Tumor suppressor TET2 promotes cancer immunity and immunotherapy efficacy.

Loss-of-function mutations in genes encoding TET DNA dioxygenase occur frequently in hematopoietic malignancy, but rarely in solid tumors which instead commonly have reduced activity. The impact of decreased TET activity in solid tumors is not known. Here we show that TET2 mediates interferon γ (IFNγ)-JAK-STAT signaling pathway to control chemokine and PD-L1 expression, lymphocyte infiltration and cancer immunity. IFNγ stimulated STAT1 to bind TET2 and recruit TET2 to hydroxymethylate chemokine and PD-L1 genes. Reduced TET activity was associated with decreased TH1-type chemokines and tumor-infiltrating lymphocytes (TILs) and the progression of human colon cancer. Deletion of Tet2 in murine melanoma and colon tumor cells reduced chemokine expression and TILs, enabling tumors to evade anti-tumor immunity and to resist anti-PD-L1 therapy. Conversely, stimulating TET activity by systematic injection of its co-factor, ascorbate/vitamin C, increased chemokine and TILs, leading to enhanced anti-tumor immunity and anti-PD-L1 efficacy and extended lifespan of tumor-bearing mice. These results suggest an IFNγ-JAK-STAT-TET signaling pathway that mediates tumor response to anti-PD-L1/PD-1 therapy and is frequently disrupted in solid tumors. Our findings also suggest TET activity as a biomarker for predicting the efficacy and patient response to anti-PD-1/PD-L1 therapy, and stimulating TET activity as an adjuvant immunotherapy of solid tumors.

Loss of the receptor tyrosine kinase Axl leads to enhanced inflammation in the CNS and delayed removal of myelin debris during experimental autoimmune encephalomyelitis.

BACKGROUND: Axl, together with Tyro3 and Mer, constitute the TAM family of receptor tyrosine kinases. In the nervous system, Axl and its ligand Growth-arrest-specific protein 6 (Gas6) are expressed on multiple cell types. Axl functions in dampening the immune response, regulating cytokine secretion, clearing apoptotic cells and debris, and maintaining cell survival. Axl is upregulated in various disease states, such as in the cuprizone toxicity-induced model of demyelination and in multiple sclerosis (MS) lesions, suggesting that it plays a role in disease pathogenesis. To test for this, we studied the susceptibility of Axl-/- mice to experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. METHODS: WT and Axl-/- mice were immunized with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide emulsified in complete Freund's adjuvant and injected with pertussis toxin on day 0 and day 2. Mice were monitored daily for clinical signs of disease and analyzed for pathology during the acute phase of disease. Immunological responses were monitored by flow cytometry, cytokine analysis and proliferation assays. RESULTS: Axl-/- mice had a significantly more severe acute phase of EAE than WT mice. Axl-/- mice had more spinal cord lesions with larger inflammatory cuffs, more demyelination, and more axonal damage than WT mice during EAE. Strikingly, lesions in Axl-/- mice had more intense Oil-Red-O staining indicative of inefficient clearance of myelin debris. Fewer activated microglia/macrophages (Iba1+) were found in and/or surrounding lesions in Axl-/- mice relative to WT mice. In contrast, no significant differences were noted in immune cell responses between naïve and sensitized animals. CONCLUSIONS: These data show that Axl alleviates EAE disease progression and suggests that in EAE Axl functions in the recruitment of microglia/macrophages and in the clearance of debris following demyelination. In addition, these data provide further support that administration of the Axl ligand Gas6 could be therapeutic for immune-mediated demyelinating diseases.

Ethyl acetate fraction of adlay bran ethanolic extract inhibits oncogene expression and suppresses DMH-induced preneoplastic lesions of the colon in F344 rats through an anti-inflammatory pathway.

Adlay ( Coix lachryma-jobi L. var. ma-yuen Stapf) is a grass crop and was reported to possess anti-inflammatory activity and an antiproliferative effect in cancer cell lines. The purpose of this study was to evaluate the effects of the ethyl acetate fraction of an adlay bran ethanolic extract (ABE-Ea) on colon carcinogenesis in an animal model and investigate its mechanism. Male F344 rats received 1,2-dimethylhydrazine (DMH) and consumed different doses of ABE-Ea. The medium-dose group (17.28 mg of ABE-Ea/day) exhibited the best suppressive effect on colon carcinogenesis and prevented preneoplastic mucin-depleted foci (MDF) formation. Moreover, RAS and Ets2 oncogenes were significantly down-regulated in this group compared to the negative control group, whereas Wee1, a gene involved in the cell cycle, was up-regulated. Cyclooxygenase-2 (COX-2) protein expression was significantly suppressed in all colons receiving the ABE-Ea, indicating that ABE-Ea delayed carcinogenesis by suppressing chronic inflammation. ABE-Ea included considerable a proportion of phenolic compounds, and ferulic acid was the major phenolic acid (5206 microg/g ABE-Ea) on the basis of HPLC analysis. Results from this study suggest that ABE-Ea suppressed DMH-indued preneoplastic lesions of the colon in F344 rats and that ferulic acid may be one of the active compounds.

