Regulation of HIF1α under Hypoxia by APE1/Ref-1 Impacts CA9 Expression: Dual-_targeting in Patient-Derived 3D Pancreatic Cancer Models

Cell Culture

Cells were maintained in culture as previously described (20–22). Patient-derived tumor cells, Pa03C, Pa02C, and Panc10.05 and CAF19 cells were a kind gift from Dr. Anirban Maitra (The Johns Hopkins University) (34). Upon receipt of the cells in 2011 as well as in June of 2015, we used STR analysis (CellCheck with IDEXX BioResearch) to confirm that we indeed received the aforementioned cells from Dr. Maitra and that they were mycoplasma-free. MIA-PaCa2 cells were purchased from and authenticated by ATCC (Manassas, VA). Cancer-associated fibroblasts, UH1303-02 were isolated using the outgrowth method from patient tumor tissue as previously described (35). All patient-derived lines were passaged only 10 times before new stocks frozen prior to authentication were resuscitated. Hypoxia exposure was achieved using a Ruskinn Invivo₂ 200 hypoxia work station. CMV-EGFP-WT APE1/Ref-1 lentiviral construct was used to overexpress APE1/Ref-1 as previously described (36). To detect the cells for imaging, a CMV-EGFP lentiviral construct was used as previously described (36). Additionally, 150 pfu/cell of the pCL7TdTOMwo lentiviral vector was incubated with Pa03C and Panc10.05 cells for 48 hours to make cells stably express TdTomato.

Western Blot Analysis

Western blots were performed as previously described (20–23) with antibodies for APE1/Ref-1 (Novus Biologicals; Littleton, CO), HIF1α (GeneTex; Irvine, CA), STAT1, STAT3, (Cell Signaling; Danvers, MA), NFκB (abcam; Cambridge, MA), CA9 (Santa Cruz; Dallas, Texas), Vinculin (Sigma; St. Louis, MO), and Actin (NeoMarkers; Fremont, CA).

Co-immunoprecipitation

Samples were co-immunoprecipitated using the Pierce Co-IP kit (Thermo Scientific) with modifications as previously described (22).

Transfection

PDAC and CAF cells were transfected with APE1/Ref -1 siRNA as previously described (20, 22, 23). siRNAs used were: #1 or scrambled control (previously reported) and two LifeTech validated siRNAs (#2: s1445 and #4: s1447) (22). APE1/Ref-1 siRNA #1 was used as the standard siRNA unless otherwise specified.

Transient Luciferase Reporter Assays

MIA-PaCa-2 cells were co-transfected with constructs containing luciferase driven by HIF1α and a Renilla luciferase reporter vector as previously described (22) using X-tremeGENE 9 DNA transfection reagent (Roche, Indianapolis, IN) along with siRNA as described above. Firefly and Renilla luciferase activities were assayed by using the Dual Luciferase Reporter Assay System (Promega Corp.) as before (20, 22).

qRT-PCR Reactions

qRT-PCR was used to measure the mRNA expression levels of CA9 as previously described (21). Cells were treated with APE1/Ref-1 siRNA or increasing amounts of APX3330 in the presence or absence of hypoxia (1% and 0.2 % O₂) for 24 h, then total RNA was extracted from cells using the QiagenRNeasy Mini kit (Valencia, CA) (37). First-strand cDNA synthesis and quantitative PCR were performed as previously described (21). The relative quantitative mRNA level was determined using the comparative C_t method using 18S rRNA, RPLP0, and B2M as reference genes (37, 38). The primers for CA9, 18S, RPLP0, and B2M are commercially available (Applied Biosystems).

Inhibitors

Compounds were prepared and used as previously described: APX3330 (32, 33) and SLC-0111 (30, 31). The concentrations of APX3330 used are within clinically tolerated levels established previously by Eisai pharmaceutical company through a prior development program that evaluated the toxicology and phase I/II safety and clinical profile in non-cancer patients. Additionally, the SLC-0111 analog FC13-555A was synthesized as described in Supplemental Methods. The structure of each inhibitor can be seen in Supplemental Figure 1.

