_:b506093800 . _:b18780962 . . _:b18780984 "at doses that effectively inhibit APE1 redox activity, and STAT3 inhibitors at doses that were utilized in the MTS-based assay for cytotoxicity (Figure 4C) and that inhibited STAT3 phosphorylation in PDAC cells (Figure 4A,B and Ref. [>>24<<]). As single agents, minimal inhibition of PDAC cells was observed with E3330 or at the doses of STATTIC or S3I-201 used (Figure 4D,E)." . _:b506093753 "2"^^ . _:b506093752 "2"^^ . _:b506093782 . _:b506093755 "2"^^ . _:b18780931 "Treatment with E3330, a small molecule redox signaling inhibitor that recognizes an alternate, redox active conformation of APE1 [>>3<<] markedly inhibits the DNA binding and transcriptional activity of NF-B, AP-1, and HIF-1 [4], [5]." . . _:b506093754 "2"^^ . _:b506093730 . _:b506093669 . _:b506093698 . _:b506093783 . _:b506093765 "2"^^ . . . _:b506093764 "2"^^ . _:b506093767 "2"^^ . _:b506093766 "2"^^ . _:b18780967 . _:b506093761 "2"^^ . _:b506093760 "2"^^ . _:b506093763 "2"^^ . _:b506093653 . . _:b506093762 "2"^^ . _:b506093789 . _:b18780971 "results" . _:b18781002 . _:b18780963 . _:b506093695 . _:b506093773 "2"^^ . . . _:b506093772 "2"^^ . _:b18780973 . _:b506093718 . . _:b506093775 "2"^^ . . _:b506093774 "2"^^ . _:b506093716 . _:b506093640 . _:b506093710 . _:b506093769 "2"^^ . . _:b506093775 . _:b18781005 . _:b506093768 "2"^^ . _:b18780969 . _:b506093673 . _:b506093649 . _:b506093642 . _:b506093771 "2"^^ . _:b18780970 . _:b506093735 . _:b506093770 "2"^^ . _:b506093781 "2"^^ . _:b18780929 . _:b506093780 "2"^^ . _:b18780955 "Sequence detection system (Applied Biosystems). The relative quantitative mRNA level was determined using the comparative Ct method using Actin (PaCa-2) or ribosomal protein large, P0 (RPLP0, patient lines) as the reference gene. [>>4<<] The primers for STAT3, survivin, Actin, and RPLP0 are commercially available (Applied Biosystems)." . _:b506093785 . _:b506093783 "2"^^ . _:b18780982 . _:b18781003 . _:b506093782 "2"^^ . _:b506093796 . _:b506093777 "2"^^ . _:b18780976 . . _:b506093776 "2"^^ . . . _:b506093779 "2"^^ . _:b18780947 . . _:b18780979 . _:b506093778 "2"^^ . . _:b506093789 "2"^^ . _:b18780977 "First we show that treatment with two previously described STAT3 antagonists, STATTIC [>>23<<] and S3I-201 [24] inhibited phosphorylation of STAT3 (Figure 4 A,B) as well as cellular proliferation in PDAC cells (Figure 4C)." . _:b506093788 "2"^^ . _:b506093791 "2"^^ . _:b506093681 . . _:b506093641 . _:b506093790 "2"^^ . _:b18781007 "Studies in animal models of PDAC confirm that dual- or multi-targeting approaches can increase anti-tumor responses [40], [>>41<<]. APE1 and STAT3 are upregulated and play important roles in cancer, suggesting that tumor cells may be \u2018addicted\u2019 to their effector functions. In addition to biochemical and molecular data linking APE1 and STAT3 in PDAC cells, our studies" . . _:b506093785 "2"^^ . . . _:b506093784 "2"^^ . . . _:b506093787 "2"^^ . . . . . _:b506093786 "2"^^ . _:b18780987 . . _:b18780939 . _:b506093797 "2"^^ . _:b18780932 . . . . _:b506093796 "2"^^ . . _:b506093799 "2"^^ . . _:b506093733 . _:b506093690 . . _:b506093798 "2"^^ . . _:b506093793 "2"^^ . . _:b506093793 . . _:b506093792 "2"^^ . . . _:b506093699 . _:b506093795 "2"^^ . . _:b506093810 . _:b506093688 . _:b506093691 . _:b506093794 "2"^^ . _:b18780992 . . . _:b18780940 . _:b506093805 "2"^^ . . . . _:b506093804 "2"^^ . . . . _:b18780979 "In addition to regulating the transcriptional activity of STAT3, APE1 also exerts redox control of other transcription factors, which have been implicated in pancreatic cancer (such as HIF-1\u03B1 and NF-\u03BAB). [>>4<<], [16], [25] We then evaluated whether the combined blockade of STAT3 and APE1 redox activity synergize to more effectively inhibit human PDAC cells." . _:b506093807 "2"^^ . . _:b506093806 "2"^^ . _:b506093627 . . _:b506093801 "2"^^ . _:b18780947 "APE1 physically interacts with STAT3 on the VEGF promoter [>>21<<] and enhances IL-6-induced DNA binding activity of STAT3 in HepG2 cells [22]." . _:b506093800 "2"^^ . _:b18780937 . . _:b506093803 "2"^^ . _:b506093802 "2"^^ . _:b18780934 "Functioning as a redox factor, APE1 stimulates the DNA binding activity of transcription factors by reducing cysteine residues in the DNA binding domain of the \u2018target\u2019 transcription factor. [>>6<<] While the organism possesses general reduction-oxidation systems (thioredoxin and glutaredoxin/glutathione), [7], [8] APE1 functions differently as it selectively regulates factors that directly govern critical cellular functions," . . _:b18780959 . . _:b506093633 . _:b506093724 . _:b506093658 . _:b18780932 "Treatment with E3330, a small molecule redox signaling inhibitor that recognizes an alternate, redox active conformation of APE1 [3] markedly inhibits the DNA binding and transcriptional activity of NF-B, AP-1, and HIF-1 [>>4<<], [5]. Functioning as a redox factor, APE1 stimulates the DNA binding activity of transcription factors by reducing cysteine residues in the DNA binding domain of the \u2018target\u2019 transcription factor. [6] While the organism possesses general" . _:b506093809 "2"^^ . _:b18780999 "STAT3, NF-\u03BAB, and