Pancreatic Cancer Cell Line MIA Research Articles

In silico evaluation and docking studies of pyrazole analogs as potential autophagy modulators against pancreatic cancer cell line MIA PaCa-2

A quantitative structure activity relationship (QSAR) model for a series of N-(1-benzyl-3,5-dimethyl-1H-pyrazole-4-yl) benzamide derivatives having autophagy inhibitory activities as potent anticancer agents was developed by the multiple linear regressions (MLR) method. In this study, previous compounds were used in the model development were divided into a set of fifteen compounds as training set and set of four compounds as test set. A model with high prediction ability and high correlation coefficients was obtained. This model showed r = 0.968, r2 = 0.937 and Q2 = 0.880, the QSAR model was also employed to predict the experimental compounds in an external test set, and to predict the activity of a new designed set of 3,5-dimethyl-4-substituted-pyrazole derivatives (1-15), result showed that compound 3 has the most promising inhibition activity (EC50 = 0.869 μM) against human pancreatic ductal adenocarcinoma cell MIA PaCa-2 compared to the reference chloroquine with (EC50 = 14 μM). Thus, the model showed good correlative and predictive ability. Docking studies was performed for designed compounds, docking analysis showed the best compound 1 with high docking affinity of -24.8616 kcal/mol.

Abstract 6343: Mitochondrial bioenergetics mediate chemo-resistance of cancer cells

Abstract Background: Resistance to chemotherapy is a major cause of the failure of treatments in many cancers including pancreatic cancer. Accumulated evidence indicates that mitochondrial metabolic alterations are associated with cancer cells growth and proliferation. In this project, we investigated the role of mitochondrial bioenergetics in drug resistance. Methods: Human pancreatic cancer cell lines MIA PaCa-2 and HPAF-II and mouse pancreatic cancer cell line UN-KPC-961 were treated with increased doses of chemotherapy drug Gemcitabine for 4 months until developing resistance. Mitochondrial proteins, cancer stemness markers and metabolic enzymes were measured by Western and RT-PCR. Cell survival was measured by MTT assay. Mitochondrial bioenergetics were measured by Seahorse Assay. Results: We found that gemcitabine resistant (GR) cells grow at a much slower rate than wild type (WT) cancer cells; however, they express a significantly higher amount of cancer stemness markers such as Nanog, Oct4 and Sox2. They also expressed higher level of pro-cancer cytokines such as IL-6 and IL-4. Seahorse analysis showed a significantly lower oxygen consumption rate (OCR) in GR cells compared to WT cells. OCR did not change much in GR cells when applying inhibitors of the mitochondrial respiratory chain complex. GR cells produced lower level of ATP and lower spare respiratory capacity. However, GR cells expressed the same amount of mitochondrial proteins such as TOM40, TOM70 and COX4 suggesting the same mass of mitochondria. Pyruvate dehydrogenase-E1 Alpha1 (PDHA1) expression level was significantly lower in GR cells compared to WT cells. PDHA1 inhibition induced WT cells to develop a partial resistance to Gemcitabine; whereas, PDH1 overexpression in GR cells made these cells less resistant to Gemcitabine. Conclusion: We showed for the first time the difference in mitochondrial bioenergetics between WT and GR cells and showed that GR cells grow independently of the mitochondrial energy production machinery. Furthermore, we identified PDHA1 as a critical mediator of cancer drug resistance. Citation Format: Fouzia Zayou, Chintan Chheda, Stephen Pandol, Mouad Edderkaoui. Mitochondrial bioenergetics mediate chemo-resistance of cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6343.

A G-quadruplex-binding compound shows potent activity in human gemcitabine-resistant pancreatic cancer cells

Gemcitabine is a drug of choice in the treatment of human pancreatic cancer. Chemo-resistance to this drug is common and has been attributed to a variety of distinct mechanisms, involving > 100 genes. A recently developed small-molecule G-quadruplex ligand, the trisubstituted naphthalene diimide compound CM03, has previously been shown to have equivalent potency to gemcitabine in the pancreatic cancer cell line MIA PaCa-2. We report here on cell lines of increased resistance to gemcitabine that have been generated from this line, with the most resistant having 1,000-fold reduced sensitivity to gemcitabine. These resistant lines retain nM sensitivity to CM03. The molecular basis for the retention of potency by this G-quadruplex ligand has been examined using whole transcriptome data analysis with RNA-seq. This has revealed that the pattern of pathways down regulated by CM03 in the parental MIA PaCa-2 cell line is largely unaffected in the gemcitabine-resistant line. The analysis has also shown that the expression patterns of numerous genes involved in gemcitabine sensitivity are down regulated in the resistant line upon CM03 treatment. These results are supportive of the concept that G-quadruplex small molecules such as CM03 have potential for clinical use in the treatment of gemcitabine-resistant human pancreatic cancer.