Cell Proliferation

PDAC cell proliferation in monolayer was measured using the Alamar Blue assay as previously described (22). Cells treated with APX3330 and SLC-0111 were exposed to hypoxia for six days followed by addition of Alamar Blue reagent (Invitrogen) and subsequent fluorescence analysis. Fold change refers to the fluorescence reading for cells treated with indicated inhibitors compared to cells growing in normal media.

pH Assay

Intracellular pH was evaluated using the pHrodo Red AM Intracellular pH Indicator (LifeTech). PDAC cells treated with APX3330 and SLC-0111 were exposed to hypoxia for 48 hours followed by analysis with pHrodo Red AM dye. Intracellular pH Calibration Buffers (LifeTech) were used to create a standard curve of fluorescence intensity for determination of pH values. Results were normalized to MTS analysis to account for changes in proliferation as before (20–22). Fluorescent images were acquired using a confocal/two-photon Olympus Fluoview FV-1000 MPE system (Olympus American) at the Indiana Center for Biological Microscopy facility (Indianapolis, IN).

Statistical Analysis

qPCR data points for scrambled, siAPE1/Ref-1, and hypoxia treatments were analyzed using the 2^−ΔΔC_T method and analysis of covariance (ANCOVA) models as previously described (22, 39). Data points in tests with multiple treatment groups were analyzed using post-hoc Multiple Comparisons Tests (Tukey, Dunnett, or Sidak as appropriate). For evaluation of data curves using multiple drugs, an extra-sum-of-squares F test was used to compare the goodness-of-fit of a nonlinear regression curve shared between groups with that of separate curves for each group. Differences between the treatment groups and control group were considered significant if p<0.05 following Bonferroni corrections as appropriate. Statistical analyses were performed using SAS (Version 9.3, Copyright ©2010 SAS Institute Inc. Cary, NC) and Prism (Version 6.0f, Copyright ©2014 GraphPad Software Inc. La Jolla, CA).

HIF-1 −/− MEF Generation

HIF-1-floxed mouse embryonic fibroblast (MEF) cells were generated as previously reported (40) and transduced with Ad-CMV-Cre (Cre adenovirus) or Ad-GFP (control) vector (Vector BioLabs; Malvern, PA) for 24 hours using 5 ng/mL polybrene to produce HIF-1-deficient cells (40). PCR was used to verify the deletion of HIF (Supplemental Figure 2).

3D Co-Culture

Ultra low attachment 96-well plates (Corning Inc., Life Sciences) were used to generate 3-dimensional tumor spheroids in the presence and absence of CAFs (75 μL/well) as described previously (41, 42). Cells were stably transduced with EGFP (green) or TdTomato (red) as indicated to preserve the genetic characteristics of the low passage patient cells (34, 42). Cells were re-suspended in colorless DMEM growth media containing 3% Reduced Growth Factor Matrigel (RGF, BD Biosciences) and 5% FBS. Following plating, cells were treated on Days 4 and 8 with media containing 5% serum, 3% RGF Matrigel, and inhibitors as indicated. On Day 12, spheroids were analyzed using Thermo ArrayScan high-content imaging system (43). Images of 3D structures were captured by ArrayScan using a 2.5× objective for TdTomato and EGFP; then 2D projections were processed to quantify differences in total intensity and total area of both CAFs and tumor.

