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Concomitant Induction of Apoptosis and Autophagy by Prostate Apoptosis Response-4 in Hypopharyngeal Carcinoma Cells

2014; Elsevier BV; Volume: 184; Issue: 2 Linguagem: Inglês

10.1016/j.ajpath.2013.10.012

ISSN

1525-2191

Autores

Ling-Jung Wang, Peir‐Rong Chen, Lee‐Ping Hsu, Wen‐Lin Hsu, Dai‐Wei Liu, Chung‐Hsing Chang, Yih‐Chih Hsu, Jeng‐Woei Lee,

Tópico(s)

RNA Interference and Gene Delivery

Resumo

The tumor-suppressive activity of prostate apoptosis response-4 (Par-4) has been demonstrated in a variety of human cancers. In this study, for the first time to our knowledge, we demonstrated that a higher intensity of Par-4 was significantly correlated with a better response in patients with hypopharyngeal carcinoma undergoing radiotherapy alone or concurrent chemoradiotherapy. Mechanistically, an elevated expression of Par-4 induced apoptosis of hypopharyngeal carcinoma cells and sensitized cells toward chemotherapeutic agents or X-ray irradiation. Along with apoptotic incitation, intriguingly, autophagic flux also increased on Par-4 stimulation and contributed to cell death. Moreover, the expressions of multiple common regulators involved in apoptosis and autophagy were regulated by Par-4. Taken together, our results suggested a prognostic role of Par-4 in hypopharyngeal carcinoma and showed novel activity of Par-4 in apoptosis and autophagy induction. The tumor-suppressive activity of prostate apoptosis response-4 (Par-4) has been demonstrated in a variety of human cancers. In this study, for the first time to our knowledge, we demonstrated that a higher intensity of Par-4 was significantly correlated with a better response in patients with hypopharyngeal carcinoma undergoing radiotherapy alone or concurrent chemoradiotherapy. Mechanistically, an elevated expression of Par-4 induced apoptosis of hypopharyngeal carcinoma cells and sensitized cells toward chemotherapeutic agents or X-ray irradiation. Along with apoptotic incitation, intriguingly, autophagic flux also increased on Par-4 stimulation and contributed to cell death. Moreover, the expressions of multiple common regulators involved in apoptosis and autophagy were regulated by Par-4. Taken together, our results suggested a prognostic role of Par-4 in hypopharyngeal carcinoma and showed novel activity of Par-4 in apoptosis and autophagy induction. Hypopharyngeal carcinoma (HPC) (International Classification of Diseases-10 code C12 to 13) is a type of head and neck malignancy. In Taiwan, the age-adjusted incidence rate of HPC has gradually increased in the past two decades from 0.84 per 100,000 individuals in 1990 to 2.94 per 100,000 individuals in 2009. Although surgery, radiotherapy alone or in conjunction with chemoradiation, is the standard treatment for HPC, understanding the molecular mechanisms is necessary for pathophysiological clarification and survival rate improvement. For instance, including p27, cyclin D1, p53, matrix metalloproteinases 2 and 9, Twist, and miRNA has been revealed to have an association with the tumorigenesis and metastasis of HPC.1Mineta H. Miura K. Suzuki I. Takebayashi S. Misawa K. Ueda Y. Ichimura K. p27 Expression correlates with prognosis in patients with hypopharyngeal cancer.Anticancer Res. 1999; 19: 4407-4412PubMed Google Scholar, 2Masuda M. Hirakawa N. Nakashima T. Kuratomi Y. Komiyama S. 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Studies have revealed that phosphorylation of Par-4 determines its activity; for example, Thr155 phosphorylation by protein kinase A is required for its pro-apoptosis function and, in contrast, modification by Akt (or protein kinase B) restrains the ability of Par-4 to reduce cell survival.14Gurumurthy S. Goswami A. Vasudevan K.M. Rangnekar V.M. Phosphorylation of Par-4 by protein kinase A is critical for apoptosis.Mol Cell Biol. 2005; 25: 1146-1161Crossref PubMed Scopus (91) Google Scholar, 15Goswami A. Burikhanov R. de Thonel A. Fujita N. Goswami M. Zhao Y. Eriksson J.E. Tsuruo T. Rangnekar V.M. Binding and phosphorylation of Par-4 by Akt is essential for cancer cell survival.Mol Cell. 2005; 20: 33-44Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar Par-4 is able to engage in extrinsic and intrinsic apoptosis pathways.16Irby R.B. Kline C.L. Par-4 as a potential target for cancer therapy.Expert Opin Ther Targets. 