• The aryl hydrocarbon receptor agonist benzo(a)pyrene reactivates LINE-1 in HepG2 cells through canonical TGF-beta 1 signaling: implications in hepatocellular carcinogenesis

      Reyes-Reyes, Elsa M; Ramos, Irma N; Tavera-Garcia, Marco A; Ramos, Kenneth S; Univ Arizona, Div Pulm Allergy Crit Care & Sleep Med, Coll Med (E-CENTURY PUBLISHING CORP, 2016)
      Long interspersed nuclear element-1 (L1) is a genetic element that mobilizes throughout the mammalian genome via retrotransposition and damages host DNA via mutational insertions, chromosomal rearrangements, and reprogramming of gene expression. The cellular mechanisms responsible for aberrant L1 expression during cancer pathogenesis are unclear. Previously, we have shown that L1 reactivation in several human cell lines is dependent upon the activation of aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor member of the PAS superfamily of proteins. We also showed that ectopic expression of L1 reprograms the HepG2 genome leading to epithelial-to-mesenchymal transition (EMT). Here we present evidence that reactivation of L1 and modulation of EMT in HepG2 cells by the AhR ligand benzo(a)pyrene (BaP) is effected through the canonical TGF-β1 signaling pathway. BaP increased TGF-β1 mRNA, SMAD2 phosphorylation and decreased expression of E-Cadherin. The functional relevance of these interactions and the involvement of TGFBR1/ALK5 and SMAD2/3 were confirmed by siRNA interference. Furthermore, expression of L1-encoded ORF1p was positively correlated with the activation of TGF-β1 signaling in human hepatocarcinoma samples at various stages of malignant progression. These results indicate that ligand-mediated AhR activation regulates L1 via canonical TGF-β1 signaling and raise important questions about the molecular etiology of human hepatocarcinomas.
    • Enrichment and characterization of ovarian cancer stem cells and its potential clinical application

      Wang, Wenxia; Zhang, Zhenbo; Zhao, Yin; Yuan, Zeng; Yang, Xingsheng; Kong, Beihua; Zheng, Wenxin; Univ Arizona, Dept Pathol (E-CENTURY PUBLISHING CORP, 2017-03-02)
      The cancer stem cell (CSC) theory proposes that a minor population in tumor cells with specific features, such as self-renewal and reproducible tumor phenotype could contribute to tumor relapse and chemotherapy resistance. Several studies have convincingly documented the existence of ovarian CSC, but questions related to the biologic behavior and specific biomarkers of ovarian CSC remain to be clarified. In the present study, we firstly established a tumor cell line with capability of regenerating tumors through serial transplantation of ovarian tumor tissue in non-obese/severe combined immunodeficient (SCID) mice. After separation of CD133+ cells with magnetic beads, we compared the phenotype and biologic behavior of CD133+ versus CD133-cells. It was found that the CD133+ cells were much more potent to produce colonies in semi-solid agar culture than CD133-cells. The proportion of the cells in G0/1 cell cycle is much higher in CD133+ cells than in CD133-cells. Furthermore, in vivo experiments demonstrated that the CD133+ cells were capable of repeatedly regenerate tumors in NOD/SCID mice, while the CD133-cells were not. Compared with CD133-cells, the CD133+ cells expressed much higher levels of the stem cell markers Oct4, Sox2, Nanog and Mcl-1. Clinically, among a total of 290 ovarian epithelial cancers, increased level of CD133 expression was positively correlated with a high cancer stage and had a worse 5-year survival rate. Taken together, the results suggest that the CD133+ cells from human ovarian cancer have the characteristics of CSC, which may contribute to ovarian cancer relapse and anti-apoptotic activity. The method of ovarian CSC enrichment we established provides a feasible and practical way of ovarian cancer research in a molecular level. In addition, CD133 may be used as a prognostic marker for ovarian epithelial cancer, which may have a role for future therapeutic effect.
    • Hypoxia-induced PIM kinase and laminin-activated integrin alpha 6 mediate resistance to PI3K inhibitors in bone-metastatic CRPC

