Loss of GATA6-mediated up-regulation of UTX promotes pancreatic tumorigenesis and progression

Ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX), also known as lysine (K)-specific demethylase 6A (KDM6A), functions as a tumor suppressor gene or oncogene depending on the tumor type and context. However, its tumor-suppressive mechanisms remain largely unknown. Here, we investigated the clinical significance and biological effects of UTX expression in pancreatic ductal adenocarcinoma (PDA) and determined the potential mechanisms of its dysregulation. UTX expression and its association with clinicopathologic characteristics of PDA patients were analyzed using immunohistochemistry. UTX mRNA and protein expression and their regulation in PDA cell lines were measured using quantitative polymerase chain reaction and Western blot analyses. The biological functions of UTX in PDA cell growth, migration, and invasion were determined using gain- and loss-of-function assays with both in vitro and in vivo animal models. UTX expression was reduced in human PDA cell lines and specimens. Low UTX expression was associated with poor differentiation and prognosis in PDA. Forced UTX expression inhibited PDA proliferation, migration, and invasion in vitro and PDA growth and metastasis in vivo, whereas knockdown of UTX expression did the opposite. Mechanistically, UTX expression was trans-activated by GATA6 activation. GATA6-mediated PDA progression could be blocked, at least partially, by silencing UTX expression. In conclusion, loss of GATA6-mediated UTX expression was evident in human PDA and restored UTX expression suppressed PDA growth and metastasis. Thus, UTX is a tumor suppressor in PDA and may serve as a prognostic biomarker and therapeutic target.


Introduction
Pancreatic ductal adenocarcinoma (PDA), generally known as pancreatic cancer, is the seventh leading cause of cancerrelated deaths worldwide.1e3 In the United States, the incidence of PDA is increasing, with an estimated 53,070 new cases and 41,780 PDA-related deaths in 2016.2e5 Recently, surgical resection, radiation therapy, chemotherapy, and immunotherapy for PDA have improved, although its 5-year survival rate remains less than 8%.3e8 Thus, further studies of the molecular mechanisms of PDA development and progression and identification of new therapeutic targets for PDA are urgently needed.9e13 Our knowledge of PDA development and progression has advanced significantly because of a rapid increase in our understanding of PDA molecular biology and genetics.14e18 Existing data provide global insights into genetic alterations in and molecular profiles of PDA.15e20 Established inherent and acquired genetic and epigenetic alterations in PDA include mutations of the oncogene K-ras and inactivation of the tumor suppressors TP53 and DPC4.15e19 Consequently, various signaling pathways are deregulated in PDA cells (e.g., Wnt/bcatenin, Kru ¨ppel-like factor 4, and Forkhead box M1 signaling axes).18e22 Presumably, these alterations critically promote cellular transformation and tumor initiation and progression.21e23 However, potential new driver pathways for PDA progression remain to be discovered and elucidated as potential targets for designing effective intervention strategies.22e25 Recently, several groups identified ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX ), also known as KDM6A, as a novel histone demethylase that catalyzes the removal of dimethyl and trimethyl groups from histone H3 lysine 27, thereby promoting target gene activation. 27,28Researchers also recently demonstrated a histone demethylation-independent role for UTX in normal and malignant T cells, mesoderm differentiation, and mouse embryonic development.29e31 Investigators identified somatic loss-of-function mutations of the UTX gene in a variety of human tumors, including multiple myeloma, esophageal cancer, and renal cancer. 32The identification of recurrent inactivating UTX mutations in several leukemia and solid tumor cases strongly supports that UTX acts as a tumor suppressor for human cancers.33e35 However, the roles of UTX in cancer development and progression appear to be more complicated than initially expected.Two independent studies demonstrated that UTX controls cell-cycle progression via the retinoblastoma protein pathway. 36,37UTX also may function as a bona fide tumor suppressor for T-cell acute lymphoblastic leukemia. 35In renal cell carcinoma cases, low expression of UTX is associated with reduced overall survival durations. 38In contrast, in patients with breast cancer, high levels of UTX expression are associated with poor prognosis, and UTX knockdown results in significant decreases in the proliferation and invasiveness of breast cancer cells in vitro and in vivo. 39he regulation of UTX expression remains unclear.Previous studies have shown estrogen receptors might be the upstream regulators of UTX. 52The importance of GATA6 in gene expression and regulation has recently been reported in many human cancers, including lung cancer, 59 gastric cancer, 60 and colorectal cancer, 61 and the downstream targets have been identified in a series of essential signaling pathways during carcinogenesis.However, whether GATA6 regulates UTX is not known.Loss of GATA6 appears to have a critical role in promoting tumorigenesis and epitheliale mesenchymal transition (EMT)-dependent metastasis in PDA. 57,58The role and mechanisms underlying the interaction between GATA6 and UTX in PDA pathogenesis are unclear.
Whole-genome sequencing and copy number-variation analyses of 100 human PDA specimens indicated that UTX was inactivated in 18% of patients. 40However, little is known about the potential function of UTX in PDA development and progression and the mechanisms underlying its potentially dysregulated expression in this tumor.Therefore, in the present study, we examined UTX expression in human PDA cells and specimens, evaluated the impact of altered expression of UTX on PDA development and progression, and explored the underlying mechanisms.

