The Multikinase Inhibitor AD80 Induces Mitotic Catastrophe and Autophagy in Pancreatic Cancer Cells

Simple Summary Pancreatic cancer is one of the most lethal human neoplasms, and its therapeutic repertoire remains limited. Advances in understanding the molecular complexity involved in the biology of the disease have paved the way for new therapeutic opportunities. AD80 is a multikinase inhibitor that inhibits S6K as well as RET, RAF, and SRC and displays antineoplastic effects in hematological and solid tumors. In the present study, we report the potential of AD80 as an antineoplastic agent for pancreatic cancer and the cellular and molecular changes induced by the drug. Abstract Significant advances in understanding the molecular complexity of the development and progression of pancreatic cancer have been made, but this disease is still considered one of the most lethal human cancers and needs new therapeutic options. In the present study, the antineoplastic effects of AD80, a multikinase inhibitor, were investigated in models of pancreatic cancer. AD80 reduced cell viability and clonogenicity and induced polyploidy in pancreatic cancer cells. At the molecular level, AD80 reduced RPS6 and histone H3 phosphorylation and induced γH2AX and PARP1 cleavage. Additionally, the drug markedly decreased AURKA phosphorylation and expression. In PANC-1 cells, AD80 strongly induced autophagic flux (consumption of LC3B and SQSTM1/p62). AD80 modulated 32 out of 84 autophagy-related genes and was associated with vacuole organization, macroautophagy, response to starvation, cellular response to nitrogen levels, and cellular response to extracellular stimulus. In 3D pancreatic cancer models, AD80 also effectively reduced growth independent of anchorage and cell viability. In summary, AD80 induces mitotic aberrations, DNA damage, autophagy, and apoptosis in pancreatic cancer cells. Our exploratory study establishes novel targets underlying the antineoplastic activity of the drug and provides insights into the development of therapeutic strategies for this disease.


Introduction
Pancreatic cancer is one of the most lethal human malignancies presenting a 5-year survival rate of less than 10% [1], indicating that current therapeutic interventions for this cancer are far from satisfactory [2]. In recent years, significant advances in understanding the molecular complexity of the development and progression of pancreatic cancer opened the opportunity for new treatments [3]. Surgical resection is a curable therapy for pancreatic cancer patients with early stages of the disease, and gemcitabine (alone or in combination with target therapies) is a backbone drug for locally advanced and metastatic pancreatic cancer patients [2,4]. The use of systemic combination chemotherapies (i.e., 5-fluorouracil, folinic acid, irinotecan, and oxaliplatin, or/and gemcitabine plus nab-paclitaxel) has gained increasing prominence in the treatment of this type of tumor. These findings highlight the importance of drugs that act on multiple targets to exert their antineoplastic activity and prevent the emergence of resistant clones that clinically culminate in disease relapse and poor survival outcomes [5,6].
AD80 is a multikinase inhibitor optimized to perform RAF-ERK inhibition without acting directly on mTOR, which prevents a reactivation loop of the MAPK pathway [7]. Furthermore, it is well established now that AD80 prevents the phosphorylation of ribosomal protein S6 (RPS6), RET, p38 MAPK, and SRC, all proteins associated with cell proliferation and survival, in different cellular models [7][8][9][10][11]. Gain-of-function KRAS mutations that lead to constitutive activation of the MAPK pathway play a critical role in the initiation and maintenance of pancreatic cancer (frequency about 65%), highlighting this pathway as a pharmacological target in this disease [3,12].
The antineoplastic effects of AD80 have been reported in various solid tumors [8,10,13]. However, using monolayer or 3D cell culture models, we identified new potential molecular targets of AD80 in pancreatic cancer models and demonstrated a promising role in activating autophagy. Thus, the present study investigated the cellular and molecular mechanisms underlying the suppressive effects of AD80 on pancreatic cellular models.

