Search

Epigenetic modifications in oncogenesis have long been an area of interest in cancer research. Histone modifications are crucial in activities such as transcriptional activation, gene silencing, and epigenetic cellular memory. In particular, lysine acetylation via lysine (K) acetyltransferases (KATs) have been implicated in cancer development. Interestingly, KAT5, also known as Tip60, has been reported to possess both tumor promoting and tumor suppressing properties depending on the context of malignancy. Herein we report that KAT5 contributes to tumorigenesis in EGFR-mutated lung cancer, and Kat5-knockout mice models demonstrate significantly reduced lung tumor burden. To probe the aberrant modification of KAT5, we demonstrated that KAT5 binds to and is phosphorylated by oncogenic EGFR in co-immunoprecipitation experiments. Next, to investigate whether KAT5 is involved in cell proliferation and survival, H1975 cells harboring L858R-T790M double-activating mutations were transfected with doxycycline inducible shRNA targeting KAT5 (shKAT5). Following treatment, shKAT5 cells were observed to have suppressed proliferation rates. Pharmacological inhibition using TH1834, a known KAT5 inhibitor, also suppressed proliferation rates in shKAT5 cells; in contrast BEAS-2B cells, an immortalized normal bronchial cell line, surprisingly exhibited increased viability compared to the transformed H1975 cells, supporting KAT5's context-dependent role in normal and abnormal cell homeostasis. To further investigate KAT5 in lung tumorigenesis in vivo, we generated EGFR-mutant conditional Kat5 knockout mice using a tetracycline-induced Cre/loxP system. Following doxycycline treatment for 10 weeks, isolated mice lungs for EGFRTL/CCSP-rtTA/Cre/Kat5F/F possessed significantly lower tumor volume compared to EGFRTL/CCSP-rtTA/Cre/Kat5W/F and EGFRTL/CCSP-rtTA/Cre/Kat5W/W mice lungs. Hemotoxylin and eosin staining showed no evident hyperproliferation in lungs isolated from EGFRTL/CCSP-rtTA/Cre/Kat5F/F mice whereas lungs isolated from EGFRTL/CCSP-rtTA/Cre/Kat5W/W and EGFRTL/CCSP-rtTA/Cre/Kat5W/F did, signifying that KAT5 has a potential regulatory role in cellular proliferation. RNA-Seq and ChIP-Seq analysis of shKAT5 H1975 cells identified downstream targets involved in tumorigenic pathways. Further studies are in progress to validate the mRNA and protein expression levels of reported targets. Taken together, these data offer insight into a KAT5 mediated oncogenic pathway that can provide novel therapeutic approaches in treating lung cancer. Citation Format: Gilbert Pan, Eunyoung Heo, Mariko Ando, Masanori Fujii, Ikei Kobayashi, Daniel G. Tenen, Hideo Watanabe, Susumu S. Kobayashi. Role of KAT5 in lung tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 354.

Wnt/ β-catenin signaling plays a key role in the pathogenesis of colon and other cancers. Emerging evidence indicates that oncogenic β-catenin regulates several biological processes essential for cancer initiation and progression. The role of β-catenin in lung cancer with epidermal growth factor receptor (EGFR) activating mutations has not been fully understood; however, recent studies suggest that co-occurring genetic alterations in CTNNB1 might cooperate with mutant EGFR in promoting tumor progression and treatment resistance to EGFR-tyrosine kinase inhibitors (EGFR-TKIs). This sheds new light on this pathway in EGFR-mutant lung cancer. We previously demonstrated that β-catenin plays an essential role in lung tumorigenesis driven by EGFR mutants, particularly EGFR-T790M. We also found that β-catenin is activated through tyrosine-phosphorylation by EGFR mutants. In this study, we identified five tyrosine phosphorylation sites (Y5) of β-catenin by mass spectrometry. Tyrosine phosphorylation at these sites may be critical in β-catenin stability, nuclear translocation and its role as a transcriptional switch in oncogenesis. Firstly, we demonstrated that tyrosine phosphorylation of β-catenin disrupts the binding of a main ubiquitin ligase β-Trcp1, leading to an escape from degradation. Secondly, in contrast to known transcriptional activity with TCF/LEF, β-catenin that is tyrosine phosphorylated by mutant EGFR was found to form a complex with a transcriptional regulator, YAP1, and a transcriptional factor, TBX5. Immunoprecipitation experiments suggested Y5 tyrosine phosphorylation of β-catenin plays a critical role in the interaction between β-catenin and TBX5. Moreover, this complex may regulate anti-apoptotic genes, including BCL2L1, which encodes Bcl-xL. Combination treatment with osimertinib, a 3rd generation EGFR-TKI, and dasatinib suppressed Y5 tyrosine phosphorylation of β-catenin, leading to reduced Bcl-xL expression. Subsequent TBX5 knockdown in vitro decreased Bcl-xL expression while Bcl2l1 knockout in vivo mouse models suppressed lung tumor development. These observations suggest that β-catenin-YAP1-TBX5 complex contributes to EGFR-T790M lung cancer and may serve to explain the underlying mechanism for EGFR-TKIs resistance and for the effectiveness of combination therapy with osimertinib and dasatinib. Citation Format: Masanori Fujii, Sohei Nakayama, Kohei Shimizu, Hisashi Takei, Mariko Ando, Eunyoung Heo, ikei Kobayashi, Gilbert Pan, Hiroyuki Inuzuka, Susumu Kobayashi. Role of tyrosine phosphorylation of β-catenin in EGFR-mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5440.

