Cytokine Signaling in Pediatric Kidney Tumor Cell Lines WT-CLS1, WT-3ab and G-401.

Renal tumors comprise ~7% of all malignant pediatric tumors. Approximately 90% of pediatric kidney tumors comprise Wilms tumors, and the remaining 10% include clear cell sarcoma of the kidney, malignant rhabdoid tumor of the kidney, renal cell carcinoma and other rare renal tumors. Over the last 30 years, the role of cytokines and their receptors has been considerably investigated in both cancer progression and anti-cancer therapy. However, more effective immunotherapies require the cytokine profiling of each tumor type and comprehensive understanding of tumor biology. In this study, we aimed to investigate the activation of signaling pathways in response to cytokines in three pediatric kidney tumor cell lines, in WT-CLS1 and WT-3ab cells (both are Wilms tumors), and in G-401 cells (a rhabdoid kidney tumor, formerly classified as Wilms tumor). We observed that interferon-alpha (IFN-α) and interferon-gamma (IFN-γ) very strongly induced the activation of the STAT1 protein, whereas IL-6 and IFN-α activated STAT3 and IL-4 activated STAT6 in all examined tumor cell lines. STAT protein activation was examined by flow cytometry and Western blot using phospho-specific anti-STAT antibodies which recognize only activated (phosphorylated) STAT proteins. Nuclear translocation of phospho-STAT proteins upon activation with specific cytokines was furthermore confirmed by immunofluorescence. Our results also showed that both IFN-α and IFN-γ caused upregulation of major histocompatibility complex (MHC) class I proteins, however, these cytokines did not have any effect on the expression of MHC class II proteins. We also observed that pediatric kidney tumor cell lines exhibit the functional expression of an additional cytokine signaling pathway, the tumor necrosis factor (TNF)-α-mediated activation of nuclear factor kappa B (NF-κB). In summary, our data show that human pediatric renal tumor cell lines are responsive to stimulation with various human cytokines and could be used as in vitro models for profiling cytokine signaling pathways.

L-Kynurenine participates in cancer immune evasion by downregulating hypoxic signaling in T lymphocytes.

Malignant tumors often escape anticancer immune surveillance by suppressing the cytotoxic functions of T lymphocytes. While many of these immune evasion networks include checkpoint proteins, small molecular weight compounds, such as the amino acid L-kynurenine (LKU), could also substantially contribute to the suppression of anti-cancer immunity. However, the biochemical mechanisms underlying the suppressive effects of LKU on T-cells remain unclear. Here, we report for the first time that LKU suppresses T cell function as an aryl hydrocarbon receptor (AhR) ligand. The presence of LKU in T cells is associated with AhR activation, which results in competition between AhR and hypoxia-inducible factor 1 alpha (HIF-1α) for the AhR nuclear translocator, ARNT, leading to T cell exhaustion. The expression of indoleamine 2,3-dioxygenase 1 (IDO1, the enzyme that leads to LKU generation) is induced by the TGF-ß-Smad-3 pathway. We also show that IDO-negative cancers utilize an alternative route for LKU production via the endogenous inflammatory mediator, the high mobility group box 1 (HMGB-1)-interferon-gamma (IFN-γ) axis. In addition, other IDO-negative tumors (like T-cell lymphomas) trigger IDO1 activation in eosinophils present in the tumor microenvironment (TME). These mechanisms suppress cytotoxic T cell function, and thus support the tumor immune evasion machinery.

T lymphocytes induce human cancer cells derived from solid malignant tumors to secrete galectin-9 which facilitates immunosuppression in cooperation with other immune checkpoint proteins.

