Multifaceted Effects of Kinase Inhibitors on Pancreatic Cancer Cells Reveals Pivotal Entities with Therapeutic Implications

Pancreatic cancer is one of the most aggressive forms of cancer and is the seventh leading cause of cancer deaths worldwide. Pancreatic ductal adenocarcinoma (PDAC) accounts for over 90% of pancreatic cancers. Most pancreatic cancers are recalcitrant to radiation, chemotherapy, and immunotherapy, highlighting the urgent need for novel treatment options for this deadly disease. To this end, we screened a library of kinase inhibitors in the PDAC cell lines PANC-1 and BxPC-3 and identified two highly potent molecules: Aurora kinase inhibitor AT 9283 (AT) and EGFR kinase inhibitor WZ 3146 (WZ). Both AT and WZ exhibited a dose-dependent inhibition of viability in both cell lines. Thus, we conducted an in-depth multilevel (cellular, molecular, and proteomic) analysis with AT and WZ in PANC-1 cells, which harbor KRAS mutation and exhibit quasimesenchymal properties representing pancreatic cancer cells as having intrinsic chemoresistance and the potential for differential response to therapy. Elucidation of the molecular mechanism of action of AT and WZ revealed an impact on the programmed cell death pathway with an increase in apoptotic, multicaspase, and caspase 3/7 positive cells. Additionally, the key survival molecule Bcl-2 was impacted. Moreover, cell cycle arrest was observed with both kinase inhibitors. Additionally, an increase in superoxide radicals was observed in the AT-treated group. Importantly, proteomic profiling revealed differentially regulated key entities with multifaceted effects, which could have a deleterious impact on PDAC. These findings suggest potential targets for efficacious treatment, including a possible increase in the efficacy of immunotherapy using PD-L1 antibody due to the upregulation of lactoferrin and radixin. Furthermore, combination therapy outcomes with gemcitabine/platinum drugs may also be more effective due to an increase in the NADH dehydrogenase complex. Notably, protein–protein interaction analysis (STRING) revealed possible enrichment of reactome pathway entities. Additionally, novel therapy options, such as vimentin-antibody--drug conjugates, could be explored. Therefore, future studies with the two kinases as monotherapy/combination therapy are warranted.


Introduction
Pancreatic cancer generally refers to pancreatic ductal adenocarcinoma (PDAC) as it accounts for 90% of pancreatic cancers identified. This malady is aggressive in nature, with a 5-year survival rate below 10% [1], and it is the seventh leading cause of cancer death worldwide and third in the United States [2,3]. It is estimated that PDAC could cause even higher cancer deaths by the year 2025 in the United States [4]. Long-term survival for patients with pancreatic cancer is rare [5]. In human PDAC, nearly all ras mutations are localized in the KRAS [6]. Apart from mutations, due to tumor and stromal interactions, novel therapeutic strategies, such as combination therapies, are needed for pancreatic cancer [7].

Library Screening and Toxicity Assay
PANC-1 cells were plated in a 96-well plate at a density of 5000 cells per well in the culture medium. After 24 h, cells were treated with various concentrations of inhibitors ranging from 0.1 µM to 10 µM for the initial screening experiment. The selected potent kinase inhibitors, AT and WZ, were further assessed for impact on the viability of PANC-1 cells at various concentrations ranging from 0.05 µM to 20 µM. Cells were incubated for 72 h at 37 • C in an atmosphere of 5% CO 2 . The assays were carried out in triplicates. Cell Counting Kit-8 assay from Bimake (Houston, TX, USA) was used to assess the cell toxicity. The media was aspirated, and 200 µL of 10% CCK-8 solution in the complete growth medium was added. Absorbance was measured at 450 nm using an Infinite 200Pro plate reader (Tecan, Männedorf, Switzerland) after 1.5 h of incubation at 37 • C. The viability of cells was expressed after normalizing the treatment group absorbance values to that of the control group. Using Prism software, version 7.0c (GraphPad, Boston, MA, USA), graphs were plotted and IC 50 was determined. Three independent experiments were performed for both screening and viability assays for AT and WZ. Identical screening and viability experiments with similar repetitions were conducted with the BxPC-3 cell line. For all mechanistic studies, PANC-1 cells were treated with AT at 8 µM and WZ at 5 µM (above IC 50 concentration).

Flow Cytometry Assays
PANC-1 cells were plated in 12-well plates at 62,500 cells per well and after 24 h cells were left either untreated or treated in triplicate for 72 h with 8 µM of AT or 5 µM of WZ. After 72 h of incubation, media were collected, and then adhered cells were trypsinized and pooled together from triplicate treatments before proceeding with Luminex Muse assay kit protocol as per manufacturer's instructions (Luminex Corporation, Austin, TX, USA). The following Luminex Muse assay kits were used: Oxidative Stress kit (MCH100111), Annexin V and dead cell kit (MCH100105), MultiCaspase kit (MCH100109), Caspase 3/7 (MCH100108), Bcl-2 activation dual detection kit (MCH200105), and Cell cycle kit (MCH100106).

Proteomic Analysis
Proteomic analysis was performed by 2D DIGE and mass spectrometry, which was conducted by Applied Biomics Inc. (Hayward, CA, USA) employing previously published methodologies [27]. PANC-1 cells were treated individually with 8 µM AT or 5 µM WZ. Control cultures (no treatment) were maintained in parallel. Cells from control samples and treated samples were collected, washed with 1×PBS, and then stored at −80 • C prior to sending the samples to Applied Biomics Inc. (Hayward, CA, USA) on dry ice for proteomic analysis. The protocol performed was as described previously [27][28][29]. STRING database version 11.5 was used to perform functional enrichment analysis and to generate protein-protein interaction networks [30].