HIF-1 signaling contribute to the crosstalk between the tumor and the tumor microenvironment (TME) [14], [>>34<<]\u2013[36]. APE1 also plays an important role in signaling within the TME through the regulation of these transcription factors. Therefore, using inhibitors that target two critical proteins, APE1 and STAT3, we can potentially disable multiple" . _:b18780959 "Drug interaction was evaluated using CalcuSyn software (Biosoft, Ferguson, MO), which is based on the Chou-Talalay method [>>44<<]." . . _:b506093737 . _:b506093808 "2"^^ . _:b18780933 . _:b506093705 . _:b18780991 "STAT family of transcription factors, STAT3 is the first STAT member to be shown to be regulated by APE1. Work by Li et al elegantly demonstrated using chimera STAT1/STAT3 proteins that STAT3, but not STAT1, is under redox control. [>>20<<] ROS can activate STAT signaling and anti-oxidants can inhibit this activation." . _:b506093810 "2"^^ . . . _:b18780956 . _:b18780935 . _:b18780934 . _:b506093759 . _:b18780958 . _:b506093728 . . _:b18780944 "STAT3 signaling has been implicated in pancreatic cancer biology, namely by mediating or regulating cell survival, tumor angiogenesis and metastasis [16]\u2013[>>18<<]. Although STAT3 signaling can be engaged and modulated by different processes, the impact of oxidative stress and its redox status are largely unknown. A recent report demonstrated that STAT3 activity is under redox control and identified" . _:b18780995 . _:b506093726 . . _:b18780948 "APE1 physically interacts with STAT3 on the VEGF promoter [21] and enhances IL-6-induced DNA binding activity of STAT3 in HepG2 cells [>>22<<]. However, it is unknown whether APE1 is involved in the redox control of STAT3 activity, and whether the cellular redox status affects STAT3 signaling in PDAC cells." . _:b18780955 . . _:b506093719 . _:b506093766 . _:b506093769 . . . _:b506093656 . . _:b506093790 . _:b506093660 . _:b506093750 . . . . "PMC0" . . _:b506093762 . _:b506093704 . . . . _:b506093634 . _:b18780996 . . _:b18780962 "All siRNA transfections were performed as previously described [10], [43], [>>45<<], [46]. STAT3 luciferase assays were conducted on day 3 following knockdown [11]." . . _:b18780975 . . . _:b506093701 . _:b506093764 . _:b506093671 . . . . . _:b506093807 . _:b506093659 . _:b18780949 . . . _:b506093643 . . _:b18780986 . _:b18780999 . _:b18780951 "E3330 was synthesized as previously described [>>3<<], [43], and STAT3 selective inhibitors S3I-201 [24] and STATTIC [23] were purchased from Calbiochem." . . . _:b506093770 . _:b18780960 . _:b506093625 . _:b18780928 . . _:b18780942 "STAT3 is a transcription factor that regulates critical cell functions and plays important roles in several cancers [12]\u2013[>>15<<]. STAT3 signaling has been implicated in pancreatic cancer biology, namely by mediating or regulating cell survival, tumor angiogenesis and metastasis [16]\u2013[18]." . _:b506093623 . _:b18780988 . . _:b506093761 . . . _:b18780988 "discussion" . _:b506093740 . . _:b18780972 "Although STAT3 DNA binding is reportedly under redox control [>>20<<], the molecular mechanism mediating this regulation is unknown. Here, we investigated whether APE1 regulates the DNA binding and transcriptional activities of STAT3 in PDAC." . _:b506093650 . _:b506093645 . . _:b506093774 . . . . _:b18780971 . _:b506093629 . _:b18780969 "Lentiviral transcriptional reporter vectors pGreenFire- STAT3 and the control pGreenFire-mCMV, (System Biosciences Inc., Mountainview, CA) were used to transduce Panc-1 cells, as previously reported. [>>4<<] For overexpression experiments, colonies were cotransfected with control pcDNA vector, pcDNA-wtAPE1 or pcDNA-C65A-APE1 and Renilla luciferase vector, pRL-CMV (Promega Corp., Madison, WI), in a 1\u223610 ratio using Lipofectamine2000." . _:b506093628 . . _:b506093631 . . _:b18780965 . _:b506093630 . _:b18780994 . _:b18780963 "STAT3 luciferase assays were conducted on day 3 following knockdown [>>11<<]." . _:b506093625 . _:b18780993 . . _:b506093624 . _:b18780986 "To demonstrate that this effect was not due to a general redox phenomenon, we also combined STAT3 inhibitors with thioredoxin inhibitor, PX-12 [27]\u2013[>>29<<]. Addition of STAT3 inhibitor S3I-201 to PX-12 treatment did not significantly enhance the effects of PX-12 (Table S1; assessed by comparison of ED25, ED50, and ED75)." . _:b506093627 . . . _:b506093626 . _:b506093670 . _:b506093637 . _:b506093651 . _:b506093636 . _:b18780966 . . _:b506093639 . _:b506093638 . _:b506093633 . _:b506093628 . . _:b506093808 . _:b506093632 . _:b506093731 . _:b506093692 . . _:b506093697 . _:b506093635 . . . _:b506093634 . . _:b506093645 . . . _:b506093644 . . _:b18780949 "materials and methods" . . _:b18780938 . _:b506093777 . _:b506093647 . . _:b18780985 . _:b506093687 . _:b506093646 . _:b506093752 . . _:b18780957 . _:b506093641 . _:b506093640 . _:b506093629 . _:b18780977 . . _:b18780943 "STAT3 signaling has been implicated in pancreatic cancer biology, namely by mediating or regulating cell survival, tumor angiogenesis and metastasis [>>16<<]\u2013[18]. Although STAT3 signaling can be engaged and modulated by different processes, the impact of oxidative