Mitomycin C and its analog trigger cytotoxicity in MCF-7 and K562 cancer cells through the regulation of RAS and MAPK/ERK pathways.

Mitomycin C (MC) is an anti-cancer drug which functions by forming interstrand crosslinks (ICLs) between opposing DNA strands. MC analog, 10-decarbamoyl mitomycin C (DMC), unlike MC, has stronger cytotoxic effects on cancer cells with TP53 mutation. We previously demonstrated that MC/DMC could activate p21WAF1/CIP1 in MCF-7 (TP53-proficient) and K562 (TP53 deficient) cells in a TP53-independent mode. We also found that MC/DMC regulate AKT activation in a TP53-dependent manner and that AKT deactivation is not associated with the activation of p21WAF1/CIP1 in response to MC/DMC treatment. RAS proteins are known players in the upstream mediated signaling of p21WAF1/CIP1 activation that leads to control of cell proliferation and cell death. Thus, this prompted us to investigate the effect of both drugs on the expression of RAS proteins and regulation of the MAPK/ERK signaling pathways in MCF-7 and K562 cancer cells. To accomplish this goal, we performed comparative label free proteomics profiling coupled to bioinformatics/complementary phosphoprotein arrays and Western blot validations of key signaling molecules. The MAPK/ERK pathway exhibited an overall downregulation upon MC/DMC treatment in MCF-7 cells but only DMC exhibited a mild downregulation of that same pathway in TP53 mutant K562 cells. Furthermore, treatment of MCF-7 and K562 cell lines with oligonucleotides containing the interstrand crosslinks (ICLs) formed by MC or DMC shows that both ICLs had a stronger effect on the downregulation of RAS protein expression in mutant TP53 K562 cells. We discuss the implication of this regulation of the MAPK/ERK pathway in relation to cellular TP53 status.

PTPN13 Participates in the Regulation of Epithelial-Mesenchymal Transition and Platinum Sensitivity in High-Grade Serous Ovarian Carcinoma Cells.

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecological cancers in Western countries. High-Grade Serous Ovarian Carcinoma (HGSOC) accounts for 60-70% of EOC and is the most aggressive subtype. Reduced PTPN13 expression levels have been previously correlated with worse prognosis in HGSOC. However, PTPN13's exact role and mechanism of action in these tumors remained to be investigated. To elucidate PTPN13's role in HGSOC aggressiveness, we used isogenic PTPN13-overexpressing clones of the OVCAR-8 cell line, which poorly expresses PTPN13, and also PTPN13 CRISPR/Cas9-mediated knockout/knockdown clones of the KURAMOCHI cell line, which strongly expresses PTPN13. We investigated their migratory and invasive capacity using a wound healing assay, their mesenchymal-epithelial transition (EMT) status using microscopy and RT-qPCR, and their sensitivity to chemotherapeutic drugs used for HGSOC. We found that (i) PTPN13 knockout/knockdown increased migration and invasion in KURAMOCHI cells that also displayed a more mesenchymal phenotype and increased expression of the SLUG, SNAIL, ZEB-1, and ZEB-2 EMT master genes; and (ii) PTPN13 expression increased the platinum sensitivity of HGSOC cells. These results suggest that PTPN13 might be a predictive marker of response to platinum salts in HGSOC.

HT-smFISH: a cost-effective and flexible workflow for high-throughput single-molecule RNA imaging.

The ability to visualize RNA in its native subcellular environment by using single-molecule fluorescence in situ hybridization (smFISH) has reshaped our understanding of gene expression and cellular functions. A major hindrance of smFISH is the difficulty to perform systematic experiments in medium- or high-throughput formats, principally because of the high cost of generating the individual fluorescent probe sets. Here, we present high-throughput smFISH (HT-smFISH), a simple and cost-efficient method for imaging hundreds to thousands of single endogenous RNA molecules in 96-well plates. HT-smFISH uses RNA probes transcribed in vitro from a large pool of unlabeled oligonucleotides. This allows the generation of individual probes for many RNA species, replacing commercial DNA probe sets. HT-smFISH thus reduces costs per targeted RNA compared with many smFISH methods and is easily scalable and flexible in design. We provide a protocol that combines oligo pool design, probe set generation, optimized hybridization conditions and guidelines for image acquisition and analysis. The pipeline requires knowledge of standard molecular biology tools, cell culture and fluorescence microscopy. It is achievable in ~20 d. In brief, HT-smFISH is tailored for medium- to high-throughput screens that image RNAs at single-molecule sensitivity.

