The cancer stem cell (CSC) concept is still very controversial; therefore identification and isolation of this specific population remain challenging. A variety of putative markers have been described and measurement of high aldehyde dehydrogenase (ALDH) activity has been defined as a characteristic of stem cells (SCs). In this study, a library of novel small molecules (1,4-di-substituted acetalanthraquinones, AAQs), containing an acetal group as protected aldehyde functionality, was designed with the aim of probing affinity for ALDH metabolism and demonstrating their potential as molecular fluorescent probes to identify CSCs. The AAQs were shown to be subjective to acidic hydrolysis using 2M HCl at 37ºC; however compounds containing secondary or tertiary amine functionalities in their sidechain were only partly hydrolysed at 70 ºC. Metabolism studies were conducted using cytosolic fractions from rat liver enriched in ALDHs, yeast ALDH and human recombinant ALDH1A1. Some evidence was demonstrated which linked ALDH metabolism with aldehyde functionalities of hydrolysed AAQs (HAAQs). The AAQs were shown to emit far-red fluorescence (600-750 nm). A close relationship between structure modifications and alteration of cellular localisation, with gained specificity for selected sub-cellular compartments were achieved when assessed in A549 and U-2 OS cell lines. Thermal DNA denaturation and chemosensitivity assays were used to obtain information about DNA binding properties and cytotoxicity of AAQs and HAAQ congeners. All compounds were shown to be weak*to*moderately binding to DNA, and symmetrical 1,4-di-substituted compounds were shown to be non*toxic (IC50 = 100 :/! with non-symmetrical analogues generating IC50 values in the 1-100 :/ range. No fundamental variation in the biological activity was observed when comparing AAQs with HAAQs in the A549 (+ALDH) and MCF7 (-ALDH) cell lines. A pilot investigation revealed that aberrant gene methylation was cell-type dependent for three ALDH isoforms (1A1, 2, 3A1). Decitabine treatment led to enhanced protein expression for ALDH1A1 (A549), ALDH2 (MCF7) and ALDH3A1 (A549). In contrast, the protein level was reduced for ALDH1A1 in HT29 cells after decitabine treatment. ALDH1A1, ALDH2 and ALDH3A1 were highly expressed in prostate cell lines, with expression linked to promoter methylation. In contrast, low levels of DNA methylation were found in primary prostate cancer cells and benign prostatic hyperplasia. Interestingly, ALDH1A1, considered a SC marker, was found to be expressed at low levels in CD133+/ α2β1hi stem cell fraction and upregulated in CD133-= α2β1lo differentiated prostate cancer cells. In summary, the results in this thesis demonstrate the complexity and tumour type specificity of ALDH expression. This creates challenges for the development of selective probes for CSC isolation, such as the AAQs discussed in this thesis. Although inconclusive results were obtained in regard to AAQs and their potential in targeting ALDHs, selected AAQs were shown to reveal interesting biological features highlighting them as potential non-invasive cytometric probes for tracking molecular interactions in live cells. / EPSRC, Biostatus / The full text was made available at the end of the re-embargo period, 1st September 2017.

No description available.

