Cardiomyocyte-specific deletion of the mitochondrial transporter Abcb10 causes cardiac dysfunction via lysosomal-mediated ferroptosis.

Heart function is highly dependent on mitochondria, which not only produce energy but also regulate many cellular functions. Therefore, mitochondria are important therapeutic targets in heart failure. Abcb10 is a member of the ABC transporter superfamily located in the inner mitochondrial membrane and plays an important role in haemoglobin synthesis, biliverdin transport, antioxidant stress, and stabilization of the iron transporter mitoferrin-1. However, the mechanisms underlying the impairment of mitochondrial transporters in the heart remain poorly understood. Here, we generated mice with cardiomyocyte-specific loss of Abcb10. The Abcb10 knockouts exhibited progressive worsening of cardiac fibrosis, increased cardiovascular risk markers and mitochondrial structural abnormalities, suggesting that the pathology of heart failure is related to mitochondrial dysfunction. As the mitochondrial dysfunction was observed early but mildly, other factors were considered. We then observed increased Hif1α expression, decreased NAD synthase expression, and reduced NAD+ levels, leading to lysosomal dysfunction. Analysis of ABCB10 knockdown HeLa cells revealed accumulation of Fe2+ and lipid peroxides in lysosomes, leading to ferroptosis. Lipid peroxidation was suppressed by treatment with iron chelators, suggesting that lysosomal iron accumulation is involved in ferroptosis. We also observed that Abcb10 knockout cardiomyocytes exhibited increased ROS production, iron accumulation, and lysosomal hypertrophy. Our findings suggest that Abcb10 is required for the maintenance of cardiac function and reveal a novel pathophysiology of chronic heart failure related to lysosomal function and ferroptosis.

Approach to Functions of BHLHE41/DEC2 in Non-Small Lung Cancer Development.

The circadian rhythm-related genes BHLHE40/DEC1 and BHLHE41/DEC2 have various functions under different cell and tissue conditions. BHLHE41/DEC2 has been reported to be both a cancer-suppressive and an oncogenic gene during cancer development. The effects of BHLHE41/DEC2 on differentiation have been examined using Bhlhe41/Dec2 knockout mice and/or in vitro differentiation models, and research has been conducted using genetic analysis of tumor cells, in vitro analysis of cancer cell lines, and immunohistochemical studies of the clinical samples. We summarize some of these studies, detail several problems, and consider possible reasons for contradictory results and the needs for further research.

MALAT1 functions as a transcriptional promoter of MALAT1::GLI1 fusion for truncated GLI1 protein expression in cancer.

BACKGROUND: The long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a cancer biomarker. Furthermore, fusion of the MALAT1 gene with glioma-associated oncogene 1 (GLI1) is a diagnostic marker of plexiform fibromyxoma and gastroblastoma; however, the function of this fusion gene remains unexplored. METHOD: In this study, we elucidate the structure and function of the MALAT1::GLI1 fusion gene. To this end, we determined a transcriptional start site (TSS) and promoter region for truncated GLI1 expression using rapid amplification of the 5' cDNA end and a luciferase reporter assay in cultured cells transfected with a plasmid harboring the MALAT1::GLI1 fusion gene. RESULTS: We found that the TATA box, ETS1 motif, and TSS were located in MALAT1 and that MALAT1 exhibited transcriptional activity and induced expression of GLI1 from the MALAT1::GLI1 fusion gene. Truncated GLI1, lacking SUMOylation and SUFU binding sites and located in the nucleus, upregulated mRNA expression of GLI1 target genes in the hedgehog signaling pathway. CONCLUSIONS: We demonstrate a distinct and alternative function of MALAT1 as a transcriptional promoter for expression of the MALAT1::GLI1 fusion gene. Our findings will aid future research on MALAT1 and its fusion gene partners.

Acteoside from Conandron ramondioides Reduces Microcystin-LR Cytotoxicity by Inhibiting Intracellular Uptake Mediated by OATP1B3.

