Publications by authors named "Satyanarayana Rachagani"

91 Publications

Endothelin-axis antagonism enhances tumor perfusion in pancreatic cancer.

Cancer Lett 2022 Jun 19:215801. Epub 2022 Jun 19.

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

Delivery of therapeutic agents in pancreatic cancer (PC) is impaired due to its hypovascular and desmoplastic tumor microenvironment. The Endothelin (ET)-axis is the major regulator of vasomotor tone under physiological conditions and is highly upregulated in multiple cancers. We investigated the effect of dual endothelin receptor antagonist bosentan on perfusion and macromolecular transport in a PC cell-fibroblast co-implantation tumor model using Dynamic Contrast Enhancement Magnetic Resonance Imaging (DCE-MRI). Following bosentan treatment, the contrast enhancement ratio and wash in rates in tumors were two- and nine times higher, respectively, compared to the controls, whereas the time to peak was significantly shorter (7.29 ± 1.29 min v/s 22.08 ± 5.88 min; p = 0.04). Importantly, these effects were tumor selective as magnitude of change for these parameters was much lower in muscles. Bosentan treatment also reduced desmoplasia and improved intratumoral distribution of high molecular weight FITC-dextran. Overall, these findings support that targeting the ET-axis can serve as a potential strategy to selectively enhance tumor perfusion and improve the delivery of therapeutic agents in pancreatic tumors.
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http://dx.doi.org/10.1016/j.canlet.2022.215801DOI Listing
June 2022

Hedgehog signaling and its molecular perspective with cholesterol: a comprehensive review.

Cell Mol Life Sci 2022 Apr 29;79(5):266. Epub 2022 Apr 29.

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

Hedgehog (Hh) signaling is evolutionarily conserved and plays an instructional role in embryonic morphogenesis, organogenesis in various animals, and the central nervous system organization. Multiple feedback mechanisms dynamically regulate this pathway in a spatiotemporal and context-dependent manner to confer differential patterns in cell fate determination. Hh signaling is complex due to canonical and non-canonical mechanisms coordinating cell-cell communication. In addition, studies have demonstrated a regulatory framework of Hh signaling and shown that cholesterol is vital for Hh ligand biogenesis, signal generation, and transduction from the cell surface to intracellular space. Studies have shown the importance of a specific cholesterol pool, termed accessible cholesterol, which serves as a second messenger, conveying signals between smoothened (Smo) and patched 1 (Ptch1) across the plasma and ciliary membranes. Remarkably, recent high-resolution structural and molecular studies shed new light on the interplay between Hh signaling and cholesterol in membrane biology. These studies elucidated novel mechanistic insight into the release and dispersal of cholesterol-anchored Hh and the basis of Hh recognition by Ptch1. Additionally, the putative model of Smo activation by cholesterol binding and/or modification and Ptch1 antagonization of Smo has been explicated. However, the coupling mechanism of Hh signaling and cholesterol offered a new regulatory principle in cell biology: how effector molecules of the Hh signal network react to and remodel cholesterol accessibility in the membrane and selectively activate Hh signaling proteins thereof. Recognizing the biological importance of cholesterol in Hh signaling activation and transduction opens the door for translational research to develop novel therapeutic strategies. This review looks in-depth at canonical and non-canonical Hh signaling and the distinct proposed model of cholesterol-mediated regulation of Hh signaling components, facilitating a more sophisticated understanding of the Hh signal network and cholesterol biology.
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http://dx.doi.org/10.1007/s00018-022-04233-1DOI Listing
April 2022

Depletion of transmembrane mucin 4 (Muc4) alters intestinal homeostasis in a genetically engineered mouse model of colorectal cancer.

Aging (Albany NY) 2022 03 7;14(5):2025-2046. Epub 2022 Mar 7.

