Genetic Deletion of a Single Immunodominant T-cell Response Confers Susceptibility to Virus-induced Demyelination

An important question in neuropathology involves determining the antigens that are targeted during demyelinating disease. Viral infection of the central nervous system (CNS) leads to T-cell responses that can be protective as well as pathogenic. In the Theiler’s murine encephalomyelitis virus (TMEV) model of demyelination it is known that the immune response to the viral capsid protein 2 (VP2) is critical for disease pathogenesis. This study shows that expressing the whole viral capsid VP2 or the minimal CD8-specific peptide VP2(121-130) as “self” leads to a loss of VP2-specific immune responses. Loss of responsiveness is caused by T cell-specific tolerance, as VP2-specific antibodies are generated in response to infection. More importantly, these mice lose the CD8 T-cell response to the immunodominant peptide VP2(121-130), which is critical for the development of demyelinating disease. The transgenic mice fail to clear the infection and develop chronic demyelinating disease in the spinal cord white matter. These findings demonstrate that T-cell responses can be removed by transgenic expression and that lack of responsiveness alters viral clearance and CNS pathology. This model will be important for understanding the mechanisms involved in antigen-specific T-cell deletion and the contribution of this response to CNS pathology

Timp1 interacts with beta-1 integrin and CD63 along melanoma genesis and confers anoikis resistance by activating PI3-K signaling pathway independently of Akt phosphorylation

Background: Anoikis resistance is one of the abilities acquired along tumor progression. This characteristic is associated with metastasis development, since tumorigenic cells must survive independently of cell-matrix interactions in this process. in our laboratory, it was developed a murine melanocyte malignant transformation model associated with a sustained stressful condition. After subjecting melan-a melanocytes to 1, 2, 3 and 4 cycles of anchorage impediment, anoikis resistant cells were established and named 1C, 2C, 3C and 4C, respectively. These cells showed altered morphology and PMA independent cell growth, but were not tumorigenic, corresponding to pre-malignant cells. After limiting dilution of 4C pre-malignant cells, melanoma cell lines with different characteristics were obtained. Previous data from our group showed that increased Timp1 expression correlated with anoikis-resistant phenotype. Timp1 was shown to confer anchorage-independent growth capability to melan-a melanocytes and render melanoma cells more aggressive when injected into mice. However, the mechanisms involved in anoikis regulation by Timp1 in tumorigenic cells are not clear yet.Methods: the beta 1-integrin and Timp1 expression were evaluated by Western blotting and CD63 protein expression by flow cytometry using specific antibodies. To analyze the interaction among Timp1, CD63 and beta 1-integrin, immunoprecipitation assays were performed, anoikis resistance capability was evaluated in the presence or not of the PI3-K inhibitors, Wortmannin and LY294002. Relative expression of TIMP1 and CD63 in human metastatic melanoma cells was analyzed by real time PCR.Results: Differential association among Timp1, CD63 and beta 1-integrins was observed in melan-a melanocytes, 4C pre-malignant melanocytes and 4C11- and 4C11+ melanoma cells. Timp1 present in conditioned medium of melanoma cells rendered melan-a melanocytes anoikis-resistant through PI3-K signaling pathway independently of Akt activation. in human melanoma cell lines, in which TIMP1 and beta-1 integrin were also found to be interacting, TIMP1 and CD63 levels together was shown to correlate significantly with colony formation capacity.Conclusions: Our results show that Timp1 is assembled in a supramolecular complex containing CD63 and beta 1-integrins along melanoma genesis and confers anoikis resistance by activating PI3-K signaling pathway, independently of Akt phosphorylation. in addition, our data point TIMP1, mainly together with CD63, as a potential biomarker of melanoma.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Dept Pharmacol, São Paulo, BrazilUniversidade Federal de São Paulo, Microbiol Immunol & Parasitol Dept, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilLudwig Inst Canc Res, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Pharmacol, São Paulo, BrazilUniversidade Federal de São Paulo, Microbiol Immunol & Parasitol Dept, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilFAPESP: 2011/12306-1FAPESP: 2010/18715-8CAPES: 2867/10Web of Scienc

