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Nano-scale protein quantification in multiple myeloma
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tified the proteins extracted at the same time as the DNA and RNA from the RLT Plus buffer, we investigated the applicability of the method to the analysis of the expres- sion of key proteins in MM biology, such as D Cyclins, c- myc, Cereblon, Ikaros, and Aiolos, among others. We also wanted to compare protein expression with the corre- sponding mRNA level, since many basic studies have revealed that only 30-40% of protein abundance can be explained by the mRNA level.34 Our results showed a moderate or low correlation between mRNA and protein levels of expression, and are consistent with the general observation that ~60% of the variation in protein concen- tration cannot be explained by measuring mRNA alone.34 We also observed that the mRNA level was less variable than protein expression among MM patients for all the mRNA and protein pairs analyzed. Indeed, protein abun- dance is regulated by a variety of complex mechanisms, such as post-transcriptional and post-translational modifi- cations, and protein degradation regulation.34,35 By measur- ing mRNA abundance, only the early steps in a long chain of regulatory events are considered.36 However, the mRNA level is still often employed as a proxy for protein abundance, mostly because of the lack of appropriate technology to quantify proteins quickly and efficiently.
Our results reproduce the well-known pattern of CCND1/Cyclin D1 and CCND2/Cyclin D2 expression in MM with t(11;14) and t(4;14).9 We also found a correla- tion between c-myc and Ikaros and Aiolos levels, analyz- ing either mRNA or protein expression, consistent with the previously demonstrated regulation of c-myc by Ikaros and Aiolos in MM.19 Interestingly, the correlation between Ikaros and Aiolos levels was stronger for the protein than for the mRNA. To our knowledge, this is the first time that the protein levels of c-myc, Ikaros and
Figure 5. Progression-free sur- vival according to levels of MRNA and protein expression. Progression-free survival in patients with low and high lev- els of mRNA (A) and protein (B) expression. The log-rank test was performed for each gene and protein and Kaplan-Meier curves represent the PFS of MM patients depending on mRNA and protein status. Cutoff Finder software (http://molpath.charite.de/cut off) was used to obtain the optimal cutoff, which was defined as that producing the most significant split that dis- criminates between good and poor survival by examining all the possible cutoffs using the log-rank test.
Aiolos have been quantified and the relationship between their expressions analyzed in MM. In T-cell acute lym- phoblastic leukemia, for example, the levels of mRNA encoding Ikaros and Aiolos were weakly, but significantly correlated.37
Among the proteins included in our study, we observed a significant association between protein level and PFS for Cereblon and Ikaros, while this association was not observed for the respective mRNA levels. Cereblon forms an E3 ubiquitin ligase complex together with the damaged DNA binding protein 1 (DDB1), Cullin4A (CUL4) and Roc1. Immunomodulatory drugs, such as lenalidomide or pomalidomide, bind to Cereblon in a region located at the C-terminus of this protein.38,39 Our results did not demon- strate a correlation between Cereblon protein and mRNA level, and showed that only high levels of Cereblon pro- tein were associated with a good prognosis in MM. These findings are concordant with those of previous studies and support the usage of protein expression to evaluate Cereblon levels.40
Several independent groups have identified Ikaros and Aiolos as the downstream targets of Cereblon after immunomodulatory drug activation.41–43 The role of the level of Ikaros in MM survival is controversial. When Ikaros expression was investigated at the RNA level, a low level of mRNA IKZF1 expression was associated with bet- ter prognosis in newly diagnosed patients treated with immunomodulatory drugs.44 On the other hand, low IKZF1 levels were found to predict a lack of responsive- ness to immunomodulatory drugs and a shorter overall survival in refractory MM patients.45 We observed that a high level of Ikaros protein was associated with longer PFS, while no significant impact on prognosis was observed when PFS was estimated from mRNA levels.
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