Immunohistochemical analysis
Two human pancreatic tissue cohorts were used in this study.The expression of UTX was analyzed in cohort #1 with 84 primary PDA, 84 adjacent pancreatic intraepithelial neoplasia (PanIN), and normal pancreatic tissue specimens (US Biomax).Correlation analysis of GATA6 and UTX protein expression was performed using cohort #2 with 60 PDA tissues.Immunohistochemical analyses of these specimens and xenografts were conducted with anti-GATA6 (R&D), anti-UTX (Bethyl Laboratories), and anti-Ki67 (Santa Cruz Biotechnology) antibodies as described previously.20e23

Cell lines
The human embryonic kidney cell line 293T, pancreatic ductal cell line HPNE, and human PDA cell lines AsPC-1, BxPC-3, CaPan-1, CaPan-2, Mia-PaCa-2, PANC-1, and Patu8902 were purchased from the ATCC.The PDA cell lines MDA28 and MDA48 were gifts from Dr. Paul J. Chiao (The University of Texas MD Anderson Cancer Center).The human PDA cell line FG was obtained from Dr. Michael P. Vezeridis (The Warren Alpert Medical School of Brown University). 41All these cell lines were maintained in plastic flasks as adherent monolayers in Eagle's minimal essential medium supplemented with 10% FBS, sodium pyruvate, nonessential amino acids, L-glutamine, and a vitamin solution (Flow Laboratories).The ATCC performs characterization and authentication of the cell lines it provides using short tandem repeat profiling, and the cell lines they provided were passaged in our laboratory for fewer than 6 months after reception.22e25 Immunofluorescent staining PDA cells were seeded on chamber slides overnight prior to experimentation.Cells were fixed with 4% paraformaldehyde and permeabilized in 0.1% Triton X-100 in PBS and sequentially blocked with 3% bovine serum albumin for 30 min.Following overnight incubation with primary antibodies against UTX, hemagglutinin (HA), or tubulin, the cells were further incubated with appropriate secondary antibodies and subjected to staining. 42

Nuclear and cytoplasmic protein extraction
The NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) were used to separate and prepare cytoplasmic and nuclear extracts of 293T and PANC-1 cells, and standard Western blotting was carried out to analyze the protein lysates. 43

Animals
Mice were purchased from the Hunan SJA Laboratory Animal CO., LTD (Changsha, Hunan, China).The animal experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the South China University of Technology.The animal protocol was approved by the Committee on Ethics of Animal Experiments of the South China University of Technology.22e25

Tumor growth and metastasis
Tumor PDA cells (1 Â 10 6 ) in 0.1 mL of Hank's balanced salt solution were injected subcutaneously into the right scapular regions of nude mice.The resulting tumors were measured every week.Tumor-bearing mice were killed when they became moribund or on day 28 after injection and their tumors were removed and weighed.22e25 To measure liver metastasis, 1 Â 10 6 tumor cells were injected intravenously into another group of mice via the ileocolic vein.The mice were killed on day 28 and their liver surface metastases were counted.

