Despite the effort of the scientific community, the etiology of PDAC is still poorly understood undermining the efforts for its prevention and cure. A large number of epidemiological studies suggested that tobacco smoking, alcohol consumptions, obesity, chronic pancreatitis, genetic risk factors and diabetes are risk factors of PDAC[7-9].

Pancreatic adenocarcinoma arises from three precursor lesions: the microscopic pancreatic intraepithelial neoplasia (PanIN) and the macroscopic intraductal papillary mucinous neoplasm and mucinous cystic neoplasm (Figure 1)[10,11].

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Figure 1 Origin of pancreatic ductal adenocarcinoma. It is widely accepted that pancreatic ductal carcinoma (PDAC) arises from precursor lesions derived from ductal cells; however, recently another model has been proposed where PDAC arises from acinar cells through a process called “acinar to ductal metaplasia” (ADM). PanIN: Intraepithelial neoplasm; IPMN: Intraductal papillary mucinous neoplasm; MCN: Mucinous cystic neoplasm.

PanINs are the more frequent preneoplastic precursors of PDAC[12]; they are classified according to the accumulation of architectural, cytologic, and genetic alterations: from PanIN 1 with the appearance of columnar cells with mucin, to PanIN3 (also called carcinoma in situ) characterized by a severe cyto-architectural athypia[10].

PDAC is a disease with a well defined progression model of accumulation of genetic alterations ranging from single point mutations to gross chromosomal abnormalities[13-17]. The most frequent and studied alterations determine the activation of epidermal growth factor receptor-KRAS pathway[15]. Although almost all patients posses at least one of these mutations, the late stage disease is characterized by increased genome instability and heterogeneity with an average of 63 genetic alterations, the majority of which are point mutations, grouped in a core set of 12 cellular signaling pathways[18]. Morphological progression from PanIN to PDAC is paralleled by the accumulation of these genetic alterations in a progression model resembling the colon cancer model. Ductal origin of PanIN and PDAC is however questioned and a new model has been proposed where the cancer ductal cells in PanIN lesions originate from metaplastic acinar cells in a process called “acinar to ductal metaplasia” (ADM)[19].

Nuclear receptors (NRs) are members of a large superfamily of evolutionarily related ligand-regulated DNA-binding transcription factors present in most metazoan[20].

The first NR was only cloned in the ‘80s by Professor Evans R[21] long after the presence of NR was detected biochemically[22,23]; so far 48 NR has been identified in human by genome sequencing. All NR share characteristic structural features or domains named A to F from the N-terminal to the C-terminal; however, defining features of NR are only the presence of two highly conserved regions: the DNA binding domain (DBD) and the ligand binding domain (LBD), that can function independently[24] and are located in the region C and in the region E of the protein, respectively (Figure 2).

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Figure 2 Domains and structural features of a classical nuclear receptor. A typical nuclear receptor (NR) consists of 6 region (A to F); region F may or may not be present. Region D (hinge) contains the nuclear localization signal (NLS); other NLSs may be present in region E. AF-1: Activator function 1; AF-2: Activator function 2.

NR are classified into six different subfamilies on homology basis: NR1 (thyroid hormone like), NR2 (HNF4-like), NR3 (estrogen like), NR4 (nerve growth factor IB-like), NR5 (fushi tarazu-F1 like), and NR6 (germ cell nuclear factor like), all originally named from the first member identified. A seventh subclass, NR0, has been introduced to classify two receptors, DAX-1 and SHP, that do not possess the DBD[25] (Table 1, information on ligands obtained from the “nuclear receptor signaling atlas”, NURSA, http://www.nursa.org).

NR ligands are small hydrophobic molecules that bind to the LBD; retinoids, fatty acids, cholesterol, lipophilic hormones and vitamins, as well as some antibiotics, xenobiotics and synthetic drugs are all NR ligands. The ligand binding induces a conformational change that modify the DBD ability to bind specific DNA sequences called response elements. NR act as monomeres, homodimers or heterodimers with the retinoid-X-receptor (RXR) (Figure 3). Upon DNA binding, the final transcriptional activity depends on the presence of co-activator or co-repressor molecules[26].

