Comparative proteomic analysis of glomerular proteins in IgA nephropathy and IgA vasculitis with nephritis.

BACKGROUND: IgA nephropathy (IgAN) and IgA vasculitis with nephritis (IgAVN) are related glomerular diseases characterized by marked similarities in immunological and histological findings. We herein performed a comparative proteomic analysis of glomerular proteins in IgAN and IgAVN. METHODS: We used renal biopsy specimens from 6 IgAN patients without nephrotic syndrome (NS) (IgAN-I subgroup), 6 IgAN patients with NS (IgAN-II subgroup), 6 IgAVN patients with 0-8.0% of glomeruli with crescent formation (IgAVN-I subgroup), 6 IgAVN patients with 21.2-44.8% of glomeruli with crescent formation (IgAVN-II subgroup), 9 IgAVN patients without NS (IgAVN-III subgroup), 3 IgAVN patients with NS (IgAN-IV subgroup), and 5 control cases. Proteins were extracted from laser microdissected glomeruli and analyzed using mass spectrometry. The relative abundance of proteins was compared between groups. An immunohistochemical validation study was also performed. RESULTS: More than 850 proteins with high confidence were identified. A principal component analysis revealed a clear separation between IgAN and IgAVN patients and control cases. In further analyses, 546 proteins that were matched with ≥ 2 peptides were selected. The levels of immunoglobulins (IgA, IgG, and IgM), complements (C3, C4A, C5, and C9), complement factor H-related proteins (CFHR) 1 and 5, vitronectin, fibrinogen chains, and transforming growth factor-ß inducible gene-h3 were higher (> 2.6 fold) in the IgAN and IgAVN subgroups than in the control group, whereas hornerin levels were lower (< 0.3 fold). Furthermore, C9 and CFHR1 levels were significantly higher in the IgAN group than in the IgAVN group. The abundance of some podocyte-associated proteins and glomerular basement membrane (GBM) proteins was significantly less in the IgAN-II subgroup than in the IgAN-I subgroup as well as in the IgAVN-IV subgroup than in the IgAVN-III subgroup. Among the IgAN and IgAVN subgroups, talin 1 was not detected in the IgAN-II subgroup. This result was supported by immunohistochemical findings. CONCLUSIONS: The present results suggest shared molecular mechanisms for glomerular injury in IgAN and IgAVN, except for enhanced glomerular complement activation in IgAN. Differences in the protein abundance of podocyte-associated and GBM proteins between IgAN and IgAVN patients with and without NS may be associated with the severity of proteinuria.

[Refractory esophageal stricture of esophageal mucous membrane pemphigoid after allogeneic hematopoietic stem cell transplantation].

Haploidentical allogeneic hematopoietic stem cell transplantation from her brother was performed on a 41-year-old lady with no prior history of pemphigoid to treat recurrent AML. On day 59 following transplantation, she experienced esophageal stenosis. During immunosuppressive therapy for graft vs. host disease, this condition was controlled with periodic esophageal dilatation (GVHD). Her esophageal stricture, which required periodic dilatation, grew worse after she stopped immunosuppressive therapy because of recurrent AML. The esophageal mucosa was easily hemorrhagic and desquamative. Histologic analysis revealed that the squamous cell layers had been divided. Indirect immunofluorescence was negative for IgG and positive for IgA on the epidermal layers, while direct immunofluorescence showed a linear deposition of IgG on the basement membrane zone. It was determined through immunoblotting utilizing recombinant protein of BP180 C-terminal domain that both IgG and IgA antibodies were present, supporting the diagnosis of mucous membrane pemphigoid with anti-BP180. After allogeneic transplantation, basal epidermal cell destruction by GVHD may result in autoimmune blistering disorders, which expose basement membrane proteins and antigen presentation. A similar mechanism could apply to our situation. For rare GVHD cases, a thorough histological diagnosis is required.

Downregulation of miR-26 promotes invasion and metastasis via targeting interleukin-22 in cutaneous T-cell lymphoma.

