Cancers adapt proteostasis to cope with the burden of misfolded proteins, stabilize key signalling nodes and sustain their malignant behaviour. Tumour stroma is subjected to similar stresses, but how they influence its aberrant status remains unclear. We show that tumour stroma presents consistent upregulation of target genes associated to the major misfolding regulator HSP90 in cancer-associated fibroblasts (CAFs), and that HSP90α is required for CAFs to remodel the extracellular matrix (ECM) and promote cancer cell motility and growth. Mechanistically, HSP90α sustains TGFβ responses and YAP protein levels required for CAF functionality. In vivo, stromal or fibroblast-specific loss of HSP90α results in reduced ECM deposition, angiogenesis, growth and dissemination of breast tumours. Clinical analyses reveal a correlation between HSP90-dependent programs and YAP activity in CAFs, that are also associated with poor patient prognosis. Our findings uncover a link between proteostasis, mechanotransduction and generation of aggressive tumour microenvironments through HSP90α. ### Competing Interest Statement The authors have declared no competing interest. Data generated during the course of this study, including RNAseq and MS-proteomics datasets of murine NF1 and CAF1 after transfection with control, Hsp90aa1 or Hps90ab1 RNAi is currently being processed for deposition in appropriate public repositories. The following public datasets have been employed in this study and acquired through NCBI GEO. Human tumour stroma: Finak (GSE9014, Breast), Yeoung (GSE40595, Ovary), and Nishida (GSE35602, Colon), Karnoub (GSE8977, Breast) and Planche (GSE26910, Breast); expression profiles of murine and human NF/CAFs: GSE20086 (human breast NF and CAFs), GSE70468 (human colon NF and CAFs), GSE35250 (human ovarian NF and CAFs), GSE45256 (murine breast NF and CAFs); expression profiles of freshly isolated murine mammary NF and CAFs (GSE195864); expression profile of MEFs subjected to control and 200 nM of NVP-AUY922 (HSP90 inhibitor) for 16h (GSE125161); expression profile and associated clinical information of colorectal cancer patients (GSE17538). METABRIC gene expression data and the associated clinical information were obtained from cBioPortal. Breast scRNAseq data was obtained from the European Genome-Phenome Archive (E-MTAB-10607).

Background: Currently there are limited methods to link disease severity and risk of disease progression in Chronic Kidney Disease (CKD). To better understand this potential relationship, we interrogated the renal transcriptomic profile of individuals with CKD with measures of CKD severity and identified FERM-domain containing protein 3 (FRMD3) as a candidate gene for follow-up study. Methods: RNA-seq was used to profile the transcriptome of CKD biopsies from the North Dublin Renal BioBank the results of which were correlated with clinical parameters. The potential function of FRMD3 was explored by interrogating the FRMD3 interactome and assessing the impact of lentiviral mediated FRMD3 knock down on human renal proximal tubule epithelial cells by assessing cell viability, metabolic activity, and structural markers. Results: We identified a subset of 93 genes which are significantly correlated with estimated glomerular filtration rate and percentage tubulointerstitial fibrosis at time of biopsy and with CKD progression 5 years post-biopsy. These results were validated against transcriptomic data from an external cohort of 432 nephrectomy samples. One of the top-ranking genes from this subset, FRMD3, has previously been associated with the risk of developing diabetic kidney disease. Interrogating the interactome of FRMD3 in tubule epithelial cells revealed interactions with cytoskeletal components of cell-cell junctions. Knockdown of FRMD3 expression in tubule epithelial cells resulted in increased pro-apoptotic activity within the cells as well as dysregulation of E-Cadherin. Conclusions: We have identified a panel of kidney-specific transcripts correlated with severity and progression of kidney disease, and from this have identified a possible role for FRMD3 in tubule cell structure and health.

ERK1/2 mitogen‐activated protein kinases (ERK) are key regulators of basic cellular processes, including proliferation, survival, and migration. Upon phosphorylation, ERK becomes activated and a portion of it dimerizes. The importance of ERK activation in specific cellular events is generally well documented, but the role played by dimerization is largely unknown. Here, we demonstrate that impeding ERK dimerization precludes cellular movement by interfering with the molecular machinery that executes the rearrangements of the actin cytoskeleton. We also show that a constitutively dimeric ERK mutant can drive cell motility per se , demonstrating that ERK dimerization is both necessary and sufficient for inducing cellular migration. Importantly, we unveil that the scaffold protein kinase suppressor of Ras 1 (KSR1) is a critical element for endowing external agonists, acting through tyrosine kinase receptors, with the capacity to induce ERK dimerization and, subsequently, to unleash cellular motion. In agreement, clinical data disclose that high KSR1 expression levels correlate with greater metastatic potential and adverse evolution of mammary tumors. Overall, our results portray both ERK dimerization and KSR1 as essential factors for the regulation of cell motility and mammary tumor dissemination.

