Qiuzhen Liu

Transcriptional patterns, biomarkers and pathways characterizing nasopharyngeal carcinoma of Southern China

BackgroundThe pathogenesis of nasopharyngeal carcinoma (NPC) is a complicated process involving genetic predisposition, Epstein-Bar Virus infection, and genetic alterations. Although some oncogenes and tumor suppressor genes have been previously reported in NPC, a complete understanding of the pathogenesis of NPC in the context of global gene expression, transcriptional pathways and biomarker assessment remains to be elucidated.MethodsTotal RNA from 32 pathologically-confirmed cases of poorly-differentiated NPC was divided into pools inclusive of four consecutive specimens and each pool (T1 to T8) was co-hybridized with pooled RNA from 24 normal non-cancerous nasopharyngeal tissues (NP) to a human 8K cDNA array platform. The reliability of microarray data was validated for selected genes by semi-quantitative RT-PCR and immunohistochemistry.ResultsStringent statistical filtering parameters identified 435 genes to be up-regulated and 257 genes to be down-regulated in NPC compared to NP. Seven up-regulated genes including CYC1, MIF, LAMB3, TUBB2, UBE2C and TRAP1 had been previously proposed as candidate common cancer biomarkers based on a previous extensive comparison among various cancers and normal tissues which did not, however, include NPC or NP. In addition, nine known oncogenes and tumor suppressor genes, MIF, BIRC5, PTTG1, ATM, FOXO1A, TGFBR2, PRKAR1A, KLF5 and PDCD4 were identified through the microarray literature-based annotation search engine MILANO, suggesting these genes may be specifically involved in the promotion of the malignant conversion of nasopharyngeal epithelium. Finally, we found that these differentially expressed genes were involved in apoptosis, MAPK, VEGF and B cell receptor signaling pathways and other functions associated with cell growth, signal transduction and immune system activation.ConclusionThis study identified potential candidate biomarkers, oncogenes/tumor suppressor genes involved in several pathways relevant to the oncogenesis of NPC. This information may facilitate the determination of diagnostic and therapeutic targets for NPC as well as provide insights about the molecular pathogenesis of NPC.

An immunologic portrait of cancer

The advent of high-throughput technology challenges the traditional histopathological classification of cancer, and proposes new taxonomies derived from global transcriptional patterns. Although most of these molecular re-classifications did not endure the test of time, they provided bulk of new information that can reframe our understanding of human cancer biology. Here, we focus on an immunologic interpretation of cancer that segregates oncogenic processes independent from their tissue derivation into at least two categories of which one bears the footprints of immune activation. Several observations describe a cancer phenotype where the expression of interferon stimulated genes and immune effector mechanisms reflect patterns commonly observed during the inflammatory response against pathogens, which leads to elimination of infected cells. As these signatures are observed in growing cancers, they are not sufficient to entirely clear the organism of neoplastic cells but they sustain, as in chronic infections, a self-perpetuating inflammatory process. Yet, several studies determined an association between this inflammatory status and a favorable natural history of the disease or a better responsiveness to cancer immune therapy. Moreover, these signatures overlap with those observed during immune-mediated cancer rejection and, more broadly, immune-mediated tissue-specific destruction in other immune pathologies. Thus, a discussion concerning this cancer phenotype is warranted as it remains unknown why it occurs in immune competent hosts. It also remains uncertain whether a genetically determined response of the host to its own cancer, the genetic makeup of the neoplastic process or a combination of both drives the inflammatory process. Here we reflect on commonalities and discrepancies among studies and on the genetic or somatic conditions that may cause this schism in cancer behavior.