Statistical Analysis
One-way ANOVA with Dunnett's multiple comparison test was used for CCK-8 assay and histogram representation of flow cytometric assays with alpha set to 0.05. The p-values are represented in the legends for the figures.

Identification of Molecules Impacting the Viability of PANC-1 and BxPC-3 Cells by Screening a Library of Kinase Inhibitors
A number of molecules were tested in the PANC-1 and BxPC-3 cell lines at various concentrations (ranging from 0.05 µM to 10 µM). As shown in Figure 1A,B, the kinase inhibitors labeled a9 and d10 showed potent inhibition of viability in PANC-1. Upon testing in the BxPC-3 cell line, a similar inhibition was observed for the inhibitors a9 and d10; see Figure 1C,D. The kinase inhibitor a9 from the library was identified as an 'Aurora see Figure 1C,D. The kinase inhibitor a9 from the library was identified as an 'Aurora kinase inhibitor,' abbreviated AT, and d10 as an 'EGFR kinase inhibitor,' abbreviated WZ. Both the kinase inhibitors showed over 50% inhibition in the initial screening experiments. Therefore, further in-depth studies were carried out with these two types of inhibitors.

Cellular Level Analysis Revealed Potent Dose-Dependent Inhibition of Viability of PANC-1 and BxPC-3 Cells by the Kinase Inhibitors AT and WZ
To investigate the effect of the kinase inhibitors AT and WZ on the viability of pancreatic adenocarcinoma cells and to determine the IC50 concentration, PANC-1 and BxPC-3 cells were treated with varying concentrations of the compound and incubated for 72 h. The effect on cell viability was assessed via CCK8 assay. A dose-dependent inhibition of cell viability was observed when the PANC-1 cells were treated with the kinase inhibitor AT. A similar dose-dependent response was observed when the cells were treated with WZ, as shown in Figure 2A, with an IC50 of 6.8 µM and 4.4 µM, respectively. A significant decrease in cell viability was observed and the p-values were ** = 0.0021, *** = 0.0002 and **** < 0.0001. When the experiment was repeated with BxPC-3 cells under identical Figure 1. A set of compounds from the library of kinase inhibitors was tested at 0.1, 0.5, 1, 5, and 10 µM concentrations for their impact on the inhibition of PANC-1 cell viability. Cells were plated at a cell density of 5000 cell/well and incubated for 72 h. Cell viability was monitored using the CCK 8 assay. (A) Compounds labeled a1, a9, d3, and d10 from the library were assessed. (B) Compounds e10, e11, h9, and h10 were the next set assayed. Similar screening assays were performed for the cell line BxPC-3, (C,D), which ranged from 0.05 to 10 µM of the kinase inhibitors. The kinase inhibitors labeled a9 and d10 exhibited an impact on cell viability in both the cell lines and was identified as an Aurora kinase inhibitor, abbreviated AT, and the EGFR kinase inhibitor, abbreviated WZ, respectively. The bars represent cell viability at various concentrations. In (A,B) concentrations from left to right are 0.1 µM, 0.5 µM, 1 µM, 5 µM, and 10 µM. In (C,D), the concentrations from left to right are 0.05 µM, 0.1 µM, 0.5 µM, 1 µM, 5 µM, and 10 µM. Details of the compounds screened and their targets are as follows: a1: Linifanib (ABT-869)-CSF-1R, PDGFR, VEGFR; a9: AT9283-Aurora Kinase, Bcr-Abl, JAK; d3: Pazopanib HCl (GW786034 HCl)-c-Kit, PDGFR,VEGFR; d10: WZ3146-EGFR; e10: CYC116-Aurora Kinase, VEGFR; e11: YM201636-PI3K; h9: SNS-314 Mesylate-Aurora Kinase; h10: Regorafenib (BAY 73-4506)-c-RET,VEGFR.

Cellular Level Analysis Revealed Potent Dose-Dependent Inhibition of Viability of PANC-1 and BxPC-3 Cells by the Kinase Inhibitors AT and WZ
To investigate the effect of the kinase inhibitors AT and WZ on the viability of pancreatic adenocarcinoma cells and to determine the IC 50 concentration, PANC-1 and BxPC-3 cells were treated with varying concentrations of the compound and incubated for 72 h. The effect on cell viability was assessed via CCK8 assay. A dose-dependent inhibition of cell viability was observed when the PANC-1 cells were treated with the kinase inhibitor AT. A similar dose-dependent response was observed when the cells were treated with WZ, as shown in Figure 2A, with an IC 50 of 6.8 µM and 4.4 µM, respectively. A significant decrease in cell viability was observed and the p-values were ** = 0.0021, *** = 0.0002 and **** < 0.0001. When the experiment was repeated with BxPC-3 cells under identical conditions, IC 50 values were 2.9 µM and 3.95 µM for AT and WZ, respectively, with p-values of ** = 0.0021,*** = 0.0002 and **** < 0.0001; see Figure 2B.
conditions, IC50 values were 2.9 µM and 3.95 µM for AT and WZ, respectively, with pvalues of ** = 0.0021,*** = 0.0002 and **** < 0.0001; see Figure 2B. Figure 2. Impact of AT and WZ on PANC-1's and BxPC-3's cell viability. (A) PANC-1 cells were treated with various concentrations of AT and WZ, and their effect on the cell viability was tested as described in 'Section 2'. Cell viability was expressed as a percentage of control cells. The experiments were performed in triplicates, and the mean of three independent experiments was calculated. A significant decrease in cell viability was observed, with p-values of ** = 0.0021, *** = 0.0002 and **** < 0.0001. (B) Similar assays were performed on BxPC-3 cells with p-values of ** = 0.0021, *** = 0.0002 and **** < 0.0001. Both PDAC cell lines showed potent dose-dependent inhibition of viability.