2013; 17: 77-87Crossref PubMed Scopus (12) Google Scholar Intriguingly, new evidence has indicated that the detailed mechanisms by which Par-4 mediates cell death involve intracellular and extracellular action modes; moreover, intracellular Par-4 is also essential for extracellular Par-4–induced apoptosis.17Hebbar N. Wang C. Rangnekar V.M. Mechanisms of apoptosis by the tumor suppressor Par-4.J Cell Physiol. 2012; 227: 3715-3721Crossref PubMed Scopus (46) Google Scholar, 18Zhao Y. Burikhanov R. Brandon J. Qiu S. Shelton B.J. Spear B. Bondada S. Bryson S. Rangnekar V.M. Systemic Par-4 inhibits non-autochthonous tumor growth.Cancer Biol Ther. 2011; 12: 152-157Crossref PubMed Scopus (23) Google Scholar, 19Burikhanov R. Zhao Y. Goswami A. Qiu S. Schwarze S.R. Rangnekar V.M. The tumor suppressor Par-4 activates an extrinsic pathway for apoptosis.Cell. 2009; 138: 377-388Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar In addition, through direct interaction with NF-κB and inhibition of NF-κB transcriptional activity, Par-4 alters DROSHA and miRNA expression and, hence, further attenuates BCL-2 mRNA translation.20Wang B.D. Kline C.L. Pastor D.M. Olson T.L. Frank B. Luu T. Sharma A.K. Robertson G. Weirauch M.T. Patierno S.R. Stuart J.M. Irby R.B. Lee N.H. Prostate apoptosis response protein 4 sensitizes human colon cancer cells to chemotherapeutic 5-FU through mediation of an NF-κB and microRNA network.Mol Cancer. 2010; 9: 98Crossref PubMed Scopus (45) Google Scholar Interestingly, evidence has also demonstrated that Par-4 is a novel substrate of caspase 3, and a cleaved fragment of Par-4 still retains pro-apoptosis activity.21Chaudhry P. Singh M. Parent S. Asselin E. 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On various stresses, such as nutrient starvation, hypoxia, or unfolded protein accumulation, autophagy is induced via double-membrane vacuole (autophagosome) formation to engulf aggregate-prone proteins or damaged organelles, which is followed by fusing with lysosomes (autolysosomes) to eliminate cytoplasmic substrates for the recycling of metabolic substances.33Ravikumar B. Futter M. Jahreiss L. Korolchuk V.I. Lichtenberg M. Luo S. Massey D.C. Menzies F.M. Narayanan U. Renna M. Jimenez-Sanchez M. Sarkar S. Underwood B. Winslow A. Rubinsztein D.C. Mammalian macroautophagy at a glance.J Cell Sci. 2009; 122: 1707-1711Crossref PubMed Scopus (148) Google Scholar Several autophagy-related proteins (Atgs) and class III phosphatidylinositol 3-kinases (alias PIK3C3 or VPS34) are required for autophagosome completion. Conversely, the activity of the autophagy machinery is negatively modulated by a master regulator of multiple signaling pathways, mammalian target of rapamycin.34Kim D.H. 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Geneste O. Kroemer G. Functional and physical interaction between Bcl-X(L) and a BH3-like domain in Beclin-1.EMBO J. 2007; 26: 2527-2539Crossref PubMed Scopus (927) Google Scholar, 40Norman J.M. Cohen G.M. Bampton E.T. The in vitro cleavage of the hAtg proteins by cell death proteases.Autophagy. 