It has been reported that certain microRNAs (miRNA) are associated with the pathogenesis of lymphoma. We have previously demonstrated that histone deacetylase inhibitors restore tumor-suppressive miRNAs, such as miR-16, miR-29, miR-150, and miR-26, in advanced cutaneous T-cell lymphoma (CTCL). Among these, the function of miR-26 remains unclear. In this study, we aimed to reveal the function of miR-26 in CTCL oncogenesis. First, we confirmed that the miR-26 family was markedly dysregulated in CTCL cell lines and primary samples. In vivo analysis using miR-26a-transduced CTCL cells injected into immunodeficient NOG mice demonstrated the significant prolonged survival of the mice, suggesting that the miRNA had a tumor-suppressive function. We performed gene expression assays and identified 12 candidate miR-26 targets, namely RGS13, FAM71F1, OAF, SNX21, CDH2, PTPLB, IL22, DNAJB5, CASZ1, CACNA1C, MYH10, and CNR1. Among these, IL22 was the most likely candidate target because the IL-22-STAT3-CCL20-CCR6 cascade is associated with tumor invasion and metastasis of advanced CTCL. In vitro analysis of IL22 and IL22RA knockdown and miR-26 transduction demonstrated inhibited CTCL cell migration. In particular, IL22 knockdown induced cell apoptosis. Finally, we conducted in vivo inoculation analysis of mice injected with shIL22-transfected CTCL cells, and found no tumor invasion or metastasis in the inoculated mice, although the control mice showed multiple tumor invasions and metastases. These results, along with our previous data, demonstrated that miR-26 is a tumor suppressor that is associated with tumor invasion and the metastasis of advanced CTCL by regulating the IL-22-STAT3-CCL20 cascade. Therefore, a IL-22-targeting therapy could be a novel therapeutic strategy for advanced CTCL.

Long-term prognosis of monoclonal immunoglobulin-associated glomerular diseases with non-organized deposits: A report of 38 cases from a Japanese single center.

Monoclonal immunoglobulin (MIg)-associated glomerular diseases with non-organized deposits are rare disorders. They have recently been categorized into light chain deposit disease (LCDD), light and heavy chain deposit disease (LHCDD), heavy chain deposit disease (HCDD), proliferative glomerulonephritis with MIg deposits (PGNMID) and its light chain only variant (PGNMID-LC), and membranous glomerulopathy with light chain-restricted deposits (MG-LC). In our Japanese cohort of more than 9,500 patients who underwent renal biopsy (1979 - 2020), we evaluated clinicopathological features and long-term outcomes in 38 patients with MIg-associated glomerular diseases with non-organized deposits: LCDD (n = 9), LHCDD (n = 8), HCDD (n = 5), PGNMID-membranoproliferative glomerulonephritis (MPGN) (n = 7), PGNMID-LC (n = 2), and MG-LC (n = 7). In patients with LCDD, a low estimated glomerular filtration rate (eGFR) at biopsy, a high detection rate of urinary MIgs, a high incidence rate of multiple myeloma, and sever tubulointerstitial and vascular lesions were significant clinicopathological characteristics. Median duration of follow-up in each group was 42 - 114 months. Most patients were treated with steroid-based therapy. Patients with LCDD, LHCDD, HCDD, and MG-LC were recently treated with bortezomib-based therapy. Renal survival rate was significantly shorter for LCDD than of PGNMID and MG-LC. Patient survival rate was significantly longer for MG-LC than HCDD and PGNMID. Major causes of death were pulmonary and cardiovascular complications. Among disease groups, significant differences were observed in eGFR at biopsy, detection rates of urinary MIgs, incidence rates of multiple myeloma, severities of tubulointerstitial and vascular lesions, and long-term outcomes.

Evaluation of a newly proposed renal risk score for Japanese patients with ANCA-associated glomerulonephritis.