Effect of Kinase Inhibitors AT and WZ on ROS Levels in PANC-1 Cells
On observing the potent inhibition of cell viability by AT and WZ in PANC-1 cells, we sought to investigate their impact on pivotal cell entities. In the present study, we assessed the reactive oxygen species (ROS) by monitoring for superoxide radicals, as many anti-cancer drugs have been reported to affect the levels of ROS upon treatment. The generation of ROS was monitored using the Luminex Muse oxidative stress kit and Muse cell analyzer. We analyzed the superoxide radicals generated in PANC-1 cells upon treatment with AT and WZ. This assay allows for quantitative measurement of superoxide radical, and at the same time, determines the count and percentage of cells undergoing oxidative stress utilizing the well-characterized reagent dihydroethidium, which has been used for the detection of ROS contributed by superoxide radical in cell populations. The assay was

Effect of Kinase Inhibitors AT and WZ on ROS Levels in PANC-1 Cells
On observing the potent inhibition of cell viability by AT and WZ in PANC-1 cells, we sought to investigate their impact on pivotal cell entities. In the present study, we assessed the reactive oxygen species (ROS) by monitoring for superoxide radicals, as many anticancer drugs have been reported to affect the levels of ROS upon treatment. The generation of ROS was monitored using the Luminex Muse oxidative stress kit and Muse cell analyzer. We analyzed the superoxide radicals generated in PANC-1 cells upon treatment with AT and WZ. This assay allows for quantitative measurement of superoxide radical, and at the same time, determines the count and percentage of cells undergoing oxidative stress utilizing the well-characterized reagent dihydroethidium, which has been used for the detection of ROS contributed by superoxide radical in cell populations. The assay was performed by treating the cells at a concentration of 8 µM AT and 5 µM, WZ, followed by incubating the cells for 72 h in the presence of these compounds. Three independent experiments were performed. Representative plots of oxidative stress levels of control and the treated groups are shown in Figure 3A, where M1 represents the ROS(−) cell population and M2 represents the ROS(+) cell population. The % gated profile of the control, AT, and WZ-treated groups are shown in Figure 3B. The impact on ROS was observed in the treated groups. Upon normalization to the control group, a statistically significant increase in ROS positive cells was observed in the AT-treated samples, with a p-value of 0.0148 for M1 and 0.0248 for the M2 population, as per 2-way ANOVA with Dunnett's multiple comparison test.
performed by treating the cells at a concentration of 8 µM AT and 5 µM, WZ, followed by incubating the cells for 72 h in the presence of these compounds. Three independent experiments were performed. Representative plots of oxidative stress levels of control and the treated groups are shown in Figure 3A, where M1 represents the ROS(−) cell population and M2 represents the ROS(+) cell population. The % gated profile of the control, AT, and WZ-treated groups are shown in Figure 3B. The impact on ROS was observed in the treated groups. Upon normalization to the control group, a statistically significant increase in ROS positive cells was observed in the AT-treated samples, with a p-value of 0.0148 for M1 and 0.0248 for the M2 population, as per 2-way ANOVA with Dunnett's multiple comparison test.

Kinase Inhibitors AT and WZ Invoked the Programmed Cell Death Pathway in PANC-1 Cells
On observing an increase in superoxide radical levels in the AT and WZ-treated cells, we proceeded to investigate whether the two kinase inhibitors invoked the programmed cell death pathway. To assess the programmed cell death pathway, the Annexin V flow cytometric assay was performed; by using this technology, the population of cells that were affected by the kinase inhibitors could be assessed. Cells were treated with 8 µM of AT and 5 µM of WZ and incubated at 37 °C for 72 h. Representative scatter plots are shown in Figure 4A. The % gated profile compared to the control is represented by a bar diagram; see Figure 4B. Upon normalization to the control group, a statistically significant increase in apoptotic cells was observed in both the AT-and the WZ-treated group; see Figure 4C.

Kinase Inhibitors AT and WZ Invoked the Programmed Cell Death Pathway in PANC-1 Cells
On observing an increase in superoxide radical levels in the AT and WZ-treated cells, we proceeded to investigate whether the two kinase inhibitors invoked the programmed cell death pathway. To assess the programmed cell death pathway, the Annexin V flow cytometric assay was performed; by using this technology, the population of cells that were affected by the kinase inhibitors could be assessed. Cells were treated with 8 µM of AT and 5 µM of WZ and incubated at 37 • C for 72 h. Representative scatter plots are shown in Figure 4A. The % gated profile compared to the control is represented by a bar diagram; see Figure 4B. Upon normalization to the control group, a statistically significant increase in apoptotic cells was observed in both the AT-and the WZ-treated group; see Figure 4C.

Multicaspase and Executioner Caspases 3/7 Were Elicited in PANC-1 Cells by AT and WZ
To further investigate the impact of the kinase inhibitors AT and WZ on the involvement of multicaspase and executioner caspases, PANC-1 cells were treated with 8 µM of AT and 5 µM of WZ. Multicaspase activation was monitored using flow cytometry and multicaspase assays. Representative scatter plots of three independent trials are shown in Figure 5A. A significant increase in multicaspase positive cells was observed in the treated group compared to the control group, and as observed in the % gated profile of each of the samples in Figure 5B. On normalizing to the control, a significant increase in cells with multicaspase was observed in AT-and WZ-treated samples; see Figure 5C. Similarly, the impact of AT and WZ on executioner caspases 3/7 was monitored using flow cytometry.

