CRISPR-Cas9 Screen Identifies DYRK1A as a Target for Radiotherapy Sensitization in Pancreatic Cancer

Although radiation therapy has recently made great advances in cancer treatment, the majority of patients diagnosed with pancreatic cancer (PC) cannot achieve satisfactory outcomes due to intrinsic and acquired radioresistance. Identifying the molecular mechanisms that impair the efficacy of radiotherapy and targeting these pathways are essential to improve the radiation response of PC patients. Our goal is to identify sensitive targets for pancreatic cancer radiotherapy (RT) using the kinome-wide CRISPR-Cas9 loss-of-function screen and enhance the therapeutic effect through the development and application of targeted inhibitors combined with radiotherapy. We transduced pancreatic cancer cells with a protein kinase library; 2D and 3D library cells were irradiated daily with a single dose of up to 2 Gy for 4 weeks for a total of 40 Gy using an X-ray generator. Sufficient DNA was collected for next-generation deep sequencing to identify candidate genes. In this study, we identified several cell cycle checkpoint kinases and DNA damage related kinases in 2D- and 3D-cultivated cells, including DYRK1A, whose loss of function sensitizes cells to radiotherapy. Additionally, we demonstrated that the harmine-targeted suppression of DYRK1A used in conjunction with radiotherapy increases DNA double-strand breaks (DSBs) and impairs homologous repair (HR), resulting in more cancer cell death. Our results support the use of CRISPR-Cas9 screening to identify new therapeutic targets, develop radiosensitizers, and provide novel strategies for overcoming the tolerance of pancreatic cancer to radiotherapy.

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Molecular Processes Involved in Pancreatic Cancer and Therapeutics

Current Chemical Biology ◽

10.2174/2212796814999201008130819 ◽

2020 ◽

Vol 14 ◽

Author(s):

Subhajit Makar ◽

Abhrajyoti Ghosh ◽

Divya ◽

Shalini Shivhare ◽

Ashok Kumar ◽

...

Keyword(s):

Pancreatic Cancer ◽

Molecular Mechanisms ◽

Early Stage ◽

Locally Advanced ◽

Therapeutic Strategies ◽

Screening Methods ◽

Pancreatic Cancer Cells ◽

Systemic Treatments ◽

Cancer Initiation ◽

Novel Therapeutic Strategies

: Despite advances in the development of cytotoxic and targeted therapies, pancreatic adenocarcinoma (PAC) remains a significant cause of cancer mortality worldwide. It is also difficult to detect it at an early stage due to numbers of factors. Most of the patients are present with locally advanced or metastatic disease, which precludes curative resection. In the absence of effective screening methods, considerable efforts have been made to identify better systemic treatments during the past decade. This review describes the recent advances in molecular mechanisms involved in pancreatic cancer initiation, progression, and metastasis. Additionally, the importance of deregulated cellular signalling pathways and various cellular proteins as potential targets for developing novel therapeutic strategies against incurable forms of pancreatic cancer is reported. The emphasis is on the critical functions associated with growth factors and their receptors viz. c-MET/HGF, CTHRC1, TGF-β, JAK-STAT, cyclooxygenase pathway, WNT, CCK, MAPK-RAS-RAF, PI3K-AKT, Notch, src, IGF-1R, CDK2NA and chromatin regulation for the sustained growth, survival, and metastasis of pancreatic cancer cells. It also includes various therapeutic strategies viz. immunotherapy, surgical therapy, radiation therapy and chemotherapy.

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Acceleration or Brakes: Which Is Rational for Cell Cycle-Targeting Neuroblastoma Therapy?

Biomolecules ◽

10.3390/biom11050750 ◽

2021 ◽

Vol 11 (5) ◽

pp. 750

Author(s):

