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.
Gemcitabine induces NURR1-dependent ATG7 and ATG12 cytoprotective autophagy in PDA cells that can be reversed by NURR1 antagonists.
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 will be the second leading cause of cancer-related deaths in United States (1). Although new therapeutic regimens against PDAC have improved treatment of this disease, the late-stage detection, lack of targeted therapies, and chemoresistance are still some of the major challenges for enhancing patient outcomes. The current treatments include surgical resection, which is followed by adjuvant chemotherapy (2) and palliative chemotherapy [with gemcitabine or FOLFIRINOX (folinic acid, 5-flouorouracil, irinotecan and oxaliplatin)] (3–5). Gemcitabine (Gemzar; 2′,2′-difluorodeoxycytidine), an FDA approved chemotherapeutic drug, is used for treatment of different cancers and overall survival and progression-free survival is enhanced by gemcitabine compared with those receiving non–gemcitabine-based therapy (6). Although, these treatments are initially effective in increasing survival of patients with pancreatic cancer, residual neoplastic cells usually result in relapse and subsequent appearance of more aggressive and lethal tumors (7). The success of cancer treatments for patients with PDA largely depends on diagnosis during initial stages of the disease. Cellular mechanisms such as apoptosis, necroptosis, and autophagy also determine the advancement and response to these cancer treatments (8). Thus, to find a remedy for treating this devastating disease, it is important to understand the mechanism through which pancreatic cancer cells acquire resistance to current therapeutics.
NURR1 (NR4A2) is an orphan nuclear receptor and a transcription factor that activates target genes by binding as monomers or dimers to cognate cis-elements in target gene promoters (9). NURR1 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) NURR1 regulates cell growth survival and metabolism (12–14) and studies in this laboratory identified 1,1-bis (3′-indolyl)-1-(p-chlorophenyl) methane (C-DIM12) as prototypical NURR1 ligand (15, 16). A recent study showed that C-DIM12 inhibited glioblastoma cell and tumor growth and also blocked NURR1-dependent prooncogenic pathways in glioblastoma (15). It was previously reported that induction of NURR1 promotes 5-fluorouracil (5-FU) resistance in squamous cell carcinoma (17); however, the mechanism of NURR1-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 NURR1 is essential for chemotherapeutic agent–induced cytoprotective autophagy through transcriptional regulation of ATGs. We show for first time that ATG7 and ATG12 are NURR1-regulated genes, and high expression of NURR1/ATG7/ATG12 corresponds to the poor survival and prognosis of patients with PDA and the NURR1-ATG7/ATG12 axis can be targeted by NURR1 antagonists.
Materials and Methods
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 CO2 incubators. CRISPR/Cas9–mediated knockout of NURR1 in MiaPaCa2 cells was accomplished using guide RNAs targeting NURR1, 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 GFP-positive 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. Cells were also authenticated using short tandem repeat analysis (Biosynthesis)
Cells were plated at 60% confluency in 6-well plates, and transient siRNA transfections (1 μmol/L) were performed using Lipofectamine 3000 (Invitrogen) and Opti-MEM (Invitrogen) according to the manufacturer's protocol; 48 hours after transfection, cells were treated or analyzed, as described previously (24, 25). siRNA oligos were purchased from Life Technologies (siNURR1, 4427038; siATG7, 4392420; siATG12, 4392420; siCTRL, AM4635).
Cell Growth Assays
Cells were plated in 96-well plates at 1 × 103 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 Trypan blue staining (Invitrogen) with a Hausser bright-line hemocytometer (Thermo Fisher Scientific; ref. 26). Annexin V/PI staining was performed using the Dead Cell Apoptosis Kit (Thermo Fisher Scientific, #V13245), according to the manufacturer's instructions. Staining was measured with an Accuri C6 flow cytometer and analyzed with FlowJo Version 10.2 software (26).
Cells were lysed using 1% Triton in TBS containing protease and phosphatase inhibitors. Tumors were lysed using 1× RIPA buffer containing protease and phosphatase inhibitors. Equal amounts of total protein were separated by electrophoresis on a 4%–12% Bis-Tris gel and transferred to a polyvinylidene difluoride membrane (23). Blots were blocked in 5% BSA, and then probed with antibodies against anti-NURR1 (sc-376984, Santa Cruz Biotechnology), anti-ATG 7 (10088–2AP, ProteinTech), anti-ATG 12(D88H11, Cell Signaling Technology), anti-LC3BI/II (3868, Cell Signaling Technology), anti-cleaved PARP (9532S, Cell Signaling Technology), and anti-α-tubulin (Sigma). Chemiluminescent (32106, Thermo Fisher Scientific) signal was captured using a Syngene G-BOX iChemi XT imager (26).