The kinesin KIF1C transports APC-dependent mRNAs to cell protrusions.

RNA localization and local translation are important for numerous cellular functions. In mammals, a class of mRNAs localize to cytoplasmic protrusions in an APC-dependent manner, with roles during cell migration. Here, we investigated this localization mechanism. We found that the KIF1C motor interacts with APC-dependent mRNAs and is required for their localization. Live cell imaging revealed rapid, active transport of single mRNAs over long distances that requires both microtubules and KIF1C. Two-color imaging directly revealed single mRNAs transported by single KIF1C motors, with the 3'UTR being sufficient to trigger KIF1C-dependent RNA transport and localization. Moreover, KIF1C remained associated with peripheral, multimeric RNA clusters and was required for their formation. These results reveal a widespread RNA transport pathway in mammalian cells, in which the KIF1C motor has a dual role in transporting RNAs and clustering them within cytoplasmic protrusions. Interestingly, KIF1C also transports its own mRNA, suggesting a possible feedback loop acting at the level of mRNA transport.

A choreography of centrosomal mRNAs reveals a conserved localization mechanism involving active polysome transport.

Local translation allows for a spatial control of gene expression. Here, we use high-throughput smFISH to screen centrosomal protein-coding genes, and we describe 8 human mRNAs accumulating at centrosomes. These mRNAs localize at different stages during cell cycle with a remarkable choreography, indicating a finely regulated translational program at centrosomes. Interestingly, drug treatments and reporter analyses reveal a common translation-dependent localization mechanism requiring the nascent protein. Using ASPM and NUMA1 as models, single mRNA and polysome imaging reveals active movements of endogenous polysomes towards the centrosome at the onset of mitosis, when these mRNAs start localizing. ASPM polysomes associate with microtubules and localize by either motor-driven transport or microtubule pulling. Remarkably, the Drosophila orthologs of the human centrosomal mRNAs also localize to centrosomes and also require translation. These data identify a conserved family of centrosomal mRNAs that localize by active polysome transport mediated by nascent proteins.

GOLT1B Activation in Hepatitis C Virus-Infected Hepatocytes Links ER Trafficking and Viral Replication.

Chronic hepatitis C carries a high risk of development of hepatocellular carcinoma (HCC), triggered by both direct and indirect effects of the virus. We examined cell-autonomous alterations in gene expression profiles associated with hepatitis C viral presence. Highly sensitive single molecule fluorescent in situ hybridization applied to frozen tissue sections of a hepatitis C patient allowed the delineation of clusters of infected hepatocytes. Laser microdissection followed by RNAseq analysis of hepatitis C virus (HCV)-positive and -negative regions from the tumoral and non-tumoral tissues from the same patient revealed HCV-related deregulation of expression of genes in the tumor and in the non-tumoral tissue. However, there was little overlap between both gene sets. Our interest in alterations that increase the probability of tumorigenesis prompted the examination of genes whose expression was increased by the virus in the non-transformed cells and whose level remained high in the tumor. This strategy led to the identification of a novel HCV target gene: GOLT1B, which encodes a protein involved in ER-Golgi trafficking. We further show that GOLT1B expression is induced during the unfolded protein response, that its presence is essential for efficient viral replication, and that its expression is correlated with poor outcome in HCC.

Lessons from past radiation accidents: Critical review of methods addressed to individual dose assessment of potentially exposed people and integration with medical assessment.

The experiences of the Chernobyl and Fukushima nuclear accidents showed that dosimetry was the essential tool in the emergency situation for decision making processes, such as evacuation and application of protective measures. However, at the consequent post-accidental phases, it was crucial also for medical health surveillance and in further adaptation to changed conditions with regards to radiation protection of the affected populations. This review provides an analysis of the experiences related to the role of dosimetry (dose measurements, assessment and reconstruction) regarding health preventive measures in the post-accidental periods on the examples of the major past nuclear accidents such as Chernobyl and Fukushima. Recommendations derived from the review are called to improve individual dose assessment in case of a radiological accident/incident and should be considered in advance as guidelines to follow for having better information. They are given as conclusions.