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Figure 3 Mechanisms of action of nuclear receptors. Type I nuclear receptors (NR) (steroid receptors) are complexed with heat shock proteins (HSP) and maintained in the cytoplasm in the absence of ligands. The other receptors are instead mainly nuclear and the ligands induce hetero- (for type II receptors) or homo-dimerization (for type III receptors). Furthermore, a group of receptors (type IV) whose regulation is poorly known act as monomers.

NR physiologic functions vary from the regulation of metabolism, to growth and development; NR are also implicated with a number of diseases such as cancer. The association of NR with major diseases has transformed these proteins in the most popular and promising drug targets thanks also the properties of their ligands that can easily cross the cell membrane[27].

PPARα sustained activation leads to the development of cancers in the liver, testis and pancreas in rodents[35,36]. Moreover, PDX-1, an oncogene for pancreatic cancer that is overexpressed in PDAC[37], is a PPARα -dependent gene and its expression is downregulated by MK886, a specific PPARα antagonist[38]. However PPARα -dependent carcinogenesis has been recently questioned[39].

Recently it has been reported that PPAR signaling, especially PPARβ/δ, is reduced in pancreatic cancer relapse, compared to primitive cancer[40], but PPARβ/δ has been suggested to be a critical component of the angiogenetic switch in pancreatic cancer[41,42]. Abdollahi[42] showed that the expression of PPARβ/δ detected by immunohistochemistry in human pancreatic specimens high-density tissue microarrays correlated with tumor staging; indeed PPARβ/δ staining intensity increased from normal pancreas to chronic pancreatitis, pancreatic cancer and metastasis and the up-regulation of PPARβ/δ is actually more enhanced in the tumor vasculature and in the tumor stroma[42]. High expression of PPARβ/δ in tumor is also confirmed by a recent paper[31]. Elevated PPARβ/δ expression levels are also highly correlated with advanced stages of tumor progression and with increased risk for tumor recurrence or distant metastasis[42] and PPARβ/δ has been proposed as a “central hub” in tumor angiogenesis given that tumor growth and angiogenesis were greatly reduced when tumor cells were implanted in PPARβ/δ null mice[42].

PPARβ/δ is also expressed in human pancreatic cancer cells and its activation regulates the metallo-protease matrix metalloproteinases (MMP)-9, decreasing cancer cells ability to transverse the basement membrane[31]. PPARβ/δ activation reduces the tumor necrosis factor (TNF)α-induced expression of various genes implicated in metastasis increasing the availability of the transcriptional repressor B-cell lymphoma (BCL)-6. BCL-6 is bound to PPARβ/δ and is released after GW501516 treatment resulting in decreased MMP-9 expression. These results suggest that increased expression of PPARβ/δ regulates pancreatic cancer cell invasion sequestering BCL-6 and hence inducing MMP-9 mediated invasion[31].

PPARγ is not only implicated in adipocyte differentiation, lipid accumulation, and glucose homeostasis but it is also an important regulator of inflammation via the inhibition of, or the interference with, proinflammatory signalings such as signal transducers and activators of transcription (STATs), nuclear factor-κB (NF-κB), and activator protein-1 (AP-1)[28,30].

PPARγ is expressed in primary PDAC[43-46] and strongly correlates with a more advanced clinical stage; PPARγ staining is also associated with shorter overall survival and its has proved to be an independent prognostic factor in uni- and multi-variates analysis[43,45]. However, whereas Kristiansen et al[45] found a strong overexpression of PPARγ by expression profiling in 19 microdissected carcinoma compared to 14 ductal epithelia, Pazienza et al[44] did not find significant alterations in the expression levels of PPARγ in pancreatic cancer. A possible explanation of this discrepancy may be that is the expression “per se” of PPARγ, and not its levels, important in pancreatic cancer progression. Interestingly, a large number of studies have demonstrated that TZD reduce the risk of PDAC[9].

