Pancreatic ductal adenocarcinoma ubiquitination profiling reveals specific prognostic and theranostic markers.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has been widely studied at multiomics level. However, little is known about its specific ubiquitination, a major post-translational modification (PTM). As PTMs regulate the final function of any gene, we decided to establish the ubiquitination profiles of 60 PDAC. METHODS: We used specific proteomic tools to establish the ubiquitin dependent proteome (ubiquitinome) of frozen PDXs (Patients' derived xenographs). Then, we performed bioinformatics analysis to identify the possible associations of these ubiquitination profiles with tumour phenotype, patient survival and resistance to chemotherapies. Finally, we used proximity ligation assays (PLA) to detect and quantify the ubiquitination level of one identified marker. FINDINGS: We identified 38 ubiquitination site profiles correlating with the transcriptomic phenotype of tumours and four had notable prognostic capabilities. Seventeen ubiquitination profiles displayed potential theranostic marker for gemcitabine, seven for 5-FU, six for oxaliplatin and thirteen for irinotecan. Using PLA, we confirmed the use of one ubiquitination profile as a drug-response marker, directly on paraffin embedded tissues, supporting the possible application of these biomarkers in the clinical setting. INTERPRETATION: These findings bring new and important insights on the relationship between ubiquitination levels of proteins and different molecular and clinical features of PDAC patients. Markers identified in this study could have a potential application in clinical settings to help to predict response to chemotherapies thereby allowing the personalization of treatments. FUNDING: Fondation ARC (PJA 20181208270 and PGA 12021010002840_3562); INCa; Canceropôle PACA; DGOS; Amidex Foundation; Fondation de France; and INSERM.

GLI1, a novel target of the ER stress regulator p97/VCP, promotes ATF6f-mediated activation of XBP1.

Upon accumulation of improperly folded proteins in the Endoplasmic Reticulum (ER), the Unfolded Protein Response (UPR) is triggered to restore ER homeostasis. The induction of stress genes is a sine qua non condition for effective adaptive UPR. Although this requirement has been extensively described, the mechanisms underlying this process remain in part uncharacterized. Here, we show that p97/VCP, an AAA+ ATPase known to contribute to ER stress-induced gene expression, regulates the transcription factor GLI1, a primary effector of Hedgehog (Hh) signaling. Under basal (non-ER stress) conditions, GLI1 is repressed by a p97/VCP-HDAC1 complex while upon ER stress GLI1 is induced through a mechanism requiring both USF2 binding and increase histone acetylation at its promoter. Interestingly, the induction of GLI1 was independent of ligand-regulated Hh signaling. Further analysis showed that GLI1 cooperates with ATF6f to induce promoter activity and expression of XBP1, a key transcription factor driving UPR. Overall, our work demonstrates a novel role for GLI1 in the regulation of ER stress gene expression and defines the interplay between p97/VCP, HDAC1 and USF2 as essential players in this process.

Nupr1 deficiency downregulates HtrA1, enhances SMAD1 signaling, and suppresses age-related bone loss in male mice.

Nuclear protein 1 (NUPR1) is a stress-induced protein activated by various stresses, such as inflammation and oxidative stress. We previously reported that Nupr1 deficiency increased bone volume by enhancing bone formation in 11-week-old mice. Analysis of differentially expressed genes between wild-type (WT) and Nupr1-knockout (Nupr1-KO) osteocytes revealed that high temperature requirement A 1 (HTRA1), a serine protease implicated in osteogenesis and transforming growth factor-ß signaling was markedly downregulated in Nupr1-KO osteocytes. Nupr1 deficiency also markedly reduced HtrA1 expression, but enhanced SMAD1 signaling in in vitro-cultured primary osteoblasts. In contrast, Nupr1 overexpression enhanced HtrA1 expression in osteoblasts, suggesting that Nupr1 regulates HtrA1 expression, thereby suppressing osteoblastogenesis. Since HtrA1 is also involved in cellular senescence and age-related diseases, we analyzed aging-related bone loss in Nupr1-KO mice. Significant spine trabecular bone loss was noted in WT male and female mice during 6-19 months of age, whereas aging-related trabecular bone loss was attenuated, especially in Nupr1-KO male mice. Moreover, cellular senescence-related markers were upregulated in the osteocytes of 6-19-month-old WT male mice but markedly downregulated in the osteocytes of 19-month-old Nupr1-KO male mice. Oxidative stress-induced cellular senescence stimulated Nupr1 and HtrA1 expression in in vitro-cultured primary osteoblasts, and Nupr1 overexpression enhanced p16ink4a expression in osteoblasts. Finally, NUPR1 expression in osteocytes isolated from the bones of patients with osteoarthritis was correlated with age. Collectively, these results indicate that Nupr1 regulates HtrA1-mediated osteoblast differentiation and senescence. Our findings unveil a novel Nupr1/HtrA1 axis, which may play pivotal roles in bone formation and age-related bone loss.