A Dual Protein-mRNA Localization Screen Reveals Compartmentalized Translation and Widespread Co-translational RNA Targeting.

Local translation allows spatial control of gene expression. Here, we performed a dual protein-mRNA localization screen, using smFISH on 523 human cell lines expressing GFP-tagged genes. 32 mRNAs displayed specific cytoplasmic localizations with local translation at unexpected locations, including cytoplasmic protrusions, cell edges, endosomes, Golgi, the nuclear envelope, and centrosomes, the latter being cell-cycle-dependent. Automated classification of mRNA localization patterns revealed a high degree of intercellular heterogeneity. Surprisingly, mRNA localization frequently required ongoing translation, indicating widespread co-translational RNA targeting. Interestingly, while P-body accumulation was frequent (15 mRNAs), four mRNAs accumulated in foci that were distinct structures. These foci lacked the mature protein, but nascent polypeptide imaging showed that they were specialized translation factories. For ß-catenin, foci formation was regulated by Wnt, relied on APC-dependent polysome aggregation, and led to nascent protein degradation. Thus, translation factories uniquely regulate nascent protein metabolism and create a fine granular compartmentalization of translation.

The Syk Kinase Promotes Mammary Epithelial Integrity and Inhibits Breast Cancer Invasion by Stabilizing the E-Cadherin/Catenin Complex.

While first discovered in immunoreceptor signaling, the Syk protein kinase behaves as a tumor and metastasis suppressor in epithelial cells. Its reduced expression in breast and other carcinomas is correlated with decreased survival and increased metastasis risk, but its action mechanism remains largely unknown. Using phosphoproteomics we found that Syk phosphorylated E-cadherin and α-, ß-, and p120-catenins on multiple tyrosine residues that concentrate at intercellular junctions. Increased Syk expression and activation enhanced E-cadherin/catenin phosphorylation, promoting their association and complex stability. In human breast cancer cells, Syk stimulated intercellular aggregation, E-cadherin recruitment and retention at adherens junctions, and promoted epithelial integrity, whereas it inhibited cell migration and invasion. Opposite effects were obtained with Syk knockdown or non-phosphorylatable mutant E-cadherin expression. Mechanistically, Syk stimulated the interaction of the E-cadherin/catenin complex with zonula occludens proteins and the actin cytoskeleton. Conditional Syk knockout in the lactating mouse mammary gland perturbed alveologenesis and disrupted E-cadherin localization at adherens junctions, corroborating the observations in cells. Hence, Syk is involved in the maintenance of the epithelial integrity of the mammary gland via the phosphorylation and stabilization of the E-cadherin/catenin adherens junction complex, thereby inhibiting cell migration and malignant tumor invasion.

A computational framework to study sub-cellular RNA localization.

RNA localization is a crucial process for cellular function and can be quantitatively studied by single molecule FISH (smFISH). Here, we present an integrated analysis framework to analyze sub-cellular RNA localization. Using simulated images, we design and validate a set of features describing different RNA localization patterns including polarized distribution, accumulation in cell extensions or foci, at the cell membrane or nuclear envelope. These features are largely invariant to RNA levels, work in multiple cell lines, and can measure localization strength in perturbation experiments. Most importantly, they allow classification by supervised and unsupervised learning at unprecedented accuracy. We successfully validate our approach on representative experimental data. This analysis reveals a surprisingly high degree of localization heterogeneity at the single cell level, indicating a dynamic and plastic nature of RNA localization.

Combining laser capture microdissection and proteomics reveals an active translation machinery controlling invadosome formation.

Invadosomes are F-actin-based structures involved in extracellular matrix degradation, cell invasion, and metastasis formation. Analyzing their proteome is crucial to decipher their molecular composition, to understand their mechanisms, and to find specific elements to target them. However, the specific analysis of invadosomes is challenging, because it is difficult to maintain their integrity during isolation. In addition, classical purification methods often suffer from contaminations, which may impair data validation. To ensure the specific identification of invadosome components, we here develop a method that combines laser microdissection and mass spectrometry, enabling the analysis of subcellular structures in their native state based on low amounts of input material. Using this combinatorial method, we show that invadosomes contain specific components of the translational machinery, in addition to known marker proteins. Moreover, functional validation reveals that protein translation activity is an inherent property of invadosomes, which is required to maintain invadosome structure and activity.