Multicaspase and Executioner Caspases 3/7 Were Elicited in PANC-1 Cells by AT and WZ
To further investigate the impact of the kinase inhibitors AT and WZ on the involvement of multicaspase and executioner caspases, PANC-1 cells were treated with 8 µM of AT and 5 µM of WZ. Multicaspase activation was monitored using flow cytometry and multicaspase assays. Representative scatter plots of three independent trials are shown in Figure 5A. A significant increase in multicaspase positive cells was observed in the treated group compared to the control group, and as observed in the % gated profile of each of the samples in Figure 5B. On normalizing to the control, a significant increase in cells with multicaspase was observed in AT-and WZ-treated samples; see Figure 5C. Similarly, the impact of AT and WZ on executioner caspases 3/7 was monitored using flow cytometry. Representative scatter plots of three independent trials are shown in Figure 5D. An increase in executioner caspases 3/7 positive cells was also observed in the AT-and WZ-treated groups, as evidenced by the % gated profile in Figure 5E. Upon normalization to the control group, a statistically significant increase in the executioner caspases was observed, Figure 5F. Representative scatter plots of three independent trials are shown in Figure 5D. An increase in executioner caspases 3/7 positive cells was also observed in the AT-and WZtreated groups, as evidenced by the % gated profile in Figure 5E. Upon normalization to the control group, a statistically significant increase in the executioner caspases was observed, Figure 5F.

The Key Cell Survival Molecule Bcl-2 Was Impacted by AT and WZ
On observing the potent impact of AT and WZ on key cell pathways such as the programmed cell death pathway, we opted to explore whether the pivotal cell survival molecule Bcl-2 was impacted. To analyze the population of cells affected, flow cytometry was performed using the Luminex Muse Bcl-2 assay. Representative scatter plots of the control and AT-and WZ-treated samples, along with the percentage of activated, inactivated, and non-expressing Bcl-2 cells, are represented in Figure 6A. The means of three independent experiments with standard deviations were analyzed and normalized to the control group and represented with a bar diagram in Figure 6B. On normalizing to the control, there was a statistically significant decrease in the Bcl-2 activated population of cells in the AT-treated group. In the WZ-treated group, there was also a statistically significant decrease in the Bcl-2 activated population of cells, as well as an increase in the non-expressing cells.

The Key Cell Survival Molecule Bcl-2 Was Impacted by AT and WZ
On observing the potent impact of AT and WZ on key cell pathways such as the programmed cell death pathway, we opted to explore whether the pivotal cell survival molecule Bcl-2 was impacted. To analyze the population of cells affected, flow cytometry was performed using the Luminex Muse Bcl-2 assay. Representative scatter plots of the control and AT-and WZ-treated samples, along with the percentage of activated, inactivated, and non-expressing Bcl-2 cells, are represented in Figure 6A. The means of three independent experiments with standard deviations were analyzed and normalized to the control group and represented with a bar diagram in Figure 6B. On normalizing to the control, there was a statistically significant decrease in the Bcl-2 activated population of cells in the AT-treated group. In the WZ-treated group, there was also a statistically significant decrease in the Bcl-2 activated population of cells, as well as an increase in the non-expressing cells.

Cell Cycle of PANC-1 Cells Was Impacted by AT and WZ
We further investigated whether AT and WZ had an effect on the different phases of the cell cycle of PANC-1 cells. To analyze the population of cells affected, a cell cycle analysis was performed. Representative plots of the control group and the AT-and WZtreated groups are represented in Figure 7A. A statistically significant decrease in the G0/G1 phase of the cell cycle was observed in the AT-treated group. A decrease in cells in the G0/G1 phase, as well as an increase in the G2/M phase, was observed in the WZtreated group, as shown in Figure 7B.

Cell Cycle of PANC-1 Cells Was Impacted by AT and WZ
We further investigated whether AT and WZ had an effect on the different phases of the cell cycle of PANC-1 cells. To analyze the population of cells affected, a cell cycle analysis was performed. Representative plots of the control group and the AT-and WZ-treated groups are represented in Figure 7A. A statistically significant decrease in the G0/G1 phase of the cell cycle was observed in the AT-treated group. A decrease in cells in the G0/G1 phase, as well as an increase in the G2/M phase, was observed in the WZ-treated group, as shown in Figure 7B.

Proteomic Analysis Reveals Entities of Pivotal Signaling Pathways That Were Differentially Regulated in PANC-1 Cells on Treatment with AT and WZ
To understand the mechanism of action and further delineate the key entities that could be impacted by AT and WZ, we employed sensitive methodologies such as 2D gel electrophoresis and mass spectrometric analysis to perform 'Protein profiling'. Two-dimensional gel electrophoresis of AT-and WZ-treated groups and the control group was performed (as described in Section 2.4). The cell lysate of the control group was combined with Cy2 dye, and the treated group was combined with Cy3 dye. An overlay of the gels exhibited differentially regulated proteins. The overlay of the control gel on the ATtreated gel is shown in Figure 8A and the overlay of the WZ-treated gel and control gel is shown in Figure 8B. The heatmaps of AT-and WZ-treated groups are shown in Figure  8C.