Polymerization of IgA has been suggested as one of the causes of mesangial deposition in IgA nephropathy. HIGA mice are an inbred model of IgA nephropathy, established by selective mating of ddY mice. This strain is characterized by a unique profile of the IgA molecule that is dominantly polymeric and has high serum levels with intense IgA deposition on the mesangium. We carried out quantitative trait loci (QTL) analysis, using F2 generations by crossing HIGA with BALB/c mice. Significant linkage of polymeric IgA in serum samples was identified around D12Mit263, which is close to the gene of the immunoglobulin heavy chain on chromosome 12. The amino acid sequence of the alpha heavy chain revealed marked differences between BALB/c and HIGA mice. Furthermore, most differences were focussed on the hinge region. The DBA/2J strain, which has the same amino acid sequence in the hinge region as the HIGA strain, also showed polymeric IgA dominance but low IgA levels in sera. Size fraction analysis revealed that these polymeric IgA showed trimer dominance in both DBA/2J and HIGA mice. In conclusion, the hinge region plays a key role in trimeric IgA formation in HIGA mice.

症例は56歳男性. 44歳時慢性糸球体腎炎と診断され50歳時に血液透析に導入された. 透析導入5年目頃より, 透析後に38度台の発熱を呈するようになり, その後, 透析以外の日にも発熱をきたすようになった. CRP上昇 (4.6mg/dL), 白血球増加 (10,700/μL), 好酸球増加 (36%), 抗核抗体陽性を認め, 透析膜アレルギーによる発熱, 透析液中の発熱物質の存在, 感染症, 膠原病, 悪性腫瘍, 薬剤熱等が疑われたが, 精査の結果いずれも否定的であった. Myeloperoxidase (MPO)-ANCA, proteinase 3 (PR3)-ANCAが陽性であり, ANCA-related vasculitisが強く疑われprednisolone 40mgを開始したところ発熱は軽快し, CRPの陰性化, 好酸球の減少を認めた. 血液透析患者の不明熱の原因としてこれまで指摘されてきたもの以外にANCA-related vasculitisも考慮する必要があると考えられた.

Background We recently developed a ddY mouse strain having high IgA levels (HIGA) that provided a murine model of IgA nephropathy. We additionally showed that administration of interleukin (IL)-12, a potent helper T (Th)1-inducing cytokine, induced an apparent reduction in serum IgA levels. In the present study, we assessed the influence of IL-12 administration on several physicochemical characteristics of nephritogenic IgA molecules in HIGA mice. Methods HIGA mice received daily intraperitoneal injections of IL-12 or control injections of phosphate-buffered saline for 3 weeks. Crescent formation and levels of circulating and glomerular IgA were analysed. Moreover, potential changes in charge, size, and glycosylation of serum and glomerular IgA were investigated. Results In the IL-12 group, glomerular IgA deposition was faint, although crescent formation was more marked than in the control group. Serum IgA levels in IL-12 mice were significantly lower than in controls. IL-12-treated mice also showed markedly decreased acidic and polymeric IgA both in sera and in glomerular eluate. A lectin-binding study revealed a markedly reduced ratio of sialylated and galactosylated IgA in the sera and in glomerular eluate from HIGA mice kidneys. IL-12 treatment significantly increased sialylation and galactosylation of circulating IgA, although glycosylation of IgA in glomerular eluate remained low. Conclusions In HIGA mice showing under-glycosylation, IL-12 administration may lead to changes in the physicochemical characteristics of IgA, and this may occur through a shift to Th1. These results suggest that the Th1 and Th2 balance might play a role in the development of immunopathologic lesions in this model of IgA nephropathy.