Cell Viability Assay
Cell viability was determined with a methylthiazoletetrazolium (MTT) assay. A total of 5 × 10 3 cells per well were plated in a 96-well plate and exposed to vehicle or increasing concentrations of AD80 (0.032, 0.16, 0.8, 4, 20, and 100 µM) for 24, 48, or 72 h. For intermittent drug exposure assays, cells were exposed to vehicle or AD80 (0.032, 0.16, 0.8, 4, 20, and 100 µM) for 6, 12, 24, or 48 h, followed by incubation in drug-free media for 72 h. For combined treatment assays, cells were exposed to vehicle AD80 (0.25, 0.5, 1, 2, and 10 µM) and/or gemcitabine (0.62, 1.25, 2.5, 5, and 10 µM) for 72 h. Then, 10 µL MTT solution (5 mg/mL) was added, and the cells were incubated at 37 • C in 5% CO 2 for 4 h. The reaction was stopped by adding 100 µL 0.1N HCl in anhydrous isopropanol. Cell viability was evaluated by measuring the absorbance at 570 nm. The IC 50 values were calculated by performing a nonlinear regression analysis in GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA). The selectivity index was calculated as the ratio of the IC 50 for the non-tumor pancreatic cell line (HPDE) to the IC 50 for pancreatic cancer cell lines.

Cell Cycle Analysis
Pancreatic cancer cell lines were seeded in 60 mm cell culture dishes (2 × 10 5 cells per plate) and cultured in the presence of a vehicle or AD80 (0.25, 0.5, and 1 µM) for 72 h. Then, cells were fixed with 70% ethanol for at least 4 h and incubated for 30 min with staining solution (0.1% Triton-X 100, 0.1 mg/mL of RNAse (Merck, Darmstadt, Germany) and 1 µg/mL of propidium iodide (Sigma-Aldrich). DNA content distribution was acquired in a FACSCalibur cytometer (Becton Dickinson, Lincoln Park, NJ, USA), and the data were analyzed using the FlowJo software v.X.0.7 (Treestar, Inc., San Carlos, CA, USA).

Morphology Analysis via Immunofluorescence
Pancreatic cancer cell lines treated with vehicle or 1 µM AD80 for 72 h were fixed with ice-cold 100% methanol, permeabilized with 0.5% Triton X-100 in PBS for 30 min at room temperature and blocked with 1% bovine serum albumin (BSA) in PBS for 1 h at room temperature. Next, the cells were incubated with anti-α-tubulin Alexa Fluor 488 conjugate (1:200 in 1% BSA in PBS; Thermo Fisher Scientific Inc., Cleveland, OH, USA) for 16 h at 4 • C protected from light, followed by washing once with PBS. Finally, the slides were mounted in ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific Inc.) for 1 h at room temperature. Images were captured using a fluorescent microscope (Lionheart FX Automated microscope; BioTek Instruments Inc., Santa Clara, CA, USA; magnification, 400×).

Cell Death Analysis
Cell death was determined using the propidium iodide (PI) and Hoechst 33342 (HO) double-staining method. Pancreatic cancer cell lines (2 × 10 5 cells per well) were seeded in six-well plates and treated with vehicle or AD80 (0.25, 0.5, and 1 µM) for 72 h. Then, cells were washed with PBS and incubated with PI (5 µg/mL, Sigma-Aldrich) and HO (10 µg/mL, Sigma-Aldrich) and diluted in PBS for 20 min at 37 • C. Images were captured using a fluorescent microscope (Lionheart FX Automated microscope; BioTek Instruments Inc.; magnification, 100×). The percentage of dead cells was determined by calculating the fraction of PI-stained or fragmented nucleus cells relative to all cells. At least 2000 cells were scored in each experiment using the ImageJ software.

Acidic Vesicular Organelle Analysis via Fluorescence Microscopy
Pancreatic cancer cell lines treated with vehicle or 1 µM AD80 for 72 h were washed with PBS, resuspended in PBS containing 0.1 µg/mL acridine orange (Sigma-Aldrich), incubated for 30 min, and evaluated for GFP and RFP channels using a fluorescent microscope (Lionheart FX Automated microscope; Agilent BioTek Instruments Inc., Santa Clara, CA, USA; magnification, 200×).

