Title: The Expression of the Endoplasmic Reticulum Stress Sensor BiP/GRP78 Predicts Response to Chemotherapy and Determines the Efficacy of Proteasome Inhibitors in Diffuse Large B-Cell Lymphoma
Abstract: Activation of the endoplasmic reticulum (ER) stress pathway is associated with poor response to doxorubicin-containing regimens, such as rituximab, cyclophosphamide, hydroxydaunorubicin (doxorubicin), vincristine and prednisone (R-CHOP), in patients with diffuse large B-cell lymphoma (DLBCL). Bortezomib, a proteasome inhibitor, interferes with ER responses and improves survival in patients with aggressive hematologic malignant tumors, although its use in DLBCL patients remains controversial. The 78-kDa glucose-regulated protein (GRP78), also known as immunoglobulin heavy chain binding protein (BiP), is an ER stress sensor involved in the resistance to doxorubicin and bortezomib, but its role in the response to chemotherapy in DLBCL has not been explored before. We show that high BiP/GRP78 expression is related to worse overall survival (median overall survival, 5.2 versus 3.4 years). Moreover, cell death after R-CHOP in DLCBL cell lines is associated with decreased BiP/GRP78 expression. Conversely, DLBCL cell lines are primarily resistant to bortezomib, probably owing to BiP/GRP78 overexpression. Small-interfering RNA silencing of BiP/GRP78 renders all cell lines sensitive to bortezomib. R-CHOP with bortezomib (R-CHOP-BZ) reduces BiP/GRP78 expression and overcomes bortezomib resistance, mimicking the small-interfering RNA silencing of BiP/GRP78. Accordingly, R-CHOP-BZ is the most effective treatment, providing a rationale for the use of this combinational therapy to improve DLBCL patient survival. Moreover, this study provides preclinical evidence that the germinal center B-cell–like subtype DLBCL is sensitive to bortezomib combined with immunochemotherapy. Activation of the endoplasmic reticulum (ER) stress pathway is associated with poor response to doxorubicin-containing regimens, such as rituximab, cyclophosphamide, hydroxydaunorubicin (doxorubicin), vincristine and prednisone (R-CHOP), in patients with diffuse large B-cell lymphoma (DLBCL). Bortezomib, a proteasome inhibitor, interferes with ER responses and improves survival in patients with aggressive hematologic malignant tumors, although its use in DLBCL patients remains controversial. The 78-kDa glucose-regulated protein (GRP78), also known as immunoglobulin heavy chain binding protein (BiP), is an ER stress sensor involved in the resistance to doxorubicin and bortezomib, but its role in the response to chemotherapy in DLBCL has not been explored before. We show that high BiP/GRP78 expression is related to worse overall survival (median overall survival, 5.2 versus 3.4 years). Moreover, cell death after R-CHOP in DLCBL cell lines is associated with decreased BiP/GRP78 expression. Conversely, DLBCL cell lines are primarily resistant to bortezomib, probably owing to BiP/GRP78 overexpression. Small-interfering RNA silencing of BiP/GRP78 renders all cell lines sensitive to bortezomib. R-CHOP with bortezomib (R-CHOP-BZ) reduces BiP/GRP78 expression and overcomes bortezomib resistance, mimicking the small-interfering RNA silencing of BiP/GRP78. Accordingly, R-CHOP-BZ is the most effective treatment, providing a rationale for the use of this combinational therapy to improve DLBCL patient survival. Moreover, this study provides preclinical evidence that the germinal center B-cell–like subtype DLBCL is sensitive to bortezomib combined with immunochemotherapy. Diffuse large B-cell lymphoma (DLBCL) is the most frequent non-Hodgkin lymphoma.1Swerdlow S.H. Campo E. Harris N.L. Jaffe E.S. Pileri S. Stein H. Thiele J. Vardiman J.W. WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues. IARC Press, Lyon, France2008Google Scholar The chemotherapeutic drugs rituximab, cyclophosphamide, hydroxydaunorubicin (doxorubicin), vincristine, and prednisone (collectively known as R-CHOP) are currently the standard regimen for patients with newly diagnosed DLBCL. Immunochemotherapy is effective in treating aggressive non-Hodgkin lymphoma, but there are still a substantial number of DLBCL patients for whom the standard treatment is insufficiently effective or has major toxic effects,2Coiffier B. Lepage E. Briere J. Herbrecht R. Tilly H. Bouabdallah R. Morel P. Van Den N.E. Salles G. Gaulard P. Reyes F. Lederlin P. Gisselbrecht C. CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma.N Engl J Med. 2002; 346: 235-242Crossref PubMed Scopus (4480) Google Scholar, 3Dumontet C. Mounier N. Munck J.N. Bosly A. Morschauser F. Simon D. Marit G. Casasnovas O. 