Melatonin controls cell proliferation and modulates mitochondrial physiology in pancreatic stellate cells.

We have investigated the effects of melatonin on major pathways related with cellular proliferation and energetic metabolism in pancreatic stellate cells. In the presence of melatonin (1 mM, 100 µM, 10 µM, or 1 µM), decreases in the phosphorylation of c-Jun N-terminal kinase and of p44/42 and an increase in the phosphorylation of p38 were observed. Cell viability dropped in the presence of melatonin. A rise in the phosphorylation of AMP-activated protein kinase was detected in the presence of 1 mM and 100 µM melatonin. Treatment with 1 mM melatonin decreased the phosphorylation of protein kinase B, whereas 100 µM and 10 µM melatonin increased its phosphorylation. An increase in the generation of mitochondrial reactive oxygen species and a decrease of mitochondrial membrane potential were noted following melatonin treatment. Basal and maximal respiration, ATP production by oxidative phosphorylation, spare capacity, and proton leak dropped in the presence of melatonin. The expression of complex I of the mitochondrial respiratory chain was augmented in the presence of melatonin. Conversely, in the presence of 1 mM melatonin, decreases in the expression of mitofusins 1 and 2 were detected. The glycolysis and the glycolytic capacity were diminished in cells treated with 1 mM or 100 µM melatonin. Increases in the expression of phosphofructokinase-1 and lactate dehydrogenase were noted in cells incubated with 100 µM, 10 µM, or 1 µM melatonin. The expression of glucose transporter 1 was increased in cells incubated with 10 µM or 1 µM melatonin. Conversely, 1 mM melatonin decreased the expression of all three proteins. Our results suggest that melatonin, at pharmacological concentrations, might modulate mitochondrial physiology and energy metabolism in addition to major pathways involved in pancreatic stellate cell proliferation.

Deciphering the Binding of the Nuclear Localization Sequence of Myc Protein to the Nuclear Carrier Importin α3.

The oncoprotein Myc is a transcription factor regulating global gene expression and modulating cell proliferation, apoptosis, and metabolism. Myc has a nuclear localization sequence (NLS) comprising residues Pro320 to Asp328, to allow for nuclear translocation. We designed a peptide comprising such region and the flanking residues (Ala310-Asn339), NLS-Myc, to study, in vitro and in silico, the ability to bind importin α3 (Impα3) and its truncated species (ΔImpα3) depleted of the importin binding domain (IBB), by using fluorescence, circular dichroism (CD), biolayer interferometry (BLI), nuclear magnetic resonance (NMR), and molecular simulations. NLS-Myc interacted with both importin species, with affinity constants of ~0.5 µM (for Impα3) and ~60 nM (for ΔImpα3), as measured by BLI. The molecular simulations predicted that the anchoring of NLS-Myc took place in the major binding site of Impα3 for the NLS of cargo proteins. Besides clarifying the conformational behavior of the isolated NLS of Myc in solution, our results identified some unique properties in the binding of this localization sequence to the nuclear carrier Impα3, such as a difference in the kinetics of its release mechanism depending on the presence or absence of the IBB domain.

