Bortezomib induces caspase-dependent apoptosis in Hodgkin lymphoma cell lines and is associated with reduced c-FLIP expression: a gene expression profiling study with implications for potential combination therapies.

The Hodgkin cells and Reed-Sternberg cells (HRS) of classical Hodgkin lymphoma (CHL) are derived from germinal center B cells. The pathogenesis of CHL is unclear but constitutive activation of NFkappaB may contribute. Proteasome inhibition aimed at inhibiting NFkappaB has been shown to result in apoptosis in HRS cells. Here we investigated the effects of bortezomib, a proteasome inhibitor, in HRS cells with a combination of functional assays and gene expression profiling (GEP). Exposure of KMH2 and L428 cells to bortezomib resulted in inhibition of proliferation and induction of apoptosis. Gene expression analysis of KMH2 cells by oligonucleotide cDNA microarrays showed that a limited set of genes were differentially expressed involving several key cellular pathways including cell cycle and apoptosis. Among them, the caspase 8 inhibitor cFLIP was down-regulated and confirmed by Q-PCR. Given the evidence that cFLIP in HRS cells contribute to cells' insensitive to death receptor-mediated apoptosis, we combined bortezomib and TRAIL. This combination caused further down-regulation of cFLIP protein and increased apoptosis in CHL cells demonstrated by PARP p85 immunohistochemistry and immunoblotting. Such apoptotic effects were inhibited by caspase inhibitor z-VAD-FMK, confirming the pro-apoptotic effects of bortezomib and TRAIL are caspase-dependent. Bortezomib has no detectable effect on expression of TRAIL receptor DR4/DR5 in these two cell lines. Tissue microarray analysis of primary Hodgkin lymphomas displayed that 82% cases (95/116) expressed cFLIP in Reed-Sternberg cells. The discovery of apoptotic pathways that can be manipulated by proteasome inhibition provides rationale for the combination of bortezomib and agents such as TRAIL in CHL treatment.

[1]  John Calvin Reed,et al.  Functional Role of Death-associated Protein 3 (DAP3) in Anoikis* , 2004, Journal of Biological Chemistry.

[2]  Marc W. Kirschner,et al.  How Proteolysis Drives the Cell Cycle , 1996, Science.

[3]  P. Baeuerle,et al.  A proteasome inhibitor prevents activation of NF‐kappa B and stabilizes a newly phosphorylated form of I kappa B‐alpha that is still bound to NF‐kappa B. , 1994, The EMBO journal.

[4]  Richard Pazdur,et al.  Velcade: U.S. FDA approval for the treatment of multiple myeloma progressing on prior therapy. , 2003, The oncologist.

[5]  P. Howley,et al.  In vivo ubiquitination and proteasome-mediated degradation of p53(1). , 1996, Cancer research.

[6]  S. Srinivasula,et al.  Death effector domain-containing proteins DEDD and FLAME-3 form nuclear complexes with the TFIIIC102 subunit of human transcription factor IIIC , 2002, Cell Death and Differentiation.

[7]  J. Adams.  The proteasome as a novel target for the treatment of breast cancer. , 2002, Breast Disease.

[8]  T. Sayers,et al.  Combining proteasome inhibition with TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) for cancer therapy , 2005, Cancer Immunology, Immunotherapy.

[9]  B. Aggarwal,et al.  Induction of Cell Cycle Arrest and Apoptosis by the Proteasome Inhibitor PS-341 in Hodgkin Disease Cell Lines Is Independent of Inhibitor of Nuclear Factor-κB Mutations or Activation of the CD30, CD40, and RANK Receptors , 2004, Clinical Cancer Research.

[10]  B. Dörken,et al.  Constitutive nuclear factor-kappaB-RelA activation is required for proliferation and survival of Hodgkin's disease tumor cells. , 1997, The Journal of clinical investigation.

[11]  E. Estey,et al.  Phase I Study of Bortezomib in Refractory or Relapsed Acute Leukemias , 2004, Clinical Cancer Research.

[12]  A. Kimchi,et al.  Structure–function analysis of an evolutionary conserved protein, DAP3, which mediates TNF‐α‐ and Fas‐induced cell death , 1999, The EMBO journal.

[13]  E. Borden,et al.  Downregulation of Bcl-2, FLIP or IAPs (XIAP and survivin) by siRNAs sensitizes resistant melanoma cells to Apo2L/TRAIL-induced apoptosis , 2004, Cell Death and Differentiation.

[14]  D. Schenkein.  Use of proteasome inhibition in the treatment of lung cancer. , 2004, Clinical lung cancer.