O carcinoma epidermóide é uma neoplasia que pode ter origem do revestimento mucoso de vários sítios das vias aerodigestivas superiores, sendo a língua o sítio primário com maior incidência. Entre os fatores de risco para a doença estão a idade, as mutações genômicas, o hábito tabagista e principalmente o consumo de etanol. O etanol é considerado um agente cocarcinogênico no processo de desenvolvimento do câncer de boca. Por outro lado, o acetaldeído, subproduto da oxidação do etanol, é tóxico e participa diretamente na carcinogênese. Assim, polimorfismos genéticos que alteram a oxidação de etanol para acetaldeído promovendo seu acúmulo podem alterar o risco de câncer oral. Os resultados sugerem que pacientes portadores do polimorfismo do gene ADH1C Ile350Val possuem maior risco de tornarem-se etilistas crônicos (OR=2,0199), mas o risco de desenvolverem câncer não é alterado quando comparado aos não portadores. Já os portadores dos polimorfismos nos genes ADH1B Arg47His (OR=0,3445), CY2E1 (ins) (OR=0,3261) e ALDH2 (GA) (OR=0,4811) apresentaram menores riscos de desenvolverem câncer oral, mas estes polimorfismos não estavam associados ao risco de tornarem-se etilistas crônicos. Observou-se também uma possível interação entre a baixa atividade da enzima ALDH2 e a expressão do gene CYP2E1 como um fator protetor no desenvolvimento do câncer de boca. Entretanto, há necessidade de mais estudos para comprovar esses achados / Squamous cell carcinoma is a neoplasm that may originate from the mucosal tissue from various sites of the upper aerodigestive tract, the tongue being the primary site with the highest incidence. Among the risk factors for the disease are age, genomic mutations, smoking habit, and especially the consumption of ethanol. Ethanol is considered a co-carcinogenic agent in the development of oral cancer. Moreover, acetaldehyde, ethanol oxidation product, is toxic and is directly involved in carcinogenesis. Thus, genetic polymorphisms that alter the oxidation of ethanol to acetaldehyde by promoting its accumulation can alter the risk of oral cancer. The results suggest that patients with the ADH1C Ile350Val polymorphism have increased risk of becoming chronic drinkers (OR = 2.0199), but the risk of developing cancer is not changed when compared to non carriers. Since the carriers of polymorphisms in genes ADH1B Arg47His (OR = 0.3445), CY2E1 (ins) (OR =0.3261) and ALDH2 (GA) (OR = 0.4811) lower risk of developing oral cancer, but these polymorphisms were not associated with risk of becoming chronic drinkers .There was also a possible interaction between the low activity of the enzymeALDH2 and CYP2E1 gene expression as a protective factor in the development of oral cancer. However, we need more studies to confirm these findings

Le Dimethyl Ampal Thiolester (DIMATE) est un inhibiteur des aldéhydes déshydrogénases (ALDHs) de type 1 et 3. L’intérêt croissant au cours de ces dernières années pour ces enzymes intra cytoplasmiques que sont les ALDHs, s’explique par leurs utilisations comme marqueurs pour distinguer les cellules souches, saines ou cancéreuses, au sein de différents tissus, comme le tissu hématopoïétique. Le traitement des Leucémies Aiguës Myéloïdes demeure une problématique clinique majeure. En effet, malgré un taux de rémission complète moyen d’environ 70% avec les traitements conventionnels, la survie moyenne des patients porteurs d’une LAM n’excède pas les 50% à 5 ans. A ce titre, le DIMATE, semble être un médicament d’avenir. Le DIMATE présente une toxicité majeure sur plusieurs lignées leucémiques humaines et sur des cellules souches leucémiques issues de patients. De manière remarquable, le DIMATE à ces doses anti-leucémiques présente une innocuité quasi-totale sur les cellules souches hématopoïétiques saines. In vivo, chez la souris, le DIMATE permet d’éradiquer spécifiquement les cellules leucémiques humaines xénogreffées et présente en monothérapie une efficacité similaire à l’association Cytarabine + Daunorubicine qui constitue le standard thérapeutique actuel. Ces résultats encourageants vont servir de support conceptuel à la mise en place prochaine d’essais cliniques. / The Dimethyl Ampal Thiolester (DIMATE) is a type 1 and 3 Aldehydes Dehydrogenases (ALDHs) inhibitor as an innovating treatment for AML. Interest in ALDH is due to its activity as a marker for identification of stem cell in different tissues. The different species of ALDHs control the levels of the endogenous apoptogenic aldehydes. Cancer cells protect themselves from the apoptogenic effect of these aldehydes by the ALDHs that oxidize them to their non-apoptogenic carboxylic acids. The vast majority of patients with AML achieve complete remission (CR) after standard induction chemotherapy. However, the majority subsequently relapses and dies of the disease. Therefore, AML remains a clinical challenge and new therapies are urgently needed. For this, DIMATE appears as a promising drug. In vitro, DIMATE is a powerful ALDH inhibitor and has a major cytotoxic activity on human AML cell lines. Moreover, DIMATE is highly active against leukemic population enriched in LSCs, but, unlike conventional chemotherapy, DIMATE is not toxic for healthy hematopoietic stem cells which retained after treatment their self-renewing and multi-lineage differentiation capacity. In immunodeficient mice, xenografted with human leukemic cells, DIMATE eradicates specifically human AML cells and spares healthy mouse hematologic cells. Moreover, DIMATE showed the same efficiency than the association Daunorubicin + Cyrabine, which is considered as the gold standard for AML induction. Results from our work open new therapeutic perspectives in AML and provide a conceptual support for initiation of a phase I-II clinical trials, but also innovating cellular therapy.

