Publications by authors named "Surinder K Batra"

371 Publications

Androgen Receptor, Although Not a Specific Marker For, Is a Novel Target to Suppress Glioma Stem Cells as a Therapeutic Strategy for Glioblastoma.

Front Oncol 2021 21;11:616625. Epub 2021 May 21.

Department of Radiation Oncology, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, United States.

Targeting androgen receptor (AR) has been shown to be promising in treating glioblastoma (GBM) in cell culture and flank implant models but the mechanisms remain unclear. AR antagonists including enzalutamide are available for treating prostate cancer patients in clinic and can pass the blood-brain barrier, thus are potentially good candidates for GBM treatment but have not been tested in GBM orthotopically. Our current studies confirmed that in patients, a majority of GBM tumors overexpress AR in both genders. Enzalutamide inhibited the proliferation of GBM cells both and . Although confocal microscopy demonstrated that AR is expressed but not specifically in glioma cancer stem cells (CSCs) (CD133+), enzalutamide treatment significantly decreased CSC population in cultured monolayer cells and spheroids, suppressed tumor sphere-forming capacity of GBM cells, and downregulated CSC gene expression at mRNA and protein levels in a dose- and time-dependent manner. We have, for the first time, demonstrated that enzalutamide treatment decreased the density of CSCs and improved survival in an orthotopic GBM mouse model. We conclude that AR antagonists potently target glioma CSCs in addition to suppressing the overall proliferation of GBM cells as a mechanism supporting their repurposing for clinical applications treating GBM.
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http://dx.doi.org/10.3389/fonc.2021.616625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8175980PMC
May 2021

Tumor- and osteoclast-derived NRP2 in prostate cancer bone metastases.

Bone Res 2021 May 14;9(1):24. Epub 2021 May 14.

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA.

Understanding the role of neuropilin 2 (NRP2) in prostate cancer cells as well as in the bone microenvironment is pivotal in the development of an effective targeted therapy for the treatment of prostate cancer bone metastasis. We observed a significant upregulation of NRP2 in prostate cancer cells metastasized to bone. Here, we report that targeting NRP2 in cancer cells can enhance taxane-based chemotherapy with a better therapeutic outcome in bone metastasis, implicating NRP2 as a promising therapeutic target. Since, osteoclasts present in the tumor microenvironment express NRP2, we have investigated the potential effect of targeting NRP2 in osteoclasts. Our results revealed NRP2 negatively regulates osteoclast differentiation and function in the presence of prostate cancer cells that promotes mixed bone lesions. Our study further delineated the molecular mechanisms by which NRP2 regulates osteoclast function. Interestingly, depletion of NRP2 in osteoclasts in vivo showed a decrease in the overall prostate tumor burden in the bone. These results therefore indicate that targeting NRP2 in prostate cancer cells as well as in the osteoclastic compartment can be beneficial in the treatment of prostate cancer bone metastasis.
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http://dx.doi.org/10.1038/s41413-021-00136-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8121836PMC
May 2021

Modeling pancreatic cancer in mice for experimental therapeutics.

Biochim Biophys Acta Rev Cancer 2021 May 1;1876(1):188554. Epub 2021 May 1.

Department of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical Center, Omaha, NE 68198-5870, USA; Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA. Electronic address:

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy that is characterized by early metastasis, low resectability, high recurrence, and therapy resistance. The experimental mouse models have played a central role in understanding the pathobiology of PDAC and in the preclinical evaluation of various therapeutic modalities. Different mouse models with targetable pathological hallmarks have been developed and employed to address the unique challenges associated with PDAC progression, metastasis, and stromal heterogeneity. Over the years, mouse models have evolved from simple cell line-based heterotopic and orthotopic xenografts in immunocompromised mice to more complex and realistic genetically engineered mouse models (GEMMs) involving multi-gene manipulations. The GEMMs, mostly driven by KRAS mutation(s), have been widely accepted for therapeutic optimization due to their high penetrance and ability to recapitulate the histological, molecular, and pathological hallmarks of human PDAC, including comparable precursor lesions, extensive metastasis, desmoplasia, perineural invasion, and immunosuppressive tumor microenvironment. Advanced GEMMs modified to express fluorescent proteins have allowed cell lineage tracing to provide novel insights and a new understanding about the origin and contribution of various cell types in PDAC pathobiology. The syngeneic mouse models, GEMMs, and target-specific transgenic mice have been extensively used to evaluate immunotherapies and study therapy-induced immune modulation in PDAC yielding meaningful results to guide various clinical trials. The emerging mouse models for parabiosis, hepatic metastasis, cachexia, and image-guided implantation, are increasingly appreciated for their high translational significance. In this article, we describe the contribution of various experimental mouse models to the current understanding of PDAC pathobiology and their utility in evaluating and optimizing therapeutic modalities for this lethal malignancy.
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http://dx.doi.org/10.1016/j.bbcan.2021.188554DOI Listing
May 2021

Role of phosphodiesterase 1 in the pathophysiology of diseases and potential therapeutic opportunities.

Pharmacol Ther 2021 Apr 22;226:107858. Epub 2021 Apr 22.

