MinD-RNase E interplay controls localization of polar mRNAs in E. coli.

The E. coli transcriptome at the cell's poles (polar transcriptome) is unique compared to the membrane and cytosol. Several factors have been suggested to mediate mRNA localization to the membrane, but the mechanism underlying polar localization of mRNAs remains unknown. Here, we combined a candidate system approach with proteomics to identify factors that mediate mRNAs localization to the cell poles. We identified the pole-to-pole oscillating protein MinD as an essential factor regulating polar mRNA localization, although it is not able to bind RNA directly. We demonstrate that RNase E, previously shown to interact with MinD, is required for proper localization of polar mRNAs. Using in silico modeling followed by experimental validation, the membrane-binding site in RNase E was found to mediate binding to MinD. Intriguingly, not only does MinD affect RNase E interaction with the membrane, but it also affects its mode of action and dynamics. Polar accumulation of RNase E in ΔminCDE cells resulted in destabilization and depletion of mRNAs from poles. Finally, we show that mislocalization of polar mRNAs may prevent polar localization of their protein products. Taken together, our findings show that the interplay between MinD and RNase E determines the composition of the polar transcriptome, thus assigning previously unknown roles for both proteins.

Down-regulation of LATS kinases alters p53 to promote cell migration.

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.

Proteomic and genomic signatures of repeat instability in cancer and adjacent normal tissues.

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.

NF-κB-miR-155 axis activation mediates ovulation-induced oncogenic effects in fallopian tube epithelium.

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.

Identification of nucleolar protein NOM1 as a novel nuclear IGF1R-interacting protein.

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.

Breast cancer tumorigenicity is dependent on high expression levels of NAF-1 and the lability of its Fe-S clusters.

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.

Reduced changes in protein compared to mRNA levels across non-proliferating tissues.

BACKGROUND: The quantitative relations between RNA and protein are fundamental to biology and are still not fully understood. Across taxa, it was demonstrated that the protein-to-mRNA ratio in steady state varies in a direction that lessens the change in protein levels as a result of changes in the transcript abundance. Evidence for this behavior in tissues is sparse. We tested this phenomenon in new data that we produced for the mouse auditory system, and in previously published tissue datasets. A joint analysis of the transcriptome and proteome was performed across four datasets: inner-ear mouse tissues, mouse organ tissues, lymphoblastoid primate samples and human cancer cell lines. RESULTS: We show that the protein levels are more conserved than the mRNA levels in all datasets, and that changes in transcription are associated with translational changes that exert opposite effects on the final protein level, in all tissues except cancer. Finally, we observe that some functions are enriched in the inner ear on the mRNA level but not in protein. CONCLUSIONS: We suggest that partial buffering between transcription and translation ensures that proteins can be made rapidly in response to a stimulus. Accounting for the buffering can improve the prediction of protein levels from mRNA levels.

Membrane chaperone SecDF plays a role in the secretion of Listeria monocytogenes major virulence factors.

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.

TMPRSS2-ERG fusion protein regulates insulin-like growth factor-1 receptor (IGF1R) gene expression in prostate cancer: involvement of transcription factor Sp1.

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.

System-wide Clinical Proteomics of Breast Cancer Reveals Global Remodeling of Tissue Homeostasis.

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.

Design and application of super-SILAC for proteome quantification.

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 MDR transporters are involved in LTA synthesis and triggering of innate immunity during infection.

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.