The hepatotoxin microcystin-LR is a strong inhibitor of serine/threonine protein phosphatase (PP) 1 and PP2A. The onset of its cytotoxicity depends on its selective uptake via the hepatocyte uptake transporters, organic anion transporting polypeptide (OATP) 1B1 and OATP1B3. Understanding and preventing the cytotoxicity of microcystin-LR is crucial to maintain human health. This chemoprevention study demonstrates that the herbal plant extract of iwajisha (20 µg/mL) reduced microcystin-LR cytotoxicity in OATP1B3-expressing cells by approximately six times. In addition, 20 µM acteoside, which is one of the major compounds in iwajisha, reduced microcystin-LR cytotoxicity by approximately 7.4 times. Acteoside could also reduce the cytotoxicity of other compounds, such as okadaic acid and nodularin, which are both substrates of OATP1B3 and inhibitors of PP1/PP2A. To investigate the mechanism by which the cytotoxicity of microcystin-LR is attenuated by acteosides, microcystin-LR and microcystin-LR-binding proteins in cells were examined after microcystin-LR and acteosides were co-exposed. Thus, acteoside noncompetitively inhibited microcystin-LR uptake by OATP1B3-expressing cells. Furthermore, acteoside inhibited the intracellular interaction of microcystin-LR with its binding protein(s), including the 22 kDa protein. Furthermore, using immunoblot analysis, acteoside induced the phosphorylation of extracellular signal-regulated kinase (ERK), which is one of the survival signaling molecules. These results suggest that acteoside reduces microcystin-LR cytotoxicity through several mechanisms, including the inhibition of microcystin-LR uptake via OATP1B3, and decreased interaction between microcystin-LR and its binding protein(s), and that ERK signaling activation contributes to the attenuation effect of acteoside against microcystin-LR cytotoxicity.

Metabolic reprograming of cancer as a therapeutic target.

Our understanding of metabolic reprogramming in cancer has tremendously improved along with the technical progression of metabolomic analysis. Metabolic changes in cancer cells proved much more complicated than the classical Warburg effect. Previous studies have approached metabolic changes as therapeutic and/or chemopreventive targets. Recently, several clinical trials have reported anti-cancer agents associated with metabolism. However, whether cancer cells are dependent on metabolic reprogramming or favor suitable conditions remains nebulous. Both scenarios are possibly intertwined. Identification of downstream molecules and the understanding of mechanisms underlying reprogrammed metabolism can improve the effectiveness of cancer therapy. Here, we review several examples of the metabolic reprogramming of cancer cells and the therapies targeting the metabolism-related molecules as well as discuss practical approaches to improve the next generation of cancer therapies focused on the metabolic reprogramming of cancer.

Nucleolar Stress Response via Ribosomal Protein L11 Regulates Topoisomerase Inhibitor Sensitivity of P53-Intact Cancers.

Nucleolar stress response is caused by perturbations in ribosome biogenesis, induced by the inhibition of ribosomal RNA processing and synthesis, as well as ribosome assembly. This response induces p53 stabilization and activation via ribosomal protein L11 (RPL11), suppressing tumor progression. However, anticancer agents that kill cells via this mechanism, and their relationship with the therapeutic efficiency of these agents, remain largely unknown. Here, we sought to investigate whether topoisomerase inhibitors can induce nucleolar stress response as they reportedly block ribosomal RNA transcription. Using rhabdomyosarcoma and rhabdoid tumor cell lines that are sensitive to the nucleolar stress response, we evaluated whether nucleolar stress response is associated with sensitivity to topoisomerase inhibitors ellipticine, doxorubicin, etoposide, topotecan, and anthracyclines. Cell proliferation assay indicated that small interfering RNA-mediated RPL11 depletion resulted in decreased sensitivity to topoisomerase inhibitors. Furthermore, the expression of p53 and its downstream target proteins via western blotting showed the suppression of p53 pathway activation upon RPL11 knockdown. These results suggest that the sensitivity of cancer cells to topoisomerase inhibitors is regulated by RPL11-mediated nucleolar stress responses. Thus, RPL11 expression may contribute to the prediction of the therapeutic efficacy of topoisomerase inhibitors and increase their therapeutic effect of topoisomerase inhibitors.