A genetic association PPARγ/PDAC has been tested by two different groups[47,48] analyzing the expression of the single nucleotide polymorphisms (SNP) Pro12Ala that has been associated with reduced risk of diabetes and some cancers[47]. Fesinmeyer et al[48] demonstrated that this SNP is associated with increased risk of PDAC in a high-risk sample of smokers randomized to high-dose vitamin A; however, two years later, a similar study in obese and diabetic patients demonstrated a protective role of the SNP, prompting the need of further studies[47].

In vitro, the role of PPARγ is controversial but it is generally accepted that the receptor acts as a tumor inhibitor at multiple levels (Figure 4). PPARγ is expressed in human pancreatic cancer cell lines[46,49-55] and its expression follows a circadian rhythmicity with a period of 24 h that could potentially influence the cell phenotype and the human disease behavior[55]. Agonists of PPARγ such as TZD, its natural ligand 15d-PGJ2, or 1,1-Bis(3-indolyl)-1-(p-substituted phenyl)methanes (C-DIMs), induce cell cycle arrest in G1, apoptosis and ductal differentiation in pancreatic cancer cells[46,53,54,56-58]. However some of the effects associated with PPARγ activation are instead receptor independent: this is especially true for TZD agonists whose receptor independent effects have been widely described[51,52,59-69].

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Figure 4 Peroxisome proliferator activated receptors and pancreatic ductal carcinoma. Peroxisome proliferator activated receptor (PPAR)γ acts as a tumor inhibitor at multiple levels, blocking cell cycle progression, inflammation, and cell invasion. NAG-1: Non steroidal anti-inflammatory drug-activated gene-1; Cox-2: Ciclooxigenase-2; NF-κB: Nuclear factor-κB.

Cell cycle arrest is associated to decreased PPARγ-dependent expression of cyclin D1[53,54,57,58] whereas the reported induction of p21 might be PPARγ-dependent or PPARγ-independent[52,54]. Deletion analysis of the p21 promoter indicates that PPARγ-dependent activation of p21 requires GC-rich sites in the proximal region of the promoter[54]. It is worth to note that some agonists of PPARγ[61] may induce a PPARγ independent down-regulation of cyclin D1, due to induction of non steroidal anti-inflammatory drug-activated gene-1 (NAG-1). NAG-1 is a member of the TGF-β superfamily involved in tumor progression acting as a pro-apoptotic gene. Interestingly, it has been reported that NAG-1 expression is positively regulated by PPARγ: MCC-555, a PPARγ agonist, induces the expression of the transcription factor KLF-4 in PPARγ-dependent manner who subsequently enhances the NAG-1 promoter activity[51,52]. PPARγ agonists reduce the invasive capacity of PDAC cells with a PPARγ dependent mechanism[50]. The PPARγ ligands 15d-PGJ2 and ciglitazone attenuate pancreatic cancer cell invasion increasing plasminogen activator inhibitor-1 and decreasing urokinase plasminogen activator levels resulting in the reduction of total urokinase activity in pancreatic cancer cells[50]. Interestingly, the PPARγ antagonist T0070907 suppresses pancreatic cancer cell motility by altering the localization of p120 catenin and by suppressing the activity of the Ras-homologous GTPases Rac1 and Cdc42[49].