stress and its redox status are largely unknown. A recent report demonstrated that STAT3 activity is under redox control and" . _:b506093643 . . _:b506093642 . _:b18781000 "STAT3, NF-\u03BAB, and HIF-1 signaling contribute to the crosstalk between the tumor and the tumor microenvironment (TME) [14], [34]\u2013[>>36<<]. APE1 also plays an important role in signaling within the TME through the regulation of these transcription factors. Therefore, using inhibitors that target two critical proteins, APE1 and STAT3, we can potentially disable multiple key" . _:b18780931 . _:b506093653 . _:b18780950 "Panc-1 and PaCa-2 were purchased from and authenticated by ATCC (Manassas, VA). Pa03C, Panc10.05, and Pa02C were obtained from Dr. Anirban Maitra at The Johns Hopkins University. [>>42<<] All cells were maintained at 37\u00B0C in 5% CO2 and grown in DMEM (Invitrogen; Carlsbad, CA) with 10% Serum (Hyclone; Logan, UT)." . _:b18780966 "Piscataway, NJ) and 0.1 pmol 5\u2032HEX-labeled double-stranded oligonucleotide DNA (Midland Certified Reagent Company, Midland, TX) containing the STAT3 direct repeat consensus sequence (5\u2032- GAT CCT TCT GGG AAT TCC TAG ATC-3\u2032) for 15 min. [>>49<<] For the experiment of APE1/STAT3 interaction, purified APE1 protein was reduced with 2 mM DTT for 10 min and diluted to a final concentration of 4 \u00B5g with 0.4 mM DTT in PBS." . _:b506093652 . _:b506093655 . _:b506093725 . _:b506093654 . _:b506093801 . _:b506093649 . _:b506093648 . _:b506093651 . _:b506093650 . . . _:b18780932 . _:b506093661 . . _:b506093683 . _:b18780933 . . _:b506093660 . _:b506093763 . _:b18780934 . _:b506093663 . . _:b18780935 . _:b506093662 . _:b506093795 . _:b18780945 . _:b506093657 . _:b18780928 . _:b506093732 . _:b18780929 . _:b506093656 . _:b18780930 . _:b506093659 . _:b18780970 "With E3330 treatment, cells were treated for 24 h in serum free media and then with IL-6 for 6 h. RLU was normalized to cell viability (MTS assay) as previously described. [>>4<<]." . _:b18781006 . _:b506093756 . _:b18780931 . _:b506093658 . . . _:b18780940 . _:b506093669 . _:b506093765 . . _:b18780952 . _:b506093665 . _:b18780941 . _:b506093668 . . _:b18780942 . _:b506093671 . . _:b18780943 . _:b506093670 . . _:b506093738 . _:b18780936 . _:b18780951 . _:b506093665 . _:b18780930 . _:b506093786 . _:b506093736 . _:b18780967 "Apoptosis was assayed 24 h after inhibitor treatment in the conditions indicated, using Annexin-V/7-AAD [>>4<<], [50] by flow cytometry. Activation of Caspase 3 was assessed using the FITC-conjugated DEVD-FMK inhibitor in permeabilized PDAC cells, by flow cytometry." . _:b18780937 . . _:b506093664 . _:b18780938 . _:b506093667 . _:b506093804 . _:b506093799 . _:b18780939 . _:b506093680 . _:b506093666 . . _:b18780948 . _:b506093677 . _:b506093747 . _:b506093746 . _:b18781005 "Monotherapies are largely ineffective, therefore one approach would be to develop two- or multi-hit approaches [>>38<<] targeting signaling pathways that critically regulate PDAC survival." . _:b18780949 . _:b18780950 . _:b506093676 . . . . _:b18780950 . . _:b506093679 . _:b506093729 . _:b18780951 . _:b506093678 . _:b506093749 . _:b18780944 . _:b506093673 . _:b18780939 "[4], [9], [10] Our previous work established APE1 as a potential molecular target in PDAC, by demonstrating that human adenocarcinoma and peri-pancreatic metastases exhibit increased APE1 expression [>>11<<], and that blockade of APE1 redox activity delays tumor progression in xenograft models of human PDAC, including patient-derived tumor cells [4]." . _:b18780945 . . _:b18780929 "APE1/Ref-1 (hereafter referred to as APE1) is a dual function protein, which in addition to DNA repair activity also exerts redox control of transcription factors, including NF-\u03BAB, p53, AP-1, HIF-1 and others [>>1<<], [2]. Treatment with E3330, a small molecule redox signaling inhibitor that recognizes an alternate, redox active conformation of APE1 [3] markedly inhibits the DNA binding and transcriptional activity of NF-B, AP-1, and HIF-1 [4], [5]." . _:b506093672 . . _:b18781006 "Studies in animal models of PDAC confirm that dual- or multi-targeting approaches can increase anti-tumor responses [>>40<<], [41]. APE1 and STAT3 are upregulated and play important roles in cancer, suggesting that tumor cells may be \u2018addicted\u2019 to their effector functions. In addition to biochemical and molecular data linking APE1 and STAT3 in PDAC cells, our" . _:b18780946 . _:b506093675 . _:b18780947 . _:b506093684 . _:b506093674 . . _:b506093760 . _:b506093623 "9"^^ . _:b18780956 . _:b506093685 . _:b506093809 . _:b18780957 . _:b506093684 . _:b18780983 . . _:b18780958 . . _:b506093687 . . _:b18780959 . _:b506093686 . _:b18780952 . _:b506093681 . _:b18780985 "To demonstrate that this effect was not due to a general redox phenomenon, we also combined STAT3 inhibitors with thioredoxin inhibitor, PX-12 [>>27<<]\u2013[29]. Addition of STAT3 inhibitor S3I-201 to PX-12 treatment did not significantly enhance the effects of PX-12 (Table S1; assessed by comparison of ED25, ED50, and ED75)." . _:b506093798 . _:b18780983 "Cell survival was assessed using the xCELLigence system, which monitors real-time changes