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The combination of bendamustine, bortezomib, and rituximab for patients with relapsed/refractory indolent and mantle cell non-Hodgkin lymphoma.Blood. 2011; 117: 2807-2812Crossref PubMed Scopus (161) Google Scholar Bortezomib induces cell death by disrupting the endoplasmic reticulum (ER) stress responses in multiple myeloma11Dong H. Chen L. Chen X. Gu H. Gao G. Gao Y. Dong B. Dysregulation of unfolded protein response partially underlies proapoptotic activity of bortezomib in multiple myeloma cells.Leuk Lymphoma. 2009; 50: 974-984Crossref PubMed Scopus (54) Google Scholar, 12Obeng E.A. Carlson L.M. Gutman D.M. Harrington Jr., W.J. Lee K.P. Boise L.H. Proteasome inhibitors induce a terminal unfolded protein response in multiple myeloma cells.Blood. 2006; 107: 4907-4916Crossref PubMed Scopus (862) Google Scholar and in mantle cell lymphoma.13Fisher R.I. Bernstein S.H. Kahl B.S. Djulbegovic B. Robertson M.J. de V.S. Epner E. Krishnan A. Leonard J.P. Lonial S. Stadtmauer E.A. 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Lee A.S. Stress induction of GRP78/BiP and its role in cancer.Curr Mol Med. 2006; 6: 45-54Crossref PubMed Scopus (510) Google Scholar, 27Li J. Ni M. Lee B. Barron E. Hinton D.R. Lee A.S. The unfolded protein response regulator GRP78/BiP is required for endoplasmic reticulum integrity and stress-induced autophagy in mammalian cells.Cell Death Differ. 2008; 15: 1460-1471Crossref PubMed Scopus (347) Google Scholar Because of its antiapoptotic role, the expression of BiP/GRP78 is important for tumor cell survival under ER stress.28Jamora C. Dennert G. Lee A.S. Inhibition of tumor progression by suppression of stress protein GRP78/BiP induction in fibrosarcoma B/C10ME.Proc Natl Acad Sci U S A. 1996; 93: 7690-7694Crossref PubMed Scopus (247) Google Scholar Nevertheless, the role of BiP/GRP78 in B-cell lymphomas remains to be determined.29Shringarpure R. Catley L. Bhole D. Burger R. Podar K. Tai Y.T. Kessler B. Galardy P. Ploegh H. Tassone P. Hideshima T. Mitsiades C. Munshi N.C. Chauhan D. 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Functional coupling of p38-induced up-regulation of BiP and activation of RNA-dependent protein kinase-like endoplasmic reticulum kinase to drug resistance of dormant carcinoma cells.Cancer Res. 2006; 66: 1702-1711Crossref PubMed Scopus (262) Google Scholar The aims of this study were to analyze the prognostic significance of BiP/GRP78 expression in DLBCL patients and to evaluate the possible role of BiP/GRP78 in the response of DLBCL cells to R-CHOP– and to R-CHOP-BZ–based regimens. Tumor specimens from 119 patients diagnosed as having DLBCL after 2002 who were treated with standard R-CHOP were retrieved from the files of the Laboratory of Pathology of the Hospital Clinic, Barcelona, Spain. In 60 of these patients, gene expression profiles were available, and 52 tumors were classified as germinal center B-cell–like (GCB)24Zhang J. Jiang Y. Jia Z. Li Q. Gong W. Wang L. Wei D. Yao J. Fang S. Xie K. Association of elevated GRP78 expression with increased lymph node metastasis and poor prognosis in patients with gastric cancer.Clin Exp Metastasis. 2006; 23: 401-410Crossref PubMed Scopus (163) Google Scholar or activated B-cell–like (ABC)8Furman R.R. Martin P. Ruan J. Cheung Y.K. Vose J.M. LaCasce A.S. Elstrom R. Coleman M. Leonard J.P. Phase 1 trial of bortezomib plus R-CHOP in previously untreated patients with aggressive non-Hodgkin lymphoma.Cancer. 