The effects of PPARγ activation or inhibition by specific molecules sinergize or interact with other pathways or other NR activations[53,56,62-64]. Combination of recombinant interferon-β (IFN-β) and the PPARγ agonist troglitazone induces a synergistic effect on the growth inhibition of pancreatic cancer cells, through the counteraction of the IFN-β-induced activation of STAT-3, MAPK and AKT and the increase in the binding of both STAT-1 related complexes and PPARγ with specific DNA responsive elements. The combination induces also an increase in autophagy and a decrease in anti-autophagic bcl-2/beclin-1 complex formation, mediated by the inactivation of the AKT/mTOR-dependent pathway[64]. PPARγ form mandatory heterodimers with RXRα and its activity is maximal in the presence of RXRα agonists; it is not surprising then that co-treatment with PPARγ and RXRα agonists exacerbates the effects of PPARγ increasing the inhibition of cell growth[53,62,63]. Synergistic effects on growth inhibition are also visible when inhibitors of ciclooxigenase-2 (Cox-2) and PPARγ agonists are used in combination[56,65]. Cox-2 is an inducible ciclooxygenase that contributes to the metabolism of arachidonic acid forming prostaglandin H2, a precursor of 15d-PGJ2[66]. Cox-2 is a downtarget of PPARγ and its expression may be either induced or repressed by the NR, depending on the cell context[30]. However, selective Cox-2 inhibitors have opposite effects in pancreatic cancer depending on Cox-2 expression: in high Cox-2-expressing cells the inhibitors reduced tumor growth; conversely, in Cox-2 negative or low expressing cancer cells the inhibitors, at very high concentrations, enhance tumor progression increasing intratumoral VEGF and tumor angiogenesis in a PPARγ-dependent way[65].

PPARγ is known to reduce tumor growth in mice in vivo[49,62,65,67] and its activation increases the gemcitabine mediated tumor suppression[68]. Tumor growth inhibition mediated by PPARγ may be due to reduced inflammation and increased activation of anti-inflammatory genes[30,67]. Genetic deletion of Ikk2, a component of the canonical NF-κB signaling pathway, in the Kras(G12D)Pdx1-cre mouse model of pancreatic cancer, substantially delays pancreatic oncogenesis and results in downregulation of the classical Notch target genes Hes1 and Hey1[67]; in the same model TNF-α stimulation resulted in increased Hes1 expression and consequent suppression of PPARγ expression facilitating the formation of a inflammatory pro-tumoral enviroment; induction of PPARγ instead may block NF-κB induced processes[30] reducing or delaying tumor formation[67].

Despite all these intriguing discoveries on PPARγ role in PDAC, clinical application of PPARγ modulation has recently suffered yet another failure when a new oral anticancer agent with LB4 antagonist and PPARγ agonist properties[69,70], the LY29311, did not demonstrate any benefit in association with gemcitabine in unpretreated patients with advanced PDAC[69].

The orphan NR subfamily 4 subgroup A (NR4A) is comprised by three members: NR4A1 (also known as Nur77, testicular receptor TR3, or nerve growth factor 1b NGFI-B), NRA42 (Nurr-related factor 1) and NR4A3 (neuron-derived orphan receptor1, Nor-1)[25,133]. The first member of NR4A family was identified by differential hybridization in the rat pheochromocytoma cell line PC-12 cell as encoded by an immediate early gene (i.e., a gene that is rapidly and transiently transcribed in response to stimuli) induced by the nerve growth factor[134]. As in the case of other NRs, the NR4A members show common features of a classic nuclear receptor and an high degree of sequence homology specifically in the DBD and LBD regions, where homology may be as high as 95% (Figure 6).

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Figure 6 Clustal sequence alignment of human NR4As. Shaded sequences correspond to the DNA binding domain where homology among NR4A1-3 is very high.

All three members are localized in the nucleus due to the presence of a nuclear localization signal in the DBD (three signals in the case of NR4A1). The three members of the family show a different and often overlapping expression in adult tissues, being Nur77 more abundantly and broadly expressed[133]. NR4A receptors might act as monomere or as homo- hetero-dimers with different affinity to DNA response elements; dimers show stronger activity over monomers[133]. The crystallographic analysis of NR4A receptors suggests that the members of this subfamily are constitutively activated: their LBD is almost completely occupied by bulky aminoacid side chains conferring a 3D structural conformation similar to that of agonist-bound receptors[132,133]. Consequently, unlike others NR, the activity of NR4As is not regulated by stimuli through a ligand binding but instead by modulation of their expression or via post-translational modifications[132,133]. Expression of these receptors is induced by a range of stimuli, including stimuli associated with metabolic functions, such as: fatty acid, growth factors, prostaglandines, membrane depolarization, cold, glucose, cholesterol, TZD and hormones[135]. Other hormones may regulate NR4A transcriptional activity interacting with the NR4 heterodimers, such 9-cis-RA on NR4A:RXR dimers, and new molecules have been identified acting as agonists or antagonists[96,97,136].