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

Mucins are components of the mucus layer overlying the intestinal epithelial cells, which maintains physiological homeostasis. Altered mucin expression is associated with disease progression. Expression of MUC4 decreases in colorectal cancer (CRC); however, its functional role and implications in the intestinal pathology in CRC are not studied well. Therefore, we generated a genetically engineered Muc4 knockout (Muc4) CRC mouse model by crossing with Muc4 and mice in the presence of colon-specific inducible Cre. We observed that deficiency of Muc4 results in an increased number of macroscopic tumors in the colon and rectal region and leads to poor survival. Further, the absence of Muc4 was associated with goblet cell dysfunction where the expression of intestinal homeostasis molecules (Muc2 and Fam3D) was downregulated. Next, we also observed that loss of Muc4 showed reduced thickness of mucus layer, leading to infiltration of bacteria, reduction in anti-microbial peptides, and upregulation of pro-inflammatory cytokines. Further, gene mutation results in activation of the Wnt/β-catenin signaling pathway that corroborated with an increased nuclear accumulation of β-catenin and activation of its target genes: cyclin D1 and c-Myc in Muc4 mice was observed. We conclude that the presence of Muc4 is essential for intestinal homeostasis, reduces tumor burden, and improves overall survival.
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http://dx.doi.org/10.18632/aging.203935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8954958PMC
March 2022

Ubiquitous Aberration in Cholesterol Metabolism across Pancreatic Ductal Adenocarcinoma.

Metabolites 2022 Jan 7;12(1). Epub 2022 Jan 7.

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

Pancreatic cancer (PC) is characterized by metabolic deregulations that often manifest as deviations in metabolite levels and aberrations in their corresponding metabolic genes across the clinical specimens and preclinical PC models. Cholesterol is one of the critical metabolites supporting PC, synthesized or acquired by PC cells. Nevertheless, the significance of the de novo cholesterol synthesis pathway has been controversial in PC, indicating the need to reassess this pathway in PC. We utilized preclinical models and clinical specimens of PC patients and cell lines and utilized mass spectrometry-based sterol analysis. Further, we also performed in silico analysis to corroborate the significance of de novo cholesterol synthesis pathway in PC. Our results demonstrated alteration in free sterol levels, including free cholesterol, across in vitro, in vivo, and clinical specimens of PC. Especially, our sterol analyses established consistent alterations in free cholesterol across the different PC models. Overall, this study demonstrates the significance and consistency in deviation of cholesterol synthesis pathway in PC while showing the aberrations in sterol metabolite intermediates and the related genes using preclinical models, in silico platforms, and the clinical specimens.
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http://dx.doi.org/10.3390/metabo12010047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8779872PMC
January 2022

Disruption of FDPS/Rac1 axis radiosensitizes pancreatic ductal adenocarcinoma by attenuating DNA damage response and immunosuppressive signalling.

EBioMedicine 2022 Jan 28;75:103772. Epub 2021 Dec 28.

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

Background: Radiation therapy (RT) has a suboptimal effect in patients with pancreatic ductal adenocarcinoma (PDAC) due to intrinsic and acquired radioresistance (RR). Comprehensive bioinformatics and microarray analysis revealed that cholesterol biosynthesis (CBS) is involved in the RR of PDAC. We now tested the inhibition of the CBS pathway enzyme, farnesyl diphosphate synthase (FDPS), by zoledronic acid (Zol) to enhance radiation and activate immune cells.

Methods: We investigated the role of FDPS in PDAC RR using the following methods: in vitro cell-based assay, immunohistochemistry, immunofluorescence, immunoblot, cell-based cholesterol assay, RNA sequencing, tumouroids (KPC-murine and PDAC patient-derived), orthotopic models, and PDAC patient's clinical study.

Findings: FDPS overexpression in PDAC tissues and cells (P < 0.01 and P < 0.05) is associated with poor RT response and survival (P = 0.024). CRISPR/Cas9 and pharmacological inhibition (Zol) of FDPS in human and mouse syngeneic PDAC cells in conjunction with RT conferred higher PDAC radiosensitivity in vitro (P < 0.05, P < 0.01, and P < 0.001) and in vivo (P < 0.05). Interestingly, murine (P = 0.01) and human (P = 0.0159) tumouroids treated with Zol+RT showed a significant growth reduction. Mechanistically, RNA-Seq analysis of the PDAC xenografts and patients-PBMCs revealed that Zol exerts radiosensitization by affecting Rac1 and Rho prenylation, thereby modulating DNA damage and radiation response signalling along with improved systemic immune cells activation. An ongoing phase I/II trial (NCT03073785) showed improved failure-free survival (FFS), enhanced immune cell activation, and decreased microenvironment-related genes upon Zol+RT treatment.