in cell adherence, morphology and viability; [4], [>>26<<] cell index (CI) was monitored over 72 h drug treatment." . . _:b506093802 . . _:b18780978 "First we show that treatment with two previously described STAT3 antagonists, STATTIC [23] and S3I-201 [>>24<<] inhibited phosphorylation of STAT3 (Figure 4 A,B) as well as cellular proliferation in PDAC cells (Figure 4C)." . _:b18780953 . _:b506093680 . . _:b506093631 . _:b18780954 . _:b506093636 . _:b506093683 . _:b506093630 "7"^^ . . _:b18780954 . _:b506093631 "6"^^ . _:b18780955 . _:b506093638 . . _:b506093682 . _:b18780964 . _:b506093628 "7"^^ . _:b18780961 "All siRNA transfections were performed as previously described [10], [>>43<<], [45], [46]. STAT3 luciferase assays were conducted on day 3 following knockdown [11]." . _:b506093693 . . _:b18780965 . _:b18780948 . _:b506093629 "7"^^ . _:b506093692 . _:b18780961 . _:b18780994 "An understanding of whether other STAT family members are regulated by APE1 is important due to their diverse roles in cellular function including cytokine and growth factor signaling, differentiation, inflammation, and senescence [>>31<<]\u2013[33], and are part of ongoing studies." . _:b18780966 . _:b506093695 . _:b506093626 "8"^^ . _:b18780953 "E3330 was synthesized as previously described [3], [43], and STAT3 selective inhibitors S3I-201 [>>24<<] and STATTIC [23] were purchased from Calbiochem." . _:b18780956 "The proliferative capacity of PDAC cells was assessed using the xCELLigence system (Roche Applied Science, Indianapolis IN) [>>4<<], [26] as well as MTS assay as previously described [4]. Combination Index Calculations." . _:b18780974 . _:b18780967 . _:b506093627 "7"^^ . _:b506093694 . _:b506093706 . _:b18780960 . _:b18780953 . . _:b506093689 . _:b506093623 . _:b506093624 "9"^^ . _:b506093707 . _:b18780961 . _:b506093688 . _:b506093625 "8"^^ . _:b18780962 . _:b506093691 . _:b506093715 . . _:b506093637 "5"^^ . _:b506093758 . _:b18780963 . _:b506093690 . _:b506093636 "5"^^ . _:b506093755 . _:b506093797 . _:b18780972 . _:b506093701 . _:b506093639 "5"^^ . . _:b18780973 . _:b506093700 . _:b506093638 "5"^^ . _:b18780974 . _:b506093703 . _:b18780975 . _:b506093702 . . _:b506093632 "6"^^ . _:b18780968 . _:b506093697 . _:b506093635 "5"^^ . . _:b506093633 "6"^^ . _:b18780969 . . _:b506093696 . _:b18780960 "All siRNA transfections were performed as previously described [>>10<<], [43], [45], [46]. STAT3 luciferase assays were conducted on day 3 following knockdown [11]." . _:b506093634 "5"^^ . . _:b18780970 . _:b506093699 . _:b506093645 "4"^^ . _:b18780965 "EMSA were performed as previously described [>>48<<] with the following modifications." . _:b506093723 . _:b18780971 . _:b506093698 . _:b506093644 "4"^^ . _:b18780980 . _:b506093709 . . _:b18780943 . _:b506093647 "4"^^ . _:b18780940 "that human adenocarcinoma and peri-pancreatic metastases exhibit increased APE1 expression [11], and that blockade of APE1 redox activity delays tumor progression in xenograft models of human PDAC, including patient-derived tumor cells [>>4<<]." . _:b18780981 . _:b506093708 . _:b18780976 "Doses were based on survival data from previously published data [>>4<<]. * p<0.05 t test, n\u200A=\u200A6 comparing DMSO control to drug-treated samples." . _:b506093646 "4"^^ . . _:b18780982 . . _:b506093711 . _:b506093641 "4"^^ . _:b506093661 . _:b18780983 . _:b506093710 . _:b506093644 . _:b506093640 "5"^^ . . _:b18780976 . . _:b506093705 . _:b506093643 "4"^^ . . _:b18780977 . _:b506093704 . _:b506093771 . _:b506093745 . _:b506093642 "4"^^ . _:b506093672 . _:b18780978 . _:b506093707 . _:b506093653 "4"^^ . _:b506093734 . _:b18780979 . . _:b506093717 . _:b506093706 . _:b506093652 "4"^^ . _:b18781003 "This is especially relevant in brain tumors, where STAT3 is important in the mesenchymal transformation [>>39<<]. As next steps, we will evaluate STAT3 and APE1 as molecular targets in xenograft models of human pancreatic cancer. A STAT3-selective inhibitor is now in a Phase I clinical trial for solid tumors and preclinical work with E3330" . . _:b18780998 "STAT3, NF-\u03BAB, and HIF-1 signaling contribute to the crosstalk between the tumor and the tumor microenvironment (TME) [>>14<<], [34]\u2013[36]." . _:b506093717 . . _:b18780988 . _:b506093655 "4"^^ . _:b18780978 . _:b18780989 . _:b506093716 . _:b506093781 . _:b506093654 "4"^^ . _:b18780989 "Previous studies demonstrated that oxidation of critical cysteines residues in STAT3 protein through peroxide treatment could decrease STAT3 (but not STAT1) DNA binding and transcriptional activity [>>20<<]. Using nuclear extracts from PDAC cells, we systematically characterized the effects of reducing and oxidizing conditions on STAT3 DNA binding. Clearly, STAT3 binds to DNA more effectively when is reduced, and the redox activity of APE1" . _:b18780930 "APE1/Ref-1 (hereafter referred to as APE1) is a dual function protein, which in addition to DNA repair activity also exerts redox control of transcription factors, including NF-\u03BAB, p53, AP-1, HIF-1 and others [1], [>>2<<]. Treatment with E3330, a small molecule redox signaling inhibitor that recognizes an alternate, redox active conformation of APE1 [3] markedly