APE1/Ref-1 interactions with HIF1α and STAT3 are stimulated by hypoxia

We previously published data demonstrating decreased STAT3, HIF1α, and NFκB activity following knockdown of APE1/Ref-1 and/or inhibition of APE1/Ref-1 redox signaling with the selective inhibitor APX3330 (also called E3330) (20, 21). Similarly, we found that inhibition of APE1/Ref-1 led to a decrease in a major HIF-1 _target within cells, Carbonic Anhydrase IX (CA9) (21, 28). To further dissect the role of APE1/Ref-1 in hypoxia signaling and more clearly determine whether hypoxia stimulates interactions between APE1/Ref-1 and its redox _targets, endogenous APE1/Ref-1 was immunoprecipitated from lysates of two low passage PDAC cell lines (Panc10.05 and Pa03C) under normoxic and hypoxic (0.2% O₂) conditions. These cell lines are representative of ductal adenocarcinoma, were previously sequenced (34), and have the common G12D mutation in KRAS as well as missense mutations in p53. Panc10.05 was derived from a primary PDAC tumor, is wt for p16(INK4A), but has a SMAD4 deletion; however Pa03C was isolated from a metastatic lesion in the liver and is wt for both p16(INK4A) and SMAD4. IPs were probed for HIF1α, STAT3, and NFκB. HIF1α and STAT3, but not NFκB were detected in the pull-down fractions under hypoxic conditions, but these interactions were not detected under normoxic conditions (Figure 1A–B). Controls of TNFα (NFκB) and IL-6 (STAT3) were performed to show that interactions between APE1/Ref-1 and the transcription factors it regulates do indeed occur under normoxic conditions with appropriate stimulation (Supplemental Figure 3). Interactions of APE1/Ref-1 with HIF1α and STAT3 were obvious under hypoxic conditions.

With overexpression of APE1/Ref-1, the interaction with HIF1α and STAT3 remained intact while NFκB was still not detected in IPs from cells overexpressing APE1/Ref-1 following exposure to hypoxia indicating that the amount of APE1/Ref-1 was not limiting in the above panels (Figure 1C–D). To demonstrate that APE1/Ref-1’s interactions with the transcription factors are specific to signaling in hypoxia, we probed for another STAT family member, STAT1. IPs from 10.05 cells were probed for STAT1, which was not detected regardless of the levels of APE1/Ref-1 or oxygen conditions (Figure 1C). Due to the complexity of the disease and the signaling between various cell types in the pancreatic tumor microenvironment, we investigated APE1/Ref-1 interactions with HIF1α, STAT3, and NFκB in CAFs. These CAFs are non-tumorigenic and although they have activated signaling pathways due to their association with the tumor, they are non-transformed. The results were identical to the result with PDAC cells: APE1/Ref-1 interacts with HIF1α and STAT3 under hypoxia, but not NFκB (Supplemental Figure 4). In light of CA9 inhibitors beginning clinical trials and our previous data demonstrating transcriptional regulation of CA9 following APE1/Ref-1 blockade (21), we focused these studies on HIF1α signaling and the regulation of the downstream molecule CA9 through APE1/Ref-1.

APE1/Ref-1 protein expression contributes to hypoxia-induced HIF1α-mediated transcription

To show that the interactions between APE1/Ref-1 and HIF1α are functionally important, we evaluated the contribution of APE1/Ref-1 to HIF-1 transcriptional activity by co-transfecting MIA PaCa-2 cells with HIF1α-driven firefly luciferase or pLuc-MCS (vector control) alongside APE1/Ref-1 siRNA or scrambled control and exposing cells to hypoxia for 24 hrs. APE1/Ref-1 knock-down resulted in a significant reduction (~47%) in hypoxia-induced HIF1α activity (Figure 2A–B).

