Experimental Evolution Generates Novel Oncolytic Vesicular Stomatitis Viruses with Improved Replication in Virus-Resistant Pancreatic Cancer Cells

ABSTRACT Vesicular stomatitis virus (VSV) based oncolytic viruses are promising agents against various cancers. We have shown that pancreatic ductal adenocarcinoma (PDAC) cell lines exhibit great diversity in susceptibility and permissibility to VSV. Here, using a directed evolution approach with our two previously described oncolytic VSV recombinants, VSV-p53wt and VSV-p53-CC, we generated novel oncolytic VSVs with an improved ability to replicate in virus-resistant PDAC cell lines. VSV-p53wt and VSV-p53-CC encode a VSV matrix protein (M) with a ΔM51 mutation (M-ΔM51) and one of two versions of a functional human tumor suppressor, p53, fused to a far-red fluorescent protein, eqFP650. Each virus was serially passaged 32 times (which accounts for more than 60 viral replication cycles) on either the SUIT-2 (moderately resistant to VSV) or MIA PaCa-2 (highly permissive to VSV) human PDAC cell lines. While no phenotypic changes were observed for MIA PaCa-2-passaged viruses, both SUIT-2-passaged VSV-p53wt and VSV-p53-CC showed improved replication in SUIT-2 and AsPC-1, another human PDAC cell line also moderately resistant to VSV, while remaining highly attenuated in nonmalignant cells. Surprisingly, two identical VSV glycoprotein (VSV-G) mutations, K174E and E238K, were identified in both SUIT-2-passaged viruses. Additional experiments indicated that the acquired G mutations improved VSV replication, at least in part due to improved virus attachment to SUIT-2 cells. Importantly, no mutations were found in the M-ΔM51 protein, and no deletions or mutations were found in the p53 or eqFP650 portions of virus-carried transgenes in any of the passaged viruses, demonstrating long-term genomic stability of complex VSV recombinants carrying large transgenes. IMPORTANCE Vesicular stomatitis virus (VSV)-based oncolytic viruses are promising agents against pancreatic ductal adenocarcinoma (PDAC). However, some PDAC cell lines are resistant to VSV. Here, using a directed viral evolution approach, we generated novel oncolytic VSVs with an improved ability to replicate in virus-resistant PDAC cell lines, while remaining highly attenuated in nonmalignant cells. Two independently evolved VSVs obtained 2 identical VSV glycoprotein mutations, K174E and E238K. Additional experiments indicated that these acquired G mutations improved VSV replication, at least in part due to improved virus attachment to SUIT-2 cells. Importantly, no deletions or mutations were found in the virus-carried transgenes in any of the passaged viruses. Our findings demonstrate long-term genomic stability of complex VSV recombinants carrying large transgenes and support further clinical development of oncolytic VSV recombinants as safe therapeutics for cancer.

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Ruxolitinib and Polycation Combination Treatment Overcomes Multiple Mechanisms of Resistance of Pancreatic Cancer Cells to Oncolytic Vesicular Stomatitis Virus

Journal of Virology ◽

10.1128/jvi.00461-17 ◽

2017 ◽

Vol 91 (16) ◽

Cited By ~ 13

Author(s):

Sébastien A. Felt ◽

Gaith N. Droby ◽

Valery Z. Grdzelishvili

Keyword(s):