Division of Cardiology, Pauley Heart Center, Virginia Commonwealth University, Richmond, VA 23298-0204, USA. Electronic address:

Cyclic nucleotide phosphodiesterases (PDEs) are superfamily of enzymes that regulate the spatial and temporal relationship of second messenger signaling in the cellular system. Among the 11 different families of PDEs, phosphodiesterase 1 (PDE1) sub-family of enzymes hydrolyze both 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) in a mutually competitive manner. The catalytic activity of PDE1 is stimulated by their binding to Ca/calmodulin (CaM), resulting in the integration of Ca and cyclic nucleotide-mediated signaling in various diseases. The PDE1 family includes three subtypes, PDE1A, PDE1B and PDE1C, which differ for their relative affinities for cAMP and cGMP. These isoforms are differentially expressed throughout the body, including the cardiovascular, central nervous system and other organs. Thus, PDE1 enzymes play a critical role in the pathophysiology of diseases through the fundamental regulation of cAMP and cGMP signaling. This comprehensive review provides the current research on PDE1 and its potential utility as a therapeutic target in diseases including the cardiovascular, pulmonary, metabolic, neurocognitive, renal, cancers and possibly others.
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http://dx.doi.org/10.1016/j.pharmthera.2021.107858DOI Listing
April 2021

Dual blockade of EGFR and CDK4/6 delays head and neck squamous cell carcinoma progression by inducing metabolic rewiring.

Cancer Lett 2021 Jul 17;510:79-92. Epub 2021 Apr 17.

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, 68198, USA; Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, 68198, USA; Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, 68198, USA. Electronic address:

Despite preclinical success, monotherapies targeting EGFR or cyclin D1-CDK4/6 in Head and Neck squamous cell carcinoma (HNSCC) have shown a limited clinical outcome. Here, we aimed to determine the combined effect of palbociclib (CDK4/6) and afatinib (panEGFR) inhibitors as an effective strategy to target HNSCC. Using TCGA-HNSCC co-expression analysis, we found that patients with high EGFR and cyclin D1 expression showed enrichment of gene clusters associated with cell-growth, glycolysis, and epithelial to mesenchymal transition processes. Phosphorylated S6 (p-S6), a downstream effector of EGFR and cyclin D1-CDK4/6 signalling, showed a progressive increase from normal oral tissues to leukoplakia and frank malignancy, and associated with poor outcome of the patients. This increased p-S6 expression was drastically reduced after combination treatment with afatinib and palbociclib in the cell lines and mouse models, suggesting its utiliy as a prognostic marker in HNSCC. Combination treatment also reduced the cell growth and induced cell senescence via increasing reactive oxygen species with concurrent ablation of glycolytic and tricarboxylic acid cycle intermediates. Finally, our findings in sub-cutaneous and genetically engineered mouse model (K14-CreER;LSL-Kras;Trp53) studies showed a significant reduction in the tumor growth and delayed tumor progression after combination treatment. This study collectively demonstrates that dual targeting may be a critical therapeutic strategy in blocking tumor progression via inducing metabolic alteration and warrants clinical evaluation.
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http://dx.doi.org/10.1016/j.canlet.2021.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8153085PMC
July 2021

Mucins reprogram stemness, metabolism and promote chemoresistance during cancer progression.

Cancer Metastasis Rev 2021 Apr 4. Epub 2021 Apr 4.

Department of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical Center, Omaha, NE, 68198-5870, USA.

Mucins are high-molecular-weight glycoproteins dysregulated in aggressive cancers. The role of mucins in disease progression, tumor proliferation, and chemotherapy resistance has been studied extensively. This article provides a comprehensive review of mucin's function as a physical barrier and the implication of mucin overexpression in impeded drug delivery to solid tumors. Mucins regulate the epithelial to mesenchymal transition (EMT) of cancer cells via several canonical and non-canonical oncogenic signaling pathways. Furthermore, mucins play an extensive role in enriching and maintaining the cancer stem cell (CSC) population, thereby sustaining the self-renewing and chemoresistant cellular pool in the bulk tumor. It has recently been demonstrated that mucins regulate the metabolic reprogramming during oncogenesis and cancer progression, which account for tumor cell survival, proliferation, and drug-resistance. This review article focuses on delineating mucin's role in oncogenic signaling and aberrant regulation of gene expressions, culminating in CSC maintenance, metabolic rewiring, and development of chemoresistance, tumor progression, and metastasis.
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http://dx.doi.org/10.1007/s10555-021-09959-1DOI Listing
April 2021

Amyloid Precursor-like Protein 2 Expression Increases during Pancreatic Cancer Development and Shortens the Survival of a Spontaneous Mouse Model of Pancreatic Cancer.

Cancers (Basel) 2021 Mar 26;13(7). Epub 2021 Mar 26.

Eppley Institute for Research in Cancer & Allied Diseases and the Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198, USA.

In the United States, pancreatic cancer is a major cause of cancer-related deaths. Although substantial efforts have been made to understand pancreatic cancer biology and improve therapeutic efficacy, patients still face a bleak chance of survival. A greater understanding of pancreatic cancer development and the identification of novel treatment targets are desperately needed. Our analysis of gene expression data from patient samples showed an increase in amyloid precursor-like protein 2 (APLP2) expression within primary tumor epithelium relative to pancreatic intraepithelial neoplasia (PanIN) epithelial cells. Augmented expression of APLP2 in primary tumors compared to adjacent stroma was also observed. Genetically engineered mouse models of spontaneous pancreatic ductal adenocarcinoma were used to investigate APLP2's role in cancer development. We found that APLP2 expression intensifies significantly during pancreatic cancer initiation and progression in the ; ; (KPC) mouse model, as shown by immunohistochemistry analysis. In studies utilizing pancreas-specific heterozygous and homozygous knockout of APLP2 in the KPC mouse model background, we observed significantly prolonged survival and reduced metastatic progression of pancreatic cancer. These results demonstrate the importance of APLP2 in pancreatic cancer initiation and metastasis and indicate that APLP2 should be considered a potential therapeutic target for this disease.
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http://dx.doi.org/10.3390/cancers13071535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8036577PMC
March 2021

ST6GalNAc-I promotes lung cancer metastasis by altering MUC5AC sialylation.

