Naïve pluripotent-like characteristics of non-tumorigenic Muse cells isolated from human amniotic membrane.

Multilineage-differentiating stress-enduring (Muse) cells are non-tumorigenic pluripotent-like stem cells that exhibit triploblastic differentiation and self-renewability at the single-cell level, and are collectable as pluripotent surface marker SSEA-3(+) from the bone marrow (BM), peripheral blood, and organ connective tissues. SSEA-3(+) cells from human amniotic membrane mesenchymal stem cells (hAMSCs) were compared with hBM-Muse cells. Similar to hBM-Muse cells, hAMSC-SSEA-3(+) cells expressed pluripotency genes (OCT3/4, NANOG, and SOX2), differentiated into triploblastic cells from a single cell, self-renewed, and exhibited non-tumorigenicity. Notably, however, they exhibited unique characteristics not seen in hBM-Muse cells, including higher expression of genes related to germline- and extraembryonic cell-lineages compared with those in hBM-Muse cells in single-cell RNA-sequencing; and enhanced expression of markers relevant to germline- (PRDM14, TFAP2C, and NANOS3) and extraembryonic cell- (CDX2, GCM1, and ID2) lineages when induced by cytokine subsets, suggesting a broader differentiation potential similar to naïve pluripotent stem cells. t-SNE dimensionality reduction and Gene ontology analysis visualized hAMSC-SSEA-3(+) cells comprised a large undifferentiated subpopulation between epithelial- and mesenchymal-cell states and a small mesenchymal subpopulation expressing genes relevant to the placental formation. The AM is easily accessible by noninvasive approaches. These unique cells are a potentially interesting target naïve pluripotent stem cell-like resource without tumorigenicity.

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.

GEP100-Arf6-AMAP1-cortactin pathway frequently used in cancer invasion is activated by VEGFR2 to promote angiogenesis.

Angiogenesis and cancer invasiveness greatly contribute to cancer malignancy.Arf6 and its effector, AMAP1, are frequently overexpressed in breast cancer, and constitute a central pathway to induce the invasion and metastasis. In this pathway, Arf6 is activated by EGFR via GEP100. Arf6 is highly expressed also in human umbilical vein endothelial cells (HUVECs) and is implicated in angiogenesis. Here, we found that HUVECs also highly express AMAP1, and that vascular endothelial growth factor receptor-2 (VEGFR2) recruits GEP100 to activate Arf6. AMAP1 functions by binding to cortactin in cancer invasion and metastasis. We demonstrate that the same GEP100-Arf6-AMAP1-cortactin pathway is essential for angiogenesis activities, including cell migration and tubular formation, as well as for the enhancement of cell permeability and VE-cadherin endocytosis of VEGF-stimulated HUVECs. Components of this pathway are highly expressed in pathologic angiogenesis, and blocking of this pathway effectively inhibits VEGF- or tumor-induced angiogenesis and choroidal neovascularization. The GEP100-Arf6-AMAP1-cortactin pathway, activated by receptor tyrosine kinases, appears to be common in angiogenesis and cancer invasion and metastasis, and provides their new therapeutic targets.

The EGFR-GEP100-Arf6-AMAP1 signaling pathway specific to breast cancer invasion and metastasis.

