Discovery of orally bioavailable inhibitors of MALT1 with in vivo activity for psoriasis.

We report the design, synthesis, and biological activity of a series of compounds that exhibit potent mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) inhibition. Structural transformation of the substructures of a starting compound gave amidomethyl derivatives and sulfonylguanidine derivatives that exhibited potent inhibition of MALT1. Compound 37 had good oral bioavailability and showed anti-psoriatic activity in an imiquimod-induced psoriasis mouse model after oral administration.

Structure-activity relationship studies of 3-substituted pyrazoles as novel allosteric inhibitors of MALT1 protease.

We report the discovery of a novel series of 1,5-bisphenylpyrazoles as potent MALT1 inhibitors. Structure-activity relationship exploration of a hit compound led to a potent MALT1 inhibitor. Compound 33 showed strong activity against MALT1 (IC50: 0.49 µM), potent cellular activity (NF-κB inhibition and inhibition of IL2 production), and high selectivity against caspase-3, -8, and -9. The results of a kinetics study suggest that compound 33 is a non-competitive inhibitor of MALT1 protein.

A prostacyclin analog prevents the regression of renal microvascular network by inhibiting mitochondria-dependent apoptosis in the kidney of rat progressive glomerulonephritis.

We have previously demonstrated that renoprotective effects of a prostacyclin analog, beraprost sodium, on the kidney of anti-glomerular basement membrane glomerulonephritis (GN) rats. The aim of this study is to address the renoprotection mechanism of beraprost sodium, especially in the terminal stage of GN. Beraprost sodium was orally administrated from 2 to 7 weeks after induction of GN, and renal function, morphology, protein and mRNA levels were analyzed. We found the beraprost sodium treatment suppressed the structural regression of renal microvascular network and decline of renal blood flow occurred in the kidney of GN rats. To address the mechanism of the structural maintenance, we focused on apoptosis because the increased number of apoptotic renal microvascular endothelial cells and tubular epithelial cells was observed in the kidneys of GN rats as compared with normal and beraprost sodium treated rats. Protein and mRNA analyses demonstrated that mitochondria-dependent apoptotic pathway was activated in the kidneys of GN rats, and beraprost sodium suppressed the activation by modulating the expression patterns of pro- and anti-apoptotic factors. These results suggest that inhibition of mitochondria-dependent apoptosis of renal cells in GN kidney and consequent maintenance of renal functional structures, including microvascular network might contribute to the renoprotective effect of beraprost sodium in GN.

Immune suppression and resistance mediated by constitutive activation of Wnt/ß-catenin signaling in human melanoma cells.

Cancer-induced immunosuppression is a major problem reducing antitumor effects of immunotherapies, but its molecular mechanism has not been well understood. We evaluated immunosuppressive roles of activated Wnt/ß-catenin pathways in human melanoma for dendritic cells (DCs) and CTLs. IL-10 expression was associated with ß-catenin accumulation in human melanoma cell lines and tissues and was induced by direct ß-catenin/TCF binding to the IL-10 promoter. Culture supernatants from ß-catenin-accumulated melanoma have activities to impair DC maturation and to induce possible regulatory DCs. Those immunosuppressive culture supernatant activities were reduced by knocking down ß-catenin in melanoma cells, partly owing to downregulation of IL-10. Murine splenic and tumor-infiltrating DCs obtained from nude mice implanted with human mutant ß-catenin-overexpressed melanoma cells had less ability to activate T cells than did DCs from mice with control melanoma cells, showing in vivo suppression of DCs by activated Wnt/ß-catenin signaling in human melanoma. This in vivo DC suppression was restored by the administration of a ß-catenin inhibitor, PKF115-584. ß-catenin-overexpressed melanoma inhibited IFN-γ production by melanoma-specific CTLs in an IL-10-independent manner and is more resistant to CTL lysis in vitro and in vivo. These results indicate that Wnt/ß-catenin pathways in human melanoma may be involved in immunosuppression and immunoresistance in both induction and effector phases of antitumor immunoresponses partly through IL-10 production, and they may be attractive targets for restoring immunocompetence in patients with Wnt/ß-catenin-activated melanoma.