Mol Oncol 2021 Mar 31. Epub 2021 Mar 31.

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA.

Lung cancer (LC) is the leading cause of cancer-related mortality. However, the molecular mechanisms associated with the development of metastasis are poorly understood. Understanding the biology of LC metastasis is critical to unveil the molecular mechanisms for designing targeted therapies. We developed two genetically engineered LC mouse models Kras ; Trp53 ; Ad-Cre (KPA) and Kras ; Ad-Cre (KA). Survival analysis showed significantly (P = 0.0049) shorter survival in KPA tumor-bearing mice as compared to KA, suggesting the aggressiveness of the model. Our transcriptomic data showed high expression of N-acetylgalactosaminide alpha-2, 6-sialyltransferase 1 (St6galnac-I) in KPA compared to KA tumors. ST6GalNAc-I is an O-glycosyltransferase, which catalyzes the addition of sialic acid to the initiating GalNAc residues forming sialyl Tn (STn) on glycoproteins, such as mucins. Ectopic expression of species-specific p53 mutants in the syngeneic mouse and human LC cells led to increased cell migration and high expression of ST6GalNAc-I, STn, and MUC5AC. Immunoprecipitation of MUC5AC in the ectopically expressing p53 cells exhibited higher affinity toward STn. In addition, ST6GalNAc-I knockout (KO) cells also showed decreased migration, possibly due to reduced glycosylation of MUC5AC as observed by low STn on the glycoprotein. Interestingly, ST6GalNAc-I KO cells injected mice developed less liver metastasis (P = 0.01) compared to controls, while colocalization of MUC5AC and STn was observed in the liver metastatic tissues of control mice. Collectively, our findings support the hypothesis that mutant p53 mediates ST6GalNAc-I expression, leading to the sialyation of MUC5AC, and thus contribute to LC liver metastasis.
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http://dx.doi.org/10.1002/1878-0261.12956DOI Listing
March 2021

Plexin-B3 Regulates Cellular Motility, Invasiveness, and Metastasis in Pancreatic Cancer.

Cancers (Basel) 2021 Feb 16;13(4). Epub 2021 Feb 16.

Department of Pathology and Microbiology, Nebraska Medical Center, Omaha, NE 68198, USA.

The Plexins family of proteins are well-characterized transmembrane receptors of semaphorins, axon guidance cue molecules, that mediate the cell attraction or repelling effects for such cues. Plexins and their ligands are involved in numerous cellular activities, such as motility, invasion, and adhesion to the basement membrane. The detachment of cells and the gain in motility and invasion are hallmarks of the cancer metastasis cascade, thus generating interest in exploring the role of plexins in cancer metastasis. Semaphorin-plexin complexes can act as tumor promoters or suppressors, depending upon the cancer type, and are under investigation for therapeutic purposes. Our group has identified Semaphorin-5A (SEMA5A)/Plexin-B3 as an attractive targetable complex for pancreatic cancer (PC) metastasis. However, our understanding of the Plexin-B3 function and pathological expression in PC is limited, and our present study delineates the role of Plexin-B3 in PC malignancy. We examined the pathological expression of Plexin-B3 in PC tumors and metastasis using a human tissue microarray, disease progression model of PDX-Cre-Kras (KC) mice, and different metastatic sites obtained from the Kras; Trp53; Pdx1-Cre (KPC) mice model. We observed a higher Plexin-B3 expression in PC tumor cores than the normal pancreas, and different metastatic sites were positive for Plexin-B3 expression. However, in the KC mice model, the Plexin-B3 expression increased initially and then decreased with the disease progression. Next, to evaluate the functional role of Plexin-B3, we utilized T3M-4- and CD18/HPAF-Control and -Plexin B3 knockdown cells for different in vivo and in vitro studies. The knockdown of Plexin-B3 enhanced the in vitro cellular migration, invasiveness, and impaired colony formation in three-dimensional culture, along with an increase in cellular spread and remodeling of the actin filaments. We also observed a higher metastasis in nude mice injected with T3M-4- and CD18/HPAF-shPlexin-B3 cells compared to their respective control cells. Furthermore, we observed a lower number of proliferating Ki-67-positive cells and higher ALDH1-A1-positive cells in the tumors formed by Plexin-B3 knockdown cells compared to tumors formed by the control cells. Together, our data suggest that the loss of Plexin-B3 is associated with the interference of cell division machinery and the induction of stem cell-like characteristics in PC cells.
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http://dx.doi.org/10.3390/cancers13040818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919786PMC
February 2021

Recent advances in organoid development and applications in disease modeling.

Biochim Biophys Acta Rev Cancer 2021 Apr 26;1875(2):188527. Epub 2021 Feb 26.

Department of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical Center, Omaha, NE 68198-5870, USA; Eppley Institute for Research in Cancer and Allied Diseases, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA. Electronic address:

An improved understanding of stem cell niches, organogenesis, and disease models has paved the way for developing a three-dimensional (3D) organoid culture system. Organoid cultures can be derived from primary tissues (single cells or tissue subunits), adult stem cells (ASCs), induced pluripotent stem cells (iPSCs), or embryonic stem cells (ESCs). As a significant technological breakthrough, 3D organoid models offer a promising approach for understanding the complexities of human diseases ranging from the mechanistic investigation of disease pathogenesis to therapy. Here, we discuss the recent applications, advantages, and limitations of organoids as in vitro models for studying metabolomics, drug development, infectious diseases, and the gut microbiome. We further discuss the use of organoids in cancer modeling using high throughput sequencing approaches.
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http://dx.doi.org/10.1016/j.bbcan.2021.188527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8068668PMC
April 2021

Reply.

Gastroenterology 2021 May 4;160(6):2225-2226. Epub 2021 Feb 4.