1,25 Dihydroxyvitamin-D3 modulates JAK-STAT pathway in IL-12/IFNgamma axis leading to Th1 response in experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis (EAE) is a Th1 cell-mediated autoimmune disease model of multiple sclerosis (MS). Vitamin D deficiency is commonly observed in MS patients and vitamin D supplements reduce the clinical symptoms of EAE and MS. Earlier studies have shown that in vivo treatment with vitamin D analogs ameliorates EAE in association with the inhibition of IL-12 production and Th1 differentiation. The mechanisms in the regulation of Th1 response by vitamin D in EAE/MS are, however, not known. We show that in vivo treatment of C57BL/6 and SJL/J mice (i.p.) with 100 ng of 1,25 dihydroxyvitamin D3, on every other day from Day 0-30, ameliorates EAE in association with the inhibition of IL-12 production and neural antigen-specific Th1 response. In vitro treatment with 1,25(OH)2D3 inhibited IFNgamma-induced tyrosine phosphorylation of STAT1, without affecting JAK2, in EOC-20 microglial cells. Treatment of activated T cells with 1,25(OH)2D3 also inhibited the IL-12-induced tyrosine phosphorylation of JAK2, TYK2, STAT3, and STAT4 in association with a decrease in T cell proliferation in vitro. These findings highlight the fact that vitamin D modulates JAK-STAT signaling pathway in IL-12/IFNgamma axis leading to Th1 differentiation and further suggest its use in the treatment of MS and other Th1 cell-mediated autoimmune diseases.

Evaluation of TNF-alpha/Bax gene therapy and radiation against C6 glioma xenografts.

Successful therapy of high-grade tumors of the brain is likely to require a combination of new therapeutic approaches. The major goal of the present study was to construct a plasmid-based bax gene vector (pGL1-Bax) and evaluate its expression in vitro and in vivo using athymic mice with subcutaneously growing C6 glioma. Preliminary experiments of efficacy and safety were also performed using pGL1-Bax alone and in combination with previously constructed pGL1-TNF-alpha, as well as with radiation. pGL1-Bax was expressed by C6 cells and was correlated with apoptosis, indicating that the construct and the bax protein were functional. Although intratumoral injections of pGL1-Bax alone, up to total doses of 450 micro g, did not significantly affect tumor growth, consistently smaller tumors were obtained when pGL1-TNF-alpha plus pGL1-Bax were injected 16-18 hr prior to tumor irradiation. Furthermore, in mice with two tumors, one treated and one untreated, progression of the untreated tumor was delayed in the animals receiving all three modalities. No prohibitive toxicities were noted, based on mouse body weights and in vitro assays of blood and spleen. Significant increases in spleen mass, total leukocyte counts, percentage of granulocytes, spontaneous blastogenesis, and CD71-expressing B cells were primarily associated with tumor presence and not treatment type. Overall, the results are promising and suggest that TNF-alpha/Bax gene therapy may be beneficial against highly malignant tumors of the brain. To our knowledge, this is the first report of bax gene therapy used together with radiation in an in vivo glioma model.

Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation.

Fc epsilon RI activation of mast cells is thought to involve Lyn and Syk kinases proximal to the receptor and the signaling complex organized by the linker for activation of T cells (LAT). We report here that Fc epsilon RI also uses a Fyn kinase-dependent pathway that does not require Lyn kinase or the adapter LAT for its initiation, but is necessary for mast cell degranulation. Lyn-deficiency enhanced Fyn-dependent signals and degranulation, but inhibited the calcium response. Fyn-deficiency impaired degranulation, whereas Lyn-mediated signaling and calcium was normal. Thus, Fc epsilon RI-dependent mast cell degranulation involves cross-talk between Fyn and Lyn kinases.

Regulation of human Fc epsilon RI alpha-chain gene expression by multiple transcription factors.

