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Mitofusin-2 over-expresses and leads to dysregulation of cell cycle and cell invasion in lung adenocarcinoma
Abstract
Mitofusin-2 (MFN2) is a mitochondrial protein associated with mitochondrial fusion process. It was initially identified as a hyperplasia suppressor and implicated in Charcot-Marie-Tooth disease. Recent studies showed that MFN2 played important roles in the development of multiple tumors. Here we examined MFN2 expression in 30 lung adenocarcinoma samples and revealed that the expression of MFN2 was significantly higher in lung adenocarcinoma tissues as compared to adjacent normal tissues. We then investigated the impact of MFN2 knockdown on A549 human lung adenocarcinoma cells and showed that cell proliferation, cell cycle and invasion behavior were all deregulated by MFN2 knockdown. And deregulation of cell cycle pathway after MFN2 knockdown was confirmed by microarray analysis. Furthermore, microarray analysis also revealed that different oncogenes such as RAP1A, RALB and ITGA2 were oppositely regulated by MFN2, which provided molecular clues for the paradoxical functions of MFN2 in tumor development. Taken together, our study unraveled the tumor-promoting functions of MFN2 in lung adenocarcinoma and implicated that the role of MFN2 in cancer development might be more complicated than expected and should be explored in detail in the future.
... Some studies have indicated several pathways regarding MFN2's significance in LC progression [53,94]. ...
... Another study by Lou et al. on patients with lung adenocarcinoma reported an increased MFN2 expression compared to normal tissues. In addition, microarray results found that other oncogenes such as J o u r n a l P r e -p r o o f RAP1A, RALB, and ITGA2, were inversely regulated by MFN2, providing molecular evidence for the paradoxical activities of MFN2 in tumor growth [94] (figure 2 and table 1). ...
... MFN2, as a tumor suppressor, appears to induce cell cycle arrest and reduce cancer progression by inhibiting mTORC2/AKT [37], increasing Bax-induced apoptosis, caspase activation, PARP cleavage, cell cycle arrest, ΔΨm reduction, and cytochrome c release [95]. On the other hand, MFN2, as an oncogene, could also increase LC cells invasion and malignancy [94,97]. This disparity appears to have two causes: 1) Most presented studies, which introduced MFN2 as a tumor suppressor, used adenoviruses to upregulate MFN2, which ultimately led to high MFN2 expression and may induce artificial in vitro phenomena that may not reflect the physiological role of MFN2 in cancer cells, whereas another used lentiviral-mediated siRNA strategy to upregulate MFN2 expression, which could give us a better view of how MFN2 works in A549 lung adenocarcinoma cells [94]. ...
Cancer is a category of disorders characterized by excessive cell proliferation with the ability to infiltrate or disseminate to other organs of the body. Mitochondrial dysfunction, as one of the most prominent hallmarks of cancer cells, has been related to the onset and development of various cancers. Mitofusin 2 (MFN2) is a major mediator of mitochondrial fusion, endoplasmic reticulum (ER)-mitochondria interaction, mitophagy and axonal transport of mitochondria. Available data have shown that MFN2, which its alterations have been associated with mitochondrial dysfunction, could affect cancer initiation and progression. In fact, it showed that MFN2 may have a double-sword effect on cancer fate. Precisely, it demonstrated that MFN2, as a tumor suppressor, induces cancer cell apoptosis and inhibits cell proliferation via Ca2+ and Bax-mediated apoptosis and increases P21 and p27 levels, respectively. It also could suppress cell survival via inhibiting PI3K/Akt, Ras-ERK1/2-cyclin D1 and mTORC2/Akt signaling pathways. On the other hand, MFN2, as an oncogene, could increase cancer invasion via snail-mediated epithelial-mesenchymal transition (EMT) and in vivo tumorigenesis. While remarkable progress has been achieved in recent decades, further exploration is required to elucidate whether MFN2 could be a friend or it's an enemy. This study aimed to highlight the different functions of MFN2 in various cancers.
... Mitochondria is dynamic organelle and the balance of mitochondrial fusion and fission is benefit to maintaining mitochondrial morphology and function, contributing to biological process, including cell proliferation, migration, invasion, apoptosis, and tumor growth [5][6][7][8][9]. Increasing evidence indicated that mitochondrial dysfunction is closely related to tumorigenesis and chemotherapy resistance of multiple tumors, including lung cancer [6,10]. ...
... Mitochondrial fusion and fission also participate in cell proliferation, migration and cell growth in A549 lung adenocarcinoma cells [7][8][9]41]. In our study, we found that HSPB8 upregulated the expression of MFN2 protein and restrained p-Drp1/Drp1 expression, which contributed to improve the mitochondrial function. ...
... In our study, we found that HSPB8 upregulated the expression of MFN2 protein and restrained p-Drp1/Drp1 expression, which contributed to improve the mitochondrial function. Our results are consistent with the former research that the increasing of mitochondrial fusion could protect A549 cells [8,9,42]. However, surprisingly, other studies got the opposite conclusion that increased fission and decreased fusion of mitochondria amplified lung cancer cell proliferation [5]. ...
Heat shock protein B8 (HSPB8) impacts on tumor proliferation and migration of malignancy. However, the role of HSPB8 in lung adenocarcinoma (LUAC) remains unclear. The aim of this study, therefore, was to clarify whether HSPB8 could bring benefits to proliferation and migration of LUAC and its underlying mechanisms. The expression of HSPB8 was first evaluated by immunohistochemistry in 35 LUAC samples. Then, A549 lung adenocarcinoma cells were transfected with pcDNA-HSPB8 or si-HSPB8 to induce HSPB8 overexpression and silence. Cellular activity was evaluated with a Cell Counting Kit-8 (CCK-8) assay. Cell proliferation and migration were observed by EdU assay and scratch assay. Mitochondria-specific reactive oxygen species (mtROS) and membrane potential were measured using MitoSOX Red probe and JC-1 staining. Superoxide dismutase (SOD) activities and malondialdehyde (MDA) level were measured using commercial kits, respectively. HSPB8 protein, mitochondrial fusion protein MFN2 and mitochondrial fission protein p-Drp1/Drp1 were measured using western blot. Compared with the normal tissues, the expression of HSPB8 protein was higher in LUAC tissues and upregulation of HSPB8 protein was related to tumor size and tumor location. Furthermore, HSPB8 overexpression aggravated cell proliferation and migration of A549 cells. Mechanistically, HSPB8 suppressed mitochondrial impairment, leading to promoting the progress of A549 lung adenocarcinoma cells. These data demonstrate that HSPB8 plays an important role in progression of LUAC and may be a new target to treat LUAC.
... abnormal expression of Mfn2 can cause adverse effects. Previous studies have reported that Mfn2 is associated with lung diseases (18,19) and the incidence of cancer (19). a limited number of studies have focused on pelvic floor diseases. ...
... abnormal expression of Mfn2 can cause adverse effects. Previous studies have reported that Mfn2 is associated with lung diseases (18,19) and the incidence of cancer (19). a limited number of studies have focused on pelvic floor diseases. ...
... The present study indicated that the cell activity in the Mfn2+ group was significantly reduced compared with that noted in the two control groups, whereas in the sh-Mfn2 group, the activity of the sh-Mfn2 fibroblasts was significantly higher compared with that of the sh-Mfn2-nc and control groups. These results were consistent with a previous study (19); however, in PoP patients, increased expression levels of Mfn2 may not be beneficial, as decreased proliferation of fibroblasts may be associated with their dysfunction, which could be the cause of PoP. ...
The present study aimed to investigate the effects of Mitofusin2 (Mfn2) on the proliferation of human uterosacral ligament fibroblasts and on the expression of procollagen. We also aimed to identify the possible signal transduction pathway involved in the development of pelvic organ prolapse (POP). For this purpose, uterosacral ligaments were harvested from POP and non‑pelvic organ prolapse (NPOP) patients for fibroblast culture. Cellular proliferation and the cell cycle were assessed following transduction with lentiviral vectors for the overexpression and suppression of Mfn2. The expression levels of the proteins Mfn2, procollagens, phosphoprotein 21 wild‑type p53 activating fragment (p21Waf1), cyclin‑dependent kinase 2 (CDK2), extracellular signal‑regulated kinase1/2 (ERK1/2) and rapidly accelerated fibrosarcoma‑1 (Raf‑1) were examined. Overexpression of Mfn2 resulted in the decreased proliferation of cells and the induction of G0/G1 phase arrest. Concomitantly, the relative expression levels of procollagen proteins, CDK2 and the phosphorylation levels of ERK1/2 and Raf‑1 proteins were notably decreased, while the levels of the p21waf1 protein were increased in the Mfn2 overexpressing group. Opposing results were reported cells following Mfn2 silencing via RNA interference. The results of the present study indicated that the cell cycle of the fibroblasts, their cellular proliferation and the levels of the procollagen proteins could be inhibited via the Ras‑Raf‑ERK axis as a result of the increased levels of Mfn2 during the development of POP.
... The MFN2 gene encodes an outer membrane GTPase that contributes to regulating the morphology [53], fission, and fusion [54] of mitochondria, critical organelles that are primarily responsible for aerobic cellular respiration. The role of MFN2 in TB-susceptibility and lung cancer etiology is still unclear. ...
... However, a previous study found that MFN2 expression levels were nominally higher in peripheral blood mononuclear cells from TB-infected cases compared to controls [55]. Furthermore, MFN2 is a known hyperplasia suppressor gene [56] and a study of clinical tumor samples found that its expression was significantly higher in lung adenocarcinoma tissues compared to adjacent normal tissues [54]. When MFN2 was knocked down in A549 lung adenocarcinoma cell lines, cellular proliferation, cell cycle and invasion behavior were all deregulated [54]. ...
... Furthermore, MFN2 is a known hyperplasia suppressor gene [56] and a study of clinical tumor samples found that its expression was significantly higher in lung adenocarcinoma tissues compared to adjacent normal tissues [54]. When MFN2 was knocked down in A549 lung adenocarcinoma cell lines, cellular proliferation, cell cycle and invasion behavior were all deregulated [54]. Given that mitochondria regulates bioenergetics, metabolism, and apoptosis [57], which are key factors in both anti-microbial immunological/inflammatory response [58] and cancer development [59][60][61], it stands to reason that a regulator of mitochondrial function such as MFN2 could influence both diseases. ...
We investigated whether genetic susceptibility to tuberculosis (TB) influences lung adenocarcinoma development among never-smokers using TB genome-wide association study (GWAS) results within the Female Lung Cancer Consortium in Asia. Pathway analysis with the adaptive rank truncated product method was used to assess the association between a TB-related gene-set and lung adenocarcinoma using GWAS data from 5512 lung adenocarcinoma cases and 6277 controls. The gene-set consisted of 31 genes containing known/suggestive associations with genetic variants from previous TB-GWAS. Subsequently, we followed-up with Mendelian Randomization to evaluate the association between TB and lung adenocarcinoma using three genome-wide significant variants from previous TB-GWAS in East Asians. The TB-related gene-set was associated with lung adenocarcinoma (p = 0.016). Additionally, the Mendelian Randomization showed an association between TB and lung adenocarcinoma (OR = 1.31, 95% CI: 1.03, 1.66, p = 0.027). Our findings support TB as a causal risk factor for lung cancer development among never-smoking Asian women.
... The role of metabolic markers in several cancers is well established [13][14][15]. Furthermore, high ROS levels are associated with tumor development and progression, and as the main sources of ROS are mitochondria, it is hypothesized that these organelles may play an essential role in tumorigenesis and impacts in the biological behavior of several cancers [16]. ...
... The exact role of MFN2 in cancer is not well established, as contradictory data, in some studies, have demonstrated an oncogenic activity, whereas other studies have researched its antioncogenic effect [13,32]. In this study's cohort of H&N melanomas, the MFN2 expression was associated with lower DFS and OS in the multivariate model for patients with CMs. ...
... This corroborates with a previous study, in which B16F10 melanoma cell knockdown for MFN2 demonstrated a higher number of lung metastasis [32]. On the other hand, for sinonasal melanomas, MFN2 was also associated with worse OS rates in the univariate model, which is validated by other studies [13,15]. Additional studies to determine the role of MFN2 in different human cancers types are highly suggested. ...
Mitochondrial dysfunction is caused by an imbalance in the fission and fusion processes, and it has been implicated in the pathogenesis of several human cancers. However, the role of mitochondrial markers in melanomas still remain poorly understood. In this study, the authors assessed the expression of three mitochondrial markers [antimitochondrial (AMT), fission protein 1 (FIS1) and mitofusin 2 (MFN2)] in a series of head and neck mucosal and cutaneous melanomas. Patients with cutaneous (n=56), and mucosal [oral – n=30 and sinonasal – n=26] melanomas of the head and neck region were enrolled in this study. Clinical and follow-up data were retrieved from medical records. The expression of three mitochondrial markers was assessed by the immunohistochemistry, then digitally quantified and correlated with clinicopathological data and outcome information. In the multivariate model, high mitochondrial content was identified as an independent prognostic value for disease-free survival (DFS) in cutaneous melanomas and overall survival (OS) in oral melanomas. FIS1 expression was significantly associated with lower OS rates in patients with oral melanomas, and strictly correlated with vascular invasion in mucosal melanomas. MFN2 was associated with high risk of distant metastasis in patients with cutaneous melanomas. In summary, the authors demonstrated that mitochondrial content, along with FIS1 and MFN2 expressions, are correlated with important clinicopathologic characteristics in patients with cutaneous and mucosal head and neck melanomas.
... In contrast, other studies have provided evidence that MFN2 functions as an oncogene in gastric cancer (25). Moreover, in vitro experiments have demonstrated that MFN2 knockdown exerts inhibitory effects on lung cancer cell proliferation, migration, and invasion (26). Thus, the impact of MFN2 on carcinogenesis and cancer progression is likely to be more complicated than expected and further research is needed. ...
... Moreover, we also found an association between CIN grade and MFN2 expression, which increased from grade I to II and III, implying that MFN2 may also be crucial in the malignant transformation of CIN. Our results are consistent with previous studies that showed that MFN2 expression is significantly higher in lung cancer tissues than in adjacent healthy tissues (26). Furthermore, significantly increased MFN2 expression has been documented in gastric cancer tissues compared to non-tumour tissues (25), and also positively correlated with depth of invasion, clinical stage, and vascular invasion in this cancer type (25). ...
... In the present study, the proliferation, migration, and invasion of cervical cancer cells were reduced in cells in which MFN2 expression had been knocked-down. In a previous investigation in A549 lung cancer cells, knockdown of MFN2 was found to exert similar significant effects on proliferation, migration, and invasion (26). The role of MFN2 in cancer development may largely depend on the cancer type or microenvironmental conditions in question. ...
Background/aim:
It remains unclear whether mitofusin-2 (MFN2) functions as a tumour suppressor or oncogene in cancer progression. In this study we, therefore, aimed to investigate the effect of MFN2 on the pathogenesis of cervical cancer.
Materials and methods:
MFN2 expression was detected in seven healthy cervical, 64 cervical intraepithelial neoplasia (CIN), and 120 cervical squamous cell carcinoma (SCC) tissues by immunohistochemistry. Moreover, biological function of MFN2 in cervical cancer was investigated in vitro.
Results:
MFN2 levels exhibited a tendency to gradually increase from healthy cervical tissue to CIN to SCC. Moreover, MFN2 expression was significantly associated with higher T-stage (p=0.008) and lymph node metastasis (p<0.001). The proliferative, migratory, and invasive abilities of MFN2-knockdown cells were significantly lower (p<0.001, p<0.001, and p<0.001, respectively) than control cells.