Culture media composition influences patient-derived organoid ability to predict therapeutic responses in gastrointestinal cancers.

BACKGROUNDA patient-derived organoid (PDO) platform may serve as a promising tool for translational cancer research. In this study, we evaluated PDO's ability to predict clinical response to gastrointestinal (GI) cancers.METHODSWe generated PDOs from primary and metastatic lesions of patients with GI cancers, including pancreatic ductal adenocarcinoma, colorectal adenocarcinoma, and cholangiocarcinoma. We compared PDO response with the observed clinical response for donor patients to the same treatments.RESULTSWe report an approximately 80% concordance rate between PDO and donor tumor response. Importantly, we found a profound influence of culture media on PDO phenotype, where we showed a significant difference in response to standard-of-care chemotherapies, distinct morphologies, and transcriptomes between media within the same PDO cultures.CONCLUSIONWhile we demonstrate a high concordance rate between donor tumor and PDO, these studies also showed the important role of culture media when using PDOs to inform treatment selection and predict response across a spectrum of GI cancers.TRIAL REGISTRATIONNot applicable.FUNDINGThe Joan F. & Richard A. Abdoo Family Fund in Colorectal Cancer Research, GI Cancer program of the Mayo Clinic Cancer Center, Mayo Clinic SPORE in Pancreatic Cancer, Center of Individualized Medicine (Mayo Clinic), Department of Laboratory Medicine and Pathology (Mayo Clinic), Incyte Pharmaceuticals and Mayo Clinic Hepatobiliary SPORE, University of Minnesota-Mayo Clinic Partnership, and the Early Therapeutic program (Department of Oncology, Mayo Clinic).

CD9 mediates the uptake of extracellular vesicles from cancer-associated fibroblasts that promote pancreatic cancer cell aggressiveness.

In pancreatic ductal adenocarcinoma (PDAC), signaling from stromal cells is implicated in metastatic progression. Tumor-stroma cross-talk is often mediated through extracellular vesicles (EVs). We previously reported that EVs derived from cancer-associated stromal fibroblasts (CAFs) that are abundant in annexin A6 (ANXA6+ EVs) support tumor cell aggressiveness in PDAC. Here, we found that the cell surface glycoprotein and tetraspanin CD9 is a key component of CAF-derived ANXA6+ EVs for mediating this cross-talk. CD9 was abundant on the surface of ANXA6+ CAFs isolated from patient PDAC samples and from various mouse models of PDAC. CD9 colocalized with CAF markers in the tumor stroma, and CD9 abundance correlated with tumor stage. Blocking CD9 impaired the uptake of ANXA6+ EVs into cultured PDAC cells. Signaling pathway arrays and further analyses revealed that the uptake of CD9+ANXA6+ EVs induced mitogen-activated protein kinase (MAPK) pathway activity, cell migration, and epithelial-to-mesenchymal transition (EMT). Blocking either CD9 or p38 MAPK signaling impaired CD9+ANXA6+ EV-induced cell migration and EMT in PDAC cells. Analysis of bioinformatic datasets indicated that CD9 abundance was an independent marker of poor prognosis in patients with PDAC. Our findings suggest that CD9-mediated stromal cell signaling promotes PDAC progression.

Bola-Amphiphilic Glycodendrimers: New Carbohydrate-Mimicking Scaffolds to Target Carbohydrate-Binding Proteins.

Dendrimers are appealing scaffolds for creating carbohydrate mimics with unique multivalent cooperativity. We report here novel bola-amphiphilic glycodendrimers bearing mannose and glucose terminals, and a hydrophobic thioacetal core responsive to reactive oxygen species. The peculiar bola-amphiphilic feature enabled stronger binding to lectin compared to conventional amphiphiles. In addition, these dendrimers are able to target mannose receptors and glucose transporters expressed at the surface of cells, thus allowing effective and specific cellular uptake. This highlights their great promise for targeted delivery.