The transcription factor MYC regulates cell proliferation, transformation, and survival in response to growth factor signaling that is mediated in part by the kinase activity of ERK2. Because ERK2 can also bind to DNA to modify gene expression, we investigated whether it more directly regulates MYC transcription. We identified ERK2 binding sites in the MYC promoter and detected ERK2 at the promoter in various serum-stimulated cell types. Expression of nuclear-localized ERK2 constructs in serum-starved cells revealed that ERK2 in the nucleus-regardless of its kinase activity-increased MYC mRNA expression and MYC protein abundance. ERK2 bound to the promoter through its amino-terminal insert domain and to the cyclin-dependent kinase CDK9 (which activates RNA polymerase II) through its carboxyl-terminal conserved docking domain. Both interactions were essential for ERK2-induced MYC expression, and depleting ERK impaired CDK9 occupancy and RNA polymerase II progression at the MYC promoter. Artificially tethering CDK9 to the MYC promoter by fusing it to the ERK2 insert domain was sufficient to stimulate MYC expression in serum-starved cells. Our findings demonstrate a role for ERK2 at the MYC promoter acting as a kinase-independent anchor for the recruitment of CDK9 to promote MYC expression.

The tumor microenvironment (TME) is reprogrammed by cancer cells and participates in all stages of tumor progression. The contribution of stromal cells to the reprogramming of the TME is not well understood. Here, we provide evidence of the role of the cytokine oncostatin M (OSM) as central node for multicellular interactions between immune and nonimmune stromal cells and the epithelial cancer cell compartment. OSM receptor (OSMR) deletion in a multistage breast cancer model halted tumor progression. We ascribed causality to the stromal function of the OSM axis by demonstrating reduced tumor burden of syngeneic tumors implanted in mice lacking OSMR. Single-cell and bioinformatic analysis of murine and human breast tumors revealed that OSM expression was restricted to myeloid cells, whereas OSMR was detected predominantly in fibroblasts and, to a lower extent, cancer cells. Myeloid-derived OSM reprogrammed fibroblasts to a more contractile and tumorigenic phenotype and elicited the secretion of VEGF and proinflammatory chemokines CXCL1 and CXCL16, leading to increased myeloid cell recruitment. Collectively, our data support the notion that the stromal OSM/OSMR axis reprograms the immune and nonimmune microenvironment and plays a key role in breast cancer progression.

Mitochondria are the main consumers of oxygen within the cell. How mitochondria sense oxygen levels remains unknown. Here we show an oxygen-sensitive regulation of TFAM, an activator of mitochondrial transcription and replication, whose alteration is linked to tumours arising in the von Hippel-Lindau syndrome. TFAM is hydroxylated by EGLN3 and subsequently bound by the von Hippel-Lindau tumour-suppressor protein, which stabilizes TFAM by preventing mitochondrial proteolysis. Cells lacking wild-type VHL or in which EGLN3 is inactivated have reduced mitochondrial mass. Tumorigenic VHL variants leading to different clinical manifestations fail to bind hydroxylated TFAM. In contrast, cells harbouring the Chuvash polycythaemia VHLR200W mutation, involved in hypoxia-sensing disorders without tumour development, are capable of binding hydroxylated TFAM. Accordingly, VHL-related tumours, such as pheochromocytoma and renal cell carcinoma cells, display low mitochondrial content, suggesting that impaired mitochondrial biogenesis is linked to VHL tumorigenesis. Finally, inhibiting proteolysis by targeting LONP1 increases mitochondrial content in VHL-deficient cells and sensitizes therapy-resistant tumours to sorafenib treatment. Our results offer pharmacological avenues to sensitize therapy-resistant VHL tumours by focusing on the mitochondria.

