Although CD138 expression is a hallmark of plasma cells and myeloma cells, decreased expression of CD138 is occasionally found. We previously reported in the last ASH meeting that 1) CD138 expression decreases in patients with relapsed/progressive disease compared with untreated MM patients and that 2) Patients with low levels of CD138 expression had a worse overall survival compared with patients with high levels of CD138 expression. However, the mechanisms of CD138 down-regulation in myeloma cells are still unclear. It is known that myeloma patient's bone marrow environment is hypoxic (Colla et al. Leukemia. 2010; 24: 1967–1970). It is also reported that tumor progression delivers hypoxic environment to MM cells in vivo (Azab et al. Blood. 2012; 119: 5782–5794). Based upon our and other reports, we hypothesized that CD138 expression may be down regulated by hypoxia. In the present study, we examined changes of CD138 and transcription factor expression in myeloma cells under hypoxic condition.
Two myeloma cell lines (RPMI 8226 and KMS-12-BM) were cultured under normoxic (20% O2) and hypoxic (1% O2) conditions for 24 hrs to 72 hrs. CD138 expression of these cell lines under normoxic and hypoxic conditions were analyzed by flow cytometry. Only viable cells were analyzed by excluding 7-AAD positive dead cells. Real time RT-PCR analysis was utilized to analyze gene expression between normoxic and hypoxic cells. Amounts of IRF4 at protein level were determined with western blotting and flow cytometry.
CD138 expression was found to decrease under hypoxic condition compared to normoxic condition in 24 hrs in culture of RPMI 8226 and in 48 hrs in KMS-12-BM when examined with flow cytometry. Real time RT-PCR analysis revealed that CD138 expression was down-regulated under hypoxic condition, indicating that CD138 was down-regulated at transcriptional level. Gene expressions of IRF4, PRDM1 and XBP1, known as plasma cell specific transcription factors, were down-regulated under hypoxic condition compared to those under normoxic condition. Western blot and flow cytometry analysis showed IRF4 was also down-regulated under hypoxic condition. Interestingly, the decreased CD138 expression rendered under hypoxic condition recovered when they were placed under normoxic condition along with an increase of IRF4 expression.
We conclude that hypoxia induces down regulation of CD138 in myeloma cells, accompanying with decreased expressions of IRF4, PRDM1 and XBP1. The present data together with our previous finding that reduced CD138 expression is frequently seen in patients with relapsed/progressive diseases suggest that the microenvironment of myeloma cells in patients with relapsed/progressive diseases may be more hypoxic comparing to that at diagnosis. Since IRF4 is a master transcription factor required for the maturation of plasma cells, the hypoxia-induced down-regulation of IRF4 suggests phenotypic shift from mature to immature state, which resembles to the report demonstrating a shift of disease status by hypoxia to more aggressive and immature phenotypes in solid tumors (Axelson et al. Semin Cell Dev Biol. 2005; 16: 554–563). Since IRF4 is also important as a target of IMiDs, further analysis of CD138-negative myeloma cells might contribute not only to better understanding of disease progression but also to drug resistance mechanisms.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.