Tumor-cell migration/invasion assay
Both cell scratch-wound (horizontal migration) and modified Boyden chamber (vertical invasion) assays were performed to determine the migratory ability and invasiveness of PDA cells with altered UTX expression as described previously. 44or the cell scratch-wound assay, cells were grown in sixwell plates until confluent.A wound was generated on the surface of the resulting cell monolayer via scraping with the 10-mL tip of a pipette, the cells in the wounded monolayer were photographed at different time points, and cell migration was assessed by measuring gap sizes in multiple fields.For the Boyden chamber assay, 24-well tissue culture plates with 12 cell culture inserts (Millipore) were used.Each insert contained an 8-mm-pore-size polycarbonate membrane with a precoated thin layer of a basement membrane matrix (ECMatrix; for the invasion assay) or without a coated matrix (for the migration assay).Ten percent FBS-containing medium was placed in the lower chambers to act as a chemoattractant.Cells (5 Â 10 5 ) in a 300-mL volume of serumfree medium were placed in the upper chambers and incubated at 37 C for 48 h.Cells on the lower surface of the polycarbonate membrane, which had invaded the ECMatrix and migrated through the membrane, were stained, counted, and photographed under a microscope. 22,24,25lymerase chain reaction analysis Polymerase chain reaction (PCR) analysis of genomic UTX was performed using total genomic DNA obtained from tumor cells.Total DNAs were purified using a QIAamp DNA Mini Kit (QIAGEN).The PCR primer sequences were as follows: UTX exon18, 5 0 -tacctcaggtggacaacaagg-3' (forward) and 5 0 -gcagatctgttttcatgggg-3' (reverse); UTX exon1, 5 0gttgtgaattcgctgcgttt-3' (forward) and 5 0 -tgccttaccttgcccagtag-3' (reverse); and actin, 5 0 -cctgcagagttccaaaggag-3' (forward) and 5 0 -ggcatcctcaccctgaagta-3' (reverse).25,42,43 Reverse transcription-PCR Reverse transcription-PCR analysis of UTX mRNA expression in tumor cells was performed using total RNA.Total RNAs were purified using an RNeasy Plus Mini Kit (QIAGEN), and cDNAs were synthesized using an iScript cDNA Synthesis Kit (Bio-Rad).The PCR primer sequences were as follows: UTX exon18, 5 0 -taaccgcacaaacctgacca-3' (forward) and 5 0tgccttgttgtccacctgag-3' (reverse); UTX exon30, 5 0 -tgtcagacattgagggaagc-3' (forward) and 5 0 -cggatggtaatggaggagct-3' (reverse); and GAPDH, 5 0 -tgcaccaccaactgcttagc-3' (forward) and 5 0 -ggcatggactgtggtcatgag-3' (reverse).24,42

Promoter reporter and dual luciferase assay
PDA cells plated in 96-well plates were transfected with UTX promoter reporters, siRNAs, or specific gene expression plasmids using Lipofectamine 2000 (Invitrogen).The UTX promoter activity in these cells was normalized via cotransfection of a b-actin/Renilla luciferase reporter containing a full-length Renilla luciferase gene.The Dualluciferase reporter assay was performed 24 h after transfection and quantified using a dual luciferase assay system (Promega).The promoter mutants were generated using the Q5 â site-directed mutagenesis kit (NEB, Singapore) according to the manufacturer's instructions.These constructs of truncated or mutated PiHL promoter were subsequently cloned into the pGL3 vector.

Chromatin immunoprecipitation assay
PDA cells (2 Â 10 6 ) were prepared for a chromatin immunoprecipitation (ChIP) assay using a ChIP assay kit (Millipore) according to the manufacturer's protocol.The resulting precipitated DNA specimens were analyzed using PCR to amplify fractions of the UTX promoter.The PCR products were resolved electrophoretically on a 2% agarose gel and visualized using ethidium bromide staining.

Statistical analysis
The two-tailed Pearson c 2 test or Fisher exact test was used to determine the significance of differences among covariates.All in vitro experiments were performed in triplicate and at least three times.Data were presented either as means AE standard deviation from one representative independent experiment of three with similar results or means AE standard error of the mean from three independent experiments.The significance of the in vitro and in vivo data was determined using the Student t-test (twotailed) or one-way analysis of variance.In all the tests, P values less than 0.05 were considered statistically significant.The SPSS software program (version 17.0; IBM Corporation) was used for statistical analysis.