2010; 116: 5432-5439Crossref PubMed Scopus (46) Google Scholar subtypes (see below), whereas 8 of them (13%) remained DLBCL unclassified. Approval for these studies was obtained from the Institutional Review Board of Hospital Clinic. Informed consent was provided according to the Declaration of Helsinki. All cases were reviewed by at least two pathologists (A.M., E.C.) and reclassified following the 2008 World Health Organization classification.1Swerdlow S.H. Campo E. Harris N.L. Jaffe E.S. Pileri S. Stein H. Thiele J. Vardiman J.W. WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues. IARC Press, Lyon, France2008Google Scholar The main clinical characteristics of the patients are summarized in Table 1. The patients had a median age of 60 years, 53% were male and 47% female, 53% presented with advanced stage disease, 52% had extranodal involvement (including bone marrow in 12.5%), and 39% registered high serum lactate dehydrogenase levels (>450 IU/L). The distribution according to the International Prognostic Index (IPI) was as follows: low risk, 29%; low/intermediate risk, 32%; high/intermediate risk, 18%; and high risk, 21%. Staging and restaging maneuvers were the standard. All patients had assessable response, and 29 (72.5%) achieved a complete response.33Cheson B.D. Horning S.J. Coiffier B. Shipp M.A. Fisher R.I. Connors J.M. Lister T.A. Vose J. Grillo-Lopez A. Hagenbeek A. Cabanillas F. Klippensten D. Hiddemann W. Castellino R. Harris N.L. Armitage J.O. Carter W. Hoppe R. Canellos G.P. Report of an international workshop to standardize response criteria for non-Hodgkin's lymphomas NCI Sponsored International Working Group.J Clin Oncol. 1999; 17: 1244Crossref PubMed Google Scholar After a median follow-up of 4.6 years for surviving patients, 16 had died, with a 5-year overall survival of 56% (95% CI, 40% to 72%).Table 1Main Clinical Features of 52 DLBCL Patients Classified by Gene Expression Profiles as ABC and GCB SubtypesFeatureFindingSex, M/F29/23Age, median (range), years60 (17–84)B-symptoms, %54Extranodal involvement, %52Advanced-stage disease, %53Positive bone marrow test result, %12.5High serum lactate dehydrogenase level, %39IPI risk, % Low29 Low/intermediate32 High/intermediate18 High21F, female; M, male; IPI, International Prognostic Index. Open table in a new tab F, female; M, male; IPI, International Prognostic Index. Immunohistochemistry (IHC) was performed on formalin-fixed, paraffin-embedded whole tissue sections and tissue microarray sections. A rabbit monoclonal antibody (C50B12) produced against a synthetic peptide corresponding to residues surrounding the Gly584 of human BiP/GRP78 (Cell Signaling Technology, Beverly, MA) was used. Briefly, paraffin sections on silane-coated slides were developed in a fully automated immunostainer (Bond Max; Vision Biosystems, Mount Waverley, Australia). Antigen retrieval was performed for 20 minutes in Bond ER1 Buffer solution (Vision Biosystems) followed by 2 hours of incubation with primary antibody (1:1000) at room temperature and 30 minutes of Bond Refine Polymer (Vision Biosystems). Diaminobenzidine (DAB) was used for 8 minutes as a chromogen. Double chromogenic immunostaining was performed by using mouse anti-human human IRF4 (Mum1p) (1:200; Dako, Carpinteria, CA), rabbit anti-human PRDM1 (Blimp1) polyclonal (Atlas Antibodies, Stockholm, Sweden), and anti-human BiP/GRP78 as primary antibodies. Sequential primary incubations and sequential detections with a peroxidase-based detection were used. DAB for IRF4 and 3-amino-9-ethylcarbazole (AEC+; Dako) for BiP/GRP78 were used as chromogen in a BondMax autostainer (Vision Biosytems). Double immunofluorescence was performed on cytospins of cell suspensions obtained after repetitive squirt of a reactive lymph node with RPMI 1640 culture medium using a fine needle as previously described.34Martinez A. Aymerich M. Castillo M. Colomer D. Bellosillo B. Campo E. Villamor N. Routine use of immunophenotype by flow cytometry in tissues with suspected hematological malignancies.Cytometry B Clin Cytom. 