Kiyohiro Ando ◽

Akira Nakagawara

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cell Cycle Progression ◽

Parp Inhibitors ◽

Cell Cycle Checkpoint ◽

Mitotic Catastrophe ◽

Malignant Neoplasms ◽

Checkpoint Kinases ◽

Molecular Features ◽

Cycle Checkpoint

Unrestrained proliferation is a common feature of malignant neoplasms. Targeting the cell cycle is a therapeutic strategy to prevent unlimited cell division. Recently developed rationales for these selective inhibitors can be subdivided into two categories with antithetical functionality. One applies a “brake” to the cell cycle to halt cell proliferation, such as with inhibitors of cell cycle kinases. The other “accelerates” the cell cycle to initiate replication/mitotic catastrophe, such as with inhibitors of cell cycle checkpoint kinases. The fate of cell cycle progression or arrest is tightly regulated by the presence of tolerable or excessive DNA damage, respectively. This suggests that there is compatibility between inhibitors of DNA repair kinases, such as PARP inhibitors, and inhibitors of cell cycle checkpoint kinases. In the present review, we explore alterations to the cell cycle that are concomitant with altered DNA damage repair machinery in unfavorable neuroblastomas, with respect to their unique genomic and molecular features. We highlight the vulnerabilities of these alterations that are attributable to the features of each. Based on the assessment, we offer possible therapeutic approaches for personalized medicine, which are seemingly antithetical, but both are promising strategies for targeting the altered cell cycle in unfavorable neuroblastomas.

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Bitter melon juice targets molecular mechanisms underlying gemcitabine resistance in pancreatic cancer cells

International Journal of Oncology ◽

10.3892/ijo.2015.2885 ◽

2015 ◽

Vol 46 (4) ◽

pp. 1849-1857 ◽

Cited By ~ 14

Author(s):

RANGANATHA R. SOMASAGARA ◽

GAGAN DEEP ◽

SANGEETA SHROTRIYA ◽

MANISHA PATEL ◽

CHAPLA AGARWAL ◽

...

Keyword(s):

Pancreatic Cancer ◽

Cancer Cells ◽

Molecular Mechanisms ◽

Bitter Melon ◽

Gemcitabine Resistance ◽

Pancreatic Cancer Cells

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Selenium Induces Pancreatic Cancer Cell Death Alone and in Combination with Gemcitabine

Biomedicines ◽

10.3390/biomedicines10010149 ◽

2022 ◽

Vol 10 (1) ◽

pp. 149

Author(s):

David J. Wooten ◽

Indu Sinha ◽

Raghu Sinha

Keyword(s):

Pancreatic Cancer ◽

Cell Lines ◽

Cancer Cell ◽

Pancreatic Cancer Cell ◽

Single Agent ◽

Cancer Cell Lines ◽

Chemotherapeutic Agents ◽

Cancer Cell Death ◽

Pancreatic Cancer Cells

Survival rate for pancreatic cancer remains poor and newer treatments are urgently required. Selenium, an essential trace element, offers protection against several cancer types and has not been explored much against pancreatic cancer specifically in combination with known chemotherapeutic agents. The present study was designed to investigate selenium and Gemcitabine at varying doses alone and in combination in established pancreatic cancer cell lines growing in 2D as well as 3D platforms. Comparison of multi-dimensional synergy of combinations’ (MuSyc) model and highest single agent (HSA) model provided quantitative insights into how much better the combination performed than either compound tested alone in a 2D versus 3D growth of pancreatic cancer cell lines. The outcomes of the study further showed promise in combining selenium and Gemcitabine when evaluated for apoptosis, proliferation, and ENT1 protein expression, specifically in BxPC-3 pancreatic cancer cells in vitro.

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ATM’s Role in the Repair of DNA Double-Strand Breaks

Genes ◽

10.3390/genes12091370 ◽

2021 ◽

Vol 12 (9) ◽

pp. 1370

Author(s):

Atsushi Shibata ◽

Penny A. Jeggo

Keyword(s):

Stress Responses ◽

Ataxia Telangiectasia ◽

Cell Cycle Checkpoint ◽

Double Strand Breaks ◽

Multiple Roles ◽

Dna Double Strand Breaks ◽

Ataxia Telangiectasia Mutated ◽

Dsb Repair ◽

Strand Breaks ◽

Cycle Checkpoint

Ataxia telangiectasia mutated (ATM) is a central kinase that activates an extensive network of responses to cellular stress via a signaling role. ATM is activated by DNA double strand breaks (DSBs) and by oxidative stress, subsequently phosphorylating a plethora of target proteins. In the last several decades, newly developed molecular biological techniques have uncovered multiple roles of ATM in response to DNA damage—e.g., DSB repair, cell cycle checkpoint arrest, apoptosis, and transcription arrest. Combinational dysfunction of these stress responses impairs the accuracy of repair, consequently leading to dramatic sensitivity to ionizing radiation (IR) in ataxia telangiectasia (A-T) cells. In this review, we summarize the roles of ATM that focus on DSB repair.