Conclusion:
MFN2 may be involved in cervical cancer pathogenesis as an oncogene and might serve as a biomarker of cervical SCC.
... Moreover, the activity of MFN2 is strictly tissueand tumor-specific and has completely different effects in different cancers. In lung adenocarcinoma, some studies have implicated MFN2 in exerting tumorpromoting effects [8,9], whereas tumor-suppressing effects of MFN2 have also been reported [10][11][12]. ...
... The role of MFN2 in lung adenocarcinoma remains highly debated [8,10,11]. To determine the expression of MFN2 in LUAD, we first accessed RNA-seq data in TPM format from UCSC XENA (https://xenabrowser. net/datapages/) based on TCGA and GTEx data. ...
Mitofusin-2 (MFN2) is a transmembrane GTPase that regulates mitochondrial fusion and thereby modulates mitochondrial function. However, the role of MFN2 in lung adenocarcinoma remains controversial. Here, we investigated the effect of MFN2 regulation on mitochondria in lung adenocarcinoma. We found that MFN2 deficiency resulted in decreased UCP4 expression and mitochondrial dysfunction in A549 and H1975 cells. UCP4 overexpression restored ATP and intracellular calcium concentration, but not mtDNA copy number, mitochondrial membrane potential or reactive oxygen species level. Furthermore, mass spectrometry analysis identified 460 overlapping proteins after independent overexpression of MFN2 and UCP4; these proteins were significantly enriched in the cytoskeleton, energy production and calponin homology (CH) domains. Moreover, the calcium signaling pathway was confirmed to be enriched in KEGG pathway analysis. We also found by protein-protein interaction network analysis that PINK1 may be a key regulator of MFN2- and UCP4-mediated calcium homeostasis. Furthermore, PINK1 increased MFN2/UCP4-mediated intracellular Ca2+ concentration in A549 and H1975 cells. Finally, we demonstrated that low expression levels of MFN2 and UCP4 in lung adenocarcinoma are associated with poor clinical prognosis. In conclusion, our data suggest not only a potential role of MFN2 and UCP4 in co-regulating calcium homeostasis in lung adenocarcinoma, but also their potential use as therapeutic targets in lung cancer.
... However, proteomics data suggest a decrease in mitochondrial fusion in luminal A subtype tumors. Similar results have been reported in gastric and lung cancer, in which Mfn2 overexpression has also been found [49,50]. Additionally, in Y. Lou et al.'s study, they also observed that silencing of Mfn2 in lung cancer results in inhibition of cell proliferation without impacting cell apoptosis [50]. ...
... Similar results have been reported in gastric and lung cancer, in which Mfn2 overexpression has also been found [49,50]. Additionally, in Y. Lou et al.'s study, they also observed that silencing of Mfn2 in lung cancer results in inhibition of cell proliferation without impacting cell apoptosis [50]. Likewise, a recent study in patients with BC provided evidence that mitochondrial fission inhibits metastasis in triple-negative BC and that genes associated with mitochondrial fission correlate with better survival. ...
Breast cancer (BC) is the most prevalent cancer and the one with the highest mortality among women worldwide. Although the molecular classification of BC has been a helpful tool for diagnosing and predicting the treatment of BC, developments are still being made to improve the diagnosis and find new therapeutic targets. Mitochondrial dysfunction is a crucial feature of cancer, which can be associated with cancer aggressiveness. Although the importance of mitochondrial dynamics in cancer is well recognized, its involvement in the mitochondrial function and bioenergetics context in BC molecular subtypes has been scantly explored. In this study, we combined mitochondrial function and bioenergetics experiments in MCF7 and MDA-MB-231 cell lines with statistical and bioinformatics analyses of the mitochondrial proteome of luminal A and basal-like tumors. We demonstrate that basal-like tumors exhibit a vicious cycle between mitochondrial fusion and fission; impaired but not completely inactive mitochondrial function; and the Warburg effect, associated with decreased oxidative phosphorylation (OXPHOS) complexes I and III. Together with the results obtained in the cell lines and the mitochondrial proteome analysis, two mitochondrial signatures were proposed: one signature reflecting alterations in mitochondrial functions and a second signature exclusively of OXPHOS, which allow us to distinguish between luminal A and basal-like tumors.
... For example, mitochondria fuse, dependent on the expression of the fusion proteins mitofusin 1 and 2 (MFN1 and 2), to form intracellular networks prior to G1-S phase. Conversely, these networks undergo fission to break these networks, dependent on the action of dynamin related protein 1 (DRP1), to facilitate the G2-M transition (13)(14)(15). Highlighting the importance of changes in dynamics, mitochondrial hyperfusion inhibits the transition from G1 to S phase, mediating cell cycle arrest and attenuating cellular proliferation (16,17). While it is well established that mitochondrial dynamics regulate cell proliferation, the factors that control mitochondrial dynamics in rapidly proliferating cells remain unclear. ...
... Westrate et al. showed that cells in cell cycle arrest have hyperfused mitochondria, while Mitra et al. demonstrated that mitochondrial fragmentation occurs during mitotic progression (16,40). Moreover, several studies show that mitofusins are downregulated in cancer cells and when over-expressed can have tumor suppressor function (15,(41)(42)(43). Our findings are consistent with the anti-proliferative effects of mitofusins in cancer cells. ...
Myoglobin is a monomeric heme protein expressed ubiquitously in skeletal and cardiac muscle and is traditionally considered to function as an oxygen reservoir for mitochondria during hypoxia. It is now well established that low concentrations of myoglobin are aberrantly expressed in a significant proportion of breast cancer tumors. Despite being expressed only at low levels in these tumors, myoglobin is associated with attenuated tumor growth and a better prognosis in breast cancer patients, but the mechanism of this myoglobin-mediated protection against further cancer growth remains unclear. Herein, we report a signaling pathway by which myoglobin regulates mitochondrial dynamics and thereby decreases cell proliferation. We demonstrate in vitro that expression of human myoglobin in MDA-MB-231, MDA-MB-468, and MCF7 breast cancer cells induces mitochondrial hyperfusion by up-regulating mitofusins 1 and 2, the predominant catalysts of mitochondrial fusion. This hyperfusion causes cell cycle arrest and subsequent inhibition of cell proliferation. Mechanistically, increased mitofusin expression was due to myoglobin-dependent free-radical production, leading to the oxidation and degradation of the E3 ubiquitin ligase parkin. We recapitulated this pathway in a murine model in which myoglobin-expressing xenografts exhibited decreased tumor volume with increased mitofusin, markers of cell cycle arrest, and decreased parkin expression. Further, in human triple-negative breast tumor tissues, mitofusin and myoglobin levels were positively correlated. Collectively, these results elucidate a new function for myoglobin as a modulator of mitochondrial dynamics and reveal a novel pathway by which myoglobin decreases breast cancer cell proliferation and tumor growth by up-regulating mitofusin levels.
... On the contrary, Lou et al. studied MFN2 expression in 30 lung adenocarcinoma samples and discovered that the expression of MFN2 was significantly greater in lung adenocarcinoma tissues as compared to adjacent normal tissues (Lou et al., 2015). Moreover, they evaluated the effect of MFN2 knockdown on A549 human lung adenocarcinoma cells and demonstrated that cell growth and invasion comportment were all altered by MFN2 knockdown. ...
... Cheng et al. (2016),Xu et al. (2017), andMa et al. al. (2012) andLou et al. (2015) ...
Mitochondria are dynamic organelles whose actions are fundamental for cell viability. Within the cell, the mitochondrial system is incessantly modified via the balance between fusion and fission processes. Among other proteins, mitofusin 2 is a central protagonist in all these mitochondrial events (fusion, trafficking, contacts with other organelles), the balance of which causes the correct mitochondrial action, shape, and distribution within the cell. Here we examine the structural and functional characteristics of mitofusin 2, underlining its essential role in numerous intracellular pathways, as well as in the pathogenesis of cancer.
... Mfn2 has been shown to have tumor-promoting functions in human cancer and may be an important therapeutic target for the treatment of urinary bladder carcinoma. 11 A number of experiments have shown that mesenchymal stem cells (MSC) possess an inherent capacity to not only improve ischemia-related organ dysfunction 12,13 but also attenuate the inflammatory condition and reduce the adaptive and intrinsic immunity 14,15 by repressing immunogenicity. 16 Recently, adipose tissue has been recognized as a convenient MSC source. ...
... Recent studies have shown that Mfn2 over-expression can result in various disorders, such as lung cancers and hypertension. 11,25 Moreover, several studies have also demonstrated that the down-regulation of miR-214 can be used to determine the diagnosis, progression and recurrence of bladder cancer. 6,7 Wang and his colleagues showed that there is downregulation of miR-214 in bladder lesion tissues, suggesting that miR-214 could exert a tumor-suppressive influence in bladder cancer through directly down-regulating oncogene PDRG1, which may act as a promising indicator for prognostic and therapeutic interventions in bladder cancer. ...
Our study aims to investigate the roles that microRNA-214 (miR-214) plays in the epithelial mesenchymal transition (EMT) process and the development of interstitial cystitis (IC) in postmenopausal women by targeting Mitofusin 2 (Mfn2). IC bladder tissues and adjacent normal bladder tissues were collected from postmenopausal women. Immunohistochemistry (IHC) staining was conducted. The target relationship between miR-214 and Mfn2 was determined by a dual luciferase reporter gene assay. Adipose-derived mesenchymal stem cells (ADMSCs) were extracted from postmenopausal rats and assigned to the blank, mimics, miR-214 inhibitors, mimics negative control (NC), inhibitors NC, Mfn2 siRNA, miR-214 inhibitors and Mfn2 siRNA groups. Exosomes secreted by transfected ADMSCs were instilled into the bladders of postmenopausal rats. The expression of miR-214 and Mfn2 mRNA and EMT markers was assessed by qRT-PCR and western blotting. It was confirmed that Mfn2 was the target gene of miR-214 in IC. Compared with the normal bladder tissues, miR-214 decreased, but Mfn2 increased in IC bladder tissues. Compared with the blank group, the expression of miR-214 and the expression levels of N-cadherin, Fibronectin, Twist1, Snail and Vimentin mRNA and protein increased, whereas the expression levels of Mfn2, E-cadherin and ZO-1 mRNA and protein decreased in the miR-214 mimics and Mfn2 groups. The expression of MiR-214 and the expression levels of N-cadherin, Fibronectin, Twist1, Snail and Vimentin mRNA and protein decreased, whereas the expression levels of Mfn2, E-cadherin and ZO-1 mRNA and protein increased in the miR-214 inhibitors group. Our findings indicate that the inhibition of miR-214 promotes the EMT process and contributes to bladder wall fibrosis by up-regulating Mfn2, thus leading to the occurrence of IC in postmenopausal women.
... These findings indicate an anti-tumor role of MFN2. However, in contrast to these studies, Lou et al. found that MFN2 was overexpressed in lung cancer [19]. It was also shown that knockdown of MFN2 halted cell proliferation and invasion. ...
... The expression of the mitochondrial GTPase MFN2 in human cancers varies according to cancer type. The survey of a public human protein atlas database indicated that MFN2 protein levels are decreased in a number of human cancers, including liver cancer and bladder cancer, whereas it is elevated in lung cancer [17,19,[24][25][26]. ...
Background: As one of the most common malignancies in the world, little is known about the molecular mechanism underlying gastric cancer (GC) and its progression. In this study, we aimed to investigate the clinical impact of the mitochondrial GTPase mitofusin 2 (MFN2) in GC.
Methods: Immunohistochemistry was used to examine the expression levels of MFN2 in gastric tissues obtained from 141 patients with GC. The correlations between MFN2 protein level and clinicopathologic parameters, as well as the significance of MFN2 protein level for overall and disease-free survival were assessed. siRNA technology was used to study the effect of MFN2 knockdown on cell proliferation and invasion.
Results: The overexpression of MFN2 was positively associated with depth of invasion (P = 0.0430), stage (P = 0.0325) and vascular invasion (P = 0.0077). Patients with high expression levels of MFN2 had a significantly lower overall survival rate and disease-free survival rate compared with those with low expression levels (P = 0.003 and 0.001, respectively). Multivariate Cox regression analysis showed that the overexpression of MFN2 was an independent prognostic marker for inferior overall survival and disease-free survival (P = 0.015 and 0.025, respectively). In addition, studies conducted in GC cells indicated that knockdown of MFN2 suppressed cell proliferation and invasion.
Conclusions: Overexpression of MFN2 can be used as a marker to predict the outcome of patients with GC. Furthermore, targeting MFN2 might provide a new therapeutic modality for the treatment of GC.
... 54 Mfn 2 (which is also known as hyperplasia suppressor gene 2) has been shown to exert antitumor effects in a diverse set of cancer cells (lung, breast, gastric, bladder, liver). [55][56][57][58][59] Mfn2 overexpression in bladder carcinoma cells significantly inhibited cell proliferation by inducing apoptosis. 58 Significant downregulation of Mfn2 was also observed in liver cancer cells and overexpression of Mfn2 inhibited proliferation of these cells and induced apoptosis by increasing the level of caspase-3. ...
... Mitofusin 2 and amphiphysin 2 have both been shown to play roles in cell cycle regulation. 38,55 In addition to its role in mitochondrial fusion, Mfn 2 has been shown to arrest cell cycle transition from G1 to S phase in gastric tumor cells, though the exact mechanism of Mfn 2 has yet to be determined. 57 Dynamin 2 was recently suggested to control c-Myc-dependent cell growth, 87 indicating that dynamins may be general regulators of cancer development by controlling cell cycle progression, cell proliferation and growth by controlling upstream signaling pathways. ...
Dynamins and BAR proteins are crucial in a wide variety of cellular processes for their ability to mediate membrane remodeling, such as membrane curvature and membrane fission and fusion. In this review, we highlight dynamins and BAR proteins and the cellular mechanisms that are involved in the initiation and progression of cancer. We specifically discuss the roles of these proteins in endocytosis, endo-lysosomal trafficking, autophagy, and apoptosis as these processes are all tightly linked to membrane remodeling and cancer.
... Similarly, increased pDRP1 S616 and a significant decrease in pDRP1 S637 at 2 mM DHA allow the repair of any damaged mitochondria through the activated fission process. An increase in Mfn2 was also noted to promote cell survival (Fig. 16) [50]. At 1 mM DHA, the significant decrease in Mfn2 and pDRP1 S616 disrupts the mitochondrial network balance, leading to further damage (Fig. 16D and D) [51]. ...
... For example, Opa1 is required for cancer angiogenesis [18], is upregulated in Cisplatin resistant lung adenocarcinomas [19], in Venetoclax-resistant acute myeloid leukemia cells [20], and in triple-negative breast cancer (TNBC), the growth of which is curtailed by genetic and pharmacological Opa1 inhibition [21]. In lung adenocarcinoma patient samples also MFN2 is overexpressed, and its downregulation in a lung adenocarcinoma cell line decreases proliferation and invasion [22]. In addition, mitochondria appear elongated upon acute treatment of lung adenocarcinoma cells with gefitinib [23]. ...
Drug resistance limits the efficacy of chemotherapy and targeted cancer treatments, calling for the identification of druggable targets to overcome it. Here we show that the mitochondria-shaping protein Opa1 participates in resistance against the tyrosine kinase inhibitor gefitinib in a lung adenocarcinoma cell line. Respiratory profiling revealed that oxidative metabolism was increased in this gefitinib-resistant lung cancer cell line. Accordingly, resistant cells depended on mitochondrial ATP generation, and their mitochondria were elongated with narrower cristae. In the resistant cells, levels of Opa1 were increased and its genetic or pharmacological inhibition reverted the mitochondrial morphology changes and sensitized them to gefitinib-induced cytochrome c release and apoptosis. In vivo, the size of gefitinib-resistant lung orthotopic tumors was reduced when gefitinib was combined with the specific Opa1 inhibitor MYLS22. The combo gefitinib-MYLS22 treatment increased tumor apoptosis and reduced its proliferation. Thus, the mitochondrial protein Opa1 participates in gefitinib resistance and can be targeted to overcome it.
