Multiplexed Real-Time NMR GTPase Assay for Simultaneous Monitoring of Multiple Guanine Nucleotide Exchange Factor Activities from Human Cancer Cells and Organoids.

Small GTPases (sGTPases) are critical switch-like regulators that mediate several important cellular functions and are often mutated in human cancers. They are activated by guanine nucleotide exchange factors (GEFs), which specifically catalyze the exchange of GTP for GDP. GEFs coordinate signaling networks in normal cells, and are frequently deregulated in cancers. sGTPase signaling pathways are complex and interconnected; however, most GEF assays do not reveal such complexity. In this Communication, we describe the development of a unique real-time NMR-based multiplexed GEF assay that employs distinct isotopic labeling schemes for each sGTPase protein to enable simultaneous observation of six proteins of interest. We monitor nucleotide exchange of KRas, Rheb, RalB, RhoA, Cdc42 and Rac1 in a single system, and assayed the activities of GEFs in lysates of cultured human cells and 3D organoids derived from pancreatic cancer patients. We observed potent activation of RhoA by lysates of HEK293a cells transfected with GEF-H1, along with weak stimulation of Rac1, which we showed is indirect. Our functional analyses of pancreatic cancer-derived organoids revealed higher GEF activity for RhoA than other sGTPases, in line with RNA-seq data indicating high expression of RhoA-specific GEFs.

Tie1 deficiency induces endothelial-mesenchymal transition.

Endothelial-mesenchymal transition (EndMT) has a significant role in embryonic heart formation and in various pathologies. However, the molecular mechanisms that regulate EndMT induction remain to be elucidated. We show that suppression of receptor tyrosine kinase Tie1 but not Tie2 induces human endothelial cells to undergo EndMT and that Slug deficiency reverts this process. We find that Erk1/2, Erk5 and Akt cascades control Slug promoter activity induced by Tie1 deficiency. Interestingly, EndMT is present in human pancreatic tumour. We propose that EndMT associated with Tie1 downregulation participates in the pathological development of stroma observed in tumours.

Nupr1: the Swiss-knife of cancer.

Nupr1 is a small, highly basic and loosely folded multifunctional protein whose expression is induced by several stresses. Its relation to cancer was first suggested by its overexpression in several human malignancies and the association of its expression with breast cancer metastasis. Accordingly, Nupr1 is structurally related to the high-mobility group (HMG) of transcriptional regulators, which play a key role in the stress response and in cancer progression. Nupr1 interacts with numerous partners to regulate cell cycle, programmed cell death, autophagy, chromatin accessibility, and transcription, and its expression is required for regulation of TGFß activity. Pleiotropic functions accomplished by Nupr1 depend on its molecular partners, its location into the cell, its expression level and on the cell-type. Nupr1 might be a new drug-targetable protein whose blockade would prevent cancer progression and metastasis development.

p8 expression controls pancreatic cancer cell migration, invasion, adhesion, and tumorigenesis.

p8 is a stress gene whose activity is necessary for tumor development and progression. The acquisition of invasive properties by transformed cells is a key event in tumor development. In order to establish whether p8 is involved or not in this phenomenon, we assessed the capacity of p8 at influencing cell adhesion, migration, invasion, and tumorigenesis of pancreatic cancer cells. p8 expression was knocked down by a small interfering RNA (siRNA) in pancreatic cancer-derived Panc-1 and MiaPaCa-2 cells and subsequent changes in cell adhesion, migration, invasion, and tumorigenesis were assessed. Influence of p8 silencing on gene expression was analyzed using cDNA microarrays. The influence of inhibiting CDC42, one of the genes most over-expressed in p8-silenced cells, on the changes observed in p8-silenced cells was also evaluated. Finally, the tumorigenic capacities of Panc-1 cells transfected with control siRNA or p8 siRNA were compared by assessing their ability to form colonies in soft agar and to grow as xenografts in nude mice. Knocking-down p8 in pancreatic cancer cells in vitro decreased migration and invasion while increasing cell adhesion; over-expression produced the opposite effect. Knocking down CDC42 reversed almost completely the effects of silencing p8 in vitro. Finally, cells transfected with p8 siRNA were almost unable to form colonies in soft agar. In addition, p8-deficient Panc-1 cells did not develop tumors when injected subcutaneously in nude mice. In conclusion, p8 expression controls pancreatic cancer cell migration, invasion and adhesion, three processes required for metastasis, at least in part, through CDC42, a major regulator of cytoskeleton organization.