The involvement of NR4A2 in pancreatic cancer in vitro has been reported by two papers[133,136]. NR4A2 is highly expressed in many cancer cell lines including Panc1 and Panc28 pancreatic cancer cells. Structure-dependent activation of NR4A2 by a series of C-DIM analogs, especially a p-bromophenyl analog (DIM-C-pPhBr), alters the expression of NR4A2 target genes; among the altered genes the drug determines a NR4A2-dependent repression of neuropilin (NP)-2 in both cell lines, whereas it induces the expression of NP-1 in PANC28 but not PANC1[136]. NPs are important in the progression of pancreatic cancer and are currently seen as potential target for PDAC treatment[137] and it is conceivable that differential expression of these molecules by NR4A2 might be important for PDAC development. Moreover, in PC3 cells NR4A2 acts as a pro-survival antiapoptotic factor[133]: silencing of the receptor greatly reduced the anchorage independent growth, with minimal effect in anchorage-dependent growth, largely due to increase anoikis; NR4A2 silencing also impaired the formation of tumors in nude mice[133].

More data link NR4A1 to pancreatic cancer, depicting a complex mechanism of action where Nur77 acts as having “two faces”, being pro-survival and anti- and pro-apoptotic at the same time.

NR4A1 is expressed as a nuclear protein in pancreatic cancer cells[136,138-142] and was found to be overexpressed in PDAC tissues primarily in the nucleus, whereas 83% of non-tumor pancreatic tissues did not express it[142].

c-DIMs are a class of molecules that activate PPARγ and might act on NR4A receptors[136,138,139]; a c-DIM, specifically the 1,1-Bis(3’-indolyl)-1-(p-anisyl)methane (DIM-C-pPhOCH3), is the first identified Nur77 agonist[139]. DIM-C-pPhOCH3 activates GAL4-Nur77 chimeras expressing wild-type and the ligand binding domain of Nur77. In Panc-28 pancreatic cancer cells, Nur77 agonists decrease cell survival activating the cell death pathways, including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and poly(ADP-ribose) polymerase (PARP) cleavage. Activation of TRAIL and PARP was further confirmed using Nur77 siRNA in Panc-28 cells. Nur77 agonists also inhibit tumor growth in vivo in athymic mice bearing Panc-28 cell xenografts[139]. DIM-C-pPhOCH3 arrests pancreatic cancer cell in G0-G1, by a Nur77-dependent, but KLF-4-independent, expression of cyclin-dependent kinase inhibitor p21. Interestingly, regulation of p21 does not require the presence of Nur77 response elements but involve a GC-rich promoter region and requires the presence of the Sp proteins Sp1 and Sp4, but not Sp3[140]. Microarray analysis of L3.6pL pancreatic cancer cells treated with DIM-C-pPhOCH3 demonstrated a NR4A1-dependent induction of genes associated with metabolism, homeostasis, signal transduction, transcription, stress, transport, immune responses, growth inhibition and apoptosis. Among the most highly induced growth inhibitory and proapoptotic genes were activating transcription factor 3 (ATF3), p21, cystathionase, dual specificity phosphatase 1 and growth differentiation factor 15. Furthermore, DIM-C-pPhOCH3 induced Fas ligand and TRAIL, the latter with a ATF3 dependent mechanism[141].