SUMMARY: The high-IgA inbred strain (HIGA) of ddY mice, an animal model of human IgA nephropathy, shows consistently high serum IgA levels, progressive mesangial sclerosis and IgA deposition, and elevated renal expression of transforming growth factor (TGF)-β. In the present study the role of the TGF-β/Smad signalling pathway in extracellular matrix (ECM) production was assessed in cultured mesangial cells derived from HIGA mice. the production of type I and type IV collagens in response to TGF-β1, expression of Smad2, Smad4, Smad7, and Sp1 and p300, and phosphorylation of Smad2 by TGF-β1 were assessed in cultured mesangial cells derived from HIGA mice at the age of 10 weeks by comparison with age-matched C57BL/6 mice as controls. In addtion, the expression of p300 and type I and type IV collagens in renal tissues of HIGA mice at the age of 60 weeks was determined. the production of type I and type IV collagens by cultured mesangial cells in HIGA mice was markedly upregulated compared with that in C57BL/6 mice. Although protein expression levels of Smad2, Smad4, Smad7, and Sp1 in the mesangial cells were simiiar in the two mouse strains, upregulation of p300 was marked in HIGA mice. Expression of p300 in renal tissues of HIGA mice was also enhanced in HIGA mice when compared with C57BL/6 mice. In HIGA mice, p300 expression was upregulated in the mesangial cells both in vitro and in vivo. It appears that the upregulation of p300 may be related to glomerular sclerosis associated with IgA nephropathy in HIGA mice.

We report a patient who had nephropathy of toxemia of pregnancy associated with membranous glomerulonephritis, which deteriorated 3 months after delivery. Electron microscopy revealed electron-dense deposits on the subepithelial surface of the glomerular basement membrane and finely granular/fibrillar materials in the subendothelial space. As a result of treatment with prednisolone and anticoagulants, marked alleviation of both proteinuria and edema was achieved. We therefore consider that the change from subclinical to clinical membranous nephropathy could be attributed to pregnancy.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) protein family consists of 11 members: APOBEC1, APOBEC2, seven APOBEC3s (A3s) (A/B/C/D/F/G/H), APOBEC4, and the activation-induced deaminase (AID). APOBEC1, A3s, and AID have cytidine deaminase activity and induce a cytidine (C) to thymidine (T) transition. The main function of A3s is to trigger an innate immune response to viral infection such as human immunodeficiency virus-1. Recently, it was reported that several APOBEC family proteins can induce somatic mutations into genomic DNA and thus promote cancer development. For example, AID-mediated somatic mutations contribute to B-cell lymphoma and A3B can be an enzymatic source in solid tumors such as breast cancer. However, little is known about other A3 member proteins. To determine the expression of A3 in hematopoietic cells, we performed quantitative PCR in leukemia cell lines. A3B, A3C, A3D, A3F and A3G were detected in all cell lines, but not A3A or A3H. Next, we analyzed published The Cancer Genome Atlas data from patients with acute myeloid leukemia to find somatic alterations in A3 genes using cBioPortal (http://www.cbioportal.org/). We found that each A3 was upregulated at 4-7 % of all samples and the total frequency of 23 %. Interestingly, overall survival of patients with A3upregulation was lower than those without upregulation, suggesting an important role of A3s in pathogenesis of leukemia. As it has been shown that A3A, A3B, and A3D show capacity to inflict DNA damage, we decided to investigate whether A3D can induce mutations in foreign and genomic DNA. During our attempt to generate A3D expression constructs, we detected multiple bands in leukemia and lung cancer cell lines. Based on our analysis and previous reports, we confirmed that there are at least 7 transcript variants (v1 to 7). Because A3D v3, v4 and v5 lack cytidine deaminase domains, we decided to examine A3Dv1, v2, v6 and v7. To determine whether A3D variants have foreign DNA editing activity, we performed differential DNA denaturation (3D)-PCR in HEK293T cells co-transfected with expression vectors for EGFP, UNG inhibitor, and pCAG-GS containing A3Dv1, v2, v6, v7, A3B or empty control. Total DNA was isolated 4 days after transfection. PCR products were detected at lower denaturation temperature (Td) in cells expressing A3B and all A3D variants vectors compared to control. Sequences of PCR products at lowest Td revealed that mutation frequencies in EGFP gene were 6.1-10.5 per 103 bps in cells transfected with all A3D variants and 18.7 per 103bps in cells transfected with A3B. Of note, 90 % of mutations were C/G to T/A transition in cells transfected with A3B, but in cells transfected with A3D these were 30-60 %. Next we performed 3D-PCR in HEK293T cells retrovirally infected with MigR1-IRES-EGFP containing A3Dv1, v2, v6, v7, A3B or empty to determine genomic DNA editing activity. Total DNA was isolated 4 weeks after infection. At lower Td, PCR products were detected only in cells expressing A3Dv1 and A3B. PCR products in cells expressing A3Dv2, v6, v7 were detected only at same Td as control. Sequences of the PCR products at lowest Td revealed that the mutation frequency in the EGFP gene was 22.2 per 103 bps in cells expressing A3Dv1, and 56.9 per 103 bps in cells expressing A3B. However mutation frequencies in cells expressing A3Dv6 or v7 were lower than 2 per 103bps and no mutations were detected in cells expressing A3Dv2. All of these mutations were C/G to T/A transition. Interestingly, we found 4 clones with deletion in cells expressing A3Dv1, v6 and A3B. In addition, all clones had several bases of micro homologous sequences in broken ends before joining. These results suggested that double strand breaks in genomic DNA were repaired by microhomology-mediated end joining. These DNA editing assays showed that all A3D variants had ability to induce mutations in foreign DNA, and A3Dv1 was major isoform which had ability to induce mutations in genomic DNA. Taken together, our experiments showed that A3D can be a DNA mutator and alternative splicing generates functional diversity of A3D. These findings suggest that A3D may contribute to development of hematopoietic malignancies. Disclosures Takaori-Kondo: Mochida Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Merck Sharp and Dohme: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Eisai: Research Funding; Takeda Pharmaceutical: Research Funding; Chugai Pharmaceutical: Research Funding; Alexion Pharmaceuticals: Research Funding; Kyowa Kirin: Research Funding; Pfizer: Research Funding; Janssen Pharmaceuticals: Speakers Bureau; Shionogi: Research Funding; Toyama Chemical: Research Funding; Cognano: Research Funding.

