Abstract We previously observed aberrant activation of NF-kB in head and neck squamous cell carcinoma (HNSCC), however, the signaling constitutively activating NF-kB in solid cancers has not been well defined. Recently, The Cancer Genome Atlas (TCGA) project has investigated 279 HNSCC tissue specimens, and uncovered significant genomic alterations of key molecules involved in inflammation, NF-kB, and death pathways. These genetic alterations include amplification of FADD and BIRC2/3, mutations of caspase 8 and RIPKs in HPV(-), or deletion of TRAF3 in HPV(+) HNSCC tissues. Using bioinformatics analyses and protein structural and interactive tools, we identified ∼60 proteins that potentially interact with these genetically altered molecules. More than 90% HNSCC tissues studied in TCGA exhibited expression or genetic alterations of these genes, of which FADD amplification and/or overexpression ranked first with 37%. We searched this group of genes among more than 20 major cancer types investigated by TCGA, among which HNSCC ranked the highest for these alterations. To further investigate and identify experimental models for functional studies of these genetic and phenotypic alterations, we have performed RNA-seq and exome DNA-seq in 15 HPV(-) and 11 HPV(+) HNSCC lines, and compared them with three normal human oral mucosa lines and 8 matched blood samples. Among the top 30 genes identified from TCGA with the alteration rate greater than 8% in cancer tissues, we found consistent expression patterns for ∼57% molecules in our cell lines. To further test the function of these molecules, we established HNSCC cell lines stably transfected with a vector that contains NF-kB transcription factor response elements upstream of the α-lactamase reporter gene (Gene BLAzer). Using these NF-kB reporter cell lines, genome-wide RNAi screening assays have been performed and the regulatory and signaling molecules involved in NF-κB and death pathways in responding to TNF-α have been identified. We further validated the screening results by knockdown of 35 genes individually using two siRNAs for each genes, and found that knockdown of 16 genes significantly modulated TNF-α and Lymphotoxin β (LTβ) induced NF-κB activity and/or cell survival. We observed that TNF-α and LTβ cross-activate classical and alternative NF-κB pathways and regulate key molecules that serve differential roles in proliferation, survival and migration in HNSCC. Our data help define key molecules modulating aberrant classical and alternative NF-kB activation, as candidate molecular biomarkers for diagnosis and prognosis and targets for further preclinical and clinical investigation in HNSCC. (Supported by NIDCD/NIH intramural projects ZIA-DC-000016, 73, 74; and NCI/NIH, under contract number HHSN261200800001E). Citation Format: Hui Cheng, Xinping Yang, Anthony Saleh, Shaleeka Cornelius, Carter Van Waes, Zhong Chen, Emine Guven-Maiorov, Ozlem Keskin, Attila Gursoy, Ruth Nussinov. RNA-seq, exome-seq, functional RNAi screening and bioinformatics analyses identify molecules promoting aberrant activation of classical and alternative NF-kB pathways in head and neck cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 81.
Abstract Activated Toll-like receptors (TLRs) cluster in lipid rafts and induce pro- and anti-tumor responses. The organization of the assembly is critical to the understanding of how these key receptors control major signaling pathways in the cell. Although several models for individual interactions were proposed, the entire TIR-domain signalosome architecture has not been worked out, possibly due to its complexity. We employ a powerful algorithm, crystal structures and experimental data to model the TLR4 and its cluster. The architecture that we obtain with 8 MyD88 molecules provides the structural basis for the MyD88-templated myddosome helical assembly and receptor clustering; it also provides clues to pro- and anti-inflammatory signaling pathways branching at the signalosome level to Mal/MyD88 and TRAM/TRIF pro- and anti-inflammatory pathways. The assembly of MyD88 death domain (DD) with TRAF3 (anti-viral/anti-inflammatory) and TRAF6 (pro-inflammatory) suggest that TRAF3/TRAF6 binding sites on MyD88 DD partially overlap, as do IRAK4 and FADD. Significantly, the organization illuminates mechanisms of oncogenic mutations, demonstrates that almost all TLR4 parallel pathways are competitive and clarifies decisions at pathway branching points. The architectures are compatible with the currently-available experimental data and provide compelling insights into signaling in cancer and inflammation pathways.