Arhgef2 regulates mitotic spindle orientation in hematopoietic stem cells and is essential for productive hematopoiesis.

How hematopoietic stem cells (HSCs) coordinate their divisional axis and whether this orientation is important for stem cell-driven hematopoiesis is poorly understood. Single-cell RNA sequencing data from patients with Shwachman-Diamond syndrome (SDS), an inherited bone marrow failure syndrome, show that ARHGEF2, a RhoA-specific guanine nucleotide exchange factor and determinant of mitotic spindle orientation, is specifically downregulated in SDS hematopoietic stem and progenitor cells (HSPCs). We demonstrate that transplanted Arhgef2-/- fetal liver and bone marrow cells yield impaired hematopoietic recovery and a production deficit from long-term HSCs, phenotypes that are not the result of differences in numbers of transplanted HSCs, their cell cycle status, level of apoptosis, progenitor output, or homing ability. Notably, these defects are functionally restored in vivo by overexpression of ARHGEF2 or its downstream activated RHOA GTPase. By using live imaging of dividing HSPCs, we show an increased frequency of misoriented divisions in the absence of Arhgef2. ARHGEF2 knockdown in human HSCs also impairs their ability to regenerate hematopoiesis, culminating in significantly smaller xenografts. Together, these data demonstrate a conserved role for Arhgef2 in orienting HSPC division and suggest that HSCs may divide in certain orientations to establish hematopoiesis, the loss of which could contribute to HSC dysfunction in bone marrow failure.

p8/nupr1 regulates DNA-repair activity after double-strand gamma irradiation-induced DNA damage.

The stress protein p8 is a small, highly basic, unfolded, and multifunctional protein. We have previously shown that most of its functions are exerted through interactions with other proteins, whose activities are thereby enhanced or repressed. In this work we describe another example of such mechanism, by which p8 binds and negatively regulates MSL1, a histone acetyl transferase (HAT)-associated protein, which in turn binds the DNA-damage-associated 53BP1 protein to facilitate DNA repair following DNA gamma-irradiation. Contrary to the HAT-associated activity, MSL1-dependent DNA-repair activity is almost completely dependent on 53BP1 expression. The picture that has emerged from our findings is that 53BP1 could be a scaffold that gets the HAT MSL1-dependent DNA-repair activity to the sites of DNA damage. Finally, we also found that, although p8 expression is transiently activated after gamma-irradiation, it is eventually submitted to sustained down-regulation, presumably to allow development of MSL1-associated DNA-repair activity. We conclude that interaction of MSL1 with 53BP1 brings MSL1-dependent HAT activity to the vicinity of damaged DNA. MSL1-dependent HAT activity, which is negatively regulated by the stress protein p8, induces chromatin remodeling and relaxation allowing access to DNA of the repair machinery.

Co-dependency between KRAS addiction and ARHGEF2 promotes an adaptive escape from MAPK pathway inhibition.

Oncogenic KRAS engages multiple effector pathways including the MAPK cascade to promote proliferation and survival of pancreatic cancer cells. KRAS-transformed cancer cells exhibit oncogene addiction to sustained activity of RAS for maintenance of malignant phenotypes. Previously, we have shown an essential role for the RHO guanine exchange factor ARHGEF2 for growth and survival of RAS-transformed pancreatic tumors. Here, we have determined that pancreatic cancer cells demonstrating KRAS addiction are significantly dependent on expression of ARHGEF2. Furthermore, enforced expression of ARHGEF2 desensitizes cells to pharmacological MEK inhibition and initiates a positive feedback loop which activates ERK phosphorylation and the downstream ARHGEF2 promoter. Therefore, targeting ARHGEF2 expression may increase the efficacy of MAPK inhibitors for treatment of RAS-dependent pancreatic cancers.

Spatiotemporal dynamics of GEF-H1 activation controlled by microtubule- and Src-mediated pathways.

Rho family GTPases are activated with precise spatiotemporal control by guanine nucleotide exchange factors (GEFs). Guanine exchange factor H1 (GEF-H1), a RhoA activator, is thought to act as an integrator of microtubule (MT) and actin dynamics in diverse cell functions. Here we identify a GEF-H1 autoinhibitory sequence and exploit it to produce an activation biosensor to quantitatively probe the relationship between GEF-H1 conformational change, RhoA activity, and edge motion in migrating cells with micrometer- and second-scale resolution. Simultaneous imaging of MT dynamics and GEF-H1 activity revealed that autoinhibited GEF-H1 is localized to MTs, while MT depolymerization subadjacent to the cell cortex promotes GEF-H1 activation in an ~5-µm-wide peripheral band. GEF-H1 is further regulated by Src phosphorylation, activating GEF-H1 in a narrower band ~0-2 µm from the cell edge, in coordination with cell protrusions. This indicates a synergistic intersection between MT dynamics and Src signaling in RhoA activation through GEF-H1.

