Search

The fallopian tube secretory epithelial cells (FTSECs) are the cell-of-origin of most high-grade serous ovarian carcinomas (HGSOC). FTSECs are repeatedly exposed to inflammation induced by follicular fluid (FF) that is released with every ovulation cycle throughout a woman’s reproductive years. Uninterrupted ovulation cycles are an established risk factor for HGSOC. Stimuli present in the FF induce an inflammatory environment which may cause DNA damage eventually leading to serous tumorigenesis. With the aim of elucidating possible mechanistic pathways, we established an ‘ex vivo persistent ovulation model’ mimicking the repeated exposure of human benign fallopian tube epithelium (FTE) to FF. We performed mass spectrometry analysis of the secretome of the ex vivo cultures as well as confirmatory targeted expressional and functional analyses. We demonstrated activation of the NF-κB pathway and upregulation of miR-155 following short-term exposure of FTE to human FF. Increased expression of miR-155 was also detected in primary HGSOC tumors compared with benign primary human FTE and corresponded with changes in the expression of miR-155 target genes. The phenotype of miR-155 overexpression in FTSEC cell line is of increased migratory and altered adhesion capacities. Overall, activation of the NF-κB-miR-155 axis in FTE may represent a possible link between ovulation-induced inflammation, DNA damage, and transcriptional changes that may eventually lead to serious carcinogenesis.

Repetitive sequences are hotspots of evolution at multiple levels. However, due to difficulties involved in their assembly and analysis, the role of repeats in tumor evolution is poorly understood. We developed a rigorous motif-based methodology to quantify variations in the repeat content, beyond microsatellites, in proteomes and genomes directly from proteomic and genomic raw data. This method was applied to a wide range of tumors and normal tissues. We identify high similarity between repeat instability patterns in tumors and their patient-matched adjacent normal tissues. Nonetheless, tumor-specific signatures both in protein expression and in the genome strongly correlate with cancer progression and robustly predict the tumorigenic state. In a patient, the hierarchy of genomic repeat instability signatures accurately reconstructs tumor evolution, with primary tumors differentiated from metastases. We observe an inverse relationship between repeat instability and point mutation load within and across patients independent of other somatic aberrations. Thus, repeat instability is a distinct, transient, and compensatory adaptive mechanism in tumor evolution and a potential signal for early detection.

SummaryRepetitive sequences are hotspots of evolution at multiple levels. However, due to technical difficulties involved in their assembly and analysis, the role of repeats in tumor evolution is poorly understood. We developed a rigorous motif-based methodology to quantify variations in the repeat content of proteomes and genomes, directly from proteomic and genomic raw sequence data, and applied it to analyze a wide range of tumors and normal tissues. We identify high similarity between the repeat-instability in tumors and their patient-matched normal tissues, but also tumor-specific signatures, both in protein expression and in the genome, that strongly correlate with cancer progression and robustly predict the tumorigenic state. In a patient, the hierarchy of genomic repeat instability signatures accurately reconstructs tumor evolution, with primary tumors differentiated from metastases. We find an inverse relationship between repeat-instability and point mutation load, within and across patients, and independently of other somatic aberrations. Thus, repeat-instability is a distinct, transient and compensatory adaptive mechanism in tumor evolution.

// Shilhav Meisel Sharon 1 , Yair Pozniak 1 , Tamar Geiger 1 , Haim Werner 1, 2 1 Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel 2 Yoran Institute for Human Genome Research, Tel Aviv University, Tel Aviv 69978, Israel Correspondence to: Haim Werner, email: hwerner@post.tau.ac.il Keywords: insulin-like growth factor-1 (IGF1), IGF1 receptor, TMPRSS2-ERG, prostate cancer, fusion proteins Received: December 29, 2015 Accepted: May 22, 2016 Published: June 06, 2016 ABSTRACT Prostate cancer is a major health issue in the Western world. The most common gene rearrangement in prostate cancer is the TMPRSS2-ERG fusion, which results in aberrant expression of the transcription factor ERG. The insulin-like growth factor-1 receptor (IGF1R) plays a key role in cell growth and tumorigenesis, and is overexpressed in most malignancies, including prostate cancer. In this study we show that TMPRSS2-ERG mediates its tumorigenic effects through regulation of IGF1R gene expression. Silencing of T-ERG in VCaP cells resulted in downregulation of both IGF1R and Sp1, a critical IGF1R regulator. Co-immunoprecipitation assays revealed a physical interaction between transcription factors ERG and Sp1, with potential relevance in IGF1R gene regulation. In addition, transactivation of the IGF1R gene by ERG was mediated at the level of transcription, as indicated by results of promoter assays. To identify new co-activators of the TMPRSS2-ERG fusion protein we performed mass spectrometry-based proteomic analyses. Among other interactors, we identified AP-2 complex subunit mu (AP2M1) and caveolin-1 (CAV1) in association with ERG in cell nuclei. These proteins play a mechanistic role in IGF1R internalization. Our analyses are consistent with a potential novel function of TMPRSS2-ERG as a major regulator of IGF1R gene expression. Results may impinge upon ongoing efforts to target the IGF1R in the clinics.