Abstract About 20% of the cancer incidences worldwide have been estimated to be associated with infections. There is a strong correlation of some pathogens with various cancer types. Although presence of commensal microbiome helps chemotherapy to be more effective, pathogenic microbiota increases the cancer risk. Invading pathogens interact with the host mainly through proteins. To subvert host defense, pathogens hijack host pathways by mimicking the binding surfaces (interfaces) of host proteins. This similarity in interfaces permits the pathogenic protein to compete with host proteins to bind to a target protein, alter the physiological signaling and cause persistent infections as well as cancer. Detection of host-pathogen interactions (HPIs) and mapping the re-wired HPI network - along with its structural details - is critical for in-depth understanding of the underlying pathogenesis mechanisms of infections and pathogen-triggered cancers, and developing efficient therapeutics. Here, we developed a novel computational approach to identify novel HPIs by employing “interface mimicry”, We applied this approach to Helicobacter pylori, dominant species in gastric microbiome that greatly increases the gastric cancer risk in order to understand how they modulate host immunity and lead to tumorigenesis. We found that its proteins interfere with the functioning of host apoptosis pathway, cytokine and chemokine pathways, and also cell-cell adhesions. Our results shed light on the molecular mechanisms of resistance to apoptosis, immune evasion and loss of cell junctions that are seen in Helicobacter pylori-infected host cells. In conclusion, HPIs can help us unravel which human pathways are targeted by the pathogenic proteins and how they contribute to pathogenesis of infections and pathogen-triggered cancer. With a better grasp on immunomodulatory strategies of pathogens, we can develop better therapies against them. Citation Format: Emine Guven Maiorov, Ruth Nussinov, Chung-Jung Tsai. Novel host-pathogen interactions of Helicobacter pylori and their implications to gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2678. doi:10.1158/1538-7445.AM2017-2678
T-cell acute lymphoblastic leukemia (T-ALL) is a complex disease, resulting from proliferation of differentially arrested immature T cells. The molecular mechanisms and the genes involved in the generation of T-ALL remain largely undefined. In this study, we propose a set of genes to differentiate individuals with T-ALL from the nonleukemia/healthy ones and genes that are not differential themselves but interconnected with highly differentially expressed ones. We provide new suggestions for pathways involved in the cause of T-ALL and show that network-based classification techniques produce fewer genes with more meaningful and successful results than expression-based approaches. We have identified 19 significant subnetworks, containing 102 genes. The classification/prediction accuracies of subnetworks are considerably high, as high as 98%. Subnetworks contain 6 nondifferentially expressed genes, which could potentially participate in pathogenesis of T-ALL. Although these genes are not differential, they may serve as biomarkers if their loss/gain of function contributes to generation of T-ALL via SNPs. We conclude that transcription factors, zinc-ion-binding proteins, and tyrosine kinases are the important protein families to trigger T-ALL. These potential disease-causing genes in our subnetworks may serve as biomarkers, alternative to the traditional ones used for the diagnosis of T-ALL, and help understand the pathogenesis of the disease.
Cytokines are messengers between tissues and the immune system. They play essential roles in cancer initiation, promotion, metastasis, and immunotherapy. Structural pathways of cytokine signaling which contain their interactions can help understand their action in the tumor microenvironment. Here, our aim is to provide an overview of the role of cytokines in tumor development from a structural perspective. Atomic details of protein-protein interactions can help in understanding how an upstream signal is transduced; how higher-order oligomerization modes of proteins can influence their function; how mutations, inhibitors or antagonists can change cellular consequences; why the same protein can lead to distinct outcomes, and which alternative parallel pathways can take over. They also help to design drugs/inhibitors against proteins de novo or by mimicking natural antagonists as in the case of interferon-γ. Since the structural database (PDB) is limited, structural pathways are largely built from a series of predicted binary protein-protein interactions. Below, to illustrate how protein-protein interactions can help illuminate roles played by cytokines, we model some cytokine interaction complexes exploiting a powerful algorithm (PRotein Interactions by Structural Matching—PRISM).