Melatonin modulates metabolic adaptation of pancreatic stellate cells subjected to hypoxia.

Pancreatic stellate cells (PSCs), the main cell type responsible for the development of fibrosis in pancreatic cancer, proliferate actively under hypoxia. Melatonin has received attention as a potential antifibrotic agent due to its anti-proliferative actions on PSCs. In this work, we investigated the activation of the PI3K/Akt/mTOR pathway and the metabolic adaptations that PSCs undergo under hypoxic conditions, as well as the probable modulation by melatonin. Incubation of cells under hypoxia induced an increase in cell proliferation, and in the expression of alpha-smooth muscle actin and of collagen type 1. In addition, an increase in the phosphorylation of Akt was observed, whereas a decrease in the phosphorylation of PTEN and GSK-3b was noted. The phosphorylation of mTOR and its substrate p70 S6K was decreased under hypoxia. Treatment of PSCs with melatonin under hypoxia diminished cell proliferation, the levels of alpha-smooth muscle actin and of collagen type 1, the phosphorylation of Akt and increased phosphorylation of mTOR. Mitochondrial activity decreased in PSCs under hypoxia. A glycolytic shift was observed. Melatonin further decreased mitochondrial activity. Under hypoxia, no increase in autophagic flux was noted. However, melatonin treatment induced autophagy activation. Nevertheless, inhibition of this process did not induce detectable changes in the viability of cells treated with melatonin. We conclude that PSCs undergo metabolic adaptation under hypoxia that might help them survive and that pharmacological concentrations of melatonin modulate cell responses to hypoxia. Our results contribute to the knowledge of the mechanisms by which melatonin could modulate fibrosis within the pancreas.

Ketogenic HMG-CoA lyase and its product ß-hydroxybutyrate promote pancreatic cancer progression.

Pancreatic ductal adenocarcinoma (PDA) tumor cells are deprived of oxygen and nutrients and therefore must adapt their metabolism to ensure proliferation. In some physiological states, cells rely on ketone bodies to satisfy their metabolic needs, especially during nutrient stress. Here, we show that PDA cells can activate ketone body metabolism and that ß-hydroxybutyrate (ßOHB) is an alternative cell-intrinsic or systemic fuel that can promote PDA growth and progression. PDA cells activate enzymes required for ketogenesis, utilizing various nutrients as carbon sources for ketone body formation. By assessing metabolic gene expression from spontaneously arising PDA tumors in mice, we find HMG-CoA lyase (HMGCL), involved in ketogenesis, to be among the most deregulated metabolic enzymes in PDA compared to normal pancreas. In vitro depletion of HMGCL impedes migration, tumor cell invasiveness, and anchorage-independent tumor sphere compaction. Moreover, disrupting HMGCL drastically decreases PDA tumor growth in vivo, while ßOHB stimulates metastatic dissemination to the liver. These findings suggest that ßOHB increases PDA aggressiveness and identify HMGCL and ketogenesis as metabolic targets for limiting PDA progression.

OncoSNIPE® Study Protocol, a study of molecular profiles associated with development of resistance in solid cancer patients.