Direct correlation of reduced UTX expression with reduced survival durations in PDA patients
To determine the roles of UTX in PDA pathogenesis, we first investigated UTX protein expression in the 84 primary PDA specimens, 84 matched adjacent PanIN specimens, and normal pancreatic tissue specimens in a TMA.The clinicopathologic characteristics of the patients from whom the 84 PDA specimens were obtained are listed in Table S1.We observed decreased expression of UTX in PDA specimens relative to the corresponding PanIN specimens (Fig. 1A).TCGA data showed that UTX expression was higher in pancreatic cancer tissues than that in the normal tissues (Fig. S1A).A similar expression pattern was observed in the colorectal cancer tissues from the TCGA cohort (Fig. S1B).Our immunohistochemical analysis confirmed that UTX protein expression was also attenuated in colorectal cancer tissues relative to the adjacent normal tissues (Fig. S1C).We analyzed the relationship between clinicopathologic parameters and UTX expression in PDA specimens (Table S1).UTX expression was negatively associated with worse histologic differentiation (P Z 0.029) (Fig. 1B, C) but with advanced T category with statistical significance at borderline (P Z 0.071) (Fig. 1D).We then investigated the relationship of UTX expression with age, sex, TNM category, and distant metastasis and found no statistically significant differences (P > 0.05).KaplaneMeier analyses demonstrated that expression of UTX was strongly associated with increased overall survival (Fig. 1E).We further examined the UTX expression in the Pdx-Cre mouse model of PDA with KRAS mutant.Consistent with our previous finding, UTX expression was higher in normal pancreatic tissue than in PanIN specimens (Fig. 1F).Collectively, UTX expression gradually decreased from normal pancreatic tissue through to pancreatic intraepithelial neoplasia and pancreatic cancer, suggesting that UTX dysregulation is an early event in the multistep progression of pancreatic carcinogenesis.

Subcellular location of UTX in PDA cells and tissues
To further determine the role of UTX in PDA development and progression, we analyzed the UTX gene integrity in PDA cell lines with a focus on exons and mRNA and protein expression using both 293T and HPNE cells as controls.According to previous findings, 26 we analyzed the UTX exon18 gene and mRNA expression and protein expression in 293T, HPNE, and PDA cell lines.UTX was highly expressed in 293T, CaPan-2, FG, MDA28, and Patu8902 cells but expressed at relatively low levels in HPNE, AsPC-1, BxPC-3, CaPan-1, MDA48, and PANC-1 cells.Importantly, we found expression of the UTX gene, mRNA, and protein was not detectable in Mia-PaCa-2 cells (Fig. S2AeC).To further examine the UTX expression in normal and malignant ductal cells, we primarily cultured PDA cells and normal pancreatic duct epithelial cells from PDA and adjacent normal tissues.Western blot showed that UTX expression was reduced in cancer cells relative to normal cells (data not shown).The general reduction of UTX in PDA may owe to genetic and epigenetic regulation, which may need further exploration.To determine the location of UTX in PDA cells, we first performed immunohistochemical staining of PDA and PanIN specimens for UTX.We found that UTX was localized in both the cytoplasm and nucleus (Fig. S3A).We confirmed this using Western blot analysis of nuclear and cytoplasmic protein extracts from PANC-1 (Fig. S3B) and 293T (Fig. S3C) cells, using H3 and tubulin as nuclear and cytoplasmic control proteins, respectively.Moreover, we confirmed the subcellular localization of UTX using a cell immunofluorescence assay in PANC-1 (Fig. S3D, E) and 293T cells (Fig. S3C).