Mechanism of miRNA-26a on the Proliferation of Pancreatic Tumor Cells by Regulating the Expression of Cycline2

Background: qRT-PCR was used to measure the expression of miRNA-26a in pancreatic epithelial cells (HPDE) and human pancreatic cancer cell lines PANC-1 and MIA PaCa-2. Methods: PANC-1 and MIA PaCa-2 cell lines were infected with lentiviruses to construct PANC-miR-26a and MIA-miR-26a, and RT-PCR was used to detect the infection efficiency. The cell proliferation ability of PANC-miR-26a and MIA-miR-26a were examined by CCK-8 assay, and apoptosis was detected by flow cytometry. Western blotting was used to detect the expressions of CyclinE2 protein and mitochondria-associated apoptotic proteins. Results: miR-26a was expressed in human normal pancreatic epithelial cells (HPDE), and not detected in PANC-1 and MIA PaCa-2; miR-26a was highly expressed in the cell lines PANC-miR-26a and MIA-miR-26a infected by the virus particles. The absorbance values of PANC-miR-26a and MIA-miR-26a were lower than those of NC1 and PANC-1 in control group. The apoptosis rates of PANC-miR-26a and MIA-miR-26a were substantially higher than those of the control group. The overexpression of miR-26a inhibited the expression of the target protein CyclinE2 in PANC-miR-26a and MIA-miR-26a. The expression of the anti-apoptotic protein Bcl-2 was decreased in PANC-miR-26a and MIA-miR-26a, while the expression of the pro-apoptotic protein Bax was increased. Compared with HPDE, miR-26a was down-regulated in PANC-1 and MIA PaCa-2. After overexpression of miR-26a, the proliferation of PANC-1 and MIA PaCa-2 cell lines was weakened. Conclusion: Molecular mechanism is the negative regulation of CyclinE2 by miR-26a as well as the expressions of downstream mitochondrial apoptosis proteins Bcl-2 and Bax.

Bcl-2/Bcl-xL inhibitor navitoclax increases the antitumor effect of Chk1 inhibitor prexasertib by inducing apoptosis in pancreatic cancer cells via inhibition of Bcl-xL but not Bcl-2.

In our previous study, we showed that prexasertib, a checkpoint kinase 1 (Chk1) inhibitor, enhances the effects of standard drugs for pancreatic cancer, including gemcitabine (GEM), S-1, and the combination of GEM and S-1 (GS). The combination of prexasertib and GS has a strong antitumor effect and induces apoptosis in pancreatic cancer cells by downregulating anti-apoptotic protein Bcl-2. In the present study, we investigated the combined effect of GEM, S-1, and prexasertib with a selective Bcl-2 inhibitor (venetoclax) and a non-selective Bcl-2 inhibitor (navitoclax) in SUIT-2 pancreatic cancer cells. An MTT assay revealed that the combination of prexasertib with navitoclax showed a synergistic effect but the combination with venetoclax did not. Investigation of the pancreatic cancer cell lines SUIT-2, MIA PaCa-2, and BxPC-3 revealed that BxPC-3 also showed a high synergistic effect when combined with prexasertib and navitoclax but not venetoclax. Mechanistic analysis of the combined effect showed that apoptosis was induced. Bcl-2 knockdown with siRNA and prexasertib treatment did not induce apoptosis, whereas Bcl-xL knockdown with siRNA and prexasertib treatment resulted in strong induction of apoptosis. In addition, among the three cell lines, the combined effect of prexasertib and navitoclax resulted in increased apoptotic cell death because the protein expression levels of Bcl-xL and Chk1 were higher. Our results demonstrate that the combination of prexasertib and navitoclax has a strong antitumor effect and induces apoptosis in pancreatic cancer cells by downregulating Bcl-xL. Simultaneous inhibition of Chk1 and Bcl-xL could be a new strategy for treating pancreatic cancer.

Synergistic Anticancer Effects of Gemcitabine with Pitavastatin on Pancreatic Cancer Cell Line MIA PaCa-2 in vitro and in vivo.