2010; 6: 1042-1056Crossref PubMed Scopus (164) Google Scholar, 41Moscat J. Diaz-Meco M.T. p62 At the crossroads of autophagy, apoptosis and cancer.Cell. 2009; 137: 1001-1004Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar Yet, the regulatory mechanisms between autophagy and apoptosis are still elusive. Extending our previous findings,11Lee J.W. Hsiao W.T. Lee K.F. Sheu L.F. Hsu H.Y. Hsu L.P. Su B. Lee M.S. Hsu Y.C. Chang C.H. Widespread expression of prostate apoptosis response-4 in nasopharyngeal carcinoma.Head Neck. 2010; 32: 877-885PubMed Google Scholar in this study, we, for the first time to our knowledge, reported a prognostic role of Par-4 in HPC, and demonstrated a novel pro-apoptosis and autophagy activity of Par-4 through modulation of multiple common regulators, thus enhancing cell death. Clinical information from 93 HPC cases, which were diagnosed from January 1991 to December 1999 in Buddhist Tzu Chi General Hospital (Hualien, Taiwan), was collected,11Lee J.W. Hsiao W.T. Lee K.F. Sheu L.F. Hsu H.Y. Hsu L.P. Su B. Lee M.S. Hsu Y.C. Chang C.H. Widespread expression of prostate apoptosis response-4 in nasopharyngeal carcinoma.Head Neck. 2010; 32: 877-885PubMed Google Scholar and the patients were followed up for an additional 6 years. The relationships between Par-4 expression11Lee J.W. Hsiao W.T. Lee K.F. Sheu L.F. Hsu H.Y. Hsu L.P. Su B. Lee M.S. Hsu Y.C. Chang C.H. Widespread expression of prostate apoptosis response-4 in nasopharyngeal carcinoma.Head Neck. 2010; 32: 877-885PubMed Google Scholar and age, sex, TNM stage, treatment, and therapeutic outcome of HPC cases were analyzed using the SPSS program (SPSS, Inc., Chicago, IL). Permission to use these clinical data for research purposes was obtained from the Research Ethics Committee at Buddhist Tzu Chi General Hospital. Human hypopharyngeal carcinoma cell line FaDu (Bioresource Collection and Research Center number 60214) was purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan). Cells were cultured in minimum essential medium supplemented with 10% fetal bovine serum, 1 mmol/L sodium pyruvate, 50 μg/mL streptomycin, and 50 U/mL penicillin at 37°C in a 5% CO2 incubator. Par-4/pCMV6-XL6 plasmid was obtained as described previously.42Lee J.W. Liu P.F. Hsu L.P. Chen P.R. Chang C.H. Shih W.L. EBV LMP-1 negatively regulates expression and pro-apoptotic activity of Par-4 in nasopharyngeal carcinoma cells.Cancer Lett. 2009; 279: 193-201Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar For microtubule-associated protein 1 light chain 3 [pGreen Fluorescent Protein (pGFP)-LC3] plasmid construction, LC3 cDNA (provided by Dr. Noboru Mizushima, Tokyo Medical and Dental University, Tokyo, Japan)43Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5433) Google Scholar was inserted into the BglII and EcoRI sites of pEGFP-C1 expression vector (Clontech Laboratories, Inc., Mountain View, CA). Plasmid Par-4–GFP, Flag-Beclin 1, and NF-κB–dependent luciferase reporter (NF-κB-luc) were generously provided by Dr. Ute Preuss (University of Bonn, Bonn, Germany), Dr. Beth Levine (University of Texas Southwestern Medical Center, Dallas), and Dr. Wen-Ling Shih (National Pingtung University of Science and Technology, Pingtung, Taiwan), respectively. ON-TARGETplus SMARTpool siRNA against human Par-4 (L-004434-00-0005) and nontargeting SMARTpool siRNA (D-001810-10−05) (Thermo Fisher Scientific, Lafayette, CO) were used for RNA silencing. Briefly, cells were placed on 35-mm plates and grown to 60% to 70% confluence. Cells were then transfected with Lipofectamine 2000 reagent (Life Technologies Corporation, Carlsbad, CA) and plasmid or siRNA mixture, according to the manufacturer's instructions. After transfection, cells were harvested for Western blot analysis or direct observation by fluorescence microscope. The percentage of GFP-LC3–positive cells with GFP-LC3 dots was calculated as the number of fluorescent punctate cells/the number of GFP-LC3 expression cells. To establish a stable GFP-LC3 expression cell line, cells were transfected with pGFP-LC3 plasmid and fluorescent colonies were picked up via 800 μg/mL G418 selection. For Western blot analysis, cell extracts were prepared as described previously.42Lee J.W. Liu P.F. Hsu L.P. Chen P.R. Chang C.H. Shih W.L. EBV LMP-1 negatively regulates expression and pro-apoptotic activity of Par-4 in nasopharyngeal carcinoma cells.Cancer Lett. 2009; 279: 193-201Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar Samples were resolved on 10% to 12% SDS-polyacrylamide gels and then subjected to immunoblotting with respective antibodies: rabbit anti–Par-4 