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LncRNA-ZFAS1 Induces Epithelial-Mesenchymal Transition of Pancreatic Cancer Cells During Nutrient Deprivation by Promoting AMPK-Mediated Phosphorylation and Stabilization Of ZEB1 Protein

10.21203/rs.3.rs-875697/v1 ◽

2021 ◽

Author(s):

ZHU ZENG ◽

Shengbo Han ◽

Yang Wang ◽

Yan Huang ◽

Yuhang Hu ◽

...

Keyword(s):

Pancreatic Cancer ◽

Epithelial Mesenchymal Transition ◽

Nutrient Deprivation ◽

Loss Of Function ◽

Western Blot Assay ◽

Normal Medium ◽

Mesenchymal Transition ◽

Glucose Depletion ◽

Pancreatic Cancer Cells ◽

Glutamine Deprivation

Abstract Background: Nutrient deprivation is a distinct feature of the tumor microenvironment that plays a crucial role in various cancers. However, the contribution and regulatory mechanism of nutrient deprivation on metastasis of pancreatic cancer (PC) have not been identified. Methods: PC cells were treated with normal medium, glucose-depletion or glutamine-depletion medium to observe the epithelial-mesenchymal transition (EMT). RT-qPCR and western blot assay were applied to evaluate the alteration of mRNA and protein of zinc finger E-box binding homeobox 1 (ZEB1), a crucial EMT regulator factor. Co-IP assay was utilized for evaluating the interaction between AMP-activated protein kinase (AMPK) and ZEB1. LncRNA microarray was adopted to detect the potential lncRNA, which facilitates the association between AMPK and ZEB1. Gain- and loss-of-function experiments were performed to evaluate the roles of ZNFX1 antisense RNA 1 (ZFAS1) in EMT and metastasis of PC. Results: The present study reveals that nutrient deprivation including glucose and glutamine deprivation significantly induces EMT of PC cells, which is dependent on stabilization of ZEB1. We further discover that nutrient deprivation induces upregulation of lncRNA ZFAS1, which promotes the association between AMPK and ZEB1 to phosphorylate and stabilize ZEB1 protein. Notably, ZEB1 reciprocally promotes the transcription of ZFAS1 by binding to the promoter of ZFAS1, forming feedback with ZFAS1. Consistently, depletion of ZFAS1 obviously inhibits nutrient deprivation-induced EMT of PC cells and lung metastasis of PC in nude mice. Meanwhile, clinical data displays that ZFAS1 is overexpressed in PC tissues and correlated with high expression of ZEB1 and Vimentin (VIM), low expression of E-cadherin (E-cad), as well as poor prognosis in PC patients. Conclusions: Our study implicates that glucose and glutamine deprivation promotes EMT and metastasis of PC through lncRNA-mediated stabilization of ZEB1.

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DNA damage checkpoint kinases in cancer

Expert Reviews in Molecular Medicine ◽

10.1017/erm.2020.3 ◽

2020 ◽

Vol 22 ◽

Cited By ~ 7

Author(s):

Hannah L. Smith ◽

Harriet Southgate ◽

Deborah A. Tweddle ◽

Nicola J. Curtin

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Environmental Dna ◽

Cell Cycle Checkpoint ◽

Checkpoint Control ◽

Integrated Network ◽

Checkpoint Kinases ◽

G1 Checkpoint ◽

Cycle Checkpoint ◽

High Level

Abstract DNA damage response (DDR) pathway prevents high level endogenous and environmental DNA damage being replicated and passed on to the next generation of cells via an orchestrated and integrated network of cell cycle checkpoint signalling and DNA repair pathways. Depending on the type of damage, and where in the cell cycle it occurs different pathways are involved, with the ATM-CHK2-p53 pathway controlling the G1 checkpoint or ATR-CHK1-Wee1 pathway controlling the S and G2/M checkpoints. Loss of G1 checkpoint control is common in cancer through TP53, ATM mutations, Rb loss or cyclin E overexpression, providing a stronger rationale for targeting the S/G2 checkpoints. This review will focus on the ATM-CHK2-p53-p21 pathway and the ATR-CHK1-WEE1 pathway and ongoing efforts to target these pathways for patient benefit.

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