Toll-like receptor 4 signaling promotes the migration of human melanoma cells.

Immune cell Toll-like receptors (TLRs) recognize conserved microbial components, leading to immune and inflammatory responses. However, TLRs are also expressed in cancer cells, including melanoma cells, which express TLR2-4. TLR4 ligands have received attention as immunotherapies; therefore, we assessed the expression of TLR4 in human melanoma specimens (29 primary lesions and 28 metastatic lesions) representing different types of melanoma. A high percentage (≥ 90%) of melanoma lesions expressed TLR4, as judged by immunohistochemistry. Next, the role of TLR4 in cell proliferation and migration was assessed using the TLR4-positive (TLR4(+)) melanoma cell lines 501mel and 888mel, and TLR4-negative (TLR4(‒)) 928mel melanoma cells. Lipopolysaccharide (LPS), a TLR4 agonist, increased the proliferation of TLR4(+) melanoma cells but not of TLR4(‒) 928mel cells. The proliferation-inducing effect of LPS in 888mel cells was abolished by blockade of TLR4 signaling via treatment with short interfering RNA (siRNA) targeting TLR4 or myeloid differentiation primary response gene 88 (MyD88), a molecule downstream of TLR4. However, knockdown of TLR4 or MyD88 expression did not affect the LPS-induced proliferation of 501mel cells, suggesting that residual TLR4 signaling is sufficient to maintain cell proliferation. By contrast, LPS increased the migration of TLR4(+) melanoma cells, and this effect was substantially inhibited by TLR4 or MyD88 knockdown. Furthermore, TLR4 knockdown decreased cell migration even in the absence of LPS, suggesting the presence of an endogenous TLR4 ligand(s) in melanoma cells. TLR4 signaling may contribute to melanoma progression, and caution should be exercised when using TLR4 ligands as adjuvant therapy for cancer.

Effect of RNA interference of BID and BAX mRNAs on apoptosis in granulosa cell-derived KGN cells.

In mitochondrion-dependent type II apoptosis, BH3-interacting domain death agonist (BID) and BCL-2-associated X protein (BAX) promote death ligand and receptor-mediated cell death. In porcine ovaries, the levels of BID and BAX increase in follicular granulosa cells during atresia. In the present study, to confirm the pro-apoptotic activity of BID and BAX in granulosa cells, we examined the effect of RNA interference of BID or BAX on apoptosis using a human ovarian granulosa tumor cell line, KGN. By reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, expression of BID and BAX was detected in KGN cells. Then, we suppressed BID and BAX mRNA expression in KGN cells using small interfering RNA (siRNA). When BID or BAX was suppressed, a significant decrease in the apoptotic cell rate was noted. In granulosa-derived cells, BID and BAX showed pro-apoptotic activity. These results suggest that BID and BAX act as signal-transducing factors in mitochondrion-dependent type II apoptosis.

Role of cell-death ligand-receptor system of granulosa cells in selective follicular atresia in porcine ovary.

In the mammalian ovary, more than 99% of follicles degenerate without ovulation and few oocytes ovulate and succeed to the next generation. Granulosa cell apoptosis plays a critical role in this process, follicular atresia. However, the molecular mechanisms responsible for the regulation of granulosa cell apoptosis have not been clarified. Death ligand and receptor systems are major apoptosis-inducing factors. This review describes the granulosa cell apoptosis via death ligand and receptor systems during follicular atresia in the porcine ovary.

Expression and function of apoptosis initiator FOXO3 in granulosa cells during follicular atresia in pig ovaries.

