Placental protein-1 (Plac1) modulates immune tolerance in mammary tumor cells
ABSTRACT: Plac1 is an X-linked (Xq26) trophoblast gene expressed at high levels in the placenta, at low levels in the testis, but not in other normal somatic tissues. However, it is re-expressed in several malignancies, including breast, colon, lung, gastric, liver and endometrial cancers as well as in most human cancer cell lines. Plac1 contains HLA-A2-restricted epitopes capable of eliciting a cytotoxic T lymphocyte (CTL) response against human breast cancer cells, and colorectal cancer patients with a Plac1-specific CTL response demonstrate long-term survival. To explore the role of Plac1 in cancer, mouse mammary tumor E0771 cells expressing high levels of Plac1 were transduced with a lentivirus expressing a Plac1 shRNA (E0771/shPlac1). The RNeasy Mini Kit was used to purify total RNA from three independent cell culture samples for gene expression analysis. After isolation in parallel, 1 μg of each of the total RNA from E0771/Scram or E0771/Shplac1 was pooled prior to microarray gene expression analysis. E0771/Scram was considered as control group in this experiment.
Project description:Efficient processing of target antigens by the ubiquitin-proteasome-system (UPS) is essential for treatment of cancers by T cell therapies. However, immune escape due to impaired expression of IFN-γ-inducible components of the antigen presentation machinery and consequent inefficient processing of HLA-dependent tumor epitopes can be one important reason for failure of such therapies. Here, we show that repeated short-term co-cultures of Melan-A/MART-1 tumor antigen-expressing melanoma cells with Melan-A/MART-1 (26-35)-specific CTL led to the generation of clones resistant to CTL-mediated cell death. To determine which of the UPS components and its associated pathways was responsible for CTL escape; three UKRV-Mel-15a clones were subjected to microarray gene expression analysis. Three UKRV-Mel-15a-derived melanoma clones were isolated following three repeated short-term exposures to Melan-A/MART (26-35) CTL and harvested for RNA extraction and hybridization on Affymetrix microarrays.
Project description:Transcriptional profiling of SAS cells transfected with pLKO.1-LYRIC shRNA-B expression vector (desinaged as B) and control SAS cells (transfected with pLKO.1 vector, designated as CTL). Goal was to determine the effects of LYRIC knockdown on global SAS cells gene expression. Two-condition experiment, SAS cells transfected with pLKO.1-LYRIC shRNA-B expression vector (desinaged as B) v.s. control SAS cells (transfected with pLKO.1 vector, designated as CTL). Biological replicates: 4 control replicates, 4 transfected replicates.
Project description:Obscurins (~70-870kDa), encoded by the single OBSCN gene, are cytoskeletal proteins originally identified in striated muscles with structural and regulatory roles. Recently, analysis of 13,023 genes in breast and colorectal cancers identified OBSCN as one of the most frequently mutated genes, implicating it in cancer formation. Herein, we studied the expression profile of obscurins in breast, colon and skin cancer cell lines, and their involvement in cell survival. Immunoblot analysis demonstrated significant reduction of obscurins in cancer cells, resulting from decreased mRNA levels and/or the presence of mutant transcripts. In normal epithelium, obscurins localize in cytoplasmic puncta, the cell membrane and the nucleus. Accordingly, subcellular fractionation demonstrated the presence of two novel nuclear isoforms of ~110 and ~120kDa. Non-tumorigenic MCF10A breast epithelial cells stably transduced with shRNAs targeting giant obscurins exhibited increased viability (~30%) and reduced apoptosis (~20%) following exposure to the DNA damaging agent, etoposide. cDNA microarrays followed by Gene Ontology profiling indicated a substantial number of transcripts related to apoptosis and survival are differentially regulated upon obscurin knock-down. Quantitative RT-PCR further indicated that the anti-apoptotic genes BAG-4 and HAX-1 were up-regulated (1.5 and 1.4 fold, respectively), whereas initiator caspase-9 and death caspase-3 transcripts were down-regulated (0.8 and 0.6 fold, respectively). Our findings are the first to pinpoint critical roles for obscurins in cancer development by contributing to the regulation of cell survival. Two-condition experiment: control shRNA- vs. obscurin shRNA-expressing MCF10A cells exposed to 150 uM etoposide. 3 independent replicates per condition.