[15]  M. Hochstrasser.  Ubiquitin-dependent protein degradation. , 1996, Annual review of genetics.

[16]  R Fischer,et al.  Hodgkin disease: Hodgkin and Reed-Sternberg cells picked from histological sections show clonal immunoglobulin gene rearrangements and appear to be derived from B cells at various stages of development. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[17]  R. Tibbetts,et al.  DNA Replication Defects, Spontaneous DNA Damage, and ATM-dependent Checkpoint Activation in Replication Protein A-deficient Cells* , 2004, Journal of Biological Chemistry.

[18]  E. Solary,et al.  Increased gadd153 messenger RNA level is associated with apoptosis in human leukemic cells treated with etoposide. , 1997, Cancer research.

[19]  O. O’Connor.  The emerging role of bortezomib in the treatment of indolent non-hodgkin’ and mantle cell lymphomas , 2004, Current treatment options in oncology.

[20]  G. Prendergast,et al.  Losses of the tumor suppressor BIN1 in breast carcinoma are frequent and reflect deficits in programmed cell death capacity , 2000, International journal of cancer.

[21]  S. Olson,et al.  Expression of c-FLIP in Classic and Nodular Lymphocyte-Predominant Hodgkin Lymphoma , 2004, Applied immunohistochemistry & molecular morphology : AIMM.

[22]  S. Alkan,et al.  Analysis of expression of nuclear factor kappa B (NF-kappa B) in multiple myeloma: downregulation of NF-kappa B induces apoptosis. , 2001, British journal of haematology.

[23]  A. Kimchi,et al.  Death-associated Protein 3 Localizes to the Mitochondria and Is Involved in the Process of Mitochondrial Fragmentation during Cell Death* , 2004, Journal of Biological Chemistry.

[24]  P. Elliott,et al.  The proteasome inhibitor PS-341 sensitizes neoplastic cells to TRAIL-mediated apoptosis by reducing levels of c-FLIP. , 2003, Blood.

[25]  J. Mier,et al.  Phosphatidylinositol 3-Kinase/Akt Activity Regulates c-FLIP Expression in Tumor Cells* , 2001, The Journal of Biological Chemistry.

[26]  V. Diehl,et al.  Hodgkin's lymphoma: biology and treatment strategies for primary, refractory, and relapsed disease. , 2003, Hematology. American Society of Hematology. Education Program.

[27]  R. Millikan,et al.  Phase I trial of the proteasome inhibitor bortezomib in patients with advanced solid tumors with observations in androgen-independent prostate cancer. , 2004, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[28]  H. Lenz,et al.  The role of proteasome inhibitors in solid tumors , 2004, Annals of medicine.

[29]  S. Alkan,et al.  Characterization of NF-κB Expression in Hodgkin’s Disease: Inhibition of Constitutively Expressed NF-κB Results in Spontaneous Caspase-Independent Apoptosis in Hodgkin and Reed-Sternberg Cells , 2001, Modern Pathology.

[30]  Michael L. Wang,et al.  Phase II study of proteasome inhibitor bortezomib in relapsed or refractory B-cell non-Hodgkin's lymphoma. , 2005, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[31]  Bart Barlogie,et al.  A phase 2 study of bortezomib in relapsed, refractory myeloma. , 2003, The New England journal of medicine.

[32]  W. Rees,et al.  Upregulation of CHOP‐10 (gadd153) expression in the mouse blastocyst as a response to stress , 1999, Molecular reproduction and development.

[33]  B. Dörken,et al.  High-level nuclear NF-kappa B and Oct-2 is a common feature of cultured Hodgkin/Reed-Sternberg cells. , 1996, Blood.

[34]  K. Anderson,et al.  Clinical update: novel targets in multiple myeloma. , 2004, Seminars in oncology.

[35]  N. Munshi,et al.  NF-κB as a Therapeutic Target in Multiple Myeloma* , 2002, The Journal of Biological Chemistry.

[36]  M. Sherman,et al.  Proteasome Inhibitors Activate Stress Kinases and Induce Hsp72 , 1998, The Journal of Biological Chemistry.

[37]  A. Goldberg,et al.  New insights into proteasome function: from archaebacteria to drug development. , 1995, Chemistry & biology.

[38]  A. Fremgen,et al.  The national cancer data base report on hodgkin's disease for 1985‐1989 and 1990‐1994 , 1998, Cancer.

[39]  H. Stein,et al.  Overexpression of I kappa B alpha without inhibition of NF-kappaB activity and mutations in the I kappa B alpha gene in Reed-Sternberg cells. , 1999, Blood.

