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, pancreasspecific 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 stressrelated pathway that is required for oncogenic Kras G12D -dependent transformation of the pancreas. IntroductionPancreatic ductal adenocarcinoma (PDAC) has the highest mortality rate and the lowest overall survival of all cancers (less than 3%-4% at 5 years). The incidence of PDAC almost equals its mortality rate and is increasing every year, with more than 38,000 predicted new cases in the United States and 65,000 in Europe. Surgery is the most effective treatment, but the mean life expectancy of the 15%-20% of patients who present with a resectable tumor is only 15-18 months (1). Even for patients eligible for surgery, aggressive metastasis often appears after operation, and survival of patients with metastatic disease is only 3-6 months (2). Chemotherapy and radiotherapy offer limited benefit for patients undergoing surgery in metastatic disease (3). Most strategies tested so far for the treatment of PDAC have consisted of using therapies that show some efficacy in other carcinomas, but none of them improved significantly the overall survival of PDAC patients. Hence, in order to develop new, efficient therapies against PDAC, future research must take into account PDAC specificities such as its remarkable resistance to cell stress, notably to the stress induced by chemotherapy and radiotherapy (4), and the preponderant presence of a fibrotic stroma, which favors tumor progression by creating a barrier against drug delivery and

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 β. 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.

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 TGFβ 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.

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.

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 HIF1a 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.

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.

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.

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.