Proteomic Analysis Reveals Entities of Pivotal Signaling Pathways That Were Differentially Regulated in PANC-1 Cells on Treatment with AT and WZ
To understand the mechanism of action and further delineate the key entities that could be impacted by AT and WZ, we employed sensitive methodologies such as 2D gel electrophoresis and mass spectrometric analysis to perform 'Protein profiling'. Twodimensional gel electrophoresis of AT-and WZ-treated groups and the control group was performed (as described in Section 2.4). The cell lysate of the control group was combined with Cy2 dye, and the treated group was combined with Cy3 dye. An overlay of the gels exhibited differentially regulated proteins. The overlay of the control gel on the AT-treated gel is shown in Figure 8A and the overlay of the WZ-treated gel and control gel is shown in Figure 8B. The heatmaps of AT-and WZ-treated groups are shown in Figure 8C Based on the fold changes obtained, we selected upregulated and downregulated proteins for further proteomic analysis using mass spectrometry, as listed in Table 1.  Based on the fold changes obtained, we selected upregulated and downregulated proteins for further proteomic analysis using mass spectrometry, as listed in Table 1.
The STRING database was utilized to conduct functional enrichment analysis and to create protein-protein interaction networks, as shown in Figure 9. The analysis revealed the enrichment of the KEGG pathway 'MicroRNAs in cancer' and the reactome pathways 'Initiation of Nuclear Envelope (NE) Reformation', 'Signaling by BRAF and RAF fusions', and 'Diseases of signal transduction by growth factor receptors and second messengers', with false discovery rates of 0.0396, 0.0353, 0.0094, and 0.0051, respectively. Results from similar studies with WZ are presented in Supplementary Figure S1.  Figure 9.  Figure 9.
The STRING database was utilized to conduct functional enrichment analysis and to create protein-protein interaction networks, as shown in Figure 9. The analysis revealed the enrichment of the KEGG pathway 'MicroRNAs in cancer' and the reactome pathways 'Initiation of Nuclear Envelope (NE) Reformation', 'Signaling by BRAF and RAF fusions', and 'Diseases of signal transduction by growth factor receptors and second messengers', with false discovery rates of 0.0396, 0.0353, 0.0094, and 0.0051, respectively. Results from similar studies with WZ are presented in Supplementary Figure S1. Figure 9. A protein-protein interaction network generated using the STRING database. Each node represents a differentially regulated protein from the AT-treated group. The blue color indicates upregulation, and the red indicates downregulation. The edges represent either known or predicted interactions, with a minimum required interaction score of 0.4 in the STRING database. The abbreviations in the above figure are mentioned in Table 1.

Discussion
The aim of this study was to discover potential pivotal molecules and treatment options for pancreatic cancer, an aggressive form of cancer with an unmet need for efficacious drugs. Screening a library of kinase inhibitors led us to two potent inhibitors: the Aurora kinase inhibitor 9283 and the EGFR Kinase inhibitor 3146, which exhibited inhibition of cell viability and potency at a low concentration in pancreatic cancer cells. PANC-1, unlike BxPC-3, harbors KRAS mutation and is described as a 'Quasimesenchymal (QM) pancreatic ductal epithelial cell line' [26]; in addition, QM PDAC cells are reported to contribute to differential response to therapy [31]. Additionally, in PANC-1, the control of the generation of stem cells has been reported [32]. Therefore, we opted to perform an indepth multilevel analysis of the impact of AT and WZ on PANC-1 cells. At the cellular level, we observed potent inhibition of the viability of PANC-1 cells. For the elucidation of the molecular mechanism of action of AT and WZ, we performed flow cytometry. Using this technology, we could assess the population of cells that were impacted in various regulatory pathways. The impact on superoxide radical formation was observed in the AT-treated groups. It is reported that Aurora kinase B inhibition led to ROS production, which was required for the cell cycle arrest in the G1 phase [33]. We observed ROS production and a smaller number of cells in the G0/G1 phase when compared to the control cells on treatment with the Aurora kinase inhibitor, AT, in the present study. Both the Figure 9. A protein-protein interaction network generated using the STRING database. Each node represents a differentially regulated protein from the AT-treated group. The blue color indicates upregulation, and the red indicates downregulation. The edges represent either known or predicted interactions, with a minimum required interaction score of 0.4 in the STRING database. The abbreviations in the above figure are mentioned in Table 1.

