The nervous system has a pivotal role in cancer biology, and pathological investigations have linked intratumoural nerve density to metastasis1. However, the precise impact of cancer-associated neurons and the communication channels at the nerve-cancer interface remain poorly understood. Previous cancer denervation models in rodents and humans have highlighted robust cancer dependency on nerves, but the underlying mechanisms that drive nerve-mediated cancer aggressivity remain unknown2,3. Here we show that cancer-associated neurons enhance cancer metabolic plasticity by transferring mitochondria to cancer cells. Breast cancer denervation and nerve-cancer coculture models confirmed that neurons significantly improve tumour energetics. Neurons cocultured with cancer cells undergo metabolic reprogramming, resulting in increased mitochondrial mass and subsequent transfer of mitochondria to adjacent cancer cells. To precisely track the fate of recipient cells, we developed MitoTRACER, a reporter of cell-to-cell mitochondrial transfer that permanently labels recipient cancer cells and their progeny. Lineage tracing and fate mapping of cancer cells acquiring neuronal mitochondria in primary tumours revealed their selective enrichment at metastatic sites following dissemination. Collectively, our data highlight the enhanced metastatic capabilities of cancer cells that receive mitochondria from neurons in primary tumours, shedding new light on how the nervous system supports cancer metabolism and metastatic dissemination.

PDF file - 112KB, Supplementary figure 3: Quantifications of Western blot and zymography analyses. The levels of Slug, vimentin and MMP-9 in scrambled or Fhit siRNA HBE4-E6/E7 transfectants were quantified either after co-transfection with Slug siRNA 1 (a) or Slug siRNA 2 (b) or in the presence of PD98059 (c), PP1 (d), Gefitinib (e) and anti-EGFR antibody (f). Western blot quantifications are provided for each target gene normalized to GAPDH. Data are expressed as fold induction relative to the respective controls. *P < 0.05; **P < 0.01; ***P < 0.001.

PDF file - 117KB, Supplementary figure 5: Rescue of EGFR inhibition by active Src in Fhit siRNA-treated cells. (a) Western blot analysis of phospho-Src, total Src, Fhit and Slug levels in HBE4-E6/E7 cells transduced with control or Src. (b) Analysis by Western blotting and zymography of the effect of Src overexpression on vimentin and MMP-9 levels in Fhit siRNA transfectants treated with DMSO or gefitinib.

PDF file - 108KB, Supplementary figure 4: Effect of pharmacological inhibitors of various signaling pathways on cell invasion induced by Fhit silencing. HBE4-E6/E7 cells were transfected with scrambled or Fhit siRNA as described in the "Materials and Methods" section. The invasive capacities of the transfectants were tested in a modified Boyden chamber invasion assay in the presence of PD98059 (ERK), PP1 (Src), SB203580 (p38), Y27632 (ROCK), KT5720 (PKA), wortmaninn (PI3K), U73122 (PLC) or SP600125 (JNK) inhibitors. A representative experiment is shown. Data are expressed as fold induction relative to the DMSO control. *P < 0.05.

The aggressive nature of certain cancers and their adverse effects on patient outcomes have been linked to cancer innervation, where neurons infiltrate and differentiate within the cancer stroma. Recently we demonstrated how cancer plasticity and TGFβ signaling could promote breast cancer innervation that is associated with increased cancer aggressivity. Despite the promising potential of cancer innervation as a target for anti-cancer therapies, there is currently a significant lack of effective methods to study cancer-induced neuronal differentiation, hindering the development of high-throughput approaches for identifying new targets or pharmacological inhibitors against cancer innervation. To overcome this challenge, we used CRISPR-based endogenous labeling of the neuronal marker β3-tubulin in neuronal precursors to investigate cancer-induced neuronal differentiation in nerve-cancer cocultures and provide a tool that allows for better standardization and reproducibility of studies about cancer-induced innervation. Our approach demonstrated that β3-tubulin gene editing did not affect neuronal behavior and enabled accurate reporting of cancer-induced neuronal differentiation dynamics in high-throughput settings, which makes this approach suitable for screening large cohorts of cells or testing various biological contexts. In a more context-based approach, by combining this method with a cell model of breast cancer epithelial-mesenchymal transition, we revealed the role of cancer cell plasticity in promoting neuronal differentiation, suggesting that cancer innervation represents an underexplored path for epithelial-mesenchymal transition-mediated cancer aggressivity.

