in the process of tumour evolution. The molecular basis for the tumour suppressor function of 14-3-3sigma is unknown. Here we report a previously unknown function for 14-3-3sigma as a regulator of mitotic translation through its direct mitosis-specific binding to a variety of translation/initiation factors, including eukaryotic initiation factor 4B in a stoichiometric manner. Cells lacking 14-3-3sigma, in marked contrast to normal cells, cannot suppress cap-dependent translation and do not stimulate cap-independent translation during and immediately after mitosis. This defective switch in the mechanism of translation results in reduced mitotic-specific expression of the endogenous internal ribosomal entry site (IRES)-dependent form of the cyclin-dependent kinase Cdk11 (p58 PITSLRE), leading to impaired cytokinesis, loss of Polo-like kinase-1 at the midbody, and the accumulation of binucleate cells. The aberrant mitotic phenotype of 14-3-3sigma-depleted cells can be rescued by forced expression of p58 PITSLRE or by extinguishing cap-dependent translation and increasing cap-independent translation during mitosis by using rapamycin. Our findings show how aberrant mitotic translation in the absence of 14-3-3sigma impairs mitotic exit to generate binucleate cells and provides a potential explanation of how 14-3-3sigma-deficient cells may progress on the path to aneuploidy and tumorigenesis..
Huang, P.H., Cavenee, W.K., Furnari, F.B. & White, F.M.
(2007). Uncovering therapeutic targets for glioblastoma: a systems biology approach. Cell cycle,
Even though glioblastoma, WHO grade IV (GBM) is one of the most devastating adult cancers, current treatment regimens have not led to any improvements in patient life expectancy or quality of life. The constitutively active EGFRvIII receptor is one of the most commonly mutated proteins in GBM and has been linked to radiation and chemotherapeutic resistance. To define the mechanisms by which this protein alters cell physiology, we have recently performed a phosphoproteomic analysis of EGFRvIII signaling networks in GBM cells. The results of this study provided important insights into the biology of this mutated receptor, including oncogene dose effects and differential utilization of signaling pathways. Moreover, clustering of the phosphoproteomic data set revealed a previously undescribed crosstalk between EGFRvIII and the c-Met receptor. Treatment of the cells with a combination employing both EGFR and c-Met kinase inhibitors dramatically decreased cell viability in vitro. In this perspective, we highlight the use of systems biology as a tool to better understand the molecular basis of GBM tumor biology as well as to uncover non-intuitive candidates for therapeutic target validation..
Huang, P.H., Mukasa, A., Bonavia, R., Flynn, R.A., Brewer, Z.E., Cavenee, W.K., Furnari, F.B. & White, F.M.
(2007). Quantitative analysis of EGFRvIII cellular signaling networks reveals a combinatorial therapeutic strategy for glioblastoma. Proc natl acad sci u s a,
Glioblastoma multiforme (GBM) is the most aggressive brain tumor in adults and remains incurable despite multimodal intensive treatment regimens. EGFRvIII is a truncated extracellular mutant of the EGF receptor (EGFR) commonly found in GBMs that confers enhanced tumorigenic behavior. To gain a molecular understanding of the mechanisms by which EGFRvIII acts, we have performed a large-scale analysis of EGFRvIII-activated phosphotyrosine-mediated signaling pathways and thereby have identified and quantified 99 phosphorylation sites on 69 proteins. Distinct signaling responses were observed as a function of titrated EGFRvIII receptor levels with the phosphatidylinositol 3-kinase pathway being dominant over the MAPK and STAT3 pathways at a high level of EGFRvIII expression. Within this data set, the activating phosphorylation site on the c-Met receptor was found to be highly responsive to EGFRvIII levels, indicating cross-activation of the c-Met receptor tyrosine kinase by EGFRvIII. To determine the significance of this finding, we devised a combined treatment regimen that used a c-Met kinase inhibitor and either an EGFR kinase inhibitor or cisplatin. This regimen resulted in enhanced cytotoxicity of EGFRvIII-expressing cells compared with treatment with either compound alone. These results suggest that the clinical use of c-Met kinase inhibitors in combination with either EGFR inhibitors or standard chemotherapeutics might represent a previously undescribed therapeutic approach to overcome the observed chemoresistance in patients with GBMs expressing EGFRvIII..
Wilding, C.P., Elms, M.L., Judson, I., Tan, A.-., Jones, R.L. & Huang, P.H.
The landscape of tyrosine kinase inhibitors in sarcomas: looking beyond pazopanib. Expert review of anticancer therapy,