Epigenetic Silencing of the Tetraspanin CD9 during Disease Progression in Multiple Myeloma Cells and Correlation with Survival

Abstract

Purpose: The purpose of this study was to investigate expression and epigenetic regulation of CD9 in multiple myeloma (MM) cells during disease progression. Experimental Design: CD9 expression was retrospectively analyzed on bone marrow myeloma samples from 81 patients by immunophenotyping. CD9 expression by murine 5TMM cells was detected by flow cytometric staining and quantitative PCR. The methylation status of the CD9 promoter was determined by bisulfite PCR sequencing. Results: Primary plasma cells in the majority of MM patients with nonactive disease (n = 28) showed CD9 expression, whereas most cases with active disease (n = 53) were CD9 negative. CD9 expression in diagnostic bone marrow samples (n = 74) correlated with survival. Moreover, CD9 expression on murine 5T33 and 5T2MM cells was significantly down-regulated during disease development. Treatment of CD9-nonexpressing 5T33MMvt cells with the clinically relevant histone deacetylase inhibitor LBH589 resulted in a significant increase in CD9 expression. In contrast, cells treated with the demethylation agent 5-aza-2 ¶deoxycytidine barely showed any increase. A combination study with both compounds resulted in a strong synergistic reactivation of CD9. CD9-expressing 5T33MMvv cells and 5T33MMvt cells stably transduced with a mCD9 lentiviral transferplasmid were shown to be more susceptible to natural killer cell^mediated cytolysis than CD9-negative 5T33MMvt cells. Conclusions: CD9 expression correlates with disease status and survival of MM patients. In the murine 5T33MM model, we show that histone modifications, and to a lesser extent CpG methylation, are key epigenetic events in CD9 down-regulation. Furthermore, as CD9 expression becomes down-regulated, 5T33MM cells become less susceptible to natural killer cell^mediated cytolysis.

Down-Regulation of CD9 Expression and its Correlation to Tumor Progression in B Lymphomas

“…The similar observations were made in lung cancer and MMs. 48,60 Although it has recently been reported that mRNA modifications (i.e., deletion, insertion, or mutations) could play a role in regulating CD9 during prostate cancer progression, 47 we could not detect any deletion and insertion in CD9 mRNA when full length of coding region of CD9 was amplified. However, because the possibility precludes CD9 detection depending on epitopes recognized by CD9 antibody used for immunohistochemical staining, it should be taken into consideration.…”

“…In addition, CD9 expression has been reported to correlate with tumor progression in various tumors but has not been studied in B lymphomas. [43][44][45][46][47][48] CD9 is highly expressed on less aggressive L3055-12 but not on more aggressive L3055-33. pression upon transition of less aggressive B lymphomas to advanced B lymphomas appeared not to be limited to L3055 cells because CD9 expressing two other B lymphoma cell lines (i.e., Wan and BL40) also gradually lost CD9 after prolonged in vitro culture.…”

“…18,19,21,35,59 Regarding B cell malignancies, CD9 expression was reported to be inversely associated with disease-free survival in diffuse B lymphoma patients and inversely correlated with disease activity and survival of MM patients. 48,59 Along with CD9, other tetraspanin members CD82 and CD63 have been reported to be inversely related with tumor progression in various tumors.…”

Roles of tetraspanin proteins in cell and tumor biology

“…CD9 also functions as a motility/migration brake (Ono et al, 1999), and this might explain the correlation detected between CD9 expression and the pattern of BM involvement. In multiple myelomas (MM), there is CD9 expression in non active MM, while in active and aggressive MM there is an epigenetic silencing of CD9 gene expression (De Bruyne et al, 2008).…”

Functional variants of TSPAN8 are associated with bipolar disorder and schizophrenia

“…Tetraspanins are a family of evolutionarily highly conserved proteins which influence a wide variety of physiologic processes and diseases, among others protein trafficking [Berditchevski and Odintsova, 2007], modulation of immune-signaling complexes [Levy and Shoham, 2005], and cancer [Z€ oller, 2009]. The regulated expression of TSPAN8 (also known as TM4SF3 or CO-029) is important for early development of the pancreas [Jarikji et al, 2009] and TSPAN8 protein abundance is known to be positively correlated with tumor progression , similar to tetraspanins CD151 (TSPAN24) [Yang et al, 2008] or TSPAN1 [Scholz et al, 2009] and opposite to, for example, CD9 (TSPAN29) [De Bruyne et al, 2008] or CD82 (TSPAN27) [Miranti, 2009], whose protein expression is negatively correlated with malignant phenotype. Besides the severe impact of expression changes of TSPAN8 and tetraspanins in general, mutations in TSPAN7 (CD231, TM4SF2) resulting in the amino acid substitution P172H lead to X-linked mental retardation [Zemni et al, 2000;Maranduba et al, 2004].…”