Mikiko Aoki

Prognostic significance of AKT/mTOR and MAPK pathways and antitumor effect of mTOR inhibitor in NF1-related and sporadic malignant peripheral nerve sheath tumors

Purpose: Malignant peripheral nerve sheath tumor (MPNST) is a rare soft tissue sarcoma with poor prognosis. MPNSTs occur frequently in patients with neurofibromatosis type 1 (NF1), in which NF1 gene deficiency leads to Ras hyperactivation. Ras activation causes the subsequent activation of the AKT/mTOR and Raf/MEK/ERK pathways and regulates cellular functions. However, the activation profiles of the AKT/mTOR and MAPK pathways in MPNSTs are poorly understood. The purposes of this study are to examine the correlation between the activation of these pathways and clinicopathologic or prognostic factors and to identify candidate target molecules in MPNST. Moreover, we assessed the antitumor effects of the inhibitor of candidate target. Experimental Design: Immunohistochemistry was conducted to evaluate the activation profiles of AKT/mTOR and MAPK pathways using 135 tumor specimens. Immunohistochemical expressions were confirmed by Western blotting. Then, an in vitro study was conducted to examine the antitumor effect of the mTOR inhibitor on MPNST cell lines. Results: Phosphorylated-AKT (p-AKT), p-mTOR, p-S6RP, p-p70S6K, p-4E-BP1, p-MEK1/2, and p-ERK1/2 expressions were positive in 58.2%, 47.3%, 53.8%, 57.1%, 62.6%, 93.4%, and 81.3% of primary MPNSTs, respectively. Positivity for each factor showed no difference between NF1-related and sporadic MPNSTs. Univariate prognostic analysis revealed that p-AKT, p-mTOR, and p-S6RP expressions were associated with poor prognosis. Furthermore, activation of each p-mTOR and p-S6RP was an independent poor prognostic factor by multivariate analysis. mTOR inhibition by Everolimus showed antitumor activity on MPNST cell lines in vitro. Conclusion: mTOR inhibition is a potential treatment option for both NF1-related and sporadic MPNSTs. Clin Cancer Res; 19(2); 450–61. ©2012 AACR.

Imatinib mesylate inhibits cell invasion of malignant peripheral nerve sheath tumor induced by platelet-derived growth factor-BB.

Malignant peripheral nerve sheath tumor (MPNST) is rare, highly aggressive, resistant to radiochemotherapy, and associated with poor prognosis. Basic research to develop new treatment regimes is critically needed. This study was designed to identify motogenic factor(s) involved in MPNST cell invasion and inhibitor(s) of such invasive activity. We profiled the invasion-inducing activities of eight motogenic growth factors on two human MPNST cell lines, FU-SFT8611 and 9817, using in vitro Matrigel invasion assays. Platelet-derived growth factor-BB (PDGF-BB) was identified as the most effective MPNST cell invasion-inducing factor. Epidermal growth factor (EGF) and hepatocyte growth factor (HGF) also stimulated invasion in one MPNST cell line. Expressions of PDGF-BB and EGF receptors (PDGFR-β and EGFR) mRNAs were detected more frequently and their proteins were expressed at higher levels in MPNST tissues than benign peripheral nerve sheath tumors (schwannomas and neurofibromas). In both MPNST cell lines, PDGF-BB induced tyrosine phosphorylation of PDGFR-β but not of PDGFR-α, and specific PDGFR-β inhibition by small interfering RNA to the receptor inhibited PDGF-BB-stimulated MPNST cell invasion, suggesting the predominant role of PDGFR-β. Inhibition of PDGFR-β phosphorylation by pretreatment with herbimycin A and imatinib mesylate effectively suppressed basement membrane invasion and cell growth in vitro. No mutations were present in exons 12 and 18 of PDGFR-β in both MPNST cell lines and 10 human MPNST tissues examined. Our results indicated that PDGF-BB enhanced the invasive activity of MPNST cells through PDGFR phosphorylation and that imatinib inhibited such activity. The results provide the ground for further assessment of the therapeutic potential of imatinib in suppressing the invasion and growth of MPNST.

Pathology of soft-tissue tumors: Daily diagnosis, molecular cytogenetics and experimental approach

This article reviews problems in diagnostic pathology and molecular cytogenetics of soft‐tissue tumors. Also discussed are the origin of soft‐tissue sarcomas and the molecular basis of effective target therapy for sarcomas. Molecular cytogenetic analysis of tumor‐specific chromosomal translocations and associated fusion gene transcripts offers a useful adjunct to the diagnosis of soft‐tissue tumors, but recent studies have indicated a growing number of fusion gene variations in each tumor type. In pleomorphic sarcoma/malignant fibrous histiocytoma, the alternative lengthening of telomeres (ALT) mechanism may result in formation of anaphase bridges and marked nuclear pleomorphism. The histogenesis of soft‐tissue sarcomas has been a matter of controversy. In the present experimental model using s.c. injection of 3‐methylcholanthrene in C57BL/6 mice pretreated with bone marrow‐transplantation from green fluorescent protein (GFP)‐positive green mice, the bone marrow‐derived mesenchymal stem cells as well as the tissue‐resident mesenchymal cells in the peripheral soft tissues are possible originators of sarcomagenesis. Little is known about a molecular basis of target therapy for sarcomas. Platelet‐derived growth factor‐BB (PDGF‐BB) enhances the invasive activity of malignant peripheral nerve sheath tumor (MPNST) cells through platelet‐derived growth factor receptor (PDGFR) phosphorylation, whereas imatinib mesylate inhibited such activity, suggesting that targeting PDGFR‐β may result in the establishment of novel treatment for MPNST. In addition, emmprin is a transmembrane glycoprotein on tumor cells that stimulates peritumoral fibroblasts to produce matrix metalloproteinases (MMP), playing a crucial role in tumor progression, invasion and metastasis. The MMP upregulation mechanism mediated by tumor‐associated emmprin may be a potentially useful target in anti‐tumor invasion therapy for sarcomas.