Cell Line ◽

Cell Lines ◽

Vesicular Stomatitis Virus ◽

Oncolytic Virus ◽

Ductal Adenocarcinoma ◽

Mechanisms Of Resistance ◽

Pdac Cell ◽

Ldl Uptake ◽

Vesicular Stomatitis ◽

Pdac Cell Line

ABSTRACT Vesicular stomatitis virus (VSV) is a promising oncolytic virus (OV). Although VSV is effective against a majority of pancreatic ductal adenocarcinoma cell (PDAC) cell lines, some PDAC cell lines are highly resistant to VSV, and the mechanisms of resistance are still unclear. JAK1/2 inhibitors (such as ruxolitinib and JAK inhibitor I) strongly stimulate VSV replication and oncolysis in all resistant cell lines but only partially improve the susceptibility of resistant PDACs to VSV. VSV tumor tropism is generally dependent on the permissiveness of malignant cells to viral replication rather than on receptor specificity, with several ubiquitously expressed cell surface molecules playing a role in VSV attachment to host cells. However, as VSV attachment to PDAC cells has never been tested before, here we examined if it was possibly inhibited in resistant PDAC cells. Our data show a dramatically weaker attachment of VSV to HPAF-II cells, the most resistant human PDAC cell line. Although sequence analysis of low-density lipoprotein (LDL) receptor (LDLR) mRNA did not reveal any amino acid substitutions in this cell line, HPAF-II cells displayed the lowest level of LDLR expression and dramatically lower LDL uptake. Treatment of cells with various statins strongly increased LDLR expression levels but did not improve VSV attachment or LDL uptake in HPAF-II cells. However, LDLR-independent attachment of VSV to HPAF-II cells was dramatically improved by treating cells with Polybrene or DEAE-dextran. Moreover, combining VSV with ruxolitinib and Polybrene or DEAE-dextran successfully broke the resistance of HPAF-II cells to VSV by simultaneously improving VSV attachment and replication. IMPORTANCE Oncolytic virus (OV) therapy is an anticancer approach that uses viruses that selectively infect and kill cancer cells. This study focuses on oncolytic vesicular stomatitis virus (VSV) against pancreatic ductal adenocarcinoma (PDAC) cells. Although VSV is effective against most PDAC cells, some are highly resistant to VSV, and the mechanisms are still unclear. Here we examined if VSV attachment to cells was inhibited in resistant PDAC cells. Our data show very inefficient attachment of VSV to the most resistant human PDAC cell line, HPAF-II. However, VSV attachment to HPAF-II cells was dramatically improved by treating cells with polycations. Moreover, combining VSV with polycations and ruxolitinib (which inhibits antiviral signaling) successfully broke the resistance of HPAF-II cells to VSV by simultaneously improving VSV attachment and replication. We envision that this novel triple-combination approach could be used in the future to treat PDAC tumors that are highly resistant to OV therapy.

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Expanding the Spectrum of Pancreatic Cancers Responsive to Vesicular Stomatitis Virus-Based Oncolytic Virotherapy: Challenges and Solutions

Cancers ◽

10.3390/cancers13051171 ◽

2021 ◽

Vol 13 (5) ◽

pp. 1171

Author(s):

Molly C. Holbrook ◽

Dakota W. Goad ◽

Valery Z. Grdzelishvili

Keyword(s):

Cell Lines ◽

Vesicular Stomatitis Virus ◽

Constitutive Expression ◽

The United States ◽

Ductal Adenocarcinoma ◽

Type I ◽

Phase I Clinical Trials ◽

Pdac Cell ◽

Viral Attachment ◽

Vesicular Stomatitis

Pancreatic ductal adenocarcinoma (PDAC) is a devastating malignancy with poor prognosis and a dismal survival rate, expected to become the second leading cause of cancer-related deaths in the United States. Oncolytic virus (OV) is an anticancer approach that utilizes replication-competent viruses to preferentially infect and kill tumor cells. Vesicular stomatitis virus (VSV), one such OV, is already in several phase I clinical trials against different malignancies. VSV-based recombinant viruses are effective OVs against a majority of tested PDAC cell lines. However, some PDAC cell lines are resistant to VSV. Upregulated type I IFN signaling and constitutive expression of a subset of interferon-simulated genes (ISGs) play a major role in such resistance, while other mechanisms, such as inefficient viral attachment and resistance to VSV-mediated apoptosis, also play a role in some PDACs. Several alternative approaches have been shown to break the resistance of PDACs to VSV without compromising VSV oncoselectivity, including (i) combinations of VSV with JAK1/2 inhibitors (such as ruxolitinib); (ii) triple combinations of VSV with ruxolitinib and polycations improving both VSV replication and attachment; (iii) combinations of VSV with chemotherapeutic drugs (such as paclitaxel) arresting cells in the G2/M phase; (iv) arming VSV with p53 transgenes; (v) directed evolution approach producing more effective OVs. The latter study demonstrated impressive long-term genomic stability of complex VSV recombinants encoding large transgenes, supporting further clinical development of VSV as safe therapeutics for PDAC.

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Investigating autophagy and glutamine metabolism as therapeutic targets for pancreatic cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2015.33.3_suppl.381 ◽

2015 ◽

Vol 33 (3_suppl) ◽

pp. 381-381

Author(s):

Mario Jardon ◽

Steve Kalloger ◽

Christina Iggulden ◽

Nancy Erro Go ◽

Paalini Sathiyaseelan ◽

...