... Cigarette smoke affects the structure and function of mitochondria thus leads to increased expression of the mitochondrial fusion protein, Mfn-2, in alveolar epithelial cells [64]. Work done by Lou et al., shows that the expression of Mfn-2 is high in lung adenocarcinoma when compared to adjacent normal lung tissue [65]. Damaged mitochondria undergo uncoupling and release of increased ROS. ...
Chronic Obstructive Pulmonary Disease (COPD) and Lung cancer are the major reasons for lung disease-related mortality worldwide. Chronic inflammation is a key attribute of COPD and a potential driver of lung carcinogenesis. Among various environmental risk factors, cigarette smoke plays a crucial role in the development and progression of COPD and lung cancer. Several epidemiological studies show that COPD patients are at a greater risk of developing lung cancer independently of cigarette smoking which suggests the role of genetic predisposition in the disease development. Uncovering the mechanistic link between these two diseases is hampered due to their heterogeneous nature: each is characterized by several sub-phenotypes of diseases. This review focuses on the nature of the link between the two diseases and specific mechanisms that occur in both COPD and lung cancer, some of the therapeutic targets which are currently employed, and the role of gene-editing technology to combat these debilitating lung-inflammatory disorders.
... Enhancing the mammalian target of rapamycin complex 2 (mTORC2)/Akt signaling pathway increases tumor cell proliferation and metastasis in MFN2-mutant cancer cells via an AktS437 phosphorylation-induced signal transduction pathway (22). In a separate investigation, MFN2 expression was considerably higher in 30 lung cancer samples compared to paired adjacent normal tissues (23). These contradictory findings suggest that the role of MFN2 in tumor growth is more complex than previously thought. ...
Background:
Mitofusin 2 (MFN2) is involved in several biological processes, including cancer. MFN2 is downregulated in some types of cancer and inhibits cancer cell proliferation, migration, and invasion. However, the relationship between MFN2 and colon cancer remains unknown.
Methods:
In this study, MFN2 expression was investigated using The Cancer Genome Atlas (TCGA) and the Human Protein Atlas (HPA), and the associations between prognostic factors and survival outcomes were assessed via univariate and multivariate analyses. Functional enrichment analyses based on Kyoto Encyclopedia of Genes and Genomes (KEGG) resource and Gene Set Enrichment Analysis (GSEA) were carried out.
Results:
MFN2 was downregulated in colon cancer tissues compared with paracancerous colon tissues (P<0.001), and low MFN2 expression was associated with an advanced tumor stage (stage IV vs. stage I, P=0.03; stage I-III vs. stage IV, P=0.003). MFN2 immunohistochemistry (IHC) staining was medium to high in colon normal tissues, but MFN2 IHC staining was faint or not identified in colorectal cancer (CRC) tumor tissues. MFN2 expression was either low or non-existent in colon cancer distinct cell clusters, according to differential gene analysis. Univariate analysis revealed that MFN2 expression in colon cancer patients was significantly associated with the stage [odds ratio (OR) =0.29 for stage IV vs. stage I, P=0.001], T-stage (OR =0.20 for T4 vs. T1, P=0.033), and distant metastasis (OR =0.31 for M1 vs. M0, P=0.000). Furthermore, Kaplan-Meier survival analysis revealed that patients with colon cancer and high MFN2 expression have a better prognosis than those with low MFN2 expression (P=0.002). MFN2 (hazard ratio =0.95, 95% confidence interval: 0.92-0.99, P=0.007) was an independent predictor of colon cancer according to univariate and multivariate Cox models. Finally, GSEA results showed that the KEGG GALACTOSE METABOLISM, APOPTOSIS, and VEGF SIGNALING pathways were activated in the high MFN2 mRNA expression group, whereas the KEGG RIBOSOME pathway was inhibited in the low MFN2 expression group.
Conclusions:
Our research revealed MFN2 to be a promising predictive biomarker and therapeutic target for colon cancer.
... Several studies have suggested that miRNA-214 is involved in the pathogenesis of BC; however, this study revealed the role of miRNA-214 in IC by targeting Mitofusin 2 (Mfn2) (Lv et al., 2017). Mfn2 is known to participate in regulating cell proliferation and apoptosis and was found to have tumour promoting effects in human cancer, which makes it one of the therapeutic targets for the treatment of BC (Lou et al., 2015). However, the study conducted by Lv et al. (2017) has investigated the interaction between miR-214 and Mfn2 in the IC of postmenopausal women. ...
Bladder diseases affect millions of patients worldwide and compromise their quality of life with a substantial economic impact. The not well understood aetiologies of bladder diseases limit the current diagnosis and therapeutic options to primarily symptomatic treatment. In addition, bladder targeted drug delivery is challenging due to its unique anatomical features and its natural physiological function of urine storage and frequent voiding. Therefore, current treatment options often fail to provide a highly effective, precisely targeted and long-lasting treatment. Thus, comprehensive studies are needed to provide a better understanding of the molecular mechanisms underpinning bladder diseases to identify novel gene therapeutic targets and biomarkers for treating bladder diseases and develop novel treatments such as gene loaded nanoparticles. This review examined the recent development on the discovery of molecular mechanisms of bladder diseases and discussed recently proposed new treatments, including novel bladder targeted gene therapies using nanoparticle-based formulations and probiotics adjuvant therapies.
... The detection process according to our previous reported [15,16]. Briefly, the transduced LUAD cell lines were analyzed by Annexin V-based flow cytometric analysis (Annexin V-APC Apoptosis Detection Kit, eBioscience) using a Guava easyCyte HT (Merck Millipore, Billerica, MA). ...
CENPU, encoding an important factor involved in kinetochore assembly during mitosis, is associated with shorter survival rates in lung adenocarcinoma (LUAD) patients. CENPU promotes growth rates and invasive behavior of LUAD cells; however, its mechanism of action in LUAD progression remains to be elucidated. CENPU mRNA and protein expression were elevated in LUAD tumors, and high CENPU gene expression was associated with inferior survival prognosis in LUAD patients. CENPU knockdown inhibited LUAD cell proliferation, clone formation, migration, invasion, and epithelial-mesenchymal transition (EMT) in addition to inducing cell cycle arrest and apoptosis in vitro and reduced LUAD xenograft tumor growth in vivo. Furthermore, we identified CENPU-regulated genes significantly enriched for proliferation and apoptosis pathways, and identified HSP Family Member C10 (DNAJC10) as putative effector of CENPU. CENPU knockdown produced DNAJC10 protein downregulation, and DNAJC10 overexpression partially rescued the phenotypic effects of CENPU knockdown in LUAD cells. Moreover, CENPU's coiled-coil domain was essential for CENPU's phenotypic effects in LUAD cells. In conclusion, the kinetochore component CENPU plays a critical role in LUAD cell proliferation and invasiveness. Targeting CENPU-DNAJC10 axis may inhibit LUAD tumor cell proliferation and metastasis.
... The results show that the expression level of Mfn2 in lung adenocarcinoma was lower than that in matched paracancerous tissues. Until now, few studies have focused on the role of Mfn2 in lung adenocarcinoma, some of which presented results inconsistent with our study showing that Mfn2 was downregulated in human lung adenocarcinoma tissues and Mfn2 exhibited antitumor activity in vivo and in vitro [26] . In addition, in our study, we did not identify a correlation between Mfn2 expression and clinical pathological parameters such as tumor size and TNM stage. ...
Aim: Chemoresistance is the biggest obstacle in cancer treatment. Our previous study demonstrated that Shenmai injection (SMI), a Chinese herbal medicine, enhanced the antitumor effect of cisplatin via glucose metabolism reprogramming. This study aimed to further determine whether the SMI sensitizes the non-small cell lung cancer (NSCLC) cells to cisplatin through regulation mitochondrial dynamics.
Methods: The Kaplan-Meier Plotter database was used to investigate the relationship between mRNA expression of mitofusin-2 (Mfn2) and the survival analysis of NSCLC patients. The protein expression of Mfn2 in a lung adenocarcinoma tissue chip was detected by immunohistochemistry staining. The expression of Mfn2 and ATAD3A were compared between cisplatin-sensitive A549 and cisplatin-resistant A549/DDP cells. Additionally, A549/DDP cells were co-treated with cisplatin and SMI to detect mitochondrial morphology by fluorescent staining, apoptosis-related protein expression with Western blotting, and mitochondrial membrane potential (ΔΨm) with flow cytometry analysis.
Results: The mean survival time of the Mfn2low group was significantly lower than that of the Mfn2high group by Kaplan-Meier Plotter database analysis, and the Mfn2 protein expression level was lower in cancer tissues than in adjacent tissues. The combination of SMI and cisplatin induced dynamic changes in A549/DDP cells, with increased mitochondrial fusion followed by upregulation of Mfn2 and downregulation of ATAD3A and reduced mitochondrial mass and ΔΨm. Moreover, SMI significantly enhanced cisplatin-induced A549/DDP apoptosis, upregulated Bax and the active subunit of caspase-3, and downregulated Bcl-2 expression, as shown via Hoechst staining and flow cytometry analysis.
Conclusion: Our findings suggest SMI enhances cisplatin-induced apoptosis through regulation of Mfn2-dependent mitochondrial dynamics in cisplatin-resistant lung adenocarcinoma cells.
... In fact, Mfn2 is downregulated in many human tumors including BC [17][18][19][20][21][22]. However, other studies reported Mfn2 overexpression in lung and gastric cancer cells compared to non-neoplastic tissues [23,24]. ...
Identification of markers predicting disease outcome is a major clinical issue for non-muscle invasive bladder cancer (NMIBC). The present study aimed to determine the role of the mitochondrial proteins Mitofusin-2 (Mfn2) and caseinolytic protease P (ClpP) in predicting the outcome of NMIBC. The study population consisted of patients scheduled for transurethral resection of bladder tumor upon the clinical diagnosis of bladder cancer (BC). Samples of the main bladder tumor and healthy-looking bladder wall from patients classified as NMIBC were tested for Mfn2 and ClpP. The expression levels of these proteins were correlated to disease recurrence, progression. Mfn2 and ClpP expression levels were significantly higher in lesional than in non-lesional tissue. Low-risk NMIBC had significantly higher Mfn2 expression levels and significantly lower ClpP expression levels than high-risk NMIBC; there were no differences in non-lesional levels of the two proteins. Lesional Mfn2 expression levels were significantly lower in patients who progressed whereas ClpP levels had no impact on any survival outcome. Multivariable analysis adjusting for the EORTC scores showed that Mfn2 downregulation was significantly associated with disease progression. In conclusion, Mfn2 and ClpP proteins were found to be overexpressed in BC as compared to non-lesional bladder tissue and Mfn2 expression predicted disease progression.
... Under-expression of Fis1 has been associated with defects in the progression of cells from the G 2 to M phase, and underexpression of regulators of the G 2 /M phase transition (Lee et al. 2014). Underexpression of MFN2 in lung adenocarcinoma has been associated with deregulation of the cell cycle and cell proliferation (Lou et al. 2015), whereas overexpression of MFN2 in the BC cell line MCF7 reduces proliferation (Li et al. 2018). In addition, cell cycle phase transition is associated with sequential activation of metabolic enzymes to meet the cells energetic requirements in every phase (Icard et al. 2019). ...
Mitochondria modify their function and morphology to satisfice the bioenergetic demand of the cells. Cancer cells take advantage of these features to sustain their metabolic, proliferative, metastatic, and survival necessities. Therefore, the understanding of mitochondrial morphologic changes of the different grades of Triple-Negative Breast Cancer (TNBC) could be relevant for the design of novel treatments. Consequently, this research aimed to explore the mitochondria morphology and gene expression of some proteins related to mitochondrial dynamics as well as proteins related to oxidative and non-oxidative metabolism of metastatic and non-metastatic TNBC. We found that mitochondrial-morphology and metabolism are different between metastatic and non-metastatic TNBC. Metastatic TNBC showed overexpression of genes related to mitochondrial dynamics, fatty acids, and glycolytic metabolism. These features were accompanied by a fused mitochondrial morphology. In contrast, the non-metastatic TNBC presented a stress-associated mitochondrial morphology, hyperfragmented mitochondria accompanied by upregulated expression of mitochondrial biogenesis-related genes, both characteristics related to the higher ROS production observed in this cell line. These differences found between metastatic and non-metastatic TNBC will allow a better understanding of the metastasis process and the improvement of the development of a specific and personalized TNBC therapy.
... High expression of Mfn1, Mfn2 and OPA1 has also been linked to cancer cell proliferation, survival and invasion, while their inhibition blocks cell growth and triggers apoptosis of different cancer cells. Conversely, mitochondrial fission occurs during apoptosis and seems important for progression of the apoptotic pathway [17,[64][65][66][67]. ...
Melanoma is the most severe type of skin cancer. Its unique and heterogeneous metabolism, relying on both glycolysis and oxidative phosphorylation, allows it to adapt to disparate conditions. Mitochondrial function is strictly interconnected with mitochondrial dynamics and both are fundamental in tumour progression and metastasis. The malignant phenotype of melanoma is also regulated by the expression levels of the enzyme acid sphingomyelinase (A-SMase). By modulating at transcriptional level A-SMase in the melanoma cell line B16-F1 cells, we assessed the effect of enzyme downregulation on mitochondrial dynamics and function. Our results demonstrate that A-SMase influences mitochondrial morphology by affecting the expression of mitofusin 1 and OPA1. The enhanced expression of the two mitochondrial fusion proteins, observed when A-SMase is expressed at low levels, correlates with the increase of mitochondrial function via the stimulation of the genes PGC-1alpha and TFAM, two genes that preside over mitochondrial biogenesis. Thus, the reduction of A-SMase expression, observed in malignant melanomas, may determine their metastatic behaviour through the stimulation of mitochondrial fusion, activity and biogenesis, conferring a metabolic advantage to melanoma cells.
... In line with the lower Mfn2 expression in cancer, several studies demonstrate that Mfn2 overexpression inhibits cancer cell proliferation and colony formation and weakens the invasion and migratory ability (106,(114)(115)(116). It is worth noting that the higher expression of Mfn2 has also been reported in lung adenocarcinoma tissues as compared to adjacent normal tissues, and Mfn2 knockdown results in impaired cancer cell proliferation (81). In ovarian cancer (82), breast cancer (83), and melanoma (84), Mfn2 knockdown suppresses oxygen consumption rate (OCR). ...
Metabolism describes the life-sustaining chemical reactions in organisms that provide both energy and building blocks for cellular survival and proliferation. Dysregulated metabolism leads to many life-threatening diseases including obesity, diabetes, and cancer. Mitochondria, subcellular organelles, contain the central energy-producing metabolic pathway, the tricarboxylic acid (TCA) cycle. Also, mitochondria exist in a dynamic network orchestrated by extracellular nutrient levels and intracellular energy needs. Upon stimulation, mitochondria undergo consistent interchange through fusion (small to big) and fission (big to small) processes. Mitochondrial fusion is primarily controlled by three GTPases, mitofusin 1 (Mfn1), Mfn2, and optic atrophy 1 (Opa1), while mitochondrial fission is primarily regulated by GTPase dynamin-related protein 1 (Drp1). Dysregulated activity of these GTPases results in disrupted mitochondrial dynamics and cellular metabolism. This review will update the metabolic roles of these GTPases in obesity, diabetes, and cancer.