Project description:Sanjuan2013 - Evolution of HIV T-cell epitope, immune activation model
Model of cellular immune response against HIV.
This model is described in the article:
Immune activation promotes evolutionary conservation of T-cell epitopes in HIV-1.
Sanjuán R, Nebot MR, Peris JB, Alcamí J.
PLoS Biol. 2013 Apr;11(4):e1001523
The immune system should constitute a strong selective pressure promoting viral genetic diversity and evolution. However, HIV shows lower sequence variability at T-cell epitopes than elsewhere in the genome, in contrast with other human RNA viruses. Here, we propose that epitope conservation is a consequence of the particular interactions established between HIV and the immune system. On one hand, epitope recognition triggers an anti-HIV response mediated by cytotoxic T-lymphocytes (CTLs), but on the other hand, activation of CD4(+) helper T lymphocytes (TH cells) promotes HIV replication. Mathematical modeling of these opposite selective forces revealed that selection at the intrapatient level can promote either T-cell epitope conservation or escape. We predict greater conservation for epitopes contributing significantly to total immune activation levels (immunodominance), and when TH cell infection is concomitant to epitope recognition (trans-infection). We suggest that HIV-driven immune activation in the lymph nodes during the chronic stage of the disease may offer a favorable scenario for epitope conservation. Our results also support the view that some pathogens draw benefits from the immune response and suggest that vaccination strategies based on conserved TH epitopes may be counterproductive.
Note from the author:
this version of the model is more general that the one described in the paper. This is because: (i) it can consider simultaneously Th-epitope escape mutants, CTL-epitope escape mutants and full T-cell (Th and CTL) escape mutants, by setting the mutation rate of Th and CTL escape mutants to zero. (ii) It allows for back mutations, which were ignored in all the simulations shown in the paper. (iii) It allows for a fitness cost of escape mutants, which was set to zero in the article. (iv) pAPCs can be infected and produce viruses (the rate of viral production for pAPCs was set to zero in the paper).
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Project description:High-resolution mass spectrometry analysis of Interleukin 2 (IL-2) and Janus kinase (JAK) controlled protein phosphorylations in cytotoxic T lymphocytes (CTL) revealed JAKs coupled IL-2 receptors to diverse and complex serine/threonine kinase-substrate networks. These involved intricate, co-ordinated phosphorylation of transcription factors, chromatin regulators within the nuclear environment, cytosolic mRNA translational machinery, regulators of GTPases, vesicle trafficking proteins and the actin and microtubule cytoskeleton. We also identified an IL-2-JAK independent SRC family Tyr kinase controlled signaling network that regulates ~10% of the CTL phosphoproteome. One key signaling pathway in CTL is mediated by phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and the serine/threonine kinase AKT. Strikingly, SRC family kinase dependent but JAK independent signaling controlled PIP3 levels and AKT activity in CTL. IL-2-JAK controlled signaling pathways thus coordinate with IL-2 independent networks of protein phosphorylation to program CTL fate.
Project description:We evaluated the gene expression profiles of the 1228- and 0316-Glioma-initiating cells (GICs), as well as the original glioblastoma tissues from which they were derived, plus neural stem cells and normal brain tissues. Short- and long-term cultures of 0316-GICs (2 and 6 months, respectively) were also included in the analysis in order to evaluate the potential effects of in vitro culture duration on gene expression. Expressional changes by EZH2 depletion and DZnep treatment were also evaluated.