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska.

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http://dx.doi.org/10.1053/j.gastro.2021.02.003DOI Listing
May 2021

Proteasomal Regulation of Mammalian SPT16 in Controlling Transcription.

Mol Cell Biol 2021 03 24;41(4). Epub 2021 Mar 24.

Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, Illinois, USA

FACT (cilitates hromatin ranscription), an essential and evolutionarily conserved heterodimer from yeast to humans, controls transcription and is found to be upregulated in various cancers. However, the basis for such upregulation is not clearly understood. Our recent results deciphering a new ubiquitin-proteasome system regulation of the FACT subunit SPT16 in orchestrating transcription in yeast hint at the involvement of the proteasome in controlling FACT in humans, with a link to cancer. To test this, we carried out experiments in human embryonic kidney (HEK293) cells, which revealed that human SPT16 undergoes ubiquitylation and that its abundance is increased following inhibition of the proteolytic activity of the proteasome, thus implying proteasomal regulation of human SPT16. Furthermore, we find that the increased abundance/expression of SPT16 in HEK293 cells alters the transcription of genes, including ones associated with cancer, and that the proteasomal degradation of SPT16 is impaired in kidney cancer (Caki-2) cells to upregulate SPT16. Like human SPT16, murine SPT16 in C2C12 cells also undergoes ubiquitylation and proteasomal degradation to regulate transcription. Collectively, our results reveal a proteasomal regulation of mammalian SPT16, with physiological relevance in controlling transcription, and implicate such proteasomal control in the upregulation of SPT16 in cancer.
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http://dx.doi.org/10.1128/MCB.00452-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8088127PMC
March 2021

Tumor microenvironment: an evil nexus promoting aggressive head and neck squamous cell carcinoma and avenue for targeted therapy.

Signal Transduct Target Ther 2021 Jan 12;6(1):12. Epub 2021 Jan 12.

Watson-Crick Centre for Molecular Medicine, Islamic University of Science and Technology, Awantipora, Jammu & Kashmir, India.

Head and neck squamous cell carcinoma (HNSCC) is a very aggressive disease with a poor prognosis for advanced-stage tumors. Recent clinical, genomic, and cellular studies have revealed the highly heterogeneous and immunosuppressive nature of HNSCC. Despite significant advances in multimodal therapeutic interventions, failure to cure and recurrence are common and account for most deaths. It is becoming increasingly apparent that tumor microenvironment (TME) plays a critical role in HNSCC tumorigenesis, promotes the evolution of aggressive tumors and resistance to therapy, and thereby adversely affects the prognosis. A complete understanding of the TME factors, together with the highly complex tumor-stromal interactions, can lead to new therapeutic interventions in HNSCC. Interestingly, different molecular and immune landscapes between HPV and HPV (human papillomavirus) HNSCC tumors offer new opportunities for developing individualized, targeted chemoimmunotherapy (CIT) regimen. This review highlights the current understanding of the complexity between HPV and HPV HNSCC TME and various tumor-stromal cross-talk modulating processes, including epithelial-mesenchymal transition (EMT), anoikis resistance, angiogenesis, immune surveillance, metastatic niche, therapeutic resistance, and development of an aggressive tumor phenotype. Furthermore, we summarize the recent developments and the rationale behind CIT strategies and their clinical applications in HPV and HPV HNSCC.
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http://dx.doi.org/10.1038/s41392-020-00419-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7804459PMC
January 2021

The Current Landscape of Antibody-based Therapies in Solid Malignancies.

Theranostics 2021 1;11(3):1493-1512. Epub 2021 Jan 1.

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, NE, 68198, USA.

Over the past three decades, monoclonal antibodies (mAbs) have revolutionized the landscape of cancer therapy. Still, this benefit remains restricted to a small proportion of patients due to moderate response rates and resistance emergence. The field has started to embrace better mAb-based formats with advancements in molecular and protein engineering technologies. The development of a therapeutic mAb with long-lasting clinical impact demands a prodigious understanding of target antigen, effective mechanism of action, gene engineering technologies, complex interplay between tumor and host immune system, and biomarkers for prediction of clinical response. This review discusses the various approaches used by mAbs for tumor targeting and mechanisms of therapeutic resistance that is not only caused by the heterogeneity of tumor antigen, but also the resistance imposed by tumor microenvironment (TME), including inefficient delivery to the tumor, alteration of effector functions in the TME, and Fc-gamma receptor expression diversity and polymorphism. Further, this article provides a perspective on potential strategies to overcome these barriers and how diagnostic and prognostic biomarkers are being used in predicting response to mAb-based therapies. Overall, understanding these interdependent parameters can improve the current mAb-based formulations and develop novel mAb-based therapeutics for achieving durable clinical outcomes in a large subset of patients.
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http://dx.doi.org/10.7150/thno.52614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7738893PMC
January 2021

Liquid Biopsy for Identification of High-Risk Cystic Lesions of Pancreas.

Gastroenterology 2021 Mar 30;160(4):1016-1018. Epub 2020 Dec 30.

Department of Biochemistry and Molecular Biology and, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska. Electronic address:

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http://dx.doi.org/10.1053/j.gastro.2020.12.039DOI Listing
March 2021

Selective inhibition of stemness through EGFR/FOXA2/SOX9 axis reduces pancreatic cancer metastasis.

Oncogene 2021 Jan 7;40(4):848-862. Epub 2020 Dec 7.

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA.