Tetraspanin CD151 is a novel prognostic marker in poor outcome endometrial cancer

BACKGROUND: Type II cancers account for 10% of endometrial cancers but 50% of recurrence. Response rates to chemotherapy at recurrence are poor and better prognostic markers are needed to guide therapy. CD151 is a small transmembrane protein that regulates cell migration and facilitates cancer metastasis. High CD151 expression confers poor prognosis in breast, pancreatic and colorectal cancer. The prognostic significance of tetraspanin CD151 expression in poor outcome endometrial cancers was evaluated, along with oestrogen receptor (ER), progesterone receptor (PR), p53, human epidermal growth factor receptor -2 (HER-2), and CD 151 staining compared with α6β1, α3β1 integrins, and E-cadherin. METHODS: Tissue microarray constructed from 156 poor outcome endometrial cancers, tested with immunohistochemistry and staining correlated with clinicopathological data were used. A total of 131 data sets were complete for analysis. RESULTS: Expression of CD151 was significantly higher in uterine papillary serous and clear cell carcinoma than in grade 3 endometrioid carcinoma, sarcoma or carcinosarcoma (P<0.001). In univariate analysis, age, stage, histology type and CD151 were significant for both recurrence free (RFS) and disease specific survival (DSS). In multivariate analyses, CD151 was significant for RFS and DSS (P=0.036 and 0.033, respectively) in triple negative (ER, PR and HER-2 negative) tumours (88/131). The HER-2, p53, ER and PR were not prognostic for survival. There was strong concordance of CD151 with E-cadherin (98%), but not with α6β1 (35%), α3β1 staining (60%). CONCLUSION: The CD151 is a novel marker in type 2 cancers that can guide therapeutic decisions. CD151 may have an important role in tumourigenesis in some histology types

Inhibition of Hedgehog Signaling Antagonizes Serous Ovarian Cancer Growth in a Primary Xenograft Model

Recent evidence links aberrant activation of Hedgehog (Hh) signaling with the pathogenesis of several cancers including medulloblastoma, basal cell, small cell lung, pancreatic, prostate and ovarian. This investigation was designed to determine if inhibition of this pathway could inhibit serous ovarian cancer growth.We utilized an in vivo pre-clinical model of serous ovarian cancer to characterize the anti-tumor activity of Hh pathway inhibitors cyclopamine and a clinically applicable derivative, IPI-926. Primary human serous ovarian tumor tissue was used to generate tumor xenografts in mice that were subsequently treated with cyclopamine or IPI-926.Both compounds demonstrated significant anti-tumor activity as single agents. When IPI-926 was used in combination with paclitaxel and carboplatinum (T/C), no synergistic effect was observed, though sustained treatment with IPI-926 after cessation of T/C continued to suppress tumor growth. Hh pathway activity was analyzed by RT-PCR to assess changes in Gli1 transcript levels. A single dose of IPI-926 inhibited mouse stromal Gli1 transcript levels at 24 hours with unchanged human intra-tumor Gli1 levels. Chronic IPI-926 therapy for 21 days, however, inhibited Hh signaling in both mouse stromal and human tumor cells. Expression data from the micro-dissected stroma in human serous ovarian tumors confirmed the presence of Gli1 transcript and a significant association between elevated Gli1 transcript levels and worsened survival.IPI-926 treatment inhibits serous tumor growth suggesting the Hh signaling pathway contributes to the pathogenesis of ovarian cancer and may hold promise as a novel therapeutic target, especially in the maintenance setting

Properties of resistant cells generated from lung cancer cell lines treated with EGFR inhibitors

