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1.
Front Cell Dev Biol ; 8: 280, 2020.
Article in English | MEDLINE | ID: mdl-32509772

ABSTRACT

HHLA2, a newly identified B7 family member, regulates T cell functions. However, the expression and prognostic value of HHLA2 in solid tumors is ill defined. This study aimed to reveal the expression landscape of HHLA2 in various solid tumors, and to evaluate its prognostic value in kidney clear cell carcinoma (KIRC). Using The Cancer Genome Atlas (TCGA) database, we investigated the expression pattern of HHLA2 across 22 types of cancer. HHLA2 and CD8 protein expression was determined via immunohistochemistry (IHC). KIRC-specific findings were further analyzed with R software and the prognostic value was validated on tissue microarrays. HHLA2 was widely expressed in cancers at both the mRNA and protein levels. Among all tested tumors, KIRC showed the highest transcript level of HHLA2, and HHLA2 levels were significantly higher in tumor tissues than in matched normal samples, as evidenced by both TCGA and IHC data. HHLA2 was also positively correlated with survival rates in KIRC based on TCGA and clinical data. Receiver operating characteristic curves data showed the prognostic value of HHLA2 for patients with KIRC in TCGA. Moreover, HHLA2 was positively correlated with immune-related genes, while HHLA2 and CD8 expression exhibited a consistent trend in KIRC tumor samples. In conclusion, HHLA2 is highly expressed in KIRC and predicts a favorable survival outcome, highlighting that it may work as a potential target for KIRC therapy.

2.
Urol Oncol ; 37(11): 810.e1-810.e5, 2019 11.
Article in English | MEDLINE | ID: mdl-31103336

ABSTRACT

BACKGROUND: High T-cell receptor (TCR) repertoire clonality is associated with clinical response to immune checkpoint blockade in bladder cancer. OBJECTIVE: To determine if TCR repertoire is more clonal in tumors than in benign inflammation. METHODS: We prospectively identified 12 patients with bladder lesions undergoing transurethral resection. Specimens were collected at time of transurethral resection and stored at -80C. DNA was extracted and high throughput DNA sequencing of the CDR3 region of the TCR beta chain using the immunoSEQ assay (Adaptive Biotechnologies) was performed. T-cell fraction, clonal dominance, and maximum frequency of TCR clone were assessed. RESULTS: Of the 12 bladder lesions resected, 3 of 12 were cT0, 3 of 12 were cTa, 3 of 12 were cT1, and 3 of 12 were cT2 or greater. The median number of T cells in urothelial carcinoma specimens (UC+) and benign (UC-) specimens was 5,569 and 25,872, respectively. The number of unique TCRs sequenced in UC+ and UC- specimens was 3,069 and 9,680, respectively. The median tumor infiltrating lymphocyte percentage in UC+ and UC- specimens was 2% and 12%, respectively. The UC+ specimens demonstrated clonality as evidenced by identification of a specific T-cell clone being present in up to 17% of the total tumor infiltrating lymphocyte pool, in contrast to 2% among UC- specimens. CONCLUSIONS: Primary urothelial tumors contain clonally expanded T-cell populations. These data support the hypothesis that bladder tumors induce an antigen-driven immunogenic host response, in contrast to the benign inflammatory response, which does not appear to demonstrate any T-cell clonal dominance.


Subject(s)
Carcinoma, Transitional Cell/immunology , Clonal Selection, Antigen-Mediated , Receptors, Antigen, T-Cell/genetics , Urinary Bladder Neoplasms/immunology , Aged , Carcinoma, Transitional Cell/pathology , Female , Humans , Lymphocytes, Tumor-Infiltrating/metabolism , Lymphocytes, Tumor-Infiltrating/pathology , Male , Prospective Studies , Receptors, Antigen, T-Cell/metabolism , T-Lymphocyte Subsets/metabolism , T-Lymphocyte Subsets/pathology , Tumor Microenvironment/immunology , Urinary Bladder Neoplasms/pathology
3.
Bladder Cancer ; 5(1): 63-71, 2019 Jan 31.
Article in English | MEDLINE | ID: mdl-30854414