... Numerous studies have confirmed that the cell cycle is a complex and strictly controlled process [27] that is frequently dysregulated in tumorigenesis, including ADC [28,29]. Furthermore, previous studies have reported that several proteins, such as FGF [30], ERBB3 [31] and MFN2 [32], may influence lung cancer progression through cell cycle pathways, Consistently, studies by Bhattacharya C et al. have demonstrated a key role for DDX11 in the proliferation and cell cycle progression of advanced melanoma. In addition, our present study found that DDX11 expression was positively associated with CCNA2, CCNB1, CCNC, CCND1, CCNE1, CDK2, CDK4 and CDK6, which are involved in the cell cycle and DNA replication [33][34][35][36]. ...
Background: Lung adenocarcinoma (ADC) is the main cause of cancer-related mortality in lung cancer patients. DEAD/DEAH box helicase 11 (DDX11) was previously shown to be dysregulated and to exert oncogenic activity in cancer. However, the diagnostic value and clinical significance of DDX11 in ADC remain unknown.
Methods: A total of 513 ADC and 59 normal tissue samples were obtained from The Cancer Genome Atlas (TCGA) database, and the mRNA expression level of DDX11 in ADC was evaluated. Additionally, a meta-analysis of 7 ADC cohorts from the Gene Expression Omnibus (GEO) database was conducted to validate the DDX11 expression pattern. Moreover, receiver operating characteristic (ROC) curve analysis was used to identify the diagnostic power of DDX11 in ADC. A tissue microarray (TMA) comprising 86 ADC specimens and their adjacent normal specimens was applied to indicate DDX11 protein expression status. In addition, Kaplan-Meier and Cox regression analyses were conducted to validate the prognostic value of DDX11 in ADC. Finally, the molecular mechanism of DDX11 action in ADC was predicted by gene set enrichment analysis (GSEA).
Results: DDX11 was upregulated in ADC tissues and was associated with worse overall survival (OS). ROC curves of DDX11 showed high values for diagnosis. Additionally, DDX11 expression has remarkable correlations with DNA replication and the cell cycle G1-S phase pathway. Consistently, it was associated with cell cycle genes, such as CCNA2, CCNB1, CCNC, CCND1, CCNE1, CDK2, CDK4 and CDK6. Moreover, high CCNA2, CCNB1, CCNE1 and CDK6 expression in ADC patients predicted worse OS and progression-free survival (PFS).
Conclusion: DDX11 was significantly upregulated and predicted poor prognosis in ADC. This gene might serve as a potential novel prognostic and diagnostic biomarker for ADC.
... A previous study revealed the antitumor effect of MFN2 in cancer cells (19). Further studies confirmed this anti-proliferative effect in a number of tumor cell lines, including hepatocellular carcinoma cells (20), breast cancer cells (10) and lung adeno- (20), breast cancer cells (10) and lung adeno- (20), breast cancer cells (10) and lung adeno-(10) and lung adeno-(10) and lung adenocarcinoma cells (21). However, to the best of our knowledge, the expression of MFN2 in bladder cancer tissues, and its potential clinical and biological significance, have not been described. ...
The present study aimed to investigate the biological role of the mitochondrial GTPase mitofusin-2 (MFN2) in bladder cancer. MFN2 mRNA expression in tumor and paired adjacent non-tumor tissues from 8 patients was investigated using reverse transcription-quantitative polymerase chain reaction analysis. Immunohistochemistry was used to investigate MFN2 expression in 117 bladder cancer specimens. The associations of MFN2 expression with clinicopathological parameters were evaluated statistically. In addition, the biological role of MFN2 in the proliferation, migration and invasion of bladder cancer cells was examined. It was identified that MFN2 expression was significantly downregulated in bladder cancer tissues compared with normal tissues. MFN2 expression was associated with tumor stage, tumor grade and lymph node status. Furthermore, patients with low MFN2 expression demonstrated a shorter overall survival time (P=0.025). MFN2 knockdown by small interfering RNA promoted cancer cell proliferation, migration and invasion in vitro, and enhanced tumor progression in vivo. Mechanistically, MFN2 was revealed to be involved in Wnt/β-catenin signaling. In conclusion, MFN2 may serve as a potential therapeutic target in the treatment of bladder cancer, and the progress of bladder cancer may be delayed by regulating MFN2 expression.
... *P < 0.05 compared with the control-untreated cells induce neuronal cell death in the presynaptic region of the aging cortex [71], in neonatal cardiomyocytes [72], and in dopaminergic neurons within the nigrostriatal circuit [73]. Studies of lung adenocarcinoma report that MFN2 overexpression induces G0/G1 phase arrest and leads to delayed cell proliferation [74][75][76]. Other research shows that OPA1 maintains inner-mitochondrial membrane morphology and structure with high mRNA expression levels in retina, brain, liver, heart and pancreas [77]. ...
Dexamethasone is an approved steroid for clinical use to activate or suppress cytokines, chemokines, inflammatory enzymes and adhesion molecules. It enters the brain, by-passing the blood brain barrier, and acts through genomic mechanisms. High levels of dexamethasone are able to induce neuronal cell loss, reduce neurogenesis and cause neuronal dysfunction. The exact mechanisms of steroid, especially the dexamethasone contribute to neuronal damage remain unclear. Therefore, the present study explored the mitochondrial dynamics underlying dexamethasone-induced toxicity of human neuroblastoma SH-SY5Y cells. Neuronal cells treatment with the dexamethasone resulted in a marked decrease in cell proliferation. Dexamethasone-induced neurotoxicity also caused upregulation of mitochondrial fusion and cleaved caspase-3 proteins expression. Mitochondria fusion was found in large proportions of dexamethasone-treated cells. These results suggest that dexamethasone-induced hyperfused mitochondrial structures are associated with a caspase-dependent death process in dexamethasone-induced neurotoxicity. These findings point to the high dosage of dexamethasone as being neurotoxic through impairment of mitochondrial dynamics.
... The results are consistent with previous studies in VSMCs (3) and in various types of cancer, including breast cancer (18,37) gastric cancer (18), urinary bladder carcinoma (38) and hepatocellular carcinoma (20,21,23) and lung adenocarcinoma (5,8,24,(38)(39)(40)(41). However, tumor-promoting functions of Mfn2 in lung adenocarcinoma have also been reported (42,43). These conflicting results may be due to differing experimental methods, but suggest that the roles of Mfn2 in different types of cancer are more complicated than expected. ...
Mitofusin 2 (Mfn2) is expressed in numerous human tissues and serves a pivotal role in cell proliferation. However, Mfn2 is considered as an anti-tumor gene, and is silenced in human malignant tumors, including those of breast cancer. However, the mechanisms contributing to Mfn2 silencing and the mechanism of its anti-tumor function in breast cancer remain unclear. In the present study, hypoexpression of Mfn2, and hypermethylation of its promoter, was confirmed in human breast cancer cells and in breast cancer tissues by reverse transcription-quantitative polymerase chain reaction (PCR) and methylation specific PCR, respectively. Chemical demethylation treatment with 5-aza-2'-deoxycytidine upregulated the mRNA expression level of Mfn2 in MCF-7 cells in a dose-dependent manner. In addition, overexpression of Mfn2 repressed the proliferation, migration and invasion of MCF-7 cells, mediated by inhibition of the Ras-extracellular signal-regulated kinase (ERK)1/2 signaling pathway. However, overexpression of Mfn2 with deletion of the p21Ras motif (Mfn2ΔRas) and proteiation site (Mfn2ΔPKA) partially reduced the anti-tumor function of Mfn2, and inhibited the Ras-ERK1/2 signaling pathway. Taken together, the present study confirmed the anti-tumor effects of Mfn2 in human breast cancer and clarified that the mechanism of its anti-tumor functions includes promoter DNA methylation, the P21Ras binding site and PKA phosphorylation.
... Besides providing energy for cells, mitochondria are involved in the processes of cell differentiation, proliferation, apoptosis, cycle, and information transmission [12][13][14]. The realization of these functions requires the constant fusion and division of membrane structures between different mitochondria, which are precisely regulated by fusion and division, and imbalance leads to disease [15][16][17]. Mitochondrial fusion requires fusion proteins (Mfn) as vectors, and outer membrane fusion requires Mfn1 and Mfn2, and intimal fusion requires OPA1 [18,19]. Abnormal expression of Mfn is involved in many diseases, including a variety of cancers, like prostate cancer [13,16]. ...
Background:
Recent studies have found that plant derived microRNA can cross-kingdom regulate the expression of genes in humans and other mammals, thereby resisting diseases. Can exogenous miRNAs cross the blood-prostate barrier and entry prostate then participate in prostate disease treatment?
Methods:
Using HiSeq sequencing and RT-qPCR technology, we detected plant miRNAs that enriched in the prostates of rats among the normal group, BPH model group and rape bee pollen group. To forecast the functions of these miRNAs, the psRobot software and TargetFinder software were used to predict their candidate target genes in rat genome. The qRT-PCR technology was used to validate the expression of candidate target genes.
Results:
Plant miR5338 was enriched in the posterior lobes of prostate gland of rats fed with rape bee pollen, which was accompanied by the improvement of BPH. Among the predicted target genes of miR5338, Mfn1 was significantly lower in posterior lobes of prostates of rats in the rape bee pollen group than control groups. Further experiments suggested that Mfn1 was highly related to BPH.
Conclusions:
These results suggesting that plant-derived miR5338 may involve in treatment of rat BPH through inhibiting Mfn1 in prostate. These results will provide more evidence for plant miRNAs cross-kingdom regulation of animal gene, and will provide preliminary theoretical and experimental basis for development of rape bee pollen into innovative health care product or medicine for the treatment of BPH.
... [4,23] This renders mitochondria function as a dynamic network that constantly undergoes fusion and fission. [4,23] However, Mfn2 overexpression affects mitochondrial fusion, [4,24] causing mitochondrial dysfunction that acts as one of the main pro-aging factors. [25] Mitochondria have an independent genetic system, and the accumulation of mitochondrial DNA mutations promotes tissue aging. ...
Background:
Both Mitofusin 2 (Mfn2) and pelvic organ prolapse (POP) are related to aging. The aim of the present study was to investigate the variations of Mfn2 expression in the uterosacral ligaments of patients with and/or without POP and their correlations with the expression of procollagen.
Methods:
Fibroblasts were cultured using tissue specimens that were harvested from the uterosacral ligaments of POP and non-POP (NPOP) patients (n = 10 for each group) from September 2016 to December 2016. The Cell Counting Kit-8 (CCK-8) assay was used to compare the differences in cell proliferation between the two groups. Relative quantitative reverse transcription-polymerase chain reaction and Western blotting assays were employed to assess the differences in the mRNA and protein expression levels of Mfn2 and procollagen 1A1/1A2/3A1 between the two groups. The changes in procollagen expression were assessed following the downregulation of Mfn2 in the POP group using RNAi. The data were assessed with independent sample t- test or general linear model univariate analysis using the SPSS 13.0 software.
Results:
The results from CCK-8 assay indicated that cell viability in the POP group was significantly lower compared with that of the NPOP group (td5, 7, 9, 11= -5.925, -6.851, -9.129, and -9.661, respectively, all P < 0.001, from D5 to D11). The mRNA and protein expression levels of Mfn2 in the cultured fibroblasts of the POP group were significantly higher compared with those of the NPOP group (mRNA: t = 2.425, P = 0.032; protein: t = 2.392, P = 0.037, respectively), whereas only the expression levels of procollagen 1A1/1A2/3A1 were significantly higher in the NPOP group (mRNA: t = -2.165, P1A1 = 0.041; t = -2.741, P1A2 = 0.026; t = -2.147, P3A1 = 0.045, respectively; protein: t = -2.418, P1A1 = 0.029; t = -2.405, P1A2 = 0.033; t = -2.470, P3A1 = 0.012, respectively). The expression levels of procollagen in the POP group increased following the downregulation of Mfn2.
Conclusions:
The proliferation rate and cell viability of the fibroblasts in the POP group were significantly lower compared with those in the NPOP group. In the POP fibroblasts, Mfn2 expression was increased, while procollagen expression was decreased.
... When this imbalance was corrected by increasing Mfn-2 expression and decreasing DRP-1 in cell lines, there was a reduction of cell proliferation and an increase in apoptosis [88]. However, LOU et al. [89] demonstrated that, in human lung adenocarcinoma, the expression of Mfn-2 was higher than in adjacent background lung tissue. ...
Chronic obstructive pulmonary disease (COPD) patients are at increased risk of developing nonsmall cell lung carcinoma, irrespective of their smoking history. Although the mechanisms behind this observation are not clear, established drivers of carcinogenesis in COPD include oxidative stress and sustained chronic inflammation. Mitochondria are critical in these two processes and recent evidence links increased oxidative stress in COPD patients to mitochondrial damage. We therefore postulate that mitochondrial damage in COPD patients leads to increased oxidative stress and chronic inflammation, thereby increasing the risk of carcinogenesis.
The functional state of the mitochondrion is dependent on the balance between its biogenesis and degradation (mitophagy). Dysfunctional mitochondria are a source of oxidative stress and inflammasome activation. In COPD, there is impaired translocation of the ubiquitin-related degradation molecule Parkin following activation of the Pink1 mitophagy pathway, resulting in excessive dysfunctional mitochondria. We hypothesise that deranged pathways in mitochondrial biogenesis and mitophagy in COPD can account for the increased risk in carcinogenesis. To test this hypothesis, animal models exposed to cigarette smoke and developing emphysema and lung cancer should be developed. In the future, the use of mitochondria-based antioxidants should be studied as an adjunct with the aim of reducing the risk of COPD-associated cancer.
... Interestingly, another study revealed that Mfn2 deficiency decreased proliferation by blocking autophagy in HeLa cells (Ding et al. 2015). Similarly, A549 human lung adenocarcinoma cells showed disturbed cell proliferation and invasion upon Mfn2 knockdown (Lou et al. 2015). A recent bioinformatics study conducted on the same cell line revealed that Mfn2 knockdown resulted in repression of genes implicated in cell-cycle progression as well as DNA replication and MAPK signalling pathway (Lou et al. 2016). ...
The execution of proper Ca²⁺ signaling requires close apposition between the endoplasmic reticulum (ER) and mitochondria. Hence, Ca²⁺ released from the ER is “quasi-synaptically” transferred to mitochondrial matrix, where Ca²⁺ stimulates mitochondrial ATP synthesis by activating the tricarboxylic acid (TCA) cycle. However, when the Ca²⁺ transfer is excessive and sustained, mitochondrial Ca²⁺ overload induces apoptosis by opening the mitochondrial permeability transition pore. A large number of regulatory proteins reside at mitochondria-associated ER membranes (MAMs) to maintain the optimal distance between the organelles and to coordinate the functionality of both ER and mitochondrial Ca²⁺ transporters or channels. In this chapter, we discuss the different pathways involved in the regulation of ER-mitochondria Ca²⁺ flux and describe the activities of the various Ca²⁺ players based on their primary intra-organelle localization.
... Interestingly, another study revealed that Mfn2 deficiency decreased proliferation by blocking autophagy in HeLa cells (Ding et al. 2015). Similarly, A549 human lung adenocarcinoma cells showed disturbed cell proliferation and invasion upon Mfn2 knockdown (Lou et al. 2015). A recent bioinformatics study conducted on the same cell line revealed that Mfn2 knockdown resulted in repression of genes implicated in cell-cycle progression as well as DNA replication and MAPK signalling pathway . ...