In mammalian ovaries, most follicles are lost by atresia before ovulation. It has become apparent that the apoptosis of granulosa cells induces follicular atresia. Forkhead box O3 (FOXO3), also called FKHRL1 (forkhead in rhabdomyosarcoma-like 1), is a proapoptotic molecule that belongs to the FOXO subfamily of forkhead transcription factors. Foxo3-deficient female mice were reported to be infertile because of abnormal ovarian follicular development, but the precise influences of FOXO3 on follicular atresia of mature ovary have not been determined. Therefore, we examined the expression and function of FOXO3 in porcine ovarian follicles and granulosa-derived cells. FOXO3 mRNA levels in granulosa cells of porcine ovaries increased during atresia, while FOXO3 protein was abundant in granulosa cells of early atretic follicles. By immunohistochemistry, the inner surface area of the granulosa layer in early atretic follicles was strongly stained with anti-FOXO3 antibody. The granulosa cells expressing FOXO3 coincided with apoptotic cells, indicating a role of FOXO3 as a proapoptotic factor in granulosa cells of porcine ovaries. In porcine (JC-410) and human (KGN) granulosa-derived cells, cell death was induced by transfection of FOXO3 expression vectors. Expression of the proapoptotic factors Fas ligand (FASLG) and BCL2-like 11 (BCL2L11) was upregulated by FOXO3 in KGN cells. In conclusion, FOXO3 is expressed in porcine ovarian follicles and induces apoptosis in granulosa cells, suggesting that it is a candidate for the initiator of follicular atresia.

Bid and Bax are involved in granulosa cell apoptosis during follicular atresia in porcine ovaries.

More than 99% of follicles undergo "atresia" during follicular development and growth. Follicular atresia is predominantly regulated by granulosa cell apoptosis. However, the intracellular signaling pathway of apoptosis in granulosa cells has not been revealed. In the present study, we examined changes in the expression of BH3-interacting domain death agonist (Bid) and Bcl-2-associated X protein (Bax), which are considered to promote the cell death ligand/receptor-mediated process in mitochondrion-dependent type II apoptosis, in porcine granulosa cells during atresia. Levels of mRNA and protein of Bid and Bax were determined by the reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting techniques, respectively. Levels of Bid and Bax mRNA and protein were markedly increased in granulosa cells of early atretic follicles compared with those of healthy follicles. In situ hybridization and immunohistochemical staining revealed that mRNA and protein of Bid and Bax were present in the granulosa cells, though only traces were found in healthy follicles; however, strong staining was noted in atretic follicles. These results indicate that Bid and Bax appear to be signal transduction factors in granulosa cells during follicular atresia and appear to play proapoptotic roles and confirm that the porcine granulosa cell is a mitochondrion-dependent type II apoptotic cell.

Characteristic distribution of melanin columns in the cornified layer of acquired acral nevus: an important clue for histopathologic differentiation from early acral melanoma.

Clinical and histopathologic differentiation between early acral melanoma and acral nevus is often difficult. Dermoscopy is helpful in this differentiation. On dermoscopy, early acral melanoma shows the parallel ridge pattern showing band-like pigmentation on the ridges of the surface skin markings, whereas a representative dermoscopic pattern in acquired acral nevus is the parallel furrow pattern showing parallel linear pigmentation along the surface furrows. The parallel furrow pattern suggests that melanocytes of acral nevus preferentially proliferate in the crista profunda limitans, an epidermal rete ridge underlying the surface furrow. In the present study, however, we found that in 13 of 18 acquired acral nevi, proliferation of melanocytes were detected not only in the crista profunda limitans but also in the crista profunda intermedia (CPI), an epidermal rete ridge underlying the surface ridge. Very interestingly, Fontana-Masson staining of these acral nevi revealed that even when proliferation of melanocytes was prominent in the CPI, melanin granules in the cornified layer were observed as regular melanin columns situated under the surface furrows and were hardly detected under the surface ridges. These findings indicate that in acral nevus, melanin granules produced by melanocytes in the CPI are not transferred to the upper epidermis. Hence, we must be careful not to overdiagnose an acral melanocytic lesion in which an increased number of melanocytes are detected in the CPI. Even in such a case, if melanin granules in the cornified layer are detected as melanin columns regularly distributed under the surface furrows, the lesion is strongly suggested to be a benign acral nevus.

mRNA expression and BRAF mutation in circulating melanoma cells isolated from peripheral blood with high molecular weight melanoma-associated antigen-specific monoclonal antibody beads.