CXCR3/CCR5 pathways in metastatic melanoma patients treated with adoptive therapy and interleukin-2

Background:Adoptive therapy with tumour-infiltrating lymphocytes (TILs) induces durable complete responses (CR) in ∼20% of patients with metastatic melanoma. The recruitment of T cells through CXCR3/CCR5 chemokine ligands is critical for immune-mediated rejection. We postulated that polymorphisms and/or expression of CXCR3/CCR5 in TILs and the expression of their ligands in tumour influence the migration of TILs to tumours and tumour regression.Methods:Tumour-infiltrating lymphocytes from 142 metastatic melanoma patients enrolled in adoptive therapy trials were genotyped for CXCR3 rs2280964 and CCR5-Δ32 deletion, which encodes a protein not expressed on the cell surface. Expression of CXCR3/CCR5 in TILs and CXCR3/CCR5 and ligand genes in 113 available parental tumours was also assessed. Tumour-infiltrating lymphocyte data were validated by flow cytometry (N=50).Results:The full gene expression/polymorphism model, which includes CXCR3 and CCR5 expression data, CCR5-Δ32 polymorphism data and their interaction, was significantly associated with both CR and overall response (OR; P=0.0009, and P=0.007, respectively). More in detail, the predicted underexpression of both CXCR3 and CCR5 according to gene expression and polymorphism data (protein prediction model, PPM) was associated with response to therapy (odds ratio=6.16 and 2.32, for CR and OR, respectively). Flow cytometric analysis confirmed the PPM. Coordinate upregulation of CXCL9, CXCL10, CXCL11, and CCL5 in pretreatment tumour biopsies was associated with OR.Conclusion:Coordinate overexpression of CXCL9, CXCL10, CXCL11, and CCL5 in pretreatment tumours was associated with responsiveness to treatment. Conversely, CCR5-Δ32 polymorphism and CXCR3/CCR5 underexpression influence downregulation of the corresponding receptors in TILs and were associated with likelihood and degree of response.

Nuclear β-catenin accumulation is associated with increased expression of Nanog protein and predicts poor prognosis of non-small cell lung cancer

BackgroundAlthough the prognostic roles of β-catenin expression in non-small cell lung cancer (NSCLC) have been reported in several immunohistochemical (IHC) studies, the results were not consistent because some studies lack sufficient number of the positive cases or did not evaluate the subcellular localization features of the protein.MethodIn this study, we have evaluated the expression levels and subcellular localization of β-catenin and Nanog proteins IHC staining in tissue specimens from 309 patients with NSCLC, and explored their association with clinicopathological features and patient outcome.ResultsWe showed that patients with negative expression of membranous beta-catenin had a trend towards shorter survival (p=0.064) than those with positive expression. In contrast to previous studies, we found that increased expression of either cytoplasmic or nuclear β-catenin was strongly associated with poor prognosis and was an independent prognosticator for overall survival (p <0.01). We further found that NSCLC cells frequently exhibited an abundance of nuclear Nanog protein which was significantly correlated with nuclear β-catenin expression (p <0.01) and poor prognosis (p <0.01). Interestingly, immunofluorescent staining results revealed that increased expression of Nanog and nuclear translocation of β-catenin occurred concomitantly in response to epidermal growth factor receptor(EGFR) signaling in A549 and H23 cells. Furthermore, western blot analysis show that nuclear β-catenin rather than cytoplasmic β-catenin expression in the A549 and H23 cells can be enhanced by adding EGF, Nanog expression in the A549 and H23 cells with knockdown of β-catenin can not be obviously enhanced by adding EGF.ConclusionWe propose that evaluation of subcellular localization of β-catenin and Nanog expression is of clinical significance for patients with NSCLC.