BACKGROUND: Nowadays, evaluation of the efficacy and the duration of treatment, in context of monitoring patients with solid tumors, is based on the RECIST methodology. With these criteria, resistance and/or insensitivity are defined as tumor non-response which does not allow a good understanding of the diversity of the underlying mechanisms. The main objective of the OncoSNIPE® collaborative clinical research program is to identify early and late markers of resistance to treatment. METHODS: Multicentric, interventional study with the primary objective to identify early and / or late markers of resistance to treatment, in 600 adult patients with locally advanced or metastatic triple negative or Luminal B breast cancer, non-small-cell lung cancer or pancreatic ductal adenocarcinoma. Patients targeted in this study have all rapid progression of their pathology, making it possible to obtain models for evaluating markers of early and / or late responses over the 2-year period of follow-up, and thus provide the information necessary to understand resistance mechanisms. To explore the phenomena of resistance, during therapeutic response and / or progression of the pathology, we will use a multidisciplinary approach including high-throughput sequencing (Exome-seq and RNAseq), clinical data, medical images and immunological profile by ELISA. Patients will have long-term follow-up with different biological samples, at baseline (blood and biopsy) and at each tumoral evaluation or tumoral progression evaluated by medical imaging. Clinical data will be collected through a dedicated Case Report Form (CRF) and enriched by semantic extraction based on the French ConSoRe (Continuum Soins Recherche) initiative, a dedicated Semantic Clinical Data Warehouse (SCDW) to cancer. The study is sponsored by Oncodesign (Dijon, France) and is currently ongoing. DISCUSSION: The great diversity of intrinsic or acquired molecular mechanisms involved in resistance to treatment constitutes a real therapeutic issue. Improving understanding of mechanisms of resistance of cancer cells to anti-tumor treatments is therefore a major challenge. The OncoSNIPE cohort will lead to a better understanding of the mechanisms of resistance and will allow to explore new mechanisms of actions and to discover new therapeutic targets or strategies making it possible to circumvent the escape in different types of cancer. TRIAL REGISTRATION: Clinicaltrial.gov. Registered 16 September 2020, https://clinicaltrials.gov/ct2/show/NCT04548960?term=oncosnipe&draw=2&rank=1 and ANSM ID RCB 2017-A02018-45.

KrasG12D induces changes in chromatin territories that differentially impact early nuclear reprogramming in pancreatic cells.

BACKGROUND: Pancreatic ductal adenocarcinoma initiation is most frequently caused by Kras mutations. RESULTS: Here, we apply biological, biochemical, and network biology methods to validate GEMM-derived cell models using inducible KrasG12D expression. We describe the time-dependent, chromatin remodeling program that impacts function during early oncogenic signaling. We find that the KrasG12D-induced transcriptional response is dominated by downregulated expression concordant with layers of epigenetic events. More open chromatin characterizes the ATAC-seq profile associated with a smaller group of upregulated genes and epigenetic marks. RRBS demonstrates that promoter hypermethylation does not account for the silencing of the extensive gene promoter network. Moreover, ChIP-Seq reveals that heterochromatin reorganization plays little role in this early transcriptional program. Notably, both gene activation and silencing primarily depend on the marking of genes with a combination of H3K27ac, H3K4me3, and H3K36me3. Indeed, integrated modeling of all these datasets shows that KrasG12D regulates its transcriptional program primarily through unique super-enhancers and enhancers, and marking specific gene promoters and bodies. We also report chromatin remodeling across genomic areas that, although not contributing directly to cis-gene transcription, are likely important for KrasG12D functions. CONCLUSIONS: In summary, we report a comprehensive, time-dependent, and coordinated early epigenomic program for KrasG12D in pancreatic cells, which is mechanistically relevant to understanding chromatin remodeling events underlying transcriptional outcomes needed for the function of this oncogene.

Design of Inhibitors of the Intrinsically Disordered Protein NUPR1: Balance between Drug Affinity and Target Function.

Intrinsically disordered proteins (IDPs) are emerging as attractive drug targets by virtue of their physiological ubiquity and their prevalence in various diseases, including cancer. NUPR1 is an IDP that localizes throughout the whole cell, and is involved in the development and progression of several tumors. We have previously repurposed trifluoperazine (TFP) as a drug targeting NUPR1 and, by using a ligand-based approach, designed the drug ZZW-115 starting from the TFP scaffold. Such derivative compound hinders the development of pancreatic ductal adenocarcinoma (PDAC) in mice, by hampering nuclear translocation of NUPR1. Aiming to further improve the activity of ZZW-115, here we have used an indirect drug design approach to modify its chemical features, by changing the substituent attached to the piperazine ring. As a result, we have synthesized a series of compounds based on the same chemical scaffold. Isothermal titration calorimetry (ITC) showed that, with the exception of the compound preserving the same chemical moiety at the end of the alkyl chain as ZZW-115, an increase of the length by a single methylene group (i.e., ethyl to propyl) significantly decreased the affinity towards NUPR1 measured in vitro, whereas maintaining the same length of the alkyl chain and adding heterocycles favored the binding affinity. However, small improvements of the compound affinity towards NUPR1, as measured by ITC, did not result in a corresponding improvement in their inhibitory properties and in cellulo functions, as proved by measuring three different biological effects: hindrance of the nuclear translocation of the protein, sensitization of cells against DNA damage mediated by NUPR1, and prevention of cancer cell growth. Our findings suggest that a delicate compromise between favoring ligand affinity and controlling protein function may be required to successfully design drugs against NUPR1, and likely other IDPs.