Inhibition of PDA cell proliferation and apoptosis in vitro and tumor growth in vivo by increased UTX expression
To determine the effect of UTX on PDA cell proliferation, we induced UTX overexpression via gene transfection and knocked down UTX expression using siRNA (SiUTX#2 with best targeting efficacy) in PANC-1, Patu8902, and MDA28 cells.We found that overexpression of UTX led to decreased PDA cell monolayer growth, whereas knockdown of UTX expression resulted in increased cell growth (Fig. 2A, B).The results of flow cytometric analysis revealed that PDA cells transfected with UTX had an obvious cell cycle arrest in the G0eG1 phase and the population of cells in the S phase was decreased (Fig. S4A).Apoptosis assays revealed that the fraction of apoptotic cells was significantly increased among the UTX-upregulated PDA cells relative to the control cells (Fig. S4B).In our animal model of PDA, transfection with HA-UTX inhibited the growth of PANC-1 and Patu8902 cells, whereas knockdown of UTX expression promoted the growth of these cells in vivo (Fig. 2CeE; Fig. S5AeC).Consistently, immunohistochemical analyses demonstrated that overexpression of UTX led to decreased Ki67 staining, whereas reduced expression of UTX led to increased Ki67 staining (Fig. S5D).These results demonstrated that UTX inhibited the tumor growth of PDA in vitro and in vivo, supporting that UTX functions as a tumor suppressor in PDA.

Inhibition of PDA cell migration and invasion in vitro and tumor metastasis in vivo by increased UTX expression
To determine the effect of UTX expression on PDA migration and invasion, we transfected PANC-1, Patu8902, and MDA28 cells with HA-UTX and UTX SiRNA, respectively, for 48 h.We wounded the monolayer of the transfected cells via scratching and maintained the cells for 6e72 h.These results demonstrated that forced expression of UTX strongly attenuated the flattening and spreading of PDA cells (Fig. 3A), whereas down-regulation of UTX promoted the flattening and spreading of the cells (Fig. 3B).Boyden chamber assay of invasiveness showed consistent results (Fig. 3C).In our animal model of PDA metastasis, liver surface metastases of HA-UTX-transfected cells were much less than those of control cells, whereas those of SiUTX-transfected cells were markedly enhanced (Fig. S6A).We further investigated several markers of cell growth and metastasis with alerted UTX expression by Western blot.Results showed that overexpression of UTX in PANC-1, Patu8902, and MDA28 cells led to decreased expression of MMP2, uPAR, and cyclinD1 and increased expression of p27, whereas knockdown of UTX expression markedly up-regulated that of MMP2, uPAR, and cyclin D1 and decreased p27 in these cell lines (Fig. S6B).UTX is known as a potent histone demethylase.We further examined whether histone methylation agents would impact UTX biofunctions in PDA cells.Treatment of GSKJ4 HCI, a selective inhibitor of the H3K27 histone demethylase, at least partially rescued the inhibitory effects on the proliferative and invasive capability of PDA cells (data not shown).These data further confirmed the tumor suppressor role of UTX in PDA development and progression.

Direct correlation between GATA6 and UTX expression in PDA cells
To identify the regulatory mechanism of UTX as a tumor suppressor during pancreatic tumorigenesis, we made a promoter analysis to find its transcription activators.By using online tools JASPAR and Animal TFDB3.0,databases for the prediction of transcription factor binding sites, we found several potent transcription regulators, including GATA6 (Fig. S7A, B).Our unpublished work demonstrated that GATA6 is a crucial suppressor for the pro-tumor immune microenvironment.GATA6 mRNA expression in PDA tissues may not accurately reflect clinical characteristics.However, its expression in the ductal compartment seems to be correlated with prolonged overall survival (data not shown).To test whether UTX acted as a transcriptional target of GATA6, a series of experiments were performed.First, by using TCGA and GEO databases, we demonstrated that mRNA expression of GATA6 and UTX in PDA were closely correlated (Fig. 4AeC).Next, we examined the protein expression of GATA6 and UTX in another cohort with 30 PDA tissues by immunohistochemical analysis.As shown in Figure 4D, UTX expression colocalized with GATA6 expression in consecutive sections of PDA tissues with different differentiation grades.Importantly, GATA6 expression in PDA specimens was highly positively correlated with UTX expression (Fig. 4E, F).Furthermore, we examined the expression of UTX and UTXmediated tumor cell aggressiveness with alerted GATA6 expression.PANC-1 cells transfected with GATA6 showed significantly elevated UTX protein and mRNA expression, while cells treated with two GATA6 specific siRNA (#1 and #2) did the opposite (Fig. 5A).Treatment in Patu8902 cells showed consistent results (Fig. 5B).To further understand the role of GATA6 in UTX-mediated PDA aggressiveness, we induced UTX knockdown in PANC-1 and Patu8902 cells with ectopic expression of GATA6 (Fig. 5C, D).Silenced UTX expression partially inhibited the GATA6-induced inhibitory effect on cell proliferation (Fig. 5E) and invasion (Fig. 5F) of PDA cells.These results clearly indicated that GATA6 upregulated UTX expression and that UTX is an important mediator of GATA6-induced PDA progression.