Association between Dysfunction of the Nucleolar Stress Response and Multidrug Resistance in Pediatric Acute Lymphoblastic Leukemia.

Approximately 20% of pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) relapse or are refractory to chemotherapy despite the low frequency of TP53 mutations. The nucleolar stress response is a P53-activating mechanism via MDM2 inhibition by ribosomal protein L11 (RPL11). We analyzed the role of the nucleolar stress response using BCP-ALL cell lines and patient samples by drug sensitivity tests, Western blotting, and reverse transcription polymerase chain reaction. We revealed that the nucleolar stress response works properly in TP53 wild-type human BCP-ALL cell lines. Next, we found that 6-mercaptopurine, methotrexate, daunorubicin, and cytarabine had anti-leukemic effects via the nucleolar stress response within BCP-ALL treatment. Comparing the samples at onset and relapse in children with BCP-ALL, RPL11 mRNA expression decreased at relapse in seven of nine cases. Furthermore, leukemia cells with relapse acquired resistance to these four drugs and suppressed P53 and RPL11 expression. Our findings suggest that the nucleolar stress response is a novel anti-leukemia mechanism in BCP-ALL. As these four drugs are key therapeutics for BCP-ALL treatment, dysfunction of the nucleolar stress response may be related to clinical relapse or refractoriness. Nucleolar stress response may be a target to predict and improve the chemotherapy effect for pediatric BCP-ALL.

Clinical significance of ALKBH4 expression in non-small cell lung cancer.

Background: Gene methylation is deeply involved in epigenetics and affects both the development and maintenance of homeostasis and carcinogenesis. ALKBH4 is a member of the AlkB homolog (ALKBH) family that controls demethylation of DNA and RNA. Methods: This study enrolled 160 patients with non-small cell lung cancer (NSCLC) who underwent complete resection. The expression of ALKBH4 in cancer tissue was evaluated by immunohistochemistry. The correlation among the expression of ALKBH4, clinicopathological factors, and prognostic outcome was evaluated. Results: In the NSCLC clinical samples, the expression of ALKBH4 was identified not only in cell membranes but also in the cytoplasm of cancer cells. In 140 of 160 cases, ALKBH4 was more highly expressed in the cancerous tissue than in the surrounding normal tissue. The proportion of cancer cells expressing ALKBH4 was higher in adenocarcinoma than in other histological types. In addition, the expression intensity of ALKBH4 in each cancer cell was also stronger in adenocarcinoma than in squamous cell carcinoma. The expression of ALKBH4 was not associated with clinicopathological factors, except for histological type. In adenocarcinoma, the recurrence-free survival (RFS) and overall survival (OS) rates were significantly lower in the ALKBH4-positive group than in the ALKBH4-negative group (P=0.008, 0.031, respectively). A multivariate logistic regression analysis indicated that the ALKBH4 expression was an independent prognostic factor for RFS (P=0.003) and OS (P=0.013). The expression of ALKBH4 was observed in all four patients with adenocarcinoma in situ. Conclusions: The ALKBH4 expression may be a useful predictor of the postoperative outcomes of lung adenocarcinoma (LUAD) patients.

BHLHE41/DEC2 Expression Induces Autophagic Cell Death in Lung Cancer Cells and Is Associated with Favorable Prognosis for Patients with Lung Adenocarcinoma.

