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Triptolide Induces the Expression of Mir-142-3P: a Negative Regulator of Heat Shock Protein 70 and Pancreatic Cancer Cell Proliferation

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Triptolide Induces the Expression of Mir-142-3P: a Negative Regulator of Heat Shock Protein 70 and Pancreatic Cancer Cell Proliferation Published OnlineFirst May 1, 2013; DOI: 10.1158/1535-7163.MCT-12-1231 Molecular Cancer Cancer Therapeutics Insights Therapeutics Triptolide Induces the Expression of miR-142-3p: A Negative Regulator of Heat Shock Protein 70 and Pancreatic Cancer Cell Proliferation Tiffany N. MacKenzie1, Nameeta Mujumdar2, Sulagna Banerjee2, Veena Sangwan2, Aaron Sarver3, Selwyn Vickers2,3, Subbaya Subramanian1,2,3, and Ashok K. Saluja1,2,3 Abstract Pancreatic ductal adenocarcinoma (PDAC), one of the deadliest malignancies, is resistant to current chemotherapies. We previously showed that triptolide inhibits PDAC cell growth in vitro and blocks metastatic spread in vivo. Triptolide downregulates HSP70, a molecular chaperone upregulated in several tumor types. This study investigates the mechanism by which triptolide inhibits HSP70. Because micro- RNAs (miRNA) are becoming increasingly recognized as negative regulators of gene expression, we tested whether triptolide regulates HSP70 via miRNAs. Here, we show that triptolide as well as quercetin, but not gemcitabine, upregulated miR-142-3p in PDAC cells (MIA PaCa-2, Capan-1, and S2-013). Ectopic expres- sion of miR-142-3p inhibited cell proliferation, measured by electric cell-substrate impedance sensing, and decreased HSP70 expression, measured by real-time PCR and immunoblotting, compared with controls. We showed that miR-142-3p directly binds to the 30UTR of HSP70, and that this interaction is important as HSP70 overexpression rescued miR–142-3p-induced cell death. We found that miR–142-3p regulates HSP70 independently of heat shock factor 1. Furthermore, Minnelide, a water-soluble prodrug of triptolide, induced the expression of miR-142-3p in vivo.ThisisthefirstdescriptionofanmiRNA- mediated mechanism of HSP70 regulation in cancer, making miR-142-3p an attractive target for PDAC therapeutic intervention. Mol Cancer Ther; 12(7); 1266–75. Ó2013 AACR. Introduction Triptolide, a diterpene triepoxide extract from the Chi- Pancreatic ductal adenocarcinoma (PDAC) is the fourth nese herb Tripterygium wilfordii, has been shown to inhibit leading cause of cancer-related death in the United States PDAC cell viability in vitro (2, 3) and to block growth and (1). At best, the 5-year survival rate for patients with metastatic spread in vivo (4). To date, in vivo studies have localized PDAC, more likely candidates for surgical resec- shown that triptolide inhibits the growth of cholangio- tion than those with metastatic disease, is 20%; however, carcinoma cells in hamsters (5) and xenografts of human when considering PDAC patients as a whole, including melanoma, breast cancer, bladder cancer, and gastric those with metastatic disease, the survival rate is only 5– carcinoma in nude mice (6). Previous data from our 6% (1). Over the past several decades, these survival rates laboratory have shown that triptolide inhibits the growth have not improved due, in large part, to aggressive of neuroblastoma cells in vitro (7) and prevents tumor growth, metastasis, and drug resistance of PDAC cells growth in vivo (8). (1). Efforts are ongoing to understand the pathobiology of Because triptolide was identified in a small-molecule PDAC and to develop innovative and effective therapies. screen to inhibit tumorigenic molecular chaperone HSP An important part of this process is to understand the gene transcription (9), our laboratory has continued to mechanism of action of potential chemotherapeutic show that triptolide, likewise, inhibits cancer cell prolif- agents. eration while concurrently inhibiting HSP70 expression in PDAC cells (2–4, 10) or neuroblastoma cells (11). Some studies suggest that triptolide inhibits the transcription Authors' Affiliations: 1Department of Pharmacology, 2Department of factor heat shock factor 1 (HSF1) and, in this way, sup- Surgery, 3Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota presses HSP70 transcription (9); however, as microRNAs (miRNA) become increasingly recognized as major neg- Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). ative regulators of gene expression, we asked whether triptolide may regulate HSP70 expression via miRNAs. Corresponding Author: Ashok K. Saluja, Department of Surgery, Univer- sity of Minnesota, MMC 195, 420 Delaware Street SE, Minneapolis, MN In cancer cells, the dysregulation of miRNAs expression 55455. Phone: 612-624-8108; Fax: 612-624-8909; E-mail: serves as an efficient means to rewire the cell gene expres- [email protected] sion map and generate a cancer phenotype. In particular, doi: 10.1158/1535-7163.MCT-12-1231 the