Project description:In order to investigate the involvement of Hsp27 in splicing, we performed a whole-genome exonic expression profiling of the castration-resistant prostate cancer PC-3 cells treated by Hsp27-siRNA or CTL-siRNA (both in biological duplicates).
Project description:Interactions with the extracellular matrix (ECM) through integrin adhesion receptors provide cancer cells with physical and chemical cues that act in concert with growth factors to support survival and proliferation. Preclinical studies testing beta1 integrin antagonists in (breast) cancer models have shown inhibition of tumor growth and sensitization to radio- or chemotherapy and these strategies are currently evaluated in clinical trials. Here, we show that disruption of beta1 integrin-mediated ECM adhesion attenuates breast tumor growth but dissemination to the lungs from such small tumors can be markedly enhanced. beta1 integrin downregulation induces compensatory upregulation of beta3 integrins, but increased beta3 expression does not lead to enhanced lung metastasis. Instead, beta1 integrin downregulation in human and mouse triple negative, E-cadherin positive breast cancer cells elicits a switch from collective invasion to individual cell migration in 3D ECM. This involves alterations in the TGFbeta-BMP signaling network shifting the balance between miR-200 and ZEB, which causes a block in E-cadherin transcription. The switch is fully reversible: restored beta1 expression reinstates E-cadherin expression and cell cohesion. Moreover, restoring the network at the level of TGFbetaR, ZEB/miR-200 balance, or E-cadherin, restores cohesion and prevents the induction of lung metastasis without affecting tumor growth. These findings reveal that integrin-mediated ECM-attachments regulate a signaling network in control of epithelial characteristics that suppress metastatic spread. This raises concerns with respect to the use of beta1 integrins as cancer drug targets
Project description:We have previously demonstrated that endoxifen is the most important tamoxifen metabolite responsible for eliciting the anti-estrogenic effects of this drug in breast cancer cells expressing estrogen receptor-alpha. However, the relevance of estrogen receptor-beta in mediating endoxifen action has yet to be explored. Therefore, the goals of this study were to determine the differences in the global gene expression profiles elicited by estradiol treatment and endoxifen between parental MCF7 breast cancer cells (expressing estrogen receptor alpha only) and MCF7 cells stably expressing estrogen receptor beta. Total RNA was isolated from parental or estrogen-receptor beta expressing MCF7 cells following 24 hour treatments with either ethanol vehicle, 1nM 17-beta-estradiol or 1nM estradiol plus 40nM endoxifen. All studies were conducted in biological replicates of 2.
Project description:Tumor metastasis remains the major cause of cancer-related death, but its molecular basis is still not well understood. Here we uncovered a splicing-mediated pathway that is essential for breast cancer metastasis. We show that the RNA-binding protein hnRNPM promotes breast cancer metastasis by activating the switch of alternative splicing that occurs during epithelial-mesenchymal transition (EMT). Genome-wide deep sequencing analysis suggests that hnRNPM potentiates TGFb signaling and identifies CD44 as a key downstream target of hnRNPM. hnRNPM ablation prevents TGFb-induced EMT and inhibits breast cancer metastasis in mice, whereas enforced expression of the specific CD44s splice isoform overrides the loss of hnRNPM and permits EMT and metastasis. Mechanistically, we demonstrate that the ubiquitously expressed hnRNPM acts in a mesenchymal-specific manner to precisely control CD44 splice isoform switching during EMT. This restricted cell-type activity of hnRNPM is achieved by competition with ESRP1, an epithelial-splicing regulator that binds to the same cis-regulatory RNA elements and is repressed during EMT. Importantly, hnRNPM is associated with aggressive breast cancer and correlates with increased CD44s in patient specimens. These findings demonstrate a novel molecular mechanism through which tumor metastasis is endowed by the hnRNPM-mediated splicing program. RNAseq for control, hnRNPM siRNA treated lung metastatic LM2 clonal line, derived from the mesenchymal MDA-MB-231 cells