inhibits the DNA binding and transcriptional activity of NF-B, AP-1, and HIF-1 [4], [5]." . _:b18780990 . _:b506093719 . _:b506093649 "4"^^ . . . . _:b18780991 . _:b506093718 . _:b506093648 "4"^^ . _:b506093689 . _:b18780984 . . _:b506093713 . _:b506093651 "4"^^ . _:b18780957 "The proliferative capacity of PDAC cells was assessed using the xCELLigence system (Roche Applied Science, Indianapolis IN) [4], [>>26<<] as well as MTS assay as previously described [4]. Combination Index Calculations." . . . _:b18780985 . _:b506093712 . _:b506093650 "4"^^ . . . _:b18780941 "STAT3 is a transcription factor that regulates critical cell functions and plays important roles in several cancers [>>12<<]\u2013[15]. STAT3 signaling has been implicated in pancreatic cancer biology, namely by mediating or regulating cell survival, tumor angiogenesis and metastasis [16]\u2013[18]." . _:b18780986 . . _:b506093715 . _:b506093661 "4"^^ . _:b18780987 . _:b506093714 . _:b506093660 "4"^^ . _:b506093674 . _:b18780996 . _:b506093741 . _:b506093725 . . . _:b506093663 "4"^^ . _:b18780997 . _:b506093724 . _:b506093662 "4"^^ . _:b18780998 . _:b506093654 . _:b506093727 . _:b506093657 "4"^^ . . _:b18780999 . _:b18780984 . _:b506093628 . _:b506093726 . _:b506093629 . _:b506093656 "4"^^ . _:b506093630 . _:b506093686 . _:b18780992 . _:b506093703 . . _:b506093631 . _:b506093624 . _:b506093721 . _:b506093625 . _:b506093659 "4"^^ . _:b506093626 . _:b506093627 . _:b18780993 . _:b506093720 . _:b18780988 _:b18780989 . _:b506093791 . _:b506093658 "4"^^ . . _:b18780988 _:b18780990 . _:b506093623 . _:b18780994 . _:b18780988 _:b18780991 . _:b506093669 "3"^^ . _:b506093723 . . _:b18780997 "However, APE1 does not regulate only the DNA binding of STAT3, but also controls the activity of NF-\u03BAB, AP-1, and HIF-1\u03B1 [1], [>>4<<]. STAT3, NF-\u03BAB, and HIF-1 signaling contribute to the crosstalk between the tumor and the tumor microenvironment (TME) [14], [34]\u2013[36]. APE1 also plays an important role in signaling within the TME through the regulation of these" . _:b18780995 . _:b506093644 . _:b506093722 . _:b506093668 "3"^^ . . _:b506093645 . _:b506093646 . _:b506093647 . _:b18781004 . _:b18780998 . _:b506093640 . _:b506093733 . _:b506093671 "3"^^ . . _:b506093641 . _:b506093642 . _:b506093643 . _:b18781005 . _:b506093679 . _:b506093636 . _:b506093732 . _:b506093670 "3"^^ . _:b506093637 . _:b506093638 . _:b18780995 "understanding of whether other STAT family members are regulated by APE1 is important due to their diverse roles in cellular function including cytokine and growth factor signaling, differentiation, inflammation, and senescence [31]\u2013[>>33<<], and are part of ongoing studies." . _:b506093639 . _:b18781006 . _:b506093632 . _:b506093735 . _:b506093633 . _:b506093665 "4"^^ . _:b506093666 . _:b506093634 . _:b506093635 . _:b18781007 . . _:b506093660 . _:b506093734 . _:b506093661 . _:b506093664 "4"^^ . _:b506093662 . _:b506093663 . _:b18781000 . _:b506093656 . _:b506093729 . _:b506093667 "3"^^ . _:b506093657 . _:b506093658 . _:b506093659 . _:b18781001 . _:b506093652 . _:b506093728 . _:b506093653 . _:b506093666 "4"^^ . _:b506093654 . _:b506093773 . _:b506093655 . _:b18781002 . _:b506093648 . _:b506093731 . _:b506093677 "3"^^ . _:b506093649 . _:b506093650 . . _:b506093651 . _:b18781003 . . _:b506093676 . _:b506093730 . _:b506093676 "3"^^ . _:b18780988 _:b18780996 . . _:b18780988 _:b18780997 . _:b506093677 . _:b506093678 . _:b18780988 _:b18780998 . _:b506093679 . _:b18780988 _:b18780999 . _:b506093672 . _:b506093741 . _:b506093679 "3"^^ . _:b18780988 _:b18780992 . _:b506093673 . _:b506093757 . _:b18780988 _:b18780993 . _:b506093674 . _:b18780988 _:b18780994 . _:b506093675 . _:b18780964 "Nuclear and cytoplasmic extracts were isolated as in [>>47<<]. Immunoblotting was performed using the following antibodies:" . _:b18780988 _:b18780995 . . _:b506093668 . _:b506093740 . _:b506093678 "3"^^ . _:b18780988 _:b18781004 . _:b506093669 . _:b506093788 . _:b506093663 . _:b18780988 _:b18781005 . _:b506093670 . _:b18780988 _:b18781006 . _:b506093671 . _:b506093678 . _:b18780988 _:b18781007 . _:b506093664 . _:b506093743 . _:b506093673 "3"^^ . . _:b18780988 _:b18781000 . _:b506093665 . _:b506093753 . _:b18780988 _:b18781001 . . _:b506093666 . _:b18780988 _:b18781002 . _:b506093667 . _:b506093720 . _:b18780988 _:b18781003 . _:b506093626 . _:b506093692 . _:b506093742 . _:b506093672 "3"^^ . . _:b506093693 . _:b18780973 "(Mountainview, CA), we generated stably expressing reporter cell lines to assay for STAT3 activity, similar to constructs used in previous studies with NF-\u03BAB, AP-1, and HIF-1 [>>4<<]. Panc-1 cells were transduced with pGF-STAT3-Luc, and single colonies were screened for basal STAT3 and IL-6-stimulated STAT3 activity. PDAC cells expressing STAT3-driven luciferase were transfected to transiently overexpress wtAPE1 or" . _:b506093694 . . _:b506093695 . _:b506093688 . _:b506093779 . _:b506093737 . _:b506093675 "3"^^ . _:b18780971 _:b18780972 . _:b506093689 . . _:b18780971 _:b18780973 . _:b506093690 . _:b506093712 . _:b18780971 _:b18780974 . _:b506093691 . _:b18780971 _:b18780975 . _:b506093684 . _:b506093736 . _:b506093674 "3"^^ . _:b506093685 . _:b18780942 . _:b506093768 . _:b506093686 . . _:b506093687 . _:b506093680 . _:b506093739 . _:b506093685 "3"^^ . _:b506093681 . _:b506093682 . . _:b506093683 . _:b506093708 . _:b506093738 . _:b506093684 "3"^^ . _:b18780971 _:b18780984 . _:b506093709 . _:b506093767 . _:b506093751 . _:b18780971 _:b18780985 . _:b506093710 . _:b506093742 . _:b18780971 _:b18780986 . . _:b18781001 "Therefore, using inhibitors that target two critical proteins, APE1 and STAT3, we can potentially disable multiple key pathways in PDAC cell survival, and to disrupt the integration of signals between tumor and the microenvironment [>>37<<]. This is particularly important in PDAC as the disease frequently presents with metastatic disease." . _:b18780971 _:b18780987 . _:b506093711 . _:b506093662 . _:b18780964 . _:b506093749 . _:b506093687 "3"^^ . _:b506093704 . _:b506093705 . _:b506093706 . . _:b506093707 . _:b18780935 "[6] While the organism possesses general reduction-oxidation systems (thioredoxin and glutaredoxin/glutathione), [>>7<<], [8] APE1 functions differently as it selectively regulates factors that directly govern critical cellular functions, including hypoxia, DNA repair, inflammation, and angiogenesis." . _:b506093700 . _:b506093748 . _:b506093686 "3"^^ . _:b18780971 _:b18780976 . . _:b506093639 . _:b18780980 . _:b18780971 _:b18780977 . _:b506093701 . . _:b18780971 _:b18780978 . _:b506093702 . _:b506093703 . _:b18780971 _:b18780979 . . _:b506093696 . _:b506093751 . _:b506093694 . _:b506093681 "3"^^ . _:b18780971 _:b18780980 . . _:b18780971 _:b18780981 . _:b506093697 . _:b506093698 . _:b18780971 _:b18780982 . _:b506093699 . _:b18780971 _:b18780983 . _:b506093724 . _:b506093792 . _:b506093750 . _:b506093680 "3"^^ . _:b506093725 . _:b506093726 . _:b506093727 . _:b506093720 . _:b18780981 . _:b506093745 . _:b506093683 "3"^^ . _:b506093721 . _:b506093722 . _:b506093630 . _:b506093723 . _:b506093716 . _:b506093794 . _:b506093744 . _:b506093682 "3"^^ . . _:b506093717 . _:b506093718 . _:b506093719 . _:b506093712 . _:b506093747 . _:b506093693 "3"^^ . _:b506093713 . _:b506093711 . _:b506093714 . _:b506093715 . _:b506093740 . _:b506093746 . _:b506093692 "3"^^ . _:b18780949 _:b18780960 . _:b506093741 . _:b18780949 _:b18780961 . _:b506093742 . _:b18780949 _:b18780962 . _:b506093743 . _:b18780982 "Cell survival was assessed using the xCELLigence system, which monitors real-time changes in cell adherence, morphology and viability; [>>4<<], [26] cell index (CI) was monitored over 72 h drug treatment." . _:b18780949 _:b18780963 . _:b506093736 . _:b506093757 . _:b506093695 "3"^^ . _:b18780949 _:b18780964 . _:b506093737 . _:b18780949 _:b18780965 . _:b506093738 . _:b506093657 . _:b18780949 _:b18780966 . _:b506093647 . _:b18780949 _:b18780967 . _:b506093739 . _:b506093732 . _:b506093772 . _:b506093756 . _:b506093694 "3"^^ . _:b18780949 _:b18780968 . _:b506093733 . _:b18780949 _:b18780969 . _:b506093734 . _:b18780936 "[6] While the organism possesses general reduction-oxidation systems (thioredoxin and glutaredoxin/glutathione), [7], [>>8<<] APE1 functions differently as it selectively regulates factors that directly govern critical cellular functions, including hypoxia, DNA repair, inflammation, and angiogenesis." . _:b18780949 _:b18780970 . _:b506093735 . . _:b506093728 . _:b506093759 . _:b506093689 "3"^^ . _:b506093729 . _:b18781001 . _:b506093730 . _:b506093731 . _:b506093756 . _:b18780968 . _:b506093758 . _:b506093688 "3"^^ . _:b506093757 . _:b506093682 . _:b506093758 . _:b506093759 . . _:b506093752 . _:b506093753 . _:b506093691 "3"^^ . _:b506093753 . _:b506093754 . _:b18780949 _:b18780950 . _:b506093755 . _:b506093806 . _:b18780949 _:b18780951 . _:b506093748 . _:b506093752 . _:b506093690 "3"^^ . _:b18780949 _:b18780952 . . _:b18780974 "address the role of the redox function of APE1 on STAT3 transcriptional activity, we performed experiments using the small molecule E3330, which selectively inhibits APE1 redox activity without affecting its endonuclease function. [>>3<<], [5] E3330 markedly inhibits STAT3 activity both in stable lines (Figure 3A; Figures S2A,B) as well as in transient luciferase assays (Figure S2C)." . _:b18780949 _:b18780953 . _:b506093749 . _:b506093750 . _:b18780949 _:b18780954 . _:b506093751 . _:b506093701 "2"^^ . _:b18780949 _:b18780955 . _:b506093744 . _:b506093755 . _:b506093708 . _:b18780949 _:b18780956 . _:b506093745 . _:b18780949 _:b18780957 . _:b506093746 . _:b18780949 _:b18780958 . . _:b506093700 "2"^^ . _:b18780949 _:b18780959 . _:b506093747 . _:b506093772 . _:b506093754 . _:b506093773 . _:b506093774 . _:b506093775 . _:b506093703 "2"^^ . _:b506093768 . _:b506093765 . _:b506093769 . _:b506093770 . _:b506093771 . _:b506093702 "2"^^ . . _:b506093764 . _:b506093764 . _:b506093765 . _:b18780968 "Apoptosis was assayed 24 h after inhibitor treatment in the conditions indicated, using Annexin-V/7-AAD [4], [>>50<<] by flow cytometry. Activation of Caspase 3 was assessed using the FITC-conjugated DEVD-FMK inhibitor in permeabilized PDAC cells, by flow cytometry." . . _:b18780928 "introduction" . _:b506093766 . _:b506093767 . _:b506093760 . _:b506093767 . _:b506093697 "3"^^ . _:b506093761 . _:b506093762 . _:b506093763 . _:b506093788 . _:b18781002 "Our approach is consistent with the idea of synthetic lethality in which the pairing of two hits is sufficient to more effectively trigger cancer cell death, markedly improving the efficacy of single-target agents [>>38<<]. Both of these proteins are upregulated in cancer and contribute to the resistance of the disease, leaving cancer cells \u201Caddicted\u201D to their effector functions. Dual targeting of STAT3 and APE1 is not only efficacious in PDAC; we also" . _:b506093766 . _:b506093696 "3"^^ . _:b506093789 . _:b506093790 . _:b506093624 . _:b506093791 . _:b506093699 "2"^^ . _:b506093784 . _:b506093761 . _:b506093785 . _:b506093786 . . _:b506093787 . _:b506093780 . _:b506093760 . _:b506093698 "3"^^ . _:b506093781 . _:b506093782 . _:b506093783 . _:b506093709 "2"^^ . _:b506093776 . _:b506093763 . _:b506093777 . _:b506093778 . . _:b506093708 "2"^^ . _:b506093779 . _:b506093804 . _:b506093762 . _:b506093805 . _:b506093806 . . _:b506093711 "2"^^ . _:b506093807 . _:b506093800 . _:b506093773 . _:b506093743 . _:b506093801 . _:b506093802 . _:b506093803 . _:b506093710 "2"^^ . _:b506093796 . _:b18780975 "address the role of the redox function of APE1 on STAT3 transcriptional activity, we performed experiments using the small molecule E3330, which selectively inhibits APE1 redox activity without affecting its endonuclease function. [3], [>>5<<] E3330 markedly inhibits STAT3 activity both in stable lines (Figure 3A; Figures S2A,B) as well as in transient luciferase assays (Figure S2C)." . _:b506093772 . . _:b506093635 . _:b506093797 . _:b506093798 . _:b506093799 . _:b506093705 "2"^^ . _:b506093792 . _:b506093775 . _:b506093793 . _:b18780958 "The proliferative capacity of PDAC cells was assessed using the xCELLigence system (Roche Applied Science, Indianapolis IN) [4], [26] as well as MTS assay as previously described [>>4<<]. Combination Index Calculations. PDAC cells were seeded into 96-well plates as described previously, and ED50s of E3330, S3I-201, or STATTIC on growth were determined using the MTS assay." . _:b506093714 . _:b506093794 . _:b18780946 . _:b506093795 . _:b506093704 "2"^^ . _:b18780928 _:b18780932 . _:b18780981 "[4], [16], [>>25<<] We then evaluated whether the combined blockade of STAT3 and APE1 redox activity synergize to more effectively inhibit human PDAC cells." . _:b506093774 . _:b18780928 _:b18780933 . _:b18780928 _:b18780934 . . _:b506093707 "2"^^ . _:b18780928 _:b18780935 . _:b506093769 . _:b18780928 _:b18780929 . _:b18780928 _:b18780930 . _:b18780928 _:b18780931 . _:b506093706 "2"^^ . _:b18780928 _:b18780940 . _:b506093768 . _:b18780928 _:b18780941 . . _:b18780928 _:b18780942 . _:b18780928 _:b18780943 . _:b506093717 "2"^^ . _:b506093808 . _:b506093771 . _:b18780928 _:b18780936 . _:b506093809 . _:b18780928 _:b18780937 . _:b506093810 . _:b506093778 . _:b18780928 _:b18780938 . _:b18780928 _:b18780939 . _:b506093744 . _:b506093716 "2"^^ . _:b18780928 _:b18780948 . _:b506093770 . _:b18780954 "E3330 was synthesized as previously described [3], [43], and STAT3 selective inhibitors S3I-201 [24] and STATTIC [>>23<<] were purchased from Calbiochem." . . _:b506093719 "2"^^ . _:b18780928 _:b18780944 . _:b18780937 "(thioredoxin and glutaredoxin/glutathione), [7], [8] APE1 functions differently as it selectively regulates factors that directly govern critical cellular functions, including hypoxia, DNA repair, inflammation, and angiogenesis. [>>4<<], [9], [10] Our previous work established APE1 as a potential molecular target in PDAC, by demonstrating that human adenocarcinoma and peri-pancreatic metastases exhibit increased APE1 expression [11], and that blockade of APE1 redox" . _:b506093781 . _:b18780928 _:b18780945 . . _:b18780928 _:b18780946 . _:b18780945 "A recent report demonstrated that STAT3 activity is under redox control and identified the critical oxidation-sensitive cysteines in the STAT3 DNA binding domain [>>19<<], [20]. However, the modifier of STAT3 which converts it from an oxidized into a reduced form has not been identified. APE1 physically interacts with STAT3 on the VEGF promoter [21] and enhances IL-6-induced DNA binding activity of STAT3" . _:b506093776 . _:b18780928 _:b18780947 . _:b506093718 "2"^^ . . _:b506093780 . _:b18780946 "A recent report demonstrated that STAT3 activity is under redox control and identified the critical oxidation-sensitive cysteines in the STAT3 DNA binding domain [19], [>>20<<]. However, the modifier of STAT3 which converts it from an oxidized into a reduced form has not been identified. APE1 physically interacts with STAT3 on the VEGF promoter [21] and enhances IL-6-induced DNA binding activity of STAT3 in" . . _:b506093713 "2"^^ . _:b506093783 . _:b18780944 . _:b506093712 "2"^^ . _:b506093782 . _:b506093715 "2"^^ . _:b506093777 . _:b506093805 . _:b506093664 . . . _:b506093787 . _:b506093714 "2"^^ . _:b506093776 . _:b506093722 . . _:b506093725 "2"^^ . . _:b506093652 . _:b506093779 . _:b506093667 . . _:b506093724 "2"^^ . _:b506093778 . _:b18780936 . _:b506093700 . _:b506093727 "2"^^ . _:b506093713 . _:b506093789 . _:b506093803 . . _:b18780980 "In addition to regulating the transcriptional