Interpretation: Our findings suggest that FDPS is a novel radiosensitization target for PDAC therapy. This study also provides a rationale to utilize Zol as a potential radiosensitizer and as an immunomodulator in PDAC and other cancers.

Funding: National Institutes of Health (P50, P01, and R01).
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http://dx.doi.org/10.1016/j.ebiom.2021.103772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8718746PMC
January 2022

Emerging Role of miR-345 and Its Effective Delivery as a Potential Therapeutic Candidate in Pancreatic Cancer and Other Cancers.

Pharmaceutics 2021 Nov 23;13(12). Epub 2021 Nov 23.

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

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with high mortality, poor prognosis, and palliative treatments, due to the rapid upregulation of alternative compensatory pathways and desmoplastic reaction. miRNAs, small non-coding RNAs, have been recently identified as key players regulating cancer pathogenesis. Dysregulated miRNAs are associated with molecular pathways involved in tumor development, metastasis, and chemoresistance in PDAC, as well as other cancers. Targeted treatment strategies that alter miRNA levels in cancers have promising potential as therapeutic interventions. miRNA-345 (miR-345) plays a critical role in tumor suppression and is differentially expressed in various cancers, including pancreatic cancer (PC). The underlying mechanism(s) and delivery strategies of miR-345 have been investigated by us previously. Here, we summarize the potential therapeutic roles of miR-345 in different cancers, with emphasis on PDAC, for miRNA drug discovery, development, status, and implications. Further, we focus on miRNA nanodelivery system(s), based on different materials and nanoformulations, specifically for the delivery of miR-345.
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http://dx.doi.org/10.3390/pharmaceutics13121987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8707074PMC
November 2021

Correction: Kaushal et al. Repurposing Niclosamide for Targeting Pancreatic Cancer by Inhibiting Hh/Gli Non-Canonical Axis of Gsk3β. 2021, , 3105.

Cancers (Basel) 2021 Nov 9;13(22). Epub 2021 Nov 9.

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

The authors would like to make a correction to their published paper [...].
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http://dx.doi.org/10.3390/cancers13225591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8616051PMC
November 2021

Differential gene expression-based connectivity mapping identified novel drug candidate and improved Temozolomide efficacy for Glioblastoma.

J Exp Clin Cancer Res 2021 Oct 25;40(1):335. Epub 2021 Oct 25.

Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, 68198-5870, USA.

Background: Glioblastoma (GBM) has a devastating median survival of only one year. Treatment includes resection, radiation therapy, and temozolomide (TMZ); however, the latter increased median survival by only 2.5 months in the pivotal study. A desperate need remains to find an effective treatment.

Methods: We used the Connectivity Map (CMap) bioinformatic tool to identify candidates for repurposing based on GBM's specific genetic profile. CMap identified histone deacetylase (HDAC) inhibitors as top candidates. In addition, Gene Expression Profiling Interactive Analysis (GEPIA) identified HDAC1 and HDAC2 as the most upregulated and HDAC11 as the most downregulated HDACs. We selected PCI-24781/abexinostat due to its specificity against HDAC1 and HDAC2, but not HDAC11, and blood-brain barrier permeability.

Results: We tested PCI-24781 using in vitro human and mouse GBM syngeneic cell lines, an in vivo murine orthograft, and a genetically engineered mouse model for GBM (PEPG - PTEN; EGFRvIII+; p16 & GFAP Cre +). PCI-24781 significantly inhibited tumor growth and downregulated DNA repair machinery (BRCA1, CHK1, RAD51, and O-methylguanine-DNA- methyltransferase (MGMT)), increasing DNA double-strand breaks and causing apoptosis in the GBM cell lines, including an MGMT expressing cell line in vitro. Further, PCI-24781 decreased tumor burden in a PEPG GBM mouse model. Notably, TMZ + PCI increased survival in orthotopic murine models compared to TMZ + vorinostat, a pan-HDAC inhibitor that proved unsuccessful in clinical trials.