Transcriptional regulation of the gene-encoding human Fc epsilon RI alpha-chain was analyzed in detail. EMSA revealed that either YY1 or PU.1 bound to the region close to that recognized by Elf-1. The alpha-chain promoter activity was up-regulated approximately 2-fold by exogenously expressed YY1 or PU.1 and approximately 7-fold by GATA-1, respectively, in KU812 cells. In contrast, coexpression of GATA-1 with either of PU.1 or YY1 dramatically activated the promoter approximately 41- or approximately 27-fold, respectively. Especially synergic activation by GATA-1 and PU.1 was surprising, because these transcription factors are known to inhibit the respective transactivating activities of each other. These up-regulating effects of PU.1 and YY1 with GATA-1 were inhibited by overexpression of Elf-1, indicating that Elf-1 serves as a repressor for the alpha-chain gene expression. Transcriptional regulation of the alpha-chain gene through four transcriptional factors is discussed.

T-cell-immunity-based inhibitory effects of orally administered herbal medicine juzen-taiho-to on the growth of primarily developed melanocytic tumors in RET-transgenic mice.

We examined the effect of oral administration of juzen-taiho-to, one of the most popular herbal medicines in Japan, on primary melanocytic tumor growth in RET-transgenic mice. There was virtually no difference between the lengths of tumor-free stages in the juzen-taiho-to-treated mice and the untreated littermate control mice. The rate of tumor growth in the juzen-taiho-to-treated mice, however, was greatly suppressed during the entire period after the initial tumor development. Correspondingly, the life span of juzen-taiho-to-treated transgenic mice was longer (over 6 mo in mean value) than that of control mice. We partially elucidated the mechanism of the antitumor effect of juzen-taiho-to. The addition of juzen-taiho-to at any of a wide range (50-1600 microg per ml) of concentrations to in vitro cultures of Mel-Ret cells, a malignant melanoma cell line derived from a RET-transgenic mouse, caused neither cell death nor cell cycle arrest directly. The addition of 50-400 microg per ml of juzen-taiho-to to cultures of murine spleen cells, however, promoted their DNA synthesis. More importantly, peritoneal exudate cells from the juzen-taiho-to-treated transgenic mice, in which the ratio and number of T cells were increased, displayed an antitumor immunity against Mel-Ret cells in vitro. Interestingly, the peritoneal-exudate-cell-associated antitumor immunity was further augmented by the addition of 200-400 microg per ml of juzen-taiho-to in vitro. This immunity, which was primarily conveyed by Thy-1+ T cells, was antigen (RET/melanoma) specific and cytotoxic. Amongst various chemical ingredients of juzen-taiho-to examined in this study, glycirrhizin displayed an action, partially replacing that of juzen-taiho-to, in promoting anti-Mel-Ret immunity when supplementarily added in vitro. These results suggest that juzen-taiho-to suppresses once-developed primary melanocytic tumors through potentiation of T-cell-mediated antitumor cytotoxic immunity in vivo.

Links between Fer tyrosine kinase expression levels and prostate cell proliferation.

In our cloning strategy to identify tyrosine kinases implicated in the regulation of prostate growth, the dog fer cDNA was obtained and shown to be highly homologous to known fer cDNAs. Using a polyclonal Fer antibody directed against a C-terminal peptide, we studied its associations with cortactin, beta-catenin and p120Cas in human prostate carcinoma PC-3 cells. In contrast to previous reports, no interactions were observed. To assess its functional role, fer cDNA constructs were transfected in PC-3 cells. Antisense clones exhibiting a marked diminution of Fer expression had a reduced growth rate (doubling time of 29 vs. 42 h) and were unable to form colonies in soft agar. In agreement with these results, Fer protein expression was linked to human prostatic proliferative diseases, with enhanced levels in extracts from cancer tissues as compared to those from normal and hyperplastic ones, and was also expressed in the human prostate carcinoma cell lines DU145 and LNCaP. In the dog model, Fer expression was up-regulated in dividing versus resting prostate epithelial cells in vitro, and also in vivo when basal cell hyperplasia and metaplasia was induced by estrogen after castration. Minimal effects were observed when renewing the luminal epithelium with androgens. Taken together, these results show that Fer expression is associated with prostate cell proliferation and enhanced in prostate cancer.

Oncoprotein expression in human synovial tissue: an immunohistochemical study of different types of arthritis.