BACKGROUND: We determined the usefulness and prognostic ability of the renal risk score (RRS), proposed in Europe, for Japanese patients with antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis (AAGN) and high myeloperoxidase (MPO)-ANCA positivity; these aspects remain to be verified. METHODS: This retrospective study was conducted on 86 Japanese patients with new, biopsy-confirmed AAGN. We calculated the RRS and analyzed the relationship between this classification, and clinicopathological features and prognosis. We also compared the predictive values between RRS for endpoints including renal death and conventional prognostic tools for patients with AAGN. RESULTS: There were 33, 37, and 16 patients in the low-, medium-, and high-risk groups, respectively. All patients were MPO-ANCA positive. The median follow-up period was 33 months; 16 (18.6%) patients progressed to end-stage renal disease (ESRD). In the high-risk group, 9/16 (56.3%) patients progressed to ESRD, and renal prognosis was significantly poorer than that in other groups (low-risk group, P < 0.001; medium-risk group, P = 0.004). In Cox multivariate regression analysis, RRS was an independent, poor renal prognostic factor (hazard ratio 5.22; 95% confidence interval 2.20-12.40; P < 0.001). The receiver-operating characteristic curves of the RRS for each endpoint were comparable with those of the 2010 histological classification and those of the severity classification of Japanese rapidly progressive glomerulonephritis. CONCLUSIONS: This is the first study to report the usefulness of the RRS for predicting renal outcomes among Japanese patients with AAGN. Our predictive value of the RRS was comparable with that of conventional prognostic tools.

Hypoxia-inducible hexokinase-2 enhances anti-apoptotic function via activating autophagy in multiple myeloma.

Multiple myeloma (MM) is an incurable hematopoietic neoplasm derived from plasma cells, and existing in the bone marrow. Recent developments in the field of myeloma onco-biology have enabled the use of proteasome inhibitors (PIs) as key drugs for MM. PIs can increase cell sensitivity to endoplasmic reticulum stress, leading to apoptosis of myeloma cells. PI cannot kill all myeloma cells, however; one reason of this might be activation of autophagy via hypoxic stress in the bone marrow microenvironment. Hypoxia-inducible gene(s) that regulate autophagy may be novel therapeutic target(s) for PI-resistant myeloma cells. Here, a hypoxia-inducible glycolytic enzyme hexokinase-2 (HK2) was demonstrated to contribute by autophagy activation to the acquisition of an anti-apoptotic phenotype in myeloma cells. We found that hypoxic stress led to autophagy activation accompanied by HK2 upregulation in myeloma cells. Under hypoxic conditions, HK2 knockdown inhibited glycolysis and impaired autophagy, inducing apoptosis. The cooperative effects of a PI (bortezomib) against immunodeficient mice inoculated with HK2-knocked down myeloma cells were examined and significant tumor reduction was observed. An HK2 inhibitor, 3-bromopyruvate (3-BrPA), also induced apoptosis under hypoxic rather than normoxic conditions. Further examination of the cooperative effects between 3-BrPA and bortezomib on myeloma cells revealed a significant increase in apoptotic myeloma cells. These results strongly suggested that HK2 regulates the activation of autophagy in hypoxic myeloma cells. Cooperative treatment using PI against a dominant fraction, and HK2 inhibitor against a minor fraction, adapted to the bone marrow microenvironment, may lead to deeper remission for refractory MM.

Clinicopathological features and outcomes of diabetic kidney disease with extracapillary hypercellularity: a Japanese single-center experience.

BACKGROUND: The prognostic significance of glomerular extracapillary hypercellularity (EXHC) in diabetic kidney disease (DKD) is unclear. The aim of this study was to investigate the clinicopathological features and outcomes of DKD patients with EXHC. METHODS: We studied 70 cases of renal biopsy-confirmed type 2 DKD that were diagnosed between 2004 and 2014 and compared the clinicopathological features and outcomes of 22 patients with EXHC (EXHC group) with those of 48 patients without EXHC (control group). All of the patients were Japanese. We assessed the renal biopsy specimens based on the Renal Pathology Society classification system. Clinical and laboratory data were collected at the time of the renal biopsy, and renal outcomes were assessed based on progression to end-stage renal disease (ESRD) requiring renal replacement therapy. The median duration of the observation period was 3 years. RESULTS: In pathological features, nodular sclerosis (Kimmelstiel-Wilson lesions) was observed more frequently in the EXHC group than in the control group (63.6% vs. 35.4%, P = 0.027). There were no significant intergroup differences in clinical features or renal outcomes. Univariate and multivariate Cox regression analyses of all patients showed that a high level of proteinuria, a low initial eGFR, and severe interstitial inflammation were poor prognostic factors. CONCLUSIONS: EXHC is related to nodular sclerosis, which is a known risk factor for ESRD. Careful observation is needed during the follow-up of DKD patients with EXHC, although there were no significant differences in renal outcomes between the EXHC and control groups.