Mitochondria are the main consumers of oxygen within the cell. How mitochondria sense oxygen levels remains unknown. Here we show an oxygen-sensitive regulation of TFAM, an activator of mitochondrial transcription and replication, whose alteration is linked to tumours arising in the von Hippel-Lindau syndrome. TFAM is hydroxylated by EGLN3 and subsequently bound by the von Hippel-Lindau tumour-suppressor protein, which stabilizes TFAM by preventing mitochondrial proteolysis. Cells lacking wild-type VHL or in which EGLN3 is inactivated have reduced mitochondrial mass. Tumorigenic VHL variants leading to different clinical manifestations fail to bind hydroxylated TFAM. In contrast, cells harbouring the Chuvash polycythaemia VHLR200W mutation, involved in hypoxia-sensing disorders without tumour development, are capable of binding hydroxylated TFAM. Accordingly, VHL-related tumours, such as pheochromocytoma and renal cell carcinoma cells, display low mitochondrial content, suggesting that impaired mitochondrial biogenesis is linked to VHL tumorigenesis. Finally, inhibiting proteolysis by targeting LONP1 increases mitochondrial content in VHL-deficient cells and sensitizes therapy-resistant tumours to sorafenib treatment. Our results offer pharmacological avenues to sensitize therapy-resistant VHL tumours by focusing on the mitochondria.

Histone post-translational modifications (PTMs) are important for regulating various DNA-templated processes. Here, we report the existence of a histone PTM in mammalian cells, namely histone H3 with hydroxylation of proline at residue 16 (H3P16oh), which is catalyzed by the proline hydroxylase EGLN2. We show that H3P16oh enhances direct binding of KDM5A to its substrate, histone H3 with trimethylation at the fourth lysine residue (H3K4me3), resulting in enhanced chromatin recruitment of KDM5A and a corresponding decrease of H3K4me3 at target genes. Genome- and transcriptome-wide analyses show that the EGLN2–KDM5A axis regulates target gene expression in mammalian cells. Specifically, our data demonstrate repression of the WNT pathway negative regulator DKK1 through the EGLN2-H3P16oh-KDM5A pathway to promote WNT/β-catenin signaling in triple-negative breast cancer (TNBC). This study characterizes a regulatory mark in the histone code and reveals a role for H3P16oh in regulating mammalian gene expression. EGLN2 hydroxylates histone H3 at proline 16, enhancing the binding of KDM5A to H3K4me3. Genome- and transcriptome-wide analyses show that the EGLN2–KDM5A axis regulates target gene expression in mammalian cells.

MNT, a transcription factor of the MXD family, is an important modulator of the oncoprotein MYC. Both MNT and MYC are basic-helix–loop–helix proteins that heterodimerize with MAX in a mutually exclusive manner, and bind to E-boxes within regulatory regions of their target genes. While MYC generally activates transcription, MNT represses it. However, the molecular interactions involving MNT as a transcriptional regulator beyond the binding to MAX remain unexplored. Here we demonstrate a novel MAX-independent protein interaction between MNT and REL, the oncogenic member of the NF-κB family. REL participates in important biological processes and it is altered in a variety of tumors. REL is a transcription factor that remains inactive in the cytoplasm in an inhibitory complex with IκB and translocates to the nucleus when the NF-κB pathway is activated. In the present manuscript, we show that MNT knockdown triggers REL translocation into the nucleus and thus the activation of the NF-κB pathway. Meanwhile, MNT overexpression results in the repression of IκBα, a bona fide REL target. Both MNT and REL bind to the IκBα gene on the first exon, suggesting its regulation as an MNT–REL complex. Altogether our data indicate that MNT acts as a repressor of the NF-κB pathway by two mechanisms: (1) retention of REL in the cytoplasm by MNT interaction, and (2) MNT-driven repression of REL-target genes through an MNT–REL complex. These results widen our knowledge about MNT biological roles and reveal a novel connection between the MYC/MXD and NF-κB pathways, two of the most prominent pathways in cancer.

Amino acid hydroxylation is a common post-translational modification, which generally regulates protein interactions or adds a functional group that can be further modified. Such hydroxylation is currently considered irreversible, necessitating the degradation and re-synthesis of the entire protein to reset the modification. Here we present evidence that the cellular machinery can reverse FIH-mediated asparagine hydroxylation on intact proteins. These data suggest that asparagine hydroxylation is a flexible and dynamic post-translational modification akin to modifications involved in regulating signalling networks, such as phosphorylation, methylation and ubiquitylation.

Fast amoeboid migration is critical for developmental processes and can be hijacked by cancer cells to enhance metastatic dissemination. This migratory behavior is tightly controlled by high levels of actomyosin contractility, but how it is coupled to other cytoskeletal components is poorly understood. Septins are increasingly recognized as novel cytoskeletal components, but details on their regulation and contribution to migration are lacking. Here, we show that the septin regulator Cdc42EP5 is consistently required for amoeboid melanoma cells to invade and migrate into collagen-rich matrices and locally invade and disseminate in vivo. Cdc42EP5 associates with actin structures, leading to increased actomyosin contractility and amoeboid migration. Cdc42EP5 affects these functions through SEPT9-dependent F-actin cross-linking, which enables the generation of F-actin bundles required for the sustained stabilization of highly contractile actomyosin structures. This study provides evidence that Cdc42EP5 is a regulator of cancer cell motility that coordinates actin and septin networks and describes a unique role for SEPT9 in melanoma invasion and metastasis.