Tumors are tissue-specific diseases, and their mechanisms of invasion and metastasis are highly diverse. In breast cancer, biomarkers that specifically correlate with the invasive phenotypes have not been clearly identified. A small GTPase Arf6 primarily regulates recycling of plasma membrane components. We have shown that Arf6 and its effector AMAP1 (DDEF1, DEF1, ASAP1 and centaurin beta4) are abnormally overexpressed in some breast cancers and used for their invasion and metastasis. Overexpression of these proteins is independent of the transcriptional upregulation of their genes, and occurs only in highly malignant breast cancer cells. We recently identified GEP100 (BRAG2) to be responsible for the Arf6 activation to induce invasion and metastasis, by directly binding to ligand-activated epidermal growth factor receptor (EGFR). A series of our studies revealed that for activation of the invasion pathway of EGFR, it is prerequisite that Arf6 and AMAP1 both are highly overexpressed, and that EGFR is activated by ligands. Pathological analyses indicate that a significant large population of human ductal cancers may utilize the EGFR-GEP100-Arf6-AMAP1 pathway for their malignancy. Microenvironments have been highly implicated in the malignancy of mammary tumors. Our results reveal an aspect of the precise molecular mechanisms of some breast cancers, in which full invasiveness is not acquired just by intracellular alterations of cancer cells, but extracellular factors from microenvironments may also be necessary. Possible translation of our knowledge to cancer therapeutics will also be discussed.

Effects and risks of radiofrequency ablation on the pulmonary tissue and vascular system: a preliminary histological study.

PURPOSE: Radiofrequency ablation (RFA) is one of the minimally invasive therapeutic modalities for malignant tumors. However, the effects and risks associated with RFA on pulmonary tumors have not yet been well investigated. We pathologically examined the effects of RFA on the healthy pulmonary parenchyma in the inflated and deflated states of swine lungs. METHODS: We used two 2-year-old female pigs. Under general anesthesia, RFA was performed twice. RESULTS: The mean duration of cauterization for the inflated lungs (93.0 +/- 65.4 s) was significantly shorter than that for the deflated lungs (390.3 +/- 75.6 seconds) (P = 0.0062). Histologically, the dilatation and congestion of the microvessels were more marked in the lungs. However, no histopathological differences were noticed between the inflated and deflated lungs. Adventitial degeneration was seen in the small vessels, but not in the wall of the large vessels. CONCLUSIONS: This study confirmed, pathologically, that RFA is effective with an acceptable degree of minimal damage to the normal lung tissue, irrespective of its conditions, i.e., inflated or deflated, and can be safely done near the pulmonary vessels. Therefore, RFA appears to be a promising therapeutic strategy to control local lesions in the field of thoracic surgery.

Perimembrane Aurora-A expression is a significant prognostic factor in correlation with proliferative activity in non-small-cell lung cancer (NSCLC).

PURPOSE: Aurora-A, also known as STK15/BTAK, is a member of the protein serine/threonine kinase family, and experimental studies have revealed that Aurora-A plays critical roles in cell mitosis and in carcinogenesis. However, no clinical studies on Aurora-A expression in non-small-cell lung cancer (NSCLC) have been reported. Thus, the present study was conducted to assess the clinical significance of Aurora-A status. EXPERIMENTAL DESIGN: A total of 189 consecutive patients with resected pathologic (p-)stage I-IIIA, NSCLC were retrospectively reviewed, and immunohistochemical staining was used to detect Aurora-A expression. RESULTS: Aurora-A expression was negative in 31 patients (16.4%); among Aurora-A positive patients, 124 patients showed pure diffuse cytoplasmic Aurora-A expression and the other 34 patients showed perimembrane Aurora-A expression. Perimembrane Aurora-A tumors showed the highest proliferative index (PI) (mean PIs for negative, diffuse cytoplasmic, and perimembrane tumors: 49.2, 41.7, and 63.5, respectively; P < .001). Five-year survival rates of Aurora-A negative, diffuse cytoplasmic, and perimembrane patients were 67.8%, 66.7%, and 47.6%, respectively, showing the poorest postoperative survival in perimembrane patients (P = .033). Subset analyses revealed that perimembrane Aurora-A expression was a significant factor to predict a poor prognosis in squamous cell carcinoma patients, not in adenocarcinoma patients. A multivariate analysis confirmed that perimembrane Aurora-A expression was an independent and significant factor to predict a poor prognosis. CONCLUSIONS: Perimembrane Aurora-A status was a significant factor to predict a poor prognosis in correlation with enhanced proliferative activity in NSCLC.

