Abnormal serum microRNA profiles in tuberous sclerosis are normalized during treatment with everolimus: possible clinical implications

Our results show that TSC is a disease with considerably altered serum profile of miRNA. In addition, these alterations seem to be mTOR-dependent, as treatment with an mTOR inhibitor partially reversed the abnormalities. Furthermore, reaction to everolimus treatment resulted in TSC-type dependent changes of miRNA expression levels, hinting at a specific regulation of miR-222 and its role in altering the phenotypes of TSC1 and TSC2.

The differential expression of several miRNAs in patients with TSC was likely given the broad spectrum of clinical features observed in these diseases. While we are aware of phenotype variability observed in TSC patients, it was impossible to draw any conclusions from deregulated miRNA depending on the type of mutation or other clinical features, such as antiepileptic drug regimens, due to the small size of the study group. In this monogenic disease, the constitutively activated mTOR signaling leads to the formation of benign tumors and additional phenotypic traits such as seizures, intellectual disability and developmental delay [13, 14]. MiRNAs were shown to be associated with several clinical features of TSC, with miR-130a and miR-130b being linked to renal organogenesis [6]. In our study, both were significantly upregulated in comparison to the control group. The second-best characterized TSC-associated miRNA was miR-142, which has already been shown to inhibit the mTOR pathway [15]. In our dataset, levels of miR-142 were lower in the TSC group at baseline than in healthy controls and increased after everolimus treatment. One may speculate that everolimus acts as a direct inhibitor of mTORC1 protein complex by binding to FKBP12 protein and alternatively, indirectly stimulating miR-142 expression. Additionally, this reversible pattern of TSC-associated alteration of serum miRNA profile of miR-142 and miR-136 shows that serum levels of microRNAs may be also used as biomarkers of treatment efficiency and perhaps as predictive factors in rare disorders, in the same way as they are used in breast and prostate cancers [16, 17]. The fact that only miR-142-3p reverted to control values after treatment with everolimus may be due to its overall higher expression, involvement of miR-142-5p in intracellular regulatory mechanisms or differences in promoter sequences – further in-depth mechanistic studies would be needed to resolve the differences between miR-142-3p and -5p expression post-treatment.

The second notable finding of our results was the interaction between the genetic background of TSC and impact of everolimus treatment on serum level of miR-222. This miRNA was previously described as being associated with the KIT/AKT pathway in gastrointestinal stromal tumors [18] and peripheral nerve regeneration processes [19]. While no functional studies on the miR-222/AKT pathway associations were performed within this work, the differential everolimus-stimulated normalization of miR-222 expression observed in TSC1 and TSC2 patients is in line with the clinical observation that patients with TSC1 have a milder form of disease than TSC2. Whether miR-222 plays a role in shaping these phenotypes remains an open question. The absence of statistical differences present in miRNA expression profile, in TSC1 vs TSC2 mutation, or other clinical features, before everolimus treatment may be attributable to the limited number of patients with variable clinical characteristics at study entry.

Our results did not indicate any dysregulation of miR-21 reported in previous in vitro studies [20]; however, this may be explained by a number of mechanisms. Firstly, the cited work used a cellular model of a lymphangioleiomyomatosis with biallelic inactivation of TSC2, which was a different genetic defect than the one observed in our group. Furthermore, cellular changes of miRNA expression do not necessarily correspond to their serum levels, as the same miRNAs may originate from different cell types. A cellular model of miRNA expression patterns changing under direct rapamycin stimulation may not be thus directly translated onto serum levels. Moreover, Trindade et al. used a 132-miRNA panel and thus may not have picked out miRNAs present in the Exiqon 752-miR panel, which may have also contributed to the between-study discrepancy.

Our work does have several limitations mostly linked to the methodology of profiling and statistical power. Firstly, no attempt was made to profile exosome-bound miRNAs, but given that most of the miRNA present in the blood is contained in the exosomes [21] and were isolated during the total miRNA extraction protocol used in our study, it is unlikely that a significant bias could result from the analysis of free miRNA rather than only its exosome-bound fraction. Secondly, our investigation of TSC1/TSC2 differences and interactions with treatment effects had a very low statistical power.

Single point measurement after three months of mTOR inhibitor treatment may be considered as a limitation of the study. However, based on our experience and previous literature we expected the most pronounced clinical response to everolimus therapy to occur within the first three months of treatment [9, 22, 23]. Thus, we decided to study the change in miRNA profile during this time. Longer follow-ups, both clinical and molecular may be useful for further confirmation of our report.

Despite the low number of patients, the fact that our patients were examined before and after treatment with mTOR inhibitors make our preliminary results a valuable reference for future studies on the role of miRNA in TSC and mTOR signaling pathway abnormalities and their functional dependencies.