An oncogenic KRAS transcription program activates the RHOGEF ARHGEF2 to mediate transformed phenotypes in pancreatic cancer.

Activating mutations of KRAS are nearly ubiquitous in pancreatic adenocarcinomas occurring in greater than 90% of cases. Cellular transformation by oncogenic RAS requires the RHO guanine exchange factor ARHGEF2 (also known as GEF-H1) for tumor growth and survival. Here, we find oncogenic KRAS activates ARHGEF2 through a minimal RAS responsive promoter. We have determined the endogenous ARHGEF2 promoter is positively regulated by the transcription factors ELK1, ETS1, SP1 and SP3 and negatively regulated by the RAS responsive element binding protein (RREB1). We find that the panel of ARHGEF2-regulating transcription factors modulates RAS transformed phenotypes including cellular viability, anchorage-independent growth and invasion-migration of pancreatic cancer cells. RREB1 knockdown activates the amplitude and duration of RHOA via increased ARHGEF2 expression. By relieving the negative regulation of RREB1 on the ARHGEF2 promoter, we determined that ETS1 and SP3 are essential for the normal expression of ARHGEF2 and contribute to the migratory behavior of pancreatic cancer cells. Furthermore, enforced expression of ARHGEF2 rescues loss of SP3 driven invasion-migration and anchorage-independent growth defective phenotypes through restored activation of RHOA. Collectively, our results identify a transcription factor program required for RAS transformation and provide mechanistic insight into the highly metastatic behavior of pancreatic cancer.

MARK3-mediated phosphorylation of ARHGEF2 couples microtubules to the actin cytoskeleton to establish cell polarity.

The PAR-1-MARK pathway controls cell polarity through the phosphorylation of microtubule-associated proteins. Rho-Rac guanine nucleotide exchange factor 2 (ARHGEF2), which activates Ras homolog family member A (RHOA), is anchored to the microtubule network and sequestered in an inhibited state through binding to dynein light chain Tctex-1 type 1 (DYNLT1). We showed in mammalian cells that liver kinase B1 (LKB1) activated the microtubule affinity-regulating kinase 3 (MARK3), which in turn phosphorylated ARHGEF2 at Ser151 This modification disrupted the interaction between ARHGEF2 and DYNLT1 by generating a 14-3-3 binding site in ARHGEF2, thus causing ARHGEF2 to dissociate from microtubules. Phosphorylation of ARHGEF2 by MARK3 stimulated RHOA activation and the formation of stress fibers and focal adhesions, and was required for organized cellular architecture in three-dimensional culture. Protein phosphatase 2A (PP2A) dephosphorylated Ser151 in ARHGEF2 to restore the inhibited state. Thus, we have identified a regulatory switch controlled by MARK3 that couples microtubules to the actin cytoskeleton to establish epithelial cell polarity through ARHGEF2.

GEF-H1 is necessary for neutrophil shear stress-induced migration during inflammation.

Leukocyte crawling and transendothelial migration (TEM) are potentiated by shear stress caused by blood flow. The mechanism that couples shear stress to migration has not been fully elucidated. We found that mice lacking GEF-H1 (GEF-H1-/-), a RhoA-specific guanine nucleotide exchange factor (GEF), displayed limited migration and recruitment of neutrophils into inflamed tissues. GEF-H1-/- leukocytes were deficient in in vivo crawling and TEM in the postcapillary venules. We demonstrated that although GEF-H1 deficiency had little impact on the migratory properties of neutrophils under static conditions, shear stress triggered GEF-H1-dependent spreading and crawling of neutrophils and relocalization of GEF-H1 to flotillin-2-rich uropods. Our results identify GEF-H1 as a component of the shear stress response machinery in neutrophils required for a fully competent immune response to bacterial infection.

Pancreatitis-associated protein does not predict disease relapse in inflammatory bowel disease patients.