GEP100 links epidermal growth factor receptor signalling to Arf6 activation to induce breast cancer invasion.

Epidermal growth factor (EGF) receptor (EGFR) signalling is implicated in tumour invasion and metastasis. However, whether there are EGFR signalling pathways specifically used for tumour invasion still remains elusive. Overexpression of Arf6 and its effector, AMAP1, correlates with and is crucial for the invasive phenotypes of different breast cancer cells. Here we identify the mechanism by which Arf6 is activated to induce tumour invasion. We found that GEP100/BRAG2, a guanine nucleotide exchanging factor (GEF) for Arf6, is responsible for the invasive activity of MDA-MB-231 breast cancer cells, whereas the other ArfGEFs are not. GEP100, through its pleckstrin homology domain, bound directly to Tyr1068/1086-phosphorylated EGFR to activate Arf6. Overexpression of GEP100, together with Arf6, caused non-invasive MCF7 cells to become invasive, which was dependent on EGF stimulation. Moreover, GEP100 knockdown blocked tumour metastasis. GEP100 was expressed in 70% of primary breast ductal carcinomas, and was preferentially co-expressed with EGFR in the malignant cases. Our results indicate that GEP100 links EGFR signalling to Arf6 activation to induce invasive activities of some breast cancer cells, and hence may contribute to their metastasis and malignancy.

The EGFR-GEP100-Arf6 pathway in breast cancer: Full invasiveness is not from the inside.

Arf6 and its effector AMAP1 are overexpressed in malignant breast cancer cells, and are involved in their invasion and metastasis. We recently revealed that GEP100, a guanine nucleotide exchanging factor, is responsible for the activation of Arf6 which induces invasion and metastasis. GEP100 associated directly with ligand-activated epidermal growth factor receptor (EGFR) to be activated. Disruption of E-cadherin-mediated cell-cell adhesion is one of the major steps involved in acquisition of invasive phenotypes of most carcinomas. The EGFR-GEP100-Arf6 pathway not only activated matrix invasion activity but also perturbed E-cadherin function. GEP100 was found to be expressed in more than 80% of invasive ductal carcinomas. However, 60% of ductal carcinomas in situ were also positive for GEP100, in which GEP100 was preferentially coexpressed with EGFR in their malignant cases. Microenvionments have been highly implicated in the development of tumor malignancy. Our results reveal an aspect of the precise molecular mechanism of cancer invasion and metastasis, in which full invasiveness is not acquired just by alterations of cancer cells themselves, but their microenvironments may also play pivotal roles.

The ratio of membrane-bound form Flt-1 mRNA to VEGF mRNA correlates with tumor angiogenesis and prognosis in non-small cell lung cancer.

Fms-like tyrosine kinase 1 (Flt-1), a receptor for vascular endothelial growth factor (VEGF), have two isoforms: membrane-bound form (mFlt-1) and soluble form. In the present study, we quantitatively evaluated expression level of mFlt-1 mRNA and VEGF mRNA in non-small cell lung cancer, and demonstrated the clinical significance of the ratio of mFlt-1 mRNA to VEGF mRNA (mFlt-1/VEGF). High mFlt-1/VEGF tumor showed a significantly lower microvessel density (P=0.004), and patients with high mFlt-1/VEGF tumor had a significantly favorable survival (P=0.037). Thus, the ratio of mFlt-1 mRNA to VEGF mRNA was inversely correlated with tumor angiogenesis, and was a significant prognostic factor.

Limited surgery and radiofrequency ablation for recurrent lung cancer.

A 72-year-old man who had been diagnosed with second recurrent lung cancer was referred for consideration of a surgical resection. He had undergone a right upper lobectomy with bronchoplasty for the primary lung cancer and stereotactic radiation therapy for the first recurrent tumor. Owing to the comorbid diseases, video-assisted wedge resection and radiofrequency ablation were performed for the second recurrent tumor. The postoperative course was uneventful and the patient was discharged home without complaints. Eighteen months after surgery, he is alive without recurrence. These procedures are minimally invasive and may decrease the local recurrence of lung cancer in properly selected patients.