Comparative proteomic analysis of renal proteins from IgA nephropathy model mice and control mice.

BACKGROUND: High-IgA ddY (HIGA) mice, an animal model of human IgA nephropathy (IgAN), spontaneously develop nephropathy with glomerular IgA deposition and markedly elevated serum IgA levels from 25 weeks of age. METHODS: We performed a comparative proteomic analysis of the renal proteins collected from HIGA mice and control C57BL/6 mice at 5 or 38 weeks of age (the H5, H38, C5, and C38 groups) (n = 4 in each group). Proteins were extracted from the left whole kidney of each mouse and analyzed using nano-liquid chromatography-tandem mass spectrometry. The right kidneys were used for histopathological examinations. RESULTS: Immunohistochemical examinations showed glomerular deposition of IgA and the immunoglobulin joining (J) chain, and increased numbers of interstitial IgA- and J-chain-positive plasma cells in the H38 group. In the proteomic analysis, > 5000 proteins were identified, and 33 proteins with H38/H5 ratios of > 5.0, H38/C38 ratios of > 5.0, and C38/C5 ratios of < 1.5 were selected. Among them, there were various proteins that are known to be involved in human IgAN and/or animal IgAN models. Immunohistochemical examinations validated the proteomic results for some proteins. Furthermore, two proteins that are known to be associated with kidney disease displayed downregulated expression (H38/H5 ratio: 0.01) in the H38 group. CONCLUSIONS: The results of comparative proteomic analysis of renal proteins were consistent with previous histopathological and serological findings obtained in ddY and HIGA mice. Various proteins that are known to be involved in kidney disease, including IgAN, and potential disease marker proteins exhibited markedly altered levels in HIGA mice.

Histone deacetylase inhibitors downregulate CCR4 expression and decrease mogamulizumab efficacy in CCR4-positive mature T-cell lymphomas.

Histone deacetylase inhibitors are promising agents for various T-cell lymphomas, including cutaneous T-cell lymphoma, peripheral T-cell lymphoma, and adult T-cell lymphoma/leukemia. CCR4 is an important therapeutic target molecule because mogamulizumab, an anti-CCR4 antibody, has shown promising efficacy against various T-cell lymphomas. In this study, we examined the in vitro synergistic effects of mogamulizumab and histone deacetylase inhibitors against various T-cell lymphomas. First, we examined the expression of CCR4 mRNA and surface CCR4 in various T-cell lymphoma cell lines and found that it was downregulated upon treatment with vorinostat, a pan-histone deacetylase inhibitor. Next, we used isoform-specific histone deacetylase inhibitors and short-interfering RNA to determine the histone deacetylase isoform involved in the regulation of CCR4, and demonstrated that romidepsin, a class I selective histone deacetylase inhibitor, reduced CCR4 most efficiently. Moreover, among class I histone deacetylases, histone deacetylase 2 knockdown led to a reduction of CCR4 in lymphoma cells, suggesting that CCR4 expression is mainly regulated by histone deacetylase 2. When we examined the CCR4 expression in skin samples from primary cutaneous T-cell lymphoma, obtained from the same patients before and after vorinostat treatment, we found that CCR4 expression was greatly reduced after treatment. Finally, when we conducted an antibody-dependent cell-mediated cytotoxicity assay with mogamulizumab by using various lymphoma cells, we found that the efficacy of mogamulizumab was significantly reduced by pretreatment with vorinostat. Altogether, our results suggest that the primary use of histone deacetylase inhibitors before treatment with mogamulizumab might not be suitable to obtain synergistic effects. Moreover, these results have potential implications for optimal therapeutic sequences in various CCR4-positive T-cell lymphomas.