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with an overall 5-year survival rate of 9.3%, and this malignancy is expected to become the second leading cause of cancer-related death by 2030. Gemcitabine resistance develops within weeks of PDAC patient’s chemotherapeutic initiation. Statins, including pitavastatin, have been indicated to have anticancer effects in numerous human cancer cell lines. Thus, in this study, we hypothesized that a combination of gemcitabine and pitavastatin may have a greater anticancer effect than gemcitabine alone on the human pancreatic carcinoma cell line MIA PaCa-2.MethodsThe anticancer effects of gemcitabine with pitavastatin were evaluated using human MIA PaCa-2 cell line in vitro and in vivo Balb/c murine xenograft tumor model. Cell viability was assessed with CCK-8, and cell migration was stained by crystal violet. Cell cycle distribution, apoptosis and mitochondrial membrane potential were examined by flow cytometry. Activation of drug transporters (hENTs, hCNTs), intracellular drug activating (dCK) and inhibition of inactivating enzymes (RRMs) pathways were assessed by Western blotting analysis. Molecular mechanisms and signaling pathways of apoptosis, necrosis and autophagy also were assessed by Western blotting.ResultsWe observed that gemcitabine and pitavastatin synergistically suppressed the proliferation of MIA PaCa-2 cells through causing sub-G1 and S phase cell cycle arrest. Activation of apoptosis/necrosis was confirmed by annexin V/propidium iodide double staining, which showed increasing levels of active caspase 3, cleaved poly(ADP-ribose) polymerase and the RIP1–RIP3–MLKL complex. Moreover, gemcitabine–pitavastatin-mediated S phase arrest downregulated cyclin A2/CDK2 and upregulated p21/p27 in MIA PaCa-2 cells. Furthermore, this combination improved drug cellular metabolism pathway, mitochondria function and activated autophagy as part of the cell death mechanism. In vivo, gemcitabine-pitavastatin effectively inhibited tumor growth in a nude mouse mode of Mia PaCa-2 xenografts without observed adverse effect.ConclusionCombined gemcitabine–pitavastatin may be an effective novel treatment option for pancreatic cancer.

Effect of N,N Coordination and RuII Halide Bond in Enhancing Selective Toxicity of a Tyramine-Based RuII (p-Cymene) Complex.

Ruthenium compounds are promising anticancer candidates owing to their lower side-effects and encouraging activities against resistant tumors. Half-sandwich piano-stool type RuII compounds of general formula [(L)RuII(η6-arene)(X)]+ (L = chelating bidentate ligand, X = halide) have exhibited significant therapeutic potential against cisplatin-resistant tumor cell lines. In RuII (p-cymene) based complexes, the change of the halide leaving group has led to several interesting features, viz., hydrolytic stability, resistance toward thiols, and alteration in pathways of action. Tyramine is a naturally occurring monoamine which acts as a catecholamine precursor in humans. We synthesized a family of N,N and N,O coordinated RuII (p-cymene) complexes, [(L)RuII(η6-arene)(X)]+ (1-4), with tyramine and varied the halide (X = Cl, I) to investigate the difference in reactivity. Our studies showed that complex 2 bearing N,N coordination with an iodido leaving group shows selective in vitro cytotoxicity against the pancreatic cancer cell line MIA PaCa-2 (IC50 ca. 5 μM) but is less toxic to triple-negative breast cancer (MDA-MB-231), hepatocellular carcinoma (Hep G2), and the normal human foreskin fibroblasts (HFF-1). Complex 2 displays stability toward hydrolysis and does not bind with glutathione, as confirmed by 1H NMR and ESI-HRMS experiments. The inert nature of 2 leads to enhancement of cytotoxicity (IC50 = 5.3 ± 1 μM) upon increasing the cellular treatment time from 48 to 72 h.

Upregulation of CD73 Confers Acquired Radioresistance and is Required for Maintaining Irradiation-selected Pancreatic Cancer Cells in a Mesenchymal State