BACKGROUND: The pancreatitis-associated protein (PAP) is increased in the serum of active inflammatory bowel disease (IBD) patients and its levels seem to be correlated with disease activity. Our aim was to evaluate the usefulness of serum and fecal PAP measurements to predict relapse in patients with inactive IBD. MATERIALS AND METHODS: We undertook a 12-month prospective study that included 66 Crohn's disease (CD) and 74 ulcerative colitis (UC) patients. At inclusion, patients were in clinical remission, defined by a Harvey-Bradshaw (HB) Index≤4 (CD) or a partial Mayo Score (MS)<3 (UC), along with a normal serum C reactive protein (CRP) and fecal calprotectin. Patients were followed every 3 months. Blood and stool samples were collected and a clinical evaluation was performed at each visit. Serum PAP and CRP levels as well as fecal concentrations of PAP and calprotectin were assessed. RESULTS: Active CD patients had an increased mean serum PAP at the diagnosis of the flare (104.1 ng/ml) and 3 months prior to activity (22.68 ng/ml) compared with patients in remission (13.26 ng/ml), p<0.05. No significant change in serum PAP levels in UC and fecal PAP levels in CD and UC were detected during disease activity. In CD, serum PAP was a poor diagnostic predictor of disease activity, with an AUC of 0.69. In patients in remission, fecal PAP was barely detectable in UC compared with CD patients. CONCLUSION: Serum PAP is increased only in active CD patients, but this marker does not predict disease activity. Inactive UC patients have marked low levels of PAP in fecal samples compared with CD patients.

The RhoGEF GEF-H1 is required for oncogenic RAS signaling via KSR-1.

Cellular transformation by oncogenic RAS engages the MAPK pathway under strict regulation by the scaffold protein KSR-1. Here, we report that the guanine nucleotide exchange factor GEF-H1 plays a critical role in a positive feedback loop for the RAS/MAPK pathway independent of its RhoGEF activity. GEF-H1 acts as an adaptor protein linking the PP2A B' subunits to KSR-1, thereby mediating the dephosphorylation of KSR-1 S392 and activation of MAPK signaling. GEF-H1 is important for the growth and survival of HRAS(V12)-transformed cells and pancreatic tumor xenografts. GEF-H1 expression is induced by oncogenic RAS and is correlated with pancreatic neoplastic progression. Our results, therefore, identify GEF-H1 as an amplifier of MAPK signaling and provide mechanistic insight into the progression of RAS mutant tumors.

Development of an ELISA detecting Tumor Protein 53-Induced Nuclear Protein 1 in serum of prostate cancer patients.

Tumor Protein 53-Induced Nuclear Protein 1 (TP53INP1) plays an important role during cell stress response in synergy with the potent "genome-keeper" p53. In human, the gene encoding TP53INP1 is expressed at very high level in some pathological situations, such as inflammation and prostate cancer (PC). TP53INP1 overexpression in PC seems to be a worse prognostic factor, particularly predictive of biological cancer relapse, making TP53INP1 a relevant specific target for molecular therapy of Castration Resistant (CR) PC. In that context, detection of TP53INP1 in patient biological fluids is a promising diagnostic avenue. We report here successful development of a new Enzyme-Linked Immunosorbent Assay (ELISA) detecting TP53INP1, taking advantage of molecular tools (monoclonal antibodies (mAbs) and recombinant proteins) generated in the laboratory during the course of basic functional investigations devoted to TP53INP1. The ELISA principle is based on a sandwich immunoenzymatic system, TP53INP1 protein being trapped by a first specific mAb coated on microplate then recognized by a second specific mAb. This new assay allows specific detection of TP53INP1 in serum of several PC patients. This breakthrough paves the way towards investigation of a large cohort of patients and assessment of clinical applications of TP53INP1 dosage.

NUPR1 works against the metabolic stress-induced autophagy-associated cell death in pancreatic cancer cells.

The incidence of pancreatic adenocarcinoma is increasing with more than 43,000 predicted new cases in the US and 65,000 in Europe this year. Pancreatic cancer patients have a short life expectancy with less than 3-4% 5-y survival, which results in an equivalent incidence and mortality rate. One of the major challenges in pancreatic cancer is the identification of pharmacological approaches that overcome the resistance of this cancer to therapy. Intensive research in the past decades has led to the classification of pancreatic cancers and the identification of the driver key genetic events. Despite the advances in understanding the molecular mechanisms responsible for pancreatic cancer pathogenesis, this knowledge had little impact on significantly improving the treatment for this dismal disease. In particular, we know today that the lack of therapeutic response in pancreatic cancer is due to the intrinsic high resistance of these tumors to chemotherapy and radiation, rather than to the inappropriate design of these therapeutic approaches. Thus, in order to ensure a better outcome for pancreatic cancer patients, there is a strong need for research focused on the mechanism that determines this resistant phenotype and the means that might drive enhanced response to therapy.