      Toth, Rachel K; Tran, Jack D; Muldong, Michelle T; Nollet, Eric A; Schulz, Veronique V; Jensen, Corbin C; Hazlehurst, Lori A; Corey, Eva; Durden, Donald; Jamieson, Christina; et al. (E-CENTURY PUBLISHING CORP, 2019)
      Bone-metastatic castration-resistant prostate cancer (CRPC) is lethal due to inherent resistance to androgen deprivation therapy, chemotherapy, and targeted therapies. Despite the fact that a majority of CRPC patients (approximately 70%) harbor a constitutively active PI3K survival pathway, targeting the PI3K/mTOR pathway has failed to increase overall survival in clinical trials. Here, we identified two separate and independent survival pathways induced by the bone tumor microenvironment that are hyperactivated in CRPC and confer resistance to PI3K inhibitors. The first pathway involves integrin α6β1-mediated adhesion to laminin and the second involves hypoxia-induced expression of PIM kinases. In vitro and in vivo models demonstrate that these pathways transduce parallel but independent signals that promote survival by reducing oxidative stress and preventing cell death. We further demonstrate that both pathways drive resistance to PI3K inhibitors in PTEN-negative tumors. These results provide preclinical evidence that combined inhibition of integrin α6β1 and PIM kinase in CRPC is required to overcome tumor microenvironment-mediated resistance to PI3K inhibitors in PTEN-negative tumors within the hypoxic and laminin-rich bone microenvironment.
    • Laminin-binding integrin gene copy number alterations in distinct epithelial-type cancers.

      Harryman, William L; Pond, Erika; Singh, Parminder; Little, Andrew S; Eschbacher, Jennifer M; Nagle, Raymond B; Cress, Anne E; Univ Arizona, Ctr Canc (E-CENTURY PUBLISHING CORP, 2016)
      The laminin-binding integrin (LBI) family are cell adhesion molecules that are essential for invasion and metastasis of human epithelial cancers and cell adhesion mediated drug resistance. We investigated whether copy number alteration (CNA) or mutations of a five-gene signature (ITGB4, ITGA3, LAMB3, PLEC, and SYNE3), representing essential genes for LBI adhesion, would correlate with patient outcomes within human epithelial-type tumor data sets currently available in an open access format.
    • Overexpression and oncogenic function of HMGA2 in endometrial serous carcinogenesis

      Wei, Linxuan; Liu, Xiaolin; Zhang, Wenjing; Wei, Yuyan; Li, Yingwei; Zhang, Qing; Dong, Ruifen; Kwon, Jungeun Sarah; Liu, Zhaojian; Zheng, Wenxin; et al. (E-CENTURY PUBLISHING CORP, 2016)
      The high-mobility group A protein 2 (HMGA2) is a non-histone chromatin factor highly expressed in fetal tissue and malignant tumors but rarely detected within normal adult tissues. The clinical implications and biological functions of HMGA2 in endometrial carcinoma are largely unknown. Here we report that HMGA2 expression was barely detected in benign endometrium samples (2 of 28 samples). However, HMGA2 expression increased significantly from precancerous lesion endometrial glandular dysplasia (7 of 17, 41.2%), to serous endometrial intraepithelial carcinoma (5 of 8, 62.5%) and to full blown endometrial serous carcinoma (39 of 59, 66.1%). Functional characterization of HMGA2 revealed that the gene has both tumor growth promotion and metastasis. In addition, HMGA2 induced epithelial-mesenchymal transition (EMT) through modulation vimentin and β-catenin. Furthermore, HMGA2 overexpression started from endometrial serous precancers, non-invasive cancers, as well as in full blown carcinomas in a p53 knockout mouse model we recently established in our laboratory. Our findings suggest that HMGA2 may serve as a useful diagnostic marker in the assessment of endometrial serous cancer and its precursor lesions.
    • A pilot clinical trial of the cytidine deaminase inhibitor tetrahydrouridine combined with decitabine to target DNMT1 in advanced, chemorefractory pancreatic cancer

      Sohal, Davendra; Krishnamurthi, Smitha; Tohme, Rita; Gu, Xiaorong; Lindner, Daniel; Landowski, Terry H; Pink, John; Radivoyevitch, Tomas; Fada, Sherry; Lee, Zhenghong; et al. (E-CENTURY PUBLISHING CORP, 2020-09-01)
      DNA methyltransferase 1 (DNMT1) is scientifically validated as a molecular target to treat chemo-resistant pancreatic ductal adenocarcinoma (PDAC). Results of clinical studies of the pyrimidine nucleoside analog decitabine to target DNMT1 in PDAC have, however, disappointed. One reason is high expression in PDAC of the enzyme cytidine deaminase (CDA), which catabolizes decitabine within minutes. We therefore added tetrahydrouridine (THU) to inhibit CDA with decitabine. In this pilot clinical trial, patients with advanced chemorefractory PDAC ingested oral THU ~10 mg/kg/day combined with oral decitabine ~0.2 mg/kg/day, for 5 consecutive days, then 2X/week. We treated 13 patients with extensively metastatic chemo-resistant PDAC, including 8 patients (62%) with ascites: all had received ≥ 1 prior therapies including gemcitabine/nab-paclitaxel in 9 (69%) and FOLFIRINOX in 12 (92%). Median time on THU/decitabine treatment was 35 days (range 4-63). The most frequent treatment-attributable adverse event was anemia (n=5). No deaths were attributed to THU/decitabine. Five patients had clinical progressive disease (PD) prior to week 8. Eight patients had week 8 evaluation scans: 1 had stable disease and 7 PD. Median overall survival was 3.1 months. Decitabine systemic exposure is expected to decrease neutrophil counts; however, neutropenia was unexpectedly mild. To identify reasons for limited systemic decitabine effect, we measured plasma CDA enzyme activity in PDAC patients, and found a > 10-fold increase in those with metastatic vs resectable PDAC. We concluded that CDA activity is increased not just locally but also systemically in metastatic PDAC, suggesting a need for even higher CDA-inhibitor doses than used here.
    • ST2-104 attenuates neuronal injuries in A beta(25-35)-induced AD rats by inhibiting CRMP2-NMDAR2B signaling pathways