BACKGROUND: The detection of circulating tumor cells (CTCs) in the peripheral blood of melanoma patients by quantitative real-time reverse-transcription PCR (qRT-PCR) analysis correlates with a poor prognosis. The assessment of CTCs from blood has been difficult because of lack of a good monoclonal antibody (mAb) directed against surface cell antigens to capture melanoma cells. METHODS: Blood was collected prospectively from 57 melanoma patients (43 test and 14 test-development cases) and 5 healthy donors. High molecular weight melanoma-associated antigen (HMW-MAA)-specific mAbs bound to immunomagnetic beads were used to isolate CTCs. mRNA and/or DNA were extracted from CTCs. Testing for the expression of a melanoma-associated gene panel (MLANA, MAGEA3, and MITF) with qRT-PCR and for the presence of BRAFmt (a BRAF gene variant encoding the V600E mutant protein) verified the beads-isolated CTCs to be melanoma cells. A peptide nucleic acid-clamping PCR assay was used for BRAFmt analysis. RESULTS: Spiking of peripheral blood cells (PBCs) with melanoma cells showed that the beads-based detection assay can detect approximately 1 melanoma cell in 5 x 10(6) PBCs. qRT-PCR analysis detected MLANA, MAGEA3, and MITF expression in 19 (44%), 29 (67%), and 19 (44%) of the patients, respectively. At least one biomarker of the panel was positive in 40 (93%) of the 43 melanoma patients. BRAFmt was detected in 17 (81%) of the 21 assessed stage IV melanoma patients. CONCLUSION: The assay of bead capture coupled with the PCR has utility for assessing CTCs in melanoma patients, which can then be characterized for both genomic and transcriptome expression.

Activation of CCR9/CCL25 in cutaneous melanoma mediates preferential metastasis to the small intestine.

PURPOSE: Specific chemokines and their respective receptors have been implicated in distant tumor cell metastasis. Cutaneous melanoma has a distinct pattern of metastasis, preferentially targeting the submucosa of the small intestine. However, the underlying pathogenic mechanism remains unknown. Migration of CCR9(+) lymphocytes to the small intestine is known to occur in response to the chemoattractant effects of CCL25 (thymus-expressed chemokine). The integrin heterodimers alphabeta are also known to be important mediators of cellular adhesion. We hypothesize that the mechanism of small intestinal metastasis by melanoma is via the CCR9-CCL25 axis and specific integrins. EXPERIMENTAL DESIGN: Quantitative reverse transcription-PCR, flow cytometry, and immunohistochemistry were used to assess melanoma tumors for CCR9 and CCL25. Integrin expression was assessed using flow cytometry. CCR9 expression by quantitative reverse transcription-PCR was assessed in primary (n = 23) and metastatic (n = 198) melanomas, and melanoma lines derived from small intestinal metastases (n = 23). RESULTS: We showed CCR9 expression in 88 of 102 paraffin-embedded metastatic melanomas from the small intestine, 8 of 8 melanoma lines derived from metastases in the small intestine, and 0 of 96 metastatic melanomas from other sites. In vitro migration and invasion studies done on CCR9(+) melanoma lines showed migration in response to CCL25 that was inhibited by anti-CCR9 antibody or by short interfering RNA CCR9. Flow cytometric analysis confirmed CCR9 expression by melanomas to the small intestine and showed concomitant alpha(4)beta(1) integrin expression. CONCLUSIONS: Our findings show that functionally active CCR9 on melanoma cells facilitates metastasis to the small intestine. The CCR9-CCL25 axis may explain the high incidence of melanoma metastasis to this specific location.

Human high molecular weight-melanoma-associated antigen: utility for detection of metastatic melanoma in sentinel lymph nodes.