antibody (Cell Signaling Technology, Beverly, MA), rabbit anti-phosphate Par-4 (Thr163) antibody (Cell Signaling Technology), mouse anti-Akt1 antibody (Cell Signaling Technology), mouse anti–phospho-Akt (Ser473) antibody (Cell Signaling Technology), goat anti–phospho-Bcl-2 (Ser87) antibody (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti–Bcl-2 antibody (Cell Signaling Technology), rabbit anti-cleaved poly (ADP-ribose) polymerase (PARP) antibody (Cell Signaling Technology), rabbit anti-cleaved caspase 3 antibody (Cell Signaling Technology), rabbit anti-Atg12 antibody (Cell Signaling Technology), rabbit anti-Atg5 antibody (Epitomics, Burlingame, CA), rabbit anti-LC3B antibody (Cell Signaling Technology), mouse anti-GFP antibody (Santa Cruz Biotechnology), rabbit anti-protein kinase C (PKC) ζ antibody (Cell Signaling Technology), and rabbit anti-p62 antibody (Cell Signaling Technology) for 1 hour. After incubation with the appropriate secondary antibody, the bands were visualized using an electrochemiluminescence system (GE Healthcare Life Sciences, Buckinghamshire, UK). The densities of the bands were measured using ImageJ software version 1.47 (NIH, Bethesda, MD), and values were normalized to the densitometric values of actin in each sample. The fold change in protein amount was then calculated for the experimental sets compared with the control. Briefly, cells were incubated with 0.5 mg/mL MTT assay (Sigma-Aldrich, St. Louis, MO) in culture medium for 3 hours at 37°C, and then dimethyl sulfoxide was added to dissolve the formazan crystals. The absorbance at 540 nm was determined using a Biokinetics plate reader (Bio-Tek Instruments, Inc., Winooski, VT). The percentage of cell viability was calculated as the value of the experimental set/the value of the control. For transmission electron microscopy (TEM) examination of autophagic vacuoles, cell pellets were collected and resuspended in glutaraldehyde solution (2.5% glutaraldehyde and 1% tannic acid in 0.1 mol/L cacodylate buffer). After washing with 5% sucrose in 0.1 mol/L cacodylate buffer, cells were postfixed with 1% osmium tetroxide in 0.1 mol/L cacodylate buffer for 1 hour at 4°C. Samples were dehydrated and embedded with 100% Spurr's resin for 2 hours at room temperature, and polymerized for 1 hour at 60°C. Ultrathin sections were made and stained with 2% uranyl acetate and lead citrate. The sections were visualized using TEM (HITACHI H-7500; Hitachi High-Technologies Corp., Tokyo, Japan) at an accelerating voltage of 80 kV. To quantify the turnover of GFP-LC3, cells were transfected with pGFP-LC3 plasmid alone, or with either Par-4/pCMV6-XL6 or Flag-Beclin 1 plasmid in the presence or absence of autophagy inhibitors. At 48 hours after transfection, 1 × 104 GFP-LC3–expressing cells in each sample were harvested and analyzed using an FACScan flow cytometer (BD Biosciences, San Jose, CA), as described previously.44Shvets E. Fass E. Elazar Z. Utilizing flow cytometry to monitor autophagy in living mammalian cells.Autophagy. 2008; 4: 621-628Crossref PubMed Scopus (143) Google Scholar For quantification, the relative fluorescence intensity (percentage) was calculated by dividing the level of GFP-LC3 of each sample/the level of the mock control in the respective experimental set. For quantification of p62, cells were transfected with or without Par-4/pCMV6-XL6 plasmid, and posttransfection after 24 or 48 hours, cells were harvested and fixed with 4% formalin for 15 minutes at room temperature, then permeabilized with 90% cold methanol/PBS for 30 minutes. Subsequently, cells were immunostained using mouse anti-p62 antibody (EMD Millipore Corporation, Billerica, MA), followed by incubation with Alexa Fluor 546 goat anti-mouse IgG (Life Technologies Corporation). A total of 1 × 104 cells were then collected and analyzed using an FACScan flow cytometer, as previously described. The relative fluorescence intensity (percentage) was calculated by dividing the level of fluorescence of overexpressing Par-4 