      Ji, Yingshi; Meng, Panpan; Hu, Yang; Khanna, Rajesh; Zhang, Yuqing; Li, Qi; Ren, Jinghong; Sun, Li; Univ Arizona, Coll Med, Dept Med Pharmacol (E-CENTURY PUBLISHING CORP, 2019)
      Collapsin response mediator protein 2 (CRMP2), traditionally regarded as an axon/dendrite growth and guidance protein, plays an important role in the regulation of both post-and pre-synaptic Ca2+ channels, such as N-methyl-d-aspartate receptors (NMDARs). The Ca2+ channel-binding domain 3 (CBD3) peptide derived from CRMP2 has recently emerged as a Ca2+ channel blocker, suppressing neuropathic pain in a spared nerve injury (SNI) model when linked to the transduction domain of HIV TAT protein and reduced neuronal death in a middle cerebral artery occlusion model and a traumatic brain injury (TBI) model. The present study aimed to examine the neuroprotective effects and biochemical mechanisms of ST2-104 (a non-arginine-conjugated CBD3 peptide) in an A beta(25-35)-induced Alzheimer's disease (AD) rat model. This study demonstrated that CRMP2 and NMDARs subunit NMDAR2B form a direct biochemical complex, which regulates NMDAR activity in a rat model. ST2-104 peptide given via tail vein injections significantly reduced spatial learning and memory impairment. ST2-104 relieved neuronal injuries by suppressing expression of NMDAR2B and p-CRMP2 and increasing expression of CRMP2 in the hippocampus. Remarkably, ST2-104 attenuated levels of intracellular Ca2+ by disrupting the interaction between p-CRMP2 and NMDAR2B. Taken together, these findings support ST2-104 as a novel neuroprotective agent, potentially representing a novel direction for a therapeutic targeting channel in AD.
    • Synergistic antitumor effect of 5-fluorouracil and withaferin-A induces endoplasmic reticulum stress-mediated autophagy and apoptosis in colorectal cancer cells

      Alnuqaydan, Abdullah M; Rah, Bilal; Almutary, Abdulmajeed G; Chauhan, Shailender Singh; Univ Arizona, Dept Cellular & Mol Med (E-CENTURY PUBLISHING CORP, 2020-03-01)
      The development of chemo-resistance against 5-fluorouracil (5-FU) in tumor cells is one of the main debacles in colorectal cancer (CRC) patients. A recent combination of 5-FU with oxaliplatin or cetuximab drastically improves the survival rate in CRC patients; however, the toxicity issue cannot be evaded completely. Thus, searching for novel drug combinations with high specificity and low toxicity is seemingly important. Owing to the less undesirable effects of natural products on normal cells, here we investigated the synergistic antitumor effect of withaferin-A (WA) in combination with 5-FU. Our results demonstrate that the combination of WA and 5-FU induces a significant antiproliferative effect and modulates endoplasmic reticulum (ER) stress in favor of cell death in colorectal cancer (CRC) cells. Mechanistically, the combination upregulates the expression of ER stress sensors (BiP, PERK, CHOP, ATF-4, and eIF2α) and executes PERK axis mediated apoptosis in CRC cells. Additionally, the combined treatment of WA and 5-FU mediated ER stress induces autophagy and apoptosis, which were confirmed by immunoblotting, acridine orange (AO) staining and annexin-V FITC by flow cytometry. In contrast, inhibition of ER stress with salubrinal significantly decreases both autophagic and apoptotic cell populations. Moreover, pharmacological inhibition of either autophagy or apoptosis by their respective inhibitors 3-methyladenine (3-MA) or carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoro-methyl ketone (Z-VAD-FMK) decreases their respective population of cells but could not affect either of the population significantly. Finally, the combination attenuates the expression of β-catenin pathway associated proteins and arrests cell cycle at the G2M phase in CRC cells. In summary, the combination of WA and 5-FU decreases cell viability by inducing ER stress-mediated induction of autophagy and apoptosis, inhibiting the β-catenin pathway and arresting the cell cycle at a G2M phase in CRC cells.