PURPOSE: Detection of micrometastasis in melanoma-draining lymph nodes is important for staging and prognosis. Immunohistochemical staining (IHC) using S-100p-HMB-45-, and MART-1-specific antibodies is used for detecting metastases in sentinel lymph nodes (SLN). However, improvement in IHC is needed for melanoma micrometastasis detection. EXPERIMENTAL DESIGN: Paraffin-embedded archival tissue (PEAT) specimens were obtained from 42 non-SLN macrometastases, 42 SLN metastases, and 16 tumor-negative SLNs of 100 melanoma patients who underwent SLN biopsy. PEAT specimens were assessed by IHC with high molecular weight-melanoma-associated antigen (HMW-MAA)-specific monoclonal antibodies (mAb) and with S-100p-, HMB-45-, and MART-1-specific antibodies. Quantitative real-time reverse-transcriptase PCR assay was used for HMW-MAA and MART-1 mRNA detection. RESULTS: Expression frequency and immunostaining intensity were higher for HMW-MAA than MART-1 in nodal macrometastases (P < 0.0001 and P < 0.0001, respectively) and micrometastases (P < 0.0001 and P = 0.004, respectively). All 52 (100%) macrometastases were positive with HMW-MAA-specific mAbs, whereas 43 (83%) were positive with MART-1-specific mAbs. In a comparison analysis, 23 of 23 (100%) micrometastases were HMW-MAA-positive, whereas 21 (91%) and 18 (78%) specimens were S-100p- and HMB-45-positive, respectively. Quantitative real-time reverse-transcriptase PCR analysis of 48 nodal metastases showed HMW-MAA mRNA detection in SLNs with metastases. CONCLUSIONS: HMW-MAA is more sensitive and specific than MART-1, S-100p, and HMB-45 for IHC-based detection of SLN micrometastases. SLN PEAT-based detection specificity of melanoma micrometastases can be improved by IHC with HMW-MAA-specific mAbs.

Activation of Toll-like receptors 2, 3, and 4 on human melanoma cells induces inflammatory factors.

Toll-like receptors (TLR) have been shown to be expressed on various types of cancers; however, their functional activity is not known. We examined TLR profiles of human melanoma cells and showed that TLR2, TLR3, and TLR4 were found to be highly expressed. By PCR array analysis, specific stimulation of TLR2, TLR3, and TLR4 on melanoma cells showed significant activation of the adaptor protein MyD88, as well as downstream signal transduction factors nuclear factor-kappaB and inflammatory response-related factors. Specific ligand activation of TLR2, TLR3, and TLR4 was shown to induce cell migration. Peripheral blood lymphocytes and melanoma purified RNA was shown to activate TLR3 on melanoma cells. These studies show expression and functional activity of specific TLRs on melanoma cells and as potential therapeutic targets to control tumor progression.

Detection of circulating tumor cells in early-stage breast cancer metastasis to axillary lymph nodes.

PURPOSE: Clinical and pathologic prognostic factors do not always accurately predict disease outcome. Patients with early-stage breast cancer may harbor clinically significant but undetected systemic disease. We hypothesized that a multimarker quantitative real-time reverse transcription-PCR (qRT) assay could detect circulating tumor cells (CTC) in patients with early-stage breast cancer and correlate with sentinel lymph node (SLN) and non-SLN metastasis status. EXPERIMENTAL DESIGN: Blood samples from 90 women with the American Joint Committee on Cancer stages I to III breast cancer and 39 age-matched normal healthy volunteers were assessed by qRT for mRNA expression of three markers: stanniocalcin-1 (STC-1), N-acetylgalactosaminyltransferase (GalNacT), and melanoma antigen gene family-A3 (MAGE-A3). CTC biomarker detection was correlated with overall axillary LN (ALN), SLN, and non-SLN histopathology status. RESULTS: CTCs were detected in 39 of 90 (43%) patients, but not in normal volunteers. At least one CTC biomarker was detected in 10 of 35 (29%) stage I patients, 19 of 42 (45%) stage II patients, and 10 of 13 (77%) stage III patients. In multivariate analysis, only lymphovascular invasion and >or=2 CTC biomarkers detected significantly correlated with ALN metastasis [odds ratio (OR), 12.42; 95% confidence interval (95% CI), 3.52-43.77, P<0.0001; and OR, 3.88; 95% CI, 1.69-8.89, P=0.001, respectively]. The number of CTC biomarkers detected similarly correlated with SLN and non-SLN metastasis status (P=0.0004). At least one CTC biomarker was detected in 10 of 11 (91%) patients with non-SLN metastases. CONCLUSION: The detection of CTCs offers a novel means to assess the presence of systemic disease spreading relative to SLN and ALN histopathology status.