Effects of APE1/Ref-1 on HIF transcriptional activity were further evaluated by examining hypoxia-mediated transcription of HIF-1 _target, CA9. We compared CA9 mRNA levels in two PDAC cell lines and one pancreatic CAF cell line following APE1/Ref-1 knock-down and exposure to hypoxia. Bar graph represents the fold change of mRNA expression level of CA9. Hypoxia-induced CA9 mRNA levels were attenuated by APE1/Ref-1 knock-down in all cell lines at both levels of hypoxia (Figure 2C–E). Variability in the amount of induction in different cell lines may be partially attributable to the extremely low baseline CA9 expression under normoxic conditions. APE1/Ref-1 knock-down similarly attenuated CA9 mRNA levels under hypoxic conditions in two additional primary PDAC cell lines (Supplemental Figure 5). These results were validated in MIA-PaCa-2 cells exposed to hypoxia using two additional APE1/Ref-1-_targeting siRNAs, and similar results were obtained (Figure 2F–G). To verify the reduction in CA9 also occurred at the protein level, hypoxia-induced CA9 protein levels were evaluated via western blot following APE1/Ref-1 knock-down in PDAC cells and pancreatic CAF cells. APE1/Ref-1 knock-down resulted in a ~70% reduction in hypoxia-induced CA9 protein levels (Figure 2H–I).

Hypoxia-induced CA9 transcription is HIF-1-dependent

To confirm that the effects of APE1/Ref-1 and hypoxia on CA9 transcription were mediated by HIF-1 activity, we evaluated hypoxia-induced CA9 mRNA levels in HIF-1-deficient (−/−) MEFs following APE1/Ref-1 knock-down. As expected, in HIF-1 proficient MEFs, CA9 is induced 30-fold compared to normal oxygen controls. In HIF-1 −/− MEFs CA9 mRNA levels were not induced by exposure to hypoxia (Figure 3A), or affected by APE1/Ref-1 knock-down (Figure 3B), indicating that CA9 transcription is HIF-1-dependent, regardless of APE1/Ref-1 expression or oxygen levels. HIF-1 depletion in these cells was confirmed by PCR (Supplemental Figure 2).

Inhibition of APE1/Ref-1 redox signaling affects CA9 transcription

As a multi-functional protein, APE1/Ref-1 is also involved in base excision repair (BER) of DNA lesions, RNA quality control, and reduction-oxidation (redox) regulation. Knock-down of APE1/Ref-1 affects all of these functions. We therefore examined whether the redox function is responsible for the APE1/Ref-1-mediated regulation of hypoxia signaling pathways using an APE1/Ref-1 specific redox inhibitor that does not affect other APE1/Ref-1 functions (44, 45) and is slated for clinical trial in the summer of 2016. We previously showed that APX3330 decreases CA9 mRNA levels in Panc-1 and MIA-PaCa2 cells exposed to hypoxia (21). Here we expand these results to primary cells and CAF cells, as well as 3D co-cultures. Following treatment with APX3330 and exposure to hypoxia, CA9 mRNA levels in Pa03C cells and in pancreatic CAF cells were attenuated in a dose-dependent manner (Figure 3C–D). Additionally, CA9 protein expression was measured in a 3D co-culture model following inhibition of APE1/Ref-1 with APX3330. While CA9 was not detected under normoxic conditions in the patient-derived Pa03C cells in monolayer, when grown as spheroids, these cells now express CA9. Tumor spheroids grown in the presence of CAFs more strongly upregulated CA9 expression ~3-fold, likely due to larger spheroids containing larger regions of hypoxia, as well as the more complex signaling present with the stromal elements. Inhibition of APE1/Ref-1 redox signaling with APX3330 led to decreased CA9 expression in 3D tumor cultures in a dose-dependent manner, both in the presence and absence of CAF cells (Figure 3E). These data support the use of the 3D co-culture system for preclinical studies validating novel _targets like CA9 and APE1/Ref-1 in PDAC.

Importantly, we evaluated CA9 and APE1/Ref-1 protein expression following exposure to hypoxia (0.2% oxygen) in three PDAC cell lines and found that, while CA9 levels increased over time, APE1/Ref-1 levels did not change significantly (Supplemental Figure 6), indicating that hypoxia-induced CA9 expression is not secondary to APE1/Ref-1 upregulation.

