RARα2 Expression Confers Myeloma Stem Cell Features

Despite the major advances in the therapy of MM, many patients ultimately relapse and die of this disease. This is most likely due to the persistence of multiple myeloma stem cells (MMSCs), which survive even after the most intensive treatments and are insensitive to non-transplant therapy. We have previously demonstrated that RARα2 expression increased in CD138 selected plasma cells of relapsed myelomas (MM) and that increased expression was linked to poor prognosis in newly diagnosed MM patients. In this study, we further explore the relationship between RARα2 and MMSCs.

RT-PCR was used to detect RARα2, induced pluripotent stem cell (iPS) gene, Wnt and Hedgehog (Hh) gene expression in MMSCs and bulk cells. Clonogenic formation assays were performed by plating RARα2 over-expressing ARP1 and OCI-MY5 MM cell lines in soft agar, treated with bortezomib, doxorubicin, etoposide, and MK-571. Side population (SP) fraction, ALDH activity and cell apoptosis in MM cells were detected by flow cytometry. Western blot were utilized to dissect RARα2-related signaling pathway. Effect of RARα2 on MM progression in vivo was also analyzed in 5TGM1 myeloma mice.

Higher expression of RARα2 was identified in the multiple myeloma stem cell (MMSC) fraction. Over-expression of RARα2 in MM cell lines resulted in 1) increased drug-resistance 2) increased clonogenic potential 3) activation of both Wnt and Hedgehog (Hh) pathways 4) increased side population (SP) and aldehyde dehydrogenase1 (ALDH1) levels and 5) increased expression of iPS genes. The opposite effects were seen with RARα2 knockdown. RARα2 induced drug resistance by activating the drug efflux pump gene ABCC3 and anti-apoptotic Bcl-2 family members. Inhibition of Wnt signaling or ABCC3 function could overcome drug resistance in RARα2 over-expressing MM cells. Our In vivo study indicated that targeting Wnt and Hh pathways using CAY10404, cyclopamine or itraconazole could significantly reduce tumor burden and increase survival time in the 5TGM1 mouse model. We confirmed these findings in primary myeloma stem cells. Finally, we showed that primary myelomas with high RARα2 expression possess similar features as myeloma stem cells.

This study demonstrates that RARα2 plays a crucial role in MMSCs by activating Wnt and Hh pathways, regulating the transcription of iPS genes and conferring drug resistance.

No relevant conflicts of interest to declare.