activity of STAT3, APE1 also exerts redox control of other transcription factors, which have been implicated in pancreatic cancer (such as HIF-1\u03B1 and NF-\u03BAB). [4], [>>16<<], [25] We then evaluated whether the combined blockade of STAT3 and APE1 redox activity synergize to more effectively inhibit human PDAC cells." . _:b506093726 "2"^^ . _:b506093675 . _:b506093788 . _:b506093696 . _:b506093648 . _:b506093739 . _:b506093721 "2"^^ . _:b506093754 . _:b18780938 "[4], [9], [>>10<<] Our previous work established APE1 as a potential molecular target in PDAC, by demonstrating that human adenocarcinoma and peri-pancreatic metastases exhibit increased APE1 expression [11], and that blockade of APE1 redox activity delays" . _:b506093791 . . _:b506093676 . . . _:b506093720 "2"^^ . _:b506093790 . _:b506093668 . _:b506093723 "2"^^ . _:b506093785 . _:b506093685 . _:b506093722 "2"^^ . _:b506093637 . _:b506093784 . _:b506093733 "2"^^ . _:b506093787 . _:b506093732 "2"^^ . _:b18780997 . _:b506093786 . _:b18780992 "to be regulated by APE1. Work by Li et al elegantly demonstrated using chimera STAT1/STAT3 proteins that STAT3, but not STAT1, is under redox control. [20] ROS can activate STAT signaling and anti-oxidants can inhibit this activation. [>>30<<] We have previously shown that knocking down of APE1 in PDAC cells does not increase ROS levels [11], and therefore its effects on STAT3 DNA-binding activity here reported cannot be attributed to changes in ROS activity." . _:b506093735 "2"^^ . . _:b18781007 . _:b506093797 . _:b506093734 "2"^^ . _:b506093677 . _:b506093796 . . . _:b506093729 "2"^^ . _:b506093799 . . _:b506093728 "2"^^ . _:b506093798 . _:b506093731 "2"^^ . _:b506093793 . . _:b506093730 "2"^^ . _:b506093792 . _:b18780991 . _:b18780996 "However, APE1 does not regulate only the DNA binding of STAT3, but also controls the activity of NF-\u03BAB, AP-1, and HIF-1\u03B1 [>>1<<], [4]. STAT3, NF-\u03BAB, and HIF-1 signaling contribute to the crosstalk between the tumor and the tumor microenvironment (TME) [14], [34]\u2013[36]. APE1 also plays an important role in signaling within the TME through the regulation of these" . _:b506093741 "2"^^ . _:b18781004 . _:b506093795 . _:b18780993 "[30] We have previously shown that knocking down of APE1 in PDAC cells does not increase ROS levels [>>11<<], and therefore its effects on STAT3 DNA-binding activity here reported cannot be attributed to changes in ROS activity." . _:b18780990 "its phosphorylation or nuclear translocation. Taken together, these data support the hypothesis that APE1 directly controls the DNA binding activity of STAT3. With the high levels of APE1 expression in PDAC tumors (Figure 1A, Ref. [>>11<<]), stimulation of STAT3 signaling through APE1\u2019s redox activity may contribute to the threshold of STAT3 activity in the tumor leading to a more aggressive phenotype." . _:b506093740 "2"^^ . . _:b506093794 . _:b18780989 . . _:b506093743 "2"^^ . _:b506093805 . . _:b18780941 . _:b506093742 "2"^^ . _:b506093632 . _:b506093804 . . _:b506093737 "2"^^ . . _:b506093807 . _:b506093748 . . _:b18780990 . _:b506093736 "2"^^ . _:b506093806 . _:b506093739 "2"^^ . _:b506093801 . _:b506093738 "2"^^ . _:b506093800 . . _:b18780952 "E3330 was synthesized as previously described [3], [>>43<<], and STAT3 selective inhibitors S3I-201 [24] and STATTIC [23] were purchased from Calbiochem." . _:b506093749 "2"^^ . _:b506093803 . "10.1371%2Fjournal.pone.0047462" . _:b506093693 . _:b506093748 "2"^^ . _:b506093802 . . _:b506093751 "2"^^ . _:b18781000 . . _:b506093750 "2"^^ . . _:b18780987 "synergisms, the Chou-Talalay method was used with increasing dose combinations of one STAT3 antagonist plus the APE1 inhibitor E3330. The ED50 for the three compounds was determined as shown above, using MTS assays (Figure 4 and Ref. [>>4<<]). Panc-1 or PaCa-2 cells were treated with two-drug combinations at fixed ratio of doses corresponding to 0.125, 0.25, 0.5, 0.75, 1, 1.125, and 1.25-fold of their individual ED50 values." . . _:b506093784 . . _:b18780933 "Treatment with E3330, a small molecule redox signaling inhibitor that recognizes an alternate, redox active conformation of APE1 [3] markedly inhibits the DNA binding and transcriptional activity of NF-B, AP-1, and HIF-1 [4], [>>5<<]. Functioning as a redox factor, APE1 stimulates the DNA binding activity of transcription factors by reducing cysteine residues in the DNA binding domain of the \u2018target\u2019 transcription factor. [6] While the organism possesses general" . _:b506093745 "2"^^ . . _:b506093744 "2"^^ . _:b506093780 . _:b506093655 . _:b506093747 "2"^^ . _:b506093809 . . _:b506093746 "2"^^ . . _:b506093808 . _:b18781004 "A STAT3-selective inhibitor is now in a Phase I clinical trial for solid tumors and preclinical work with E3330 demonstrates its efficacy against patient-derived PDAC xenografts [>>4<<]. These studies will set the framework for a future clinical study using this dual-targeting STAT3\u2013 APE1 strategy." . . . . _:b506093757 "2"^^ . _:b506093646 . _:b506093721 . _:b506093756 "2"^^ . _:b506093702 . _:b506093810 . . _:b506093727 . _:b506093759 "2"^^ . _:b506093709 . _:b18780972 . _:b506093758 "2"^^ .