LDL receptor-peptide conjugate as in vivo tool for specific targeting of pancreatic ductal adenocarcinoma.

Despite clinical advances in diagnosis and treatment, pancreatic ductal adenocarcinoma (PDAC) remains the third leading cause of cancer death, and is still associated with poor prognosis and dismal survival rates. Identifying novel PDAC-targeted tools to tackle these unmet clinical needs is thus an urgent requirement. Here we use a peptide conjugate that specifically targets PDAC through low-density lipoprotein receptor (LDLR). We demonstrate by using near-infrared fluorescence imaging the potential of this conjugate to specifically detect and discriminate primary PDAC from healthy organs including pancreas and from benign mass-forming chronic pancreatitis, as well as detect metastatic pancreatic cancer cells in healthy liver. This work paves the way towards clinical applications in which safe LDLR-targeting peptide conjugate promotes tumor-specific delivery of imaging and/or therapeutic agents, thereby leading to substantial improvements of the PDAC patient's outcome.

Inactivation of the Euchromatic Histone-Lysine N-Methyltransferase 2 Pathway in Pancreatic Epithelial Cells Antagonizes Cancer Initiation and Pancreatitis-Associated Promotion by Altering Growth and Immune Gene Expression Networks.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, painful disease with a 5-year survival rate of only 9%. Recent evidence indicates that distinct epigenomic landscapes underlie PDAC progression, identifying the H3K9me pathway as important to its pathobiology. Here, we delineate the role of Euchromatic Histone-lysine N-Methyltransferase 2 (EHMT2), the enzyme that generates H3K9me, as a downstream effector of oncogenic KRAS during PDAC initiation and pancreatitis-associated promotion. EHMT2 inactivation in pancreatic cells reduces H3K9me2 and antagonizes Kras G12D -mediated acinar-to-ductal metaplasia (ADM) and Pancreatic Intraepithelial Neoplasia (PanIN) formation in both the Pdx1-Cre and P48 Cre/+ Kras G12D mouse models. Ex vivo acinar explants also show impaired EGFR-KRAS-MAPK pathway-mediated ADM upon EHMT2 deletion. Notably, Kras G12D increases EHMT2 protein levels and EHMT2-EHMT1-WIZ complex formation. Transcriptome analysis reveals that EHMT2 inactivation upregulates a cell cycle inhibitory gene expression network that converges on the Cdkn1a/p21-Chek2 pathway. Congruently, pancreas tissue from Kras G12D animals with EHMT2 inactivation have increased P21 protein levels and enhanced senescence. Furthermore, loss of EHMT2 reduces inflammatory cell infiltration typically induced during Kras G12D -mediated initiation. The inhibitory effect on Kras G12D -induced growth is maintained in the pancreatitis-accelerated model, while simultaneously modifying immunoregulatory gene networks that also contribute to carcinogenesis. This study outlines the existence of a novel KRAS-EHMT2 pathway that is critical for mediating the growth-promoting and immunoregulatory effects of this oncogene in vivo, extending human observations to support a pathophysiological role for the H3K9me pathway in PDAC.

Crowding Effects on the Structure and Dynamics of the Intrinsically Disordered Nuclear Chromatin Protein NUPR1.