Lung cancer constitutes a threat to human health. BHLHE41 plays important roles in circadian rhythm and cell differentiation as a negative regulatory transcription factor. This study investigates the role of BHLHE41 in lung cancer progression. We analyzed BHLHE41 function via in silico and immunohistochemical studies of 177 surgically resected non-small cell lung cancer (NSCLC) samples and 18 early lung squamous cell carcinoma (LUSC) cases. We also examined doxycycline (DOX)-inducible BHLHE41-expressing A549 and H2030 adenocarcinoma cells. BHLHE41 expression was higher in normal lung than in lung adenocarcinoma (LUAD) tissues and was associated with better prognosis for the overall survival (OS) of patients. In total, 15 of 132 LUAD tissues expressed BHLHE41 in normal lung epithelial cells. Staining was mainly observed in adenocarcinoma in situ and the lepidic growth part of invasive cancer tissue. BHLHE41 expression constituted a favorable prognostic factor for OS (p = 0.049) and cause-specific survival (p = 0.042) in patients with LUAD. During early LUSC, 7 of 18 cases expressed BHLHE41, and this expression was inversely correlated with the depth of invasion. DOX suppressed cell proliferation and increased the autophagy protein LC3, while chloroquine enhanced LC3 accumulation and suppressed cell death. In a xenograft model, DOX suppressed tumor growth. Our results indicate that BHLHE41 expression prevents early lung tumor malignant progression by inducing autophagic cell death in NSCLC.

Significance of Mitochondrial DNA Haplogroup on Epidermal Growth Factor Receptor Mutation in Japanese Patients With Lung Adenocarcinoma.

BACKGROUND/AIM: The frequency of somatic mutations of epidermal growth factor receptor (EGFR) in primary lung adenocarcinoma varies among populations and countries. The aim of the present study was to clarify whether the frequency of EGFR mutations in patients with lung adenocarcinoma depends on their mitochondrial DNA haplogroup, which reflects their maternal lineage. PATIENTS AND METHODS: Using normal lung tissue specimens, the mitochondrial DNA haplogroup was determined by multiplex polymerase chain reaction in 135 Japanese patients who underwent surgery for primary lung adenocarcinoma. RESULTS: The 135 patients were divided into two groups according to the two primitive haplotypes (N group, n=32; M group, n=103). The frequency of EGFR mutations in the N group was significantly higher than that in the M group (69% vs. 48%, p=0.044). The difference was prominent when the analysis was restricted to non-smokers (95% vs. 57%, p<0.01). CONCLUSION: The frequency of EGFR mutations in lung adenocarcinoma patients depends on their mitochondrial lineage.

ALKBH4 promotes tumourigenesis with a poor prognosis in non-small-cell lung cancer.

The human AlkB homolog family (ALKBH) of proteins play a critical role in some types of cancer. However, the expression and function of the lysine demethylase ALKBH4 in cancer are poorly understood. Here, we examined the expression and function of ALKBH4 in non-small-cell lung cancer (NSCLC) and found that ALKBH4 was highly expressed in NSCLC, as compared to that in adjacent normal lung tissues. ALKBH4 knockdown significantly induced the downregulation of NSCLC cell proliferation via cell cycle arrest at the G1 phase of in vivo tumour growth. ALKBH4 knockdown downregulated E2F transcription factor 1 (E2F1) and its target gene expression in NSCLC cells. ALKBH4 and E2F1 expression was significantly correlated in NSCLC clinical specimens. Moreover, patients with high ALKBH4 expression showed a poor prognosis, suggesting that ALKBH4 plays a pivotal tumour-promoting role in NSCLC.

Development of a highly sensitive method for the quantitative analysis of modified nucleosides using UHPLC-UniSpray-MS/MS.

There are more than 150 types of naturally occurring modified nucleosides, which are believed to be involved in various biological processes. Recently, an ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) technique has been developed to measure low levels of modified nucleosides. A comprehensive analysis of modified nucleosides will lead to a better understanding of intracellular ribonucleic acid modification, but this analysis requires high-sensitivity measurements. In this perspective, we established a highly sensitive and quantitative method using the newly developed ion source, UniSpray. A mass spectrometer was used with a UniSpray source in positive ion mode. Our UHPLC-UniSpray-MS/MS methodology separated and detected the four major nucleosides, 42 modified nucleosides, and dG15N5 (internal standard) in 15 min. The UniSpray method provided good correlation coefficients (>0.99) for all analyzed nucleosides, and a wide range of linearity for 35 of the 46 nucleosides. Additionally, the accuracy and precision values satisfied the criteria of <15% for higher concentrations and <20% for the lowest concentrations of all nucleosides. We also investigated whether this method could measure nucleosides in biological samples using mouse tissues and non-small cell lung cancer clinical specimens. We were able to detect 43 and 31 different modified nucleosides from mouse and clinical tissues, respectively. We also found significant differences in the levels of N6-methyl-N6-threonylcarbamoyladenosine (m6t6A), 1-methylinosine (m1I), 2'-O-methylcytidine (Cm), 5-carbamoylmethyluridine (ncm5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5S2U), and 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um) between cancerous and noncancerous tissues. In conclusion, we developed a highly sensitive methodology using UHPLC-UniSpray-MS/MS to simultaneously detect and quantify modified nucleosides, which can be used for analysis of biological samples.

