Here we present diverse lines of evidence that long-term molecular effects of doxorubicin action in ALL survivors include persistently altered transcription factor activity, which results in changes of miRNA presence in the circulation that may contribute to cardiomyopathy development, the major life-threatening long-term effects of anthracycline treatment41,42. We demonstrate that especially miRNA circulating in EVs may be a useful source of information on transcriptomic processes that are changed due to anticancer treatment.
Indeed, our KEGG and GO enrichment analyses for differentially expressed vesicular miRNA targets indicate that processes including dilated cardiomyopathy and related processes (e.g. ERBB signalling43,44) are ongoing in ALL survivors. Hence, we decided to search for TFs that regulate the expression of differentially expressed miRNAs.
Among TFs regulating miRNAs in EVs, we found multiple important players in EMT - TGFB1, SLUG, JAG1, SNAI1, ZEB-1, SIP-1, TWIST, and BMP445–49. TGFB1, having the highest statistical significance, was reported to be a master regulator of EMT 50,51, a process linked to therapy-triggered senescence52 and fibrosis53,54. Senescence, proposed as an alternative to entering apoptosis in response to genotoxic treatment55,56, is also suggested to reinforce long-term cardiac complications of anticancer treatment57. It is noteworthy that TGFB expression can be increased in the heart tissue many weeks after doxorubicin treatment58.
Moreover, TGFB1- related factors including TWIST1 and SLUG (SNAI2), were previously reported as related to cardiac morphogenesis including atrioventricular cushion formation or cardiac septation59,60. Recent data revealed that TWIST2 is expressed in specific populations of interstitial heart cells, which contributes to cardiac homeostasis and regeneration61. Additionally, TWIST1 as well as TWIST1-regulated mir-199a (miRNA correlated with IVS thickness in our study) were reported as down-regulated in the heart tissue of patients with severe cardiomyopathy62. Dysfunction within this system may result in poor cell renewal and proliferation, as was shown for stem cells63.
Additionally, our analysis of miRNA expression data from individuals with dilated and idiopathic cardiomyopathy revealed that except for some similarities in miRNA differential expression, vesicular miRNAs in the former ALL patients share Tfs regulators with miRNA related with cardiomyopathy (TGB, ZEB2, SNAI1, TWIST1).
Moreover, one of the miRNA common for these analyses, mir-31, significantly upregulated in extracellular vesicles of ALL survivors, and which we found to be involved in the genetics of heart failure is also the top miRNA correlated with the highest number of Tfs regulating miRNA in A2 analysis, including factors well known for significant role in both response to doxorubicin and cardiomyocyte functioning (NRF2, TP53, HIF1A) as well as EMT process (ZEB1, SNAI1). Mir-31 has been previously reported to be upregulated in cardiomyocytes due to hypoxia or oxidative stress and in hearts after myocardial infarction. Its silencing upregulated troponin T in cardiomyocytes, while administration of mir-31 inhibitor in rats post-myocardial infarction improved heart function64. Moreover, a recent paper has shown that doxorubicin treatment induced mir-31 expression in cultured cardiomyocytes as well as in heart tissue, causing downregulation of RNA binding protein quaking playing a significant role in doxorubicin-induced cardiotoxicity65, what imply that pathways related to mir-31 might also be valuable targets for future studies.