O carcinoma epidermóide é uma neoplasia que pode ter origem do revestimento mucoso de vários sítios das vias aerodigestivas superiores, sendo a língua o sítio primário com maior incidência. Entre os fatores de risco para a doença estão a idade, as mutações genômicas, o hábito tabagista e principalmente o consumo de etanol. O etanol é considerado um agente cocarcinogênico no processo de desenvolvimento do câncer de boca. Por outro lado, o acetaldeído, subproduto da oxidação do etanol, é tóxico e participa diretamente na carcinogênese. Assim, polimorfismos genéticos que alteram a oxidação de etanol para acetaldeído promovendo seu acúmulo podem alterar o risco de câncer oral. Os resultados sugerem que pacientes portadores do polimorfismo do gene ADH1C Ile350Val possuem maior risco de tornarem-se etilistas crônicos (OR=2,0199), mas o risco de desenvolverem câncer não é alterado quando comparado aos não portadores. Já os portadores dos polimorfismos nos genes ADH1B Arg47His (OR=0,3445), CY2E1 (ins) (OR=0,3261) e ALDH2 (GA) (OR=0,4811) apresentaram menores riscos de desenvolverem câncer oral, mas estes polimorfismos não estavam associados ao risco de tornarem-se etilistas crônicos. Observou-se também uma possível interação entre a baixa atividade da enzima ALDH2 e a expressão do gene CYP2E1 como um fator protetor no desenvolvimento do câncer de boca. Entretanto, há necessidade de mais estudos para comprovar esses achados / Squamous cell carcinoma is a neoplasm that may originate from the mucosal tissue from various sites of the upper aerodigestive tract, the tongue being the primary site with the highest incidence. Among the risk factors for the disease are age, genomic mutations, smoking habit, and especially the consumption of ethanol. Ethanol is considered a co-carcinogenic agent in the development of oral cancer. Moreover, acetaldehyde, ethanol oxidation product, is toxic and is directly involved in carcinogenesis. Thus, genetic polymorphisms that alter the oxidation of ethanol to acetaldehyde by promoting its accumulation can alter the risk of oral cancer. The results suggest that patients with the ADH1C Ile350Val polymorphism have increased risk of becoming chronic drinkers (OR = 2.0199), but the risk of developing cancer is not changed when compared to non carriers. Since the carriers of polymorphisms in genes ADH1B Arg47His (OR = 0.3445), CY2E1 (ins) (OR =0.3261) and ALDH2 (GA) (OR = 0.4811) lower risk of developing oral cancer, but these polymorphisms were not associated with risk of becoming chronic drinkers .There was also a possible interaction between the low activity of the enzymeALDH2 and CYP2E1 gene expression as a protective factor in the development of oral cancer. However, we need more studies to confirm these findings

Les cellules souches cancéreuses (CSC) représentent une sous-population de cellules tumorales à l’origine de l’hétérogénéité et de la croissance tumorale. Les CSC sont plus résistantes aux traitements, et à l’origine de la rechute et des métastases. L’identification des CSC constitue actuellement un enjeu majeur dans le développement de nouvelles thérapies ciblées pour inhiber la croissance tumorale et éradiquer le cancer. Dans ce travail, nous avons cherché à identifier, caractériser, et cibler les CSC dans l’adénocarcinome gastrique. Des modèles murins de xénogreffe de tumeurs primaires de patients atteints d'adénocarcinome gastrique hors cardia de types intestinal et diffus ont été développés, ainsi qu’un modèle de tumorsphere in vitro afin d’évaluer les capacités tumorigéniques de sous-populations tumorales. Nous avons identifié CD44 et l'aldéhyde déshydrogénase (ALDH) comme marqueurs d’enrichissement des CSC dans les 2 types d’adénocarcinomes gastriques, l’ALDH représentant un marqueur plus spécifique que CD44. Nous avons ensuite étudié l'effet de l’acide rétinoïque tout trans (ATRA), et nous avons montré que l'ATRA inhibe la formation et la croissance des tumorspheres in vitro ainsi que la croissance tumorale in vivo. Cet effet de l’ATRA passe par l’inhibition de l’expression des marqueurs souches et des capacités d'auto-renouvèlement des CSC. En conclusion, CD44 et ALDH sont des marqueurs de CSC dans les adénocarcinomes gastriques hors cardia de types intestinal et diffus, et le traitement par l’ATRA constituerait une stratégie commune de traitement pour cibler spécifiquement les CSC et inhiber la croissance tumorale dans ces deux types de cancer gastrique. / Cancer stem cells (CSCs) are a subpopulation of tumor cells at the origin of the heterogeneity and growth of tumors. CSCs are more resistant to treatment, and are responsible for relapse and metastasis. The identification of CSCs is a major challenge for the development of new targeted therapies to inhibit tumor growth and