A new melanoma antigen fatty acid-binding protein 7, involved in proliferation and invasion, is a potential target for immunotherapy and molecular target therapy.

The identification of molecules that are preferentially expressed in melanoma cells and involved in their malignant phenotypes is important for understanding melanoma biology and the development of new diagnostic and therapeutic methods. By comparing the expression profile of a melanoma cell line with those of various normal tissues using GeneChip and by confirming the actual expression of the selected genes by reverse transcription-PCR and Northern and Western blot analyses, fatty acid-binding protein 7 (FABP7), which is frequently expressed in melanomas, was identified. Immunohistochemical examination revealed that FABP7 was expressed in 11 of 15 melanoma tissues. By down-regulating the FABP7 expression with FABP7-specific small interfering RNAs, in vitro cell proliferation and Matrigel invasion were suppressed in two of six melanoma cell lines. Overexpression of FABP7 in a FABP7-negative embryonic kidney cell line 293T by transfecting with the FABP7 cDNA resulted in enhanced cell proliferation and Matrigel invasion, indicating that FABP7 plays a role in the malignant phenotype of some melanoma cell lines. IgG antibodies specific for the phage or bacterial recombinant FABP7 protein were detected in 14 of 25 (56%) or in 8 of 31 (26%) sera from melanoma patients, respectively, but not in sera from healthy individuals, indicating that FABP7 is an immunogenic antigen in melanoma patients. These results showed that FABP7 is frequently expressed in melanoma, may be involved in cell proliferation and invasion, and may be a potential target for development of diagnostic and therapeutic methods.

A novel cancer testis antigen that is frequently expressed in pancreatic, lung, and endometrial cancers.

PURPOSE: To isolate cancer testis antigens that are expressed in pancreatic cancers and may be useful in clinical applications. EXPERIMENTAL DESIGN: To efficiently isolate cancer testis antigens, a testis cDNA library was immunoscreened (SEREX) with serum from a patient with pancreatic ductal adenocarcinoma. The expression of isolated antigens in various cancer cell lines and tissues was evaluated by reverse transcription-PCR and Northern blot analyses. The immunogenicity of the antigen in cancer patients was evaluated by detection of the IgG antibody in sera from patients with various cancers. RESULTS: Of the three clones isolated through screening of a total of 2 x 10(6) cDNA library clones, one clone (KU-CT-1) was found to be expressed in various cancers but only in testis among normal tissues, indicating that it was a novel cancer testis antigen. The KU-CT-1 gene is located on chromosome 10p12 and produces two splice variants, which encode proteins of 397 and 872 amino acids, respectively. KU-CT-1 was expressed in pancreatic cancer tissues (3 of 9, 33%), lung cancer tissues (9 of 24, 38%), and endometrial cancer tissues (7 of 11, 64%). Specific serum IgG antibodies were detected in 3 of 20 pancreatic cancer patients, 2 of 12 endometrial cancer patients, 1 of 18 colon cancer patients, and 1 of 10 prostate cancer patients but not detected in 30 healthy individuals. CONCLUSIONS: KU-CT-1 is a new cancer testis antigen that is expressed in pancreatic, lung, and endometrial cancers and may be useful for diagnosis and immunotherapy for patients with various cancers.