IRF-1 responsiveness to IFN- γ predicts different cancer immune phenotypes

Background:Several lines of evidence suggest a dichotomy between immune active and quiescent cancers, with the former associated with a good prognostic phenotype and better responsiveness to immunotherapy. Central to such dichotomy is the master regulator of the acute inflammatory process interferon regulatory factor (IRF)-1. However, it remains unknown whether the responsiveness of IRF-1 to cytokines is able to differentiate cancer immune phenotypes.Methods:IRF-1 activation was measured in 15 melanoma cell lines at basal level and after treatment with IFN-γ, TNF-α and a combination of both. Microarray analysis was used to compare transcriptional patterns between cell lines characterised by high or low IRF-1 activation.Results:We observed a strong positive correlation between IRF-1 activation at basal level and after IFN-γ and TNF-α treatment. Microarray demonstrated that three cell lines with low and three with high IRF-1 inducible translocation scores differed in the expression of 597 transcripts. Functional interpretation analysis showed mTOR and Wnt/β-cathenin as the top downregulated pathways in the cell lines with low inducible IRF-1 activation, suggesting that a low IRF-1 inducibility recapitulates a cancer phenotype already described in literature characterised by poor prognosis.Conclusion:Our findings support the central role of IRF-1 in influencing different tumour phenotypes.

IRF5 gene polymorphisms in melanoma

BackgroundInterferon regulatory factor (IRF)-5 is a transcription factor involved in type I interferon signaling whose germ line variants have been associated with autoimmune pathogenesis. Since relationships have been observed between development of autoimmunity and responsiveness of melanoma to several types of immunotherapy, we tested whether polymorphisms of IRF5 are associated with responsiveness of melanoma to adoptive therapy with tumor infiltrating lymphocytes (TILs).Methods140 TILs were genotyped for four single nucleotide polymorphisms (rs10954213, rs11770589, rs6953165, rs2004640) and one insertion-deletion in the IRF5 gene by sequencing. Gene-expression profile of the TILs, 112 parental melanoma metastases (MM) and 9 cell lines derived from some metastases were assessed by Affymetrix Human Gene ST 1.0 array.ResultsLack of A allele in rs10954213 (G > A) was associated with non-response (p < 0.005). Other polymorphisms in strong linkage disequilibrium with rs10954213 demonstrated similar trends. Genes differentially expressed in vitro between cell lines carrying or not the A allele could be applied to the transcriptional profile of 112 melanoma metastases to predict their responsiveness to therapy, suggesting that IRF5 genotype may influence immune responsiveness by affecting the intrinsic biology of melanoma.ConclusionsThis study is the first to analyze associations between melanoma immune responsiveness and IRF5 polymorphism. The results support a common genetic basis which may underline the development of autoimmunity and melanoma immune responsiveness.

Melanoma NOS1 expression promotes dysfunctional IFN signaling

In multiple forms of cancer, constitutive activation of type I IFN signaling is a critical consequence of immune surveillance against cancer; however, PBMCs isolated from cancer patients exhibit depressed STAT1 phosphorylation in response to IFN-α, suggesting IFN signaling dysfunction. Here, we demonstrated in a coculture system that melanoma cells differentially impairs the IFN-α response in PBMCs and that the inhibitory potential of a particular melanoma cell correlates with NOS1 expression. Comparison of gene transcription and array comparative genomic hybridization (aCGH) between melanoma cells from different patients indicated that suppression of IFN-α signaling correlates with an amplification of the NOS1 locus within segment 12q22-24. Evaluation of NOS1 levels in melanomas and IFN responsiveness of purified PBMCs from patients indicated a negative correlation between NOS1 expression in melanomas and the responsiveness of PBMCs to IFN-α. Furthermore, in an explorative study, NOS1 expression in melanoma metastases was negatively associated with patient response to adoptive T cell therapy. This study provides a link between cancer cell phenotype and IFN signal dysfunction in circulating immune cells.

The immune‐related role of BRAF in melanoma

The existence of a dichotomy between immunologically active and quiescent tumor phenotypes has been recently recognized in several types of cancer. The activation of a Th1 type of immune signature has been shown to confer better prognosis and likelihood to respond to immunotherapy. However, whether such dichotomy depends on the genetic make‐up of individual cancers is not known yet. BRAF and NRAS mutations are commonly acquired during melanoma progression. Here we explored the role of BRAF and NRAS mutations in influencing the immune phenotype based on a classification previously identified by our group.