Cancer type­SLCO1B3 promotes epithelial­mesenchymal transition resulting in the tumour progression of non­small cell lung cancer.

Non­small cell lung cancer (NSCLC) is one of the most common histologically defined subtypes of lung cancer. To identify a promising molecular target for NSCLC therapy, we performed gene expression analysis at the exon level using postoperative specimens of NSCLC patients. Exon array and real­time PCR analyses revealed that an alternative splicing variant of solute carrier organic anion transporter family member 1B3 (SLCO1B3) called cancer type­SLCO1B3 (Ct­SLCO1B3) was significantly upregulated in the NSCLC samples. SLCO1B3 expressed in the liver [liver type (Lt)­SLCO1B3] was found to be localised in the cell membrane, whereas Ct­SLCO1B3 was detected in the cytoplasm of NSCLC cells. RNAi­mediated knockdown of Ct­SLCO1B3 inhibited in vitro anchorage­independent cell growth, cell migration, and in vivo tumour growth of A549 cells. Overexpression of Ct­SLCO1B3 but not Lt­SLCO1B3 upregulated anchorage­independent cell growth and cell migration of NCI­H23 cells. Mechanistically, Ct­SLCO1B3 was found to regulate the expression of epithelial­mesenchymal transition (EMT)­related genes. The upregulation of E­cadherin was discovered to be especially pivotal to phenotypes of Ct­SLCO1B3­suppressed A549 cells. These findings suggest that Ct­SLCO1B3 functions as a tumour­promoting factor via regulating EMT­related factors in NSCLC.

Involvement of ribosomal protein L11 expression in sensitivity of gastric cancer against 5-FU.

5-Fluorouracil (5-FU) is widely used in the treatment of various types of solid cancer. Our study showed that ribosomal protein L11 (RPL11) was a crucial factor affecting sensitivity of gastric cancer to 5-FU, implying that RPL11 expression is a potential biomarker for predicting 5-FU sensitivity. Kaplan-Meier survival analysis indicated that high RPL11 expression in gastric cancer patients treated with 5-FU was significantly associated with good prognosis. It was therefore investigated whether RPL11 affected the sensitivity of gastric cancer against 5-FU using four human gastric cancer cell lines, MKN45 (wild-type TP53 gene), NUGC4 (wild-type), MKN7 (mutated), and KE39 cells (mutated). In vitro assays demonstrated that RPL11 knockdown in gastric cancer cell lines carrying the TP53 wild-type gene attenuated 5-FU-induced cell growth suppression and activation of the P53 pathway, but not in cells carrying mutated TP53, suggesting that 5-FU suppresses tumor progression via RPL11-mediated activation of the P53 pathway in gastric cancer. The present study provides a potential therapeutic strategy for improving 5-FU resistance in gastric cancer by elevating RPL11 expression.

FARP1 boosts CDC42 activity from integrin αvß5 signaling and correlates with poor prognosis of advanced gastric cancer.