The genomic and transcriptomic landscapes of breast cancer have been extensively studied, but the proteomes of breast tumors are far less characterized. Here, we use high-resolution, high-accuracy mass spectrometry to perform a deep analysis of luminal-type breast cancer progression using clinical breast samples from primary tumors, matched lymph node metastases, and healthy breast epithelia. We used a super-SILAC mix to quantify over 10,000 proteins with high accuracy, enabling us to identify key proteins and pathways associated with tumorigenesis and metastatic spread. We found high expression levels of proteins associated with protein synthesis and degradation in cancer tissues, accompanied by metabolic alterations that may facilitate energy production in cancer cells within their natural environment. In addition, we found proteomic differences between breast cancer stages and minor differences between primary tumors and their matched lymph node metastases. These results highlight the potential of proteomic technology in the elucidation of clinically relevant cancer signatures.

The insulin-like growth factor-1 receptor (IGF1R) mediates the biological actions of both IGF1 and IGF2. In recent years, evidence has accumulated showing that, in addition to its classical cell-surface distribution, IGF1R translocates to cell nucleus via an apparently SUMO-1-dependent mechanism. While the role of IGF1R in nucleus has not yet been settled, available information suggests that the nuclear receptor displays activities usually linked to transcription factors, including DNA binding and transcription regulation. To gain insight into the biological pathways associated with nuclear IGF1R action we conducted a mass spectrometry-based proteomic analysis aimed at identifying interactors of IGF1R in nucleus of both benign and malignant breast cells. The nucleolar NOM1 molecule belongs to a family of proteins that contain the middle domain of eukaryotic initiation factor 4G (MIF4G) and/or interaction module (MA3), and functions in translation, cell growth and proliferation. Using a combination of co-immunoprecipitation and silencing assays we provide evidence of a complex, bi-directional interplay between nuclear IGF1R and nucleolar protein NOM1. Inhibition of nuclear IGF1R translocation by dansylcadaverine reduced NOM1 levels in nuclei of MCF7 cells. On the other hand, IGF1R overexpression enhanced NOM1 levels in the nuclear fraction. Of interest, NOM1 silencing led to a major increase in IGF1R biosynthesis. In summary, results are consistent with a physiologically-relevant interplay between the nuclear IGF1 signaling pathway and nucleolar protein NOM1.

Although cysteine cathepsins have been identified as key regulators of cancer growth, their specific role in tumor development remains unclear. Recent studies have shown that high activity levels of tumor cathepsins are primarily a result of increased cathepsin activity in cancer-promoting tumor-associated macrophages (TAMs). To further investigate the role of cysteine cathepsin activity in normal and polarized macrophages, we established in vitro and in vivo models of macrophage differentiation and polarization and used a novel cysteine cathepsin inhibitor, GB111-NH2, to block the activity of cathepsins B, L and S. Here we show that in vitro, cysteine cathepsin inhibition yields both apoptosis and proliferation of macrophages, owing to increased oxidative stress. Proteomic analysis of cathepsin- inhibited macrophages demonstrates inhibition of autophagy, suggesting a likely cause of elevated reactive oxygen species (ROS) levels. In vivo models of mammary cancer further show that cathepsin inhibition yields TAM death owing to increased ROS levels. Strikingly, apoptosis in TAMs yields a seemingly cell non-autonomous death of neighboring cancer cells, and regression of the primary growth. These results show that cysteine cathepsin inhibitors can specifically trigger macrophage cell death and may function as an effective anticancer therapy in tumors with high levels of TAMs.

