Yutaro Otsuka

Engagement of Overexpressed Her2 with GEP100 Induces Autonomous Invasive Activities and Provides a Biomarker for Metastases of Lung Adenocarcinoma

Overexpression of Her2/ErbB2/Neu in cancer is often correlated with recurrent distant metastasis, although the mechanism still remains largely elusive. We have previously shown that EGFR, when tyrosine-phosphorylated, binds to GEP100/BRAG2 to activate Arf6, which induces cancer invasion and metastasis. We now show that overexpressed Her2 in lung adenocarcinoma cells also employs GEP100. Like EGFR-GEP100 binding, this association is primarily mediated by the pleckstrin homology (PH) domain of GEP100 and Tyr1139/Tyr1196 of Her2. Tyr1139/Tyr1196 are autonomously phosphorylated, when Her2 is overexpressed. Accordingly, invasive activities mediated by the Her2-GEP100 pathway are not dependent on external factors. Blocking Her2-GEP100 binding, as well as its signaling pathway all inhibit cancer invasive activities. Moreover, our clinical study indicates that co-overexpression of Her2 with GEP100 in primary lung adenocarcinomas of patients is correlated with the presence of their node-metastasis with a statistical significance. Since the GEP100 PH domain interacts with both Her2 and EGFR, targeting this domain may provide novel cancer therapeutics.

P53- and mevalonate pathway–driven malignancies require Arf6 for metastasis and drug resistance

The mevalonate pathway (MVP) is a metabolic pathway associated with tumor invasiveness and is known to prenylate proteins, but which prenylated proteins are critical for MVP-driven cancers is unknown. Hashimoto et al. show that MVP-driven cancers require activation of the GTPase Arf6 for invasion and that the MVP substrate Rab11 is required for Arf6 activation.

Acquired platinum resistance involves epithelial to mesenchymal transition through ubiquitin ligase FBXO32 dysregulation

To identify the molecules involved in epithelial to mesenchymal transition (EMT) in urothelial carcinoma (UC) after acquisition of platinum resistance, here we examined the changes in global gene expression before and after platinum treatment. Four invasive UC cell lines, T24, 5637, and their corresponding sublines T24PR and 5637PR with acquired platinum resistance, were assessed by microarray, and the ubiquitin E3 ligase FBXO32 was newly identified as a negative regulator of EMT in UC tumors after acquisition of platinum resistance. In vitro and in vivo studies showed an intimate relationship between FBXO32 expression and EMT, demonstrating that FBXO32 dysregulation in T24PR cells results in elevated expression of the mesenchymal molecules SNAIL and vimentin and decreased expression of the epithelial molecule E-cadherin. The association between FBXO32 expression and EMT was further validated using clinical samples. Knockdown of MyoD expression, a specific target of FBXO32 polyubiquitination, revealed upregulation of E-cadherin expression and downregulation of SNAIL and vimentin expression in T24PR cells. Comparative genomic hybridization array analysis demonstrated loss of heterozygosity at 8q24.13 in T24PR cells, which harbors FBXO32. Our findings suggest the importance of the association between EMT and ubiquitin-proteasome regulation when tumors develop acquired platinum resistance.

High expression of EPB41L5, an integral component of the Arf6-driven mesenchymal program, correlates with poor prognosis of squamous cell carcinoma of the tongue

BackgroundSquamous cell carcinoma of the tongue (tongue SCC) is a major subtype of head and neck squamous cell carcinoma (HNSCC), which is an intractable cancer under current therapeutics. ARF6 and its effector AMAP1 are often overexpressed in different types of cancers, such as breast cancer and renal cancer, and in these cancers, AMAP1 binds to EPB41L5 to promote invasion, metastasis, and drug resistance. EPB41L5 is a mesenchymal-specific protein, normally induced during epithelial-mesenchymal transition (EMT) to promote focal adhesion dynamics. Similarly to breast cancer and renal cancer, the acquisition of mesenchymal phenotypes is the key process that drives the malignancy of HNSCC. We previously showed that the overexpression of AMAP1 in tongue SCC is statistically correlated with the poor outcome of patients. In this study, we examined whether tongue SCC also expresses EPB41L5 at high levels.ResultsImmunohistochemical staining of clinical specimens of tongue SCC demonstrated that high expression levels of EPB41L5 statistically correlate with poor disease-free survival and poor overall survival rates of patients. The tongue SCC cell line SCC-9, which overexpress Arf6 and AMAP1, also expressed EPB41L5 at high levels to promote invasiveness, whereas the weakly invasive SCC-25 cells did not express EPB41L5 at notable levels. Among the different EMT-associated transcriptional factors, ZEB1 was previously found to be most crucial in inducing EPB41L5 in breast cancer and renal cancer. In contrast, expression levels of ZEB1 did not correlate with the expression levels of EPB41L5 in tongue SCC, whereas KLF8 and FOXO3 levels showed positive correlations with EPB41L5 levels. Moreover, silencing of EPB41L5 only marginally improved the drug resistance of SCC-9 cells, even when coupled with ionizing radiation.ConclusionOur results indicate that activation of the cancer mesenchymal program in tongue SCC, which leads to EPB41L5 expression, closely correlates with the poor prognosis of patients. However, ZEB1 was not the major inducer of EPB41L5 in tongue SCC, unlike in breast cancer and renal cancer. Thus, processes that trigger the mesenchymal program of tongue SCC, which drives their malignancies, seem to be substantially different from those of other cancers.