eradicate cancer. In this work, we aimed to identify, characterize, and target CSCs in gastric adenocarcinoma. Mouse models of primary tumor xenografts from intestinal and diffuse type non-cardia gastric adenocarcinomas from patients were developed, as well as an in vitro tumorsphere assay, to assess the tumorigenic capacity of subpopulations of tumor cells. We identified CD44 and aldehyde dehydrogenase (ALDH) as CSC enrichment markers in the two types of gastric adenocarcinoma, ALDH representing a more specific marker than CD44. We then studied the effect of All-trans retinoic acid (ATRA), and showed that it inhibited the formation and growth of tumorspheres in vitro and tumor growth in vivo. This effect of ATRA is due to the inhibition of stem marker expression and the self-renewal capacity of CSCs. In conclusion, CD44 and ALDH are effective CSC markers in intestinal and diffuse type non-cardia gastric adenocarcinomas, and treatment with ATRA provides a common treatment strategy to specifically target CSCs and inhibit tumor growth in both subtypes of this gastric cancer.

Indiana University-Purdue University Indianapolis (IUPUI) / The human genome encodes 19 members of the aldehyde dehydrogenase (ALDH) superfamily, critical enzymes involved in the metabolism of aldehyde substrates. A major function of the ALDH1A subfamily is the oxidation of retinaldehyde to retinoic acid, a key regulator of numerous cell growth and differentiation pathways. ALDH1A1 has been identified as a biomarker for both normal stem cells and cancer stem cells. Small molecule probes are needed to better understand the role of this enzyme in both normal and disease states. However, there are no commercially available, small molecules that selectively inhibit ALDH1A1. Our goal is to identify and characterize small molecule inhibitors of ALDH1A1 as chemical tools and as potential therapeutics. To better understand the basis for selective inhibition of ALDH1A1, we characterized N,N-diethylaminobenzaldehyde (DEAB), which is a commonly used inhibitor of ALDH1A1 and purported to be selective. DEAB serves as the negative control for the Aldefluor assay widely utilized to identify stem cells. Rather than being a selective inhibitor for ALDH1A1, we found that DEAB is a slow substrate for multiple ALDH isoenzymes, and depending on the rate of turnover, DEAB behaves as either a traditional substrate or as an inhibitor. Due to its very slow turnover, DEAB is a potent inhibitor of ALDH1A1 with respect to propionaldehyde oxidation, but it is not a good candidate for the development of selective ALDH1A1 inhibitors because of its promiscuity. Next, to discover novel selective inhibitors, we used an in vitro, high-throughput screen of 64,000 compounds to identify 256 hits that either activate or inhibit ALDH1A1 activity. We have characterized two structural classes of compounds, CM026 and CM037, using enzyme kinetics and X-ray crystallographic structural data. Both classes contained potent and selective inhibitors for ALDH1A1. Structural studies of ALDH1A1 with CM026 showed that CM026 binds at the active site, and its selectivity is achieved by a single residue substitution. Importantly, CM037 selectively inhibits proliferation of ALDH+ ovarian cancer cells. The discovery of these two selective classes of ALDH1A1 inhibitors may be useful in delineating the role of ALDH1A1 in biological processes and may seed the development of new chemotherapeutic agents.

Indiana University-Purdue University Indianapolis (IUPUI) / Breast cancer is the second leading cause of cancer death for women in the United States. Approximately, 1 in 5 women will recur with cancer within 10 years of completing treatment and recent publications have suggested that breast cancer stem cells confer resistance to therapy. These reports highlight aldehyde dehydrogenase 1A1 (ALDH1A1) and Yes-associated protein (YAP) as a biomarker and key mediator of the stem cell phenotype respectively. To further understand how YAP and ALDH1A1 facilitate chemoresistance, this study investigated how ALDH1A1 specific inhibition affected YAP activity and growth of basal-like breast cancer cells, which are enriched in cancer stem cells. Intriguingly, attenuation of growth by ALDH1A1 inhibition was observed when cells were plated on a reconstituted basement membrane. Further, the inhibition of cell growth correlated with cytosolic retention of YAP and a reduction in YAP signaling. In a complementary analysis, the overexpression of YAP correlated with an increased level of ALDH1A1 transcript. Results from this study indicate a novel mechanism by which basal-like breast cancer cells utilize YAP to maintain the stem cell phenotype and also suggest ALDH1A1 as a potential therapeutic target for breast cancer therapy.