The molecular mechanisms underlying exceptional radioresistance in pancreatic cancer remain elusive. In the present study, we established a stable radioresistant pancreatic cancer cell line MIA PaCa-2-R by exposing the parental MIA PaCa-2 cells to fractionated ionizing radiation (IR). Systematic proteomics and bioinformatics analysis of protein expression in MIA PaCa-2 and MIA PaCa-2-R cells revealed that several growth factor-/cytokine-mediated pathways, including the OSM/STAT3, PI3K/AKT, and MAPK/ERK pathways, were activated in the radioresistant cells, leading to inhibition of apoptosis and increased epithelial-mesenchymal plasticity. In addition, the radioresistant cells exhibited enhanced capabilities of DNA repair and antioxidant defense compared with the parental cells. We focused functional analysis on one of the most up-regulated proteins in the radioresistant cells, ecto-5'-nucleotidase (CD73), which is a cell surface protein that is overexpressed in different types of cancer. Ectopic overexpression of CD73 in the parental cells resulted in radioresistance and conferred resistance to IR-induced apoptosis. Knockdown of CD73 re-sensitized the radioresistant cells to IR and IR-induced apoptosis. The effect of CD73 on radioresistance and apoptosis is independent of the enzymatic activity of CD73. Further studies demonstrate that CD73 up-regulation promotes Ser-136 phosphorylation of the proapoptotic protein BAD and is required for maintaining the radioresistant cells in a mesenchymal state. Our findings suggest that expression alterations in the IR-selected pancreatic cancer cells result in hyperactivation of the growth factor/cytokine signaling that promotes epithelial-mesenchymal plasticity and enhancement of DNA repair. Our results also suggest that CD73, potentially a novel downstream factor of the enhanced growth factor/cytokine signaling, confers acquired radioresistance by inactivating proapoptotic protein BAD via phosphorylation of BAD at Ser-136 and by maintaining the radioresistant pancreatic cancer cells in a mesenchymal state.

Rivaroxaban does not affect growth of human pancreatic tumors in mice

BackgroundSome clinical studies have shown that low‐molecular‐weight heparins (LMWHs) prolong the survival of cancer patients. In addition, various anticoagulants have been shown to reduce growth of tumors in mice. However, there are no studies on the effect of the factor Xa inhibitor rivaroxaban on growth of human pancreatic tumors in nude mice. ObjectivesTo test the hypothesis that the factor Xa inhibitor rivaroxaban reduces the growth of tissue factor (TF)‐positive pancreatic tumors but not TF‐negative pancreatic tumors in mice. MethodsThe TF‐positive human pancreatic cancer cell line BxPc‐3 and the TF‐negative human pancreatic cancer cell line MIA PaCa‐2 were injected subcutaneously into nude mice and tumors grown to a mean volume of ~100 mm3. Mice were then divided into two groups. One group was fed chow containing rivaroxaban (0.5 g/kg chow) whereas the other group was fed chow without rivaroxaban. ResultsRivaroxaban significantly prolonged prothrombin time in tumor‐bearing mice. Rivaroxaban did not affect cell proliferation or growth of either BxPc‐3 or MIA PaCa‐2 tumors grown subcutaneously in nude mice. ConclusionOur results indicate that inhibition of factor Xa with rivaroxaban does not affect the growth of two human pancreatic tumors in nude mice.

MicroRNA-296-5p Promotes Cell Invasion and Drug Resistance by Targeting Bcl2-Related Ovarian Killer, Leading to a Poor Prognosis in Pancreatic Cancer

Background/Aims: Pancreatic ductal adenocarcinoma (PDAC) is characterized by aggressive invasion, early metastasis, and resistance to chemotherapy, leading to a poor prognosis. To clarify the molecular mechanism of these malignant characteristics, we performed a genome-wide microRNA (miRNA) array analysis utilizing micro-cancer tissues from patients with unresectable PDAC (stage IV), obtained by endoscopic ultrasound-fine needle aspiration (EUS-FNA). Methods: The expression profiles of 2,042 miRNAs were determined using micro-cancer tissues from 13 patients with unresectable PDAC obtained by EUS-FNA. The relationship between individual miRNA levels and overall survival (OS) was analyzed. Possible target genes for miRNAs were bioinformatically analyzed using the online database miRDB. Pancreatic cancer cell lines PANC-1, MIA PaCa-2, and PK-8 were transfected with miRNA mimic or small interfering RNA, and cell invasion, epithelial-mesenchymal transition (EMT), and apoptosis markers were examined. miRNA and mRNA expressions were examined by quantitative polymerase chain reaction. Results: Of 2,042 miRNAs, the 10 that exhibited the lowest correlation coefficient (p ≤ 0.005) between miRNA expression level and OS among the patients were identified. The miRDB and expression analysis in cancer cell lines for the 10 miRNAs identified miR-296-5p and miR-1207-5p as biomarkers predictive of shorter survival (p < 0.0005). Bioinformative target gene analysis and transfection experiments with miRNA mimics showed that Bcl2-related ovarian killer (BOK), a pro-apoptotic gene, is a target for miR296-5p in pancreatic cancer cells; transfection of miR-296-5p mimic into PANC-1, MIA PaCa-2, and PK-8 cells resulted in significant suppression of BOK mRNA and protein expression. These transfectants showed significantly higher invasion capability compared with control cells, and knock down of BOK in pancreatic cancer cells similarly enhanced invasion capability. Transfectants of miR-296-5p mimic also exhibited aberrant expression of EMT markers, including vimentin and N-cadherin. Moreover, these transfectants showed a significantly lower apoptosis rate in response to 5-fluorouracil and gemcitabine with a decrease of BOK expression, suggesting a role of miR-296-5p in drug resistance. Conclusion: These results suggest that miR-296-5p is a useful biomarker for a poor prognosis in patients with PDAC, and that the miR-296-5p/BOK signaling axis plays an important role in cell invasion, drug resistance, and EMT in PDACs.

