The Heme Oxygenase-1/Carbon Monoxide Pathway Activates TLR4 Signaling Promoting Bortezomib Resistance in Multiple Myeloma Cells

Heme oxygenase (HO)-1 catalyzes the conversion of heme to biliverdin, iron an d carbon monoxide. In myeloma plasma cells, its expression has been demonstrated to increase during bortezomib (BTZ) treatment and localize into the nucleus conferring drug resistance. Recently, our group demonstrated that BTZ also induces up-regulation of Toll like receptor 4 (TLR4) which acts as a stress-responsive mechanism protecting mitochondria during BTZ exposure and sustaining mitochondrial metabolism. Since two studies independently demonstrated that both HO-1 and TLR4 protect myeloma cells from BTZ-induced apoptotic signals, specific functional connections between these two proteins were considered herein. MM cell lines were treated with BTZ alone or in combination with TAK-242, a selective inhibitor of TLR4. We observed a significant increase of apoptosis in TAK-242/BTZ treated cells compared to BTZ alone. Drug combination also led to higher mitochondrial depolarization and decreased mitochondrial mass evaluated using flow cytometry. Accordingly, TAK-242/BTZ treatment act ivated mitophagy as demonstrated by evaluating co-localization of the autophagosome marker LC3 with mitochondria using confocal microscopy. Since it is known BTZ treated cells increased HO-1 expression as protective mechanism, we next evaluated if BTZ combination with TAK-242 could affect HO-1 expression. Western blot showed a down-regulation of HO-1 after TAK-242/BTZ treatment. Immunofluorescence analysis confirmed that drug combination decreased nuclear HO-1 and increased its cytoplasmic localization compared to BTZ alone. To address this controversy, we administered tin protoporphyrin (SnPP), a well-characterized HO-1 enzymatic inhibitor, alone or in combination with BTZ. Interestingly, SnPP/BTZ treated cells showed lower expression of TLR4 compared to BTZ treated ones. To better investigate if HO-1 enzymatic activity could regulate TLR4 expression, MM cells were exposed to hemin, an inducer of HO-1. We observed a significant up-regulation of TLR4 and NF-kB nuclear localization. Treating cells with rapid or slowly carbon monoxide-releasing molecules (CORM-3 and CORM-A1), an increase of TLR4 expression was observed after 3h with the consequent activation of p-p38, p-ERK and NF-kB nuclear translocation. Moreover, silencing HO-1 confirmed its role in the regulation of TLR4 expression. Moreover, compared to U266 cells, shHO-1/U266 cells howed higher apoptosis after treatment with BTZ, confirming that HO-1/TLR4 signaling protect MM cells from BTZ-induced apoptosis. Our data demonstrate that a functional regulatory link exists between HO1 and TLR4 which in turn impact on drug response. Specifically, inhibition of HO-1/TLR4 axis augmented cytotoxicity of BTZ against MM cells. In conclusion the HO-1/TLR4 axis is involved in BTZ mediated chemoresistance thus providing an important tool to improve the clinical outcome of MM patients resistant to BTZ.