Although activation of Nur77 by specific c-DIM suggests that Nur77 is a tumor suppressor, experiments with the antagonist 1,1-bis(3’-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) suggest the contrary[142]. Blocking of endogenous Nur77 results in increased cell death and reduced cell proliferation; moreover expression of antiapoptotic genes Bcl-2 and Survivin is also reduced. When administered in vivo, DIM-C-pPhOH inhibits tumor growth, acting on the same antiapoptotic markers observed in vitro. Survivin is overexpressed in pancreatic cancer[143,144] and its expression increases during PanIN progression to PDAC[144]. Transcriptional regulation of Survivin by Nur77 is Sp1-dependent, paralleling the p21 regulation[140], and it is co-regulated by p300. Thus, activation of nuclear Nur77 by the agonist DIM-C-pPhOCH3 or inactivation by the antagonist DIM-C-pPhOH reduces proliferation and induces apoptosis through two different transcription pathways: the first involves the induction of expression of apoptosis promoter genes such as p21 and TRAIL, whereas the latter is dependent on suppression of pro-survival genes. Consequently, Nur77 acts both as a tumor suppressor and as a tumor promoting gene in pancreatic cancer (Figure 7).

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Figure 7 Nur77 acts as a tumor suppressor and as tumor promoting gene in pancreatic ductal carcinoma, inducing pro-apototic and anti-proliferative genes or repressing pro-survival genes. Dashed lines: Effects yet to be demonstrated in pancreatic ductal adenocarcinoma (PDAC). TRAIL: Tumor necrosis factor-related apoptosis-inducing ligand; PARP: Poly(ADP-ribose) polymerase; RXR: Retinoid X receptor; ER: Endoplasmic reticulum.

Interestingly, Nur77 may act as an apoptotic inducer agent in several cancer cells after nuclear export[96,97,145-147]; although not yet described in PDAC it is conceivable that this extra-nuclear action may also be present in pancreatic cancer cells. Inducers of apoptosis, including 5’-fluorouracil which is used in PDAC treatment, stimulate nuclear export of Nur77 mediated by the export receptor CRM1[145,147]. NR4A1 nuclear export may also be induced by 9-cis-RA, requires RXRα as a carrier[97], and targets Nur77 to mitochondria[96]. Despite lacking classical mitochondria targeting sequences, Nur77 might translocate to mitochondria in response to cell death stimuli, through interaction with anti-apoptotic Bcl-2[147]. Bcl-2 acts forming channels in the mitochondria membrane to regulate apoptosis[148]. The interaction Bcl-2/Nur77 is mediated by the N-terminal loop of Bcl-2 and by the NR4A1 LBD: this binding induces a conformational change that exposes the BH3 region of Bcl-2, resulting in its transformation in an inducer of apoptosis[147]. Interestingly, Nur77 may also translocate to other organelles, specifically endoplasmic reticulum (ER). Cell treatment with CD437 induces a nucleus-cytoplasmic translocation of Nur77, followed by ER localization: this requires again the interaction with Bcl-2 and triggers the release of Ca²⁺ from ER inducing apoptosis. Again, this effect has been demonstrated in human neuroblastoma, esophageal squamous carcinoma and hepatocarcinoma cells[145,146] but not in PDAC cells (Figure 7).

LHR-1 (NR5A2) is an orphan NR that is essential during development and necessary in the adult for the function of the pancreas, liver, intestine, and ovary[149,150]. LHR-1 recognizes specific DNA sequences to whom it binds as monomere[151].

The status of LHR-1 as “orphan” is debated and some scientists suggest that it may be classified as “adopted”[132,151]. The structure of the mouse LHR-1 LDB shows an active conformation with a large hydrophobic, but empty, ligand binding pocket resulting in a constitutive active receptor[132]; instead, the crystallographic analysis of the human LHR-1 revealed the presence of phosphatidyl inositol in the binding pocket[151]. Phosphatidyl inositol is required for activation[151] but it is not clear if it may enter and leave the pocket acting as a proper ligand[132]. Nonetheless, new molecules acting as antagonists have been recently identified by screening of commercially available compounds[152].

Chromatin immunoprecipitation-seq and RNA-seq analyses revealed that LRH-1 directly induces expression of genes encoding digestive enzymes and secretory and mitochondrial proteins and cooperates with the pancreas transcription factor 1-L complex in regulating exocrine pancreas-specific gene expression[153].