2003; 56: 8-15Crossref PubMed Scopus (31) Google Scholar An enrichment of B cells was performed by CD19 labeled to magnetic beads (130-050-30; Miltenyi Biotech, Bergisch Gladbach, Germany) according to manufacturer's recommendations and positive sorting using the Automacs system (Miltenyi Biotech). Thereafter, multiples cytospins were obtained for immunofluorescence. An anti-BiP/GRP78 antibody was incubated as described above. After washing, the slides were then incubated at room temperature for 1 hour with a goat anti-rabbit antibody labeled with fluorescein isothiocyanate (Jackson ImmunoResearch Laboratories Inc, West Grove, PA). The slides were rinsed again and a secondary incubation with anti-Calnexin antibody (Clone H-70; Santa Cruz Biotechnology, Santa Cruz, CA) was performed for 2 hours at room temperature. A goat anti-mouse antibody labeled with sulforhodamine 101 acid chloride (Texas Red; Jackson ImmunoResearch Laboratories Inc) was applied for 1 hour in the dark and mounted with an aqueous mounting media that contains DAPI as nuclear counterstain (Fluorescent Mounting Medium; Dako). Samples were visualized on a Olympus BX51 microscope (Olympus GmbH, Hamburg Germany) by means of DP70 cooled CCD camera (Olympus) with the use of CellB̂ imaging software (Olympus). The 4 human DLBCL cell lines used in this study (SUDHL-4, SUDHL-6, SUDHL-16, and OCI-LY8) were grown in RPMI 1640 or Dulbecco's minimal essential medium, supplemented with 10% to 20% fetal calf serum, 2 mmol/L glutamine (GIBCO, Gaithersburg, MD), and 50 μg/mL of penicillin-streptomycin (GIBCO). Cells were incubated for 8 to 16 hours with the proteasome inhibitor bortezomib, either alone or in combination with R-CHOP (R-CHOP-BZ). For the R-CHOP combinations, cells were supplemented with nondescomplemented serum. Cyclophosphamide was used at a concentration ranging from 0.01 to 25 nmol/L, doxorubicin from 0.01 to 100 nmol/L, vincristine from 0.5 to 100 nmol/L, prednisone from 1 to 50 μmol/L, rituximab at 50 μg/mL, and bortezomib from 5 to 10 nmol/L. All experiments were performed in triplicate. The sources and dilutions of the drugs used in the study are specified in Table 2.Table 2Drugs, Doses, and Source Used in the StudyDrugDosesSourceRituximab50 μg/mLRoche, Basel, SwitzerlandCyclophosphamide0.01 nmol/L-0.5 nmol/L-25 nmol/LASTAMedica, Frankfurt, GermanyDoxorubicin0.01 nmol/L-1 nmol/L-100 nmol/LPfizer, New York, NYVincristine0.5 nmol/L-10 nmol/L-100 nmol/LSTADA, Barcelona, SpainPrednisone1 μmol/L-10 μmol/L-50 μmol/LPfizerBortezomib5 nmol/L-10 nmol/LMillennium Pharmaceuticals, Cambridge, MA Open table in a new tab Cell viability was analyzed by quantification of phosphatidylserine exposure by double staining with Annexin V–fluorescein isothiocyanate and propidium iodide (Bender Medsystems Gmbh, Vienna, Austria). A minimum of 104 cells per sample were acquired in a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ), and the labeled populations were analyzed with the Paint-A-Gate software (Becton Dickinson). Total RNA for real-time PCR was extracted using RNeasy minikit (Qiagen, Germantown, MD) and the RNase-Free DNase Set (Qiagen). Afterward, it was reverse transcribed using 1 μg of total RNA and the QuantiTect Reverse Transcription Kit (Qiagen). The product was amplified and quantified using complementary DNA TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA) and TaqMan Gene Expression Assay for BIP/HSPA5 (Hs99999174_m1) in an ABI Prism 7900HT Fast Sequence Detection System (Applied Biosystems). Relative quantification of gene expression was performed as described in the Taqman user's manual, and the expression levels were analyzed with the 2-ΔΔCt method using human β-glucuronidase (Hs999999058_m1) as endogenous control and the Universal Human Reference RNA (Stratagene, Agilent Technologies, Santa Clara, CA) as calibrator. Total RNA was extracted with the DNA/RNA Mini kit following the manufacturer's recommendations (Qiagen). RNA integrity was examined with the Agilent 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA), and only high-quality RNA samples were hybridized to HU133plus2.0 GeneChips (Affymetrix, Santa Clara, CA), according to Affymetrix standard