ABSTRACT

BACKGROUND: B7x (B7-H4/B7S1/VTCN1), an inhibitory immune checkpoint molecule is a potential therapeutic target owing to its immunosuppressive effect and well-known expression in cancers. Immune checkpoints in canine bladder cancer are largely undefined. Here, we report the first evaluation on expression of B7x in spontaneous canine invasive bladder cancer, a novel model system for the study of invasive human urothelial carcinoma. OBJECTIVE: This work aims to study expression of immune checkpoint B7x in spontaneous canine invasive bladder cancer. METHODS: RNA-seq analysis was performed to determine B7x expression in tumor versus normal bladder. Gene ontology (GO) study was used to explore the biological role of B7x. B7x protein expression was evaluated by immunohistochemistry (IHC). TCGA and GTEx were used to examine B7x expression in 599 human bladder urothelial carcinoma (BLCA). RESULTS: RNA-seq analysis indicated 5.72 and 7.04 fold up regulation of B7x in tumors, using DESeq2 and edge R respectively (p < 0.00008). B7x was closely associated with immune processes in GO analysis. IHC results revealed 60% of cases as B7x positive. B7x intensity was scored as negative in 40% (n = 20/50), low in 24% (n = 12/50), medium in 14% (n = 7/50) and high in 22% (n = 11/50) of cases. In human BLCA, B7x expression was significantly associated with worse overall survival (p = 0.02). CONCLUSIONS: Our results suggest that B7x is over expressed in canine bladder cancer. Thus canine model can be vital in advancing the translational research on B7x, a new potential therapeutic target in human bladder cancer.

4.
Article in English | MEDLINE | ID: mdl-29907598

ABSTRACT

Tumors with anaplastic lymphoma kinase (ALK) fusion rearrangements, including non-small-cell lung cancer and anaplastic large cell lymphoma, are highly sensitive to ALK tyrosine kinase inhibitors (TKIs), underscoring the notion that such cancers are addicted to ALK activity. Although mutations in ALK are heavily implicated in childhood neuroblastoma, response to the ALK TKI crizotinib has been disappointing. Embryonal tumors in patients with DNA repair defects such as Fanconi anemia (FA) often have a poor prognosis, because of lack of therapeutic options. Here we report a child with underlying FA and ALK mutant high-risk neuroblastoma responding strongly to precision therapy with the ALK TKI ceritinib. Conventional chemotherapy treatment caused severe, life-threatening toxicity. Genomic analysis of the initial biopsy identified germline FANCA mutations as well as a novel ALK-I1171T variant. ALK-I1171T generates a potent gain-of-function mutant, as measured in PC12 cell neurite outgrowth and NIH3T3 transformation. Pharmacological inhibition profiling of ALK-I1171T in response to various ALK TKIs identified an 11-fold improved inhibition of ALK-I1171T with ceritinib when compared with crizotinib. Immunoaffinity-coupled LC-MS/MS phosphoproteomics analysis indicated a decrease in ALK signaling in response to ceritinib. Ceritinib was therefore selected for treatment in this child. Monotherapy with ceritinib was well tolerated and resulted in normalized catecholamine markers and tumor shrinkage. After 7.5 mo treatment, the residual primary tumor shrunk, was surgically removed, and exhibited hallmarks of differentiation together with reduced Ki67 levels. Clinical follow-up after 21 mo treatment revealed complete clinical remission including all metastatic sites. Therefore, ceritinib presents a viable therapeutic option for ALK-positive neuroblastoma.


Subject(s)
Anaplastic Lymphoma Kinase/antagonists & inhibitors , Brain Neoplasms/drug therapy , Neuroblastoma/drug therapy , Protein Kinase Inhibitors/therapeutic use , Pyrimidines/therapeutic use , Sulfones/therapeutic use , 3T3 Cells , Adolescent , Anaplastic Lymphoma Kinase/genetics , Animals , Brain Neoplasms/genetics , Fanconi Anemia/complications , Fanconi Anemia/genetics , Fanconi Anemia Complementation Group A Protein/genetics , Humans , Male , Mice , Mutation, Missense , Neuroblastoma/complications , Neuroblastoma/genetics , PC12 Cells , Rats
5.
Cancers (Basel) ; 9(11)2017 Oct 30.
Article in English | MEDLINE | ID: mdl-29084134