loss of tumor-suppressive miRNAs upregulates onco- Ó2013 American Association for Cancer Research. genic targets (12). No previous reports have examined 1266 Mol Cancer Ther; 12(7) July 2013 Downloaded from mct.aacrjournals.org on September 23, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst May 1, 2013; DOI: 10.1158/1535-7163.MCT-12-1231 Triptolide Induces miR-142-3p in PDAC the effect of triptolide on the PDAC miRNAome nor Assay). To verify HSPA1B (HSP70) overexpression, evaluated miRNA-mediated regulation of HSP70 in HSP70 primers targeting the HSP70 ORF region (2) were DD PDAC cells. Consequently, the aims of this study are to: used. Quantification was done using the Ct method. (i) examine the effect of triptolide on the miRNAome of PDAC cells in vitro and verify this in vivo; (ii) evaluate Transfection of miR-142-3p mimic or inhibitor whether miRNAs upregulated by triptolide play a tumor- Cells were seeded in 6-well (8  104 cells/well) or 24- suppressive role in PDAC; and (iii) validate that predict- well plates (1.5  104 cells/well) and incubated overnight ed miRNAs regulate HSP70 independent of the HSF1 before transfection. Transfection mastermixes were dilut- pathway. We hypothesize that triptolide will upregulate ed in Reduced-Serum Medium (Opti-MEM, Life Technol- tumor-suppressive miRNAs, which directly regulate ogies) containing HiPerFect (Qiagen), miR-142-3p mimic, HSP70. This is the first evaluation of an miRNA-mediated miR-142-3p inhibitor, or negative control (NC) miRIDIAN mechanism of HSP70 regulation in cancer. reagents (Thermo Scientific Dharmacon). Transfected cells were analyzed for Western blotting or cell viability Materials and Methods as previously reported (2). Cell culture and drug treatment Cells from the MIA PaCa-2, Capan-1, and HEK-293 Cell proliferation using electric cell-substrate lines were obtained from the American Type Culture impedance sensing Collection (ATCC) and cultured in Dulbecco’s Modified Using the electric cell-substrate impedance sensing Eagle Medium (DMEM; Life Technologies) containing (ECIS) method, cells are grown on the surface of planar 10% FBS (Life Technologies). S2-013 cells were kindly gold-film electrodes and the AC impedance of the cell- provided by Professor Masato Yamamoto (University of covered electrode is measured continuously at a frequen- Minnesota at Minneapolis, Minneapolis, MN) and cul- cy of 4,000 Hz. Because of the insulating properties of cell tured in RPMI medium (Life Technologies) containing membranes, the impedance increases with increasing 10% FBS. Multiple aliquots of cells were cryopreserved coverage of the electrode. MIA PaCa-2 or Capan-1 cells  4  5 when initially grown. All the cell lines were used within 6 (6 10 cells/well) and S2-013 (1 10 cells/well) were þ months of resuscitation. No authentication was done by plated in 8-well, gold-film electrode-coated 10E arrays the authors, but ATCC authenticates cells using short (Applied Biophysics). Proliferation rates were normalized tandem repeat profiling. For all drug treatments, cells to 6 hours following transfection.  5 were seeded in 6-well plates (2.5 10 cells/well) and 0 incubated for the periods indicated in the figure legends Dual-Luciferase reporter assay and 3 UTR binding- before RNA or protein analysis. Triptolide treatment site mutagenesis (Calbiochem) was carried out as previously described HEK-293 cells were seeded in seeded in 24-well plates  4 (2, 3). Quercetin (Sigma-Aldrich) or gemcitabine (Eli Lilly (6 10 cells/well). Mastermixes were diluted in serum- Corporation) treatments were carried out as previously free media (Life Technologies) containing Attractene, reported (13, 14). Cells were maintained as previously pGL4.73 control vector expressing firefly luciferase described (2, 3). (Promega), GoClone (HSPA1B) containing the wild-type HSPA1B 30UTR expressing renilla luciferase (SwitchGear miRNA expression profiling Genomics), miR-142-3p mimic, or NC (Thermo Scientific). miRNA was isolated using the mirVana RNA Isolation Mutagenesis was done using the QuickChange Site-Direct- Kit (Ambion) and quantified using a Nanodrop spectro- ed Mutagenesis Kit (Agilent Stratagene). The Dual-Lucif- photometer (Thermo Fisher). RNA quality (RNA index erase Reporter Assay System (Promega) was used on a number of 5) was verified by an Agilent 6000 nanochip Synergy2 luminometer (BioTek). (Agilent) before miRNA analysis using miRNA BeadAr- rays (Illumina). Arrays were imaged using an Illumina Transfection of HSPA1B (HSP70) or HSF1 ORF BeadArray Reader, and the fluorescent intensity of vector miRNA probes were analyzed using BeadStudio version Cells were seeded in 24-well plates (1.5  104 cells/well) 3.3.3 (Illumina). Quality control and statistical analyses and were incubated overnight before transfection. Trans- ofmiRNAprofilingwerecarriedoutaspreviously fection mastermixes were diluted in
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