PCR Array Analysis
Total RNA from PANC-1 cells treated with vehicle or AD80 (1 µM) for 72 h was obtained using TRIzol reagent (Thermo Fisher Scientific). The cDNA was synthesized from 2 µg RNA using an RT 2 First Strand Kit (Qiagen Sciences Inc., Germantown, MD, USA). PCR array analysis was performed using a Human Autophagy RT 2 Profiler PCR Array kit (#PAHS-084ZA; Qiagen) according to the manufacturer's instructions. The mRNA levels were normalized to those in vehicle-treated cells, and genes that presented p < 0.05 (Student's t-test) were included in the heatmap using Multiple Experiment Viewer (MeV) 4.9.0 software [14]. Amplification was performed using QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, Inc.). A network for AD80 modulated genes was constructed using the GeneMANIA database (https://genemania.org/, (accessed on 1 June 2023)).

Quantitative PCR
Total RNA from MIA PaCa-2, PANC-1, and AsPC-1 cells treated with vehicle or AD80 (1 µM) for 72 h was obtained using TRIzol reagent (Thermo Fisher Scientific, Inc.). cDNA was synthesized from 1 µg RNA using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Inc.). Quantitative PCR (qPCR) was performed using a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, Inc.), a SybrGreen System, and specific primers (Supplementary Table S1). HPRT1 and ACTB were used as reference genes. Relative quantification values were calculated using the 2 −∆∆CT equation [15]. A negative 'No Template Control' was included for each primer pair.

Soft Agar Assay
In a 12-well plate, 500 µL of 0.5% agarose was added to form the bottom layer and 500 µL was used for the upper layer, which contained the pancreatic cancer cells with 0.3% agarose. A total of 200 µL of the medium was added to the surface to prevent drying, and plates were kept for approximately 21 days until the visualization of colony formation. Then, plates were stained with MTT (5 mg/mL) for 45 min. Images were acquired using a G: BOX Chemi XRQ (Syngene) and analyzed using ImageJ v.1.45s software.

Statistical Analysis
Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, Inc.). ANOVA followed by the post hoc Bonferroni's test was used for multiple comparisons. The paired Student's t-test was used to compare the two groups. Results with p-values of <0.05 were considered statistically significant.

AD80 Exhibits Antineoplastic Activity in Pancreatic Cancer Cells
First, the effects of AD80 on viability were investigated in pancreatic cancer cells. As shown in Figure 1A, AD80 reduced cell viability in a concentration-and time-dependent manner, with MIA PaCa-2 cells being the most sensitive to the drug. The IC 50 values ranged from 0.08 to 12.3 µM for MIA PaCa-2, 4.46 to 30.38 µM for PANC-1, and 0.33 to 43.24 µM for AsPC-1. In HPDE cells, IC 50 values ranged from 1.48 to 4.78 µM and showed a favorable selectivity index (SI > 1) for MIA PaCa-2 and AsPC-1 cells after the 24 and 48 h treatments (Supplementary Figure S2). Similarly, clonal growth was strongly inhibited through exposure to AD80 in all pancreatic cell lines tested (p < 0.05, Figure 1B,C). Next, an intermittent exposure assay was performed to better elucidate the role of drug exposure time in this context. A 6 h exposure to AD80 is enough to impact cell viability negatively, but the longer the exposure time, the more potent AD80 is at reducing cell viability ( Figure 1D).
Next, the cellular events involved in the reduction of viability were investigated. AD80 treatment increased the cell population in the G 2 /M phases of the cell cycle and led to the appearance of a polyploidy population (>4 N) (all p < 0.05), which was most evident in PANC-1 cells (Figure 2A). An increase in subG1 cells was also observed, suggesting induction of apoptosis (p < 0.05, Figure 2A). The morphological analysis confirmed the increase in nuclear size and volume of MIA PaCa-2, PANC-1, and AsPC-1 cells ( Figure 2B). Notably, in PANC-1 cells, it was possible to observe the presence of multiple nuclei, which confirms the most prominent findings in the assay for evaluating DNA content ( Figure 2B). In addition, the number of apoptotic cells increased after AD80 exposure, corroborating the subG 1 data from the flow cytometry findings ( Figure 2C). Together these findings suggest that AD80 induces mitotic aberrations and cell death in pancreatic cancer cells.