Discussion
The aim of this study was to discover potential pivotal molecules and treatment options for pancreatic cancer, an aggressive form of cancer with an unmet need for efficacious drugs. Screening a library of kinase inhibitors led us to two potent inhibitors: the Aurora kinase inhibitor 9283 and the EGFR Kinase inhibitor 3146, which exhibited inhibition of cell viability and potency at a low concentration in pancreatic cancer cells. PANC-1, unlike BxPC-3, harbors KRAS mutation and is described as a 'Quasimesenchymal (QM) pancreatic ductal epithelial cell line' [26]; in addition, QM PDAC cells are reported to contribute to differential response to therapy [31]. Additionally, in PANC-1, the control of the generation of stem cells has been reported [32]. Therefore, we opted to perform an in-depth multilevel analysis of the impact of AT and WZ on PANC-1 cells. At the cellular level, we observed potent inhibition of the viability of PANC-1 cells. For the elucidation of the molecular mechanism of action of AT and WZ, we performed flow cytometry. Using this technology, we could assess the population of cells that were impacted in various regulatory pathways. The impact on superoxide radical formation was observed in the AT-treated groups. It is reported that Aurora kinase B inhibition led to ROS production, which was required for the cell cycle arrest in the G1 phase [33]. We observed ROS production and a smaller number of cells in the G0/G1 phase when compared to the control cells on treatment with the Aurora kinase inhibitor, AT, in the present study. Both the kinase inhibitors invoked the programmed cell death pathway, leading to an increase in apoptotic, multicaspase, and the executioner caspases 3/7-positive cells. Furthermore, the key cell survival molecule Bcl-2 was impacted by both inhibitors. Moreover, both inhibitors affected the cell cycle phases of PANC-1 cells. Importantly, we performed highly sensitive assays such as 2D gel electrophoresis and mass spectrometry, which are more sensitive than Western blots and ELISA and can detect molecules in the femtomolar range [34]. On treatment with AT and WZ, various upregulated and downregulated entities were identified. One of the upregulated proteins was lactotransferrin, also termed lactoferrin (LF). LF has been reported to inhibit the proliferation/migration of cancer cells [35], induce apoptosis [36], and inhibit growth in vitro [37] and retard tumor growth in vivo [38] and is considered a 'Miracle molecule' [39]. The impact of LF on the human genome and its effects has been discussed recently [40]. The action of LF has been described at various fronts, including apoptosis via the Bcl-2 pathway [41]. Additionally, there are reports of the action of LF on caspases [42], including cell cycle arrest in the G0/G1 phase [37]. In fact, in the molecularlevel analysis in our present study, we observed an increase in apoptotic, multicaspase, and caspase 3/7 cells, as well as a decrease in the Bcl-2 levels and also impact on the phases of the cell cycle of PANC-1 cells. An increase in LF observed on proteomic profiling could be one of the contributing factors to these observations. Additionally, the action of LF on the immune system is considered as a major contributor to its anticancer activity in the in vivo systems [43,44]. Moreover, LF has been reported to be effective when delivered by novel strategies such as the liposomalization of LF, which enhanced antitumoral effects in melanoma, and thermally responsive elastin-like polypeptide fused to an LF-derived peptide in pancreatic cancer cells [45,46]. Importantly, oncolytic peptide LTX-315 derived from bovine LF has been reported to induce anti-pancreatic cancer immunity via the ATP11BPD-L1 axis and downregulates PD-L1 [47], as well as immunity, via the natural killer (NK) cells in breast cancer [48]. Therefore, the increase in LF induced by both the kinase inhibitors in PANC-1 cells that we observed in the present study could be harnessed to sensitize and implement synergistic novel treatments with PD-L1 antibody with improved efficacy of immunotherapy for pancreatic cancers.
It is reported that a family of proteins, in which radixin is a key moiety, is involved in crosslinking the transmembrane protein PD-L1 to the actin cytoskeleton [49]. The evaluation of the expression levels of PD-L1 in various pancreatic cancer cell lines has shown weak to moderate levels of expression [50]. Interestingly, radixin is reported to possibly act as a scaffold protein responsible for cell surface localization of PD-L1 in a pancreatic cancer cell line [51]. In the present study, the kinase inhibitors AT and WZ upregulated the levels of radixin. We envision that this could lead to an increase in PD-L1, which could then be targeted by PD-L1 antibodies. Although radixin is reported to play a role in pancreatic cancer progression [52], its essential role in regulating the transmembrane molecule PD-L1 could lead to success in targeting pancreatic cancer by immunotherapy with PD-L1 antibody in the presence of kinase inhibitors such as AT or WZ, which increase radixin. Thus, apart from the increase in LF observed in the present study, which could sensitize PDAC cells to checkpoint inhibitors, an increase in radixin could also possibly aid in increasing immunotherapy by immune checkpoint inhibitors of PD-L1 in pancreatic cancer cells.
Among the protein phosphatases, serine/threonine-protein phosphatase 2A (PP2A) was one of the molecules upregulated by AT and WZ. Serine/threonine protein phosphatases play a major role in controlling various pivotal pathways. Additionally, phosphatases such as PP2C are reported to be tumor suppressors [53]. Further, small molecules activating phosphatases and reducing tumor cell invasion have been reported [54]. Notably, a molecule termed FTY720 is reported to activate phosphatase PP2A and lead to apoptosis and cell cycle arrest in lymphoma cells [55]. In the molecular mode of action study, we observed an increase in apoptotic cells, as well as an effect on phases of the cell cycle. Thus, an increase in the levels of PP2A could be one of the factors involved in this process in the PANC-1 cells.
Vimentin is expressed in mesenchymal tissue and is a marker of mesenchymal differentiation, but occasionally, epithelial cells that acquire mesenchymal phenotype express vimentin [56]. Studies carried out on patients with pancreatic cancer have shown poor survival in the presence of vimentin. Interestingly, a subset of patients exhibit autoantibodies to isoforms of vimentin, and their detection has been utilized for early diagnosis of pancreatic cancer [57]. One of the entities upregulated by AT and WZ in the present study was vimentin. Therefore, antibodies against vimentin could be harnessed for early diagnosis and also as treatment with vimentin-drug conjugates in combination with the kinase inhibitors AT and WZ.
Phosphatidylethanolamine-binding protein 1 (PEBP1), also termed RKIP, was another protein upregulated by both the inhibitors in the present study. PEBP1 has been reported in cancers of various tissue origins [58]. Reduced expression of RKIP is shown to correlate with poor prognosis in pancreatic cancer [59]. Further, the loss of RKIP leads to phenotypes with aggressive nature in PDAC [60]. Furthermore, this molecule has been reported as a metastasis suppressor in pancreatic cancer [61]. Thus, the upregulation of this pivotal molecule by AT and WZ could lead to a beneficial effect in reducing metastasis if used as a therapeutic agent for pancreatic cancers.
For cancers in general, and pancreatic cancers in particular, resistance to chemotherapeutic drugs poses a major problem in efficiently treating the disease [62]. To overcome this hurdle, cosensitizing agents are considered as options. Clinical trials have been conducted with cisplatin, gemcitabine, and other agents in patients with PDAC [63]. Moreover, the NADH dehydrogenase complex, which is one of the key enzymes involved in oxidative phosphorylation and cellular respiration and a key regulator of NAD+/NADH ratio, impacts sensitivity to platinum drugs. Notably, it has been reported that the knockout of NADH dehydrogenase complex subunit genes results in decreased sensitivity to oxaliplatin in pancreatic cells [64]. In the present study, an increase in the NADH dehydrogenase complex subunit was observed. The increase in this pivotal entity could be harnessed to increase the sensitivity of platinum drugs against pancreatic cancers if used in combination with the kinase inhibitors AT and WZ.
One of the downregulated entities by AT and WZ was the key molecule, transitional endoplasmic reticulum ATPase. The transitional endoplasmic reticulum ATPase (p97) plays a major role in various pathological processes. Notably, p97, an antiapoptotic molecule, leads to metastasis to lymph nodes resulting in a poor prognosis of pancreatic cancer [65]. The downregulation of this key moiety by AT and WZ in PANC-1 cells could be enormously beneficial as it would reduce metastasis and also lead to apoptosis. In PANC-1 cells, in the present study, we did observe apoptosis in the studies of the molecular mode of action with AT and WZ.
Vinculin was another molecule that was downregulated in the current study. An increase in vinculin expression leads to poor prognosis in pancreatic cancer patients and is considered an unfavorable prognostic indicator [66]. A decrease in vinculin by AT and WZ could lead to better outcomes in the survival of pancreatic cancer patients.
Prelamin A/C is processed into lamin A and is one of the mechano-regulating molecules in pancreatic cancers. An increase in this molecule leads to stiffness and invasiveness [67]. The downregulation of prelamin A/C by AT and WZ could prevent pancreatic cells from becoming invasive and reduce the aggressiveness of this lethal cancer.
T-complex protein 1 (TCP1) is a molecular chaperone. In cancers such as breast cancer and small cell lung cancer, it is reported that the upregulation of its expression is correlated with chemoresistance and metastasis [68]. In the present protein profiling study, we observed the downregulation of this entity in PANC-1 cells, and we envision that the downregulation of TCP1 could reduce metastasis as well as chemoresistance of PDAC upon treatment with AT and WZ.
Basic transcription factor (BTF3) was also one of the factors reduced by AT and WZ. As this transcription factor is involved in the expression of various cancer-associated genes, its decrease in AT-and WZ treated cells could lead to the decrease in cell viability that we observed at the cellular level analysis. In fact, the silencing of this entity is reported to lead to a decrease in cancer-associated genes in pancreatic cancer [69].
Stathmin was another one of the molecules downregulated by AT and WZ. Stathmin belongs to the microtubule dynamic destabilizing protein family. It is overexpressed in pancreatic cancer. It is reported that the knockdown of stathmin leads to a decrease in cell viability, colony-forming ability, and cell cycle arrest in the G2/M phase [70]. In fact, we observed a decrease in cell viability in the cellular-level analysis in the present study. Furthermore, in the analysis of the molecular mode of action, we observed cell cycle arrest in the G2/M phase in WT-treated samples.
The details of the cell signaling pathways and the entities impacted by AT and WZ are represented in the schematic diagram in Figure 10. that we observed at the cellular level analysis. In fact, the silencing of this entity is reported to lead to a decrease in cancer-associated genes in pancreatic cancer [69]. Stathmin was another one of the molecules downregulated by AT and WZ. Stathmin belongs to the microtubule dynamic destabilizing protein family. It is overexpressed in pancreatic cancer. It is reported that the knockdown of stathmin leads to a decrease in cell viability, colony-forming ability, and cell cycle arrest in the G2/M phase [70]. In fact, we observed a decrease in cell viability in the cellular-level analysis in the present study. Furthermore, in the analysis of the molecular mode of action, we observed cell cycle arrest in the G2/M phase in WT-treated samples.
The details of the cell signaling pathways and the entities impacted by AT and WZ are represented in the schematic diagram in Figure 10.