Clinicopathological and long-term prognostic features of membranous nephropathy with crescents: a Japanese single-center experience.

BACKGROUND: Three recent studies from the United States and China reported the clinicopathological features and short-term prognosis in patients with membranous nephropathy (MN) and crescents in the absence of secondary MN, anti-glomerular basement membrane (GBM) antibodies, and anti-neutrophil cytoplasmic antibodies (ANCA). METHODS: We compared clinicopathological and prognostic features in 16 MN patients with crescents (crescent group) and 38 MN patients without crescents (control group), in the absence of secondary MN, anti-GBM antibodies, and ANCA. Median follow-up periods in the crescent and control groups were 79 and 50 months, respectively. RESULTS: Decreased estimated glomerular filtration rates (<50 mL/min/1.73 m2), glomerulosclerosis, and moderate-to-severe interstitial fibrosis were more frequently observed in the crescent group than in the control group (P = 0.043, P = 0.004, and P = 0.035, respectively). Positive staining rates for glomerular IgG2 and IgG4 were significantly different between the 2 groups (P = 0.032, P = 0.006, respectively). Doubling of serum creatinine during follow-up was more frequently observed in the crescent group than in the control group (P = 0.002), although approximately two-thirds of patients in the crescent group were treated with immunosuppressive therapy. Crescent formation and interstitial fibrosis were risks for doubling of serum creatinine [hazard ratio (HR) = 10.506, P = 0.012; HR = 1.140, P = 0.009, respectively]. CONCLUSIONS: This is the first Japanese study demonstrating significant differences in clinicopathological and prognostic features between the 2 groups. Most patients in the crescent group may develop a long-term decline in renal function despite immunosuppressive therapy.

Hypoxia-inducible microRNA-210 regulates the DIMT1-IRF4 oncogenic axis in multiple myeloma.

Multiple myeloma (MM) is characterized by the accumulation of a population of malignant plasma cells within the bone marrow and its microenvironment. A hypoxic niche is located within the microenvironment, which causes myeloma cells to become quiescent, anti-apoptotic, glycolytic, and immature. Cell heterogeneity may be related to distinct gene expression profiles under hypoxic and normoxic conditions. During hypoxia, myeloma cells acquire these phenotypes by downregulating interferon regulatory factor 4 (IRF4), an essential transcription factor in myeloma oncogenesis. To identify essential microRNAs and their targets regulated under hypoxic conditions, we undertook microRNA and cDNA microarray analyses using hypoxia-exposed primary MM samples and myeloma cell lines. Under hypoxia, only miR-210 was highly upregulated and was accompanied by direct downregulation of an 18S rRNA base methyltransferase, DIMT1. This inverse expression correlation was validated by quantitative RT-PCR for primary MM samples. We further determined that DIMT1 has an oncogenic potential as its knockdown reduced tumorigenicity of myeloma cells through regulation of IRF4 expression. Notably, by analyzing gene expression omnibus datasets in the National Center for Biotechnology Information database, we found that DIMT1 expression increased gradually with MM progression. In summary, by screening for targets of hypoxia-inducible microRNA-210, we identified DIMT1 as a novel diagnostic marker and therapeutic target for all molecular subtypes of MM.

Refractory IgG4-related disease complicated with organising pneumonia and hypertrophic pachymeningitis.

Immunoglobulin G4 (IgG4)-related disease (IgG4-RD) involves multiple organs, including the lungs and central nervous system. Lung lesions are frequently reported as mass lesions or non-specific interstitial pneumonia, whereas organising pneumonia (OP) due to IgG4-RD is rare. Furthermore, limited information is currently available on hypertrophic pachymeningitis (HP). We herein report a case of IgG4-RD complicated with OP and HP. The diagnosis was confirmed based on the serum concentration of IgG4 and the results of salivary gland and transbronchial lung biopsies. HP did not respond to steroid monotherapy and was also resistant to rituximab and intravenous cyclophosphamide; however, the combination therapy of methotrexate and dexamethasone was effective.

Adult-onset Still's Disease during Pregnancy Treated with Tocilizumab.