AbstractFast amoeboid migration in the invasive fronts of melanoma is controlled by high levels of actomyosin contractility, which underlie its highly metastatic potential. How this migratory behaviour is coupled to other cytoskeletal components is poorly understood. Septins are increasingly recognized as novel cytoskeletal components, but details on their regulation and contribution to cancer migration and metastasis are lacking. Here, we show that the septin regulator Cdc42EP5 is consistently required for melanoma cells to migrate and invade into collagen-rich matrices, and to locally invade and disseminate in vivo. Cdc42EP5 associates with actin structures leading to increased actomyosin contractility and amoeboid migration. Cdc42EP5 effects these functions through SEPT9-dependent F-actin crosslinking, which enables the generation of F-actin bundles required for the sustained stabilisation of highly contractile actomyosin structures. This study provides evidence for Cdc42EP5 as a regulator of cancer cell motility that coordinates actin and septin networks. It also describes a unique role for SEPT9 in invasion and metastasis, and illustrates a mechanism that regulates its function in melanoma.

Protein hydroxylation affects protein stability, activity, and interactome, therefore contributing to various diseases including cancers. However, the transiency of the hydroxylation reaction hinders the identification of hydroxylase substrates. By developing an enzyme-substrate trapping strategy coupled with TAP-TAG or orthogonal GST- purification followed by mass spectrometry, we identify adenylosuccinate lyase (ADSL) as an EglN2 hydroxylase substrate in triple negative breast cancer (TNBC). ADSL expression is higher in TNBC than other breast cancer subtypes or normal breast tissues. ADSL knockout impairs TNBC cell proliferation and invasiveness in vitro and in vivo. An integrated transcriptomics and metabolomics analysis reveals that ADSL activates the oncogenic cMYC pathway by regulating cMYC protein level via a mechanism requiring ADSL proline 24 hydroxylation. Hydroxylation-proficient ADSL, by affecting adenosine levels, represses the expression of the long non-coding RNA MIR22HG, thus upregulating cMYC protein level. Our findings highlight the role of ADSL hydroxylation in controlling cMYC and TNBC tumorigenesis.

Despite the discovery of the oxygen-sensitive regulation of HIFα by the von Hippel-Lindau (VHL) protein, the mechanisms underlying the complex genotype/phenotype correlations in VHL disease remain unknown. Some germline VHL mutations cause familial pheochromocytoma and encode proteins that preserve their ability to down-regulate HIFα. While type 1, 2A, and 2B VHL mutants are defective in regulating HIFα, type 2C mutants encode proteins that preserve their ability to down-regulate HIFα. Here, we identified an oxygen-sensitive function of VHL that is abolished by VHL type 2C mutations. We found that BIM-EL, a proapoptotic BH3-only protein, is hydroxylated by EglN3 and subsequently bound by VHL. VHL mutants fail to bind hydroxylated BIM-EL, regardless of whether they have the ability to bind hydroxylated HIFα or not. VHL binding inhibits BIM-EL phosphorylation by extracellular signal-related kinase (ERK) on serine 69. This causes BIM-EL to escape from proteasomal degradation, allowing it to enhance EglN3-induced apoptosis. BIM-EL was rapidly degraded in cells lacking wild-type VHL or in which EglN3 was inactivated genetically or by lack of oxygen, leading to enhanced cell survival and chemotherapy resistance. Combination therapy using ERK inhibitors, however, resensitizes VHL- and EglN3-deficient cells that are otherwise cisplatin-resistant.

Modern omics technologies allow us to obtain global information on different types of biological networks. However, integrating these different types of analyses into a coherent framework for a comprehensive biological interpretation remains challenging. Here, we present a conceptual framework that integrates protein interaction, phosphoproteomics, and transcriptomics data. Applying this method to analyze HRAS signaling from different subcellular compartments shows that spatially defined networks contribute specific functions to HRAS signaling. Changes in HRAS protein interactions at different sites lead to different kinase activation patterns that differentially regulate gene transcription. HRAS-mediated signaling is the strongest from the cell membrane, but it regulates the largest number of genes from the endoplasmic reticulum. The integrated networks provide a topologically and functionally resolved view of HRAS signaling. They reveal distinct HRAS functions including the control of cell migration from the endoplasmic reticulum and TP53-dependent cell survival when signaling from the Golgi apparatus.