Pancreatic cancer (PC) is difficult to defeat due to mechanism (s) driving metastasis and drug resistance. Cancer stemness is a major challenging phenomenon associated with PC metastasis and limiting therapy efficacy. In this study, we evaluated the pre-clinical and clinical significance of eradicating pancreatic cancer stem cells (PCSC) and its components using a pan-EGFR inhibitor afatinib in combination with gemcitabine. Afatinib in combination with gemcitabine significantly reduced Kras; Pdx-1 Cre (KC) (P < 0.01) and Kras; p53; Pdx-1 Cre (KPC) (P < 0.05) derived mouse tumoroids and KPC-derived murine syngeneic cell line growth compared to gemcitabine/afatinib alone treatment. The drug combination also reduced PC xenograft tumor burden (P < 0.05) and the incidence of metastasis by affecting key stemness markers, as confirmed by co-localization studies. Moreover, the drug combination significantly decreases the growth of various PC patient-derived organoids (P < 0.001). We found that SOX9 is significantly overexpressed in high-grade PC tumors (P < 0.05) and in chemotherapy-treated patients compared to chemo-naïve patients (P < 0.05). These results were further validated using publicly available datasets. Moreover, afatinib alone or in combination with gemcitabine decreased stemness and tumorspheres by reducing phosphorylation of EGFR family proteins, ERK, FAK, and CSC markers. Mechanistically, afatinib treatment decreased CSC markers by downregulating SOX9 via FOXA2. Indeed, EGFR and FOXA2 depletion reduced SOX9 expression in PCSCs. Taken together, pan-EGFR inhibition by afatinib impedes PCSCs growth and metastasis via the EGFR/ERK/FOXA2/SOX9 axis. This novel mechanism of pan-EGFR inhibitor and its ability to eradicate CSC may serve as a tailor-made approach to enhance chemotherapeutic benefits in other cancer types.
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http://dx.doi.org/10.1038/s41388-020-01564-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848971PMC
January 2021

Nanoscale platform for delivery of active IRINOX to combat pancreatic cancer.

J Control Release 2021 Feb 18;330:1229-1243. Epub 2020 Nov 18.

Department of Pharmaceutical Sciences, Center for Drug Delivery and Nanomedicine, College of Pharmacy, University of Nebraska Medical Center, 985830 Nebraska Medical Center, Omaha, NE 68198, USA. Electronic address:

Due to its late diagnosis and dismal prognosis, pancreatic ductal adenocarcinoma (PDAC) is one of the most devastating solid malignancies, with only 9% of patients surviving after being diagnosed. A multidrug chemotherapeutic regimen FOL-F-IRIN-OX (combination of 5-fluorouracil, leucovorin, irinotecan, and oxaliplatin) offers survival benefits superior to that of gemcitabine single agent, but the treatment-related side effects are also severe. To overcome this therapeutic barrier, we developed polymeric micelles bearing active formats of irinotecan and oxaliplatin, SN38 and 1,2-diaminocyclohexane‑platinum (II), DACHPt. Crosslinked micelles were prepared using amphiphilic PEG-b-poly(L-glutamic acid)/SN38 conjugates and subsequently loaded with DACHPt. The dual drug-loaded micelles exhibited improved colloidal stability, prolonged drug release and remarkable cytotoxicity in human pancreatic cancer cell lines and Kras; Trp52; Pdx-1 Cre murine tumor organoids models. In vivo, (SN38 + DACHPt)-loaded micelles displayed superior antitumor and antimetastatic activities without impairing safety. Our results suggest that nanomedicine mimicking irinotecan and oxaliplatin as parts of FOLFIRINOX regimen may further improve the feasibility of this multidrug treatment for patients with advanced pancreatic cancer.
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http://dx.doi.org/10.1016/j.jconrel.2020.11.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8008503PMC
February 2021

Secretory Mucin 5AC Promotes Neoplastic Progression by Augmenting KLF4-Mediated Pancreatic Cancer Cell Stemness.

Cancer Res 2021 01 30;81(1):91-102. Epub 2020 Oct 30.

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska.

Secreted mucin 5AC (MUC5AC) is the most abundantly overexpressed member of the mucin family during early pancreatic intraepithelial neoplasia stage I (PanIN-I) of pancreatic cancer. To comprehend the contribution of Muc5ac in pancreatic cancer pathology, we genetically ablated it in an autochthonous murine model (KrasG12D; Pdx-1cre, KC), which mirrors the early stages of pancreatic cancer development. Neoplastic onset and the PanIN lesion progression were significantly delayed in Muc5ac knockout (KrasG12D; Pdx-1 cre; Muc5ac-/-, KCM) animals with a 50% reduction in PanIN-2 and 70% reduction in PanIN-3 lesions compared with KC at 50 weeks of age. High-throughput RNA-sequencing analysis from pancreatic tissues of KCM animals revealed a significant decrease in cancer stem cell (CSC) markers Aldh1a1, Klf4, EpCAM, and CD133. Furthermore, the silencing of MUC5AC in human pancreatic cancer cells reduced their tumorigenic propensity, as indicated by a significant decline in tumor formation frequency by limiting dilution assay upon subcutaneous administration. The contribution of MUC5AC in CSC maintenance was corroborated by a significant decrease in tumor burden upon orthotopic implantation of MUC5AC-depleted pancreatic cancer cells. Mechanistically, MUC5AC potentiated oncogenic signaling through integrin αvβ5, pSrc (Y416), and pSTAT3 (Y705). Phosphorylated STAT3, in turn, upregulated Klf4 expression, thereby enriching the self-renewing CSC population. A strong positive correlation of Muc5ac with Klf4 and pSTAT3 in the PanIN lesions of KC mouse pancreas reinforces the crucial involvement of MUC5AC in bolstering the CSC-associated tumorigenic properties of Kras-induced metaplastic cells, which leads to pancreatic cancer onset and progression. SIGNIFICANCE: This study elucidates that expression of MUC5AC promotes cancer cell stemness during Kras-driven pancreatic tumorigenesis and can be targeted for development of a novel therapeutic regimen.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-1293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990052PMC
January 2021

Neutrophil Gelatinase-Associated Lipocalin Protects Acinar Cells From Cerulein-Induced Damage During Acute Pancreatitis.