Castration-resistant prostate cancer (CRPC) remains an aggressive incurable disease in men mainly due to treatment resistance. Current treatments do not effectively eradicate cancer stem cells (CSCs), which play a pivotal role in tumour maintenance, progression and drug resistance. Aldehyde dehydrogenases (ALDHs) have been used in some tumour types as CSC markers. Their high expression and high functional activity found in CSCs is also associated with drug resistance. Emerging evidence suggests deregulation of certain ALDH isoforms have implications in cancer. The role of ALDHs in prostate cancer as potential biomarkers and therapeutic targets has not been fully explored yet. Accordingly, this study investigated the expression, regulation and function of selected ALDH isoforms in prostate cancer. This study showed that ALDH1A3, ALDH1B1, ALDH2 and ALDH7A1 are highly expressed in primary prostate cancer cells (n=9) compared to benign (n=9) prostate cells. The expression of ALDH1A3 was high in the stem cells (SCs) (n=3) as well as the more differentiated counterparts (n=16). Treatment of both benign and malignant primary prostate cancer cells with all-trans retinoic acid (atRA) also resulted in increased expression of ALDH1A3 and ALDH3A1, supporting a feedback loop between atRA and ALDHs. Furthermore, SerBob, Bob and LNCaP cells were sensitive to treatment with epigenetic drugs and led to significantly higher expression of ALDH1A2, ALDH3A1 and ALDH7A1 respectively. Importantly, siRNA suppression of ALDH1A3 and ALDH7A1 led to reduced SC properties of primary prostate cultures including reduced cell viability, migration and colony formation, and increased differentiation of transit amplifying (TA) cells to committed basal (CB) cells. Novel ALDH-affinic probes showed reduced cell viability of primary prostate epithelial cultures as a single agent and also when used in combination with docetaxel. The results indicate the potential of using ALDH-affinic compounds as single agents for therapeutic intervention or in combination with docetaxel to sensitise resistant cells to this anticancer drug. The data in this thesis provides novel findings, which supports ALDH1A2, -1A3 and -7A1 as potential biomarkers and/or therapeutic targets for drug intervention. Although, a study analysing a larger number of samples is necessary to fully understand ALDH isoform expression in CSC, TA and CB cells it is envisaged that an ALDH-targeted therapy have potential in future treatment strategies for prostate cancer. / Prostate Cancer UK

A cardiomiopatia Chagásica Crônica (CCC) é a principal complicação decorrente da infecção pelo protozoário hemoflagelado Trypanosoma cruzi (T. cruzi). Trata-se de uma cardiomiopatia dilatada, caracterizada por um intenso infiltrado inflamatório, fibrose, dilatação das câmaras cardíacas, hipertrofia de cardiomiócitos e anormalidades de condução. Sua fisiopatologia é complexa e ainda não se consegue explicar porque apenas 30% dos pacientes infectados desenvolvem essa complicação. Nesse contexto, nosso laboratório descreveu pela primeira vez uma redução na expressão de microRNAs (miRNAs) enriquecidos em músculo (myomiRs) no miocárdio de pacientes com CCC. Sabendo-se que disfunções na biogênese de miRNAs em modelos animais levam ao desenvolvimento de cardiomiopatia do tipo dilatada com redução da expressão de myomiRs, hipotetizou-se que a CCC em humanos estaria associada a um prejuízo na biogênese de miRNAs no miocárdio. Dessa forma, amostras de ventrículo esquerdo de miocárdio de pacientes com CCC (n=16) e controles não-cardiomiopatas (n=6) foram utilizadas para avaliar: 1) a expressão gênica e proteica da maquinaria da biogênese de miRNAs (Drosha, Exportina-5, RAN, Dicer1, TRBP, PACT e Argonauta2), por qPCR e western blotting, respectivamente; 2) a expressão do transcrito primário (pri-miRNA), precursor (pré-miRNA) e miRNA maduro de myomiRs (miR-1, -133a, -133b, -208a, -208b, e -499); 3) o perfil de miRNAs diferencialmente expressos em CCC utilizando qPCR array; e 4) a interação dos miRNAs diferencialmente expressos com disfunções características da CCC (fibrose, miocardite, arritmia e hipertrofia) por meio de análises de bioinformática. Nossos resultados apontam