Functional Characterization of Nupr1L, A Novel p53-Regulated Isoform of the High-Mobility Group (HMG)-Related Protumoral Protein Nupr1.

We have previously demonstrated a crucial role of nuclear protein 1 (NUPR1) in tumor development and progression. In this work, we report the functional characterization of a novel Nupr1-like isoform (NUPR1L) and its functional interaction with the protumoral factor NUPR1. Through the use of primary sequence analysis, threading, and homology-based molecular modeling, as well as expression and immunolocalization, studies reveal that NUPR1L displays properties, which are similar to member of the HMG-like family of chromatin regulators, including its ability to translocate to the cell nucleus and bind to DNA. Analysis of the NUPR1L promoter showed the presence of two p53-response elements at positions -37 and -7, respectively. Experiments using reporter assays combined with site-directed mutagenesis and using cells with controllable p53 expression demonstrate that both of these sequences are responsible for the regulation of NUPR1L expression by p53. Congruently, NUPR1L gene expression is activated in response to DNA damage induced by oxaliplatin treatment or cell cycle arrest induced by serum starvation, two well-validated methods to achieve p53 activation. Interestingly, expression of NUPR1L downregulates the expression of NUPR1, its closely related protumoral isoform, by a mechanism that involves the inhibition of its promoter activity. At the cellular level, overexpression of NUPR1L induces G1 cell cycle arrest and a decrease in their cell viability, an effect that is mediated, at least in part, by downregulating NUPR1 expression. Combined, these experiments constitute the first functional characterization of NUPR1L as a new p53-induced gene, which negatively regulates the protumoral factor NUPR1.

Chloroquine plays a cell-dependent role in the response to treatment of pancreatic adenocarcinoma.

In this study, our aim is to assess the role played by autophagy and its inhibition in the different PDAC cellular compartments, and its involvement in chemo-resistance using primary human pancreatic cancer-derived cells (PCC) and Cancer Associated Fibroblasts (CAF). Autophagy flux, as measured by LC3-I and -II in the presence of Chloroquine, showed a variable level in PCC and CAFs. We found no correlation between autophagy level and degree of tumor differentiation. Association of Chloroquine with gemcitabine, 5FU, oxaliplatin, irinotecan and docetaxel revealed that its effect on survival is cell- and drug-dependent in vitro and in vivo. In addition, we demonstrated that autophagy in CAFs can play an important role in sensitizing PDAC to anticancer treatments since its inhibition increased the resistance of PCCs to gemcitabine. In conclusion, this work clearly shows a heterogeneity in the effect of Chloroquine and highlights a role of CAFs autophagy in sensitizing tumors to treatments. It also reveals that the role of autophagy is more complex than expected in PDAC as well as its sensitivity to treatments.

Pivotal Role of the Chromatin Protein Nupr1 in Kras-Induced Senescence and Transformation.

Nupr1 is a chromatin protein, which cooperates with Kras(G12D) to induce PanIN formation and pancreatic cancer development in mice, though the molecular mechanisms underlying this effect remain to be fully characterized. In the current study, we report that Nupr1 acts as a gene modifier of the effect of Kras(G12D)-induced senescence by regulating Dnmt1 expression and consequently genome-wide levels of DNA methylation. Congruently, 5-aza-2'-deoxycytydine, a general inhibitor of DNA methylation, reverses the Kras(G12D)-induced PanIN development by promoting senescence. This requirement of Nupr1 expression, however, is not restricted to the pancreas since in lung of Nupr1(-/-) mice the expression of Kras(G12D) induces senescence instead of transformation. Therefore, mechanistically this data reveals that epigenetic events, at least at the level of DNA methylation, modulate the functional outcome of common genetic mutations, such as Kras(G12D), during carcinogenesis. The biomedical relevance of these findings lies in that they support the rational for developing similar therapeutic interventions in human aimed at controlling either the initiation or progression of cancer.