Pancreas 2020 Nov/Dec;49(10):1297-1306

From the Department of Biochemistry and Molecular Biology.

Objectives: Elevated neutrophil gelatinase-associated lipocalin (NGAL) is a promising marker for severe acute pancreatitis (SAP) and multiple organ failure, suggesting systemic and local contributions during pancreatitis. We investigated the role of NGAL locally on acinar cell biology.

Methods: Western blot, reverse transcriptase-polymerase chain reaction, and immunohistochemistry analysis were performed to analyze the levels of NGAL receptors, apoptotic and regeneration markers, and 4-hydroxynonenal (4HNE) levels, 3-[4,5-Dimethylthiazole-2-yl]-2, 5-diphenyltetrazolium bromide assay, and annexin V/propidium iodide staining were used to evaluate cell viability, and effect on endothelial cells was accessed by endothelial permeability assay.

Results: Cerulein treatment at 20 μM for 12 hours significantly reduced acinar cell viability by 40%, which was rescued by NGAL at 800 and 1600 ng/mL concentrations, observed during mild and SAP, respectively. Mechanistically, NGAL significantly reduced the levels of reactive oxygen species and 4HNE adduct formation in a 24p3R-dependent manner and upregulated the expression of acinar cell regeneration markers, like CDK-2, CDK-4, and C-myc. However, SAP levels of NGAL significantly increased endothelial permeability and downregulated the levels of ZO-1, and cerulein treatment in NGAL knockout mice showed increased levels of 4HNE adducts.

Conclusions: Neutrophil gelatinase-associated lipocalin rescues intracellular reactive oxygen species during pancreatitis and promotes survival and regeneration of acinar cells.
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http://dx.doi.org/10.1097/MPA.0000000000001690DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8056863PMC
October 2020

Metabolic programming of distinct cancer stem cells promotes metastasis of pancreatic ductal adenocarcinoma.

Oncogene 2021 01 27;40(1):215-231. Epub 2020 Oct 27.

Department of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical Center, Omaha, NE, 68198-5870, USA.

Pancreatic ductal adenocarcinoma (PDAC) metastasizes to distant organs, which is the primary cause of mortality; however, specific features mediating organ-specific metastasis remain unexplored. Emerging evidence demonstrates that cancer stem cells (CSCs) and cellular metabolism play a pivotal role in metastasis. Here we investigated the role of distinct subtypes of pancreatic CSCs and their metabolomic signatures in organ-specific metastatic colonization. We found that PDAC consists of ALDH+/CD133+ and drug-resistant (MDR1+) subtypes of CSCs with specific metabolic and stemness signatures. Human PDAC tissues with gemcitabine treatment, autochthonous mouse tumors from Kras; Pdx1-Cre (KC) and Kras; Trp53; Pdx-1 Cre (KPC) mice, and KPC- Liver/Lung metastatic cells were used to evaluate the CSC, EMT (epithelial-to-mesenchymal transition), and metabolic profiles. A strong association was observed between distinct CSC subtypes and organ-specific colonization. The liver metastasis showed drug-resistant CSC- and EMT-like phenotype with aerobic glycolysis and fatty acid β-oxidation-mediated oxidative (glyco-oxidative) metabolism. On the contrary, lung metastasis displayed ALDH+/CD133+ and MET-like phenotype with oxidative metabolism. These results were obtained by evaluating FACS-based side population (SP), autofluorescence (AF+) and Alde-red assays for CSCs, and Seahorse-based oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and fatty acid β-oxidation (FAO)-mediated OCR assays for metabolic features along with specific gene signatures. Further, we developed in vitro human liver and lung PDAC metastasis models by using a combination of liver or lung decellularized scaffolds, a co-culture, and a sphere culture methods. PDAC cells grown in the liver-mimicking model showed the enrichment of MDR1+ and CPT1A+ populations, whereas the PDAC cells grown in the lung-mimicking environment showed the enrichment of ALDH+/CD133+ populations. In addition, we observed significantly elevated expression of ALDH1 in lung metastasis and MDR1/LDH-A expression in liver metastasis compared to human primary PDAC tumors. Our studies elucidate that distinct CSCs adapt unique metabolic signatures for organotropic metastasis, which will pave the way for the development of targeted therapy for PDAC metastasis.
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http://dx.doi.org/10.1038/s41388-020-01518-2DOI Listing
January 2021

Blocking c-MET/ERBB1 Axis Prevents Brain Metastasis in ERBB2+ Breast Cancer.

Cancers (Basel) 2020 10 1;12(10). Epub 2020 Oct 1.

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA.