Based on the fact that synovial lining cells have some properties of transformed-appearing cells, we have examined the expression of Myc, Myb, Fos, Jun and Ras oncoproteins in synovial tissues from patients with different types of arthritis. Formalin-fixed and paraffin-embedded sections of synovial tissue from 12 patients with rheumatoid arthritis (RA), 14 with reactive arthritis (ReA), nine with other seronegative arthritis (OSA), seven with bacterial arthritis (BA), eight with probable bacterial arthritis (PBA) and eight with osteoarthritis (OA) were studied using the immunoperoxidase staining technique. The oncoproteins studied were expressed both in the synovial lining layer and in the sublining layer, consisting of lymphocytes, other inflammatory cells and blood vessels. Among the six disease entities, RA and OA appeared to be the most distinct, whereas the results obtained for ReA and OSA, and on the other hand for BA and PBA, closely resembled each other. The expression of Myc, Myb, Fos and Jun was significantly correlated both to the degree of synovial hypercellularity and the synovial lymphocytic infiltration. For Ras, such a correlation could not be seen. We conclude that we find no evidence of a cell lineage-specific or a disease-specific abnormality of proto-oncogene products in RA, and the expression of these oncoproteins is consistent with inflammation rather than with any primary abnormality of cell growth.

Analysis of the trk NGF receptor tyrosine kinase using recombinant fusion proteins.

Nerve growth factor (NGF) represents a family of structurally related trophic factors, including brain-derived neurotrophin factor (BDNF), neurotrophin-3 (NT-3), NT-4, and NT-5. These neurotrophin factors interact with two classes of receptors, the trk receptor tyrosine kinase family, and the low affinity p75 neurotrophin receptor. To study potential ligand-receptor interactions, recombinant trk fusion proteins have been constructed, and pan-trk polyclonal antisera directed against the cytoplasmic tyrosine kinase domain have been generated. The recombinant proteins were assessed for in vitro kinase activity and for the ability of K-252a to inhibit phosphorylation. Antibodies made against the fusion protein recognize all trk family members, and are effective in immunoprecipitation of affinity-crosslinked receptors. Comparative crosslinking indicates that NGF can recognize all trk receptor members, illustrating the large number of potential ligand-receptor interactions between neurotrophins and their receptors.

c-fos mRNA, Fos, and Fos-related antigen induction by hypertonic saline and stress.

The induction of c-fos mRNA was assessed using Northern blots and in situ hybridization in adult rats administered hypertonic saline (HS) and isotonic saline (IS). HS induced c-fos mRNA in magnocellular paraventricular nucleus (PVNm), parvocellular paraventricular nucleus (PVNp), supraoptic nucleus (SON), and lamina terminalis (LMT). This occurred within 5 min, peaked at 30-60 min, and disappeared by 180 min. Fos protein, detected using a specific monoclonal antibody, was maximal at 1-2 hr and disappeared 4-8 hr after HS administration. This confirms observations showing that the c-fos gene response is transient even in the presence of a continuing stimulus. In contrast, Fos-like immunoreactivity (FLI), detected using two polyclonal antisera, was observed in PVNm, PVNp, SON, and LMT for 1-24 hr during continuous osmotic stimulation. Moreover, FLI was observable in these structures for 7 d in rats administered HS and allowed to drink water ad libitum beginning 24 hr later. At times greater than 8 hr, FLI presumably represents Fos-related antigens (FRA), proteins immunologically and functionally related to Fos, whose expression is much more prolonged than authentic Fos following the osmotic stimulus. In addition to induction of c-fos expression in regions specifically involved in osmotic regulation, HS injections also induced c-fos in many other forebrain regions. In order to assess the induction of c-fos mRNA due to the "stress" of the injections, rats injected with isotonic saline were compared to uninjected controls. Isotonic saline injections induced c-fos mRNA in the PVNp, anterior hypothalamus, suprachiasmatic nucleus, cingulate gyrus, neocortex, ventral lateral septal nucleus, piriform cortex, hippocampal pyramidal and dentate granule neurons, paraventricular and intralaminar thalamic nuclei, bed nuclei of stria terminalis, cortical and medial amygdaloid nuclei, and other structures. In accord with other work, we interpret this pattern of c-fos expression to result from the stress of handling and injections. Since Fos and FRA probably bind to the promoters of target genes and regulate their expression, they likely mediate biochemical changes in the cells activated by the osmotic and stressful stimuli. Whereas the Fos signal is transient, FRA may act on target genes for the duration of the stimulus or longer.