Conclusion: PCI-24781 is a novel GBM-signature specific HDAC inhibitor that works synergistically with TMZ to enhance TMZ efficacy and improve GBM survival. These promising MGMT-agnostic results warrant clinical evaluation.
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http://dx.doi.org/10.1186/s13046-021-02135-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8543939PMC
October 2021

Nuclear factor kappa-B contributes to cigarette smoke tolerance in pancreatic ductal adenocarcinoma through cysteine metabolism.

Biomed Pharmacother 2021 Dec 19;144:112312. Epub 2021 Oct 19.

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

Background: Retrospective studies revealed that cigarette smoking enhances risk of incidence and worsens prognosis in pancreatic cancer (PC) patients. Poor prognosis in smoker cohort of PC patients indicates prevalence of cigarette smoke stimulated survival mechanisms yet to be explored in PC. In this study, cigarette smoke induced metabolic pathways were explored and targeted in PC.

Methods: Human pancreatic ductal adenocarcinoma cell (PDAC) lines, genetically engineered mice models (GEMMs), mass spectrometry based heavy isotope-based metabolite analysis, cytotoxicity assays and Nuclear factor kappa-B (NF-kB) targeting were utilized in this study. Cigarette smoke extract (CSE) was prepared fresh each day by bubbling cell culture media with the smoke emitted from 85 mm, filtered, Code 1R6F reference cigarettes and used for in vitro procedures. High dose cigarette smoke exposure of GEMMs was achieved by daily exposure of animals to similar cigarettes, 6 h/day for a total period of 180 days.

Findings: We observed that PDAC cells upregulate glutathione anabolism through cysteine uptake and glutamate cysteine ligase (GCLM), supporting survival, upon CSE exposure. In vivo, cigarette smoke exposure leads to concomitant upregulation of GCLM and activated NF-kB in the PDAC consistent with in vitro, in CSE-exposed PDAC. Finally, either inhibition of NF-kB or depletion of cysteine impaired PDAC cell survival in cigarette smoke exposed conditions through suppression of glutathione and ROS enhancement, reverted by glutathione supplementation.

Interpretation: Our findings demonstrate scope for targeting smoke induced, NF-kB mediated, cysteine and glutathione metabolism for improving the survival of smoke addicted PDAC.
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http://dx.doi.org/10.1016/j.biopha.2021.112312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8599650PMC
December 2021

Reduction in O-glycome induces differentially glycosylated CD44 to promote stemness and metastasis in pancreatic cancer.

Oncogene 2022 01 21;41(1):57-71. Epub 2021 Oct 21.

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

Aberrant protein glycosylation has been shown to have a significant contribution in aggressive cancer, including pancreatic cancer (PC). Emerging evidence has implicated the involvement of cancer stem cells (CSCs) in PC aggressiveness; however, the contribution of glycosylation on self-renewal properties and maintenance of CSC is understudied. Here, using several in vitro and in vivo models lacking C1GALT1 expression, we identified the role of aberrant O-glycosylation in stemness properties and aggressive PC metastasis. A loss in C1GALT1 was found to result in the truncation of O-glycosylation on several glycoproteins with an enrichment of Tn carbohydrate antigen. Mapping of Tn-bearing glycoproteins in C1GALT1 KO cells identified significant Tn enrichment on CSC glycoprotein CD44. Notably, a loss of C1GALT1 in PC cells was found to enhance CSC features (side population-SP, ALDH1+, and tumorspheres) and self-renewal markers NANOG, SOX9, and KLF4. Furthermore, a loss of CD44 in existing C1GALT1 KO cells decreased NANOG expression and CSC features. We determined that O-glycosylation of CD44 activates ERK/NF-kB signaling, which results in increased NANOG expression in PC cells that facilitated the alteration of CSC features, suggesting that NANOG is essential for PC stemness. Finally, we identified that loss of C1GALT1 expression was found to augment tumorigenic and metastatic potential, while an additional loss of CD44 in these cells reversed the effects. Overall, our results identified that truncation of O-glycans on CD44 increases NANOG activation that mediates increased CSC activation.
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http://dx.doi.org/10.1038/s41388-021-02047-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8727507PMC
January 2022

Mucins, gut microbiota, and postbiotics role in colorectal cancer.

