Nuclear Receptor 4A2 (NR4A2/NURR1) Regulates Autophagy and Chemoresistance in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor prognosis and chemotherapy with gemcitabine has limited effects and is associated with development of drug resistance. Treatment of Panc1 and MiaPaca2 pancreatic cancer cells with gemcitabine induced expression of the orphan nuclear receptor 4A2 (NURR1) and analysis of The Cancer Genome Atlas indicated the NURR1 is overexpressed in pancreatic tumors and is a negative prognostic factor for patient survival. Results of NURR1 knockdown or treatment with the NURR1 antagonist 1,1-bis(3΄-indolyl)-1-(p-chlorophenyl)methane (C-DIM 12) demonstrated that NURR1 was prooncogenic in pancreatic cancer cells and regulated cancer cell and tumor growth and survival. NURR1 is induced by gemcitabine and serves as a key drug resistance factor and is also required for gemcitabine-induced cytoprotective autophagy. NURR1-regulated genes were determined by RNA sequencing of mRNAs expressed in MiaPaCa2 cells expressing NURR1 and in CRISPR/Cas9 gene–edited cells for NURR1 knockdown and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the differentially expressed genes showed that autophagy was the major pathway regulated by NURR1. Moreover, NURR1 regulated expression of two major autophagic genes, ATG7 and ATG12, which are also overexpressed in pancreatic tumors and like NURR1 are negative prognostic factors for patient survival. Thus, gemcitabine-induced cytoprotective autophagy is due to the NURR1–ATG7/ATG12 axis and this can be targeted and disrupted by NURR1 antagonist C-DIM12 demonstrating the potential clinical applications for combination therapies with gemcitabine and NURR1 antagonists. Significance: Gemcitabine induces NURR1-dependent ATG7 and ATG12 cytoprotective autophagy in PDA cells that can be reversed by NURR1 antagonists.


Introduction
Pancreatic ductal adenocarcinoma (PDAC) is a lethal human malignancy with a five-year survival rate of 9% and it is estimated that by 2030 pancreatic cancer cis-elements in target gene promoters (9). NURR has been characterized as an important regulator in neuronal development and there is also increasing evidence of a prooncogenic role for their receptor in solid tumors (9-11) NURR regulates cell growth survival and metabolism (12)(13)(14) and studies in this laboratory identified 1,1-bis (3 -indolyl)-1-(p-chlorophenyl) methane (C-DIM12) as prototypical NURR ligand (15,16). A recent study showed that C-DIM12 inhibited glioblastoma cell and tumor growth and also blocked NURR-dependent prooncogenic pathways in glioblastoma (15). It was previously reported that induction of NURR promotes 5-fluorouracil (5-FU) resistance in squamous cell carcinoma (17); however, the mechanism of NURR-mediated chemoresistance and its role in pancreatic cancer has not been determined.
Autophagy is an evolutionary cellular response to diverse stress and physiologic conditions during cancer progression and has been reported to induce tumor cell survival and drug resistance in cancer cells (18,19). Autophagy is initiated with the formation of double layer membrane around the cytoplasmic components known as autophagosome which fuses with lysosomes to recycle these components for protein synthesis and energy production. It is closely regulated by a highly conserved set of genes known as autophagy-related genes (ATG; ref. 18). A recent study demonstrates that autophagy regulates the unique properties of cancer stem cells, such as differentiation and self-renewal, which contributes to tumor metastasis, tumor reoccurrence, and chemoresistance (20). A study, combining an autophagy inhibitor with photodynamic therapy, significantly reduced the colorectal tumor size suggesting that autophagy is a cytoprotective process (21), and in another study autophagy inhibition increased the drug sensitivity (22). Thus, understanding the mechanism of autophagy and drug resistance is an unsolved problem and could lead to enhanced therapeutic efficacy.
This study demonstrates that NURR is essential for chemotherapeutic agentinduced cytoprotective autophagy through transcriptional regulation of ATGs. We show for first time that ATG and ATG are NURR-regulated genes, and high expression of NURR/ATG/ATG corresponds to the poor survival and prognosis of patients with PDA and the NURR-ATG/ATG axis can be targeted by NURR antagonists.