Necessity of p53-binding to the CDH1 locus for its expression defines two epithelial cell types differing in their integrity

TP53 mutation (i.e., loss of normal-p53) may evoke epithelial-mesenchymal transition (EMT), which was previously attributed to loss of certain miRNAs. However, not all epithelial cells undergo EMT upon TP53 mutation, and the p53-miRNA axis may not fully explain p53 function in epithelial integrity. We here show two modes of epithelial integrity: one involves p53-binding to a nucleotide region and the other does not. In the former, p53 binds to the CDH1 (encoding E-cadherin) locus to antagonize EZH2-mediated H3K27 trimethylation (H3K27me3) to maintain high levels of acetylation of H3K27 (H3K27ac). In the latter, the same locus is not highly acetylated at H3K27, and does not allow p53-binding, nor needs to antagonize EZH2. We moreover demonstrated that although the CDH1 locus in the p53-independent cells, but not in fibroblasts, becomes high-H3K27ac by butyrate and allows p53-biniding, their CDH1 expression does not become dependent on p53. Our results identified novel modes of the epithelial integrity, in which the same epithelial-specific gene locus exhibits different requirement for p53 with different histone modifications among different epithelial cells to warrant its expression.

Frequent overexpression of AMAP1, an Arf6 effector in cell invasion, is characteristic of the MMTV-PyMT rather than the MMTV-Neu human breast cancer model

BackgroundThe small GTPase Arf6 and its downstream effector AMAP1 (also called ASAP1/DDEF1) constitute a signaling pathway promoting cell invasion, in which AMAP1 interacts with several different proteins, including PRKD2, EPB41L5, paxillin, and cortactin. Components of this pathway are often overexpressed in human breast cancer cells, to be correlated with poor prognosis of the patients, whereas overexpression of the Arf6 pathway did not correlate with the four main molecular classes of human breast tumors. In this pathway, receptor tyrosine kinases, including EGFR and Her2, activate Arf6 via GEP100. MMTV-PyMT mice and MMTV-Neu mice are well-established models of human breast cancer, and exhibit the early dissemination and the lung metastasis, by utilizing protein tyrosine phosphorylation for oncogenesis. PyMT-tumors and Neu-tumors are known to have overlapping gene expression profiles, which primarily correspond to the luminal B-type of human mammary tumors, although they differ in the time necessary for tumor onset and metastasis. Given the common usage of protein tyrosine phosphorylation, as well as the frequent use of these animal models for studying breast cancer at the molecular level, we here investigated whether mammary tumors in these mouse models utilize the Arf6-based pathway for invasion.MethodsExpression levels of Arf6, AMAP1, and GEP100 were analyzed in PyMT-tumors and Neu-tumors by western blotting. Expression of Arf6 and AMAP1 was also analyzed by immunohistochemistry. The involvement of AMAP1 in invasion, and the possible correlation of its high expression levels with cancer mesenchymal properties were also investigated.ResultsWe found that PyMT-tumors, but not Neu-tumors, frequently overexpress AMAP1 and use it for invasion, whereas both types of tumors expressed Arf6 and GEP100 at different levels. High levels of the AMAP1 expression among PyMT-tumor cells were frequently correlated with loss of the epithelial marker CK8 and also with expression of the mesenchymal marker vimentin both at the primary sites and at sites of the lung metastases.ConclusionsPyMT-tumors appear to frequently utilize the Arf6-based invasive machinery, whereas Neu-tumors do not. Our results suggest that MMTV-PyMT mice, rather than MMTV-Neu mice, are useful to study the Arf6-based mammary tumor malignancies, as a representative model of human breast cancer.

mitoNEET Regulates Mitochondrial Iron Homeostasis Interacting with Transferrin Receptor

Iron is an essential trace element for regulation of redox and mitochondrial function, and then mitochondrial iron content is tightly regulated in mammals. We focused on a novel protein localized at the outer mitochondrial membrane. Immunoelectron microscopy revealed transferrin receptor (TfR) displayed an intimate relationship with the mitochondria, and mass spectrometry analysis also revealed mitoNEET interacted with TfR in vitro. Moreover, mitoNEET was endogenously coprecipitated with TfR in the heart, which indicates that mitoNEET also interacts with TfR in vivo. We generated mice with cardiac-specific deletion of mitoNEET (mitoNEET-knockout). Iron contents in isolated mitochondria were significantly increased in mitoNEET-knockout mice compared to control mice. Mitochondrial reactive oxygen species (ROS) were higher, and mitochondrial maximal capacity and reserve capacity were significantly decreased in mitoNEET-knockout mice, which was consistent with cardiac dysfunction evaluated by echocardiography. The complex formation of mitoNEET with TfR may regulate mitochondrial iron contents via an influx of iron. A disruption of mitoNEET could thus be involved in mitochondrial ROS production by iron overload in the heart.