Conclusions
Multilevel analysis on treatment of PANC-1 cells with the Aurora kinase inhibitor (AT) and the EGFR kinase inhibitor (WZ), has highlighted pivotal entities that are impacted that belong to key cell signaling pathways. Notably, treatment with these kinase inhibitors could be harnessed in future studies for the efficacious treatment of pancreatic cancer as monotherapy due to their deleterious impact on cell-survival-related signaling pathways such as apoptosis, cell cycle, ROS, and Bcl-2 and also upregulation of entities that inhibit metastasis, decrease chemoresistance, increase immunogenic cell death, and inhibit cancer gene transcription. Moreover, proteomics analysis has revealed novel targets that can be exploited to design combination therapy with kinase inhibitors. Notably, an increase in LF could sensitize and also lead to better efficacious outcomes if synergized with immunotherapy against PD-1/PD-L1. Moreover, vimentin antibody-drug conjugates and increased efficacy with platinum drugs (because of the increase in NADH dehydrogenase complex) in combination with AT and WZ could be pursued. Therefore, future studies for the treatment of pancreatic cancer with AT and WZ are warranted.
Supplementary Materials: The following supporting information can be downloaded at www.mdpi.com/xxx/s1, Figure S1: A protein-protein interaction network generated using the STRING database. Each node represents a differentially regulated protein from the WZ-treated group. The blue color indicates upregulation, and the red indicates downregulation. The edges represent either known or predicted interactions, with a minimum required interaction score of 0.4 in the STRING database. The abbreviations in the above figure are mentioned in Table 1. that we observed at the cellular level analysis. In fact, the silencing of this entity is reported to lead to a decrease in cancer-associated genes in pancreatic cancer [69]. Stathmin was another one of the molecules downregulated by AT and WZ. Stathmin belongs to the microtubule dynamic destabilizing protein family. It is overexpressed in pancreatic cancer. It is reported that the knockdown of stathmin leads to a decrease in cell viability, colony-forming ability, and cell cycle arrest in the G2/M phase [70]. In fact, we observed a decrease in cell viability in the cellular-level analysis in the present study. Furthermore, in the analysis of the molecular mode of action, we observed cell cycle arrest in the G2/M phase in WT-treated samples.
The details of the cell signaling pathways and the entities impacted by AT and WZ are represented in the schematic diagram in Figure 10.