Brain metastasis (BrM) remains a significant cause of cancer-related mortality in epidermal growth factor receptor 2-positive (ERBB2) breast cancer (BC) patients. We proposed here that a combination treatment of irreversible tyrosine kinase inhibitor neratinib (NER) and the c-MET inhibitor cabozantinib (CBZ) could prevent brain metastasis. To address this, we first tested the combination treatment of NER and CBZ in the brain-seeking ERBB2 cell lines SKBrM3 and JIMT-1-BR3, and in ERBB2 organoids that expressed the c-MET/ERBB1 axis. Next, we developed and characterized an orthotopic mouse model of spontaneous BrM and evaluated the therapeutic effect of CBZ and NER in vivo. The combination treatment of NER and CBZ significantly inhibited proliferation and migration in ERBB2 cell lines and reduced the organoid growth in vitro. Mechanistically, the combination treatment of NER and CBZ substantially inhibited ERK activation downstream of the c-MET/ERBB1 axis. Orthotopically implanted SKBrM3 cells formed primary tumor in the mammary fat pad and spontaneously metastasized to the brain and other distant organs. Combination treatment with NER and CBZ inhibited primary tumor growth and predominantly prevented BrM. In conclusion, the orthotopic model of spontaneous BrM is clinically relevant, and the combination therapy of NER and CBZ might be a useful approach to prevent BrM in BC.
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http://dx.doi.org/10.3390/cancers12102838DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7601177PMC
October 2020

A Non-genetic Mechanism Involving the Integrin β4/Paxillin Axis Contributes to Chemoresistance in Lung Cancer.

iScience 2020 Aug 22;23(9):101496. Epub 2020 Aug 22.

Department of Medical Oncology & Therapeutics Research, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, CA 91010-3000, USA. Electronic address:

Tumor heterogeneity and cisplatin resistance are major causes of tumor relapse and poor survival. Here, we show that in lung cancer, interaction between paxillin (PXN) and integrin β4 (ITGB4), components of the focal adhesion (FA) complex, contributes to cisplatin resistance. Knocking down PXN and ITGB4 attenuated cell growth and improved cisplatin sensitivity, both in 2D and 3D cultures. PXN and ITGB4 independently regulated expression of several genes. In addition, they also regulated expression of common genes including USP1 and VDAC1, which are required for maintaining genomic stability and mitochondrial function, respectively. Mathematical modeling suggested that bistability could lead to stochastic phenotypic switching between cisplatin-sensitive and resistant states in these cells. Consistently, purified subpopulations of sensitive and resistant cells re-created the mixed parental population when cultured separately. Altogether, these data point to an unexpected role of the FA complex in cisplatin resistance and highlight a novel non-genetic mechanism.
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http://dx.doi.org/10.1016/j.isci.2020.101496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7502350PMC
August 2020

CXCR3 and Cognate Ligands are Associated with Immune Cell Alteration and Aggressiveness of Pancreatic Ductal Adenocarcinoma.

Clin Cancer Res 2020 11 1;26(22):6051-6063. Epub 2020 Sep 1.

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska.

Purpose: The cytokine milieu in pancreatic ductal adenocarcinoma (PDAC) promotes tumor progression and immune suppression, contributing to the dismal prognosis of patients with PDAC. The roles of many of these cytokines, however, have not been thoroughly investigated in PDAC.

Experimental Design: PDAC microarray and The Cancer Genome Atlas datasets were analyzed to identify cytokines and cognate receptors overexpressed in PDAC and associated with survival. Pathway and CIBERSORT analyses were used to elucidate potential mechanisms of altered patient survival. Comparative analysis of cytokine expression in KPC (K-ras; TP53; Pdx-1cre) and KC (K-ras; Pdx-1cre) PDAC models and multicolor immunofluorescence (IF) staining of human PDAC-resected samples were used to validate these findings.

Results: and were among the most highly overexpressed cytokines by bioinformatics analyses, while their receptor, CXCR3, was significantly overexpressed by IHC analysis. Higher CXCR3 ligand expression was associated with shorter overall survival, while high expression was associated with better survival. The CXCR3 ligands, , , and , were overexpressed in KPC compared with KC mice. Pathway analysis of CXCR3- and CXCR3 ligand-associated genes showed that is a marker of antitumor immunity, while its ligands may promote immunosuppression. CIBERSORT and IF studies of PDAC tissues demonstrated that high CXCR3 expression was associated with increased CD8 T-cell and naïve B-cell signatures and loss of plasma cell signatures. CXCR3 ligand expression was associated with increased CD8 T-cell signatures and loss of natural killer-cell signatures.

Conclusions: CXCR3 ligands are overexpressed in PDAC and are associated with poor survival likely related to alterations in tumor immune infiltrate/activity.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-1359DOI Listing
November 2020

Sildenafil Potentiates the Therapeutic Efficacy of Docetaxel in Advanced Prostate Cancer by Stimulating NO-cGMP Signaling.

Clin Cancer Res 2020 11 26;26(21):5720-5734. Epub 2020 Aug 26.

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska.

Purpose: Docetaxel plays an indispensable role in the management of advanced prostate cancer. However, more than half of patients do not respond to docetaxel, and those good responders frequently experience significant cumulative toxicity, which limits its dose duration and intensity. Hence, a second agent that could increase the initial efficacy of docetaxel and maintain tolerability at biologically effective doses may improve outcomes for patients.

Experimental Design: We determined phosphodiesterase 5 (PDE5) expression levels in human and genetically engineered mouse (GEM) prostate tissues and tumor-derived cell lines. Furthermore, we investigated the therapeutic benefits and underlying mechanism of PDE5 inhibitor sildenafil in combination with docetaxel using , Pten conditional knockout (cKO), derived tumoroid and xenograft prostate cancer models.

Results: PDE5 expression was higher in both human and mouse prostate tumors and cancer cell lines compared with normal tissues/cells. In GEM prostate-derived cell lines, PDE5 expression increased from normal prostate (wild-type) epithelial cells to androgen-dependent and castrated prostate-derived cell lines. The addition of physiologically achievable concentrations of sildenafil enhanced docetaxel-induced prostate cancer cell growth inhibition and apoptosis , reduced murine 3D tumoroid growth, and tumorigenicity as compared with docetaxel alone. Furthermore, sildenafil enhanced docetaxel-induced NO and cGMP levels thereby augmenting antitumor activity.