Cell Lines and Transfection with siRNA and Plasmids
MiaPaCa2 and Panc1 pancreatic cancer cell lines were obtained from ATCC and were cultured in in DMEM supplemented with 10% FBS (Gibco/Invitrogen), 1% l-glutamine (Gibco/Invitrogen), and 1% penicillin-streptomycin (Invitrogen) at 37°C in 5% humidified CO 2 incubators. CRISPR/Cas9-mediated knockout of NURR1 in MiaPaCa2 cells was accomplished using guide RNAs targeting NURR, fused with CRISPR/Cas9 and GFP protein (23). CRISPR Universal Negative Control plasmid (CRISPR06-1EA) was purchased from Sigma-Aldrich. Cells were harvested after 48 hours of transfection and GFPpositive cells were single sorted by using FACSCalibur flow cytometer. The guide RNA sequences used were: For all cell culture experiments, Mycoplasma testing (MycoAlert Mycoplasma Detection Kit, Lonza) was performed after each thawing and at least monthly.

Cell Growth Assays
Cells were plated in 96-well plates at 1 × 10 3 cells per well. After 5 days of incubation, cell growth was measured using Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen). To estimate cell death, cells were trypsinized and counted after

Clonogenic Assay
1000-2000 cells per well were plated in a 6-well plate. The media were not changed during experiments unless indicated. Upon completion of the experiments, colonies were fixed in reagent containing 80% methanol and stained with 0.5% crystal violet. To determine relative growth, dye was extracted from stained colonies with 10% acetic acid and the associated absorbance measured at 600 nm using a Microplate Reader/Synergy HT BioTek plate reader (23,27).

Luciferase Reporter Assays
Cells were seeded at 60% confluency in 6-well dishes, then transfected using lipofectamine 3000 (Thermo Fisher Scientific) with 2 μg of the promoter dual

Electron Microscopy
Cells were cultured in permanox petri dish and treated with gemcitabine (1 μmol/L) and C-DIM12 (15 μmol/L) for 24 hours. The cells were fixed with 2.5% paraformaldehyde, 2% glutaraldehyde, 0.1 mol/L cacodylate buffer, and embedded using Epon 812. The ultra-thin sections (∼100 nm) were cut using a Leica EM UC6 ultramicrotome and diamond knife. The sections were then placed on copper grids, poststained with saturated Uranyl Acetate and Reynolds Lead Citrate, and imaged using an FEI Morgagni 268 transmission electron microscope equipped with a MegaView III CCD camera.

RNA Sequencing
RNA quality was assessed via the Agilent 2100 Bioanalyzer (Agilent Technologies). Strand-specific RNA-sequencing (RNA-seq) library was prepared using NEBNext Ultra II Directional RNA Library Prep Kit (NEB) according to the manufacturer's protocols (27). RNA-seq was performed using 150-bp pairedend format on a NovaSeq 6000 (Illumina) sequencer. RNA-seq quality was checked by running FastQC, and TrimGalore was used for adapter and quality trimming (27). Sequence reads were aligned to the hg19 human genome build using the STAR aligning program (28). Quantification of all genes and their isoforms was performed using FPKM normalized values using Cufflinks v2.2.1, DESeq2 analysis with an P adj < 0.05 was used to get a list of differentially expressed genes (29).

Immunofluorescence Assay
Cells were seeded in 24-well plate (50,000 cells/well). After 24 hours, the media were discarded and the cells were then washed with 1× PBS. The cells were then fixed with 10% formaldehyde and permeabilized with 0.1% Triton-X 100. The cells were then incubated with primary antibody, overnight at 4°C and then with secondary antibody (Thermo Fisher Scientific #A11001 and Cell Signaling Technology #4412S)] for 2 hours at room temperature. The images were captured using Zeiss Imager.Z1 AXIO at 40× magnification.