ABSTRACT

Numerous mutations have been observed in the Anaplastic Lymphoma Kinase (ALK) receptor tyrosine kinase (RTK) in both germline and sporadic neuroblastoma. Here, we have investigated the Y1278S mutation, observed in four patient cases, and its potential importance in the activation of the full length ALK receptor. Y1278S is located in the 1278-YRASYY-1283 motif of the ALK activation loop, which has previously been reported to be important in the activation of the ALK kinase domain. In this study, we have characterized activation loop mutations within the context of the full length ALK employing cell culture and Drosophila melanogaster model systems. Our results show that the Y1278S mutant observed in patients with neuroblastoma harbors gain-of-function activity. Secondly, we show that the suggested interaction between Y1278 and other amino acids might be of less importance in the activation process of the ALK kinase than previously proposed. Thirdly, of the three individual tyrosines in the 1278-YRASYY-1283 activation loop, we find that Y1283 is the critical tyrosine in the activation process. Taken together, our observations employing different model systems reveal new mechanistic insights on how the full length ALK receptor is activated and highlight differences with earlier described activation mechanisms observed in the NPM-ALK fusion protein, supporting a mechanism of activation more in line with those observed for the Insulin Receptor (InR).

6.
Oncotarget ; 8(7): 11566-11578, 2017 Feb 14.
Article in English | MEDLINE | ID: mdl-28030793

ABSTRACT

Activating mutations in full length anaplastic lymphoma kinase (ALK) have been reported in neuroblastoma and in anaplastic thyroid cancer. ALK-L1198F and ALK-G1201E mutations were originally identified in anaplastic thyroid cancer (ATC) and characterized as constitutively activating mutations. In this study, we employed in vitro cell culture assays together with biochemical and in vivo Drosophila analyses to characterize their sensitivity to either activation by the FAM150A (AUG-ß) and FAM150B (AUG-α) ALK ligands or inhibition by ALK inhibitors. Here we report that neither ALK-L1198F nor ALK-G1201E mutations result in ligand independent gain-of-function (GOF) activity in either in vitro biochemical analysis or the various model systems employed. ALK-L1198F is activated by the FAM150 (AUG) ligands and its ligand-dependant activity is similar to the wild type full length ALK receptor. ALK-G1201E is only very weakly activated by the FAM150 (AUG) ligands, most likely due to impaired protein stability. We conclude that neither ALK-L1198F nor ALK-G1201E displays ligand independent kinase activity, with ALK-L1198F belonging to the class of ligand dependent ALK mutations which are not constitutively active but that responds to ligand activation, while the ALK-G1201E mutation generates an unstable receptor with very low levels of kinase activity.


Subject(s)
Drosophila/genetics , Mutation , Receptor Protein-Tyrosine Kinases/genetics , Thyroid Carcinoma, Anaplastic/genetics , Thyroid Neoplasms/genetics , Anaplastic Lymphoma Kinase , Animals , Humans , Ligands , Thyroid Carcinoma, Anaplastic/enzymology , Thyroid Neoplasms/enzymology
7.
FEBS J ; 280(21): 5269-82, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23889739

ABSTRACT

Activation of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase is a key oncogenic mechanism in a growing number of tumor types. In the majority of cases, ALK is activated by fusion with a dimerizing partner protein as a result of chromosomal translocation events, most studied in the case of the nucleophosmin-ALK and echinoderm microtubule-associated protein-like 4-ALK oncoproteins. It is now also appreciated that the full-length ALK receptor can be activated by point mutations and by deletions within the extracellular domain, such as those observed in neuroblastoma. Several studies have employed phosphoproteomics approaches to find substrates of ALK fusion proteins. In this study, we used MS-based phosphotyrosine profiling to characterize phosphotyrosine signaling events associated with the full-length ALK receptor. A number of previously identified and novel targets were identified. One of these, signal transducer and activator of transcription 3 (STAT3), has previously been observed to be activated in response to oncogenic ALK signaling, but the significance of this in signaling from the full-length ALK receptor has not been explored further. We show here that activated ALK robustly activates STAT3 on Tyr705 in a number of independent neuroblastoma cell lines. Furthermore, knockdown of STAT3 by RNA interference resulted in a reduction in myelocytomatosis neuroblastom (MYCN) protein levels downstream of ALK signaling. These observations, together with a decreased level of MYCN and inhibition of neuroblastoma cell growth in the presence of STAT3 inhibitors, suggest that activation of STAT3 is important for ALK signaling activity in neuroblastoma.