Visualization of single endogenous polysomes reveals the dynamics of translation in live human cells.

Translation is an essential step in gene expression. In this study, we used an improved SunTag system to label nascent proteins and image translation of single messenger ribonucleoproteins (mRNPs) in human cells. Using a dedicated reporter RNA, we observe that translation of single mRNPs stochastically turns on and off while they diffuse through the cytoplasm. We further measure a ribosome density of 1.3 per kilobase and an elongation rate of 13-18 amino acids per second. Tagging the endogenous POLR2A gene revealed similar elongation rates and ribosomal densities and that nearly all messenger RNAs (mRNAs) are engaged in translation. Remarkably, tagging of the heavy chain of dynein 1 (DYNC1H1) shows this mRNA accumulates in foci containing three to seven RNA molecules. These foci are translation sites and thus represent specialized translation factories. We also observe that DYNC1H1 polysomes are actively transported by motors, which may deliver the mature protein at appropriate cellular locations. The SunTag should be broadly applicable to study translational regulation in live single cells.

smiFISH and FISH-quant - a flexible single RNA detection approach with super-resolution capability.

Single molecule FISH (smFISH) allows studying transcription and RNA localization by imaging individual mRNAs in single cells. We present smiFISH (single molecule inexpensive FISH), an easy to use and flexible RNA visualization and quantification approach that uses unlabelled primary probes and a fluorescently labelled secondary detector oligonucleotide. The gene-specific probes are unlabelled and can therefore be synthesized at low cost, thus allowing to use more probes per mRNA resulting in a substantial increase in detection efficiency. smiFISH is also flexible since differently labelled secondary detector probes can be used with the same primary probes. We demonstrate that this flexibility allows multicolor labelling without the need to synthesize new probe sets. We further demonstrate that the use of a specific acrydite detector oligonucleotide allows smiFISH to be combined with expansion microscopy, enabling the resolution of transcripts in 3D below the diffraction limit on a standard microscope. Lastly, we provide improved, fully automated software tools from probe-design to quantitative analysis of smFISH images. In short, we provide a complete workflow to obtain automatically counts of individual RNA molecules in single cells.

Foci of cyclin A2 interact with actin and RhoA in mitosis.

Cyclin A2 is a key player in the regulation of the cell cycle. Its degradation in mid-mitosis depends primarily on the ubiquitin-proteasome system (UPS), while autophagy also contributes. However, a fraction of cyclin A2 persists beyond metaphase. In this work, we focus on cyclin A2-rich foci detected in mitosis by high resolution imaging and analyse their movements. We demonstrate that cyclin A2 interacts with actin and RhoA during mitosis, and that cyclin A2 depletion induces a dramatic decrease in active RhoA in mitosis. Our data suggest cyclin A2 participation in RhoA activation in late mitosis.

Cyclin A2: At the crossroads of cell cycle and cell invasion.

Cyclin A2 is an essential regulator of the cell division cycle through the activation of kinases that participate to the regulation of S phase as well as the mitotic entry. However, whereas its degradation by the proteasome in mid mitosis was thought to be essential for mitosis to proceed, recent observations show that a small fraction of cyclin A2 persists beyond metaphase and is degraded by autophagy. Its implication in the control of cytoskeletal dynamics and cell movement has unveiled its role in the modulation of RhoA activity. Since this GTPase is involved in both cell rounding early in mitosis and later, in the formation of the cleavage furrow, this suggests that cyclin A2 is a novel actor in cytokinesis. Taken together, these data point to this cyclin as a potential mediator of cell-niche interactions whose dysregulation could be taken as a hallmark of metastasis.

High-resolution live-cell imaging reveals novel cyclin A2 degradation foci involving autophagy.

Cyclin A2 is a key player in the regulation of the cell cycle. Its degradation in mid-mitosis relies on the ubiquitin-proteasome system (UPS). Using high-resolution microscopic imaging, we find that cyclin A2 persists beyond metaphase. Indeed, we identify a novel cyclin-A2-containing compartment that forms dynamic foci. Förster (or fluorescence) resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) analyses show that cyclin A2 ubiquitylation takes place predominantly in these foci before spreading throughout the cell. Moreover, inhibition of autophagy in proliferating cells induces the stabilisation of a subset of cyclin A2, whereas induction of autophagy accelerates the degradation of cyclin A2, thus showing that autophagy is a novel regulator of cyclin A2 degradation.