Tissue factor initiates the extrinsic coagulation pathway by activating coagulation factor X to factor Xa, and factor V is a cofactor for the prothrombin activation by factor Xa. As factor Xa is known to promote the proliferation of mesangial cells in culture, the roles of the coagulation pathway and factor Xa were studied in an animal model of mesangioproliferative glomerulonephritis (MsPGN). MsPGN was induced in Wistar rats by an intravenous injection of anti-Thy 1.1 monoclonal antibody, OX-7. To clarify the role of factor Xa in MsPGN, a specific factor Xa inhibitor, DX-9065a, was injected intravenously at 2.5 or 10 mg/kg at the same time as OX-7, and kidney involvement was assessed by immunohistological analyses. We also examined p44/42 mitogen-activated protein (MAP) kinase activation. Time-course study revealed that expressions of tissue factor, factor V, and protease-activated receptor 2 (PAR2) were peaked on day 3, followed by factor X accumulation and mesangial proliferation. DX-9065a treatment significantly ameliorated proteinuria in a dose-dependent manner on day 8. Histological analyses showed a significant reduction in the size of glomeruli, the total number of glomerular cells, and crescent formation by DX-9065a treatment. Macrophage infiltration, which was rapidly observed on day 1 in disease control rats was not inhibited on days 1-3 by DX-9065a treatment, however it was suppressed on days 5-8. The deposition of fibrin, the number of PCNA-positive cells, and phosphorylation of p44/42 MAP kinase were markedly increased in the disease control group, whereas they were significantly reduced in the treatment group. Tissue factor and factor V induction may accelerate MsPGN through the activation and accumulation of factor X via proinflammatory and procoagulant mechanisms, and the inhibition of factor Xa would be a promising method to regulate the disease process.