<p>Abstract</p> <p>Background</p> <p>Epidermal growth factor receptor (EGFR) signaling plays an important role in non-small cell lung cancer (NSCLC) and therapeutics targeted against EGFR have been effective in treating a subset of patients bearing somatic EFGR mutations. However, the cancer eventually progresses during treatment with EGFR inhibitors, even in the patients who respond to these drugs initially. Recent studies have identified that the acquisition of resistance in approximately 50% of cases is due to generation of a secondary mutation (T790M) in the EGFR kinase domain. In about 20% of the cases, resistance is associated with the amplification of MET kinase. In the remaining 30-40% of the cases, the mechanism underpinning the therapeutic resistance is unknown.</p> <p>Methods</p> <p>An erlotinib resistant subline (H1650-ER1) was generated upon continuous exposure of NSCLC cell line NCI-H1650 to erlotinib. Cancer stem cell like traits including expression of stem cell markers, enhanced ability to self-renew and differentiate, and increased tumorigenicity <it>in vitro </it>were assessed in erlotinib resistant H1650-ER1 cells.</p> <p>Results</p> <p>The erlotinib resistant subline contained a population of cells with properties similar to cancer stem cells. These cells were found to be less sensitive towards erlotinib treatment as measured by cell proliferation and generation of tumor spheres in the presence of erlotinib.</p> <p>Conclusions</p> <p>Our findings suggest that in cases of NSCLC accompanied by mutant EGFR, treatment targeting inhibition of EGFR kinase activity in differentiated cancer cells may generate a population of cancer cells with stem cell properties.</p

Inhibition of Hedgehog Signaling Decreases Proliferation and Clonogenicity of Human Mesenchymal Stem Cells

Human mesenchymal stem cells (hMSC) have the ability to differentiate into osteoblasts, adipocytes and chondrocytes. We have previously shown that hMSC were endowed with a basal level of Hedgehog signaling that decreased after differentiation of these cells. Since hMSC differentiation is associated with growth-arrest we investigated the function of Hh signaling on cell proliferation. Here, we show that inhibition of Hh signaling, using the classical inhibitor cyclopamine, or a siRNA directed against Gli-2, leads to a decrease in hMSC proliferation. This phenomenon is not linked to apoptosis but to a block of the cells in the G0/G1 phases of the cell cycle. At the molecular level, it is associated with an increase in the active form of pRB, and a decrease in cyclin A expression and MAP kinase phosphorylation. Inhibition of Hh signaling is also associated with a decrease in the ability of the cells to form clones. By contrast, inhibition of Hh signaling during hMSC proliferation does not affect their ability to differentiate. This study demonstrates that hMSC are endowed with a basal Hedgehog signaling activity that is necessary for efficient proliferation and clonogenicity of hMSC. This observation unravels an unexpected new function for Hedgehog signaling in the regulation of human mesenchymal stem cells and highlights the critical function of this morphogen in hMSC biology

Association of CD99 short and long forms with MHC class I, MHC class II and tetraspanin CD81 and recruitment into immunological synapses

<p>Abstract</p> <p>Background</p> <p>CD99, a leukocyte surface glycoprotein, is broadly expressed in many cell types. On the cell surface, CD99 is expressed as two distinct isoforms, a long form and a short form. CD99 has been demonstrated to play a key role in several biological processes, including the regulation of T cell activation. However, the molecular mechanisms by which CD99 participates in such processes are unclear. As CD99 contains a short cytoplasmic tail, it is unlikely that CD99 itself takes part in its multi-functions. Association of CD99 with other membrane proteins has been suggested to be necessary for exerting its functions.</p> <p>Results</p> <p>In this study, we analyzed the association of CD99 with other cell surface molecules involved in T cell activation. We demonstrate the association of MHC class I, MHC class II and tetraspanin CD81 with CD99 molecules on the cell surface. Association of CD99 with its partners was observed for both isoforms. In addition, we determined that CD99 is a lipid raft-associated membrane protein and is recruited into the immunologic synapse during T cell activation. The implication of CD99 on T cell activation was investigated. Inhibition of anti-CD3 induced T cell proliferation by an anti-CD99 monoclonal antibody was observed.</p> <p>Conclusions</p> <p>We provide evidence that CD99 directly interact and form the complex with the MHC class I and II, and tetraspanin CD81, and is functionally linked to the formation of the immunologic synapse. Upon T cell activation, CD99 engagement can inhibit T cell proliferation. We speculate that the CD99-MHC-CD81 complex is a tetraspanin web that plays an important role in T cell activation.</p