Gut Microbes 2021 Jan-Dec;13(1):1974795

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

An imbalance in the crosstalk between the host and gut microbiota affects the intestinal barrier function, which results in inflammatory diseases and colorectal cancer. The colon epithelium protects itself from a harsh environment and various pathogenic organisms by forming a double mucus layer, primarily comprising mucins. Recent studies are focusing on how dietary patterns alter the gut microbiota composition, which in turn regulates mucin expression and maintains the intestinal layers. In addition, modulation of gut microbiota by microbiotic therapy (involving fecal microbiota transplantation) has emerged as a significant factor in the pathologies associated with dysbiosis. Therefore, proper communication between host and gut microbiota different dietary patterns (prebiotics and probiotics) is needed to maintain mucus composition, mucin synthesis, and regulation. Here, we review how the interactions between diet and gut microbiota and bacterial metabolites () regulate mucus layer functionalities and mucin expression in human health and disease.
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http://dx.doi.org/10.1080/19490976.2021.1974795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8489937PMC
January 2022

Pancreatic Tumor Microenvironment Factor Promotes Cancer Stemness via SPP1-CD44 Axis.

Gastroenterology 2021 12 19;161(6):1998-2013.e7. Epub 2021 Aug 19.

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

Background & Aims: Tumor-microenvironment factors and cancer stem cells (CSCs) play a critical role in the aggressiveness of pancreatic cancer (PC). However, the degree to which tumor-microenvironment factors promote stemness remains unexplored. Here, we examined whether cancer-associated fibroblasts (CAFs) promote CSC features in PC.

Methods: PC cells were treated long-term (30, 60, and 90 days) with conditioned media (CM)-derived from normal human fibroblasts (NFs) and CAFs. The stemness features of tumorsphere formation and stemness populations, along with CSCs markers, were analyzed using 2-dimensional and 3-dimensional sodium alginate bead-based co-culture models. Immunohistochemistry and immunofluorescence staining were performed for CSCs and fibroblast markers in autochthonous Kras; Trp53; Pdx1-Cre mice and human pancreatic tumors. Polymerase chain reaction array and gene knockdown were performed to identify the mechanism of stemness enrichment.

Results: Long-term treatment of PC cells with CAF-CM enriched stemness, as indicated by significantly higher CD44, ALDH, and AF populations in PC cells. Increased tumorsphere formation and elevated CSC, self-renewal, and drug-resistance markers in CAF-CM-treated PC cells were observed. In addition, CAFs co-cultured with PC cells in the 3-dimensional model showed a substantial increase in stemness features. CD44 and α-smooth muscle actin were positively correlated and their expressions progressively increased from the early to late stages of Kras; Trp53; Pdx1-Cre mouse and human pancreatic tumors. Osteopontin/secreted phosphoprotein 1 was identified as the top differentially overexpressed gene in CAF-CM-treated PC cells and knockdown of osteopontin/secreted phosphoprotein 1 significantly reduced stemness characteristics in CAF-CM-treated PC cells.

Conclusions: Our data uncovered novel insight into the interplay between CAF and enrichment of stemness population through the osteopontin/secreted phosphoprotein 1-CD44 axis in PC.
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http://dx.doi.org/10.1053/j.gastro.2021.08.023DOI Listing
December 2021

MASTL regulates EGFR signaling to impact pancreatic cancer progression.

Oncogene 2021 09 30;40(38):5691-5704. Epub 2021 Jul 30.