Introduction Reprogrammed energy metabolism is one among the distinguished cancer hallmarks that unravels the dysregulated bioenergetics of cancer cells. Isocitrate dehydrogenase 2 (IDH2), a mitochondrial metabolic enzyme has been reported to be overexpressed in several cancers, yet the mechanisms of wildtype IDH2-induced transformation are still obscure. We previously identified IDH2, as a novel poor prognosis marker of breast cancer progression. In agreement, transcriptomics (TCGA) and proteomics data of breast cancer clinical samples showed similar expression changes of increased wildtype IDH2 from luminal, to triple-negative and to HER-2 positive, which correlated with tumour aggressiveness. Material and methods We performed intracellular metabolite tracing with heavy labelled glucose (13C6) and glutamine (13C5) followed by ultra-high performance liquid chromatography separation and mass spectrometric analysis on the Q-Exactive Plus instrument. Genome-scale metabolic modelling analysis was performed to identify the IDH2-dependent metabolic genes using Minimization of Metabolic Adjustment (MOMA) algorithm. Results and discussions Overexpression of IDH2 increased serine biosynthesis, increased glycolytic rate and oxidative stress resistance. Genome-scale metabolic modelling of breast cancer tumour data identified dependence on serine biosynthesis as one of the metabolic vulnerability of IDH2 overexpressing cells. CRISPR-Cas9 mediated knockout of phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT) specifically inhibited IDH2 overexpressing cells, even in the presence of exogenous serine and glycine, and reduced the glycolysis rate and glucose uptake. Furthermore, loss of serine synthesis pathway hampered the flux from glycolysis, into the TCA cycle. Moreover the metabolic perturbations of synthetic dosage lethal partners, PHGDH and PSAT sensitised the IDH2 overexpressing cells to oxidative stress. Conclusion This study reveals WT-IDH2 as a critical metabolic regulator in breast cancer that supports the pro-tumorigenic Warburg effect. These findings may contribute to translate the metabolic dependency to improve targeted therapeutics.

Iron-sulfur (Fe-S) proteins are thought to play an important role in cancer cells mediating redox reactions, DNA replication, and telomere maintenance. Nutrient-deprivation autophagy factor-1 (NAF-1) is a 2Fe-2S protein associated with the progression of multiple cancer types. It is unique among Fe-S proteins because of its 3Cys-1His cluster coordination structure that allows it to be relatively stable, as well as to transfer its clusters to apo-acceptor proteins. Here, we report that overexpression of NAF-1 in xenograft breast cancer tumors results in a dramatic augmentation in tumor size and aggressiveness and that NAF-1 overexpression enhances the tolerance of cancer cells to oxidative stress. Remarkably, overexpression of a NAF-1 mutant with a single point mutation that stabilizes the NAF-1 cluster, NAF-1(H114C), in xenograft breast cancer tumors results in a dramatic decrease in tumor size that is accompanied by enhanced mitochondrial iron and reactive oxygen accumulation and reduced cellular tolerance to oxidative stress. Furthermore, treating breast cancer cells with pioglitazone that stabilizes the 3Cys-1His cluster of NAF-1 results in a similar effect on mitochondrial iron and reactive oxygen species accumulation. Taken together, our findings point to a key role for the unique 3Cys-1His cluster of NAF-1 in promoting rapid tumor growth through cellular resistance to oxidative stress. Cluster transfer reactions mediated by the overexpressed NAF-1 protein are therefore critical for inducing oxidative stress tolerance in cancer cells, leading to rapid tumor growth, and drugs that stabilize the NAF-1 cluster could be used as part of a treatment strategy for cancers that display high NAF-1 expression.

p53 is a pivotal tumor suppressor and a major barrier against cancer. We now report that silencing of the Hippo pathway tumor suppressors LATS1 and LATS2 in nontransformed mammary epithelial cells reduces p53 phosphorylation and increases its association with the p52 NF-κB subunit. Moreover, it partly shifts p53's conformation and transcriptional output toward a state resembling cancer-associated p53 mutants and endows p53 with the ability to promote cell migration. Notably, LATS1 and LATS2 are frequently down-regulated in breast cancer; we propose that such down-regulation might benefit cancer by converting p53 from a tumor suppressor into a tumor facilitator.