Surprisingly, the results of our analyses support the role of NRF2 as one of the key regulators of the transcriptomic network in ALL survivors treated with doxorubicin. NRF2 is associated with doxorubicin resistance and short-term effects of chemotherapy66,67. Down-regulation of NRF2, which physiologically plays the main role in the protection against oxidative stress through the activation of transcription of antioxidative response genes, causes excessive doxorubicin-induced cardiotoxicity 68,69. Additionally, prolonged oxidative stress and unbalanced NRF2 activity may induce senescence and premature age-related diseases, which have been attributed to the long-term effects of anticancer treatment70,71. To investigate the detailed role of NRF2 in the transcriptional network upon doxorubicin treatment, we performed NRF2 silencing experiments on cultured cardiomyocytes. It reveals that even without drug exposure, down-regulation of NRF2 is accompanied by changes in the functioning of NOTCH1 (present also among TFs regulating plasma miRNAs) and TGFB-related TFs (present among TFs regulating miRNAs in EVs’) involved in EMT: TGFB, TWIST1, TWIST2, ZEB1, SNAI2, which have been previously reported as related to cardiac morphogenesis including atrioventricular cushions formation or cardiac septation59,60. We also observed significant alterations of NOTCH signaling targets: HEY2, HEYL, and HES factors are required for efficient chromatin binding by HEY172, which we show is further dysregulated in shNRF2 cells upon doxorubicin treatment. HEY1, HEY2, and HEYL together with HES family of TFs are crucial for the formation of valves and septa during embryogenesis59. HEY1, the second significant TF regulating plasma miRNA expression, turns up as differentially active upon NRF downregulation as well as during doxorubicin stimulation in control cells. It was previously shown that inactivation of HEY1 and HEYL causes congenital heart defects including ventricular septal defects35. Whereas in aging muscle stem cells, HEY1 rescues cells from death due to mitotic catastrophe during muscle regeneration73. Moreover, in the case of NRF2 loss, HEY1 downregulation was accompanied by a delay in repair after chemical injury63. Second of Hey genes - HEY2 is specifically expressed in the interventricular septum, ventricular compact myocardium, the atrioventricular canal outflow tract, at the base of trabeculae, and in epicardial cells74. It was also postulated that HEY2 is necessary to maintain ventricular identity by mature cells74. Indeed, cardiomyocyte-specific deletion of HEY2 results in the transcription of atrial genes in the ventricular myocardium accompanied by impairment of cardiac contractility and changes in the morphology of the right ventricle75,76, and its deficiency causes septal defects and cardiomyopathy in mice75.
Additionally, TWIST1 as well as TWIST1-regulated mir-199a (miRNA correlated with IVS thickness in our study) were reported as down-regulated in the heart tissue of patients with severe cardiomyopathy62. Notably, GRHL1, a regulator of gene expression upon doxorubicin treatment in the case of diminished NRF2 activity, is a member of the grainyhead family of TFs, which acts as a suppressor of TGFβ-induced, and TWIST-induced EMT77. The finding of co-regulation between NRF2 activity and NOTCH signaling in ALL survivors remains in line with previous reports suggesting reciprocal regulation between these these Tfs78. Dysfunction within this system may result in poor cell renewal and proliferation, as was shown for stem cells63.
Here we also show that doxorubicin treatment of cardiomyocytes alters the activity of RBPJ, an important transcriptional regulator of NOTCH79. Additionally, since TWIST, HEY, and HES proteins all belong to the basic helix-loop-helix (bHLH) transcription factors family80,81, it seems reasonable to hypothesize that the co-regulation of these TFs with NRF2 may be related to the 60 amino acid region which contains two highly conserved domains, considered as involved in interactions with TFs82.
In summary, our approach allowed to identify dysregulated mechanisms, including altered NRF2 activity, that might be involved in late cardiac complications due to doxorubicin treatment.
However, to the best of our knowledge, it is still unclear how the damage caused by anthracyclines, often during childhood, can be propagated until adulthood. Doxorubicin-induced DSBs are especially frequent around active promoters83 and are known to cause transcriptional repression84. Gene expression silencing was shown to be converted to long-term stable silencing through modifications like CpGs or histone methylation, which may be heritable 85,86. Thus, NRF2 gene with high transcriptional activity upon exposure to oxidative stress, could be at particularly high risk of modification during doxorubicin treatment. Although further investigations are required to prove such possibility.
Our approach using differential expression of circulating miRNA allowed to identify dysregulated mechanisms, which may be involved in the development of long-term consequences of doxorubicin treatment in ALL survivors. Additionally, microRNA encapsulated in extracellular vesicles significantly indicated EMT-related processes which seem to be related to cardiomyopathy development, which although needs further study.
In summary, we present data proving that NRF2-related molecular changes initiated by doxorubicin treatment may cause alterations in the functioning of transcription factors related to cardiac cells identity and regeneration. Among them, especially HEY as well as TWIST Tfs could be considered as potential targets in future studies.
Our approach using differential expression of circulating miRNA allowed to identify dysregulated mechanisms, which may be involved in the development of long-term consequences of doxorubicin treatment in ALL survivors. Additionally, microRNA encapsulated in extracellular vesicles significantly indicated EMT-related processes which seem to be related to cardiomyopathy development, which although needs further study.