PDF file - 297KB, Supplementary figure 2: Representative fields of cell invasion assays. The invasive capacities of scrambled or Fhit siRNA HBE4-E6/E7 transfectants were tested in a modified Boyden chamber invasion assay either after co-transfection with Slug siRNA 1 (a) or Slug siRNA 2 (b) or in the presence of PD98059 (c), PP1 (d), Gefitinib (e) and anti-EGFR antibody (f). The respective controls for Slug siRNA, inhibitor and neutralizing antibody experiments were scrambled siRNA, DMSO and IgG1. Bar = 100 ?m.

PDF file - 99KB, Supplementary figure 1: Cell invasion induced by Fhit is Slug-dependent. (a) Analysis of the effect of Slug siRNA 2 or a scrambled siRNA on the invasive capacities of HBE4-E6/E7 transfectants in a modified Boyden chamber invasion assay. (b) Western blot and zymography analysis of the effect of Slug siRNA 2 or a scrambled siRNA on Fhit, Slug, vimentin and MMP-9 levels in HBE4-E6/E7 transfectants.

The epithelial to mesenchymal transition (EMT), a process that is aberrantly activated in cancer and facilitates metastasis to distant organs, requires coordinated transcriptional and post-transcriptional control of gene expression. The tumor-suppressive RNA binding protein, hnRNP-E1, regulates splicing and translation of EMT-associated transcripts and it is thought that it plays a major role in the control of epithelial cell plasticity during cancer progression. We have utilized yeast 2 hybrid screening to identify novel hnRNP-E1 interactors that play a role in regulating hnRNP-E1; this approach led to the identification of the E3 ubiquitin ligase ARIH1. Here, we demonstrate that hnRNP-E1 protein stability is increased upon ARIH1 silencing, whereas, overexpression of ARIH1 leads to a reduction in hnRNP-E1. Reduced ubiquitination of hnRNP-E1 detected in ARIH1 knockdown (KD) cells compared to control suggests a role for ARIH1 in hnRNP-E1 degradation. The identification of hnRNP-E1 as a candidate substrate of ARIH1 led to the characterization of a novel function for this ubiquitin ligase in EMT induction and cancer progression. We demonstrate a delayed induction of EMT and reduced invasion in mammary epithelial cells silenced for ARIH1. Conversely, ARIH1 overexpression promoted EMT induction and invasion. ARIH1 silencing in breast cancer cells significantly attenuated cancer cell stemness in vitro and tumor formation in vivo. Finally, we utilized miniTurboID proximity labeling to identify novel ARIH1 interactors that may contribute to ARIH1’s function in EMT induction and cancer progression.

Tumor axonogenesis is an emerging hallmark of cancer and TGF-beta is a well-known cytokine involved in the control of cancer progression. In this study we identify a novel function for the TGF-beta signaling in cancer aggressivity by promoting cancer axonogenesis. Metastasis is the leading driver of cancer-related death. Tumor cell plasticity associated with the epithelial–mesenchymal transition (EMT), an embryonic program also observed in carcinomas, has been proposed to explain the colonization of distant organs by the primary tumor cells. Many studies have established correlations between EMT marker expression in the primary tumor and metastasis in vivo. However, the longstanding model of EMT-transitioned cells disseminating to secondary sites is still actively debated and hybrid states are presently considered as more relevant during tumor progression and metastasis. Here, we describe an unexplored role of EMT on the tumor microenvironment by controlling tumor innervation. Using in vitro and in vivo breast tumor progression models, we demonstrate that TGFβ-mediated tumor cell EMT triggers the expression of the embryonic LincRNA Platr18 those elevated expression controls the expression of the axon guidance protein semaphorin-4F and other neuron-related molecules such as IGSF11/VSIG-3. Platr18/Sema4F axis silencing abrogates axonogenesis and attenuates metastasis. Our observations suggest that EMT-transitioned cells are also locally required in the primary tumor to support distant dissemination by promoting axonogenesis, a biological process known for its role in metastatic progression of breast cancer.

Heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) is a tumor suppressor protein that binds site- and structure-specifically to RNA sequences to regulate mRNA stability, facilitate alternative splicing, and suppress protein translation on several metastasis-associated mRNAs. Here, we show that hnRNP E1 binds polycytosine-rich DNA tracts present throughout the genome, including those at promoters of several oncogenes and telomeres and monitors genome integrity. It binds DNA in a site- and structure-specific manner. hnRNP E1-knockdown cells displayed increased DNA damage signals including γ-H2AX at its binding sites and also showed increased mutations. UV and hydroxyurea treatment of hnRNP E1-knockdown cells exacerbated the basal DNA damage signals with increased cell cycle arrest, activation of checkpoint proteins, and monoubiquitination of proliferating cell nuclear antigen despite no changes in deubiquitinating enzymes. DNA damage caused by genotoxin treatment localized to hnRNP E1 binding sites. Our work suggests that hnRNP E1 facilitates functions of DNA integrity proteins at polycytosine tracts and monitors DNA integrity at these sites.

BACKGROUND:The effect of treatment delay on survival in pancreatic ductal adenocarcinoma (PDAC) remains unclear. AIMS:This study aimed to assess the prognostic impact of time to diagnosis and chemotherapy in advanced PDAC and factors influencing the time intervals. METHODS:advanced PDAC patients receiving chemotherapy in five centers in the decade 2007-2016 were included. Key time points during care pathway from clinical presentation to beginning of chemotherapy were retrospectively collected. Multivariate Cox proportional hazard model was performed. RESULTS:A total of 409 patients were included (mean age 66.1 ± 10.3 years; 250 metastatic (61%); 139 received FOLFIRINOX chemotherapy (34%). The median overall survival (OS) was 7.2 months. The median times from first symptoms and from first specialist visit to the beginning of chemotherapy were respectively 100 days and 47 days. None of time intervals was significantly associated with OS. Significant prognostic factors were FOLFIRINOX chemotherapy (HR 0.6 [0.5-0.8]; P < 0.001), metastasis (HR 1.6 [1.3-2.0]; P = 0.001), WHO PS ≥ 2 (HR 1.6 [1.2-2.1]; P < 0.001) and acute pancreatitis as first symptom (HR 2.9 [1.7-4.9]; P < 0.001). Jaundice shortened time to diagnosis (P < 0.001). Acute pancreatitis (P < 0.001) and diabetes (P = 0.01) increased time to treatment. CONCLUSION:Wait times from clinical presentation to beginning of chemotherapy do not influence survival in advanced PDAC.

FAM3C/Interleukin-like EMT Inducer (ILEI) is an oncogenic member of the FAM3 cytokine family and serves essential roles in both epithelial-mesenchymal transition (EMT) and breast cancer metastasis. ILEI expression levels are regulated through a non-canonical TGFβ signaling pathway by 3′-UTR-mediated translational silencing at the mRNA level by hnRNP E1. TGFβ stimulation or silencing of hnRNP E1 increases ILEI translation and induces an EMT program that correlates with enhanced invasion and migration. Recently, EMT has been linked to the formation of breast cancer stem cells (BCSCs) that confer both tumor cell heterogeneity as well as chemoresistant properties. Herein, we demonstrate that hnRNP E1 knockdown significantly shifts normal mammary epithelial cells to mesenchymal BCSCs in vitro and in vivo. We further validate that modulating ILEI protein levels results in the abrogation of these phenotypes, promoting further investigation into the unknown mechanism of ILEI signaling that drives tumor progression. We identify LIFR as the receptor for ILEI, which mediates signaling through STAT3 to drive both EMT and BCSC formation. Reduction of either ILEI or LIFR protein levels results in reduced tumor growth, fewer tumor initiating cells and reduced metastasis within the hnRNP E1 knock-down cell populations in vivo. These results reveal a novel ligand-receptor complex that drives the formation of BCSCs and represents a unique target for the development of metastatic breast cancer therapies.