Autophagy is biological mechanism allowing recycling of long-lived proteins, abnormal protein aggregates, and damaged organelles under cellular stress conditions. Following sequestration in double- or multimembrane autophagic vesicles, the cargo is delivered to lysosomes for degradation. ATG5 is a key component of an E3-like ATG12-ATG5-ATG16 protein complex that catalyzes conjugation of the MAP1LC3 protein to lipids, thus controlling autophagic vesicle formation and expansion. Accumulating data indicate that ATG5 is a convergence point for autophagy regulation. Here, we describe the scaffold protein RACK1 (receptor activated C-kinase 1, GNB2L1) as a novel ATG5 interactor and an autophagy protein. Using several independent techniques, we showed that RACK1 interacted with ATG5. Importantly, classical autophagy inducers (starvation or mammalian target of rapamycin blockage) stimulated RACK1-ATG5 interaction. Knockdown of RACK1 or prevention of its binding to ATG5 using mutagenesis blocked autophagy activation. Therefore, the scaffold protein RACK1 is a new ATG5-interacting protein and an important and novel component of the autophagy pathways. Autophagy is biological mechanism allowing recycling of long-lived proteins, abnormal protein aggregates, and damaged organelles under cellular stress conditions. Following sequestration in double- or multimembrane autophagic vesicles, the cargo is delivered to lysosomes for degradation. ATG5 is a key component of an E3-like ATG12-ATG5-ATG16 protein complex that catalyzes conjugation of the MAP1LC3 protein to lipids, thus controlling autophagic vesicle formation and expansion. Accumulating data indicate that ATG5 is a convergence point for autophagy regulation. Here, we describe the scaffold protein RACK1 (receptor activated C-kinase 1, GNB2L1) as a novel ATG5 interactor and an autophagy protein. Using several independent techniques, we showed that RACK1 interacted with ATG5. Importantly, classical autophagy inducers (starvation or mammalian target of rapamycin blockage) stimulated RACK1-ATG5 interaction. Knockdown of RACK1 or prevention of its binding to ATG5 using mutagenesis blocked autophagy activation. Therefore, the scaffold protein RACK1 is a new ATG5-interacting protein and an important and novel component of the autophagy pathways.
Abstract Head and neck squamous cell carcinoma (HNSCC) arises from the oral cavity, pharynx and larynx, and is the 6th most common cancer worldwide. The incidence of oropharyngeal SCC (OSCC) due to HPV is rising despite a decline in tobacco related HNSCC. However, the molecular mechanisms promoting HPV+ OSCC has not been clearly established. Recently, The Cancer Genome Atlas (TCGA) HNSCC project revealed a higher frequency of homozygous and heterozygous deletions in chromosome 14q32.32 associated with lower expression of the TNF receptor-associated factor 3 (TRAF3) gene, unique to HPV+ tumors. Deleterious nonsense and frame shift mutations of TRAF3 are also exclusively observed in HPV+ HNSCC. Structural interactions of TRAF3 with other signaling molecules were modeled three dimensionally using the PRISM algorithm (PRotein Interactions by Structural Matching). The interactions revealed that these TRAF3 mutations abolished key inhibitory interactions at the interface between receptors and NF-κB inducing kinase (NIK) in the alternative NF-κB pathway. Consistent with TCGA findings, we found varying TRAF3 expression in HPV+ HNSCC cell lines. Overexpression of TRAF3 in HPV+ cell lines suppressed the alternative NF-κB pathway and p52/p100 nuclear localization, and key target genes, while increasing expression of IFN genes implicated in anti-viral immunity. Further, overexpression of TRAF3 inhibited cell growth, colony formation, and migration, as well as sensitized cells to TNF-α and cisplatin induced cell death. Our data reveal that decreased TRAF3 promotes the malignant phenotype via alternative NF-κB signaling, and attenuates antiviral IFN expression, important in pathogenesis of HPV+ HNSCC. Supported by NIDCD intramural project ZIA-DC-000016, 73, 74 Citation Format: Jialing Zhang, Hui Cheng, Xinping Yang, Emine Guven Maiorov, Ozlem Keskin, Attila Gursoy, Ruth Nussinov, Zhong Chen, Carter Van Waes. Alternative NF-κB pathway activation enhanced by deficient TRAF3 in human papillomavirus (HPV)-associated head and neck cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3170. doi:10.1158/1538-7445.AM2014-3170