Considering the poor prognosis of most advanced cancers, prevention of invasion and metastasis is essential for disease control. Ras homologous (Rho) guanine exchange factors (GEFs) and their signaling cascade could be potential therapeutic targets in advanced cancers. We conducted in silico analyses of The Cancer Genome Atlas expression data to identify candidate Rho-GEF genes showing aberrant expression in advanced gastric cancer and found FERM, Rho/ArhGEF, and pleckstrin domain protein 1 (FARP1) expression is related to poor prognosis. Analyses in 91 clinical advanced gastric cancers of the relationship of prognosis and pathological factors with immunohistochemical expression of FARP1 indicated that high expression of FARP1 is significantly associated with lymphatic invasion, lymph metastasis, and poor prognosis of the patients (P = 0.025). In gastric cancer cells, FARP1 knockdown decreased cell motility, whereas FARP1 overexpression promoted cell motility and filopodium formation via CDC42 activation. FARP1 interacted with integrin ß5, and a potent integrin αvß5 inhibitor (SB273005) prevented cell motility in only high FARP1-expressing gastric cancer cells. These results suggest that the integrin αvß5-FARP1-CDC42 axis plays a crucial role in gastric cancer cell migration and invasion. Thus, regulatory cascade upstream of Rho can be a specific and promising target of advanced cancer treatment.

Formin-like 1 (FMNL1) Is Associated with Glioblastoma Multiforme Mesenchymal Subtype and Independently Predicts Poor Prognosis.

Glioblastoma multiforme (GBM), the most common primary malignant brain tumor in adults, is characterized by rapid proliferation, aggressive migration, and invasion into normal brain tissue. Formin proteins have been implicated in these processes. However, the role of formin-like 1 (FMNL1) in cancer remains unclear. We studied FMNL1 expression in glioblastoma samples using immunohistochemistry. We sought to analyze the correlation between FMNL1 expression, clinicopathologic variables, and patient survival. Migration and invasion assays were used to verify the effect of FMNL1 on glioblastoma cell lines. Microarray data were downloaded from The Cancer Genome Atlas and analyzed using gene set enrichment analysis (GSEA). FMNL1 was an independent predictor of poor prognosis in a cohort of 217 glioblastoma multiforme cases (p < 0.001). FMNL1 expression was significantly higher in the mesenchymal subtype. FMNL1 upregulation and downregulation were associated with mesenchymal and proneural markers in the GSEA, respectively. These data highlight the important role of FMNL1 in the neural-to-mesenchymal transition. Conversely, FMNL1 downregulation suppressed glioblastoma multiforme cell migration and invasion via DIAPH1 and GOLGA2, respectively. FMNL1 downregulation also suppressed actin fiber assembly, induced morphological changes, and diminished filamentous actin. FMNL1 is a promising therapeutic target and a useful biomarker for GBM progression.

Continuous Cytostatic Effects of BCR-ABL Tyrosine Kinase Inhibitors (TKIs) after Washout in Human Leukemic K562 Cells.

Tyrosine kinase inhibitors (TKIs) are used as the first choice for chronic myeloid leukemia (CML) pharmacotherapeutics. Some patients taking these drugs showed good therapeutic reactivity despite the disappearance of drugs from blood. We investigated whether these drugs have sustained effects even after their disappearance and whether their effects depend on their amounts of intracellular accumulation. Cell proliferation after exposure of K562 cells or Multidrug resistance-1 (MDR-1)-transfected K562 cells was determined by a cell counting kit-8 assay. The intracellular accumulation amount of the drug showing a sustained cytostatic effect was measured by ultra high performance liquid chromatography mass spectrometry. Cell viability decreased in a culture time-dependent manner after washing out nilotinib and dasatinib. The sustained cytostatic effect of dasatinib, but not that of nilotinib, correlated with the intracellular accumulation level. In contrast, imatinib showed continuous a cytostatic effect after drug washout for long-term exposure but not after drug washout for short-term exposure. These results suggest that a good response in patients with a low serum concentration of imatinib, nilotinib or dasatinib may be due to the cytostatic effect of that drug continues even after its disappearance in plasma.

5-Aza-2-deoxycytidine Enhances the Sensitivity of 5-Fluorouracil by Demethylation of the Thymidine Phosphorylase Promoter.