The epithelial-mesenchymal transition (EMT), in which cells undergo a switch from a polarized, epithelial phenotype to a highly motile fibroblastic or mesenchymal phenotype is fundamental during embryonic development and can be reactivated in a variety of diseases including cancer. Spatio-temporally-regulated mechanisms are constantly orchestrated to allow cells to adapt to their constantly changing environments when disseminating to distant organs. Although numerous transcriptional regulatory factors are currently well-characterized, the post-transcriptional control of EMT requires continued investigation. The hnRNP E1 protein displays a major role in the control of tumor cell plasticity by regulating the translatome through multiple non-redundant mechanisms, and this role is exemplified when E1 is absent. hnRNP E1 binding to RNA molecules leads to direct or indirect translational regulation of specific sets of proteins: (1) hnRNP E1 binding to specific targets has a direct role in translation by preventing elongation of translation; (2) hnRNP E1-dependent alternative splicing can prevent the generation of a competing long non-coding RNA that acts as a decoy for microRNAs (miRNAs) involved in translational inhibition of EMT master regulators; (3) hnRNP E1 binding to the 3' untranslated region of transcripts can also positively regulate the stability of certain mRNAs to improve their translation. Globally, hnRNP E1 appears to control proteome reprogramming during cell plasticity, either by direct or indirect regulation of protein translation.

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for protein synthesis by translating codons in mRNA into amino acid sequences. tRNAs were long considered as house-keeping molecules that lacked regulatory functions. However, a growing body of evidence indicates that cellular tRNA levels fluctuate in correspondence to varying conditions such as cell type, environment, and stress. The fluctuation of tRNA expression directly influences gene translation, favoring or repressing the expression of particular proteins. Ultimately comprehending the dynamic of protein synthesis requires the development of methods able to deliver high-quality tRNA profiles. The method that we present here is named SPOt, which stands for Streamlined Platform for Observing tRNA. SPOt consists of three steps starting with metabolic labeling of cell cultures with radioactive orthophosphate, followed by guanidinium thiocyanate-phenol-chloroform extraction of radioactive total RNAs and finally hybridization on in-house printed macroarrays. tRNA levels are estimated by quantifying the radioactivity intensities at each probe spot. In the protocol presented here we profile tRNAs in Mycobacterium smegmatis mc(2)155, a nonpathogenic bacterium often used as a model organism to study tuberculosis.