Abstract 1821: LINC00346 regulates c-Myc transcriptional activity through CTCF in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC), a most challenging malignant disease, is estimated to become the second leading cause of cancer-related deaths in the United States between the year 2020 and 2030. Although the aberrant activation of KRAS has been demonstrated as the driving force of PDAC progression, the contribution of other molecules, especially long non-coding RNAs (lncRNAs), to PDAC progression remain largely unknown. In the present study, we interrogated the pancreatic adenocarcinoma dataset of the Cancer Genome Atlas (TCGA) consisting of 186 cases. Among 2,730 lncRNAs analyzed, LINC00346 revealed the highest alterations among these lncRNAs with 11% frequency at the RNA level. In addition, its upregulation was significantly associated with overall survival (OS) and disease-free survival (DFS), respectively. To determine the biological function of LINC00346 in pancreatic cancer, we next performed loss-of-function assay in pancreatic cancer cell lines MIA PaCa-2 and BxPC-3 by CRISPR/Cpf1 technology using dual gRNA approach. The results showed that knockout (KO) of LINC00346 impaired pancreatic cancer cell proliferation, tumorigenesis, migration and invasion ability, while these phenotypes were restored by LINC00346 re-expression in KO cells (i.e., rescue experiment). Mechanistically, LINC00346 functions as a positive regulator of c-Myc. Further characterizations demonstrated that LINC00346 transcriptionally activates c-Myc expression by interaction with CTCF. Together, these results suggest that LINC00346 contributes to PDAC pathogenesis by activating c-Myc, and thus, LINC00346 may serve as a potential target for pancreatic cancer therapy. Note: This abstract was not presented at the meeting. Citation Format: Wan-Xin Peng, Yin-Yuan Mo. LINC00346 regulates c-Myc transcriptional activity through CTCF in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1821.

Abstract 1735: Cathepsin B knocked down in pancreatic cancer cell lines fail to establish tumor in nude mice and influences the differential expression of circular RNA

Abstract Most malignant tumors show increased transcriptional activation of cathepsin B. Over expression of cathepsin B has been associated with different malignancies and this has made it an attractive pharmacological target. Tumor progression can be defined in several steps with respect to proteases. The first step requires that surrounding tissue be cleared to create a pathway for growth by modification and degradation of the extracellular matrix and basement membrane. Cathepsin B is one of the cathepsins that has been directly linked to extracellular matrix degradation. The second is a proteolytic cascade where activation of matrix metalloproteinases and urokinase plasminogen activator (uPA), results in further tissue invasion, this activation is known to be mediated by Cathepsin B. The last is cleavage of E-cadherin at adherin junctions, detaching cells to enter circulation and migrate. Cathepsin B plays a central role in these processes. Methods: In this study, we used two pancreatic cancer cell lines PANC-1 and MIA PaCa-2 that show differential sensitivity to gemcitabine. We knocked down the expression of cathepsin B in both PANC-1 and MIA PaCa-2 cells using CrisperCas9. We determined the tumorogenic potential of these down regulated cells and determined changes in gemcitabine sensitivity. Further, we determined the global expression profiles of circular RNA to predict the post-transcriptional regulation of associated microRNAs associated with chemo-sensitivity. Results: From the tumorogenic studies, we observed that both PANC-1 and MIA PaCa-2 cells downregulated for Cathepsin B failed to develop tumors in nude mice subcutaneous tumor models. Further, from the gemcitabine sensitivity studies using the MTT assay we observed that Cathepsin B down regulation reduced IC50 in both PANC and MIA PaCa-2 by at least 25%. Global analysis of circular RNA expression showed that Cathepsin B downregulated PANC-1 cells showed a 4.2 fold increase in the expression of hsa_circRNA_081069 and a 3.8 fold decrease in the expression of hsa_circRNA_104169. In case of MIA PaCa-2 cells downregulated for Cathepsin B, we observed that hsa_circRNA_042488 was upregulated by 15.9 fold and hsa_circRNA_101692 was decreased by 20.5 fold. Conclusions: From our observations, we conclude that Cathepsin B expression is necessary for (1) tumor establishment and development; (2) gemcitabine resistance and (3) modulation of circular RNA expression that regulate oncogenic and metabolic pathways necessary for tumorigenesis. Citation Format: Manu Gnanamony, Liang Ge, Tulika Chatterjee, Christopher S. Gondi. Cathepsin B knocked down in pancreatic cancer cell lines fail to establish tumor in nude mice and influences the differential expression of circular RNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1735.