Stable isotope labeling with amino acids in cell culture (SILAC) is considered the most accurate method for proteome quantification by mass spectrometry. As it relies on active protein translation, it was traditionally limited to cells in culture and was not applicable to tissues. We have previously developed the super-SILAC mix, which is a mixture of several cell lines that serves as an internal spike-in standard for the study of human tumor tissue. The super-SILAC mix greatly improves the quantification accuracy while lowering error rates, and it is a simple, economic, and robust technique. Here we describe the design and application of super-SILAC to a broad range of biological systems, for basic biological research as well as clinical one.

Multi-drug resistance (MDR) transporters are known eponymously for their ability to confer resistance to various antimicrobial drugs. However, it is likely that this is not their primary function and that MDR transporters evolved originally to play additional roles in bacterial physiology. In Listeria monocytogenes a set of MDR transporters was identified to mediate activation of innate immune responses during mammalian cell infection. This phenotype was shown to be dependent on c-di-AMP secretion, but the physiological processes underlying this phenomenon were not completely resolved. Here we describe a genetic approach taken to screen for L. monocytogenes genes or physiological pathways involved in MDR transporter-dependent triggering of the type I interferon response. We found that disruption of L. monocytogenes lipoteichoic acid (LTA) synthesis results in enhanced triggering of type I interferon responses in infected macrophage cells yet does not impact bacterial intracellular growth. This innate immune response required the MDR transporters and could be recapitulated by exposing macrophage cells to culture supernatants derived from LTA mutant bacteria. Notably, we found that the MDR transporters themselves are required for full production of LTA, an observation that links MDR transporters to LTA synthesis for the first time. In light of our findings, we propose that the MDR transporters play a role in regulating LTA synthesis, possibly via c-di-AMP efflux, a physiological function in cell wall maintenance that triggers the host innate immune system.

Stable isotope labeling with amino acids in cell culture (SILAC) is considered the most accurate method for proteome quantification by mass spectrometry. As it relies on active protein translation, it was traditionally limited to cells in culture and was not applicable to tissues. We have previously developed the super-SILAC mix, which is a mixture of several cell lines that serves as an internal spike-in standard for the study of human tumor tissue. The super-SILAC mix greatly improves the quantification accuracy while lowering error rates, and it is a simple, economic, and robust technique. Here we describe the design and application of super-SILAC to a broad range of biological systems, for basic biological research as well as clinical one.

Listeria monocytogenes is a Gram-positive human intracellular pathogen that infects diverse mammalian cells. Upon invasion, L. monocytogenes secretes multiple virulence factors that target host cellular processes and promote infection. It has been presumed, but was not empirically established, that the Sec translocation system is the primary mediator of this secretion. Here, we validate an important role for SecDF, a component of the Sec system, in the secretion of several critical L. monocytogenes virulence factors. A ΔsecDF mutant is demonstrated to exhibit impaired membrane translocation of listeriolysin O (LLO), PlcA, PlcB, and ActA, factors that mediate L. monocytogenes phagosomal escape and spread from cell to cell. This impaired translocation was monitored by accumulation of the factors on the bacterial membrane and by reduced activity upon secretion. This defect in secretion is shown to be associated with a severe intracellular growth defect of the ΔsecDF mutant in macrophages and a less virulent phenotype in mice, despite normal growth in laboratory medium. We further show that SecDF is upregulated when the bacteria reside in macrophage phagosomes and that it is necessary for efficient phagosomal escape. Taken together, these data support the premise that SecDF plays a role as a chaperone that facilitates the translocation of L. monocytogenes virulence factors during infection.