Metastasis is a complex mechanism – that accounts for the majority of cancer related-deaths. The ability of primary tumors to develop metastases represents a major roadblock against the action anti-tumor therapeutics. Metastatic cells, on their journey to colonize distant tissues, rely on coordinated and timely molecular programs to adapt to constantly changing environments. High-throughput ‘omics’ methods for profiling DNA, RNA or other analytes are invaluable tools to highlight underlying mechanisms driving cancer progression and to single out potential targets for drug design. From these technologies, both academia and the pharmaceutical industry expected quick breakthroughs in the development of antitumor therapies. Unfortunately, translational research efforts remain too often dampened by drug resistance [1]. The lesson learned through success and failure is that the most significant attribute that works against anti-tumor therapeutics is undoubtedly the acquired capacity of primary tumors to show extraordinary plasticity. Each cell is unique – at any given time and as the tumor progresses, it occupies an exclusive spatiotemporal position consistent with its own molecular program. Tumor cells harbor distinct phenotypes and susceptibility to drugs [2]. These distinct features are regulated through complex biological processes that are orchestrated by transcriptional and post-transcriptional mechanisms. Single-cell sequencing technologies are currently providing significant insights in tumor biology at unprecedented resolution. These analyses allow the dissection of tumor cell heterogeneity by overcoming biases inherent to the analysis of whole tissue samples or cell populations and permit refinement of molecular mechanisms mediating tumor progression. However, to appreciate the extent of tumor cell heterogeneity and fully grasp its clinical relevance, time also has to be considered. Numerous studies have underscored the critical role of the transforming growth factor beta (TGF-ß) during tumor progression [3]. From the existing literature, and our own experimental observations, it is readily appreciated that the overall cellular reprograming induced by TGF-ß is built around a succession of interrelated regulatory mechanisms that are gradually set in place and dismantled over time. Time frames of TGF-ß biological activities vary from minutes to months. Capturing all these steps requires careful and often challenging experimental design. A straightforward illustration of the importance of time as a significant variable is the so-called dichotomous nature of TGF-ß during tumorigenesis, a phenomenon now well known as the “TGF-beta paradox” and described by Roberts et al. some 30 years ago [4]. During the early phase of carcinoma progression, TGF-ß inhibits primary tumor development by inducing cell cycle arrest and apoptosis. In later stages, TGF-ß exerts a pro-tumor effect by stimulating invasion and migration. On a time scale in the order of several days, numerous studies have demonstrated its role as a transcriptional regulator of both epithelial and mesenchymal markers and, ultimately as an effector of cell plasticity. A few years ago, in our laboratory, Chaudhury et al. uncovered a mechanism operating on a time scale in the order of only a few hours in which TGF-ß quickly initiates the translation of a cohort of mesenchymal transcripts including Dab2 and ILEI [5]. Such activation occurs within three to six hours of TGF-ß signaling and results directly from the Akt2-dependent phosphorylation of hnRNP E1 happening within 30 minutes after TGF-ß treatment. More recently, we identified an alternative splicing mechanism activated by TGF-ß and mediated by hnRNP E1, which regulates EMT and metastasis through the generation of a lncRNA acting as a decoy for miRNA-205 [6]. This mechanism occurs within minutes after cytokine addition and persists for only a few hours. Despite its transient nature, the lncRNA-PNUTS induces EMT. Its expression allows for the translation of ZEB proteins which in turn maintain their own expression over time by transcriptionally repressing members of the miRNA-200 family in a feedback loop as previously proposed by Brabletz et al. [7]. This elaborate mechanism illustrates the temporal connection between molecular processes such as transcription, translation and splicing and places ZEB as the central molecular switch of EMT. Such model of regulation allows the cell to respond to immediate and sudden environmental changes. Contrary to the classical model of differentiation in which cell commit to a particular fate in response to regulation of

Epithelial-mesenchymal transition (EMT) is a spatially- and temporally-regulated process involved in physiological and pathological transformations, such as embryonic development and tumor progression. While the role of TGF-β as an EMT-inducer has been extensively documented, the molecular mechanisms regulating this transition and their implications in tumor metastasis are still subjects of intensive debates and investigations. TGF-β regulates EMT through both transcriptional and post-transcriptional mechanisms, and recent advances underline the critical roles of non-coding RNAs in these processes. Although microRNAs and lncRNAs have been clearly identified as effectors of TGF-β-mediated EMT, the contributions of other atypical non-coding RNA species, such as piRNAs, snRNAs, snoRNAs, circRNAs, and even housekeeping tRNAs, have only been suggested and remain largely elusive. This review discusses the current literature including the most recent reports emphasizing the regulatory functions of non-coding RNA in TGF-β-mediated EMT, provides original experimental evidence, and advocates in general for a broader approach in the quest of new regulatory RNAs.