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 reporter plasmid (pCheck2–promoter ATG7 [TSS = 11272324; Upstream = 1265, Downstream = 295; Length = 1561] or ATG12 [TSS = 115841851; Upstream = 1271, Downstream = 228; Length = 1500]), purchased from GeneCopoeia (23). The luciferase activity of the cultured supernatant was measured 48 hours after transfection using Luciferase Assay Reporter Kit (Promega) according to the manufacturer's instructions (23).
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.
Total RNA was extracted using RNAeasy Kit (Qiagen). cDNA was synthesized from 1 μg of total RNA using TagMan probes (NURR1, Hs00428691, Hs01117525; ATG7, Hs00893766, Hs04969948; ATG12, Hs00740818, Hs01047860; 18s, 99999901; Actin, Hs00157387, Life Technologies) and MultiScribe Reverse Transcriptase (Life Technologies). qRT-PCR analysis was performed using the Applied Biosystems 7500 Fast Real-Time PCR System (Life Technologies) and TagMan RT-PCR Master Mix (Life Technologies).
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 paired-end 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 Padj < 0.05 was used to get a list of differentially expressed genes (29).
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).
Crosslinked cells were lysed with lysis buffer 1 (50 mmol/L HEPES pH 7.5, 140 mmol/L NaCl, 1 mmol/L EDTA, 10% glycerol, 0.5% NP-40, and 0.25% Triton X-100), pelleted, and resuspended in lysis buffer 2 (10 mmol/L TrisHCl pH 8.0, 200 mmol/L NaCl, 1 mmol/L EDTA, 0.5 mmol/L EGTA), and pelleted again (30). The pellet was resuspended in sonication buffer (50 mmol/L HEPES pH 7.5, 140 mmol/L NaCl, 1 mmol/L EDTA pH 8.0, 1 mmol/L EGTA, 0.1% sodium deoxycholate, 0.1% SDS, and 1% Triton X-100), and sonicated (30) using a Branson Sonifier (power setting 5) for 10 cycles at 30 seconds each on ice (18–21 W) with 60 seconds on ice between cycles.
Dynal magnetic beads (Sigma) (50 μL) were blocked with 0.5% BSA (w/v) in PBS, and then bound with 10 μg of antibody against NURR1, Abcam (AB41917). 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).
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, NURR1.KO cells, were prepared in 100 μL solution comprised of 70% DPBS and 30% Matrigel. Suspensions of 3 × 106 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 × Width2/2), along with body weight. Mice with established tumors (after 25 days, mean tumor volume of ∼100 mm3) 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.
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.
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 NURR1 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 NURR1 expression was significantly higher in patients with N0 and N1 PDA compared with normal individuals (Fig. 1B). NURR1 was also overexpressed in grade 2 tumor samples confirming that high levels of NURR1 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 NURR1 expression pancreatic cancer cells were treated with gemcitabine and analysis by immunoblot and immunofluorescence showed that gemcitabine induced expression of NURR1 in both MiaPaCa2 and Panc1 cells (Fig. 1D and E). This demonstrates that NURR1 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 NURR1 in drug resistance, we treated the MiaPaCa2 cells with the NURR1 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 NURR1 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
NURR1 regulates specific gene activity and mediates cell survival, migration, invasion, and transformation (9–13, 15, 31). To identify NURR1-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 NURR1 expression and in control cells expressing NURR1 (Fig. 3A). To investigate pathways altered by the NURR1 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 ATG7 and ATG12 genes (18) was downregulated in NURR1.KO cells when compared with NURR1.CTRL cells (Fig. 3A–C) and therefore the potential roles of ATG7 and ATG12 in NURR1-mediated chemoresistance was further investigated because autophagy plays a key role in cell survival and drug resistance (32). Immunoblot analysis confirmed that NURR1 regulates the expression of ATG7 and ATG12 because knockdown of NURR1 significantly downregulated levels of ATG7 and 12 proteins (Fig. 3D). Further analysis by chromatin immunoprecipitation (ChIP) revealed that NURR1 is associated with the ATG7 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 ATG7 and ATG12 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 NURR1 expression significantly decreased luciferase activity in both AGT7 and AGT12 promoter-luciferase constructs (Fig. 3F) confirming that ATG7 and ATG12 are key autophagic genes regulated by NURR1. Therefore, the role of NURR1 and ATG7/ATG12 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 NURR1 expression and autophagy was further investigated in MiaPaCa2 cells treated with gemcitabine. Figure 1D and E show that gemcitabine induced NURR1 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 LC3B-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 NURR1 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 NURR1 was inhibited (Fig. 4D) and as a control we show that chloroquine increased LC3-II punctate staining (Fig. 4E). These data suggest that NURR1 is essential for induction of autophagy in pancreatic cancer cells. We next investigated the effects of the NURR1 antagonist C-DIM12 on expression of ATG7 and ATG12 in MiaPaCa2 cells and showed that C-DIM12 decreased expression