NOS1 S -nitrosylates PTEN and inhibits autophagy in nasopharyngeal carcinoma cells

Autophagy is a cellular survival mechanism that involves the catabolic degradation of damaged proteins and organelles during periods of metabolic stress, and when overly stimulated, commonly contributes to cell death. Nitric oxide (NO), a potent cellular messenger, participates in a complex mechanism which assists in controlling autophagy. However, the mechanism by which endogenous NO formed by distinct isoforms of nitric oxide synthase (NOS) helps to regulate autophagy in cancer cells remains unclear. Here we report that NOS1 reduces excessive levels of autophagy and promotes the survival of nasopharyngeal carcinoma cells. We found that inhibition of NOS1 increased cell death resulting from siRNA or the use of pharmacologic agents; and this effect was reversed by the autophagy inhibitor, chloroquine. The role of NOS1 in the autophagy process depended on the activation of AKT/mTOR signaling by S-nitrosylation of phosphatase and tensin homolog (PTEN) proteins. The mechanism by which NOS1 modifies PTEN protein might involve a direct interaction between these two molecules. Moreover, in an in vivo study, the NOS1 inhibitor N(G)-nitro-L-arginine methyl ester activated AKT/mTOR signaling and promoted autophagy in xenograph tumors. Our studies demonstrated that NOS1 prevents excessive autophagy via S-nitrosylation of PTEN, and activation of the AKT/mTOR signaling pathway. PTEN and the AKT/mTOR signaling pathway are promising targets for improving the chemotherapeutic treatment of cancer.

IRF-1 responsiveness to immune cytokines predicts different cancer phenotypes

A dichotomy between immunologically active and quiescent cancer phenotypes has been demonstrated for several types of cancer. Central to such dichotomy is the master regulator of the acute inflammatory process interferon regulatory factor (IRF)-1. Although the relevance of IRF-1 to the immune biology of cancer is emerging, it remains unknown whether the responsiveness of IRF-1 to immune cytokines is able to point out different cancer behaviours. Here, we explored the significance of the IFN-γ- and TNF-α-induced activation of IRF-1 in 15 melanoma cell lines. We measured IRF-1 activation in 15 melanoma cell lines at basal level and after treatment with IFN-γ, TNF-α and a combination of both using ImageStream technique (Amnis Corp). Microarray analysis was applied to compare transcriptional patterns between cell lines characterised by high and low IRF-1 activation. Functional interpretation analysis was performed using the Ingenuity Pathway Analysis system (IPA) tools 3.0. We observed a strong positive correlation between IRF-1 activation at basal level and after IFN-γ and TNF-α treatment (ρ=0.65, p=0.007 and ρ=0.66, p=0.007, respectively) suggesting that both the cytokines have a strong effect in inducing IRF-1 activation. Given that different cell lines have different amplitude of response to cytokine stimulation, we next asked whether the inducibility of IRF-1 by IFN-γ and TNF-α was able to distinguish a parallel different behaviour of cancer cells. Toward this aim, we measured the difference of nuclear translocation score between controls and stimulated cells and further selected 3 cell lines with low and 3 cell lines with high delta IRF-1 nuclear translocation. Microarray demonstrated that the 3 cell lines with low and the 3 with high IRF-1 inducible translocation scores differed in the expression of 597 annotated transcripts. Functional interpretation analysis showed mTOR and Wnt/β-cathenin as the top upregulated pathways in the cell lines with low inducible IRF-1 activation, suggesting that a low IRF-1 inducibility recapitulates a cancer phenotype already described in literature characterised by poor prognosis. In conclusion, our findings suggest that the responsiveness of IRF-1 to IFN-γ and TNF-α is able to point out different cancer phenotypes and support the central role of this transcription factor in influencing different tumor behaviours.