Nupr1-aurora kinase A pathway provides protection against metabolic stress-mediated autophagic-associated cell death.

PURPOSE: The limited supply of oxygen and nutrients is thought to result in rigorous selection of cells that will eventually form the tumor. EXPERIMENTAL DESIGN: Nupr1 expression pattern was analyzed in human tissue microarray (TMA) and correlated with survival time of the patient. Microarray analysis was conducted on MiaPaCa2 cells subjected to metabolic stress in Nupr1-silenced conditions. DNA repair and cell cycle-associated gene expression was confirmed by real-time quantitative PCR (qRT-PCR). Nupr1 and AURKA protective role were analyzed using RNA interference (RNAi) silencing or overexpression. DNA damage and autophagy were analyzed by Western blot analysis and immunofluorescence. RESULTS: We showed that both Nupr1 and HIF1α are coexpressed in human pancreatic ductal adenocarcinoma (PDAC) samples and negatively correlate with survival time. PDAC-derived cells submitted to hypoxia and/or glucose starvation induce DNA damage-dependent cell death concomitantly to the overexpression of stress protein Nupr1. Affymetrix-based transcriptoma analysis reveals that Nupr1 knockdown enhances DNA damage and alters the expression of several genes involved in DNA repair and cell-cycle progression. Expression of some of these genes is common to hypoxia and glucose starvation, such as Aurka gene, suggesting that Nupr1 overexpression counteracts the transcriptional changes occurring under metabolic stress. The molecular mechanism by which hypoxia and glucose starvation induce cell death involves autophagy-associated, but not caspase-dependent, cell death. Finally, we have found that AURKA expression is partially regulated by Nupr1 and plays a major role in this response. CONCLUSIONS: Our data reveal that Nupr1 is involved in a defense mechanism that promotes pancreatic cancer cell survival when exposed to metabolic stress.

Homotypic cell cannibalism, a cell-death process regulated by the nuclear protein 1, opposes to metastasis in pancreatic cancer.

Pancreatic adenocarcinoma (PDAC) is an extremely deadly disease for which all treatments available have failed to improve life expectancy significantly. This may be explained by the high metastatic potential of PDAC cells, which results from their dedifferentiation towards a mesenchymal phenotype. Some PDAC present cell-in-cell structures whose origin and significance are currently unknown. We show here that cell-in-cells form after homotypic cell cannibalism (HoCC). We found PDAC patients whose tumours display HoCC develop less metastasis than those without. In vitro, HoCC was promoted by inactivation of the nuclear protein 1 (Nupr1), and was enhanced by treatment with transforming growth factor ß. HoCC ends with death of PDAC cells, consistent with a metastasis suppressor role for this phenomenon. Hence, our data indicates a protective role for HoCC in PDAC and identifies Nupr1 as a molecular regulator of this process.

Nuclear protein 1 promotes pancreatic cancer development and protects cells from stress by inhibiting apoptosis.

Pancreatic ductal adenocarcinoma (PDAC) has the lowest survival rate of all cancers and shows remarkable resistance to cell stress. Nuclear protein 1 (Nupr1), which mediates stress response in the pancreas, is frequently upregulated in pancreatic cancer. Here, we report that Nupr1 plays an essential role in pancreatic tumorigenesis. In a mouse model of pancreatic cancer with constitutively expressed oncogenic Kras(G12D), we found that loss of Nupr1 protected from the development of pancreatic intraepithelial neoplasias (PanINs). Further, in cultured pancreatic cells, nutrient deprivation activated Nupr1 expression, which we found to be required for cell survival. We found that Nupr1 protected cells from stress-induced death by inhibiting apoptosis through a pathway dependent on transcription factor RelB and immediate early response 3 (IER3). NUPR1, RELB, and IER3 proteins were coexpressed in mouse PanINs from Kras(G12D)-expressing pancreas. Moreover, pancreas-specific deletion of Relb in a Kras(G12D) background resulted in delayed in PanIN development associated with a lack of IER3 expression. Thus, efficient PanIN formation was dependent on the expression of Nupr1 and Relb, with likely involvement of IER3. Finally, in patients with PDAC, expression of NUPR1, RELB, and IER3 was significantly correlated with a poor prognosis. Cumulatively, these results reveal a NUPR1/RELB/IER3 stress-related pathway that is required for oncogenic Kras(G12D)-dependent transformation of the pancreas.