Crucial relationship of Neural Wiskott Aldrich syndrome protein(N-WASP) and Lysyl oxidase-like 2(LOXL2) in the promotion of pancreatic cancer metastasis

Introduction: Pancreatic cancer has been widely known to have aggressive malignancy and poor prognosis. N-WASP is part of the WASP family that regulate actin polymerization by activating Arp2/3 complex. FAK(Focal adhesion kinase) promotes N-WASP and epithelial mesenchymal transition(EMT) in cancer metastasis. The overexpression of LOXL2 promotes distant metastasis in pancreatic cancer. However, there is insufficient evidence of the relationship between LOXL2 and N-WASP in pancreatic cancer. In this study, we aimed to confirm the expression of N-WASP and relation between LOXL2 and N-WASP in pancreatic cancer. Method: Between June 2002 and December 2012, 81 patients underwent radical curative resection for pancreatic cancer at Gangnam Severance Hospital. Pancreatic cancer cell lines MIA PaCa-2, PANC-1 were used for in vitro study. To evaluate the endogenous expression level of N-WASP, we purified the whole RNA and protein to perform the qPCR, RT-PCR and Western blot. And we confirmed the motility and invasiveness of MIA PaCa-2 and PANC-1. Result: High expression of LOXL2 and N-WASP was observed and correlated with the expression of mesenchymal markers (Snail, L1CAM, Vimentin). We generated stably LOXL2 silenced pancreatic cancer cell which exhibited significant changes of factors related to invasiveness and N-WASP expression. Importantly, silencing of N-WASP markedly decrease in motility and invasiveness of MIA PaCa-2 and PANC-1 as determined from wound healing assay. Conclusion: Our results demonstrated that N-WASP is a regulator of EMT for migration and invasion in pancreatic cancer. LOXL2 and N-WASP have a powerful relationship for activating the promotion of EMT in pancreatic cancer metastasis. These findings suggest that N-WASP can be a target for the determining of distant metastasis in pancreatic cancer.

Application of Integrated Drug Screening/Kinome Analysis to Identify Inhibitors of Gemcitabine-Resistant Pancreatic Cancer Cell Growth

Continuous exposure of a pancreatic cancer cell line MIA PaCa-2 (MiaS) to gemcitabine resulted in the formation of a gemcitabine-resistant subline (MiaR). In an effort to discover kinase inhibitors that inhibited MiaR growth, MiaR cells were exposed to kinase inhibitors (PKIS-1 library) in a 384-well screening format. Three compounds (UNC10112721A, UNC10112652A, and UNC10112793A) were identified that inhibited the growth of MiaR cells by more than 50% (at 50 nM). Two compounds (UNC10112721A and UNC10112652A) were classified as cyclin-dependent kinase (CDK) inhibitors, whereas UNC10112793A was reported to be a PLK inhibitor. Dose–response experiments supported the efficacy of these compounds to inhibit growth and increase apoptosis in 2D cultures of these cells. However, only UNC10112721A significantly inhibited the growth of 3D spheroids composed of MiaR cells and GFP-tagged cancer-associated fibroblasts. Multiplexed inhibitor bead (MIB)–mass spectrometry (MS) kinome competition experiments identified CDK9, CLK1-4, DYRK1A, and CSNK1 as major kinase targets for UNC10112721A in MiaR cells. Another CDK9 inhibitor (CDK-IN-2) replicated the growth inhibitory effects of UNC10112721A, whereas inhibitors against the CLK, DYRK, or CSNK1 kinases had no effect. In summary, these studies describe a coordinated approach to discover novel kinase inhibitors, evaluate their efficacy in 3D models, and define their specificity against the kinome.