Keyword(s):

Cell Lines ◽

Therapeutic Targets ◽

Glutamine Metabolism ◽

Ductal Adenocarcinoma ◽

Pdac Cell ◽

Multiple Cancer ◽

Tissue Micro Array ◽

Cancer Hallmarks ◽

Functional Studies ◽

New Therapeutic Approaches

381 Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest types of cancer, for which new therapeutic approaches are urgently needed. We are developing novel combination therapy approaches based on the inhibition of glutamine metabolism and autophagy, to improve current treatments for PDAC. Since both processes are key mediators of multiple cancer hallmarks, and have inter-related but non-redundant roles, this combination may result in a more efficient disruption of cancer cell resistance to treatments. Methods: We interrogated the Pancreas Centre BC tissue micro-array, containing the epithelial component of 252 PDAC samples, for expression of three key glutamine-metabolizing proteins and two autophagy-related proteins: glutamine synthetase (GLUL), asparagine synthetase (ASNS), glutaminase C (GLS-GAC), microtubule-associated protein 1 light chain 3 beta (MAP1-LC3B or LC3B) and autophagy-related protein 4B (ATG4B). While previous efforts by other groups have focused on GLS-GAC, the role of GLUL in cancer has remained less well understood. We thus investigated the functional relevance of GLUL using a panel of PDAC cell lines. We are also investigating interactions between glutamine metabolism and autophagy, including the exploration of strategies to target GLUL and the evaluation of a new class of ATG4B inhibitors for the treatment of pancreatic cancers. Results: We found that GLUL, ASNS, GLS-GAC, LC3B and ATG4B were expressed in 31%, 58%, 99%, 51% and 73%, respectively, of PDAC samples. Furthermore, higher LC3B expression correlated with poor outcome. Our functional studies revealed that various PDAC cells express GLUL, which can be upregulated upon glutamine deprivation. In all cell lines tested, GLUL knockdown sensitized them to gemcitabine, as assessed by a long-term recovery assay. We also found that the candidate ATG4B inhibitors, shown to inhibit this target in cell-free assays, effectively inhibit PDAC cell proliferation at micromolar concentrations. Conclusions: Our study reveals that glutamine metabolism and autophagy are clinically relevant in PDAC and may have potential as therapeutic targets. Supported by Pancreas Centre BC, BC Cancer Foundation and VGH Foundation.

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Robo2 Predicts a Better Prognosis and Inhibits Malignant Behavior in Vivo in Pancreatic Ductal Adenocarcinoma

10.21203/rs.3.rs-148994/v1 ◽

2021 ◽

Author(s):

Cheng Ding ◽

Yatong Li ◽

Shunda Wang ◽

Cheng Xing ◽

Lixin Chen ◽

...

Keyword(s):

Cell Proliferation ◽

Western Blot ◽

Pancreatic Ductal Adenocarcinoma ◽

Rna Sequencing ◽

Cell Lines ◽

Ductal Adenocarcinoma ◽

Migration And Invasion ◽

Pdac Cell ◽

Pancreatic Cell

Abstract BackgroundPancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with an extremely poor prognosis and a high mortality rate. Genome-wide studies have shown that the SLIT/ROBO signaling pathway plays an important role in pancreatic tumor development and progression. However, the effect and mechanism of ROBO2 in the progression of pancreatic cancer remains largely unknown.MethodsIn this study, real-time polymerase chain reaction (RT-PCR) and western blot analyses were adopted to evaluate the expression level of ROBO2 and proteins in pancreatic cell lines. Cell migration and invasion and cell proliferation were conducted in AsPC-1 and MIA PaCa-2 cell lines. RNA sequencing and western blot were undertaken to explore the mechanisms and potential targeted molecules. ROBO2 expression in tumor tissues was evaluated by immunohistochemistry in 95 patients.ResultsROBO2 expression was downregulated in PDAC cell lines and tissue samples. A high level of ROBO2 was associated with good overall survival. Upregulation of ROBO2 inhibited PDAC cell proliferation, migration, and invasion, whereas the opposite results were found in the ROBO2 downregulation group. In addition, xenograft animal models further confirmed the effect of ROBO2 on proliferation. Finally, the RNA sequencing results indicated that ROBO2 facilitates anti-tumorigenicity partly via inhibiting ECM1 in PDAC. ConclusionsOur work suggests that ROBO2 inhibits tumor progression in PDAC and may serve as a predictive biomarker and therapeutic target in PDAC.