Systemic inflammatory response syndrome and surgical stress in thoracic surgery.

PURPOSE: To evaluate the clinical usefulness of postoperative systemic inflammatory response syndrome (SIRS) as an index of surgical stress in patients undergoing thoracic surgery. METHODS: Forty-five consecutive patients who underwent thoracic surgery with thoracotomy were enrolled. The SIRS criteria were examined daily during the first 7 postoperative days. The serum interleukin-6 (IL-6) level, operation time, intraoperative blood loss, amount of thoracic drainage, and C-reactive protein levels were also measured. RESULTS: Sixteen cases were categorized into the SIRS group, whereas 29 cases were categorized into the non-SIRS group. Among the patients who underwent thoracic surgery, the physiological responses of the patients to the surgery, such as serum IL-6 levels and C-reactive protein levels, were significantly higher in the SIRS group than in the non-SIRS group (P = .002 and .024, respectively). The serum IL-6 level on the first postoperative day was an independent factor associated with SIRS (95% CI 1.002-1.041; P = .030). Furthermore, there was a correlation between the number of SIRS days and the duration of the postoperative hospital stay (r = 0.379, P = .012). CONCLUSIONS: Our results demonstrated that SIRS reflected the degree of surgical stress, especially thoracotomic procedures, through the IL-6 levels, and affected the postoperative hospital stay. Systemic inflammatory response syndrome can be useful for the postoperative management of patients undergoing thoracic surgery.

Targeting AMAP1 and cortactin binding bearing an atypical src homology 3/proline interface for prevention of breast cancer invasion and metastasis.

Invasive potentials of carcinomas greatly contribute to their metastasis, which is a major threat in most cancers. We have recently shown that Arf6 plays a pivotal role in breast cancer invasive activities and identified AMAP1 as an effector of GTP-Arf6 in invasion. Expression of AMAP1 correlates well with invasive phenotypes of primary tumors of the human breast. We also have shown that AMAP1 functions by forming a trimeric protein complex with cortactin and paxillin. In this complex, AMAP1 binds to the src homology 3 (SH3) domain of cortactin via its proline-rich peptide, SKKRPPPPPPGHKRT. SH3 domains are known to bind generally to the proline-rich ligands with a one-to-one stoichiometry. We found that AMAP1/cortactin binding is very atypical in its stoichiometry and interface structure, in which one AMAP1 proline-rich peptide binds to two cortactin SH3 domains simultaneously. We made a cell-permeable peptide derived from the AMAP1 peptide, and we show that this peptide specifically blocks AMAP1/cortactin binding, but not other canonical SH3/proline bindings, and effectively inhibits breast cancer invasion and metastasis. Moreover, this peptide was found to block invasion of other types of cancers, such as glioblastomas and lung carcinomas. We also found that a small-molecule compound, UCS15A, which was previously judged as a weak inhibitor against canonical SH3/proline bindings, effectively inhibits AMAP1/cortactin binding and breast cancer invasion and metastasis. Together with fine structural analysis, we propose that the AMAP1/cortactin complex, which is not detected in normal mammary epithelial cells, is an excellent drug target for cancer therapeutics.

A novel classification of MUC1 expression is correlated with tumor differentiation and postoperative prognosis in non-small cell lung cancer.