Abstract 585: Exosomal EphA2 transmits chemoresistance and predicts pancreatic cancer patient responses to therapy

Abstract Exosomes are small extracellular vesicles secreted by most cells that are found in blood and other bodily fluids, and which contain cytoplasmic material and membrane factors corresponding to their cell type of origin. Exosome membrane factors and contents have been reported to alter adjacent and distant cell behavior in multiple studies, but the impact of cancer-derived exosomes on chemoresistance is less clear. We therefore analyzed exosomes from the human pancreatic cancer (PC) cell lines PANC-1, MIA PaCa-2 and BxPC-3 for their ability to transmit chemoresistance, finding that PANC-1 exosomes increased the gembicitabine (GEM) resistance of MIA PaCa-2 and BxPC-3 cell cultures. Comparative proteomics determined that PANC-1 exosomes overexpressed Ephrin type-A receptor 2 (EphA2) versus exosomes of less chemoresistant PC cell lines MIA PaCa-2 and BxPC-3. EphA2-knockdown in PANC-1 cells inhibited their ability to transmit exosome-mediated chemoresistance to MIA PaCa-2 and BxPC-3, while treatment of MIA PaCa-2 and BxPC-3 cells with soluble EphA2 did not promote chemoresistance, indicating that membrane carried EphA2 was important for the EphA2 chemoresistance effect. EphA2 levels on circulating exosomes were markedly increased in mice bearing PANC-1 tumors and PC patients who subsequently revealed poor responses to treatment. We thus concluded that exosomal EphA2 expression could transmit chemoresistance and may serve as a minimally-invasive predictive biomarker for PC treatment response. Further work should address whether additional exosomal factors regulate resistance to other cancer therapeutic agents for PC or other cancer types. Citation Format: Jia Fan, Qian Wei, Eugene J. Koay, Yang Liu, Zhen Zhao, Ye Hu. Exosomal EphA2 transmits chemoresistance and predicts pancreatic cancer patient responses to therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 585.

Small-molecule screening yields a compound that inhibits the cancer-associated transcription factor Hes1 via the PHB2 chaperone

The transcription factor Hes family basic helix-loop-helix transcription factor 1 (Hes1) is a downstream effector of Notch signaling and plays a crucial role in orchestrating developmental processes during the embryonic stage. However, its aberrant signaling in adulthood is linked to the pathogenesis of cancer. In the present study, we report the discovery of small organic molecules (JI051 and JI130) that impair the ability of Hes1 to repress transcription. Hes1 interacts with the transcriptional corepressor transducing-like enhancer of split 1 (TLE1) via an interaction domain comprising two tryptophan residues, prompting us to search a chemical library of 1,800 small molecules enriched for indole-like π-electron-rich pharmacophores for a compound that blocks Hes1-mediated transcriptional repression. This screening identified a lead compound whose extensive chemical modification to improve potency yielded JI051, which inhibited HEK293 cell proliferation with an EC50 of 0.3 μm Unexpectedly, using immunomagnetic isolation and nanoscale LC-MS/MS, we found that JI051 does not bind TLE1 but instead interacts with prohibitin 2 (PHB2), a cancer-associated protein chaperone. We also found that JI051 stabilizes PHB2's interaction with Hes1 outside the nucleus, inducing G2/M cell-cycle arrest. Of note, JI051 dose-dependently reduced cell growth of the human pancreatic cancer cell line MIA PaCa-2, and JI130 treatment significantly reduced tumor volume in a murine pancreatic tumor xenograft model. These results suggest a previously unrecognized role for PHB2 in the regulation of Hes1 and may inform potential strategies for managing pancreatic cancer.

Detection and proteomic characterization of extracellular vesicles in human pancreatic juice

AimsThe prognosis of patients with pancreatic cancer has remained virtually unchanged with a high mortality rate compared to other types of cancers. An earlier detection would provide a time window of opportunity for treatment and prevention of deaths.In the present study we investigated extracellular vesicle (EV)-associated potential biomarkers for pancreatic cancer by directly assessing EV size-based subpopulations in pancreatic juice samples of patients with chronic pancreatitis or pancreatic cancer. In addition, we also studied blood plasma and pancreatic cancer cell line-derived EVs. MethodsComparative proteomic analysis was performed of 102 EV preparations from human pancreatic juices, blood, and pancreatic cancer cell lines Capan-1 and MIA PaCa-2. EV preparations were also characterized by electron microscopy, tunable resistive pulse sensing, and flow cytometry. ResultsHere we describe the presence of EVs in human pancreatic juice samples. Pancreatic juice EV-associated proteins that we identified as possible candidate markers for pancreatic cancer included mucins, such as MUC1, MUC4, MUC5AC, MUC6 and MUC16, CFTR, and MDR1 proteins. These candidate biomarkers could also be detected by flow cytometry in EVs found in pancreatic juice and those secreted by pancreatic cancer cell lines. ConclusionsTogether our data show that detection and characterization of EVs directly in pancreatic juice is feasible and may prove to be a valuable source of potential biomarkers of pancreatic cancer.

