Tetraspanin-induced death of myeloma cell lines is autophagic and involves increased UPR signalling

Abstract

BACKGROUND: Multiple myeloma (MM) therapy is hindered by the interaction of the heterogeneous malignant plasma cells with their microenvironment and evolving drug resistance. We have previously shown that the membranal tetraspanins, CD81 and CD82, are under-expressed in MM cells and that their reintroduction causes massive non-apoptotic death. In this study, we aimed to characterise the tetraspanin-induced MM death. METHODS: Multiple myeloma cell lines were transiently transfected with eGFP -CD81N1/CD82N1 fusion proteins and assessed for death mode by flow cytometry (propidium iodide, ZVAD-fmk, 3MA), activation of unfolded protein response (UPR), and autophagy (immunoblot, RT -PCR). RESULTS: Cell death induced by CD81N1 and CD82N1 in MM cell lines was autophagic and involved endoplasmic reticulum (ER)-stress manifested by activation of UPR pathways, PERK (protein kinase-like ER kinase) and IRE1 (inositol-requiring 1). We also established the relative X-box binding protein 1 baseline expression levels in a panel of MM cell lines and their general dependence on autophagy for survival. Timeline of UPR cascades and cell fate supported our results. INTERPRETATION: This is the first publication implicating tetraspanins in UPR signalling pathways, autophagy, and autophagic death. Integration of our findings with published data highlights the unifying dependence of MM cells on ER -Golgi homoeostasis, and underscores the potential of tetraspanin complexes and ER-stress as leverage for MM therapy.

Tetraspanin CD82: a suppressor of solid tumors and a modulator of membrane heterogeneity

“…The hypermethylation of their gene promoters contributes to the silencing [32]. Overexpression of CD81-GFP and CD82-GFP fusions in MM cells causes massive autophagic, nonapoptotic death [33,34] by attenuating Akt/mTOR signaling [35], activating the unfolded protein response [34], and stimulating protein synthesis [36]. Notably, overexpression of GFP-CD81 and GFP-CD82 fusions in MM cells reduces cell-matrix adhesion and cell movement [33].…”

Bevacizumab attenuates major signaling cascades and eIF4E translation initiation factor in multiple myeloma cells

“…ARP-1 displayed a decreased death rate similar to RPMI 8226 (7%k in 2 mg/ml; 6%k in 4 mg/ml; Po0.05; Figure 3c), yet similar to ARK, no change was observed in U266. Previously, we showed that MM cell lines depend on autophagy for survival; 15 thus, we speculated that the cells might have induced an autophagic response to the VEGF inhibition promoting survival, but at a metabolic cost evidenced in the viability assay. By immunoblotting, we determined increased LC3-II (autophagy-induced lipidated form of LC3-I) 28 in the VEGF-inhibited cells (2.7-and 1.5-fold change in 2 mg/ml-treated U266 and ARP-1, respectively, Po0.05), indicating elevated autophagy.…”

“…15 Nuclear and cytosolic proteins were extracted with NucBuster protein extraction kit (Novagen, Madison, WI, USA) according to manufacturer's instructions. Rabbit anti-human antibodies were used for detection of the following: pmTOR(Ser2448)/total mTOR, peIF4E(Ser209)/total eIF4E, pAkt(Ser473)/total Akt, pSTAT3 (Ser727/Tyr705)/total STAT3, pERK1/2(Thr202/Tyr204)/ total ERK1/2, pMNK(Thr 197/202)/total MNK, p4EBP (Ser65)/total 4EBP, Cyclin D, c-Myc (Cell Signaling Technology), HSC-70, total VEGFR1 (Santa Cruz, CA, USA), pVEGFR1(Tyr 1213; R&D Systems, Minneapolis, MN, USA) and LC3/LC3II (Sigma).…”

“…Future studies should address the combined application of bevacizumab with chemotherapy that targets cells in their G1 phase, such as dexamethasone commonly used for myeloma treatment. 48 Moreover, the co-administration of VEGF inhibitors and drugs that block autophagy or, on the contrary, critically elevate ER-golgi stress may also be beneficial (bortezomib 15 for instance).…”

“…We explored the effect of VEGF inhibition on the viability, proliferation, and survival of the MM cells, as well as multiple downstream signaling cascades of established importance to MM cell biology, with specific emphasis on protein translation initiation. MM cell lines RPMI 8226, U266 (ATCC, Manassas, VA, USA), ARP1, and ARK (Professor Epstein, Little Rock, AR, USA) were cultured in RPMI 1640, as described previously 15 (Biological Industries, Kibbutz Beit-Haemek, Israel).…”