BACKGROUND: MUC1 is a transmembrane mucin that plays an important role in tumor progression. Many clinical studies have suggested that the expression pattern of MUC1 core protein can be a useful prognostic marker in various malignancies, but the prognostic significance in non-small cell lung cancer (NSCLC) remains uncertain. We performed a study to assess clinical significance, especially prognostic impact, of MUC1 expression in NSCLC. METHODS: A total of 62 patients with completely resected pathologic stage I to IIIA NSCLC were retrospectively reviewed. Histologic sections cut from primary tumors were immunohistochemically stained with an anti-MUC1 monoclonal antibody (CA15-3, clone DF3), which recognizes unglycosylated epitope of MUC1 core protein. According to MUC1 expression pattern, each patient was classified into the high-grade polarized expression (HP), the low-grade polarized expression (LP), or the depolarized expression (D) group. RESULTS: Twenty-four (38.7%), 21 (33.9%), and 17 (27.4%) patients were classified into the HP group, the LP group, and the D group, respectively. HP was exclusively seen in adenocarcinoma, mostly in well-differentiated adenocarcinoma. D was correlated with progressive stage and lymph node metastasis. Postoperative survival of the D group seemed to be poorer than that of the HP group for all NSCLC patients, and the difference was enhanced in adenocarcinoma patients. CONCLUSION: A novel classification of MUC1 expression pattern (HP, LP, and D) was correlated with tumor differentiation and postoperative survival in NSCLC, especially in lung adenocarcinoma.

Carbonyl reductase expression and its clinical significance in non-small-cell lung cancer.

Carbonyl reductase (CBR) is a cytosolic NADPH-dependent oxidoreductase metabolizing prostaglandins, steroids, quinines, and anthracycline antibiotics. Many experimental studies have shown that CBR plays important roles in the regulation of tumor progression, but clinical significance of CBR status remains unclear. Thus, we conducted a retrospective study on CBR mRNA expression in lung cancer. Tumor tissues obtained from 59 non-small-cell lung cancer patients were analyzed by quantitative real-time reverse transcription-PCR assay to reveal clinical significance of CBR expression. Angiogenesis was measured immunohistochemically as intratumoral microvessel density (IMVD) using anti-CD34 monoclonal antibody CD34-IMVD) and anti-CD105 monoclonal antibody (CD105-IMVD). CBR mRNA expression was significantly reduced along with progression of primary tumors (the mean CBR mRNA/GAPDH mRNA, 3.288x10(-2) for pT1, 1.628x10(-2) for pT2, and 1.175x10(-2) for pT3-4 disease, respectively; P=0.02). Moreover, CBR mRNA expression in tumor with nodal involvement seemed to be reduced as compared with that in tumor without nodal involvement (the mean CBR mRNA/GAPDH mRNA, 1.446x10(-2) and 2.531x10(-2), respectively), whereas the difference did not reach a statistical significance (P=0.09). The mean CD105-IMVD for CBR-high tumor was 59.2, which was significantly lower than that for CBR-low tumor (130.6, P=0.02), whereas no significant difference between the mean CD34-IMVDs for CBR-high tumor and CBR-low tumor was found. The 5-year survival rate of CBR-high patients was 68.3%, significantly higher than that of CBR-low patients (36.5%; P=0.03). A multivariate analysis confirmed that CBR-high expression was a significant factor to predict a favorable prognosis (P=0.04; relative risk, 0.39; 95% confidence interval, 0.16-0.98). Expression of CBR mRNA was a significant prognostic factor in non-small-cell lung cancer and was inversely associated with tumor progression and angiogenesis.

Expression of AMAP1, an ArfGAP, provides novel targets to inhibit breast cancer invasive activities.

Identification of the molecular machinery employed in cancer invasion, but not in normal adult cells, will greatly contribute to cancer therapeutics. Here we found that an ArfGAP, AMAP1/PAG2, is expressed at high levels in highly invasive breast cancer cells, but at very low levels in noninvasive breast cancer cells and normal mammary epithelial cells. siRNA-mediated silencing of AMAP1 effectively blocked the invasive activities. AMAP1 expression in human breast primary tumors also indicated its potential correlation with malignancy. Paxillin and cortactin have been shown to colocalize at invadopodia and play a pivotal role in breast cancer invasion. We found that AMAP1 is also localized at invadopodia, and acts to bridge paxillin and cortactin. This AMAP1-mediated trimeric protein complex was detected only in invasive cancer cells, and blocking this complex formation effectively inhibited their invasive activities in vitro and metastasis in mice. Our results indicate that AMAP1 is a component involved in invasive activities of different breast cancers, and provide new information regarding the possible therapeutic targets for prevention of breast cancer invasion and metastasis.