Inter-organellar contact sites establish microdomains for localised Ca²⁺-signalling events. One of these microdomains is established between the ER and the mitochondria. Importantly, the so-called mitochondria-associated ER membranes (MAMs) contain, besides structural proteins and proteins involved in lipid exchange, several Ca²⁺-transport systems, mediating efficient Ca²⁺ transfer from the ER to the mitochondria. These Ca²⁺ signals critically control several mitochondrial functions, thereby impacting cell metabolism, cell death and survival, proliferation and migration. Hence, the MAMs have emerged as critical signalling hubs in physiology, while their dysregulation is an important factor that drives or at least contributes to oncogenesis and tumour progression. In this book chapter, we will provide an overview of the role of the MAMs in cell function and how alterations in the MAM composition contribute to oncogenic features and behaviours.
... Another group also found that overexpression of Mfn2 reduces PASMC proliferation [32]. Evidence is emerging that in human lung adenocarcinoma, cell cycle and invasion behavior were all deregulated by Mfn2 knockdown [33]. In endothelial cells, Mfn1 and Mfn2 ablation diminished cell viability, migration and differentiation [22]. ...
Abnormal pulmonary arterial smooth muscle cells (PASMCs) proliferation is an important pathological process in hypoxic pulmonary arterial hypertension. Mitochondrial dynamics and quality control have a central role in the maintenance of the cell proliferation-apoptosis balance. However, the molecular mechanism is still unknown. We used hypoxic animal models, cell biology, and molecular biology to determine the effect of mitofusin 1 (Mfn1) on hypoxia-mediated PASMCs mitochondrial homeostasis. We found that Mfn1 expression was increased in hypoxia, which was crucial for hypoxia-induced mitochondrial dysfunction and smooth muscle cell proliferation as well as hypoxia-stimulated cell-cycle transition from the G0/G1 phase to S phase. Subsequently, we studied the role of microRNAs in mitochondrial function associated with PASMC proliferation under hypoxic conditions. The promotive effect of Mfn1 on pulmonary vascular remodeling was alleviated in the presence of miR-125a agomir, and miR-125a antagomir mimicked the hypoxic damage effects to mitochondrial homeostasis. Moreover, in vivo and in vitro treatment with miR-125a agomir protected the pulmonary vessels from mitochondrial dysfunction and abnormal remodeling. In the present study, we determined that mitochondrial homeostasis, particularly Mfn1, played an important role in PASMCs proliferation. MiR-125a, an important underlying factor, which inhibited Mfn1 expression and decreased PASMCs disordered growth during hypoxia. These results provide a theoretical basis for the prevention and treatment of pulmonary vascular remodeling.
Key messages:
Hypoxia leads to upregulation of mitofusin 1 (Mfn1) both in vivo and in vitro. Mfn1 is involved in hypoxia-induced PASMCs proliferation. Mfn1-mediated mitochondrial homeostasis is regulated by miR-125a. MiR-125a plays a role in PASMCs oxidative phosphorylation and glycolysis.
... Higher expression of Mfn1 and Mfn2 has been linked with cancer cell proliferation, enhanced cell survival and invasion. Conversely, inhibition of Mfn1 and Mfn2 expression inhibits cell growth and induces apoptosis of various cancer cells 29,30,43,50 . Expression of Opa1 also has been shown to enhance cancer cell proliferation and survival whereas its inhibition leads to apoptosis 51,52 . ...
Dysregulated mitochondrial dynamics and biogenesis have been associated with various pathological conditions including cancers. Here, we assessed the therapeutic effect of cryptolepine, a pharmacologically active alkaloid derived from the roots of Cryptolepis sanguinolenta, on melanoma cell growth. Treatment of human melanoma cell lines (A375, Hs294t, SK-Mel28 and SK-Mel119) with cryptolepine (1.0, 2.5, 5.0 and 7.5 μM) for 24 and 48 h significantly (P < 0.001) inhibited the growth of melanoma cells but not normal melanocytes. The inhibitory effect of cryptolepine was associated with loss of mitochondrial membrane potential and reduced protein expression of Mfn1, Mfn2, Opa1 and p-Drp1 leading to disruption of mitochondrial dynamics. A decrease in the levels of ATP and mitochondrial mass were associated with activation of the metabolic tumor suppressor AMPKα1/2-LKB1, and a reduction in mTOR signaling. Decreased expression of SDH-A and COX-I demonstrated that cryptolepine treatment reduced mitochondrial biogenesis. In vivo treatment of A375 xenograft-bearing nude mice with cryptolepine (10 mg/Kg body weight, i.p.) resulted in significant inhibition of tumor growth, which was associated with disruption of mitochondrial dynamics and a reduction in mitochondrial biogenesis. Our study suggests that low toxicity phytochemicals like cryptolepine may be tested for the treatment of melanoma.
... Mitofusin 2 was first recognized as a key protein not only regulating mitochondria fusion but also participating in tumor cell proliferation. Although MFN2 has not been proven to act as a tumor suppressor gene in cancer cells [32], its antitumor function, as revealed in various tumors, continues to be accepted. Previously, we proved that overexpression of the MFN2 gene in HCC resulted in tumor cell apoptosis via mitochondrial pathways mediated by calcium influx [14][15][16]. ...
Background:
The mitochondrial GTPase mitofusin-2 (MFN2) gene encodes a mitochondrial membrane protein that can induce apoptosis of hepatocellular carcinoma (HCC) via the mitochondrial apoptotic pathway, as validated in our previous research. However, little is known of the clinical significance of MFN2 expression and its signaling pathways in HCC.
Methods:
MFN2 mRNA expression in tumor and adjacent non-tumor tissues from 115 patients with HCC was investigated using quantitative real-time PCR. The association of the MFN2 mRNA expression level with clinical and pathological parameters was evaluated statistically, while a comparative microarray analysis was used to identify MFN2 signaling pathways in HepG2 cells.
Results:
MFN2 was significantly (p < 0.0001) downregulated in HCC tissues. Low MFN2 expression was significantly correlated with sex and preoperative alpha-fetoprotein (p < 0.05). Both a Kaplan-Meier survival curve and multivariate analyses showed that MFN2 was related to overall survival. A comparative gene expression microarray revealed 211 upregulated (58 %) and 153 downregulated (42 %) genes. Eighteen pathways were identified as the most significant pathways correlated with MFN2.
Conclusions:
Low MFN2 expression in HCC indicated a worse overall survival. Crucial signaling molecules such as PI3K-AKT, cytokine receptor, and focal adhesion may participate in MFN2-mediated signaling pathway changes in HCC.
... It has been reported that MFN2 expression was significantly higher in lung adenocarcinoma tissues than in adjacent normal tissues. 11 However, another report showed that MFN2 immunostaining was very weak in HCC tissues, and patients with HCC with lower MFN2 expression had a poorer prognosis. 12 MFN2 has been shown to induce apoptosis in HCC cells, but the exact molecular mechanism remains unclear. ...
Background:
The ability to evaluate the prognosis of hepatocellular carcinoma (HCC) following hepatectomy using biological markers is of great importance.
Materials and methods:
In this study, we collected samples from 54 patients with HCC after hepatectomy. Immunohistochemistry was used to detect the expression of Sema3C and MFN2 in the HCC samples.
Results:
Immunohistochemistry results showed that Sema3C and MFN2 co-expression was significantly associated with tumor size. In addition, a significant association between high Sema3C and low MFN2 levels and shorter overall survival was noted, when Sema3C and MFN2 co-expression was analyzed.
Conclusion:
The results suggest that the correlative expression level of Sema3C and MFN2 has a strong value in the prognosis of patients with HCC following hepatectomy.
... For example, mutations in mitofusin-2 (MFN2), a mitochondrial protein, were found to be causative of Charcot-Marie-Tooth disease type 2A (Züchner et al. 2004). It was recently found that MFN2 is a key player in the cell cycle and cell invasion in lung adenocarcinoma (Lou et al. 2015). Similarly, Parkin, a mitochondrial protein is shown to be involved in the development of Parkinson's disease as well as in human cancers (Shimura et al. 2000;Matsuda et al. 2015. ...
Gigaxonin, the product of GAN gene localized to chromosome 16, is associated with the early onset neuronal degeneration disease giant axonal neuropathy (GAN). Gigaxonin is an E3 ubiquitin ligase adaptor protein involved in intermediate filament processing in neural cells, and vimentin filaments in fibroblasts. Mutations of the gene cause pre-neural filaments to accumulate and form giant axons resulting in the inhibition of neural cell signaling. Analysis of the catalog of somatic mutations in cancer, driver DB and IDGC data portal databases containing 21,000 tumor genomic sequences has identified GAN patient mutations in cancer cell lines and primary tumors. The database search has also shown the presence of identical missense and nonsense gigaxonin mutations in GAN and colon cancer. These mutations frequently occur in the domains associated with protein homodimerization and substrate interaction such as Broad-Complex, Tramtrack and Bric a brac (BTB), BTB associated C-terminal KELCH (BACK), and KELCH repeats. Analysis of the International HapMap Project database containing 1200 normal genomic sequences has identified a single nucleotide polymorphism (SNP), rs2608555, in exon 8 of the gigaxonin sequence. While this SNP is present in >40 % of Caucasian population, it is present in less than 10 % of Japanese and Chinese populations. Although the role of gigaxonin polymorphism is not yet known, CFTR and MDR1 gene studies have shown that silent mutations play a role in the instability and aberrant splicing and folding of mRNAs. We believe that molecular and functional investigation of gigaxonin mutations including the exon 8 polymorphism could lead to an improved understanding of the relationship between GAN and cancer.
Background:
Mitochondrial fission and fusion processes are known as mitochondrial dynamics and the occurrence of imbalances in the mitochondrial activity is related to the pathogenesis of many human cancers. However, the importance of mitochondrial dynamics in malignant salivary gland tumours remains unknown. Therefore, we aimed to investigate its prognostic significance in adenoid cystic carcinoma.
Methods:
Fifty-seven formalin-fixed paraffin-embedded cases were retrieved and disposed in tissue microarray. Histological sections were submitted to immunohistochemical reactions against AMT, DRP1, FIS1, MFN1, MFN2 and OPA1 proteins. Clinical data were retrieved from the patients' medical files, including specific and disease-free survival data.
Results:
It was observed that 50.9% of the cases were strongly positive for AMT and DRP1, and 49.1%, 21.1%, 22.8% and 24.6% strongly positive for FIS1, MFN1, MFN2 and OPA1, respectively. Reactions were observed in both epithelial and myoepithelial components of the tumour. The higher expression of MFN2 was associated with solid microscopic pattern (p = 0.016). DRP1 overexpression showed a trend towards a shorter overall survival (p = 0.054), while negative/weak OPA1 showed a trend towards a lower disease-free survival (p = 0.051) in the univariate analysis, but no mitochondrial marker represented an independent prognostic determinant under multivariate analysis.
Conclusion:
In conclusion, mitochondrial dynamics markers do not seem to carry a prognostic significance for adenoid cystic carcinoma patients, but these proteins may play an important role in its pathogenesis.
Introduction
Gestational diabetes mellitus (GDM) is a metabolic disease that endangers pregnant women and their offspring. Insights into biomarkers and GDM pathogenesis are crucial. Ectopic expression of microRNA-17-5p was found in GDM, but its the diagnostic value and role of miR-17-5p remain unclear.
Material and methods
Detection of miRNA microarray and quantitative PCR (qPCR) found that miR-17-5p was significantly increased and positively associated with biochemical indicators of GDM in 30 GDM plasma samples and 28 matched control plasma samples.
Results
The area under the ROC curve was 0.827 (P < 0.01), which showed good diagnostic potential. Mitochondrial staining showed that compared with controls, trophoblasts exhibited more mitochondrial fusion and endothelial cells exhibited more mitochondrial fission in GDM than these in controls. Western blot and qPCR assays further revealed that expression of the mitofusin Mfn1/Mfn2 was lower in primary endothelial cells from GDM patients, whereas their expression was significantly higher in primary trophoblasts from GDM patients compared with those from controls. Conversely, miR-17-5p expression was higher in primary endothelial cells from GDM patients, whereas their expression was significantly lower in primary trophoblasts from GDM patients compared with those from controls. Bioinformatics and luciferase reporter assays confirmed that both Mfn1 and Mfn2 are targets of miR-17-5p. Last, decreased Mfn1/2 was observed not only to increase the apoptotic rate of primary endothelial cells from GDM, but also to reverse anti-apoptotic effects of miR-17-5p inhibitor.
Conclusions
MiR-17-5p regulates Mfn1/Mfn2-mediated mitochondrial dynamics involved in GDM. MiR-17-5p may serve as a promising biomarker and therapeutic target for GDM.
Circular RNA (circRNA) have been found to play an important role in the progression of many diseases, including interstitial cystitis (IC). However, the role of circTHBS1 in IC progression is still unclear. Exploring the role and potential molecular mechanism of circTHBS1 in the development of IC. The enzyme-linked immunosorbent assay was used to assess the levels of inflammatory cytokines. The expression levels of circTHBS1, microRNA (miR)-139-5p, and mitofusin 2 (MFN2) were evaluated using quantitative real-time PCR. Cell proliferation and migration were determined using MTT assay, Edu staining, and transwell assay. The protein levels of epithelial-mesenchymal transition (EMT) markers and MFN2 were examined using western blot analysis. The relationship between miR-139-5p and circTHBS1 or MFN2 was confirmed using the dual-luciferase reporter assay and RIP assay. CircTHBS1 was highly repressed in IC tissues and cells, and its expression was positively correlated with the inflammatory response of IC patients. CircTHBS1 could promote the proliferation, migration, EMT process, and inflammation of IC cells, while its knockdown had an opposite effect. CircTHBS1 could serve as a sponge of miR-139-5p, and miR-139-5p could participate in the regulation of circTHBS1 on IC cell progression. In addition, miR-139-5p could target MFN2, and it could inhibit the progression of IC cells by targeting MFN2. Furthermore, circTHBS1 sponged miR-139-5p to positively regulate MFN2. CircTHBS1 promoted IC cell proliferation, migration, EMT process, and inflammation by regulating the miR-139-5p/MFN2 axis indicating that circTHBS1 might be a potential target for IC treatment.
Isobavachalcone (IBC) has been shown to induce apoptosis and differentiation of acute myeloid leukemia (AML) cells. However, the underlying molecular mechanisms are not fully understood. Herein, IBC exhibited significant inhibition on the cell viability, proliferation, and the colony formation ability of AML cells. Moreover, IBC induced mitochondrial apoptosis evidenced by reduced mitochondrial membrane potential, increased Bax level, decreased Bcl‐2, Bcl‐xL, and Mcl‐1 levels, elevated cytochrome c level in the cytosol and increased cleavage of caspase‐9, caspase‐3, and PARP. Furthermore, IBC obviously promoted the differentiation of AML cells, accompanied by the increase of the phosphorylation of MEK and ERK and the C/EBPα expression as well as the C/EBPβ LAP/LIP isoform ratio, which was significantly reversed by U0126, a specific inhibitor of MEK. Notably, IBC enhanced the intracellular ROS level. More importantly, IBC‐induced apoptosis and differentiation of HL‐60 cells were significantly mitigated by NAC. In addition, IBC also exhibited an obvious anti‐AML effect in NOD/SCID mice with the engraftment of HL‐60 cells. Together, our study suggests that the ROS‐medicated signaling pathway is highly involved in IBC‐induced apoptosis and differentiation of AML cells.