protocols. The analysis of the scanned images and the determination of the signal value for each probe set of the array were obtained with the GeneChip Operating Software (Affymetrix). The data normalization was performed by the global scaling method with the target intensity set at 150. We used the bayesian compound covariate predictor of the ABC/GCB DLBCL previously described by Lenz et al.35Lenz G. Wright G. Dave S.S. Xiao W. Powell J. Zhao H. Xu W. Tan B. Goldschmidt N. Iqbal J. Vose J. Bast M. Fu K. Weisenburger D.D. Greiner T.C. Armitage J.O. Kyle A. May L. Gascoyne R.D. Connors J.M. Troen G. Holte H. Kvaloy S. Dierickx D. Verhoef G. Delabie J. Smeland E.B. Jares P. Martinez A. Lopez-Guillermo A. Montserrat E. Campo E. Braziel R.M. Miller T.P. Rimsza L.M. Cook J.R. Pohlman B. Sweetenham J. Tubbs R.R. Fisher R.I. Hartmann E. Rosenwald A. Ott G. Muller-Hermelink H.K. Wrench D. Lister T.A. Jaffe E.S. Wilson W.H. Chan W.C. Staudt L.M. Stromal gene signatures in large-B-cell lymphomas.N Engl J Med. 2008; 359: 2313-2323Crossref PubMed Scopus (1336) Google Scholar All samples predicted as ABC DLBCL with greater than 90% were called ABC DLBCL. The samples that showed less than 10% of probability of being called ABC DLBCL were classified as GCB DLBCL. For transient down-regulation assays, cell lines were electroporated in a Nucleofector device (Amaxa-Lonza, Cologne, Germany) with two different sets of small-interfering RNA (siRNA) targeting the exons 1 (s6980) and 3 (s6981) of the BiP/GRP78 gene (Applied Biosystems, Foster City, CA). As control, irrelevant nonsilencing siRNA (5′-UUCUCCGAACGUGUCACGU-3′) was used as previously described.36Roue G. Perez-Galan P. Lopez-Guerra M. Villamor N. Campo E. Colomer D. Selective inhibition of IkappaB kinase sensitizes mantle cell lymphoma B cells to TRAIL by decreasing cellular FLIP level.J Immunol. 2007; 178: 1923-1930PubMed Google Scholar Briefly, 7 × 106 cells were resuspended in 100 μL of Ingenio Electroporation Solution (Mirius, Madison, WI) containing 0.5 or 2.5 μmol/L double-stranded siRNA and electroporated using the Nucleofector program O-017. Cells were then transferred to culture plates and cultivated at a final concentration of 3 × 106 cells/mL for 3 hours. Then, cells were transferred to a new plate and cultured at a concentration of 1 × 106 cells/mL for 3 hours. The electroporation efficiency ranged from 81.2% to 99.3% for SUDHL16 and from 69.7% to 79.6% in OCI-LY8. Cytotoxicity due to electroporation process was low in both cell lines, approximately 20%. The nontargeting (scramble) siRNA (5′-UUCUCCGAACGUGUCACGU-3′; Qiagen) was used as a negative control. Western blot was performed as described previously.17Balague O. Mozos A. Martinez D. Hernandez L. Colomo L. Mate J.L. Teruya-Feldstein J. Lin O. Campo E. Lopez-Guillermo A. Martinez A. Activation of the endoplasmic reticulum stress-associated transcription factor x box-binding protein-1 occurs in a subset of normal germinal-center B cells and in aggressive B-cell lymphomas with prognostic implications.Am J Pathol. 2009; 174: 2337-2346Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar Briefly, exponentially growing cells (approximately 106 cells/mL) were lysed in a nondenaturing detergent (M-PER; Pierce, Rockford, IL) containing protease inhibitors (Complete Mini; Roche, Manheim, Germany) and phosphatase inhibitors (Cocktails 1 and 2; Sigma, St Louis, MO). The protein content was determined using a bicinchoninic acid protein assay kit (Pierce), according to the manufacturer's instructions. Identical amounts of total extracted protein were heated 10 minutes at 70°C in NuPAGE LDS Sample buffer (Invitrogen, Carlsbad, CA) and separated by electrophoresis on 4% to 20% (wt/vol) polyacrylamide gradient gels (Novex NuPAGE). After transference to a 0.45-μm pore size nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA), the immunoblotting was performed as follows. The membrane was blocked 2 hours at room temperature in 50 mmol/L Tris-buffered saline (pH 7.6) with 0.05% Tween-20 containing 5% nonfat dry milk. The nitrocellulose membranes were incubated with