Cellular decision-making involves integration of inputs from multiple signaling pathways, including tumor suppressor pathways. It is, therefore, of interest to elucidate how tumor suppressor pathways talk with each other to coordinate their activities in normal cells, and whether and how this coordination is perturbed in cancer. To that end, we have been investigating the crosstalk between two pivotal tumor suppressor pathways: the extensively studied p53 pathway and the Hippo pathway, whose relevance to cancer is becoming increasingly appreciated. In mammalian cells, two of the key members of the Hippo pathway are the LATS1 and LATS2 protein kinases. In earlier studies, we found that LATS2 binds to MDM2, the major negative regulator of p53, and inhibits MDM2's E3 ubiquitin ligase activity. Consequently, the binding of LATS2 to MDM2 leads to stabilization p53. The LATS2-MDM2 axis contributes to the activation of p53 in a number of biological stress-response settings, particularly in the context of mitotic machinery dysfunction as well as upon excessive activity of some oncogenes (e.g. H-Ras and K-Ras), where LATS2 undergoes extensive nuclear translocation with a consequent increase in its association with MDM2. Interestingly, the LATS2 gene itself is a bona fide transcriptional target of p53, but its induction by p53 is observed only under particular conditions, most notably those that involve p53 activation via the LATS2-MDM2 axis. These findings position LATS2 in the interface between the p53 and Hippo pathways, and suggest that it may play a role in coordinating the output of those two tumor suppressor pathways in response to stress. We next wished to determine whether the Hippo pathway impacts p53 activity also under nonstressed conditions. To that end, we silenced simultaneously LATS1 and LATS2 in nontransformed MCF10A human mammary epithelial cells. Although LATS downregulation did not affect p53 protein levels, it led to a change in its phosphorylation pattern, particularly a reduction in p53 phosphorylation on serines 15 and 315. Concomitantly, a fraction of the p53 molecules assumed a mutant-like conformation, reflected by increased reactivity with the mutant-specific PAb240 antibody. Remarkably, although p53 did not affect the migratory properties of control MCF10A cells, it turned into a positive regulator of cell migration in LATS-depleted cells. Indeed, RNA-seq analysis revealed that in such cells p53 acquired an increased ability to modulate the expression of genes that are implicated in regulation of cell motility. Of particular note, the genes upregulated by p53 in LATS-silenced cells displayed a significant overlap with genes reported to be upregulated by gain-of-function mutant p53 in breast cancer cells. Interestingly, one of the genes whose expression was selectively upregulated by p53 in LATS-depleted cells was PTGS2, better known as COX2. Moreover, PTGS2 upregulation was found to contribute to the migratory features of such cells, and PTGS2 depletion could mimic to some extent the impact of p53 depletion on migration. In an attempt to understand the molecular basis for the transcriptional rewiring of the “altered” p53 in LATS-depleted cells, we sought to identify proteins that associate preferentially with p53 in cells where LATS expression has been silenced. One of the top hits was the p52 subunit of NF-κB. Furthermore, we found that p52 contributed to the upregulation of PTGS2 in cells harboring the “altered” p53. Together, these findings imply that LATS proteins play an active role in maintaining wild-type p53 in its canonical state, ensuring that it is preferentially dedicated to the transcriptional regulation of canonical wild-type p53 target genes. Once LATS activity is compromised, as occurs in some cancers as a consequence of epigenetic silencing of LATS1 and/or LATS2, part of the p53 population shifts to a mutant-like state, potentially favoring the activation of genes that contribute to cancer rather than suppressing it. Hence, optimal performance of key players in one tumor suppressor pathway (the Hippo pathway) is required in order to sustain optimal performance of another tumor suppressor pathway (the p53 pathway). It seems highly plausible that this tumor suppressor crosstalk is not restricted to LATS kinases only, and input from additional tumor suppressor pathways may potentially also serve to keep p53 in a fully competent tumor suppressive state. Conversely, when such “support group” tumor suppressor pathways are rendered dysfunctional in some cancer cells, this may enable the endogenous wild type to acquire mutant-like properties without the need to accrue real genetic mutations in the TP53 locus. Further identification of such pathways and elucidation of the mechanisms by which they bolster the canonical biochemical features of p53 may provide clues for new strategies to restore tumor suppressive p53 function in cancers that retain a wild type TP53 gene. [N. Furth and N. Bossel Ben-Moshe contributed equally to this work.] Citation Format: Moshe Oren, Noa Furth, Noa Bossel Ben-Moshe, Ziv Porat, Eytan Domany, Yair Pozniak, Tamar Geiger, Yael Aylon. Tumor suppressor crosstalk: Modulation of p53 activity by the Hippo pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr SY26-03.