Conclusions: Our results demonstrate that sildenafil's addition could sensitize docetaxel chemotherapy in prostate cancer cells at much lesser concentration than needed for inducing cell death. Thus, the combinatorial treatment of sildenafil and docetaxel may improve anticancer efficacy and reduce chemotherapy-induced side-effects among patients with advanced prostate cancer.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-1569DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642013PMC
November 2020

Alcohol and Smoking Mediated Modulations in Adaptive Immunity in Pancreatitis.

Cells 2020 08 11;9(8). Epub 2020 Aug 11.

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198-5870, USA.

Pancreatitis is a condition of pancreatic inflammation driven by injury to the pancreatic parenchyma. The extent of acinar insult, intensity, and type of immune response determines the severity of the disease. Smoking, alcohol and autoimmune pancreatitis are some of the predominant risk factors that increase the risk of pancreatitis by differentially influencing the adaptive immune system. The overall decrease in peripheral lymphocyte (T-, B- and (natural killer T-) NKT-cell) count and increased infiltration into the damaged pancreatic tissue highlight the contribution of adaptive immunity in the disease pathology. Smoking and alcohol modulate the responsiveness and apoptosis of T- and B-cells during pancreatic insult. Acute pancreatitis worsens with smoking and alcohol, leading to the development of systemic inflammatory response syndrome and compensatory anti-inflammatory response syndrome, suggesting the critical role of adaptive immunity in fatal outcomes such as multiple organ dysfunction. The presence of CD4 and CD8 T-lymphocytes and perforin-expressing cells in the fibrotic tissue in chronic pancreatitis modulate the severity of the disease. Due to their important role in altering the severity of the disease, attempts to target adaptive immune mediators will be critical for the development of novel therapeutic interventions.
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http://dx.doi.org/10.3390/cells9081880DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463831PMC
August 2020

Extracellular Vesicle and Particle Biomarkers Define Multiple Human Cancers.

Cell 2020 08 13;182(4):1044-1061.e18. Epub 2020 Aug 13.

School of Life Science and Technology, Tokyo Institute of Technology, Yokohama, Japan.

There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.
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http://dx.doi.org/10.1016/j.cell.2020.07.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7522766PMC
August 2020

RNA Polymerase II-Associated Factor 1 Regulates Stem Cell Features of Pancreatic Cancer Cells, Independently of the PAF1 Complex, via Interactions With PHF5A and DDX3.

Gastroenterology 2020 11 8;159(5):1898-1915.e6. Epub 2020 Aug 8.

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska; Eppley Institute for Research in Cancer and Allied Diseases and Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska. Electronic address:

Background & Aims: It is not clear how pancreatic cancer stem cells (CSCs) are regulated, resulting in ineffective treatments for pancreatic cancer. PAF1, a RNA polymerase II-associated factor 1 complex (PAF1C) component, maintains pluripotency of stem cells, by unclear mechanisms, and is a marker of CSCs. We investigated mechanisms by which PAF1 maintains CSCs and contributes to development of pancreatic tumors.

Methods: Pancreatic cancer cell lines were engineered to knockdown PAF1 using inducible small hairpin RNAs. These cells were grown as orthotopic tumors in athymic nude mice and PAF1 knockdown was induced by administration of doxycycline in drinking water. Tumor growth and metastasis were monitored via IVIS imaging. CSCs were isolated from pancreatic cancer cell populations using flow cytometry and characterized by tumor sphere formation, tumor formation in nude mice, and expression of CSC markers. Isolated CSCs were depleted of PAF1 using the CRISPR/Cas9 system. PAF1-regulated genes in CSCs were identified via RNA-seq and PCR array analyses of cells with PAF1 knockdown. Proteins that interact with PAF1 in CSCs were identified by immunoprecipitations and mass spectrometry. We performed chromatin immunoprecipitation sequencing of CSCs to confirm the binding of the PAF1 sub-complex to target genes.

Results: Pancreatic cancer cells depleted of PAF1 formed smaller and fewer tumor spheres in culture and orthotopic tumors and metastases in mice. Isolated CSCs depleted of PAF1 downregulated markers of self-renewal (NANOG, SOX9, and β-CATENIN), of CSCs (CD44v6, and ALDH1), and the metastasis-associated gene signature, compared to CSCs without knockdown of PAF1. The role of PAF1 in CSC maintenance was independent of its RNA polymerase II-associated factor 1 complex component identity. We identified DDX3 and PHF5A as proteins that interact with PAF1 in CSCs and demonstrated that the PAF1-PHF5A-DDX3 sub-complex bound to the promoter region of Nanog, whose product regulates genes that control stemness. Levels of the PAF1-DDX3 and PAF1-PHF5A were increased and co-localized in human pancreatic tumor specimens, human pancreatic tumor-derived organoids, and organoids derived from tumors of KPC mice, compared with controls. Binding of DDX3 and PAF1 to the Nanog promoter, and the self-renewal capacity of CSCs, were decreased in cells incubated with the DDX3 inhibitor RK-33. CSCs depleted of PAF1 downregulated genes that regulate stem cell features (Flot2, Taz, Epcam, Erbb2, Foxp1, Abcc5, Ddr1, Muc1, Pecam1, Notch3, Aldh1a3, Foxa2, Plat, and Lif).

Conclusions: In pancreatic CSCs, PAF1 interacts with DDX3 and PHF5A to regulate expression of NANOG and other genes that regulate stemness. Knockdown of PAF1 reduces the ability of orthotopic pancreatic tumors to develop and progress in mice and their numbers of CSCs. Strategies to target the PAF1-PHF5A-DDX3 complex might be developed to slow or inhibit progression of pancreatic cancer.
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http://dx.doi.org/10.1053/j.gastro.2020.07.053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7680365PMC
November 2020