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904 Repurposing CD26 (DPP4) inhibitors to enhance immunotherapy response in pancreatic ductal adenocarcinoma

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.904 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A948-A949

Author(s):

Maggie Phillips ◽

Michael Ware ◽

Cameron Herting ◽

Thomas Mace ◽

Shishir Maithel ◽

...

Keyword(s):

Flow Cytometry ◽

Protein Expression ◽

Pancreatic Ductal Adenocarcinoma ◽

Cell Lines ◽

Tumor Volume ◽

Fold Change ◽

Ductal Adenocarcinoma ◽

Murine Models ◽

Pdac Cell ◽

Cd26 Expression

BackgroundPancreatic ductal adenocarcinoma (PDAC) is refractory to immunotherapy due in part to cellular cross-talk with cancer associated fibroblasts (CAFs). These interactions shape the microenvironment in a manner that is profoundly immunosuppressive. Our group is identifying novel targets in the PDAC stroma that can be manipulated to enhance immunotherapy efficacy. We hypothesize dysregulation of the serine protease, CD26/DPP4 in PDAC contributes to the limited efficacy of immunotherapy. Further, we posit targeting CD26 enzymatic activity using inhibitors that are FDA-approved for adult patients with Type 2 Diabetes Mellitus can enhance the efficacy of immunotherapy in PDAC.MethodsPrimary CAFs isolated from patient PDAC resection specimens under an IRB-approved protocol, were subject to NanoString analysis.1 CD26 protein expression was measured in primary and immortalized CAFs and PDAC cells by immunoblot, flow cytometry and immunofluorescence, while ELISA detected soluble CD26. For in vivo efficacy, luciferase-expressing KPC-tumor cells were implanted orthotopically in the pancreas of immune-competent C57BL/6 mice. Bioluminescence imaging (BLI) confirmed established tumors and mice were randomized to sitagliptin (75 mg/kg in drinking water, CD26/DPP4 inhibitor), anti-PD-L1 Ab (200 ug 2x/week), or both combined for 3 weeks. Controls received vehicle or isotype control Ab. BLI utilized to track tumor progression and tissues harvested for analysis at study endpoint (day 18 of treatment).ResultsNanoString analysis identified CD26/DPP4 as significantly upregulated in RNA transcripts from primary CAFs vs. fibroblasts from normal pancreas (figure 1). We confirmed abundant CD26 expression on patient-derived CAFs and immortalized CAF cell lines, however, lower CD26 expression was observed on human PDAC cell lines (HPAC, PANC-1) by immunoblot, flow cytometry and immunofluorescence (figure 5).Abstract 904 Figure 1(A) Schema for analysis of transcript from n=10 primary CAFs (PSC) from PDAC patients vs. normal human pancreatic fibroblasts (HPPFC) via NanoString nCounter PanCancer Immune Profiling Panel. (B) Heat map of gene expression with upregulate DPP4 or CD26 transcript detected. Adapted from Mace et al., 2016.Abstract 904 Figure 2Validation of CD26 protein expression in human PDAC-derived CAF and PDAC cell lines by immunoblot analysisAbstract 904 Figure 3Analysis of surface human CD26 expression in PBMCs, PDAC-derived CAFs (h-iPSC-PDAC-1), and PDAC cells (PANC-1) by flow cytometry. Histograms representing human surface CD26 expressionAbstract 904 Figure 4Immunofluorescence analysis of CD26/DPP4 cellular localization in a human PDAC-derived CAF cell lineAbstract 904 Figure 5Combined Sitagliptin and PD-L1 blockade in a murine orthotopic model of PDAC. Fold change in tumor volume, determined by BLI, comparing baseline (Day 0 of treatment) to Day 18 of treatment. Each bar represents fold change in BLI determined tumor volume for each animalConclusionsOur results are the first to describe CD26 expression on PDAC-derived CAFs and indicate that sitagliptin augments anti-tumor activity of anti-PD-L1 in PDAC tumor-bearing mice. Our ongoing work will provide insight into specific immune cell populations responsible for efficacy of immunotherapy in murine models of PDAC, and the role of CD26 in various cellular compartments within the PDAC microenvironment.ReferencesMace TA, Shakya R, Pitarresi JR, Swanson B, McQuinn CW, Loftus S, Nordquist E, Cruz-Monserrate Z, Yu L, Young G, Zhong X, Zimmers TA, Ostrowski MC, Ludwig T, Bloomston M, Bekaii-Saab T, Lesinski GB. IL-6 and PD-L1 antibody blockade combination therapy reduces tumour progression in murine models of pancreatic cancer. Gut 2018;67(2):320–32.