In order to better understand the process of breast cancer metastasis, we have generated a mammary epithelial progression series of increasingly aggressive cell lines that metastasize to lung. Here we demonstrate that upregulation of an endoplasmic reticulum (ER) to Golgi trafficking gene signature in metastatic cells enhances transport kinetics, which promotes malignant progression. We observe increased ER–Golgi trafficking, an altered secretome and sensitivity to the retrograde transport inhibitor brefeldin A (BFA) in cells that metastasize to lung. CREB3 was identified as a transcriptional regulator of upregulated ER–Golgi trafficking genes ARF4, COPB1, and USO1, and silencing of these genes attenuated the metastatic phenotype in vitro and lung colonization in vivo. Furthermore, high trafficking gene expression significantly correlated with increased risk of distant metastasis and reduced relapse-free and overall survival in breast cancer patients, suggesting that modulation of ER–Golgi trafficking plays an important role in metastatic progression.

Recent studies have placed transfer RNA (tRNA), a housekeeping molecule, in the heart of fundamental cellular processes such as embryonic development and tumor progression. Such discoveries were contingent on the concomitant development of methods able to deliver high-quality tRNA profiles. The present study describes the proof of concept obtained in Escherichia coli (E. coli) for an original tRNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). This approach comprises three steps. First, E. coli cultures are spiked with radioactive orthophosphate; second, labeled total RNAs are trizol-extracted; third, RNA samples are hybridized on in-house printed microarrays and spot signals, the proxy for tRNA levels, are quantified by phosphorimaging. Features such as reproducibility and specificity were assessed using several tRNA subpopulations. Dynamic range and sensitivity were evaluated by overexpressing specific tRNA species. SPOt does not require any amplification or post-extraction labeling and can be adapted to any organism. It is modular and easily streamlined with popular techniques such as polysome fractionation to profile tRNAs interacting with ribosomes and actively engaged in translation. The biological relevance of these data is discussed in regards to codon usage, tRNA gene copy number, and position on the genome.

Zonula occludens‐1 (ZO‐1) is a submembrane scaffolding protein that may display proinvasive functions when it relocates from tight junctions into the cytonuclear compartment. This article examines the functional involvement of ZO‐1 in CXCL8/IL‐8 chemokine expression in lung and breast tumor cells. ZO‐1 small interfering RNA and cDNA transfection experiments emphasized regulation of CXCL8/IL‐8 expression via a cytonuclear pool of ZO‐1. Luciferase reporter assays highlighted a 173‐bp region of CXCL8/IL‐8 promoter that responded to ZO‐1. Moreover, by using mutated promoter constructs, we identified a NF‐κB site as critical in this activation. Furthermore, NF‐κB pathway signaling analysis revealed both IκBα and p65 phosphorylation in ZO‐1‐overexpressing cells, and subsequent p65 silencing validated its requirement for CXCL8/IL‐8 induction. Investigation of the functional implication of this regulatory axis next showed the proangiogenic activity of ZO‐1 in both ex vivo and in vivo angiogenesis assays. Finally, we found that non–small‐cell lung carcinoma that presented a cytonuclear ZO‐1 pattern was significantly more angiogenic that that without detectable cytonuclear ZO‐1 expression. Taken together, our results demonstrate that ZO‐1 regulates CXCL8/IL‐8 expression via the NF‐κB signaling pathway and its p65 subunit, which subsequently modulates the transcription of IL‐8. We also provide evidence of a newly identified regulatory pathway that could promote angiogenesis. Thus, our results support the concept that the ZO‐1 shuttle from the cell junction to the cytonuclear compartment may affect both the intrinsic invasive properties of tumor cells and the establishment of the protumoral microenvironment. —Lesage, J., Suarez‐Carmona, M., Neyrinck‐Leglantier, D., Grelet, S., Blacher, S., Hunziker, W., Birembaut, P., Noël, A., Nawrocki‐Raby, B., Gilles, C., Polette, M. Zonula occludens‐1/NF‐κB/CXCL8: a new regulatory axis for tumor angiogenesis. FASEB J. 31, 1678–1688 (2017) www.fasebj.org