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

Pancreatic cancer (PC) remains a major cause of cancer-related deaths primarily due to its inherent potential of therapy resistance. Checkpoint inhibitors have emerged as promising anti-cancer agents when used in combination with conventional anti-cancer therapies. Recent studies have highlighted a critical role of the Greatwall kinase (microtubule-associated serine/threonine-protein kinase-like (MASTL)) in promoting oncogenic malignancy and resistance to anti-cancer therapies; however, its role in PC remains unknown. Based on a comprehensive investigation involving PC patient samples, murine models of PC progression (Kras;PdxCre-KC and Kras;p53;PdxCre-KPC), and loss and gain of function studies, we report a previously undescribed critical role of MASTL in promoting cancer malignancy and therapy resistance. Mechanistically, MASTL promotes PC by modulating the epidermal growth factor receptor protein stability and, thereupon, kinase signaling. We further demonstrate that combinatorial therapy targeting MASTL promotes the efficacy of the cell-killing effects of Gemcitabine using both genetic and pharmacological inhibitions. Taken together, this study identifies a key role of MASTL in promoting PC progression and its utility as a novel target in promoting sensitivity to the anti-PC therapies.
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http://dx.doi.org/10.1038/s41388-021-01951-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8817225PMC
September 2021

Repurposing Niclosamide for Targeting Pancreatic Cancer by Inhibiting Hh/Gli Non-Canonical Axis of Gsk3β.

Cancers (Basel) 2021 Jun 22;13(13). Epub 2021 Jun 22.

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

Niclosamide (Nic), an FDA-approved anthelmintic drug, is reported to have anti-cancer efficacy and is being assessed in clinical trials for various solid tumors. Based on its ability to target multiple signaling pathways, in the present study, we evaluated the therapeutic efficacy of Nic on pancreatic cancer (PC) in vitro. We observed an anti-cancerous effect of this drug as shown by the G0/G1 phase cell cycle arrest, inhibition of PC cell viability, colony formation, and migration. Our results revealed the involvement of mitochondrial stress and mTORC1-dependent autophagy as the predominant players of Nic-induced PC cell death. Significant reduction of Nic-induced reactive oxygen species (ROS) and cell death in the presence of a selective autophagy inhibitor spautin-1 demonstrated autophagy as a major contributor to Nic-mediated cell death. Mechanistically, Nic inhibited the interaction between BCL2 and Beclin-1 that supported the crosstalk of autophagy and apoptosis. Further, Nic treatment resulted in Gsk3β inactivation by phosphorylating its Ser-9 residue leading to upregulation of Sufu and Gli3, thereby negatively impacting hedgehog signaling and cell survival. Nic induced autophagic cell death, and p-Gsk3b mediated Sufu/Gli3 cascade was further confirmed by Gsk3β activator, LY-294002, by rescuing inactivation of Hh signaling upon Nic treatment. These results suggested the involvement of a non-canonical mechanism of Hh signaling, where p-Gsk3β acts as a negative regulator of Hh/Gli1 cascade and a positive regulator of autophagy-mediated cell death. Overall, this study established the therapeutic efficacy of Nic for PC by targeting p-Gsk3β mediated non-canonical Hh signaling and promoting mTORC1-dependent autophagy and cell death.
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http://dx.doi.org/10.3390/cancers13133105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8269055PMC
June 2021

PGC1α-Mediated Metabolic Reprogramming Drives the Stemness of Pancreatic Precursor Lesions.

Clin Cancer Res 2021 10;27(19):5415-5429

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

Purpose: Metabolic reprogramming and cancer stem cells drive the aggressiveness of pancreatic ductal adenocarcinoma (PDAC). However, the metabolic and stemness programs of pancreatic precursor lesions (PPL), considered early PDAC development events, have not been thoroughly explored.

Experimental Design: Meta-analyses using gene expression profile data from NCBI Gene Expression Omnibus and IHC on tissue microarrays (TMA) were performed. The following animal and cellular models were used: cerulean-induced KrasG12D; Pdx1 Cre (KC) acinar-to-ductal metaplasia (ADM) mice, KrasG12D; Smad4Loss; Pdx-1 Cre (KCSmad4-) intraductal papillary mucinous neoplasm (IPMN) mice, LGKC1 cell line derived from the doxycycline-inducible Gnas IPMN model, and human IPMN organoids. Flow cytometry, Seahorse extracellular flux analyzer, qRT-PCR, and sphere assay were used to analyze metabolic and stemness features. SR18292 was used to inhibit PGC1α, and short hairpin RNA was used to knockdown (KD) PGC1α.