Flt-4-positive endothelial cell density and its clinical significance in non-small cell lung cancer.

PURPOSE: Experimental studies have revealed that fms-like tyrosine kinase (Flt)-4 plays important roles in lymphangiogenesis in malignant tumors, but the clinical significance remains unclear. We assessed Flt-4 expression in tumor cells and in endothelial cells in correlation with clinical outcomes in non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: A total of 206 consecutive patients with resected pathological stage I-IIIA NSCLC were reviewed. Expression of Flt-4 was examined immunohistochemically, and Flt-4-positive microvessels were quantitatively evaluated (Flt-4-positive endothelial cell density). RESULTS: There was no significant correlation between Flt-4-positive endothelial cell density and any characteristic of patients including nodal metastases. A significant correlation between Flt-4-positive endothelial cell density and Flt-4 status in tumor cells was documented (P < 0.001), but there was no significant difference in the mean Flt-4-positive endothelial cell density according to vascular endothelial growth factor-C or -D status in tumor cells. The 5-year survival rate for higher Flt-4-positive endothelial cell density tumor (56.4%) was significantly lower than that of lower Flt-4-positive endothelial cell density tumor (69.0%, P = 0.046); the prognostic significance was enhanced in pIIIA-N2 patients (5-year survival rates, 18.8% for higher Flt-4-positive endothelial cell density tumor and 50.0% for lower Flt-4-positive endothelial cell density tumor, respectively; P = 0.012). A multivariate analysis confirmed that higher Flt-4-positive endothelial cell density was a significant and independent prognostic factor (P = 0.019). CD34-positive vessel density or Flt-4 status in tumor cells was not a significant prognostic factor. CONCLUSIONS: Flt-4-positive endothelial cell density, not Flt-4 status in tumor cells, was a significant prognostic factor in NSCLC.

Ultrasonographic evaluation of small nodules in the peripheral lung during video-assisted thoracic surgery (VATS).

OBJECTIVE: We examined the possibility of intraoperative ultrasonography during video-assisted thoracic surgery (VATS) to localize and make a qualitative diagnosis of small peripheral pulmonary nodules. METHODS: Ultrasonography during VATS and conventional thoractomy was performed on 25 and 18 nodules, respectively, all which were localized in the peripheral lung, were less than 30 mm in diameter and for which there was no definitive diagnosis. RESULTS: All 25 nodules, including 10 invisible but palpable and three both invisible and non-palpable, could be localized by ultrasonography during VATS. If nodules were located less than 15 mm from the pleural surface, ultrasonography during VATS could detect nodules 10 mm or less in diameter. The rate of malignant tumors among 11 of 12 pulmonary nodules (91.6%) showing both heterogeneous and ill-defined patterns was significantly higher than 6 of 16 nodules (37.5%) showing both homogeneous and well-defined patterns on ultrasonography. CONCLUSIONS: Our study suggested that ultrasonography during VATS is useful for the detection of peripheral pulmonary nodules, even when they are not identified on video images or palpation, and may enable a differential diagnosis between malignant and non-malignant lesions.

Endobronchial closure of postoperative bronchopleural fistula using vascular occluding coils and n-butyl-2-cyanoacrylate.

We report herein 2 patients with intractable postoperative bronchopleural fistula with empyema after lobectomy or subsegmentectomy. The patients underwent several treatments including thoracotomy, but the fistula closure was not successful. Finally, the bronchopleural fistula was successfully treated by endobronchial closure using vascular occluding coils and n-butyl-2-cyanoacrylate (Histoacryl).