BACKGROUND: Galectin-9 is a member of the family of lectin proteins and crucially regulates human immune responses, particularly because of its ability to suppress the anticancer activities of T lymphocytes and natural killer cells. Recent evidence demonstrated that galectin-9 is highly expressed in a wide range of human malignancies including the most aggressive tumors, such as high-grade glioblastomas and pancreatic ductal adenocarcinomas, as well as common malignancies such as breast, lung and colorectal cancers. However, solid tumor cells at rest are known to secrete either very low amounts of galectin-9 or, in most of the cases, do not secrete it at all. Our aims were to elucidate whether T cells can induce galectin-9 secretion in human cancer cells derived from solid malignant tumors and whether this soluble form displays higher systemic immunosuppressive activity compared with the cell surface-based protein. METHODS: A wide range of human cancer cell lines derived from solid tumours, keratinocytes and primary embryonic cells were employed, together with helper and cytotoxic T cell lines and human as well as mouse primary T cells. Western blot analysis, ELISA, quantitative reverse transcriptase-PCR, on-cell Western and other measurement techniques were used to conduct the study. Results were validated using in vivo mouse model. RESULTS: We discovered that T lymphocytes induce galectin-9 secretion in various types of human cancer cells derived from solid malignant tumors. This was demonstrated to occur via two differential mechanisms: first by translocation of galectin-9 onto the cell surface followed by its proteolytic shedding and second due to autophagy followed by lysosomal secretion. For both mechanisms a protein carrier/trafficker was required, since galectin-9 lacks a secretion sequence. Secreted galectin-9 pre-opsonised T cells and, following interaction with other immune checkpoint proteins, their activity was completely attenuated. As an example, we studied the cooperation of galectin-9 and V-domain Ig-containing suppressor of T cell activation (VISTA) proteins in human cancer cells. CONCLUSION: Our results underline a crucial role of galectin-9 in anticancer immune evasion. As such, galectin-9 and regulatory pathways controlling its production should be considered as key targets for immunotherapy in a large number of cancers.

Expression of the Immune Checkpoint Protein VISTA Is Differentially Regulated by the TGF-ß1 - Smad3 Signaling Pathway in Rapidly Proliferating Human Cells and T Lymphocytes.

Immune checkpoint proteins play crucial roles in human embryonic development but are also used by cancer cells to escape immune surveillance. These proteins and biochemical pathways associated with them form a complex machinery capable of blocking the ability of cytotoxic immune lymphoid cells to attack cancer cells and, ultimately, to fully suppress anti-tumor immunity. One of the more recently discovered immune checkpoint proteins is V-domain Ig-containing suppressor of T cell activation (VISTA), which plays a crucial role in anti-cancer immune evasion pathways. The biochemical mechanisms underlying regulation of VISTA expression remain unknown. Here, we report for the first time that VISTA expression is controlled by the transforming growth factor beta type 1 (TGF-ß)-Smad3 signaling pathway. However, in T lymphocytes, we found that VISTA expression was differentially regulated by TGF-ß depending on their immune profile. Taken together, our results demonstrate the differential biochemical control of VISTA expression in human T cells and various types of rapidly proliferating cells, including cancer cells, fetal cells and keratinocytes.

Chromosomal Heterogeneity of the G-401 Rhabdoid Tumor Cell Line: Unusual Partial 7p Trisomy.

Rhabdoid tumor is a very aggressive and hardly curable pediatric malignancy. It commonly starts in the kidneys but also can occur in the brain, liver, and other organs. The treatment of this tumor usually involves a combination of surgery, radiation, and chemotherapy. Because this tumor is rare, there is still limited experience with a defined standard of care. Cytogenetic analysis is an important routine method to monitor chromosomal aberrations. We have analyzed metaphases of the G-401 rhabdoid tumor cell line. In these cells we have observed metaphases with derivative chromosome 12 arising from partial trisomy 7p. With increasing passage number the numbers of metaphases having this derivative chromosome 12 were found to be higher. In passage number 2 only one metaphase had this pathological chromosome 12. By passage number 10 and passage number 15 about 25 and 95% of this derivative chromosome 12 were found, respectively. We were able to subclone G-401 cells by limiting dilutions and successfully separated cells having apparently normal karyotypes from cells having derivative chromosome 12. Using the cell proliferation assay we showed that clones possessing the derivative chromosome 12 grew more rapidly than clones with normal chromosomes. The cell cycle analysis confirmed this observation. Overall, in this study we describe for the first time a 7p triplication in a rare rhabdoid tumor of kidney. Both types of clones described in this study could be used as a preclinical model to study the involvement of partial chromosome 7 alterations in the development of rhabdoid tumors.

The Retinal Pigment Epithelial Cell Line (ARPE-19) Displays Mosaic Structural Chromosomal Aberrations.