A 28-year-old woman exhibited a spiking fever, arthritis, and liver disfunction when she was 22 weeks pregnant. She was diagnosed with adult-onset Still's disease (AOSD). As her condition was resistant to corticosteroid therapy, tocilizumab (TCZ) was selected. The TCZ treatment was effective, and she delivered a healthy child while receiving TCZ treatment. Cases in which AOSD first arises during pregnancy are rare, and there have been no reports of TCZ treatment for AOSD being initiated during pregnancy. Although the safety of TCZ treatment during pregnancy has not been established, it may be effective against severe AOSD that develops during pregnancy.

Successful Pregnancies in a Patient with Childhood-onset Steroid-dependent Nephrotic Syndrome during Rituximab Maintenance Therapy.

There are an increasing number of reports on the safe use of rituximab (RTX), a chimeric anti-CD20 monoclonal antibody, in pregnant women with hematological malignancies or refractory autoimmune diseases. In 2014, the use of RTX for patients with complicated steroid-dependent nephrotic syndrome (SDNS) was approved in Japan. We herein report a woman with childhood-onset complicated SDNS due to focal and segmental glomerulosclerosis, who had two successful pregnancies while receiving RTX maintenance therapy. No adverse complications were observed during the pregnancies, and she delivered healthy newborns. This case suggested that RTX may be used safely in pregnant women complicated with SDNS.

A Pediatric Case of Relapsing Eosinophilic Granulomatosis with Polyangiitis Successfully Treated with Mepolizumab.

We herein report the first pediatric case (a 13-year-old girl) of relapsing eosinophilic granulomatosis with polyangiitis (EGPA) successfully treated with mepolizumab (anti-interleukin-5). She was classified as having EGPA based on the presence of asthma, eosinophilia, pulmonary infiltrates, and extravascular eosinophil infiltration confirmed by a biopsy. She achieved remission after initial oral prednisolone (PSL) therapy, but EGPA relapsed during PSL tapering. Subsequent combined therapy with PSL and tacrolimus did not improve the recurrent disease. Intravenous methylprednisolone pulse therapy was started, followed by oral PSL. During PSL tapering, mepolizumab was added to the treatment, which resulted in sustained remission and successful PSL tapering.

Immunoglobulin Light Chain Amyloidosis with Severe Liver Dysfunction Accompanied by Factor X Deficiency.

Severe hepatic failure is rarely a cause of death in patients with immunoglobulin light chain (AL) amyloidosis. We herein report a case of AL amyloidosis involving a bleeding tendency due to factor X deficiency and marked hepatic involvement of amyloidosis. The patient died due to severe liver dysfunction two weeks after admission. The diagnosis was confirmed histologically by AL-λ amyloidosis, with the liver and spleen as the main lesions, on an autopsy. As treatment-related toxicity is strong in advanced cases, appropriate treatments are required to improve the prognosis of AL amyloidosis with severe liver dysfunction.

Hypoxia-inducible KDM3A addiction in multiple myeloma.

In multiple myeloma (MM), the bone marrow (BM) microenvironment may contain a myeloma cell fraction that has acquired treatment resistance by undergoing an epigenetic gene expression change. Hypoxic stress is an important factor in the BM microenvironment. Recently, we demonstrated that miR-210 was upregulated in hypoxia and downregulated IRF4, which is known as an essential factor in myeloma oncogenesis in normoxia. In the study, we demonstrated that myeloma cells still showed a strong antiapoptotic phenotype despite IRF4 downregulation, suggesting that another antiapoptotic factor might be involved under hypoxic stress. To determine the factor or factors, we conducted gene expression analysis on myeloma cells (primary samples and cell lines) that were exposed to chronic hypoxia and observed upregulation of glycolytic genes and genes encoding H3K9 demethylases in myeloma cells with hypoxia. Among these, KDM3A was most significantly upregulated in all examined cells, and its knockdown induced apoptosis of myeloma cells in chronic hypoxia. Expression of KDM3A was dependent on HIF-1α, which is a transcription factor specifically upregulated in hypoxia. We further demonstrated that an essential target of KDM3A was a noncoding gene, MALAT1, whose upregulation contributed to acquisition of an antiapoptotic phenotype by accumulation of HIF-1α, leading to upregulation of glycolytic genes under hypoxia. This process was independent from IRF4. These results led us to conclude that the hypoxia-inducible HIF-1α-KDM3A-MALAT1 axis also contributes to acquisition of the antiapoptotic phenotype via upregulation of glycolysis-promoting genes. Thus, this axis is a promising therapeutic target against myeloma cells in the BM microenvironment.