AD80 Induces Mitotic Catastrophe and Autophagy Molecular Marker Expression in Pancreatic Cancer Cells
The effects of AD80 on the activation of proteins involved in proliferation, cell cycle progression, autophagy, apoptosis, and DNA damage were evaluated. AD80 reduced RPS6 activation, an expected on-target effect since the drug directly inhibits S6K [7]. The effects on ERK1/2 phosphorylation varied widely, from inhibition in AsPC-1 cells to activation in PANC1 cells. AD80 markedly reduced AURKA/B/C phosphorylation and AURKA expression ( Figure 3A). PARP1 cleavage and γHA2X expression were observed in all cell lines but were more intense in MIA PaCa-2 cells, corroborating the greater sensitivity of this cell line to drug-induced cell death ( Figure 3A). Confirming the presence of mitotic aberrations and increased γHA2X, increased levels of CHK1 and/or CHK2 phosphorylation, as well as the induction of genes involved in the response to DNA damage, were observed in pancreatic cells upon AD80 exposure (Supplementary Figure S3). In PANC-1 cells, AD80 drastically reduced the expression of SQSTM1/p62 and LC3B indicating intense autophagic flux. In MIA PaCa-2 cells but not in AsPC-1 cells, a reduction in SQSTM1/p62 was also observed ( Figure 3A).   Considering that the relationship between AD80 and autophagy is a little-explored topic, we deepen the investigations in this context. Acridine orange staining suggests an increase in acidic vesicular organelles (AVOs) in MIA PaCa-2 and PANC-1 cells but not in ASPC-1 cells, corroborating the molecular findings ( Figure 3B). In PANC-1 cells, treatment with bafilomycin A1, a potent autophagic flux inhibitor, prevented the consumption of LC3B and SQSTM1 triggered by AD80, corroborating the hypothesis that AD80 is an autophagy inducer (Supplementary Figure S4). Using a PCR array for autophagy-related genes, we observed that 32 out of 84 genes were modulated (28 upregulated and 4 downregulated) after exposure of PANC-1 cells to AD80 (all p < 0.05, Figure 3C and Supplementary Table S2). An integrated analysis of the modulated genes (fold-change > 1.5, p < 0.05) indicates that AD80 impacts several stages of autophagy, from sensitization to nutrient deprivation and vacuole organization to autophagy (all FDR q < 0.05, Figure 3D).

AD80 Reduces Anchorage-Independent Growth and Spheroid Cell Viability in Pancreatic Cancer
We performed anchorage-independent growth and spheroid generation assays to provide evidence of the antineoplastic potential of AD80 in more complex conditions. The anchorage-independent growth assay mimics the stress that the cells that leave the primary tumor go through to generate distal metastases, with most cells spontaneously entering a process of detachment-induced cell death known as anoikis [16,17]. Cells that survive this process are usually resistant to cell death. Interestingly, AD80 significantly inhibited anchorage-independent growth in all pancreatic cell lines tested (all p < 0.05, Figure 4A). On the other hand, the 3D model mimics an already-formed tumor, with cellular communications, nutrients gradient, and spheroid architecture, establishing a greater complexity compared to monolayer cellular models [18]. In this context, AD80 reduced the cell viability of pancreatic cancer spheroid models (all p < 0.05, Figure 4B). These results suggest that AD80 could treat primary tumors and attenuate their ability to spread to other sites.

AD80 Potentiates Gemcitabine-Reduced Cell Viability in PANC-1 and AsPC-1 Cells
Lastly, the effects of AD80 combined with gemcitabine, a frontline therapy for pancreatic cancer, were investigated in cellular models of the disease. In PANC-1 and AsPC-1 cells, but not MIA PaCa-2 cells, AD80 exposure potentiated the antineoplastic effects of gemcitabine on the reduction of cell viability (p < 0.05, Figure 5).   Lastly, the effects of AD80 combined with gemcitabine, a frontline therapy for pancreatic cancer, were investigated in cellular models of the disease. In PANC-1 and AsPC-1 cells, but not MIA PaCa-2 cells, AD80 exposure potentiated the antineoplastic effects of gemcitabine on the reduction of cell viability (p < 0.05, Figure 5).