Chromosomal mapping of hyperserum IgA and glomerular IgA deposition in a high IgA (HIGA) strain of DdY mice. Background The high IgA (HIGA) strain of ddY mice is an inbred model of IgA nephropathy (IgAN), established by selective mating of outbred ddY mice. HIGA mice show high levels of serum IgA and glomerulonephritis with mesangial IgA deposition. To identify the genetic loci responsible for hyperserum IgA and glomerular IgA deposition in this strain, quantitative trait loci analysis was carried out. Methods By crossing HIGA with BALB/c mice, 244 F2 generations were produced. Serum IgA levels and glomerular IgA deposition were examined at 40 weeks of age. Genetic markers were typed at 105 microsatellites and the quantitative trait loci of hyperserum IgA and glomerular IgA deposition were confirmed using Map Manager QTX software. Results Two significant quantitative trait loci of hyperserum IgA were identified on chromosome 2 [logarithm of odds (LOD) = 5.01] and chromsome 4 (LOD = 4.45), and a suggestive quantitative trait locus of hyperserum IgA was located on chromosome 1 (LOD = 3.49). On chromosome 15, a significant quantitative trait locus of glomerular IgA deposition was identified (LOD = 4.40) without the hyperserum IgA locus. Serum IgA level was weakly correlated with the intensity of glomerular IgA in 244 F2 mice; however, the quantitative trait loci of hyperserum IgA were not significantly associated with glomerular IgA deposition. Conclusion These findings indicate that, in HIGA mice, glomerular IgA deposition is mainly regulated by a quantitative trait locus on chromosome 15, and hyperserum IgA synergistically but weakly affect glomerular IgA deposition. The immune disturbance similar to IgAN was revealed to be under multigenic control in HIGA mice.

BACKGROUND Our recently established high immunoglobulin (Ig)A inbred strain (HIGA) of ddY mice showed constantly high serum IgA levels, progressive mesangial sclerosis accompanied by IgA deposits, and elevated renal expression of transforming growth factor (TGF)-beta, mimicking IgA nephropathy. In the present study, we assessed the role of the immune system, especially of T cells, in this strain. METHODS The in vitro production of interferon (IFN)-gamma, interleukin (IL)-4 and TGF-beta1 by splenic CD4+ T cells was assessed in HIGA mice at 14 and 28 weeks of age by comparison with age-matched C57BL/6 and BALB/c mice, T-helper (Th) 1, and Th2 prone controls respectively. Moreover, recombinant murine IL-12 was administered intraperitoneally to HIGA mice and serum IgA and renal lesions were analysed. RESULTS The production of IFN-gamma by splenic CD4+ T cells was markedly upregulated in HIGA mice at both ages as compared with age-matched C57BL/6 and BALB/c mice. Although splenic CD4+ T cells from HIGA mice produced less IL-4 than those from BALB/c mice at both ages, the former produced significantly more IL-4 with age, which contrasted with the age-associated decrease in the latter. Moreover, TGF-beta1 production of these cells in HIGA mice was equal to or greater than that in the two groups of control mice at both ages. Daily intraperitoneal administration of IL-12 for 1 week significantly enhanced crescent formation with glomerular macrophage accumulation and interstitial cell infiltration, whereas it reduced the serum IgA level. CONCLUSIONS In HIGA mice, Th1 is markedly upregulated from a young age and there is an age-associated Th2 increase with TGF-beta1 upregulation in helper T cells. The former may be related to the exacerbation of inflammatory renal lesions on IL-12 administration, while the latter may contribute to increased IgA production, leading to glomerular IgA deposition and progressive glomerulosclerosis in HIGA mice. The pathogenic role of T cell function and fluctuation of these subsets, especially the Th1/Th2 balance, is crucial to the immunopathological phenotype of the renal lesions in HIGA mice.

Because abnormalities of mucosal immunity have been suggested in human IgA nephropathy, we examined the involvement of mucosal immunity in IgA deposition to the kidney in hyper IgA (HIGA) mice, which was established as a mouse model for human IgA nephropathy with hyperserum IgA. The number of surface IgA+B220− lymphocytes in the intestinal lamina propria (LP) of HIGA mice increased 2.7-fold at 30 wk of age as compared with those at 10 wk of age, whereas normal mice did not show such increase. The surface IgA+B220− LP lymphocytes spontaneously secreted IgA in culture. Morphological studies showed that the surface IgA+B220− lymphocytes of murine intestinal LP are identical with plasma cells (PCs). About 20% of IgA+B220− PC in LP expressed both Mac-1 and CD19, suggesting that they may derive from peritoneal B-1 cells. Cell cycle study on intestinal IgA-PCs using bromodeoxyuridine revealed no difference between HIGA mice and normal mice, suggesting that the high frequency of IgA-producing PCs in HIGA mice is not due to enhanced proliferation or prolonged survival of IgA-producing PCs in LP. In addition, IgA secretion into the gut lumen of HIGA mice decreased drastically (to one forth) with aging. These data suggest that the increased number of intestinal IgA-producing PCs and the down-regulation of IgA excretion into the intestinal lumen might synergistically contribute to the hyperserum IgA in HIGA mice and resultant IgA deposition to the kidney.