The homeostatic oxygen sensing system (HOSS) optimizes systemic oxygen delivery. Specialized tissues utilize a conserved mitochondrial sensor, often involving NDUFS2 in complex I of the mitochondrial electron transport chain, as a site of pO2-responsive production of reactive oxygen species (ROS). These ROS are converted to a diffusible signaling molecule, hydrogen peroxide (H2O2), by superoxide dismutase (SOD2). H2O2 exits the mitochondria and regulates ion channels and enzymes, altering plasma membrane potential, intracellular Ca²⁺ and Ca²⁺-sensitization and controlling acute, adaptive, responses to hypoxia that involve changes in ventilation, vascular tone and neurotransmitter release. Subversion of this O2-sensing pathway creates a pseudohypoxic state that promotes disease progression in pulmonary arterial hypertension (PAH) and cancer. Pseudohypoxia is a state in which biochemical changes, normally associated with hypoxia, occur despite normal pO2. Epigenetic silencing of SOD2 by DNA methylation alters H2O2 production, activating hypoxia-inducible factor 1α, thereby disrupting mitochondrial metabolism and dynamics, accelerating cell proliferation and inhibiting apoptosis. Other epigenetic mechanisms, including dysregulation of microRNAs (miR), increase pyruvate dehydrogenase kinase and pyruvate kinase muscle isoform 2 expression in both diseases, favoring uncoupled aerobic glycolysis. This Warburg metabolic shift also accelerates cell proliferation and impairs apoptosis. Disordered mitochondrial dynamics, usually increased mitotic fission and impaired fusion, promotes disease progression in PAH and cancer. Epigenetic upregulation of dynamin-related protein 1 (Drp1) and its binding partners, MiD49 and MiD51, contributes to the pathogenesis of PAH and cancer. Finally, dysregulation of intramitochondrial Ca²⁺, resulting from impaired mitochondrial calcium uniporter complex (MCUC) function, links abnormal mitochondrial metabolism and dynamics. MiR-mediated decreases in MCUC function reduce intramitochondrial Ca²⁺, promoting Warburg metabolism, whilst increasing cytosolic Ca²⁺, promoting fission. Epigenetically disordered mitochondrial O2-sensing, metabolism, dynamics, and Ca²⁺ homeostasis offer new therapeutic targets for PAH and cancer. Promoting glucose oxidation, restoring the fission/fusion balance, and restoring mitochondrial calcium regulation are promising experimental therapeutic strategies.
Mitofusin‐2 (MFN2) is primarily involved in mitochondrial fusion and participates in diverse biological processes. Several reports show that MFN2 is a target of different miRNAs; however, the transcriptional regulation of MFN2 has not been extensively studied. To gain insight into the transcriptional regulation of MFN2, we expressed E2F1 exogenously and observed that it increased the endogenous expression of MFN2 by binding to its putative promoter region. Although the levels of E2F1 were shown to vary during the cell cycle, the expression of MFN2 and its regulator SP1 did not change throughout the different phases, suggesting that E2F1 regulates MFN2 in a cell cycle‐independent manner. In the cell‐cycle phases where expression of E2F1 was reduced, SP1 might act in its place to regulate the expression of MFN2.We showed that E2F1 and SP1 are present as a complex on the promoter of MFN2 during the S‐phase as well as in E2F1 overexpressing cells, suggesting that they may regulate the expression of MFN2 synergistically. Furthermore, we found that E2F1 modulated mitochondrial fusion and mitophagy, likely through regulation of MFN2. Bioinformatic analysis revealed that several potential targets of E2F1 are localized in mitochondria and associated with autophagy. Collectively, these data identify the E2F1‐MFN2 axis as a regulator of mitochondrial morphology and mitophagy, suggesting a potential therapeutic target for the treatment of mitochondrial disorders. This article is protected by copyright. All rights reserved.
Background
Head and neck mucosal melanomas (MMs) are rare tumors with adverse outcomes and poorer prognoses than their more common cutaneous counterparts (cutaneous melanomas–CMs). Few studies have compared the expression of mitochondrial dynamic markers in these tumors. This study aimed to access the correlations of mitochondrial markers with melanoma progression and their potential as predictors of lymph node involvement and distant metastasis.
Methods
Immunohistochemistry against anti‐mitochondrial (AMT), dynamin‐related protein 1 (DRP1), mitochondrial fission protein 1 (FIS1), mitofusin‐1 (MFN1) and mitofusin‐2 (MFN2) antibodies was performed in 112 cases of head and neck CM and MM. A Cox regression multivariate model was used to assess the correlation of AMT, FIS1 and MFN2 expressions considering the risk for nodal and distant metastasis.
Results
All markers studied presented higher staining in tumor cells than normal adjacent tissues. Higher mitochondria content was observed in MM than in CM, and it was significantly associated with nodal metastasis in oral melanomas. Both FIS1 and DRP1 expression were related to advanced Clark's levels in CM, and they were overexpressed in oral melanomas. Moreover, increased immunoexpression of MFN2 was significantly associated with a higher risk of metastasis in CM, and it was also overexpressed in sinonasal melanomas.
Conclusions
Our results suggest that mitochondrial fission and fusion processes can play an important role during multiple stages of tumorigenesis and the development of nodal and distant metastasis in cutaneous and mucosal melanomas.
This article is protected by copyright. All rights reserved.
Emerging data demonstrates that the monomeric heme protein myoglobin is aberrantly expressed in breast cancer tumors and is associated with slower tumor growth and better patient prognosis. However, the mechanism by which myoglobin slows tumor growth is unknown. In this study we hypothesized that myoglobin regulates mitochondrial structure/function to inhibit cell proliferation. Using a model of MDA-MB-231 breast cancer cells, we showed that stably expressing human myoglobin decreased cell proliferation and induced cell cycle arrest (characterized by increase p21 and decreased cyclin E expression). We demonstrate that mechanistically this cell cycle arrest is due to the myoglobin-dependent oxidation and degradation of the ubiquitin ligase parkin, which results in an increase in the expression of mitofusin-1. This protein is known to induce mitochondrial fusion and inhibit cell cycle progression. These data are recapitulated in vivo in a murine xenograft model. These data suggest a novel role for myoglobin as a modulator of mitochondrial dynamics, cell proliferation, and tumor growth.
Mitochondria are bioenergetic, biosynthetic, and signaling organelles that control various aspects of cellular and organism homeostasis. Quality control mechanisms are in place to ensure maximal mitochondrial function and metabolic homeostasis at the cellular level. Dysregulation of these pathways is a common theme in human disease. In this mini-review, we discuss how alterations of the mitochondrial network influences mitochondrial function, focusing on the molecular regulators of mitochondrial dynamics (organelle’s shape and localization). We highlight similarities and critical differences in the mitochondrial network of cancer and type 2 diabetes, which may be relevant for treatment of these diseases.
Circular RNAs (circRNAs) have recently shown capabilities as gene regulators in mammals. Some of them interact with microRNAs (miRNAs) and function as sponges to affect related miRNAs' activities. In this study, the molecular function of circRNA_0009910 and its potential downstream miRNA targets were explored. The expression levels of hsa_circ_0009910 were found to be overexpressed in osteosarcoma (OS) cells. Knockdown of circ_0009910 induced cell proliferation inhibition, cell cycle arrest, and apoptosis in OS cells. The target miRNA was predicted to be miR-449a, whose expression was downregulated in OS cells. Inhibition of miR-449a abolished the effect of circ_0009910 knockdown on cell growth and apoptosis. The expression of miR-449a were found to be negatively correlated with that of circ_0009910 in OS tissues. Direct interaction of circ_0009910 and miR-449a was confirmed through dual-luciferase assays. Moreover, IL6R was predicted as a potential target of miR-449a. Overexpression of miR-449a decreased the mRNA and protein levels of IL6R. Restoration of IL6R impaired the miR-449a induced inhibition of cell proliferation, cell cycle arrest, and apoptosis. The mRNA expression of IL6R was inversely correlated with miR-449a in OS tissues. In addition, JAK1/STAT3 signaling pathway was regulated by circ_0009910/miR-449a/IL6R axis. Taken together, we suggested that circ_0009910 acted as a sponge of miR-449a and upregulated miR-449a functional target IL6R, thereby contributed to carcinogenesis of OS.
Cancer is a complex disease marked by uncontrolled cell growth and invasion. These processes are driven by the accumulation of genetic and epigenetic alterations that promote cancer initiation and progression. Contributing to genome changes are the regulation of oxidative stress and reactive species-induced damage to molecules and organelles. Redox regulation, metabolic plasticity, autophagy, and mitophagy play important and interactive roles in cancer hallmarks including sustained proliferation, activated invasion, and replicative immortality. However, the impact of these processes can differ depending on the signaling pathways altered in cancer, tumor type, tumor stage, and/or the differentiation state. Here, we highlight some of the representative studies on the impact of oxidative and nitrosative activities, mitochondrial bioenergetics, metabolism, and autophagy and mitophagy in the context of tumorigenesis. We discuss the implications of these processes for cellular activities in cancer for anti-cancer-based therapeutics.
Cancer results from processes prone to selective pressure and dysregulation acting along the sequence-to-phenotype continuum DNA --> RNA --> protein --> disease. However, the extent to which cancer is a manifestation of the proteome is unknown. Here we present an integrated omic map representing non-small cell lung carcinoma. Dysregulated proteins not previously implicated as cancer drivers are encoded throughout the genome including, but not limited to, regions of recurrent DNA amplification/deletion. Clustering reveals signatures composed of metabolism proteins particularly highly recapitulated between patient-matched primary and xenograft tumours. Interrogation of The Cancer Genome Atlas reveals cohorts of patients with lung and other cancers that have DNA alterations in genes encoding the signatures, and this was accompanied by differences in survival. The recognition of genome and proteome alterations as related products of selective pressure driving the disease phenotype may be a general approach to uncover and group together cryptic, polygenic disease drivers.
Gastric cancer is a leading cause of cancer deaths, but analysis of its molecular and clinical characteristics has been complicated by histological and aetiological heterogeneity. Here we describe a comprehensive molecular evaluation of 295 primary gastric adenocarcinomas as part of The Cancer Genome Atlas (TCGA) project. We propose a molecular classification dividing gastric cancer into four subtypes: tumours positive for Epstein–Barr virus, which display recurrent PIK3CA mutations, extreme DNA hypermethylation, and amplification of JAK2, CD274 (also known as PD-L1) and PDCD1LG2 (also known as PD-L2); microsatellite unstable tumours, which show elevated mutation rates, including mutations of genes encoding targetable oncogenic signalling proteins; genomically stable tumours, which are enriched for the diffuse histological variant and mutations of RHOA or fusions involving RHO-family GTPase-activating proteins; and tumours with chromosomal instability, which show marked aneuploidy and focal amplification of receptor tyrosine kinases. Identification of these subtypes provides a roadmap for patient stratification and trials of targeted therapies.
Adenocarcinoma of the lung is the leading cause of cancer death worldwide. Here we report molecular profiling of 230 resected lung adenocarcinomas using messenger RNA, microRNA and DNA sequencing integrated with copy number, methylation and proteomic analyses. High rates of somatic mutation were seen (mean 8.9 mutations per megabase). Eighteen genes were statistically significantly mutated, including RIT1 activating mutations and newly described loss-of-function MGA mutations which are mutually exclusive with focal MYC amplification. EGFR mutations were more frequent in female patients, whereas mutations in RBM10 were more common in males. Aberrations in NF1, MET, ERBB2 and RIT1 occurred in 13% of cases and were enriched in samples otherwise lacking an activated oncogene, suggesting a driver role for these events in certain tumours. DNA and mRNA sequence from the same tumour highlighted splicing alterations driven by somatic genomic changes, including exon 14 skipping in MET mRNA in 4% of cases. MAPK a
To evaluate the diagnostic value of a novel fully automated immunohistochemistry (IHC) assay for detection of anaplastic lymphoma kinase (ALK) fusion in a large number of ALK-positive lung adenocarcinoma (ADC) patients.
We tested 196 lung ADCs for ALK rearrangement by two IHC assays (Ventana pre-diluted ALK D5F3 antibody with the Optiview DAB IHC detection kit and Optiview Amplification kit, D5F3 by Cell Signaling Technology (CST) with Ultraview DAB detection kit by Ventana), fluorescence in situ hybridization (FISH) and real-time reverse transcription-PCR (RT-PCR). CST ALK IHC was scored using the scoring scheme of 0, no staining; 1+, faint; 2+, moderate; and 3+, strong cytoplasmic reactivity in ≥10% of tumor cells. As for Ventana IHC, a binary scoring system (positive or negative for ALK status) was adopted for evaluating the staining results.
Among 196 cases tested, 63 (32%), 65 (33%), 70 (36%), and 69 (35%) cases were ALK positive by FISH, Ventana IHC, CST IHC, and RT-PCR, respectively. The sensitivity and specificity of Ventana IHC were 100% and 98%, respectively. Two Ventana IHC-positive cases, which were also CST IHC score of 3+, showed FISH negative, but their ALK rearrangement was confirmed by RT-PCR and direct sequencing. The sensitivity and specificity of CST IHC with staining intensity score of 1+ or more were 100% and 95%, respectively. Five (25%, of 20) patients with CST IHC score of 1+ were both FISH and RT-PCR negative. The sensitivity and specificity of RT-PCR for detection of ALK fusion were 98% and 95%, respectively. The total accordance rate between ALK RT-PCR and Ventana IHC was 97%.
The novel fully automated IHC assay is a reliable screening tool in routine pathologic laboratories for identification of patients with ALK rearrangement for targeted therapy in lung ADC.
Mitofusin-2 (Mfn2) is a mitochondrial outer membrane protein involved in mitochondrial fusion. Its mutation can cause Charcot-Marie-Tooth disease. Recent studies of Mfn2 in cancer research have not included gastric cancer. We confirmed that Mfn2 expression was lower in tumor tissue than in normal gastric mucosal tissue and that it was negatively correlated with tumor size, indicating an anti-tumor role for Mfn2. In vitro experiments showed that Mfn2 overexpression suppressed gastric cancer cell proliferation and colony formation, weakened the invasion and migratory ability of cancer cells by downregulating MMP-2 and MMP-9, halted the cell cycle and induced apoptosis. Western blotting indicated the likely involvement of P21 and PI3K/Akt signaling. Therefore, Mfn2 is a potential anti-tumor gene and a potential therapeutic target for treating gastric cancer. The progress of gastric cancer may be delayed by controlling Mfn2 expression.
In the present study, we investigated whether estrogen receptor (ER) β affected the proliferation and migration of the human breast cancer cell line MCF-7 through regulation of mitofusin 2 (mfn2). A previous study reported that mfn2 may be regulated by ER through a non-classical pathway; in this pathway, the ER modulates the activities of other transcription factors by stabilizing their binding to DNA and/or recruiting coactivators to the complex. However, the previous study, unlike the study presented here, did not directly explore the interactions between ER and mfn2. Here, RT-PCR and western blot analysis were used to test the expression of mfn2 in MCF-7 cells after exposure to different doses of estradiol (E2). The ability of cells to proliferate and migrate was determined by MTT assay and a monolayer-wounding protocol, respectively. Finally, changes in MCF-7 cell biology after transfection with ERβ or mfn2 expression vectors were investigated, and the role of ERβ in mfn2 expression was also explored. Our results showed that E2 attenuated mfn2 expression in a dose-dependent manner, concomitant with the activation of proliferation and migration of MCF-7 cells. The mfn2 expression vector effectively suppressed E2-induced upregulation of PCNA and migration in MCF-7 cells. ERβ inhibited the E2-induced mfn2 downregulation that accompanied the inhibition of proliferation and migration in MCF-7 cells. Briefly, ERβ may inhibit E2-induced proliferation and migration of MCF-7 cells through regulation of mfn2.