Dual-_targeting of CA9 and APE1/Ref-1 acidifies PDAC cells and inhibits cell viability under hypoxia

CA9 regulates intracellular pH under hypoxic conditions, and APE1/Ref-1 redox activity contributes to hypoxia-induced CA9 expression. We analyzed intracellular pH in hypoxia-exposed PDAC cells following treatment with CA9 inhibitor, SLC-0111 and the APE1/Ref-1 redox inhibitor, APX3330 using the pHrodo Red AM fluorescent pH indicator as a functional endpoint for carbonic anhydrase activity under hypoxic conditions. The analysis of intracellular pH was performed at an early timepoint (48 hr exposure to inhibitors and hypoxia) to avoid drastic changes in cell survival. Still, some viability changes were observed (Supplemental Figure 7), so these changes were taken into account in the analysis and normalization of the intracellular pH data. Dual-treatment with SLC-0111 and APX3330 results in a greater decrease in intracellular pH than treatment with either inhibitor alone (Figure 4A–B).

Inhibition of APE1/Ref-1 redox activity results in a dose-dependent decrease in PDAC cell viability following treatment of cells with APX3330 and hypoxia. Remarkably, the effect of APE1/Ref-1 inhibition on cell viability is greatly enhanced by treating with the CA9 inhibitor, SLC-0111 in addition to APX3330 treatment under hypoxia (Figure 4C). In support of these results, new CA9 inhibitors are being developed and the combination of APX3330 with SLC-0111 analog, FC13-555A is also significantly effective at killing PDAC cells in monolayer (Figure 4D), supporting the hypothesis that blockade of hypoxia signaling proteins will be deleterious to PDAC cells.

Dual-_targeting of CA9 and APE1/Ref-1 inhibits PDAC tumor growth in a 3D co-culture model

In order to more accurately mimic the tumor microenvironment, we utilized a three-dimensional co-culture model of PDAC that included the low passage patient derived tumor cells as well as cancer-associated fibroblasts. As demonstrated above, the levels of CA9 were greater in these tumor spheroids when grown with CAF cells, and CA9 expression was attenuated by treatment with APX3330 (Figure 3E). Inhibition of CA9 with SLC-0111 was more potent in the 3D model with dramatic effects on tumor cell killing observed at lower doses than in monolayer, as measured by reductions in area of patient-derived cells (Figure 5A–B). Cell killing was more dramatic in the tumor cells than in the CAFs, especially when CAF19 cells were in co-culture with Pa03C cells. Although these CAFs have aberrantly activated signaling pathways, they are non-tumorigenic and suggest that tumor cells are more greatly affected by the dual _targeting approach as compared to normal cells. Similar trends were seen when measuring fluorescence intensity (data not shown). Importantly, inhibition of CA9 can effectively kill tumor cells even when in the protective environment of the CAFs.

To determine if blockade of STAT3 and HIF-mediated transcription alongside the inhibition of CA9 activity would potentiate PDAC cell death, we combined APX3330 and SLC-0111 in the 3D co-culture model. We can assess the effects of dual _targeting on both the tumor alone and the tumor and CAFs in co-culture due to the different fluorescent labels in each cell type. As seen in hypoxia-exposed 2D cultures, addition of CA9 inhibition to APE1/Ref-1 redox inhibition resulted in dramatic potentiation of the cell killing in the tumor spheroids. Spheroids composed of patient-derived PDAC cells (Pa03C or Panc10.05 – labeled red) and CAF cells (labeled green) were treated with APX3330 and SLC-0111 (Figure 5E–F), and the graphical representation i.e. area of red and green fluorescence were evaluated separately as markers for each cell type and is shown in Figure 5C–D. Dramatic enhancement of the APX3330-induced blockade of spheroid growth was observed with the addition of CA9 inhibition. The observed decrease in tumor cell area with APX3330 treatment was significantly different in the presence of SLC-0111, validating the effects seen in hypoxia-exposed 2D cultures. Similar trends were seen when measuring red and green fluorescence intensity (data not shown).