Results: The meta-analysis revealed a significant upregulation of specific stemness genes in ADM-mediated pancreatic intraepithelial neoplasms (PanIN) and IPMN. Meta- and TMA analyses followed by in vitro and in vivo validation revealed that ADM/PanIN exhibit increased PGC1α and oxidative phosphorylation (OXPhos) but reduced CPT1A. IPMN showed elevated PGC1α, fatty acid β-oxidation (FAO) gene expression, and FAO-OXPhos. PGC1α was co-overexpressed with its coactivator NRF1 in ADM/PanINs and with PPARγ in IPMN. PGC1α KD or SR18292 inhibited the specific metabolic and stemness features of PPLs and repressed IPMN organoid growth.

Conclusions: ADM/PanINs and IPMNs show specific stemness signatures with unique metabolisms. Inhibition of PGC1α using SR18292 diminishes the specific stemness by targeting FAO-independent and FAO-dependent OXPhos of ADM/PanINs and IPMNs, respectively.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-5020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8709878PMC
October 2021

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

Cancer Lett 2021 07 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

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 07 1;15(7):1866-1881. Epub 2021 May 1.

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
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8253099PMC
July 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

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

Oncogene 2021 01 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 02 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

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

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

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

Acinar transformed ductal cells exhibit differential mucin expression in a tamoxifen-induced pancreatic ductal adenocarcinoma mouse model.

Biol Open 2020 09 7;9(9). Epub 2020 Sep 7.

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

Pancreatic cancer (PC) is acquired postnatally; to mimic this scenario, we developed an inducible Kras; Ptf1a-CreER™ (iKC) mouse model, in which Kras is activated postnatally at week 16 upon tamoxifen (TAM) administration. Upon TAM treatment, iKC mice develop pancreatic intraepithelial neoplasia (PanIN) lesions and PC with metastasis at the fourth and fortieth weeks, respectively, and exhibited acinar-to-ductal metaplasia (ADM) and transdifferentiation. Kras activation upregulated the transcription factors Ncoa3, p-cJun and FoxM1, which in turn upregulated expression of transmembrane mucins (Muc1, Muc4 and Muc16) and secretory mucin (Muc5Ac). Interestingly, knockdown of Kras in multiple PC cell lines resulted in downregulation of MUC1, MUC4, MUC5AC and MUC16. In addition, iKC mice exhibited ADM and transdifferentiation. Our results show that the iKC mouse more closely mimics human PC development and can be used to investigate pancreatic ductal adenocarcinoma (PDAC) biomarkers, early onset of PDAC, and ADM. The iKC model can also be used for preclinical strategies such as targeting mucin axis alone or in combination with neo-adjuvant, immunotherapeutic approaches and to monitor chemotherapy response.
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http://dx.doi.org/10.1242/bio.052878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7502593PMC
September 2020

CXCR2 signaling promotes secretory cancer-associated fibroblasts in pancreatic ductal adenocarcinoma.

FASEB J 2020 07 26;34(7):9405-9418. Epub 2020 May 26.