Histone deacetylase inhibitors inhibit metastasis by restoring a tumor suppressive microRNA-150 in advanced cutaneous T-cell lymphoma.

Tumor suppressive microRNA (miR)-150 inhibits metastasis by combining with the C-C chemokine receptor 6 (CCR6) "seed sequence" mRNA of the 3'-untranslated region (3'-UTR) in advanced cutaneous T-cell lymphoma (CTCL). Because the histone deacetylase inhibitor (HDACI) vorinostat showed excellent outcomes for treating advanced CTCL, HDACIs may reduce the metastasis of CTCL by targeting miR-150 and/ or CCR6. To examine whether these candidate molecules are essential HDACI targets in advanced CTCL, we used the My-La, HH, and HUT78 CTCL cell lines for functional analysis because we previously demonstrated that their xenografts in NOD/Shi-scid IL-2γnul mice (CTCL mice) induced multiple metastases. We found that pan- HDACIs (vorinostat and panobinostat) inhibited the migration of CTCL cells and downregulated CCR6. The miRNA microarray analysis against CTCL cell lines demonstrated that these pan-HDACIs commonly upregulated 161 miRNAs, including 34 known tumor suppressive miRNAs such as miR-150. Although 35 miRNAs possessing the CCR6 "seed sequence" were included in these 161 miRNAs, miR-150 and miR-185-5p were downregulated in CTCL cells compared to in normal CD4+ T-cells. The transduction of 12 candidate miRNAs against CTCL cells revealed that miR-150 most efficiently inhibited their migration capabilities and downregulated CCR6. Quantitative reverse transcriptase-polymerase chain reaction demonstrated that miR-150 was downregulated in advanced but not early CTCL primary cases. Finally, we injected miR-150 or siCCR6 into CTCL mice and found that mouse survival was significantly prolonged. These results indicate that miR-150 and its target, CCR6, are essential therapeutic targets of pan-HDACIs in advanced CTCL with metastatic potential.

Disruption of CCL20-CCR6 interaction inhibits metastasis of advanced cutaneous T-cell lymphoma.

We recently demonstrated that upregulation of a chemokine receptor CCR6 and its ligand CCL20 led to metastasis of advanced cutaneous T-cell lymphoma (CTCL) cells, suggesting the involvement of CCL20-CCR6 interaction in initiating CTCL cell metastasis. In this study, we determined whether this interaction is functional in metastatic CTCL cells. We first demonstrated increased STAT3 expression during the progression of primary CTCL. STAT3 was spontaneously activated and mediated the transcription of CCL20 in CTCL cell lines. Next, to determine whether the transient knockdown of STAT3, CCL20, or CCR6 or treatment with neutralizing antibody against CCL20 (neutralizing CCL20 antibody) could reduce the migration ability of CTCL cells, we conducted an in vitro migration assay. All treatments reduced the nutrition-dependent migration activity of CTCL cells. Notably, treatment with neutralizing CCL20 antibody reduced the migration ability of the cells without decreasing the expression of CCL20 and CCR6. This demonstrated that the CCL20-CCR6 interaction is actually functional in metastatic CTCL cells. Finally, to examine the in vivo effect of neutralizing CCL20 antibody, we used NOD/Shi-scid IL-2γnul mice inoculated with CTCL cells. These mice were expected to die due to metastasis of CTCL cells into multiple organs. However, administration of neutralizing CCL20 antibody significantly prolonged the survival of the xenografted mice. These findings suggested that automatic activation of the STAT3/CCL20/CCR6 cascade was involved in CTCL lymphomagenesis and that disruption of CCL20-CCR6 interaction could be a key therapeutic strategy against advanced CTCL.