Fast amoeboid migration in the invasive fronts of melanoma is controlled by high levels of actomyosin contractility, which underlie its highly metastatic potential. How this migratory behaviour is coupled to other cytoskeletal components is poorly understood. Septins are increasingly recognized as novel cytoskeletal components, but details on their regulation and contribution to cancer migration and metastasis are lacking. Here, we show that the septin regulator Cdc42EP5 is consistently required for melanoma cells to migrate and invade into collagen-rich matrices, and to locally invade and disseminate in vivo . Cdc42EP5 associates with actin structures leading to increased actomyosin contractility and amoeboid migration. Cdc42EP5 effects these functions through SEPT9-dependent F-actin crosslinking, which enables the generation of F-actin bundles required for the sustained stabilisation of highly contractile actomyosin structures. This study provides evidence for Cdc42EP5 as a regulator of cancer cell motility that coordinates actin and septin networks. It also describes a unique role for SEPT9 in invasion and metastasis, and illustrates a mechanism that regulates its function in melanoma.

Carbon dioxide (CO2) is a physiological gas produced as a by‐product of oxidative metabolism. Physiologically, CO2 levels within the blood of respiring animals are significantly higher than the external environment at approximately 35mmHg or 5% CO2. This creates a CO2 gradient around respiring organisms that many species have evolved the ability to sense. For instance, mosquitos sense the CO2 gradient around organisms in order to locate a blood meal. However, while CO2 is acutely sensed via chemosensitive neurons, CO2 also impacts on a transcriptional level. Notably, studies in Drosophila have demonstrated that flies exposed to hypercapnia (elevated CO2) have significantly increased infection‐induced mortality due to down‐regulation of multiple Rel‐dependent genes. These genes code for anti‐microbial peptides which are key immune effectors regulated by an NFκB orthologue Relish. In humans, hypercapnia is a feature of many lung pathologies such as chronic obstructive pulmonary disease (COPD) and is associated with a poor prognosis in these patients at least in part due to their susceptibility to infection. Thus, the current state of the art is that hypercapnia is detrimental in the context of infection due to immunosuppression. Interestingly however, recent studies suggest that exploiting the anti‐inflammatory effects of CO2 might be of benefit clinically e.g. during one lung ventilation. Importantly, the mechanisms of action for these effects are poorly understood. Thus, the aim of this project is to gain novel, mechanistic insight into CO2‐dependent alterations of NFκB signalling and inflammatory gene expression.In our previously published work we have shown that in the absence of an inflammatory stimulus, hypercapnia results in altered RelB processing, nuclear translocation and protein‐protein interaction (particularly those in DNA binding). Furthermore, several other members of the non‐canonical NFκB family such as IKKα and p100 also undergo cellular re‐localisation. Here we investigate the effect of hypercapnia on inflammatory signalling against the background of an immune stimulus and determine the relative contribution of key NFkB proteins in the response. In the stimulated state (cells treated with lipopolysaccharide (LPS)), we show using a cytokine array that hypercapnia alters inflammatory cytokine expression. Our data indicates that against a background of immune stimulation hypercapnia can alter both canonical (p65) and non‐canonical (RelB) NFκB‐regulated cytokines. Taken together, this study gives exciting, novel mechanistic insight into the effects of CO2 on NFκB‐dependent inflammatory signaling which is of clinical relevance for patients who experience elevated levels of CO2.Support or Funding InformationFunded by UCD School of Medicine and Science Foundation Ireland (15/CDA/3490)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of how a stimulus regulates the dynamics of protein-protein interaction is critical for understanding how a particular protein is regulated in an intracellular signaling network. Protein hydroxylation, which is a posttranslational modification catalyzed by oxygen-dependent enzymes, is a crucial regulator of protein-protein interactions. Under low oxygen conditions, the activity of many hydroxylases is inhibited, which results in a reduction of substrate hydroxylation. These changes alter the interactome of the substrate, and this dynamic rewiring of signaling networks explains crucial aspects of the adaptive response to hypoxia. In order to fully understand the systemic role of hydroxylation, it is necessary to identify a comprehensive set of substrates, as well as to determine which residues are hydroxylated. In addition, hydroxylation-dependent changes in the interactome of the substrates are indicative of the molecular function of the modification. To identify new substrates of hydroxylases, we have developed an approach involving the use of a pharmacological substrate-trap strategy followed by label-free quantitative mass spectrometry. An overview is provided for the sample preparation, mass spectrometry techniques, and statistical analysis used for detection of new substrates, hydroxylated residue, and hydroxylation-dependent protein-protein interaction changes.