Conclusions
Multilevel analysis on treatment of PANC-1 cells with the Aurora kinase inhibitor (AT) and the EGFR kinase inhibitor (WZ), has highlighted pivotal entities that are impacted that belong to key cell signaling pathways. Notably, treatment with these kinase inhibitors could be harnessed in future studies for the efficacious treatment of pancreatic cancer as monotherapy due to their deleterious impact on cell-survival-related signaling pathways such as apoptosis, cell cycle, ROS, and Bcl-2 and also upregulation of entities that inhibit metastasis, decrease chemoresistance, increase immunogenic cell death, and inhibit cancer gene transcription. Moreover, proteomics analysis has revealed novel targets that can be exploited to design combination therapy with kinase inhibitors. Notably, an increase in LF could sensitize and also lead to better efficacious outcomes if synergized with immunotherapy against PD-1/PD-L1. Moreover, vimentin antibody-drug conjugates and increased efficacy with platinum drugs (because of the increase in NADH dehydrogenase complex) in combination with AT and WZ could be pursued. Therefore, future studies for the treatment of pancreatic cancer with AT and WZ are warranted.
Supplementary Materials: The following supporting information can be downloaded at www.mdpi.com/xxx/s1, Figure S1: A protein-protein interaction network generated using the STRING database. Each node represents a differentially regulated protein from the WZ-treated group. The blue color indicates upregulation, and the red indicates downregulation. The edges represent either known or predicted interactions, with a minimum required interaction score of 0.4 in the STRING database. The abbreviations in the above figure are mentioned in Table 1. that we observed at the cellular level analysis. In fact, the silencing of this entity is reported to lead to a decrease in cancer-associated genes in pancreatic cancer [69]. Stathmin was another one of the molecules downregulated by AT and WZ. Stathmin belongs to the microtubule dynamic destabilizing protein family. It is overexpressed in pancreatic cancer. It is reported that the knockdown of stathmin leads to a decrease in cell viability, colony-forming ability, and cell cycle arrest in the G2/M phase [70]. In fact, we observed a decrease in cell viability in the cellular-level analysis in the present study. Furthermore, in the analysis of the molecular mode of action, we observed cell cycle arrest in the G2/M phase in WT-treated samples.
The details of the cell signaling pathways and the entities impacted by AT and WZ are represented in the schematic diagram in Figure 10.

Conclusions
Multilevel analysis on treatment of PANC-1 cells with the Aurora kinase inhibitor (AT) and the EGFR kinase inhibitor (WZ), has highlighted pivotal entities that are impacted that belong to key cell signaling pathways. Notably, treatment with these kinase inhibitors could be harnessed in future studies for the efficacious treatment of pancreatic cancer as monotherapy due to their deleterious impact on cell-survival-related signaling pathways such as apoptosis, cell cycle, ROS, and Bcl-2 and also upregulation of entities that inhibit metastasis, decrease chemoresistance, increase immunogenic cell death, and inhibit cancer gene transcription. Moreover, proteomics analysis has revealed novel targets that can be exploited to design combination therapy with kinase inhibitors. Notably, an increase in LF could sensitize and also lead to better efficacious outcomes if synergized with immunotherapy against PD-1/PD-L1. Moreover, vimentin antibody-drug conjugates and increased efficacy with platinum drugs (because of the increase in NADH dehydrogenase complex) in combination with AT and WZ could be pursued. Therefore, future studies for the treatment of pancreatic cancer with AT and WZ are warranted.
Supplementary Materials: The following supporting information can be downloaded at www.mdpi.com/xxx/s1, Figure S1: A protein-protein interaction network generated using the STRING database. Each node represents a differentially regulated protein from the WZ-treated group. The blue color indicates upregulation, and the red indicates downregulation. The edges represent either known or predicted interactions, with a minimum required interaction score of 0.4 in the STRING database. The abbreviations in the above figure are mentioned in Table 1.
) entities in PANC-1 pancreatic cancer cells upon treatment with AT and WZ.

Conclusions
Multilevel analysis on treatment of PANC-1 cells with the Aurora kinase inhibitor (AT) and the EGFR kinase inhibitor (WZ), has highlighted pivotal entities that are impacted that belong to key cell signaling pathways. Notably, treatment with these kinase inhibitors could be harnessed in future studies for the efficacious treatment of pancreatic cancer as monotherapy due to their deleterious impact on cell-survival-related signaling pathways such as apoptosis, cell cycle, ROS, and Bcl-2 and also upregulation of entities that inhibit metastasis, decrease chemoresistance, increase immunogenic cell death, and inhibit cancer gene transcription. Moreover, proteomics analysis has revealed novel targets that can be exploited to design combination therapy with kinase inhibitors. Notably, an increase in LF could sensitize and also lead to better efficacious outcomes if synergized with immunotherapy against PD-1/PD-L1. Moreover, vimentin antibody-drug conjugates and increased efficacy with platinum drugs (because of the increase in NADH dehydrogenase complex) in combination with AT and WZ could be pursued. Therefore, future studies for the treatment of pancreatic cancer with AT and WZ are warranted.

Supplementary Materials:
The following supporting information can be downloaded at https:// www.mdpi.com/article/10.3390/biomedicines11061716/s1, Figure S1: A protein-protein interaction network generated using the STRING database. Each node represents a differentially regulated protein from the WZ-treated group. The blue color indicates upregulation, and the red indicates downregulation. The edges represent either known or predicted interactions, with a minimum required interaction score of 0.4 in the STRING database. The abbreviations in the above figure are mentioned in Table 1.
Author Contributions: Y.N.K. and K.P.: investigation, data curation, formal analysis, writing, final approval; J.M. and G.A.D.: investigation, experimental data analysis, final approval; S.B.P.: supervision and guidance in all phases of the study, providing resources, writing, and final approval. All authors have read and agreed to the published version of the manuscript.