Targeting CD44 as a novel therapeutic approach for treating pancreatic cancer recurrence.

Pancreatic ductal adenocarcinoma (PDAC) is an extraordinarily lethal disease, which, despite a more or less efficient chemotherapeutic treatment, systematically displays a rapid and uncontrolled progression towards a fatal recurrence. Determining which cells give rise to such tumor recurrence is thus crucial before an improved therapeutics outcome can be envisaged for patients with PDAC. In this regard, we recently reported that following a standard chemotherapy for PDAC, a heterogeneous subpopulation of CD44+ cells proliferates and is responsible for tumor recurrence, as shown by almost all recurrent tumor cells becoming CD44+. We designed a strategy to eliminate these cells based on a weekly administration of an anti-CD44 monoclonal antibody to human PDAC-derived xenografts in mice. We demonstrate that xenografts, which were unresponsive to gemcitabine treatment, are however sensitive to this strategy. In conclusion, CD44 represents an efficient therapeutic target in patients with recurrent PDAC.

IL17 Functions through the Novel REG3ß-JAK2-STAT3 Inflammatory Pathway to Promote the Transition from Chronic Pancreatitis to Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) offers an optimal model for discovering "druggable" molecular pathways that participate in inflammation-associated cancer development. Chronic pancreatitis, a common prolonged inflammatory disease, behaves as a well-known premalignant condition that contributes to PDAC development. Although the mechanisms underlying the pancreatitis-to-cancer transition remain to be fully elucidated, emerging evidence supports the hypothesis that the actions of proinflammatory mediators on cells harboring Kras mutations promote neoplastic transformation. Recent elegant studies demonstrated that the IL17 pathway mediates this phenomenon and can be targeted with antibodies, but the downstream mechanisms by which IL17 functions during this transition are currently unclear. In this study, we demonstrate that IL17 induces the expression of REG3ß, a well-known mediator of pancreatitis, during acinar-to-ductal metaplasia and in early pancreatic intraepithelial neoplasia (PanIN) lesions. Furthermore, we found that REG3ß promotes cell growth and decreases sensitivity to cell death through activation of the gp130-JAK2-STAT3-dependent pathway. Genetic inactivation of REG3ß in the context of oncogenic Kras-driven PDAC resulted in reduced PanIN formation, an effect that could be rescued by administration of exogenous REG3ß. Taken together, our findings provide mechanistic insight into the pathways underlying inflammation-associated pancreatic cancer, revealing a dual and contextual pathophysiologic role for REG3ß during pancreatitis and PDAC initiation.

Deciphering the cellular source of tumor relapse identifies CD44 as a major therapeutic target in pancreatic adenocarcinoma.

It has been commonly found that in patients presenting Pancreatic Ductal Adenocarcinoma (PDAC), after a period of satisfactory response to standard treatments, the tumor becomes non-responsive and patient death quickly follows. This phenomenon is mainly due to the rapid and uncontrolled development of the residual tumor. The origin and biological characteristics of residual tumor cells in PDAC still remain unclear. In this work, using PDACs from patients, preserved as xenografts in nude mice, we demonstrated that a residual PDAC tumor originated from a small number of CD44+ cells present in the tumor. During PDAC relapse, proliferating CD44+ cells decrease expression of ZEB1, while overexpressing the MUC1 protein, and gain morphological and biological characteristics of differentiation. Also, we report that CD44+ cells, in primary and residual PDAC tumors, are part of a heterogeneous population, which includes variable numbers of CD133+ and EpCAM+ cells. We confirmed the propagation of CD44+ cells in samples from cases of human relapse, following standard PDAC treatment. Finally, using systemic administration of anti-CD44 antibodies in vivo, we demonstrated that CD44 is an efficient therapeutic target for treating tumor relapse, but not primary PDAC tumors. We conclude that CD44+ cells generate the relapsing tumor and, as such, are themselves promising therapeutic targets for treating patients with recurrent PDAC.

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.