Discussion
Herein, we have investigated the effects of AD80 on malignant phenotype in monolayer and 3D pancreatic cancer cellular models. Remarkable antineoplastic effects, including the reduction of cell viability, clonal growth, cell cycle progression, and anchorage-free growth, were observed, in addition to the induction of cell death, autophagy, and mitotic aberrations. AD80 acts as a multikinase inhibitor and exerts antineoplastic effects in a variety of cancer types, including lymphoma [8], glioma [8], lung cancer [11], hepatocellular carcinoma [10], ovarian cancer [13], prostate cancer [19], acute leukemia [9], and colorectal cancer [20]. Although reduced cell viability is a similar aspect between different types of cancer, the versatility of AD80 allows it to act on various molecular targets, including S6K, RET, RAF, and p38 MAPK [7][8][9][10][11], making it an attractive multitarget compound. The use of combination therapies or drugs that act on multiple targets in antineoplastic therapy has been proposed to avoid the emergence of chemoresistant clones and subsequent refractoriness or disease relapse [5,6]. In our study, a 6 h AD80 exposure generated irreversible data that reflected in the reduction of long-term cell viability, indicating the high sensitivity of this type of cancer to the drug.
In the present study, aurora kinases (AURKs) were downregulated with AD80 in pancreatic cancer cells. The relevant role of AURKs in this disease has been described in previous studies, especially for AURKA. AURKA expression is stimulated via a KRAS mutation, and high AURKA expression predicts unfavorable clinical outcomes in patients with pancreatic cancer. AURKA inhibition with siRNA or pharmacological inhibitors reduces the in vitro and in vivo proliferation, clonogenicity, and survival of MIA PaCa-2, PANC-1, and/or AsPC-1 cells [21,22]. The inhibition of AURKs via AD80 was reported in the initial characterization of the drug. In vitro, the drug reduces AURKA, AURKB, and AURKC activities by 76%, 87%, and 58%, respectively [7]. AURKs are essential proteins for the correct segregation of chromosomes and the success of mitosis and cytokinesis [23,24]. Thus, the cellular effects noticed in pancreatic cancer models upon AD80 exposure indicate that AURKs may be one of the main targets associated with the observed phenotype.
AD80-induced autophagic flow activation was a breakthrough finding in our study. mTOR-mediated signaling is one of the main physiological inhibitors of autophagy [25]. AD80 was specifically designed not to directly inhibit mTOR, preventing the reactivation of the mTOR/RAF/ERK regulatory loop, but the drug inhibits S6K, a critical downstream target of mTOR [7]. In our study, the induction of autophagy in pancreatic cancer cells was independent of the inhibition of RPS6 phosphorylation status, indicating that other molecular mechanisms may be involved. Our exploratory analysis based on gene expression suggests that AD80 alters the expression of genes involved in multiple stages of autophagy, from sensitization through nutrient deprivation to the organization of the autophagic vacuole.
Our study opens perspectives for novel antineoplastic agents in pancreatic cancer; however, it has limitations. The scope of the present study was based on in vitro studies using pancreatic cancer cell lines, which provided cellular and molecular information. Investigating the effects in animal models of pancreatic cancer (e.g., ectopic or orthotropic tumor models) is needed to define the selectivity and toxicity of the drug. The results could lead to clinical trials.

Conclusions
In summary, our results indicate that AD80 exerts multiple antineoplastic effects in monolayer and 3D pancreatic cancer cellular models, leading to mitotic catastrophe and autophagy. Our preclinical findings highlight AD80 as a multitarget drug that may contribute to the therapeutic repertoire against pancreatic cancer.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/cancers15153866/s1, Supplementary Figure S1: Whole gel images. Supplementary Figure S2: Effects of AD80 on cell viability of HPDE cells (non-tumor pancreatic cell line) and its selectivity index for pancreatic cancer cell lines. Supplementary Figure S3: AD80 increases levels of CHK1 and/or CHK2 phosphorylation and induces the expression of genes involved in response to DNA damage in pancreatic cells. Supplementary Figure S4: Bafilomycin A1 prevents the consumption of LC3B and SQSTM1 triggered by AD80 in PANC-1 cells. Supplementary Table S1: PCR array results for autophagy-related genes upon AD80 exposure in PANC-1 cells. Supplementary  Table S2. PCR array results for autophagy-related genes upon AD80 exposure in PANC-1 cells.