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

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most challenging malignancies. Desmoplasia and tumor-supporting inflammation are hallmarks of PDAC. The tumor microenvironment contributes significantly to tumor progression and spread. Cancer-associated fibroblasts (CAFs) facilitate therapy resistance and metastasis. Recent reports emphasized the concurrence of multiple subtypes of CAFs with diverse roles, fibrogenic, and secretory. C-X-C motif chemokine receptor 2 (CXCR2) is a chemokine receptor known for its role during inflammation and its adverse role in PDAC. Oncogenic Kras upregulates CXCR2 and its ligands and, thus, contribute to tumor proliferation and immunosuppression. CXCR2 deletion in a PDAC syngeneic mouse model produced increased fibrosis revealing a potential undescribed role of CXCR2 in CAFs. In this study, we demonstrate that the oncogenic Kras-CXCR2 axis regulates the CAFs function in PDAC and contributes to CAFs heterogeneity. We observed that oncogenic Kras and CXCR2 signaling alter CAFs, producing a secretory CAF phenotype with low fibrogenic features; and increased secretion of pro-tumor cytokines and CXCR2 ligands, utilizing the NF-κB activity. Finally, using syngeneic mouse models, we demonstrate that oncogenic Kras is associated with secretory CAFs and that CXCR2 inhibition promotes activation of fibrotic cells (myofibroblasts) and impact tumors in a mutation-dependent manner.
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http://dx.doi.org/10.1096/fj.201902990RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7501205PMC
July 2020

Correction: Global analysis of human glycosyltransferases reveals novel targets for pancreatic cancer pathogenesis.

Br J Cancer 2020 May;122(11):1726

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

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41416-020-0842-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7251069PMC
May 2020

Global analysis of human glycosyltransferases reveals novel targets for pancreatic cancer pathogenesis.

Br J Cancer 2020 05 19;122(11):1661-1672. Epub 2020 Mar 19.

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

Background: Several reports have shown the role of glycosylation in pancreatic cancer (PC), but a global systematic screening of specific glycosyltransferases (glycoTs) in its progression remains unknown.

Methods: We demonstrate a rigorous top-down approach using TCGA-based RNA-Seq analysis, multi-step validation using RT-qPCR, immunoblots and immunohistochemistry. We identified six unique glycoTs (B3GNT3, B4GALNT3, FUT3, FUT6, GCNT3 and MGAT3) in PC pathogenesis and studied their function using CRISPR/Cas9-based KD systems.

Results: Serial metastatic in vitro models using T3M4 and HPAF/CD18, generated in house, exhibited decreases in B3GNT3, FUT3 and GCNT3 expression on increasing metastatic potential. Immunohistochemistry identified clinical significance for GCNT3, B4GALNT3 and MGAT3 in PC. Furthermore, the effects of B3GNT3, FUT3, GCNT3 and MGAT3 were shown on proliferation, migration, EMT and stem cell markers in CD18 cell line. Talniflumate, GCNT3 inhibitor, reduced colony formation and migration in T3M4 and CD18 cells. Moreover, we found that loss of GCNT3 suppresses PC progression and metastasis by downregulating cell cycle genes and β-catenin/MUC4 axis. For GCNT3, proteomics revealed downregulation of MUC5AC, MUC1, MUC5B including many other proteins.

Conclusions: Collectively, we demonstrate a critical role of O- and N-linked glycoTs in PC progression and delineate the mechanism encompassing the role of GCNT3 in PC.
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http://dx.doi.org/10.1038/s41416-020-0772-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7251111PMC
May 2020

Mechanistic and Functional Shades of Mucins and Associated Glycans in Colon Cancer.

Cancers (Basel) 2020 Mar 11;12(3). Epub 2020 Mar 11.

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

Mucus serves as the chief protective barrier against pathogenic and mechanical insults in respiratory, gastrointestinal, and urogenital tracts. Altered mucin expression, the major component of mucus, in conjunction with differential glycosylation has been strongly associated with both benign and malignant pathologies of colon. Mucins and their associated glycans arbitrate their impact sterically as well as mechanically by altering molecular and microbial spectrum during pathogenesis. Mucin expression in normal and pathological conditions is regulated by nonspecific (dietary factors and gut microbiota) and specific (epigenetic and transcriptional) modulators. Further, recent studies highlight the impact of altering mucin glycome (cancer-associated carbohydrate antigens including Tn, Sialyl-Tn, Sialyl-Lew A, and Sialyl-Lewis X) on host immunomodulation, antitumor immunity, as well as gut microbiota. In light of emerging literature, the present review article digs into the impact of structural organization and of expressional and glycosylation alteration of mucin family members on benign and malignant pathologies of colorectal cancer.
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http://dx.doi.org/10.3390/cancers12030649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139953PMC
March 2020
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