Transcriptional activation of UTX expression by GATA6
To determine whether GATA6 transcriptionally activates UTX expression, we first explored Cistrome Data Browser (cistrome.org),an online data portal for ChIP-Seq data.Prominent GATA6 peaks in UTX suggested strong binding (Fig. 6A).Next, we analyzed the UTX promoter sequence for the presence of potential GATA6 binding sites containing (A/T) GATA (A/G) motif (Fig. 6B) and identified two putative GATA6-binding elements (referred to as GBE#1 and #2).Accordingly, we constructed the deletion mutant reporters p1799 (containing GBE#1 and #2), p899 (containing GBE#2), and p262 (containing no GBE).We co-transfected the deletion mutant reporters with or without GATA6 expression vectors into 293T cells.Luciferase reporter assay results demonstrated that deletion of the region covering binding site#2 markedly decreased the promoter activity of UTX activated by GATA6 (Fig. 6C).In addition, we determined the activity of wide-type promoter, instead of GBE#2 mutant one, got promoted with enforced GATA6 expression (Fig. 6D).To further determine whether GATA6 regulates UTX promoter transcriptional activity in PDA cells, we cotransfected a full-length reporter with GATA6 expression   6E and F, increased GATA6 expression in PANC-1 cells induced UTX promoter activity, whereas knockdown of GATA6 expression showed an inhibiting effect.A similar result was found in Patu8902 cells (Fig. S8A, B).ChIP assay also confirmed GATA6 directly bound GBE#2 in PDA cells (Fig. 6J; Fig. S8C).Taken together, these results suggested that GATA6 can not only directly bind to the UTX promoter but also influence the biofunctions of UTX in PDA.