p53-Dependent and -Independent Epithelial Integrity: Beyond miRNAs and Metabolic Fluctuations

In addition to its classical roles as a tumor suppressor, p53 has also been shown to act as a guardian of epithelial integrity by inducing the microRNAs that target transcriptional factors driving epithelial–mesenchymal transition. On the other hand, the ENCODE project demonstrated an enrichment of putative motifs for the binding of p53 in epithelial-specific enhancers, such as CDH1 (encoding E-cadherin) enhancers although its biological significance remained unknown. Recently, we identified two novel modes of epithelial integrity (i.e., maintenance of CDH1 expression): one involves the binding of p53 to a CDH1 enhancer region and the other does not. In the former, the binding of p53 is necessary to maintain permissive histone modifications around the CDH1 transcription start site, whereas in the latter, p53 does not bind to this region nor affect histone modifications. Furthermore, these mechanisms likely coexisted within the same tissue. Thus, the mechanisms involved in epithelial integrity appear to be much more complex than previously thought. In this review, we describe our findings, which may instigate further experimental scrutiny towards understanding the whole picture of epithelial integrity as well as the related complex asymmetrical functions of p53. Such understanding will be important not only for cancer biology but also for the safety of regenerative medicine.

Small-Conductance Ca2+-Activated K+ Channel Activation Deteriorates Hypoxic Ventricular Arrhythmias via CaMKII in Cardiac Hypertrophy

The molecular and electrophysiological mechanisms of acute ischemic ventricular arrhythmias in hypertrophied hearts are not well known. We hypothesized that small-conductance Ca2+-activated K+ (SK) channels are activated during hypoxia via the Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent pathway. We used normotensive Wistar-Kyoto (WKY) rats and spontaneous hypertensive rats (SHRs) as a model of cardiac hypertrophy. The inhibitory effects of SK channels and ATP-sensitive K+ channels on electrophysiological changes and genesis of arrhythmias during simulated global hypoxia (GH) were evaluated. Hypoxia-induced abbreviation of action potential duration (APD) occurred earlier in ventricles from SHRs versus. WKY rats. Apamin, a SK channel blocker, prevented this abbreviation in SHRs in both the early and delayed phase of GH, whereas in WKY rats only the delayed phase was prevented. In contrast, SHRs were less sensitive to glibenclamide, a ATP-sensitive K+ channel blocker, which inhibited the APD abbreviation in both phases of GH in WKY rats. SK channel blockers (apamin and UCL-1684) reduced the incidence of hypoxia-induced sustained ventricular arrhythmias in SHRs but not in WKY rats. Among three SK channel isoforms, SK2 channels were directly coimmunoprecipitated with CaMKII phosphorylated at Thr286 (p-CaMKII). We conclude that activation of SK channels leads to the APD abbreviation and sustained ventricular arrhythmias during simulated hypoxia, especially in hypertrophied hearts. This mechanism may result from p-CaMKII-bound SK2 channels and reveal new molecular targets to prevent lethal ventricular arrhythmias during acute hypoxia in cardiac hypertrophy. NEW & NOTEWORTHY We now show a new pathophysiological role of small-conductance Ca2+-activated K+ channels, which shorten the action potential duration and induce ventricular arrhythmias during hypoxia. We also demonstrate that small-conductance Ca2+-activated K+ channels interact with phosphorylated Ca2+/calmodulin-dependent protein kinase II at Thr286 in hypertrophied hearts.

ArfGAPs: Not Only for the Termination

While Arf-family small GTPases (Arf-GTPases) consist of 5 members in humans, 31 human genes have been identified that encode proteins bearing the GTPase-activating protein (GAP) domain for Arf-GTPases. Interestingly, Arf1, the first identified Arf, was shown to substantially lack intrinsic GTPase activity, which other Ras-superfamily members of small GTPases generally bear. Likewise, ArfGAP domains primarily consist of zinc-finger structures, and do not resemble GAP domains for other small GTPases. Arfs primarily function in intracellular vesicle/membrane trafficking. A general model shows that Arfs play roles in membrane budding, in which GTP-Arfs recruit coatomer proteins to generate and maintain membrane curvature to initiate the budding. Coatomers are thought to be separated from Arf-mediated vesicles before they reach the target membrane, while this separation may or may not be coupled with the GTP hydrolysis activity. We have shown that several ArfGAPs, such as AMAP1 and AMAP2, have the ability to bind stably to GTP-Arf6, without immediate GTP hydrolysis. They each contain a BAR domain and hence may act as coatomers for Arf-mediated vesicles. These ArfGAPs moreover act to recruit their binding proteins to sites of Arf6 activation, which are not coatomer components. These findings have amended the classical, general model of the functions of ArfGAPs, as well as Arf-GTPases. In this review, we will describe the recent information revealed about ArfGAPs, with the aim to decipher and discuss their fundamental roles.