[40]  L. Pham,et al.  Inhibition of Constitutive NF-κB Activation in Mantle Cell Lymphoma B Cells Leads to Induction of Cell Cycle Arrest and Apoptosis1 , 2003, The Journal of Immunology.

[41]  L. Schwartz,et al.  Phase II trial of bortezomib for patients with advanced renal cell carcinoma. , 2004, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[42]  H. Stein,et al.  Hodgkin and reed-sternberg cells represent an expansion of a single clone originating from a germinal center B-cell with functional immunoglobulin gene rearrangements but defective immunoglobulin transcription. , 2000, Blood.

[43]  G. Prendergast,et al.  Bin1 mediates apoptosis by c-Myc in transformed primary cells. , 2001, Cancer research.

[44]  Ingo Schmitz,et al.  The Role of c-FLIP in Modulation of CD95-induced Apoptosis* , 1999, The Journal of Biological Chemistry.

[45]  J. Berenson,et al.  The role of nuclear factor-?b in the biology and treatment of multiple myeloma , 2001 .

[46]  S. Alkan,et al.  Analysis of expression of nuclear factor κB (NF‐κB) in multiple myeloma: downregulation of NF‐κB induces apoptosis , 2001 .

[47]  C. Peschle,et al.  Increased death receptor resistance and FLIPshort expression in polycythemia vera erythroid precursor cells. , 2006, Blood.

[48]  K. Rajewsky,et al.  Hodgkin and Reed-Sternberg cells in Hodgkin's disease represent the outgrowth of a dominant tumor clone derived from (crippled) germinal center B cells , 1996, The Journal of experimental medicine.

[49]  H. Schulman,et al.  Inhibition of CaMKII-mediated c-FLIP expression sensitizes malignant melanoma cells to TRAIL-induced apoptosis. , 2005, Experimental cell research.

[50]  W. El-Deiry,et al.  Direct Repression of FLIP Expression by c-myc Is a Major Determinant of TRAIL Sensitivity , 2004, Molecular and Cellular Biology.

[51]  D. Budman.  Dose and schedule as determinants of outcomes in chemotherapy for breast cancer. , 2004, Seminars in oncology.

[52]  D. Esseltine,et al.  A phase 2 study of two doses of bortezomib in relapsed or refractory myeloma , 2004, British journal of haematology.

[53]  Tom Maniatis,et al.  The ubiquitinproteasome pathway is required for processing the NF-κB1 precursor protein and the activation of NF-κB , 1994, Cell.

[54]  Kesava Reddy.  Activity of bortezomib in advanced non-small-cell lung cancer. , 2004, Clinical lung cancer.

[55]  E. Campo,et al.  Selective Inhibition of IκB Kinase Sensitizes Mantle Cell Lymphoma B Cells to TRAIL by Decreasing Cellular FLIP Level1 , 2007, The Journal of Immunology.

[56]  M. Sporn,et al.  An Inducible Pathway for Degradation of FLIP Protein Sensitizes Tumor Cells to TRAIL-induced Apoptosis* , 2002, The Journal of Biological Chemistry.

[57]  G. Cohen,et al.  DEDD regulates degradation of intermediate filaments during apoptosis , 2002, The Journal of cell biology.

[58]  J. Crocker,et al.  Expression of the cellular FLICE-inhibitory protein (c-FLIP) protects Hodgkin's lymphoma cells from autonomous Fas-mediated death. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[59]  V. Diehl,et al.  Constitutive expression of c-FLIP in Hodgkin and Reed-Sternberg cells. , 2002, The American journal of pathology.

[60]  Pengbo Zhou.  Targeted protein degradation. , 2005, Current opinion in chemical biology.

[61]  M. Smyth,et al.  TRAIL and its receptors as targets for cancer therapy , 2004, Cancer science.

[62]  A. Zelenetz,et al.  Phase II clinical experience with the novel proteasome inhibitor bortezomib in patients with indolent non-Hodgkin's lymphoma and mantle cell lymphoma. , 2005, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[63]  H. Stein,et al.  c-FLIP Mediates Resistance of Hodgkin/Reed-Sternberg Cells to Death Receptor–induced Apoptosis , 2004, The Journal of experimental medicine.

[64]  Xiaolu Yang,et al.  DEDD and DEDD2 associate with caspase-8/10 and signal cell death , 2003, Oncogene.

[65]  John Calvin Reed,et al.  A GTP-binding adapter protein couples TRAIL receptors to apoptosis-inducing proteins , 2001, Nature Immunology.