BACKGROUND/AIM: 5-Aza-2-deoxycytidine (5-Aza-CdR) enhances the sensitivity to 5-fluorouracil (5-FU), but the molecular mechanism is not fully understood. The aim of this study was to investigate the molecular mechanism that enhances the sensitivity to 5-FU treated with 5-Aza-CdR via thymidine phosphorylase (TP). MATERIALS AND METHODS: The sensitivity to drugs was determined on several cancer cell lines by the MTT assay. Protein and mRNA levels were examined by immunoblot and RT-PCR, respectively. Gene silencing, binding of Sp1 to DNA and methylation of DNA was performed by siRNA, ChIP assay and sodium bisulfate genomic sequencing, respectively. RESULTS: Sp1-binding sites in the TP promoter were methylated in epidermoid carcinoma. 5-Aza-CdR demethylated Sp1-binding sites and enhanced sensitivity to 5-FU. CONCLUSION: Demethylation of Sp1-binding sites by 5-Aza-CdR was a key factor enhancing 5-FU sensitivity, which may enable more effective treatments for cancer patients with the combination of 5-Aza-CdR and 5-FU.

mRNA Therapy Improves Metabolic and Behavioral Abnormalities in a Murine Model of Citrin Deficiency.

Citrin deficiency is an autosomal recessive disorder caused by loss-of-function mutations in SLC25A13, encoding the liver-specific mitochondrial aspartate/glutamate transporter. It has a broad spectrum of clinical phenotypes, including life-threatening neurological complications. Conventional protein replacement therapy is not an option for these patients because of drug delivery hurdles, and current gene therapy approaches (e.g., AAV) have been hampered by immunogenicity and genotoxicity. Although dietary approaches have shown some benefits in managing citrin deficiency, the only curative treatment option for these patients is liver transplantation, which is high-risk and associated with long-term complications because of chronic immunosuppression. To develop a new class of therapy for citrin deficiency, codon-optimized mRNA encoding human citrin (hCitrin) was encapsulated in lipid nanoparticles (LNPs). We demonstrate the efficacy of hCitrin-mRNA-LNP therapy in cultured human cells and in a murine model of citrin deficiency that resembles the human condition. Of note, intravenous (i.v.) administration of the hCitrin-mRNA resulted in a significant reduction in (1) hepatic citrulline and blood ammonia levels following oral sucrose challenge and (2) sucrose aversion, hallmarks of hCitrin deficiency. In conclusion, mRNA-LNP therapy could have a significant therapeutic effect on the treatment of citrin deficiency and other mitochondrial enzymopathies with limited treatment options.

MicroRNA-130b functions as an oncomiRNA in non-small cell lung cancer by targeting tissue inhibitor of metalloproteinase-2.

Non-small cell lung cancer (NSCLC) is the most frequent cause of cancer-related death worldwide. Although many molecular-targeted drugs for NSCLC have been developed in recent years, the 5-year survival rate of patients with NSCLC remains low. Therefore, an improved understanding of the molecular mechanisms underlying the biology of NSCLC is essential for developing novel therapeutic strategies for the treatment of NSCLC. In this study, we examined the role of miR-130b in NSCLC. Our results showed that high expression of miR-130b in clinical specimens was significantly associated with poor overall survival in patients with NSCLC. Moreover, miR-130b expression was significantly increased in NSCLC clinical specimens from patients with vascular and lymphatic invasion. Consistent with this, overexpression of miR-130b promoted invasion and matrix metalloproteinase-2 (MMP-2) activity in A549 cells. Argonaute2 immunoprecipitation and gene array analysis identified tissue inhibitor of metalloproteinase-2 (TIMP-2) as a target of miR-130b. Invasion activity promoted by miR-130b was attenuated by TIMP-2 overexpression in A549 cells. Furthermore, TIMP-2 concentrations in serum were inversely correlated with relative miR-130b expression in tumor tissues from the same patients with NSCLC. Overall, miR-130b was found to act as an oncomiR, promoting metastasis by downregulating TIMP-2 and invasion activities in NSCLC cells.