Chromatin Immunoprecipitation Assay
Cells were cross-linked for 10 minutes at room temperature by the addition of one-tenth volume of 11% formaldehyde (11% formaldehyde, 50 mmol/L HEPES pH 7.4, 100 mmol/L NaCl, 1 mmol/L EDTA pH 8.0, 0.5 mmol/L EGTA pH 8.0), followed by 5-minute quenching with 1/20th volume of 2.5 mol/L glycine. Cells were washed twice with PBS, with spins after each rinse, the supernatant was aspirated, and the cell pellet was flash frozen in liquid nitrogen. Frozen crosslinked cells were stored at −80°C (30).
Sonicated crosslinked lysates were incubated overnight at 4°C with magnetic beads bound with antibody. Beads were pelleted, and then washed several times: two times with sonication buffer, one time with sonication buffer with 500 mmol/L NaCl, one time with LiCl wash buffer (10 mmol/L TrisHCl pH 8.0, 1 mmol/L EDTA, 250 mmol/L LiCl, 0.5% NP-40, 0.5% sodium deoxycholate) and one time with TE (10 mmol/L TrisHCl pH 8.0, 1 mmol/L EDTA). Bound protein and cross-linked DNA were eluted in elution buffer (50 mmol/L TrisHCl pH 8.0, 10 mmol/L EDTA, 1% SDS), and cross-links were reversed by overnight incubation using RNase A (10 mg/mL) and Proteinase K (20 mg/mL) for 1 hour, respectively, and DNA was purified with phenol/chloroform extraction and ethanol precipitation and used for qPCR (primers summarized in Supplementary Table S1).

Mouse Studies
All experiments involving mice were approved by the Texas A&M University's Animal Care and Use Committee. Six-week-old, female, athymic nude mice (Nude-Foxn1nu) were purchased from ENVIGO. MiaPaCa2 (ctrl), CTRL.KO, NURR.KO cells, were prepared in 100 μL solution comprised of 70% DPBS and 30% Matrigel. Suspensions of 3 × 10 6 cells were then injected subcutaneously into the left and right flanks of mice. Tumor volumes were measured three times per week using calipers (Volume = Length × Width 2 /2), along with body weight. Mice with established tumors (after 25 days, mean tumor volume of ∼100 mm 3 ) were randomly divided into four groups, which were then treated with vehicle (20 μL/g of 0.9% NaCl), gemcitabine (5 mg/mL, prepared in vehicle solution) 100 mg/kg intraperitoneally i.p.) twice biweekly, C-DIM12 (30 mg/kg i.p.; Monday/Wednesday/Friday; LC Laboratories #R-5000) or a combination of both. Upon termination of mouse experiments, mice were euthanized using carbon dioxide inhalation followed by cervical dislocation, and tumors were harvested.

Statistical Analysis
Data were expressed as mean ± SEM of at least three independent experiments. An unpaired, two-tailed Student t test was used to determine the differences between groups (* P < 0.05; ** P < 0.01; *** P < 0.001). ANOVA test was used for the analysis of tumor measurements among treated groups.

Data Availability
RNA-seq files have been deposited in Gene Expression Omnibus (GEO) with accession number GSE159099.

Prognostic Significance of NURR1 in Patients with PDAC
Previous studies showed that NURR1 is highly expressed in glioblastoma (15) and analysis of the published The Cancer Genome Atlas (TCGA) database showed that NURR was also more highly expressed in tumor samples from a patient with PDA. Kaplan-Meier analysis of NURR1 expression data showed that NURR1 overexpression of PDA patients' tumor samples was also significantly associated with their poor survival (Fig. 1A). Lymph node metastasis was studied in 173 patients with PDA and NURR expression was significantly higher in patients with N0 and N1 PDA compared with normal individuals (Fig. 1B). NURR was also overexpressed in grade 2 tumor samples confirming that high levels of NURR are associated with the severity of PDAC (Fig. 1C).
PDA is a prime example of a tumor that develops chemoresistance and to investigate the possible relationship between chemotherapeutic drugs and NURR

AACRJournals.org
Cancer Res Commun; 1(2) November 2021 expression pancreatic cancer cells were treated with gemcitabine and analysis by immunoblot and immunofluorescence showed that gemcitabine induced expression of NURR in both MiaPaCa2 and Panc1 cells (Fig. 1D and E). This demonstrates that NURR expression is increased during chemotherapeutic treatment and the mechanisms of gemcitabine induction of NURR1 and GEM -C-DIM12 interactions are unknown and are currently being investigated.