Kizuna is a novel mitotic substrate for CDC25B phosphatase.

CDC25 dual-specificity phosphatases play a central role in cell cycle control through the activation of Cyclin-Dependent Kinases (CDKs). Expression during mitosis of a stabilized CDC25B mutant (CDC25B-DDA), which cannot interact with the F-box protein ßTrCP for proteasome-dependent degradation, causes mitotic defects and chromosome segregation errors in mammalian cells. We found, using the same CDC25B mutant, that stabilization and failure to degrade CDC25B during mitosis lead to the appearance of multipolar spindle cells resulting from a fragmentation of pericentriolar material (PCM) and abolish mitotic Plk1-dependent phosphorylation of Kizuna (Kiz), which is essential for the function of Kiz in maintaining spindle pole integrity. Thus, in mitosis Kiz is a new substrate of CDC25B whose dephosphorylation following CDC25B stabilization leads to the formation of multipolar spindles. Furthermore, endogenous Kiz and CDC25B interact only in mitosis, suggesting that Kiz phosphorylation depends on a balance between CDC25B and Plk1 activities. Our data identify a novel mitotic substrate of CDC25B phosphatase that plays a key role in mitosis control.

REV, A BRET-Based Sensor of ERK Activity.

Networks of signaling molecules are activated in response to environmental changes. How are these signaling networks dynamically integrated in space and time to process particular information? To tackle this issue, biosensors of single signaling pathways have been engineered. Bioluminescence resonance energy transfer (BRET)-based biosensors have proven to be particularly efficient in that matter due to the high sensitivity of this technology to monitor protein-protein interactions or conformational changes in living cells. Extracellular signal-regulated kinases (ERK) are ubiquitously expressed and involved in many diverse cellular functions that might be encoded by the strength and spatio-temporal pattern of ERK activation. We developed a BRET-based sensor of ERK activity, called Rluc8-ERKsubstrate-Venus (REV). As expected, BRET changes of REV were correlated with ERK phosphorylation, which is required for its kinase activity. In neurons, the nature of the stimuli determines the strength, the location, or the moment of ERK activation, thus highlighting how acute modulation of ERK may encode the nature of initial stimulus to specify the consequences of this activation. This study provides evidence for suitability of REV as a new biosensor to address biological questions.

A novel function for Cyclin A2: control of cell invasion via RhoA signaling.

Cyclin A2 plays a key role in cell cycle regulation. It is essential in embryonic cells and in the hematopoietic lineage yet dispensable in fibroblasts. In this paper, we demonstrate that Cyclin A2-depleted cells display a cortical distribution of actin filaments and increased migration. These defects are rescued by restoration of wild-type Cyclin A2, which directly interacts with RhoA, or by a Cyclin A2 mutant unable to associate with Cdk. In vitro, Cyclin A2 potentiates the exchange activity of a RhoA-specific guanine nucleotide exchange factor. Consistent with this, Cyclin A2 depletion enhances migration of fibroblasts and invasiveness of transformed cells via down-regulation of RhoA activity. Moreover, Cyclin A2 expression is lower in metastases relative to primary colon adenocarcinoma in matched human tumors. All together, these data show that Cyclin A2 negatively controls cell motility by promoting RhoA activation, thus demonstrating a novel Cyclin A2 function in cytoskeletal rearrangements and cell migration.

Cyclin A2: a genuine cell cycle regulator?

Abstract Cyclin A2 belongs to the core cell cycle regulators and participates in the control of both S phase and mitosis. However, several observations suggest that it is also endowed with other functions, and our recent data shed light on its involvement in cytoskeleton dynamic and cell motility. From the transcription of its gene to its posttranslational modifications, cyclin A2 regulation reveals the complexity of the regulatory network shaping cell cycle progression. We summarize our current knowledge on this cell cycle regulator and discuss recent findings raising the possibility that cyclin A2 might play a much broader role in epithelial tissues homeostasis.