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Contribution of nuclear BCL10 expression to tumor progression and poor prognosis of advanced and/or metastatic pancreatic ductal adenocarcinoma by activating NF-κB-related signaling

Cancer Cell International ◽

10.1186/s12935-021-02143-z ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Sung-Hsin Kuo ◽

Shih-Hung Yang ◽

Ming-Feng Wei ◽

Hsiao-Wei Lee ◽

Yu-Wen Tien ◽

...

Keyword(s):

Cell Cycle ◽

Tumor Progression ◽

Pancreatic Ductal Adenocarcinoma ◽

Cell Lines ◽

Poor Prognosis ◽

Cyclin B1 ◽

Ductal Adenocarcinoma ◽

Clinical Samples ◽

Wild Type ◽

Pdac Cell

Abstract Background We previously demonstrated that nuclear BCL10 translocation participates in the instigation of NF-κB in breast cancer and lymphoma cell lines. In this study, we assessed whether nuclear BCL10 translocation is clinically significant in advanced and metastatic pancreatic ductal adenocarcinoma (PDAC). Method and materials We analyzed the expression of BCL10-, cell cycle-, and NF-κB- related signaling molecules, and the DNA-binding activity of NF-κB in three PDAC cell lines (mutant KRAS lines: PANC-1 and AsPC-1; wild-type KRAS line: BxPC-3) using BCL10 short hairpin RNA (shBCL10). To assess the anti-tumor effect of BCL10 knockdown in PDAC xenograft model, PANC-1 cells treated with or without shBCL10 transfection were inoculated into the flanks of mice. We assessed the expression patterns of BCL10 and NF-κB in tumor cells in 136 patients with recurrent, advanced, and metastatic PDAC using immunohistochemical staining. Results We revealed that shBCL10 transfection caused cytoplasmic translocation of BCL10 from the nuclei, inhibited cell viability, and enhanced the cytotoxicities of gemcitabine and oxaliplatin in three PDAC cell lines. Inhibition of BCL10 differentially blocked cell cycle progression in PDAC cell lines. Arrest at G1 phase was noted in wild-type KRAS cell lines; and arrest at G2/M phase was noted in mutant KRAS cell lines. Furthermore, shBCL10 transfection downregulated the expression of phospho-CDC2, phospho-CDC25C, Cyclin B1 (PANC-1), Cyclins A, D1, and E, CDK2, and CDK4 (BxPC-3), p-IκBα, nuclear expression of BCL10, BCL3, and NF-κB (p65), and attenuated the NF-κB pathway activation and its downstream molecule, c-Myc, while inhibition of BCL10 upregulated expression of p21, and p27 in both PANC-1 and BxPC-3 cells. In a PANC-1-xenograft mouse model, inhibition of BCL10 expression also attenuated the tumor growth of PDAC. In clinical samples, nuclear BCL10 expression was closely associated with nuclear NF-κB expression (p < 0.001), and patients with nuclear BCL10 expression had the worse median overall survival than those without nuclear BCL10 expression (6.90 months versus 9.53 months, p = 0.019). Conclusion Nuclear BCL10 translocation activates NF-κB signaling and contributes to tumor progression and poor prognosis of advanced/metastatic PDAC.

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FUS-induced circRHOBTB3 facilitates cell proliferation via miR-600/NACC1 mediated autophagy response in pancreatic ductal adenocarcinoma

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-02063-w ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Taoyue Yang ◽

Peng Shen ◽

Qun Chen ◽

Pengfei Wu ◽

Hao Yuan ◽

...