NURR1 has a Cytoprotective Role Against Chemotherapeutic Drug-Induced Cell Death in Pancreatic Cancer Cells
To study the role of NURR in drug resistance, we treated the MiaPaCa2 cells with the NURR antagonist C-DIM12 (DIM-C-pPhCl; Fig. 2A) and gemcitabine and in parallel studies we also determined the effects of gemcitabine alone and after NURR1 knockout (by RNA interference) in MiaPaCa cells. C-DIM12 has been characterized as an NURR1 antagonist in cancer cells and has been used as a model compound for studying the actions of NURR1 (15,16). Crystal violet assay, phase contrast microscopy, and Annexin V staining showed that gemcitabine and C-DIM12 alone were effective; however, treatment with gemcitabine in combination with C-DIM12 was a more potent inhibitor of cell proliferation and survival than the individual compounds alone (Fig. 2B-D) and similar results were observed in Panc1 cells (Supplementary Figs. S1A -S1D). Moreover, treating the cells with C-DIM12 in combination with gemcitabine increased apoptosis as determined by cleaved PARP in MiaPaCa2 (Fig. 2E) and Panc1 cells ( Supplementary Fig. S2A and S2B). Results summarized in Fig. 2E and 2F also show that cleaved PARP (marker of apoptosis) and inhibition of cell growth by gemcitabine were enhanced by cotreatment with C-DIM12 or by NURR knockout using CRISPR/Cas9 gene-edited cells and this response can be attenuated by overexpression of Nurr1 (NOE; Fig. 2G). Electron microscopy images also demonstrate that pancreatic cancer cells were more sensitive to gemcitabine when treated in combination with C-DIM12 and this is evidenced by enhanced cell shrinkage and nuclear condensation in treated compared with control cells. We also observed progressive fragmentation and increase in number of apoptotic bodies, which are indicators of apoptosis in the cells treated with gemcitabine and C-DIM12 ( Supplementary Fig. S1C).

ATG7 and ATG12 are the Key Targets of NURR1
NURR regulates specific gene activity and mediates cell survival, migration, invasion, and transformation (9-13, 15, 31). To identify NURR-regulated genes that may be involved in drug resistance, we performed RNA-seq and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway analysis in MiaPaCa2 cells, modified by CRISPR/Cas9 gene editing for NURR expression and in control cells expressing NURR (Fig. 3A). To investigate pathways altered by the NURR knockout, we performed KEGG enrichment pathway analysis and observed that regulation of genes associated with autophagy was enriched ( Fig. 3B; Supplementary Table S2). The expression of the autophagic ATG and ATG genes (18) was downregulated in NURR.KO cells when compared with NURR.CTRL cells (Fig. 3A-C) and therefore the potential roles of ATG and ATG in NURR-mediated chemoresistance was further investigated because autophagy plays a key role in cell survival and drug resistance (32). Immunoblot analysis confirmed that NURR regulates the expression of ATG and ATG because knockdown of NURR significantly downregulated levels of ATG and 12 proteins (Fig. 3D). Further analysis by chromatin immunoprecipitation (ChIP) revealed that NURR1 is associated with the ATG and 12 promoters (Fig. 3E). Nurr1 was also associated with the Sp1 but not the ATG13 (negative control) promoter in a ChIP assay (Fig. 3E). We also subcloned the ATG and ATG promoter sequences into a luciferase reporter plasmid and after transfection into MiaPaCa2 cells the effects of NURR1 knockdown on luciferase activity was determined. Knockdown of NURR expression significantly decreased luciferase activity in both AGT and AGT promoter-luciferase constructs (Fig. 3F) confirming that ATG and ATG are key autophagic genes regulated by NURR1. Therefore, the role of NURR and ATG/ATG in gemcitabine resistance was further investigated.

