RESUMEN
Wilson's disease (WD, MIM#277900) is an autosomal recessive disorder resulting in copper excess caused by biallelic variants in the ATP7B gene (MIM#606882) encoding a copper transporting P-type ATPase. ATP7B variants of unknown significance (VUS) are detected frequently, sometimes impeding a clear diagnosis. Functional analyses can help to classify these variants as benign or pathogenic. Additionally, variants already classified as (likely) pathogenic benefit from functional analyses to understand their pathomechanism, thus contribute to the development of personalized treatment approaches in the future. We described clinical features of six WD patients and functionally characterized five ATP7B missense variants (two VUS, three yet uncharacterized likely pathogenic variants), detected in these patients. We determined the protein level, copper export capacity, and cellular localization in an in vitro model and potential structural consequences using an ATP7B protein model based on AlphaFold. Our analyses give insight into the pathomechanism and allowed reclassification for the two VUS to likely pathogenic and for two of the three likely pathogenic variants to pathogenic.
Asunto(s)
ATPasas Transportadoras de Cobre , Degeneración Hepatolenticular , Humanos , Cobre , ATPasas Transportadoras de Cobre/genética , Degeneración Hepatolenticular/diagnóstico , Degeneración Hepatolenticular/genética , Mutación Missense/genéticaRESUMEN
BACKGROUND: In hepatocellular carcinoma (HCC), histone deacetylases (HDACs) are frequently overexpressed. This results in chromatin compaction and silencing of tumor-relevant genes and microRNAs. Modulation of microRNA expression is a potential treatment option for HCC. Therefore, we aimed to characterize the epigenetically regulated miR-129-5p regarding its functional effects and target genes to understand its relevance for HCC tumorigenesis. METHODS: Global miRNA expression of HCC cell lines (HLE, HLF, Huh7, HepG2, Hep3B) and normal liver cell lines (THLE-2, THLE-3) was analyzed after HDAC inhibition by miRNA sequencing. An in vivo xenograft mouse model and in vitro assays were used to investigate tumor-relevant functional effects following miR-129-5p transfection of HCC cells. To validate hepatoma-derived growth factor (HDGF) as a direct target gene of miR-129-5p, luciferase reporter assays were performed. Survival data and HDGF expression were analyzed in public HCC datasets. After siRNA-mediated knockdown of HDGF, its cancer-related functions were examined. RESULTS: HDAC inhibition induced the expression of miR-129-5p. Transfection of miR-129-5p increased the apoptosis of HCC cells, decreased proliferation, migration and ERK signaling in vitro and inhibited tumor growth in vivo. Direct binding of miR-129-5p to the 3'UTR of HDGF via a noncanonical binding site was validated by luciferase reporter assays. HDGF knockdown reduced cell viability and migration and increased apoptosis in Wnt-inactive HCC cells. These in vitro results were in line with the analysis of public HCC datasets showing that HDGF overexpression correlated with a worse survival prognosis, primarily in Wnt-inactive HCCs. CONCLUSIONS: This study provides detailed insights into the regulatory network of the tumor-suppressive, epigenetically regulated miR-129-5p in HCC. Our results reveal for the first time that the therapeutic application of mir-129-5p may have significant implications for the personalized treatment of patients with Wnt-inactive, advanced HCC by directly regulating HDGF. Therefore, miR-129-5p is a promising candidate for a microRNA replacement therapy to prevent HCC progression and tumor metastasis.
RESUMEN
Chromosomal instability (CIN) can be a driver of tumorigenesis but is also a promising therapeutic target for cancer associated with poor prognosis such as triple negative breast cancer (TNBC). The treatment of TNBC cells with defects in DNA repair genes with poly(ADP-ribose) polymerase inhibitor (PARPi) massively increases CIN, resulting in apoptosis. Here, we identified a previously unknown role of microRNA-449a in CIN. The transfection of TNBC cell lines HCC38, HCC1937 and HCC1395 with microRNA-449a mimics led to induced apoptosis, reduced cell proliferation, and reduced expression of genes in homology directed repair (HDR) in microarray analyses. EME1 was identified as a new target gene by immunoprecipitation and luciferase assays. The reduced expression of EME1 led to an increased frequency of ultrafine bridges, 53BP1 foci, and micronuclei. The induced expression of microRNA-449a elevated CIN beyond tolerable levels and induced apoptosis in TNBC cell lines by two different mechanisms: (I) promoting chromatid mis-segregation by targeting endonuclease EME1 and (II) inhibiting HDR by downregulating key players of the HDR network such as E2F3, BIRC5, BRCA2 and RAD51. The ectopic expression of microRNA-449a enhanced the toxic effect of PARPi in cells with pathogenic germline BRCA1 variants. The newly identified role makes microRNA-449a an interesting therapeutic target for TNBC.
Asunto(s)
Antineoplásicos , MicroARNs , Neoplasias de la Mama Triple Negativas , Antineoplásicos/farmacología , Línea Celular Tumoral , Cromátides/metabolismo , Reparación del ADN/genética , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
BACKGROUND: Patients with hepatocellular carcinoma (HCC) have very limited treatment options. For the last fourteen years, the multi-tyrosine kinase inhibitor sorafenib has been used as standard-of-care therapeutic agent in advanced HCC. Unfortunately, drug resistance develops in many cases. Therefore, we aimed to find a way to mitigate drug resistance and to improve the sorafenib efficacy in HCC cells. MicroRNAs play a significant role in targeting genes involved in tumor control suggesting microRNA/sorafenib combination therapy as a promising treatment option in advanced HCC. METHODS: MiR-449a-5p target genes were identified by Ago-RIP sequencing and validated by luciferase reporter assays and expression analyses. Target gene expression and survival data were analyzed in public HCC datasets. Tumor-relevant functional effects of miR-449a-5p and its target genes as well as their impact on the effects of sorafenib were analyzed using in vitro assays. An indirect transwell co-culture system was used to survey anti-angiogenic effects of miR-449a-5p. RESULTS: PEA15, PPP1CA and TUFT1 were identified as direct target genes of miR-449a-5p. Overexpression of these genes correlated with a poor outcome of HCC patients. Transfection with miR-449a-5p and repression of miR-449a-5p target genes inhibited cell proliferation and angiogenesis, induced apoptosis and reduced AKT and ERK signaling in HLE and Huh7 cells. Importantly, miR-449a-5p potentiated the efficacy of sorafenib in HCC cells via downregulation of PEA15, PPP1CA and TUFT1. CONCLUSIONS: This study provides detailed insights into the targetome and regulatory network of miR-449a-5p. Our results demonstrate for the first time that targeting PEA15, PPP1CA and TUFT1 via miR-449a overexpression could have significant implications in counteracting sorafenib resistance suggesting miR-449a-5p as a promising candidate for a microRNA/sorafenib combination therapy.