Keyword(s):

Cell Proliferation ◽

Pancreatic Ductal Adenocarcinoma ◽

Cell Lines ◽

Rna Binding ◽

Ductal Adenocarcinoma ◽

Luciferase Reporter ◽

Circular Rnas ◽

Pdac Cell

Abstract Background Circular RNAs (circRNAs) are becoming a unique member of non-coding RNAs (ncRNAs) with emerging evidence of their regulatory roles in various cancers. However, with regards to pancreatic ductal adenocarcinoma (PDAC), circRNAs biological functions remain largely unknown and worth investigation for potential therapeutic innovation. Methods In our previous study, next-generation sequencing was used to identify differentially expressed circRNAs in 3 pairs of PDAC and adjacent normal tissues. Further validation of circRHOBTB3 expression in PDAC tissues and cell lines and gain-and-loss function experiments verified the oncogenic role of circRHOBTB3. The mechanism of circRHOBTB3 regulatory role was validated by pull-down assays, RIP, luciferase reporter assays. The autophagy response of PANC-1 and MiaPaca-2 cells were detected by mCherry-GFP-LC3B labeling and confocal microscopy, transmission electron microscopy and protein levels of LC3B or p62 via Western blot. Results circRHOBTB3 is highly expressed in PDAC cell lines and tissues, which also promotes PDAC autophagy and then progression in vitro and in vivo. Mechanistically, circRHOBTB3 directly binds to miR-600 and subsequently acts as a miRNA-sponge to maintain the expression level of miR-600-targeted gene NACC1, which facilitates the autophagy response of PDAC cells for adaptation of proliferation via Akt/mTOR pathway. Moreover, the RNA-binding protein FUS (FUS) directly binds to pre-RHOBTB3 mRNA to mediate the biogenesis of circRHOBTB3. Clinically, circRHOBTB3, miR-600 and NACC1 expression levels are correlated with the prognosis of PDAC patients and serve as independent risk factors for PDAC patients. Conclusions FUS-mediated circRHOBTB3 functions as a tumor activator to promote PDAC cell proliferation by modulating miR-600/NACC1/Akt/mTOR axis regulated autophagy.

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WEE1 Inhibition in Combination With Targeted Agents and Standard Chemotherapy in Preclinical Models of Pancreatic Ductal Adenocarcinoma

Frontiers in Oncology ◽

10.3389/fonc.2021.642328 ◽

2021 ◽

Vol 11 ◽

Author(s):

Sarah J. Hartman ◽

Stacey M. Bagby ◽

Betelehem W. Yacob ◽

Dennis M. Simmons ◽

Morgan MacBeth ◽

...

Keyword(s):

Cell Cycle ◽

Cell Lines ◽

Ductal Adenocarcinoma ◽

Cell Cycle Distribution ◽

Targeted Agents ◽

Preclinical Models ◽

Standard Chemotherapy ◽

Pdac Cell ◽

P53 Status ◽

Pdx Models

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer with high incidences of p53 mutations. AZD1775 (adavosertib, previously MK-1775) is a small molecule WEE1 inhibitor that abrogates the G2M checkpoint and can potentially synergize with DNA damaging therapies commonly used in PDAC treatment. The purpose of this study was to identify combination partners for AZD1775, including standard chemotherapy or targeted agents, in PDAC preclinical models. Low powered preliminary screens demonstrated that two of the four PDX models responded better to the combinations of AZD1775 with irinotecan or capecitabine than to either single agent. Following the screens, two full powered PDAC PDX models of differing p53 status were tested with the combinations of AZD1775 and irinotecan or capecitabine. The combinations of AZD1775 and SN38 or 5-FU were also tested on PDAC cell lines. Cellular proliferation was measured using an IncuCyte Live Cell Imager and apoptosis was measured using a Caspase-Glo 3/7 assay. Flow cytometry was conducted to measure alterations in cell cycle distribution. Western blot analysis was used to determine the effects of the drug combinations on downstream effectors. In PDX models with mutated p53 status, there was significant tumor growth inhibition from the combination of AZD1775 with irinotecan or capecitabine (P ≤ 0.03), while PDX models with wild type p53 did not show anti-tumor synergy from the same combinations (P ≥ 0.08). The combination of AZD1775 with SN38 or 5-FU significantly decreased proliferation in all PDAC cell lines, and enhanced apoptosis in multiple cell lines. Cell cycle distribution was disrupted from the combination of AZD1775 with SN38 or 5-FU which was recorded as G2M arrest and decreased G1 phase. AZD1775 inhibited phospho-CDC2 and increased the expression of γH2AX that was either maintained or enhanced after combination with SN38 or 5-FU. The combination of AZD1775 with irinotecan/SN38 or capecitabine/5-FU showed anti-tumor effects in vivo and in vitro in PDAC models. These results support further investigation for these combination strategies to enhance outcomes for PDAC patients.