The discovery of RET fusions in lung cancers has uncovered a new therapeutic target for patients whose tumors harbor these changes. In an unselected population of non–small cell lung carcinomas (NSCLCs), RET fusions are present in 1% to 2% of cases. This incidence increases substantially, however, in never-smokers with lung adenocarcinomas that lack other known driver oncogenes. Although preclinical data provide experimental support for the use of RET inhibitors in the treatment of RET fusion-positive tumors, clinical data on response are lacking. We report preliminary data for the first three patients treated with the RET inhibitor cabozantinib on a prospective phase II trial for patients with RET fusion-positive NSCLCs (NCT01639508). Confirmed partial responses were observed in 2 patients, including one harboring a novel TRIM33–RET fusion. A third patient with a KIF5B–RET fusion has had prolonged stable disease approaching 8 months (31 weeks). All three patients remain progression-free on treatment.
Significance: Driver oncogene discovery in lung cancers has dramatically changed today's therapeutic landscape. This report of the activity of cabozantinib in RET fusion-positive disease provides early clinical validation of RET fusions as drivers in lung cancers and suggests that RET inhibition may represent a new treatment paradigm in this molecular cohort. Cancer Discov; 3(6); 630–5. ©2013 AACR.
See related commentary by Gainor and Shaw, p. 604
This article is highlighted in the In This Issue feature, p. 591
Mitochondria exist in dynamic networks that undergo fusion and fission. Mitochondrial fusion and fission are mediated by several GTPases in the outer mitochondrial membrane, notably mitofusin-2 (Mfn-2), which promotes fusion, and dynamin-related protein (Drp-1), which promotes fission. We report that human lung cancer cell lines exhibit an imbalance of Drp-1/Mfn-2 expression, which promotes a state of mitochondrial fission. Lung tumor tissue samples from patients demonstrated a similar increase in Drp-1 and decrease in Mfn-2 when compared to adjacent healthy lung. Complementary approaches to restore mitochondrial network formation in lung cancer cells by overexpression of Mfn-2, Drp-1 inhibition, or Drp-1 knockdown resulted in a marked reduction of cancer cell proliferation and an increase in spontaneous apoptosis. The number of cancer cells in S phase decreased from 32.4 ± 0.6 to 6.4 ± 0.3% with Drp-1 inhibition (P<0.001). In a xenotransplantation model, Mfn-2 gene therapy or Drp-1 inhibition could regress tumor growth. The tumor volume decreased from 205.6 ± 59 to 70.6 ± 15 mm(3) (P<0.05) with Mfn-2 overexpression and from 186.0 ± 19 to 87.0 ± 6 mm(3) (P<0.01) with therapeutic Drp-1 inhibition. Impaired fusion and enhanced fission contribute fundamentally to the proliferation/apoptosis imbalance in cancer and constitute promising novel therapeutic targets.
In melanoma, the extracellular protein SPARC (secreted protein acidic and rich in cysteine) is related to tumor progression. Some of the evidence that links SPARC to melanoma progression indicates that SPARC may be involved in the acquisition of mesenchymal traits that favor metastatic dissemination. However, no molecular pathways that link extracellular SPARC to a mesenchymal phenotype have been described. In this study, global protein expression analysis of the melanoma secretome following enforced downregulation of SPARC expression led us to elucidate a new molecular mechanism by which SPARC promotes cathepsin B-mediated melanoma invasiveness using collagen I and α2β1 integrins as mediators. Interestingly, we also found that the transforming growth factor (TGF)-β1 contribution to cathepsin B-mediated invasion is highly SPARC dependent. In addition, induction of the E-cadherin to N-cadherin switch by SPARC enabled melanoma cells to transmigrate across an endothelial layer through a mechanism independent to that of enhancing invasion. Finally, SPARC also enhanced the extracellular expression of other proteins involved in epithelial-mesenchymal transformation, such as family with sequence similarity 3, member C/interleukin-like EMT-inducer. Our findings demonstrate a previously unreported molecular pathway for SPARC activity on invasion and support an active role of SPARC in the mesenchymal transformation that contributes to melanoma dissemination.
Oncogenic fusion genes consisting of EML4 and anaplastic lymphoma kinase (ALK) are present in a subgroup of non-small-cell lung cancers, representing 2 to 7% of such tumors. We explored the therapeutic efficacy of inhibiting ALK in such tumors in an early-phase clinical trial of crizotinib (PF-02341066), an orally available small-molecule inhibitor of the ALK tyrosine kinase.
After screening tumor samples from approximately 1500 patients with non-small-cell lung cancer for the presence of ALK rearrangements, we identified 82 patients with advanced ALK-positive disease who were eligible for the clinical trial. Most of the patients had received previous treatment. These patients were enrolled in an expanded cohort study instituted after phase 1 dose escalation had established a recommended crizotinib dose of 250 mg twice daily in 28-day cycles. Patients were assessed for adverse events and response to therapy.
Patients with ALK rearrangements tended to be younger than those without the rearrangements, and most of the patients had little or no exposure to tobacco and had adenocarcinomas. At a mean treatment duration of 6.4 months, the overall response rate was 57% (47 of 82 patients, with 46 confirmed partial responses and 1 confirmed complete response); 27 patients (33%) had stable disease. A total of 63 of 82 patients (77%) were continuing to receive crizotinib at the time of data cutoff, and the estimated probability of 6-month progression-free survival was 72%, with no median for the study reached. The drug resulted in grade 1 or 2 (mild) gastrointestinal side effects.
The inhibition of ALK in lung tumors with the ALK rearrangement resulted in tumor shrinkage or stable disease in most patients. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00585195.).
Chloroquine (CQ), the worldwide used anti-malarial drug, has recently being focused as a potential anti-cancer agent as well as a chemosensitizer when used in combination with anti-cancer drugs. It has been shown to inhibit cell growth and/or to induce cell death in various types of cancer. 5-Fluorouracil (5-FU) is the chemotherapeutic agent of first choice in colorectal cancer, but in most cases, resistance to 5-FU develops through various mechanisms. Here, we focused on the combination of CQ as a mechanism to potentiate the inhibitory effect of 5-FU on human colon cancer cells.
HT-29 cells were treated with CQ and/or 5-FU, and their proliferative ability, apoptosis and autophagy induction effects, and the affection of the cell cycle were evaluated. The proliferative ability of HT-29 was analyzed by the MTS assay. Apoptosis was quantified by flow-cytometry after double-staining of the cells with AnnexinV/PI. The cell cycle was evaluated by flow-cytometry after staining of cells with PI. Autophagy was quantified by flow-cytometry and Western blot analysis. Finally, to evaluate the fate of the cells treated with CQ and/or 5-FU, the colony formation assay was performed.
5-FU inhibited the proliferative activity of HT-29 cells, which was mostly dependent on the arrest of the cells to the G0/G1-phase but also partially on apoptosis induction, and the effect was potentiated by CQ pre-treatment. The potentiation of the inhibitory effect of 5-FU by CQ was dependent on the increase of p21Cip1 and p27Kip1 and the decrease of CDK2. Since CQ is reported to inhibit autophagy, the catabolic process necessary for cell survival under conditions of cell starvation or stress, which is induced by cancer cells as a protective mechanism against chemotherapeutic agents, we also analyzed the induction of autophagy in HT-29. HT-29 induced autophagy in response to 5-FU, and CQ inhibited this induction, a possible mechanism of the potentiation of the anti-cancer effect of 5-FU.
Our findings suggest that the combination therapy with CQ should be a novel therapeutic modality to improve efficacy of 5-FU-based chemotherapy, possibly by inhibiting autophagy-dependent resistance to chemotherapy.
OBJECTIVE
Type 2 diabetes is associated with insulin resistance and skeletal muscle mitochondrial dysfunction. We have found that subjects with early-onset type 2 diabetes show incapacity to increase Vo2max in response to chronic exercise. This suggests a defect in muscle mitochondrial response to exercise. Here, we have explored the nature of the mechanisms involved.
RESEARCH DESIGN AND METHODS
Muscle biopsies were collected from young type 2 diabetic subjects and obese control subjects before and after acute or chronic exercise protocols, and the expression of genes and/or proteins relevant to mitochondrial function was measured. In particular, the regulatory pathway peroxisome proliferator–activated receptor γ coactivator (PGC)-1α/mitofusin-2 (Mfn2) was analyzed.
RESULTS
At baseline, subjects with diabetes showed reduced expression (by 26%) of the mitochondrial fusion protein Mfn2 and a 39% reduction of the α-subunit of ATP synthase. Porin expression was unchanged, consistent with normal mitochondrial mass. Chronic exercise led to a 2.8-fold increase in Mfn2, as well as increases in porin, and the α-subunit of ATP synthase in muscle from control subjects. However, Mfn2 was unchanged after chronic exercise in individuals with diabetes, whereas porin and α-subunit of ATP synthase were increased. Acute exercise caused a fourfold increase in PGC-1α expression in muscle from control subjects but not in subjects with diabetes.
CONCLUSIONS
Our results demonstrate alterations in the regulatory pathway that controls PGC-1α expression and induction of Mfn2 in muscle from patients with early-onset type 2 diabetes. Patients with early-onset type 2 diabetes display abnormalities in the exercise-dependent pathway that regulates the expression of PGC-1α and Mfn2.
Ras and Rap proteins are closely related small GTPases. Whereas Ras is known for its role in cell proliferation and survival, Rap1 is predominantly involved in cell adhesion and cell junction formation. Ras and Rap are regulated by different sets of guanine nucleotide exchange factors and GTPase-activating proteins, determining one level of specificity. In addition, although the effector domains are highly similar, Rap and Ras interact with largely different sets of effectors, providing a second level of specificity. In this review, we discuss the regulatory proteins and effectors of Ras and Rap, with a focus on those of Rap.
Although changes in mitochondrial size and arrangement accompany both cellular differentiation and human disease, the mechanisms that mediate mitochondrial fusion, fission and morphogenesis in mammalian cells are not understood. We have identified two human genes encoding potential mediators of mitochondrial fusion. The mitofusins (Mfn1 and Mfn2) are homologs of the Drosophila protein fuzzy onion (Fzo) that associate with mitochondria and alter mitochondrial morphology when expressed by transient transfection in tissue culture cells. An internal region including a predicted bipartite transmembrane domain (TM) is sufficient to target Mfn2 to mitochondria and requires hydrophobic residues within the TM. Co-expression of Mfn2 with a dominant interfering mutant dynamin-related protein (Drp1(K38A)) proposed to block mitochondrial fission resulted in long mitochondrial filaments and networks. Formation of mitochondrial filaments and networks required a wild-type Mfn2 GTPase domain, suggesting that the Mfn2 GTPase regulates or mediates mitochondrial fusion and that mitofusins and dynamin related GTPases play opposing roles in mitochondrial fusion and fission in mammals, as in yeast.
In many cells and specially in muscle, mitochondria form elongated filaments or a branched reticulum. We show that Mfn2 (mitofusin
2), a mitochondrial membrane protein that participates in mitochondrial fusion in mammalian cells, is induced during myogenesis
and contributes to the maintenance and operation of the mitochondrial network. Repression of Mfn2 caused morphological and
functional fragmentation of the mitochondrial network into independent clusters. Concomitantly, repression of Mfn2 reduced
glucose oxidation, mitochondrial membrane potential, cell respiration, and mitochondrial proton leak. We also show that the
Mfn2-dependent mechanism of mitochondrial control is disturbed in obesity by reduced Mfn2 expression. In all, our data indicate
that Mfn2 expression is crucial in mitochondrial metabolism through the maintenance of the mitochondrial network architecture,
and reduced Mfn2 expression may explain some of the metabolic alterations associated with obesity.
Cancer cells possess a broad spectrum of migration and invasion mechanisms. These include both individual and collective cell-migration strategies. Cancer therapeutics that are designed to target adhesion receptors or proteases have not proven to be effective in slowing tumour progression in clinical trials--this might be due to the fact that cancer cells can modify their migration mechanisms in response to different conditions. Learning more about the cellular and molecular basis of these different migration/invasion programmes will help us to understand how cancer cells disseminate and lead to new treatment strategies.
We report missense mutations in the mitochondrial fusion protein mitofusin 2 (MFN2) in seven large pedigrees affected with Charcot-Marie-Tooth neuropathy type 2A (CMT2A). Although a mutation in kinesin family member 1B-β (KIF1B) was associated with CMT2A in a single Japanese family, we found no mutations in KIFIB in these seven families. Because these families include all published pedigrees with CMT2A and are ethnically diverse, we conclude that the primary gene mutated in CMT2A is MFN2.
Receptor tyrosine kinase genes were sequenced in non–small cell lung cancer (NSCLC) and matched normal tissue. Somatic mutations
of the epidermal growth factor receptor gene EGFR were found in 15of 58 unselected tumors from Japan and 1 of 61 from the United States. Treatment with the EGFR kinase inhibitor
gefitinib (Iressa) causes tumor regression in some patients with NSCLC, more frequently in Japan. EGFR mutations were found in additional lung cancer samples from U.S. patients who responded to gefitinib therapy and in a lung
adenocarcinoma cell line that was hypersensitive to growth inhibition by gefitinib, but not in gefitinib-insensitive tumors
or cell lines. These results suggest that EGFR mutations may predict sensitivity to gefitinib.
Vascular proliferative disorders, such as atherosclerosis and restenosis, are the most common causes of severe cardiovascular diseases, but a common molecular mechanism remains elusive. Here, we identify and characterize a novel hyperplasia suppressor gene, named HSG (later re-named rat mitofusin-2). HSG expression was markedly reduced in hyper-proliferative vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rat arteries, balloon-injured Wistar Kyoto rat arteries, or ApoE-knockout mouse atherosclerotic arteries. Overexpression of HSG overtly suppressed serum-evoked VSMC proliferation in culture, and blocked balloon injury induced neointimal VSMC proliferation and restenosis in rat carotid arteries. The HSG anti-proliferative effect was mediated by inhibition of ERK/MAPK signalling and subsequent cell-cycle arrest. Deletion of the p21(ras) signature motif, but not the mitochondrial targeting domain, abolished HSG-induced growth arrest, indicating that rHSG-induced anti-proliferation was independent of mitochondrial fusion. Thus, rHSG functions as a cell proliferation suppressor, whereas dysregulation of rHSG results in proliferative disorders.
Hyperplasia suppressor gene (HSG), also called human mitofusin 2, is a novel gene that markedly suppresses the cell proliferation of hyperproliferative vascular smooth muscle cells from spontaneously hypertensive rat arteries. This gene encodes a mitochondrial membrane protein that participates in mitochondrial fusion and contributes to the maintenance and operation of the mitochondrial network. In this report, we showed that an adenovirus vector encoding human HSG (Ad5-hHSG) had an antitumor activity in a wide range of cancer cell lines. We further focused on the lung cancer cell line A549 and the colon cancer cell line HT-29 and then observed that Ad5-hHSG induced apoptosis both in vitro and in vivo. Confocal laser scanning microscopy and electron microscopy revealed that cells infected with Ad5-hHSG formed dose-dependent perinuclear clusters of fused mitochondria. Adenovirus-mediated hHSG overexpression induced apoptosis, cell cycle arrest, mitochondrial membrane potential (DeltaPsim) reduction and release of cytochrome c, caspase-3 activation, and cleavage of PARP in vitro. Overexpression of hHSG also significantly suppressed the growth of subcutaneous tumors in nude mice both ex vivo and in vivo. In addition, Ad5-hHSG increased the sensitivity of these cell lines to two chemotherapeutic agents, VP16 and CHX, and radiation. These results suggest that Ad5-hHSG may serve as an effective therapeutic drug against tumors.