NURR1 Induces Autophagy, ATG7, and ATG12 in Pancreatic Cancer Cells
Autophagy is a cellular self-degradation process that reduces cellular damage in response to stressful conditions and the link between NURR expression and autophagy was further investigated in MiaPaCa2 cells treated with gemcitabine. Figure 1D and E show that gemcitabine induced NURR expression in Panc1 and MiaPaCa2 cells and this is also observed in Fig. 4A where gemcitabine-induced NURR1 expression is accompanied by an increase in the autophagic marker LCB-II, whereas in NURR-KO cells ± gemcitabine, low levels of LC3B-II were expressed (Fig. 4A). Next, electron microscopy supports the immunoblot data showing that gemcitabine induced autophagy, which is characterized by autophagosomes and autophagic compartments; however, treatment with gemcitabine in combination with the NURR1 antagonist C-DIM12 reduced evidence for autophagy (Fig. 4B). Confocal microscopy also demonstrated that gemcitabine induced autophagy and that knockdown of NURR or treating the cells with C-DIM12 significantly reduced autophagy marked by decreased punctate staining of LC3-II (Fig. 4C). The bar graph representation revealed that LC3-II punctate staining was decreased 2-3-fold when expression of NURR was inhibited (Fig. 4D) and as a control we show that chloroquine increased LC3-II punctate staining (Fig. 4E). These data suggest that NURR is essential for induction of autophagy in pancreatic cancer cells. We next investigated the effects of the NURR antagonist C-DIM12 on expression of ATG and ATG in MiaPaCa2 cells and showed that C-DIM12 decreased expression of ATG and ATG mRNA levels (Fig. 5A). Treatment with C-DIM12 in combination with gemcitabine significantly downregulated expression of ATG and ATG in immunoblot analysis in MiaPaCa2 and Panc1 cells ( Fig. 5B and C) and in the latter cell line, C-DIM12 decreased Nurr1 expression. Gemcitabine alone induced PARP cleavage in MiaPac2 cells ( Fig. 5D and E), whereas knockdown of ATG7 or ATG12 did not induce this response.
In contrast, gemcitabine alone and gemcitabine plus knockdown of ATG7 (Fig. 5D) or ATG12 (Fig. 5E) induced PARP cleavage and p62, and these responses were not enhanced by C-DIM12. The unexpected synergistic interactions of gemcitabine plus knockdown of ATG7 and ATG12 on induction of p62 are being further investigated. We also observed that silencing of ATG7 or ATG12 enhanced the cytotoxicity of gemcitabine (Fig. 5F) and these results are consistent with enhanced gemcitabine cytotoxicity after treatment with C-DIM12 or knockdown of Nurr1 (Fig. 2F).

In Vivo Confirmation of NURR1/ATG7/ATG12 Axis
In athymic nude mice bearing MiaPaCa2 cells (Ctrl and NURR-KO), gemcitabine alone decreased tumor volume ( Fig. 6A and B), and both tumor volumes and weights were decreased in mice bearing NURR1-KO cells alone and after treatment with gemcitabine. Compared with the vehicle-treated group, the combination of gemcitabine, and NURR knockdown, also extended the    F, Average tumor volume of MiaPacA2 (vehicle, gemcitabine, C-DIM, and combination of both) xenografts at the end of the experiment (day 30; n = 10 tumors per group; **, P < 0.01; ***, P < 0.001). G, Survival was assessed after treatment cessation. Mice were removed from the group when tumors achieved a volume of 1,500 mm 3 . Statistical significance was determined by log-rank test. H, Tumor RNA was extracted from vehicle or combination of gemcitabine and C-DIM xenografts and analyzed for mRNA expression of NURR1, ATG7, or ATG12. β-Actin was used as normalization control. Normalized values are shown as mean ± SEM (n = 6 tumors per group; *, P < 0.05; **, P < 0.01).
median survival of the mice from 20 to 97 days (Fig. 6C). Both ATG and ATG mRNA levels were lower in tumors derived from mice bearing NURR-KO cells and treated with gemcitabine compared with control cells (Fig. 6D).
A comparable set of data were obtained in tumors from mice bearing wildtype MiaPaCa2 cells and treated with vehicle, gemcitabine, C-DIM12 and their combination. Both gemcitabine and C-DIM12 and their combination inhibited tumor growth (Fig. 6E and F) to a similar extent; however, posttreatment survival (Fig. 6G) shows that gemcitabine plus C-DIM12 was more effective than either compound alone. Gemcitabine plus C-DIM12 also inhibited ATG and ATG mRNA levels (Fig. 6H) and this corresponds to results observed in the in vitro studies (Fig. 5).

Prognostic Significance of ATG7 and ATG12 and Association of NURR1 and ATG7 and ATG12 Expressions in Clinical Specimens
To study the prognostic significance of ATG and ATG in PDA, we examined TCGA, which showed a significant increase in expression of ATG in pancre- NURR expression. These data coupled with mechanistic studies suggest a role for NURR in gemcitabine-induced resistance, which is linked to enhanced autophagy and the potential clinical applications of combination therapies using gemcitabine and NURR1 antagonists (Fig. 7E). The linkage between NURR1 and ATG7/ATG12 has been clearly demonstrated; however, the possible interactions with other important autophagic factors (e.g., TFEB) has not been determined and is currently been investigated.