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Sorafenib in Combination with Betulinic Acid Synergistically Induces Cell Cycle Arrest and Inhibits Clonogenic Activity in Pancreatic Ductal Adenocarcinoma Cells

International Journal of Molecular Sciences ◽

10.3390/ijms19103234 ◽

2018 ◽

Vol 19 (10) ◽

pp. 3234 ◽

Cited By ~ 6

Author(s):

Justyna Kutkowska ◽

Leon Strzadala ◽

Andrzej Rapak

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Pancreatic Ductal Adenocarcinoma ◽

Signaling Pathways ◽

Cell Lines ◽

Betulinic Acid ◽

Combined Treatment ◽

Ductal Adenocarcinoma ◽

Pdac Cell ◽

Cycle Arrest

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers in the world due to late diagnosis and poor response to available treatments. It is important to identify treatment strategies that will increase the efficacy and reduce the toxicity of the currently used therapeutics. In this study, the PDAC cell lines AsPC-1, BxPC-3, and Capan-1 were treated with sorafenib and betulinic acid alone and in combination. We examined the effect of combined treatments on viability (MTS test), proliferation and apoptosis (annexin V staining), cell cycle arrest (PI staining), alterations in signaling pathways (Western blotting), and colony-forming ability. The combination of sorafenib with betulinic acid inhibited the viability and proliferation of PDAC cells without the induction of apoptosis. The antiproliferative effect, caused by G2 cell cycle arrest, was strongly associated with increased expression of p21 and decreased expression of c-Myc and cyclin D1, and was induced only by combined treatment. Additionally, decreased proliferation could also be associated with the inhibition of the P13K/Akt and MAPK signaling pathways. Importantly, combination treatment reduced the colony-forming ability of PDAC cells, as compared to both compounds alone. Collectively, we showed that combined treatment with low concentrations of sorafenib and betulinic acid had the capacity to inhibit proliferation and abolish clonogenic activity in PDAC cell lines.

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H2A.Z overexpression suppresses senescence and chemosensitivity in pancreatic ductal adenocarcinoma

Oncogene ◽

10.1038/s41388-021-01664-1 ◽

2021 ◽

Author(s):

P. A. Ávila-López ◽

G. Guerrero ◽

H. N. Nuñez-Martínez ◽

C. A. Peralta-Alvarez ◽

G. Hernández-Montes ◽

...

Keyword(s):

Cell Cycle ◽

Pancreatic Ductal Adenocarcinoma ◽

Cell Lines ◽

Fatty Acid Biosynthesis ◽

Xenograft Model ◽

Histone Variants ◽

Ductal Adenocarcinoma ◽

Potential Candidate ◽

Pdac Cell ◽

Mouse Xenograft Model

AbstractPancreatic ductal adenocarcinoma (PDAC) is one of the most intractable and devastating malignant tumors. Epigenetic modifications such as DNA methylation and histone modification regulate tumor initiation and progression. However, the contribution of histone variants in PDAC is unknown. Here, we demonstrated that the histone variant H2A.Z is highly expressed in PDAC cell lines and PDAC patients and that its overexpression correlates with poor prognosis. Moreover, all three H2A.Z isoforms (H2A.Z.1, H2A.Z.2.1, and H2A.Z.2.2) are highly expressed in PDAC cell lines and PDAC patients. Knockdown of these H2A.Z isoforms in PDAC cell lines induces a senescent phenotype, cell cycle arrest in phase G2/M, increased expression of cyclin-dependent kinase inhibitor CDKN2A/p16, SA-β-galactosidase activity and interleukin 8 production. Transcriptome analysis of H2A.Z-depleted PDAC cells showed altered gene expression in fatty acid biosynthesis pathways and those that regulate cell cycle and DNA damage repair. Importantly, depletion of H2A.Z isoforms reduces the tumor size in a mouse xenograft model in vivo and sensitizes PDAC cells to gemcitabine. Overexpression of H2A.Z.1 and H2A.Z.2.1 more than H2A.Z.2.2 partially restores the oncogenic phenotype. Therefore, our data suggest that overexpression of H2A.Z isoforms enables cells to overcome the oncoprotective barrier associated with senescence, favoring PDAC tumor grow and chemoresistance. These results make H2A.Z a potential candidate as a diagnostic biomarker and therapeutic target for PDAC.

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