Mutated in colorectal cancer (MCC) is a novel oncogene in B lymphocytes

Abstract

BackgroundIdentification of novel genetic risk factors is imperative for a better understanding of B lymphomagenesis and for the development of novel therapeutic strategies. TRAF3, a critical regulator of B cell survival, was recently recognized as a tumor suppressor gene in B lymphocytes. The present study aimed to identify novel oncogenes involved in malignant transformation of TRAF3-deficient B cells.MethodsWe used microarray analysis to identify genes differentially expressed in TRAF3−/− mouse splenic B lymphomas. We employed lentiviral vector-mediated knockdown or overexpression to manipulate gene expression in human multiple myeloma (MM) cell lines. We analyzed cell apoptosis and proliferation using flow cytometry, and performed biochemical studies to investigate signaling mechanisms. To delineate protein-protein interactions, we applied affinity purification followed by mass spectrometry-based sequencing.ResultsWe identified mutated in colorectal cancer (MCC) as a gene strikingly up-regulated in TRAF3-deficient mouse B lymphomas and human MM cell lines. Aberrant up-regulation of MCC also occurs in a variety of primary human B cell malignancies, including non-Hodgkin lymphoma (NHL) and MM. In contrast, MCC expression was not detected in normal or premalignant TRAF3−/− B cells even after treatment with B cell stimuli, suggesting that aberrant up-regulation of MCC is specifically associated with malignant transformation of B cells. In elucidating the functional roles of MCC in malignant B cells, we found that lentiviral shRNA vector-mediated knockdown of MCC induced apoptosis and inhibited proliferation in human MM cells. Experiments of knockdown and overexpression of MCC allowed us to identify several downstream targets of MCC in human MM cells, including phospho-ERK, c-Myc, p27, cyclin B1, Mcl-1, caspases 8 and 3. Furthermore, we identified 365 proteins (including 326 novel MCC-interactors) in the MCC interactome, among which PARP1 and PHB2 were two hubs of MCC signaling pathways in human MM cells.ConclusionsOur results indicate that in sharp contrast to its tumor suppressive role in colorectal cancer, MCC functions as an oncogene in B cells. Our findings suggest that MCC may serve as a diagnostic marker and therapeutic target in B cell malignancies, including NHL and MM.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-014-0056-6) contains supplementary material, which is available to authorized users.

Signaling mechanisms of bortezomib in TRAF3-deficient mouse B lymphoma and human multiple myeloma cells

“…Rabbit polyclonal antibody against Noxa was purchased from AXXORA (Farmingdale, NY). Other antibodies and reagents used in this study were described previously [14,18–20]. …”

“…MTT assay, annexin V staining and cell cycle analyses were performed as previously described [14,18–20]. For caspase cleavage assays, total protein lysates were prepared from cells at different time points after treatment with bortezomib, and cleavage of caspases 3, 8, and 9 was subsequently examined by immunoblot analysis.…”

“…Immunoblot analysis was performed using various antibodies as described [14,18–20]. Images of immunoblots were acquired using a low-light imaging system (LAS-4000 mini, FUJIFILM Medical Systems USA, Inc., Stamford, CT).…”

“…Lentiviruses of the pUB-SBP-Noxa expression vector and an empty pUB-Thy1.1 vector were packaged, titered, and used to transduce human MM cells as previously described [18–20]. At 24 hours post transduction, the transduction efficiency of cells was analyzed by Thy1.1 immunofluorescence staining and flow cytometry.…”

“…At 24 hours post transduction, the transduction efficiency of cells was analyzed by Thy1.1 immunofluorescence staining and flow cytometry. Transduced cells were subsequently analyzed for apoptosis and cell cycle distribution using annexin V staining and propidium iodide (PI) staining as previously described [14,18–20]. …”