The intracellular environment is crowded with macromolecules, including sugars, proteins and nucleic acids. In the cytoplasm, crowding effects are capable of excluding up to 40% of the volume available to any macromolecule when compared to dilute conditions. NUPR1 is an intrinsically disordered protein (IDP) involved in cell-cycle regulation, stress-cell response, apoptosis processes, DNA binding and repair, chromatin remodeling and transcription. Simulations of molecular crowding predict that IDPs can adopt compact states, as well as more extended conformations under crowding conditions. In this work, we analyzed the conformation and dynamics of NUPR1 in the presence of two synthetic polymers, Ficoll-70 and Dextran-40, which mimic crowding effects in the cells, at two different concentrations (50 and 150 mg/ml). The study was carried out by using a multi-spectroscopic approach, including: site-directed spin labelling electron paramagnetic resonance spectroscopy (SDSL-EPR), nuclear magnetic resonance spectroscopy (NMR), circular dichroism (CD), small angle X-ray scattering (SAXS) and dynamic light scattering (DLS). SDSL-EPR spectra of two spin-labelled mutants indicate that there was binding with the crowders and that the local dynamics of the C and N termini of NUPR1 were partially affected by the crowders. However, the overall disordered nature of NUPR1 did not change substantially in the presence of the crowders, as shown by circular dichroism CD and NMR, and further confirmed by EPR. The changes in the dynamics of the paramagnetic probes appear to be related to preferred local conformations and thus crowding agents partially affect some specific regions, further pinpointing that NUPR1 flexibility has a key physiological role in its activity.

Human Endogenous Retrovirus (HERV)-K env Gene Knockout Affects Tumorigenic Characteristics of nupr1 Gene in DLD-1 Colorectal Cancer Cells.

Human endogenous retroviruses (HERVs) are suggested to be involved in the development of certain diseases, especially cancers. To elucidate the function of HERV-K Env protein in cancers, an HERV-K env gene knockout (KO) in DLD-1 colorectal cancer cell lines was generated using the CRISPR-Cas9 system. Transcriptome analysis of HERV-K env KO cells using next-generation sequencing (NGS) was performed to identify the key genes associated with the function of HERV-K Env protein. The proliferation of HERV-K env KO cells was significantly reduced in in vitro culture as well as in in vivo nude mouse model. Tumorigenic characteristics, including migration, invasion, and tumor colonization, were also significantly reduced in HERV-K env KO cells. Whereas, they were enhanced in HERV-K env over-expressing DLD-1 cells. The expression of nuclear protein-1 (NUPR1), an ER-stress response factor that plays an important role in cell proliferation, migration, and reactive oxygen species (ROS) generation in cancer cells, significantly reduced in HERV-K env KO cells. ROS levels and ROS-related gene expression was also significantly reduced in HERV-K env KO cells. Cells transfected with NUPR1 siRNA (small interfering RNA) exhibited the same phenotype as HERV-K env KO cells. These results suggest that the HERV-K env gene affects tumorigenic characteristics, including cell proliferation, migration, and tumor colonization through NUPR1 related pathway.

Intrinsically disordered protein NUPR1 binds to the armadillo-repeat domain of Plakophilin 1.

Plakophilin 1 (PKP1), a member of the armadillo repeat family of proteins, is a scaffold component of desmosomes, which are key structural components for cell-cell adhesion. However, PKP1 can be also found in the nucleus of several cells. NUPR1 is an intrinsically disordered protein (IDP) that localizes throughout the whole cell, and intervenes in the development and progression of several cancers. In this work, we studied the binding between PKP1 and NUPR1 by using several in vitro biophysical techniques and in cellulo approaches. The interaction occurred with an affinity in the low micromolar range (~10 µM), and involved the participation of at least one of the tryptophan residues of PKP1 (as shown by fluorescence and molecular docking). The binding region of NUPR1, mapped by NMR and molecular modelling, was a polypeptide patch at the 30s region of its sequence. The association between PKP1 and NUPR1 also occurred in cellulo and was localized in the nucleus, as tested by protein ligation assays (PLAs). We hypothesize that NUPR1 plays an active role in carcinogenesis modulating the function of PKP1.