LHR-1 is important in maintaining acinar identity but is not required for acinar development[154] and mice with a selective deletion of LHR-1 in the pancreas did not display histological abnormalities[153-155]. A genome-wide association study conducted in pancreatic cancer patients and unaffected controls identified 5 SNP in the vicinity of NR5A2 associated with the risk of PDAC[156], that were confirmed by later studies[47,157,158].

In normal human pancreas, the LRH-1 protein is expressed at low levels in the nucleus and cytoplasm of both acini and ducts cells; in contrast PDAC show heightened levels of the protein and in some neoplastic cells the receptor appeared to localize predominantly in the cytoplasm[159]. An increased presence of LRH-1 was also detected in the acinar cells affected by pancreatitis and in PanIN lesions[159]. Overexpression of the receptor was also detected in pancreatic cancer cells in vitro[159].

Treatment of pancreatic cancer cells in vitro with LHR-1 antagonists or with LHR-1 siRNA significantly inhibits cell proliferation inducing a G0/G1 block associated with a reduction of of cyclins D1 and E1[152,159], suggesting that in vitro LHR-1 promotes tumor proliferation. In vivo, selective inactivation of one NR5A2 allele in pancreatic epithelial cells is sufficient to cause impaired recovery from pancreatitis[154] and conditional pancreatic deletion of LHR-1 leads to destabilization of the mature acinar differentiation state, increased inflammation, ADM and loss of regenerative capacity following acute caerulein pancreatitis[154,155]; loss of both alleles also dramatically accelerates the development of oncogenic Kras driven ADM and PanIN lesions[154,155].

The in vivo studies clearly show that LHR-1 inhibits the ductal transformation of adult acinar cells by mutant Kras and prevent PDAC progression; however in vitro studies show that NR5A2 promotes, instead of inhibiting, tumorigenesis. It has been hypothesized that NR5A2 exercises an inhibitory action in the early phase of PDAC development, blocking RAS with unknown mechanisms, while it will have an opposite effect later on[160].

COUP-TFII is a orphan nuclear receptor encoded by the NR2F2 gene localized in the chromosome region 15q26, a region frequently amplified in pancreatic cancer[14], and it is a down target of multiple pathways altered in pancreatic cancer[46,161-163]. In mouse two different transcription variants are described whereas in human at least four different variant are expressed. They differ in the N-terminal region and only one variant presents the structural features of NR being the others without the DBD. The role of these variants is not fully understood and two recent papers gave contradictory results describing one of the DBD lacking forms acting either as enhancer of COUP-TFII transcriptional activity or as a repressor, increasing the cytoplasmic localization of full length COUP-TFII, suggesting a cell specific function for this truncated NR[164,165]. COUP-TFII exists in a autorepressed conformation that prevents recruitment of coactivators, and might respond to retinoids that promote COUP-TFII to recruit coactivators[166]. Full length COUP-TFII exerts an important role during development and in adulthood[167], and it is implicated in the progression of various type of cancers[168]. COUP-TFII is expressed at low levels in adult normal exocrine pancreas[168,169] and recently we demonstrated its involvement in pancreatic cancer in vitro and in vivo[168] (Figure 8). COUP-TFII was expressed in 69% of tested primary samples correlating with the presence of lymph and distant metastasis as well as clinical stage; PDAC patients stained positive for the NR showed a significant reduction of survival compared to NR-negative patients. In vitro silencing of COUP-TFII reduces the cell growth and invasiveness and it strongly inhibits angiogenesis, an effect mediated by the regulation of VEGF-C. The reduced proliferation is associated with a block in G1 and decreased expression of E2F1, but not apoptosis; moreover, COUP-TFII silencing reduces OCT4 and increases Nanog expression. In vitro effects were confirmed in nude mice where COUP-TFII silencing reduces tumor growth by 40%[168].

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Figure 8 Chicken ovalbumin upstream promoter transcription factor II is involved in the regulation of angiogenesis, invasion and tumor proliferation. Expression of chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is induced by several pathways altered in pancreatic ductal carcinoma, including Wnt/β-catenin, RAS-MAPK and Hedgehog.