The retinal pigment epithelial cell line ARPE-19 was established in 1996 and remains widely used today for biomedical and in particular ophthalmology research. We have analyzed the chromosomes of the ARPE-19 cell line and found cultured cells exist as a heterogeneous mixture having both normal karyotypes and chromosomal rearrangements. In ARPE-19 cells, we observed metaphases with a single translocation t(15;19) and metaphases with two translocations t(5;15) and t(15;19) and a derivative chromosome 9. Aneuploidies have also been detected (monosomy: -16; trisomy: +11, +18). Multiple attempts to isolate clones with a normal karyotype from those with aberrant karyotypes failed due to senescence of cells of normal karyotypes. We could, however, isolate clones with the translocation t(15;19) and clones with two translocations t(5;15) and t(15;19). In continued cell culture after second subcloning for 30 passages, all clones maintained their cytogenetic integrity.We have further investigated the chromosomal profiles of the ARPE-19 cell line from another laboratory and observed cells with a normal karyotype as well as abnormalities in chromosomes 6p and 11q. The DNA profiles of the ARPE-19 cells from both labs were identical to the ATCC profiles, excluding contamination with other cell lines. Since chromosomal translocations in ARPE-19 cells differ from lab to lab and display a mosaicism for structural chromosomal aberrations, researchers dealing with ARPE-19 cells should screen their stocks for chromosomal aberrations and proceed with caution against misinterpretations during experimental manipulations with this cell line. This chapter describes in detail our laboratory methods for single cell cloning, karyotype analysis and fluorescence in situ hybridization (FISH), which we used for the identification and characterization of chromosomal translocations in the retinal pigment epithelial cell line ARPE-19.

Cleavage of extracellular matrix in periodontitis: gingipains differentially affect cell adhesion activities of fibronectin and tenascin-C.

Gingipains are cysteine proteases that represent major virulence factors of the periodontopathogenic bacterium Porphyromonas gingivalis. Gingipains are reported to degrade extracellular matrix (ECM) of periodontal tissues, leading to tissue destruction and apoptosis. The exact mechanism is not known, however. Fibronectin and tenascin-C are pericellular ECM glycoproteins present in periodontal tissues. Whereas fibronectin mediates fibroblast adhesion, tenascin-C binds to fibronectin and inhibits its cell-spreading activity. Using purified proteins in vitro, we asked whether fibronectin and tenascin-C are cleaved by gingipains at clinically relevant concentrations, and how fragmentation by the bacterial proteases affects their biological activity in cell adhesion. Fibronectin was cleaved into distinct fragments by all three gingipains; however, only arginine-specific HRgpA and RgpB but not lysine-specific Kgp destroyed its cell-spreading activity. This result was confirmed with recombinant cell-binding domain of fibronectin. Of the two major tenascin-C splice variants, the large but not the small was a substrate for gingipains, indicating that cleavage occurred primarily in the alternatively spliced domain. Surprisingly, cleavage of large tenascin-C variant by all three gingipains generated fragments with increased anti-adhesive activity towards intact fibronectin. Fibronectin and tenascin-C fragments were detected in gingival crevicular fluid of a subset of periodontitis patients. We conclude that cleavage by gingipains directly affects the biological activity of both fibronectin and tenascin-C in a manner that might lead to increased cell detachment and loss during periodontal disease.

SMOC1 is a tenascin-C interacting protein over-expressed in brain tumors.

Tenascin-C is an extracellular matrix protein over-expressed in a large variety of cancers. In the present study, we aimed at identifying new interactors of tenascin-C by purifying secreted proteins on a tenascin-C affinity column. Analysis of eluates by mass spectrometry revealed phosphoglycerate kinase 1, clusterin, fibronectin, SPARC-related modular calcium-binding protein 1 (SMOC1) and nidogen-2 as potential interactors of tenascin-C. The interaction between tenascin-C and SMOC1 was confirmed by co-immunoprecipitation and further analyzed by Surface Plasmon Resonance Spectroscopy, which revealed an apparent dissociation constant (K(D)) value of 2.59∗10(-9)M. Further analyses showed that this binding is reduced in the presence of EDTA. To investigate whether SMOC1 itself could be over-expressed in the context of tumorigenesis, we analyzed data of two independent RNA profiling studies and found that mRNA levels of SMOC1 are significantly increased in oligodendrogliomas compared to control brain samples. In support of these data, western blot analysis of protein extracts from 12 oligodendrogliomas, 4 astrocytomas and 13 glioblastomas revealed elevated levels compared to healthy brain extract. Interestingly, cell migration experiments revealed that SMOC1 can counteract the chemo-attractive effect of tenascin-C on U87 glioma cells. The present study thus identified SMOC1 as a new cancer-associated protein capable of interacting with tenascin-C in vitro.