Discussion
In this study, we determined the role of UTX expression and its regulatory mechanism during PDA development and progression.We found that UTX expression was drastically reduced in human PDA cell lines and specimens.Low UTX expression was associated with worse differentiation and poor prognosis of PDA.Forced UTX expression inhibited PDA proliferation, migration, and invasion in vitro and growth and metastasis in vivo, whereas knockdown of UTX expression did the opposite.UTX attenuated PDA cell aggressiveness by serving as an important target of transcriptional factor GATA6, as demonstrated in PDA cell lines, the orthotopic mouse model, and human PDA specimens.Mechanistically, GATA6 bound directly to the promoter region of UTX and activated its transcriptional activity.Our clinical and mechanistic data has established that UTX is a tumor suppressor in PDA and GATA6-UTX axis may serve as a potential target for PDA therapy.respectively, for 48 h.Invading cells were examined as described in Materials and methods.The data represent the means AE standard error of the mean from three independent experiments with similar results.*P < 0.05 in comparisons of the HA-UTX-and SiUTX-treated groups with the control groups (Student t-test); **P < 0.01 in comparisons of the HA-UTX-and SiUTXtreated groups with the control groups (Student t-test).
The conflicting role of UTX in cancer gains increasing attention recently.Low expression of UTX is associated with reduced overall and disease-free survival durations in clear cell renal cell carcinoma patients and appears to be an independent prognostic factor. 38A similar result is reported in glioblastoma. 45However, in a study of estrogen receptor-positive breast cancer, no clinical parameters are significantly associated with UTX expression. 46These discrepancies suggest the complex role of UTX in tumor progression, likely stage-and context-dependent.In the present study, we delineated the relationship between UTX expression in PanIN and PDA and the patients' clinicopathologic characteristics.We found that reduced UTX expression was associated with poor tumor differentiation.Decreased UTX expression was directly associated with reduced overall survival time in the PDA patients.Consistently, PDA mouse models produced similar results.Interestingly, UTX was localized in both nuclei and cytoplasm of PDA cells.However, the significance and potential mechanisms of the subcellular localization of UTX protein and its translocation to the cytoplasm and nucleus remain unclear, warranting further study.
UTX is frequently mutated in myeloma cells, and overall survival durations are shorter in myeloma patients with a UTX mutation or deletion than in those without mutation. 47 recent study has shown that UTX is inactivated in 18% of PDA patients (eight homozygous deletions, five structural variants, four frame shifts, two missense mutations, and one in-frame deletion).40 In most cases with inactivated UTX, both alleles of UTX are affected.In the present study, we determined the UTX expression pattern in nine PDA cell lines and found that expression of UTX mRNA and protein was substantially reduced, while a genetic loss of UTX occurred only in one cell line, suggesting that down-regulation of UTX gene is mostly through an epigenetic mechanism.
UTX exhibits pro-tumor and anti-tumor activities.49e52 However, a systematic screen by using CRISPR/ Cas9-mediated somatic gene knockout in a Kras G12D/þ mouse model has confirmed that loss of UTX significantly promotes lung tumorigenesis. 50UTX mutant cells show an increased in vitro and in vivo sensitivity to inhibition of EZH2, a histone methyltransferase that generates H3K27me3. 53UTX deficiency renders lymphoma sensitive to cytarabine treatment. 54UTX could restrain Kras G12D -driven PDA and confers its sensitivity to BET inhibitors. 55In our biologic studies, knockdown of UTX expression markedly enhanced PDA tumor growth and metastasis in vitro and in vivo, whereas increased UTX did the opposite.All these findings consistently and strongly support that UTX functions as a tumor suppressor gene.
UTX was identified as X chromosome-encoded histone demethylase.Therefore, its roles in regulating methylation and demethylation in tumor cells are widely investigated.UTX could directly influence the methylation level or indirectly through targeting other epigenetic regulators.Knockout of UTX increases the EZH2 level, thus up-regulating the H3K27me3 level.UTX could coordinate with JHDM1D and CBP to direct the H3K27 methylatione acetylation transition and to create a permissive chromatin state on ER targets in breast cancer. 52The most significant network of genes bound by UTX is tied to cellcycle pathways centered on the tumor suppressor gene retinoblastoma and retinoblastoma-binding proteins. 36A similar finding is made in a genetic screen of Drosophila for ectopic cell growth mutants. 37However, UTX appears to play an oncogenic role in breast cancer and TAL1-driven Tcell acute lymphoblastic leukemia. 39,48Estrogen receptorinduced UTX could activate ER expression, forming a feedforward loop in the regulation of hormone response of breast cancer cells. 52Despite the wide mechanism exploration of UTX in other types of cancer, understanding the role of UTX in PDA development and progression is limited.UTX loss could deregulate the COMPASS-like complex and aberrant activation of super-enhancers regulating DNp63, MYC, and RUNX3 oncogenes selectively in female patients. 55However, the mechanism of aberrant loss of UTX during pancreatic tumorigenesis is still unknown.
Recent studies have identified GATA6 as a transcriptional factor and tumor suppressor in a variety of human cancers, including PDA.GATA6 contributes to complete pancreatic acinar differentiation and maintains its exocrine function. 56n vivo evidence from genetically engineered mouse models shows that acinar GATA6 suppresses Kras G12V -driven pancreatic tumorigenesis. 57GATA6 is also critical for canonical pancreatic epithelial differentiation.Loss of GATA6 promotes epithelialemesenchymal transition and metastasis. 58Given the important role of GATA6 during pancreatic differentiation and malignancy transition, key downstream target genes and signaling pathways warrant further exploration.In the current study, we provided several lines of evidence that UTX is a direct target of GATA6 in PDA and that GATA6-UTX signaling is critical in PDA progression.
In summary, UTX is a potential biomarker for PDA and functions as a tumor suppressor gene, and inhibits PDA growth and metastasis.Mechanistically, GATA6 activates UTX expression by direct binding to its promoter region and activating its transcription.Thus, the UTX-GATA6 signaling axis offers a potentially novel therapeutic target for PDA treatment.