Subject(s)
Neuroblastoma/metabolism , Phosphoproteins/metabolism , Proteome/analysis , Receptor Protein-Tyrosine Kinases/metabolism , STAT3 Transcription Factor/metabolism , Anaplastic Lymphoma Kinase , Animals , Apoptosis , Blotting, Western , Cell Proliferation , Humans , Immunoprecipitation , Luciferases , Neuroblastoma/genetics , Neuroblastoma/pathology , PC12 Cells , Phosphorylation , Phosphotyrosine/metabolism , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Rats , Real-Time Polymerase Chain Reaction , Receptor Protein-Tyrosine Kinases/genetics , Reverse Transcriptase Polymerase Chain Reaction , STAT3 Transcription Factor/antagonists & inhibitors , STAT3 Transcription Factor/genetics , Signal Transduction
8.
Dis Model Mech ; 6(2): 373-82, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23104988

ABSTRACT

Neuroblastoma is a childhood extracranial solid tumour that is associated with a number of genetic changes. Included in these genetic alterations are mutations in the kinase domain of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK), which have been found in both somatic and familial neuroblastoma. In order to treat patients accordingly requires characterisation of these mutations in terms of their response to ALK tyrosine kinase inhibitors (TKIs). Here, we report the identification and characterisation of two novel neuroblastoma ALK mutations (A1099T and R1464STOP), which we have investigated together with several previously reported but uncharacterised ALK mutations (T1087I, D1091N, T1151M, M1166R, F1174I and A1234T). In order to understand the potential role of these ALK mutations in neuroblastoma progression, we have employed cell culture-based systems together with the model organism Drosophila as a readout for ligand-independent activity. Mutation of ALK at position 1174 (F1174I) generates a gain-of-function receptor capable of activating intracellular targets such as ERK (extracellular signal regulated kinase) and STAT3 (signal transducer and activator of transcription 3) in a ligand-independent manner. Analysis of these previously uncharacterised ALK mutants and comparison with ALK(F1174) mutants suggests that ALK mutations observed in neuroblastoma fall into three classes. These classes are: (i) gain-of-function ligand-independent mutations such as ALK(F1174l), (ii) kinase-dead ALK mutants, e.g. ALK(I1250T) (Schönherr et al., 2011a) and (iii) ALK mutations that are ligand-dependent in nature. Irrespective of the nature of the observed ALK mutants, in every case the activity of the mutant ALK receptors could be abrogated by the ALK inhibitor crizotinib (Xalkori/PF-02341066), albeit with differing levels of sensitivity.


Subject(s)
Drosophila melanogaster/enzymology , Mutation/genetics , Neuroblastoma/enzymology , Neuroblastoma/genetics , Receptor Protein-Tyrosine Kinases/genetics , Anaplastic Lymphoma Kinase , Animals , Cell Culture Techniques , Cell Proliferation/drug effects , Cell Transformation, Neoplastic/drug effects , Cell Transformation, Neoplastic/pathology , Crizotinib , Disease Models, Animal , Humans , Inhibitory Concentration 50 , Mutant Proteins/metabolism , Neurites/drug effects , Neurites/metabolism , Neurites/pathology , Neuroblastoma/drug therapy , Neuroblastoma/pathology , PC12 Cells , Phenotype , Phosphorylation/drug effects , Protein Structure, Tertiary , Pyrazoles/pharmacology , Pyrazoles/therapeutic use , Pyridines/pharmacology , Pyridines/therapeutic use , Rats , Receptor Protein-Tyrosine Kinases/chemistry
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