To enhance therapeutic efficacy and reduce adverse effects, practitioners of traditional Chinese medicine (TCM) prescribe a combination of plant species/minerals, called formulae, based on clinical experience. Nearly 100,000 formulae have been recorded, but the working mechanisms of most remain unknown. In trying to address the possible beneficial effects of formulae with current biomedical approaches, we use Realgar-Indigo naturalis formula (RIF), which has been proven to be very effective in treating human acute promyelocytic leukemia (APL) as a model. The main components of RIF are realgar, Indigo naturalis, and Salvia miltiorrhiza, with tetraarsenic tetrasulfide (A), indirubin (I), and tanshinone IIA (T) as major active ingredients, respectively. Here, we report that the ATI combination yields synergy in the treatment of a murine APL model in vivo and in the induction of APL cell differentiation in vitro. ATI causes intensified ubiquitination/degradation of promyelocytic leukemia (PML)-retinoic acid receptor α (RARα) oncoprotein, stronger reprogramming of myeloid differentiation regulators, and enhanced G1/G0 arrest in APL cells through hitting multiple targets compared with the effects of mono- or biagents. Furthermore, ATI intensifies the expression of Aquaglyceroporin 9 and facilitates the transportation of A into APL cells, which in turn enhances A-mediated PML-RARα degradation and therapeutic efficacy. Our data also indicate A as the principal component of the formula, whereas T and I serve as adjuvant ingredients. We therefore suggest that dissecting the mode of action of clinically effective formulae at the molecular, cellular, and organism levels may be a good strategy in exploring the value of traditional medicine.
• traditional chinese medicine
• active ingredient
• tetraarsenic tetrasulfide
• synergism
• systems biology
As with other books in the Molecular Pathology Library Series, Molecular Pathology of Lung Cancer bridges the gap between the molecular specialist and the clinical practitioner, including the surgical pathologist who now has a key role in decisions regarding molecular targeted therapy for lung cancer. Molecular Pathology of Lung Cancer provides the latest information and current insights into the molecular basis for lung cancer, including precursor and preinvasive lesions, molecular diagnosis, molecular targeted therapy, molecular prognosis, molecular radiology and related fields for lung cancer generally and for the specific cell types. As many fundamental concepts about lung cancer have undergone revision in only the past few years, this book will likely be the first to comprehensively cover the new molecular pathology of lung cancer. It provides a foundation in this field for pathologists, medical oncologists, radiation oncologists, thoracic surgeons, thoracic radiologists and their trainees, physician assistants, and nursing staff.
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Lung adenocarcinoma, the most common subtype of non-small cell lung cancer, is responsible for more than 500,000 deaths per year worldwide. Here, we report exome and genome sequences of 183 lung adenocarcinoma tumor/normal DNA pairs. These analyses revealed a mean exonic somatic mutation rate of 12.0 events/megabase and identified the majority of genes previously reported as significantly mutated in lung adenocarcinoma. In addition, we identified statistically recurrent somatic mutations in the splicing factor gene U2AF1 and truncating mutations affecting RBM10 and ARID1A. Analysis of nucleotide context-specific mutation signatures grouped the sample set into distinct clusters that correlated with smoking history and alterations of reported lung adenocarcinoma genes. Whole-genome sequence analysis revealed frequent structural rearrangements, including in-frame exonic alterations within EGFR and SIK2 kinases. The candidate genes identified in this study are attractive targets for biological characterization and therapeutic targeting of lung adenocarcinoma.
The hepatitis B virus X (HBx) protein has many significant roles in hepatocellular carcinoma (HCC). Our previous research demonstrated that mitofusion-2 (Mfn2), a potential tumor suppressor gene in HCC, is a novel direct target of p53 that exerts apoptotic effects via the mitochondrial apoptotic pathway. However, the relationship between HBx and Mfn2 expression in the development of HCC is unknown. We found that HBx had little direct effect on the expression of Mfn2 or p53 in HCC cells not treated with doxorubicin. However, HBx inhibited the upregulation of Mfn2 in HBx-transfected HCC cells simultaneously treated with doxorubicin or cotransfected with p53 plasmid, as evidenced by Western Blot and real-time PCR. Through electrophoretic mobility shift analysis, we confirmed that HBx interfered with the binding event of the p53 protein and the p53 binding site-oligo of the Mfn2 promoter. Moreover, luciferase assays revealed that the activity of the Mfn2 promoter did not increase when transfected with HBx plasmid in doxorubicin-treated HepG2 cells. These results indicate that HBx impacts p53-mediated transcription of Mfn2, providing insight into the negative effect of HBx against p53-dependent chemotherapeutic agents, such as doxorubicin, used in the treatment of HCC.
Malignant glioma is the most common brain tumor with 16,000 new cases diagnosed annually in the United States. We performed a systematic large-scale transcriptomics data mining study of 9783 tissue samples from the GeneSapiens database to systematically identify genes that are most glioma-specific. We searched for genes that were highly expressed in 322 glioblastoma multiforme tissue samples and 66 anaplastic astrocytomas as compared with 425 samples from histologically normal central nervous system. Transcription cofactor HES6 (hairy and enhancer of split 6) emerged as the most glioma-specific gene. Immunostaining of a tissue microarray showed HES6 expression in 335 (98.8%) out of the 339 glioma samples. HES6 was expressed in endothelial cells of the normal brain and glioma tissue. Recurrent grade 2 astrocytomas and grade 2 or 3 oligodendrogliomas showed higher levels of HES6 immunoreactivity than the corresponding primary tumors. High HES6 mRNA expression correlated with the proneural subtype that generally has a favorable outcome but is prone to recur. Functional studies suggested an important role for HES6 in supporting survival of glioma cells, as evidenced by reduction of cancer cell proliferation and migration after HES6 silencing. The biological role and consequences of HES6 silencing and overexpression was explored with genome-wide analyses, which implicated a role for HES6 in p53, c-myc and nuclear factor-κB transcriptional networks. We conclude that HES6 is important for glioma cell proliferation and migration, and may have a role in angiogenesis.
The hallmarks of cancer comprise six biological capabilities acquired during the multistep development of human tumors. The hallmarks constitute an organizing principle for rationalizing the complexities of neoplastic disease. They include sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis. Underlying these hallmarks are genome instability, which generates the genetic diversity that expedites their acquisition, and inflammation, which fosters multiple hallmark functions. Conceptual progress in the last decade has added two emerging hallmarks of potential generality to this list-reprogramming of energy metabolism and evading immune destruction. In addition to cancer cells, tumors exhibit another dimension of complexity: they contain a repertoire of recruited, ostensibly normal cells that contribute to the acquisition of hallmark traits by creating the "tumor microenvironment." Recognition of the widespread applicability of these concepts will increasingly affect the development of new means to treat human cancer.
Estimates of the worldwide incidence and mortality from 27 cancers in 2008 have been prepared for 182 countries as part of the GLOBOCAN series published by the International Agency for Research on Cancer. In this article, we present the results for 20 world regions, summarizing the global patterns for the eight most common cancers. Overall, an estimated 12.7 million new cancer cases and 7.6 million cancer deaths occur in 2008, with 56% of new cancer cases and 63% of the cancer deaths occurring in the less developed regions of the world. The most commonly diagnosed cancers worldwide are lung (1.61 million, 12.7% of the total), breast (1.38 million, 10.9%) and colorectal cancers (1.23 million, 9.7%). The most common causes of cancer death are lung cancer (1.38 million, 18.2% of the total), stomach cancer (738,000 deaths, 9.7%) and liver cancer (696,000 deaths, 9.2%). Cancer is neither rare anywhere in the world, nor mainly confined to high-resource countries. Striking differences in the patterns of cancer from region to region are observed.
Mitochondrial GTPase mitofusin-2 (Mfn2) is a novel gene that remarkably suppresses the injury-mediated proliferation of vascular smooth muscle cells (VSMCs) and has a potential apoptotic effect via the mitochondrial apoptotic pathway. Hepatocellular carcinoma (HCC) tissues and matched normal tissues were examined for mfn2 expression. HCC cells were infected with adenovirus carrying Mfn2 (Ad-mfn2) or green fluorescent protein (Ad-GFP), used as a control. Short hairpin RNA (shRNA) was formed by shR-mfn2 and shR-Bax to repress mfn2 and Bax transcription, respectively. The effects of mfn2 on cell cycle distribution and apoptosis were measured by flow cytometric analysis. Significant downregulation of mfn2 was observed in HCC tissues compared with nearby normal tissues. Overexpression of mfn2 inhibited HCC cell proliferation and induced apoptosis by increasing the level of active caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage. Overexpression of mfn2 also induced cytochrome c release to the cytoplasm by enhancing Bax translocation from the cytoplasm to the mitochondrial membrane. Upregulation of mfn2 promoted apoptosis of HCC cells, and this was dramatically suppressed by shR-Bax. Our results show that the mfn2 gene is a potential tumor suppressor target that may significantly promote apoptosis via Bax and may inhibit proliferation in HCC cells. This gene may be an important therapeutic target for the treatment of tumors or hyperproliferative diseases.
Mitochondrial GTPase mitofusin-2 gene (Mfn2) is a novel gene characterised as a cell proliferation inhibitor. Mfn2 protein over-expression, mediated by an adenovirus, has a significant anti-tumour effect in A548 and HT-29 cells. However, there is no report on the effect of Mfn2 on urinary bladder carcinoma (UBCC). In this study, we sought to investigate the function of Mfn2 in UBCC. Mfn2 expression in 36 paired UBCC samples was investigated by reverse transcription-polymerase chain reaction and Western blot analyses. An adenovirus encoding the complete Mfn2 open reading frame (Ad-Mfn2) was used to infect UBCC cells, and an adenoviral vector encoding green fluorescent protein (Ad-GFP) was used as a control. The effects of Mfn2 on cell-cycle distribution and apoptosis were assessed by flow cytometry and Western blot analyses. The Mfn2 protein showed significantly lower expression in UBCC tissues than nearby non-tumourous tissues. Ad-Mfn2 exhibited a significant anti-tumour effect in T24 and 5,637 cells. Mfn2 overexpression in T24 cells significantly inhibited cell proliferation, by arresting the transition of the cell cycle from the G(1) to S phase, and induced apoptosis by upregulating active caspase-3 and cleaved PARP levels. Mfn2 also induced increased p21 and p27 expression levels, but down-regulated PCNA levels. These findings indicate that Mfn2 is a potential UBCC tumour suppressor gene, which showed significantly lower expression in tumour tissues than adjacent non-tumourous tissues and could promote apoptosis and inhibit the proliferation of UBCC cells. Mfn2 may become an important therapeutic target for treating UBCC.
Hyperplasia suppressor gene (HSG) is a novel gene that markedly suppresses the mitogenetic stimuli or injury mediated by vascular smooth muscle cell proliferation. Herein we provide experimental evidence to show that HSG can also play a key role in tumor proliferation. Down-regulation of HSG protein in hepatocellular carcinoma tissues compared to adjacent tissues. Overexpression of HSG suppressed the growth of liver cancer cell lines, resulted in cell cycle arrest in the G0/G1 phase, increased expression of the cyclin dependent kinase inhibitors (CKIs), and reduced expression of proliferating cell nuclear antigen (PCNA). It also showed that adenovirus-mediated HSG overexpression induced apoptosis. Up-regulation of HSG by adenovirus also significantly suppressed the growth of subcutaneous tumors in nude mice both ex vivo and in vivo. Collectively, our data suggest that HSG is a potential therapy for tumors and possibly other proliferative diseases as well and it has antitumor efficacy on hepatocellular carcinoma by using adenovirus vectors, which may be a new therapeutic target for liver cancer prevention.
Ras and Rap proteins are closely related small GTPases. Whereas Ras is known for its role in cell proliferation and survival, Rap1 is predominantly involved in cell adhesion and cell junction formation. Ras and Rap are regulated by different sets of guanine nucleotide exchange factors and GTPase activating proteins, determining one level of specificity. In addition, although the effector domains are highly similar, Rap and Ras interact with largely different sets of effectors, providing a second level of specificity. In this review we discuss the regulatory proteins and effectors of Ras and Rap with a focus on those of Rap.
Therapy and prognosis of lung cancer depend crucially on tumor size, tumor stage, and the histomorphological tumor type at the time of primary diagnosis. A tumor weighing only 1 g and barely detectable by clinical examination consists of about 1 x 10(9) tumor cells. The primary histological diagnosis is generally based on small biopsies 1-2 mm in diameter, which allow the assessment of only a few up to some hundred tumor cells in a section of 4 microns. Until 20 years ago light microscopic and histochemical investigations were the basis for sophisticated morphological tumor diagnosis. In recent years electron microscopy, immunohistochemistry, cytometry, and molecular biology have extended our knowledge of the complex tumor biology, with the range of phenotypes and genotypes indicating enormous tumor heterogeneity. The value, expressiveness, and prognostic importance of these laborious and expensive techniques must be examined in studies, keeping in mind new aspects of tumor classification. Histological and cytological findings are still the decisive basis for the primary diagnosis of the pathologist in any given case.
The hyperplasia suppressor gene HSG, previously characterized for its function in mitochondrial fusion, has now been identified as a novel inhibitor of the Ras signalling pathway during smooth muscle cell proliferation. Because of the critical function of this process in atherosclerotic heart disease, these findings may have important clinical implications.
In order to evaluate the effect of mitofusin-2 gene (mfn2) on proliferation and chemotherapy sensitivity of human breast carcinoma cell line MCF-7 in vitro, pEGFPmfn2 plasmid carrying full length of mitofusin-2 gene was transfected, by using sofast, into MCF-7 cells. Mitofusin-2 gene expression in MCF-7 cells transfected by sofast after 48 h was detected by PCR and Western blotting, and the stable expression of GFP protein in MCF-7 cells by Western blot analysis. The proliferation of MCF-7 cells was assayed by MTT and cell counting. By using PI method, the effects of mfn2 on the cell cycle distribution of MCF-7 were measured. Annexin-V/PI double labeling method was employed to detect the changes in apoptosis induced by chemotherapeutics before and after transfection. The results showed that the MCF-7 cells transfected with mfn2 gene could stably and highly express GFP protein. MTT assay revealed that after transfection of mfn2 cDNA, the proliferation of MCF-7 cells was significantly inhibited. DNA histogram showed that cells arrested in S phase, and the percentage of S phase cells was 42.7, 17.2 and 19.6 in mfn2 cDNA transfection group, blank plasmid transfection group and blank control group, respectively (P<0.05). The apoptosis ratio of the cells transfected with mfn2 gene was increased from 3.56% to 15.95%, that of the cells treated with camptothecin (CAMP) followed by mfn2 gene transfection was 69.6%, and that in blank plasmid transfection group and blank control group was 31.0% and 23.4% respectively (P<0.05). It was suggested that transfection of mfn2 gene could significantly inhibit the proliferation of MCF-7 cells and promote their sensitivity to CAMP with a synergic effect.
Cancer Genome Atlas Research Network. Comprehensive molecular profiling of lung adenocarcinoma
- Cancer Genome Atlas Research Network
Small molecules inhibit the RAS-like small GTPases RALA and RALB
Pro-apoptotic and anti-proliferative effects of mitofusin-2 via Bax signaling in hepatocellular carcinoma cells
- W L Wang
- J J Lu
- F Zhu
- J F Wei
- C K Jia
- Y B Zhang
- WL Wang
Molecular pathology of lung cancer. Molecular pathology library
- P T Cagle
- T C Allen
- M B Beasley
- L R Chirieac
- S Dacic
- A C Borczuk
- PT Cagle
SPARC promotes cathepsin B-mediated melanoma invasiveness through a collagen I/alpha 2 beta 1 integrin axis
- M R Girotti
- M Fernandez
- J A Lopez
- E Camafeita
- E A Fernandez
- J P Albar
- MR Girotti