Figure 1
Figure 1 Reduced and lost UTX expression in human PDA specimens.PDA and PanIN specimens in TMAs were immunostained with a specific anti-UTX antibody.(A) Representative images of UTX expression in PDA and PanIN specimens.(B, C) Positive association of UTX expression with tumor differentiation in PDAs.(D) Graphs of the UTX expression in PDA specimens at different T categories.(E) KaplaneMeier analysis of the overall survival times in high and low UTX-expressing PDA patients.(F) UTX expression in normal pancreatic tissue ("N") and PanIN ("P") specimens obtained from a Pdx-Cre mouse with PDA.

Figure 2
Figure 2 Increased UTX expression suppressed PDA cell proliferation in vitro and in vivo.PANC-1, Patu8902, and MDA28 cells were transfected with HA-UTX (A) or SiUTX (B) and control vectors and siRNAs, respectively, for 48 h.The cell growth was assessed using a Cell Counting Kit-8 (according to an optical density at 450 nm [OD 450 ]) at the indicated time points.PANC-1 cells with UTX overexpression or knockdown of UTX expression transfected with HA-UTX or SiUTX were injected subcutaneously into the right scapular regions of nude mice (1 Â 10 6 cells per mouse, four mice per group).Gross tumors in the mice (C), tumor growth curves (D), and tumor weights (E) are shown.*P < 0.05; **P < 0.01.

Figure 3
Figure 3 Increased UTX expression suppressed PDA cell migration and invasion in vitro.PANC-1, Patu8902, and MDA28 cells were transfected with HA-UTX (A) or SiUTX (B) and control vectors and siRNAs, respectively, for 48 h.(A) For a cell scratch-wound assay, cells in each group were placed in six-well plates, wounded via scratching, and maintained at 37 C for 12, 24, or 48 h.Cell cultures were photographed, and cell migration was assessed by measuring the cell-free areas in multiple fields (the inset numbers are the mean percentage gap areas AE standard error of the mean from three independent experiments with similar results).(C) For an invasion assay, PANC-1, Patu8902, and MDA28 cells were transfected with HA-UTX or SiUTX and control vectors and siRNAs,

Figure 4
Figure 4 Co-expression of UTX and GATA6 in PDA tissues.(A) The correlation analysis between UTX and GATA6 mRNA level made in pancreatic cancer from TCGA database (www.LinkedOmics.org).(B, C) Data from the GEO database (GSE71729 and GSE6629) illustrated that GATA6 was positively correlated with UTX mRNA expression.(D) Immunohistochemical analysis of GATA6 and UTX correlative expression in human PDA surgical samples (magnification,Â200 and Â400 in the inserts).(E) The distribution of immunohistochemical results between GATA6 and UTX expression.(F) Statistical analysis of immunohistochemical results of GATA6 and UTX expression in human PDA surgical samples.

Figure 6
Figure 6 Direct activation of UTX transcription by GATA6.(A) Representation of ChIP-Seq peaks on UTX promoters.The online data portal for ChIP-Seq data, Cistrome Data Browser (cistrome.org),was used to test the possibility of GATA6 binding to the UTX promoter.(B) Two GATA6-binding sites located at different sites in the UTX promoter sequence.(C)UTX promoter reporters p1977, p899, and p262 were transfected into 239T cells in triplicate with GATA6 expression or control vectors for 24 h.The UTX promoter activity was then examined using a dual luciferase assay kit.(D) Luciferase-based reporter assay showing the activity of UTX promoter reporters in 293T cells co-transfected with GATA6 plasmid and empty vector or with vectors expressing either wild-type or GATA6-binding site#2 mutated UTX promoter.(E) PANC-1 cells were co-transfected with p1799, GATA6 plasmid, or control vector.(F) PANC-1 cells were co-transfected with p1799, SiGATA6 (#1 or #2), or a non-targeting siRNA.The promoter activities in the cells determined using a dual luciferase assay are shown.(G) The results of the ChIP assay conducted using chromatins isolated from PANC-1 cells.The immunoprecipitated DNA was analyzed by PCR followed by agarose gel electrophoresis.Genomic DNA input was 1%.The experiments were performed three times independently.*P < 0.05.