para uma não-alteração nas etapas nucleares da biogênese de miRNAs (transcrição, edição e transporte), já que não foram encontradas alterações na expressão de pri- e pré-miRNAs de myomiRs, bem como dos componentes protéicos da biogênese, Drosha, Exportina-5 e RAN. Entretanto, observou-se uma disfunção na segunda etapa de edição da biogênese, citoplasmática, caracterizada por uma redução de 2/3 nos níveis protéicos de Dicer1, a qual não foi acompanhada por uma redução na expressão de seu RNA mensageiro. Evidenciou-se ainda, uma redução na expressão de 97,5% dos miRNAs maduros diferencialmente expressos no miocárdio de pacientes com CCC, incluindo myomiRs. As análises in silico revelaram haver participação dos miRNAs diferencialmente expressos em disfunções associadas a CCC, com destaque para a fibrose miocárdica, nodo central da rede. Experimentos adicionais preliminares sugeriram o acúmulo de adutos de 4-hidroxi-2-nonenal, decorrente do estresse oxidativo e de uma menor atividade da enzima aldeído desidrogenase 2, como uma possível causa para as alterações encontradas. Este é o primeiro estudo a caracterizar a biogênese de microRNAs em uma cardiomiopatia. Além disso, demonstrou-se que uma redução global do perfil dos miRNAs maduros diferencialmente expressos, decorrente uma disfunção na enzima Dicer1, está associada a eventos patológicos característicos da CCC. Estes mecanismos apresentam relevância biológica e terapêutica, podendo ser possivelmente compartilhados com cardiomiopatias de outras etiologias / Chronic Chagas disease cardiomyopathy (CCC) is the most severe complication of the infection by the haemoflagellate protozoan Trypanosoma cruzi. This dilated cardiomyopathy is characterized by an intense inflammatory infiltrate, fibrosis, dilation of cardiac chambers, cardiomyocyte hypertrophy and conduction abnormalities. Its pathophysiology is complex and why only 30% of patients experience this complication remains an open question. In this regard, our laboratory described for the first time a reduction in the expression of muscle-enriched microRNAs (myomiRs) in human CCC myocardium. Knowing that biogenesis dysfunction and myomiR reduced expression have been associated to the development of dilated cardiomyopathy in animal models, we hypothesized that an impairment of myocardial microRNA biogenesis would be associated to CCC. Hence, left ventricle tissue samples from CCC patients (16) and non-cardiomyopathy donors (6) were used to analyze: 1) mRNA and protein expression, by qPCR and western blotting, of canonical microRNA biogenesis machinery (Drosha, Exportin-5, RAN, Dicer1, TRBP, PACT, AGO2); 2) primary transcript (pri-miR), precursor (pre-miR) and mature microRNA expression of myomiRs (miR-1, -133a, -133b, -208a, -208b, e -499); 3) mature microRNA profile using qPCR array; and 4) the interaction between differentially expressed mature microRNAs and hallmark CCC dysfunctions (fibrosis, myocarditis, hypertrophy and arrhythmia) using bioinformatics tools. Our results point to a non-dysfunction of biogenesis nuclear steps (transcription, editing and transport), since expression of pri-, pre-microRNAs, Drosha, Exportin-5 and Ran are similar between CCC patients and controls. However, we observed an alteration in the cytoplasmic editing step, characterized by a 2/3 reduction in Dicer1 protein levels. In addition, a major downregulation of differentially expressed mature microRNAs (97,5%) was noticed. In silico analysis revealed an association between differentially expressed microRNAs and CCC hallmarks, particularly fibrosis, a central node in the network. Additional preliminary data suggest 4-hydroxi-2-nonenal myocardial accumulation, resulting from oxidative stress and aldehyde dehydrogenase 2 lower activity, as a possible cause for the alterations here described. This is the first study to conduct a comprehensive analysis of microRNA biogenesis machinery in a cardiomyopathy. Moreover, we have shown a major reduction in the expression of mature microRNAs, due to lower Dicer1 protein levels, to be associated to CCC hallmark dysfunctions. These mechanisms are biologically and therapeutically relevant, and may be shared with cardiomyopathies from different etiologies