TRAF6 Silencing Attenuates Multiple Myeloma Cell Adhesion to Bone Marrow Stromal Cells

Abstract

The bone marrow (BM) microenvironment plays an important role in supporting proliferation, survival and drug resistance of Multiple Myeloma (MM) cells. MM cells adhere to bone marrow stromal cells leading to the activation of tumour-promoting signaling pathways. Activation of the NFκB pathway, in particular, is central to the pathogenesis of MM. Tumour necrosis factor receptor-associated factor 6 (TRAF6) is a key mediator of NFκB activation and has previously been highlighted as a potential therapeutic target in MM. Here, we demonstrate that adherence of MM cell lines to stromal cells results in a reciprocal increase in TRAF6 expression. Knockdown of TRAF6 expression attenuates the ability of MM cells to bind to stromal cells and this is associated with a decrease in NFκB-induced expression of the adhesion molecules ICAM1 and VCAM1. Finally, we show that knockdown of TRAF6 sensitizes MM cells to treatment with bortezomib when co-cultured with stromal cells. Inhibiting TRAF6 represents a promising strategy to target MM cells in the BM microenvironment.

Keywords: BMSCs; Multiple Myeloma; TRAF6.

Figure 1

Tumour necrosis factor receptor-associated factor 6 (TRAF6) expression is enhanced in bone marrow stromal cell (BMSC) co-cultures: (A) TRAF6 protein expression in KMS-11 and U266 cells cultured on their own or in co-culture with HS-5 cells; optical density normalized to GAPDH and expressed as a percentage of KMS-11 or U266 cells cultured alone (n = 3). (B) TRAF6 protein expression in HS-5 cells cultured on their own or in co-cultures with KMS-11 or U266 cells; optical density normalized to GAPDH and expressed as a percentage of HS-5 cells cultured alone (n = 3). (C) Proliferation of KMS-11 cells transduced with non-targeting control (NTC) shRNA or shRNA targeting TRAF6 (shTRAF6), cultured in isolation (left panel) or in co-culture with HS-5 cells (right panel), n = 4; (D) Proliferation of U266 cells transduced with NTC shRNA or shRNA targeting TRAF6, cultured in isolation (left panel) or in co-culture with HS-5 cells (right panel), n = 4. * p ≤ 0.05, ** p ≤ 0.01.

Figure 2

TRAF6 knockdown disrupts adhesion to BMSCs: (A) Proportion of suspension and adherent cells in KMS-11 TRAF6 knockdown cells (shTRAF6) compared to non-targeting control (NTC) cells; (B) Effect of TRAF6 knockdown on the ability of KMS-11 and U266 cells to adhere to HS-5 cells; (C) Effect of TRAF6 knockdown on the ability of KMS-11 and U266 cells to adhere to BMSCs from MM patients. The data is presented as mean (± st dev) of three independent experiments. * p ≤ 0.05, ** p ≤ 0.01.

Figure 3

TRAF6 knockdown inhibits NFκB signaling and reduces NFκB-dependent transcription of adhesion molecules: (A) Protein expression of TRAF6 and key proteins in the activation of NFκB signaling. q-PCR analysis of TRAF6, VCAM1 and ICAM1 gene expression in (B) KMS-11 TRAF6 knockdown (shTRAF6) and non-targeting control (NTC) cells, (C) U266 shTRAF6 and NTC cells, (D) HS-5 cells co-cultured with KMS-11 NTC or shTRAF6 cells and (E) HS-5 cells co-cultured with U266 NTC or shTRAF6 cells. The data is presented as mean (± st. dev) of three independent experiments. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 4

TRAF6 knockdown enhances the efficacy of bortezomib in MM–BMSC co-cultures: (A) Dose response of KMS-11 NTC and shTRAF6 cells co-cultured with HS-5; (B) Dose response of U266 NTC and shTRAF6 cells co-cultured with HS-5. The data is presented as mean (±st. dev) of three independent experiments, * p ≤ 0.05.