cells/the level of the mock control in the respective experiment. To measure the percentage of cells undergoing apoptosis and autophagy, Par-4 transiently transfected cells were treated with or without apoptotic stimulus in the presence or absence of an autophagy inhibitor. Cells were then fixed and permeabilized using BD Cytofix/Cytoperm solution (BD Biosciences) for 30 minutes at room temperature. After washing, cells were incubated with phycoerythrin-conjugated rabbit anti-LC3B antibody (Cell Signaling Technology) and fluorescein isothiocyanate–conjugated rabbit anti-active caspase 3 antibody (BD Biosciences) for 1 hour at 4°C. Subsequently, 1 × 104 cells were harvested using an FACScan flow cytometer and further analyzed with CELLQuest version 3.0.1 and ModFitLT version 2.0 software. Briefly, cells were labeled with 50 μmol/L monodansylcadaverine (MDC) reagent (Sigma-Aldrich) for 15 minutes. After washing with PBS, the fluorescence signal was immediately observed by fluorescence microscope with an excitation wavelength of 360 nm and an emission wavelength of 457 nm. Autolysosomal distribution was quantified as the proportion of cells with predominant perinuclear localization of fluorescent vesicles. For protein de novo synthesis, cells were pretreated with 40 μmol/L cycloheximide (CHX) for 3 hours, then transfected with or without Par-4/pCMV6-XL6 plasmid in the absence of CHX (0 hours). At 24 hours after transfection, cell lysates were prepared and subjected to Western blot analysis using a primary antibody corresponding to Par-4, cleaved PARP, LC3B, or tubulin. The densities of the bands were measured using ImageJ software version 1.47, and values were normalized to the densitometric values of tubulin in each sample. The fold change in protein amount was then calculated for the experimental sets compared with the vector control (0 hours). Cells were transfected with Par-4/pCMV6-XL6 or pGFP-LC3 plasmid, or cotransfected with both. At 48 hours after transfection, cells were harvested and lysed in lysis buffer. After preclearing with protein A–Sepharose beads (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), cell lysates were incubated with antibody beads overnight at 4°C. The immunocomplex was precipitated using protein A–Sepharose and washed three times in lysis buffer. The immunoprecipitates were resuspended in sample buffer and subjected to Western blot analysis. The density of the band was measured using ImageJ software version 1.47. For whole cell extracts, values were normalized to the densitometric values of actin in each sample. The fold change in protein amount was calculated for the experimental set compared with the vector control. FaDu cells were cotransfected with both NF-κB-luc and pRL-TK vector (Promega, Madison, WI) plus either Par-4/pCMV6-XL6 or Par-4–GFP expression plasmid. At 48 hours after transfection, the luciferase activity of each sample was measured using the Dual Luciferase Reporter Assay System (Promega), according to the manufacturer's instructions, and calculated by dividing the value of luciferase activity/the value of Renilla luciferase activity. The relative luciferase activity was represented as the fold of activation via the experimental set over the NF-κB–dependent luciferase reporter alone. Briefly, FaDu cells were transfected with or without Par-4/pCMV6-XL6 plasmid, and at 48 hours after transfection, diluent cell lysates were prepared and subjected to a protein kinase array using a Proteome Profiler Human Phospho-Kinase Array Kit (R&D Systems, Minneapolis, MN), according to the manufacturer's instructions. The array membranes were washed, followed by incubation with a mixture of biotinylated detection antibodies and streptavidin–horseradish peroxidase antibodies. Phospho-kinase signals were then detected using a chemiluminescent detection reagent, and the density of each spot was further measured using ImageJ software version 1.47. The fold change of each phosphoryl

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