Synergistic Anti-Cancer Effects of AKT and SRC Inhibition in Human Pancreatic Cancer Cells.

PurposeTo investigate the effect of combined inhibition of protein kinase B (AKT) and SRC on the growth and metastatic potential of human pancreatic cancer cells.Materials and MethodsAKT and SRC were inhibited using 10-DEBC and PP2, respectively. The expression of their messenger RNAs were down-regulated by specific small interfering RNA (siRNA). Changes in pancreatic cancer cell growth and metastatic potential were determined using a cell viability assay and a xenotransplant model of pancreatic cancer, as well as cell migration and invasion assays. Signal proteins were analyzed by Western blot.ResultsThe inhibitors 10-DEBC and PP2 suppressed cell proliferation in a dose-dependent fashion in pancreatic cancer cell lines MIA PaCa-2 and PANC-1. The simultaneous inhibition of AKT and SRC at low concentrations resulted in a significant suppression of cell proliferation. Knockdown of AKT2 and SRC using siRNAs also significantly decreased cell proliferation. In a pancreatic cancer model, combined treatment with 10-DEBC and PP2 also significantly suppressed the growth of pancreatic cancer. Application of 10-DEBC with PP2 significantly reduced the metastatic potential of pancreatic cancer cells by inhibiting migration and invasion. The combined inhibition suppressed the phosphorylation of mTOR and ERK in pancreatic cancer cells.ConclusionCombined targeting of AKT and SRC resulted in a synergistic efficacy against human pancreatic cancer growth and metastasis.

Targeted Delivery of Auristatin-Modified Toxins to Pancreatic Cancer Using Aptamers.

Pancreatic cancer is one of the most lethal malignancies. Treatment with the first-line agent, gemcitabine, is often unsuccessful because it, like other traditional chemotherapeutic agents, is non-specific, resulting in off-target effects that necessitate administration of subcurative doses. Alternatively, monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) are highly toxic small molecules that require ligand-targeted delivery. MMAE has already received FDA approval as a component of an anti-CD30 antibody-drug conjugate, brentuximab vedotin. However, in contrast to antibodies, aptamers have distinct advantages. They are chemicals, which allows them to be produced synthetically and facilitates the rapid development of diagnostics and therapeutics with clinical applicability. In addition, their small size allows for enhanced tissue distribution and rapid systemic clearance. Here, we assayed the toxicity of MMAE and MMAF conjugated to an anti-transferrin receptor aptamer, Waz, and an anti-epidermal growth factor receptor aptamer, E07, on the pancreatic cancer cell lines Panc-1, MIA PaCa-2, and BxPC3. In vitro, our results indicate that these aptamers are a viable option for the targeted delivery of toxic payloads to pancreatic cancer cells.

Quantitative Differences in Physical Size, H 2 O 2 -Removal, and Cellular Bioenergetics in the Human Pancreatic Cancer Cell Lines MIA PaCa-2 and Panc-1: Impact on Susceptibilities to Ascorbate/H 2 O 2

We have been using quantitative approaches to better understand how externally/internally generated reactive species, metabolism, cell signaling and death pathways lead to differential sensitivities to cancer treatment. Studying aspects of cell biology in more quantitative and absolute terms allows direct comparisons to be made and unknown relationships to be discovered that may be therapeutically actionable to enhance treatment efficacy, prevent treatment-limiting complications or therapy/cross resistance. We have focused primarily on using quantitative approaches on a set of human pancreatic cancer cell lines, MIA PaCa-2 and Panc-1, which are differentially sensitive to high concentrations of ascorbate. High-dose ascorbate acts as a pro-drug that generates extracellular H2O2 in vivo and in vitro. We have identified cell size (volume), catalase levels, changes in GSH, and quantitative differences in cellular bioenergetics (ATP) as major contributors to the differential toxicity seen in this pair of cell lines. For example, Panc-1 has a much higher rate of ATP-linked oxygen consumption compared to MIA PaCa-2 as determined using a Seahorse XF96 extracellular flux analyzer. Using our quantitative approaches along with the new proton efflux rate (PER) developed by Agilent/Seahorse, we are able to quantitatively estimate the basal and maximal ATP generation rate from respiration and glycolysis on a per cell basis for this cell line pair. These quantitative approaches to cellular bioenergetics allows a direct comparison of the rates of ATP generation in these two cell lines and the role that bioenergetics may play in cell susceptibility to ascorbate/ H2O2.