Hedgehog-mediated regulation of PPARγ controls metabolic patterns in neural precursors and shh-driven medulloblastoma

Sonic hedgehog (Shh) signaling is critical during development and its aberration is common across the spectrum of human malignancies. In the cerebellum, excessive activity of the Shh signaling pathway is associated with the devastating pediatric brain tumor medulloblastoma. We previously demonstrated that exaggerated de novo lipid synthesis is a hallmark of Shh-driven medulloblastoma and that hedgehog signaling inactivates the Rb/E2F tumor suppressor complex to promote lipogenesis. Indeed, such Shh-mediated metabolic reprogramming fuels tumor progression, in an E2F1- and FASN-dependent manner. Here, we show that the nutrient sensor PPARγ is a key component of the Shh metabolic network, particularly its regulation of glycolysis. Our data show that in primary cerebellar granule neural precursors (CGNPs), proposed medulloblastoma cells-of-origin, Shh stimulation elicits a marked induction of PPARγ alongside major glycolytic markers. This is also documented in the actively proliferating Shh-responsive CGNPs in the developing cerebellum, and PPARγ expression is strikingly elevated in Shh-driven medulloblastoma in vivo. Importantly, pharmacological blockade of PPARγ and/or Rb inactivation inhibits CGNP proliferation, drives medulloblastoma cell death and extends survival of medulloblastoma-bearing animals in vivo. This coupling of mitogenic Shh signaling to a major nutrient sensor and metabolic transcriptional regulator define a novel mechanism through which Shh signaling engages the nutrient sensing machinery in brain cancer, controls the cell cycle, and regulates the glycolytic index. This also reveals a dominant role of Shh in the etiology of glucose metabolism in medulloblastoma and underscores the function of the Shh → E2F1 → PPARγ axis in altering substrate utilization patterns in brain cancers in favor of tumor growth. These findings emphasize the value of PPARγ downstream of Shh as a global therapeutic target in hedgehog-dependent and/or Rb-inactivated tumors

Epithelial to Mesenchymal Transition by TGFβ-1 Induction Increases Stemness Characteristics in Primary Non Small Cell Lung Cancer Cell Line

Cancer Stem Cells (CSCs) hypothesis asserts that only a small subset of cells within a tumour is capable of both tumour initiation and sustainment. The Epithelial-Mesenchymal Transition (EMT) is an embryonic developmental program that is often activated during cancer invasion and metastasis. The aim of this study is to shed light on the relationship between EMT and CSCs by using LC31 lung cancer primary cell line.A549 and LC31 cell lines were treated with 2 ng/ml TGFβ-1 for 30 days, and 80 days, respectively. To evaluate EMT, morphological changes were assessed by light microscopy, immunofluorescence and cytometry for following markers: cytokeratins, e-cadherin, CD326 (epithelial markers) and CD90, and vimentin (mesenchymal markers). Moreover, RT-PCR for Slug, Twist and β-catenin genes were performed. On TGFβ-1 treated and untreated LC31 cell lines, we performed stemness tests such as pneumospheres growth and stem markers expression such as Oct4, Nanog, Sox2, c-kit and CD133. Western Blot for CD133 and tumorigenicity assays using NOD/SCID mice were performed.TGFβ-1 treated LC31 cell line lost its epithelial morphology assuming a fibroblast-like appearance. The same results were obtained for the A549 cell line (as control). Immunofluorescence and cytometry showed up-regulation of vimentin and CD90 and down-regulation of cytocheratin, e-cadherin and CD326 in TGFβ-1 treated LC31 and A549 cell lines. Slug, Twist and β-catenin m-RNA transcripts were up-regulated in TGFβ-1 treated LC31 cell line confirming EMT. This cell line showed also over-expression of Oct4, Nanog, Sox2 and CD133, all genes of stemness. In addition, in TGFβ-1 treated LC31 cell line, an increased pneumosphere-forming capacity and tumours-forming ability in NOD/SCID mice were detectable.The induction of EMT by TGFβ-1 exposure, in primary lung cancer cell line results in the acquisition of mesenchymal profile and in the expression of stem cell markers