of ATG7 and ATG12 mRNA levels (Fig. 5A). Treatment with C-DIM12 in combination with gemcitabine significantly downregulated expression of ATG7 and ATG12 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 NURR1-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 NURR1 knockdown, also extended the median survival of the mice from 20 to 97 days (Fig. 6C). Both ATG7 and ATG12 mRNA levels were lower in tumors derived from mice bearing NURR1-KO cells and treated with gemcitabine compared with control cells (Fig. 6D). A comparable set of data were obtained in tumors from mice bearing wild-type 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 ATG7 and ATG12 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 ATG7 and ATG12 in PDA, we examined TCGA, which showed a significant increase in expression of ATG7 in pancreatic tumors (T = 179) compared with normal tissue samples (n = 171; Fig. 7A). Kaplan–Meier analysis survival curve showed that high expression of ATG7 correlated with poor survival rate of patients with PDA (Fig. 7B). This analyses also demonstrated a significant increase in the expression level of ATG12 in pancreatic tumors, (T = 179) compared with normal tissue samples (N = 171; Fig. 7C) and Kaplan–Meier analysis shows that high expression of ATG12 corresponds to poor patient survival rate (Fig. 7D). We also investigated the correlation between the expression of NURR1 and ATGs (ATG7 and ATG12) in pancreatic tumor samples by performing correlation analysis. NURR1 expression was strongly correlated with ATG7 expression (R = 0.2, P = 6.2e-05; Supplementary Fig. S2C) and ATG12 expression (R = 0.31, P = 2e-05; Supplementary Fig. S2D) in PDA patient samples. Together, these data indicate prognostic significance of ATG7 and ATG12 in PDA and their correlation with NURR1 expression. These data coupled with mechanistic studies suggest a role for NURR1 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.
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 NURR1 was more highly expressed in pancreatic tumors compared with the normal pancreas and patients with pancreatic cancer expressing high levels of NURR1 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 NURR1 have been investigated in multiple tumors (9, 33–38) 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 NURR1 has not been identified, a recent study shows that the bis-indole–derived C-DIM12, a known NURR1 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 NURR1 in squamous cell carcinoma cells increased resistance to 5-FU treatment (17) suggesting a possible role for NURR1 in drug resistance. Gemcitabine has replaced 5-FU for treatment of pancreatic cancer and based on the prognostic and functional characteristics of NURR1 in cancer cells this study focused on determining the functions of NURR1 in pancreatic cancer and its role in gemcitabine resistance.
Knockout of NURR1 in pancreatic cancer cells or treatment with C-DIM12–induced apoptosis and inhibited proliferation (Fig. 2). Moreover, in athymic nude mice bearing MiaPaCa2 (NURR1+/+) or NURR1-KO cells, it was clear that loss of NURR1 or treatment with C-DIM12 inhibited tumor growth and enhanced survival (Fig. 6). These results confirmed the prooncogenic activity of NURR1 in pancreatic cancer cells as previously observed in other cancer cell lines (15) and demonstrated that the NURR1 antagonist C-DIM12 was an effective anticancer agent that blocked NURR1-mediated responses.
We also investigated the role of NURR1 in drug resistance and observed that treatment of pancreatic cancer cells with gemcitabine resulted in induction of NURR1 (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 NURR1, 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 NURR1 inactivation or treatment with C-DIM12 enhanced the effectiveness of gemcitabine thus demonstrating that induction of NURR1 by gemcitabine is associated with drug resistance.
The mechanism of NURR1 as a drug-resistant factor was further investigated by RNA-seq in wild-type and NURR1-KO cells and this resulted in identification of several genes/pathways regulated by NURR1; however, the major pathway was associated with changes in expression of genes involved in autophagy (Fig. 3; refs. 39–43). Because autophagy has previously been linked to cytoprotection in colon cancer cells (21), we examined this gene set and identified two genes, namely ATG7 and ATG12, that were not only regulated by NURR1 (Fig. 5A and B) but also exhibit clinical characteristics similar to that observed for NURR1 in patients with pancreatic cancer (Figs. 1 and 7). These results confirm that NURR1 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 NURR1 antagonist such as C-DIM12.
No disclosures were reported by the authors.
Mehrdad Zarei: Investigation, methodology. R. Shrestha: Investigation, methodology. S. Johnson: Investigation, methodology, writing-original draft. Z. Yu: Investigation, methodology. K. Karki: Investigation, methodology. A. Vasiri-Gohar: Investigation, methodology. J. Epps: Investigation, methodology. H. Du: Investigation, methodology. L. Suva: Funding acquisition, administration, supervision. Masha Zarei: Conceptualization, funding acquisition, supervision, administration, writing and editing. S. Safe: Funding acquisition, supervision, administration, writing and editing.
This work was supported by the Department of Defense (DOD-W81XWH-18–0592, to M. Zarei) and the NIH P30-ES029067 (to S. Safe).