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May 9, 2012

Pancreatic Cancer Target Identified that Protects Cells Against Stress

  • Scientists have identified a potential pancreatic cancer target that in a mouse model appears to play an essential role in tumorigenesis, and in human patients correlates with poor survival. Nuclear protein 1 (Nupr1) mediates stress response in the pancreas and is often upregulated in pancreatic cancer. In vitro and in vivo studies by a Centre de Recherche en Cancérologie de Marseille (CRCM)-led team showed that Nupr1 is essential both for the survival of pancreatic cancer cells exposed to stress, and for the development of precancerous PanIN lesions in KrasG12D mice.

    Mechanistically, the chromatin-binding Nupr1 protein was found to activate expression of the NF-κB family member RelB, which then induces expression of the antiapoptotic factor IER3. Evalaution of human pancreatic tissue samples demonstrated that expression of Nupr1, RelB, and IER3 was associated with poor prognosis. Juan Lucio Iovanna, Ph.D., and colleagues describe their findings in the Journal of Clinical Investigation, in a paper titled “Nuclear protein 1 promotes pancreatic cancer development and protects cells from stress by inhibiting apoptosis.”

    Pancreatic ductal adenocarcinoma (PDAC) has the highest mortality rate and the lowest overall survival of all cancers, and even patients who undergo surgery often display aggressive metastasis, after which survival averages at just 3–6 months. Notably, PDAC is particularly resistant to cellular stress induced by chemotherapy and radiotherapy, and poor vascularization that is characteristic of the disease.

    A potentially important mediator of these anti-stress survival responses is Nupr1, one of the best-characterized stress sensors in the pancreas, the researchers note. The gene is highly expressed during the progression of PDAC and other cancers, and is induced in pancreatic cancer cells by endogenous and exogenous stress agents. The CRCM team and colleagues investigated the effects of Nupr1 deficiency in a mouse model of pancreatic cancer that displays constitutively activated KrasG12D, and also carried out in vivo studies to investigate the role of Nupr1 in the survival of pancreatic cancer cells in response to stress.

    Initial studies demonstrated that while KrasG12D mice develop numerous PanIN lesions from 13 weeks of age, Nupr1-knockout mice (KrasG12D;Nupr1KO) didn’t develop any PanINs, and only about a third of the animals bred to be heterozygous for Nupr1 (Nupr1+/- ) developed PanIN lesions. Working on the hypothesis that Nupr1 responds to stress signals by triggering a protective gene expression cascade, the researchers then exposed pancreatic cells to nutrient deprivation. Under these conditions Nupr1 mRNA was increased up to 19-fold within 9 hours of stress. In contrast, and under the same conditions, pancreatic cells in which Nupr1 was inactivated using an siRNA demonstrated significantly reduced cell survival and increased apoptosis. In fact the Nupr1 knockout cells exhibited lower survival when compared with control cells, even when cultured in conventional medium.

    Upregulation of Nupr1 in response to environmental stress was associated with the activation of key survival genes, and in particular Relb, the researchers found. Nutrient deprivation and starvation led to marked increases in RELB mRNA expression in unmodified pancreatic cancer cells, but this increase was dramatically inhibited following Nupr1 inhibition. “ChIP assays demonstrated that Nupr1 directly binds the endogenous Relb promoter, supporting a role for this chromatin-associated HMG protein in the regulation of NF-κB gene expression,” the authors write. Moreover, knocking out RelB in pancreatic cells subjected to nutrient deprivation-induced stress was associated with decrease levels of Nupr1, and led to significant decreases in cell survival and apoptosis. This suggested that a positive feedback loop operates between Nupr1 and RelB, the investigators suggest.

    Further microarray and PCR analyses identified immediate early response 3 (IER3) as a key prosurvival gene triggered by Nupr1/RelB activation. Under conditions of nutrient deprivation, IER3 knockdown resulted in massive pancreatic cancer cell death even in cells that expressed Nupr1 and RelB.

    The researchers moved on to analyze Nupr1, RelB, and IER3 protein levels in the pancreas of KrasG12D mice. The results showed that increased expression of all three genes was initiated before PanIN lesions developed, and was dependent on Nupr1 expression. Moreover, KrasG12D;Nupr1KO animals failed to produce RelB and IER3 proteins, confirming the notion that the cascade is dependent on Nupr1 expression. Animals in which RelB specifically was knocked out in the pancreas seldom developed PanIN lesions, and also failed to express IER3.

    “These genetic experiments demonstrate that, as for Nupr1 deletion, inactivation of the Relb locus delays the development of PanIN lesions in KrasG12D pancreas and that this delay is associated with the absence of IER3,” the investigators state. “These data lend further support to the existence of a hierarchical pathway arrangement (Nupr1/RelB/IER3) of the key pro-survival genes.”

    In a final set of analyses the team used immunohistochemistry and tissue microarrays to evaluate expression of the three proteins in cancer tissues from 34 PDAC patients. The data confirmed a significant correlation between the intensities of expression of Nupr1, RelB, and IER3. More importantly, there was a marked inverse correlation between Nupr1 expression and time to survival.

    “This study unravels a cell stress response pathway that accounts for the remarkable resistance of pancreatic cancer cells to stress,” the authors conclude. “Together, our data indicate that Nupr1-induced RelB upregulation is sufficient to trigger an IER3-based antiapoptotic mechanism in PDAC cells … It is hoped that description of this pathway will open up new perspectives on the involvement of the alternative RelB-based NF-κB pathway during pancreatic adenocarcinoma development. We believe these findings will promote studies attempting to block the Nupr1/RelB/IER3 cascade as a novel anticancer therapy.”


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