Discussion
PDA is usually detected in later stages and patients with PDA have low survival rates and current treatment options are limited in their effectiveness.
Improvements in PDA patient survival will require development of validated biomarkers that appear early in the formation of these tumors and also new mechanism-based drugs and drug combinations that increase efficacy and decrease drug resistance. We initially examined the TCGA database and observed that expression of the orphan nuclear receptor NURR was more highly expressed in pancreatic tumors compared with the normal pancreas and patients with pancreatic cancer expressing high levels of NURR exhibited decreased survival (Fig. 1) and this was similar to our recent studies on the expression and prognostic value of NURR1 in glioblastoma (15). The functions of NURR have been investigated in multiple tumors (9,(33)(34)(35)(36)(37)(38) and results of knockdown or overexpression studies have characterized this receptor as a prooncogenic factor that regulates cancer cell proliferation, survival, migration, and invasion. Although an endogenous ligand for NURR has not been identified, a recent study shows that the bis-indole-derived C-DIM12, a known NURR ligand, acted as a receptor antagonist in glioblastoma cells and inhibited cell growth and invasion and increased apoptosis (15). Another report showed that overexpression of NURR in squamous cell carcinoma cells increased resistance to 5-FU treatment (17) suggesting a possible role for NURR in drug resistance. Gemcitabine has replaced 5-FU for treatment of pancreatic cancer and based on the prognostic and functional characteristics of NURR in cancer cells this study focused on determining the functions of NURR in pancreatic cancer and its role in gemcitabine resistance.
Knockout of NURR in pancreatic cancer cells or treatment with C-DIM12induced apoptosis and inhibited proliferation (Fig. 2). Moreover, in athymic nude mice bearing MiaPaCa2 (NURR +/+ ) or NURR-KO cells, it was clear that loss of NURR or treatment with C-DIM12 inhibited tumor growth and enhanced survival (Fig. 6). These results confirmed the prooncogenic activity of NURR in pancreatic cancer cells as previously observed in other cancer cell lines (15) and demonstrated that the NURR antagonist C-DIM12 was an effective anticancer agent that blocked NURR-mediated responses.
We also investigated the role of NURR in drug resistance and observed that treatment of pancreatic cancer cells with gemcitabine resulted in induction of NURR (Fig. 1). Thus, although gemcitabine alone induced apoptosis and inhibited growth of pancreatic cancer cells and tumors (Figs. 1 and 6) this was accompanied by induction of NURR, which exhibits tumor promoter-like activity. This counterintuitive effect of gemcitabine could be a component of drug resistance and this was confirmed in combination treatments of cells and mice with gemcitabine alone and in combination with NURR1 knockdown or receptor antagonist (C-DIM12; Figs. 1 and 6). Results of the in vitro and in vivo effects of these combination therapies are complementary and demonstrate the NURR inactivation or treatment with C-DIM12 enhanced the effectiveness of gemcitabine thus demonstrating that induction of NURR by gemcitabine is associated with drug resistance.
The mechanism of NURR as a drug-resistant factor was further investigated by RNA-seq in wild-type and NURR-KO cells and this resulted in identification of several genes/pathways regulated by NURR; however, the major pathway AACRJournals.org Cancer Res Commun; 1(2) November 2021 was associated with changes in expression of genes involved in autophagy ( Fig. 3; refs. [39][40][41][42][43]. Because autophagy has previously been linked to cytoprotection in colon cancer cells (21), we examined this gene set and identified two genes, namely ATG and ATG, that were not only regulated by NURR ( Fig. 5A and B) but also exhibit clinical characteristics similar to that observed for NURR in patients with pancreatic cancer (Figs. 1 and 7). These results confirm that NURR exhibits prooncogenic-like activity in pancreatic cancer cells and this receptor also plays a role in gemcitabine-induced drug resistance (Fig. 7E). These observations coupled with the anticarcinogenic effects of C-DIM12 suggest that the effectiveness of therapeutic regimens including gemcitabine for treatment of PDAC can be significantly enhanced using combination therapies that include a NURR antagonist such as C-DIM12.