Overexpression of IQGAP1 in advanced colorectal cancer correlates with poor prognosis—critical role in tumor invasion

IQGAP1 is a multifunctional protein involved in actin cytoskeleton assembly and E‐cadherin‐mediated cell adhesion. We reported previously IQGAP1 overexpression in human colorectal carcinomas especially at the invasion front (IF) and that such overexpression tended to correlate with lymph node metastasis in advanced cases. Thus, in this study, we investigated the clinicopathological significance of IQGAP1 expression in 85 cases of pT2–3 colorectal carcinomas with special reference to its expression pattern and prognosis, followed by analysis of the role of IQGAP1 in cancer invasion in vitro. Quantitative reverse transcription‐PCR showed significant upregulation of IQGAP1 in colorectal carcinomas compared with normal mucosa. Immunohistochemically, IQGAP1 expression pattern was classified into diffuse (20%), IF‐associated (35.3%) and focal (44.7%). The diffuse pattern was associated with higher rates of distant metastasis. Patients with IQGAP1 overexpression and diffuse pattern had significantly shorter survival (p < 0.0001) than others, and the diffuse pattern was an independent predictor of poor survival by multivariate analysis. In vitro invasion assays using three human colon carcinoma cell lines showed that IQGAP1 siRNA significantly suppressed hepatocyte growth factor (HGF)‐stimulated cell invasion. HGF reduced membranous localization of α‐catenin, but did not alter localization of E‐cadherin, β‐catenin and IQGAP1 in membranes. Suppression of IQGAP1 expression by siRNA did not alter membranous localization of α‐catenin even in the presence of HGF. Our results indicate that IQGAP1 plays a critical role in colon cancer cell invasion, and therefore diffuse and high expression of IQGAP1 predicts poor prognosis in patients with colorectal carcinoma.

Expression of laminin 5-γ2 chain in cutaneous squamous cell carcinoma and its role in tumour invasion

Background:Laminin-5 (Ln5), a heterotrimer composed of three chains (α3, β3, and γ2), is a major component of the basement membrane in most adult tissues. One of the chains, Ln5-γ2, is a marker of invasive tumours because it is frequently expressed as a monomer in malignant tumours. Recent studies from our laboratories detected higher levels of Ln5-γ2 expression in basal cell carcinoma (BCC) than in trichoblastoma. Furthermore, Ln5-γ2 overexpression tended to correlate with aggressiveness in BCC.Methods:In this study, we compared the expression of Ln5-γ2 in invasive squamous cell carcinoma (SCC, n=62) of the skin to that in preinvasive Bowen’s disease (BD, n=51), followed by analysis of the role of Ln5-γ2 in cancer invasion in vitro.Results:Immunohistochemically, the proportion of SCC cases (86%) strongly positive for Ln5-γ2 expression was higher than that of BD (16%). Real-time RT–PCR showed Ln5-γ2 overexpression in SCC cell line, A431, compared with normal keratinocyte cell line, HaCaT. Ln5-γ2 monomer and proteolytically cleaved, biologically active fragments of Ln5-γ2 were identified in SCC tumour extracts. In in vitro raft cultures, which simulate in vivo conditions, Ln5-γ2 siRNA significantly suppressed epidermal growth factor (EGF)-stimulated A431 cell invasion.Conclusion:Our results indicate that Ln5-γ2 has a role in cutaneous SCC invasion.

Emmprin in epithelioid sarcoma : Expression in tumor cell membrane and stimulation of MMP-2 production in tumor-associated fibroblasts

Emmprin is a transmembrane glycoprotein on tumor cells that stimulates peritumoral fibroblasts to produce matrix metalloproteinases (MMPs). Emmprin and the induced MMPs play a crucial role in tumor progression, invasion and metastasis of human carcinomas (epithelial malignancies). However, only a few reports have addressed its role in soft tissue sarcomas. This study investigated the expression and role of emmprin in epithelioid sarcoma (ES). Immunoblot studies of 2 ES cell lines showed that they express emmprin, and co‐culture of these ES cells with dermal fibroblasts resulted in upregulation of gelatinase A (MMP‐2) in fibroblasts, as shown by zymography, immunoblotting and enzyme immunoassay. This stimulation was inhibited by an activity‐blocking peptide against emmprin and by antiemmprin antibody. In addition, in vivo, immunohistochemical analysis of 5 ES patient cases demonstrated diffuse emmprin expression in ES cells and MMP‐2 expression in both ES cells and peritumoral fibroblasts. The histopathological findings that peritumoral fibroblasts that were not in direct contact with emmprin‐expressing ES cells exhibit upregulated MMP‐2 prompted us to look for a soluble form of emmprin. Soluble full‐length emmprin released from ES cells was detected in conditioned medium and shown to stimulate MMP‐2 production by fibroblasts. In conclusion, emmprin is expressed in ES in both membrane and soluble forms and stimulates MMP‐2 production via interactions with fibroblasts, which could play a role in ES cell stromal invasion and vascular involvement.

Association of c-Met phosphorylation with micropapillary pattern and small cluster invasion in pT1-size lung adenocarcinoma

Lung adenocarcinomas with micropapillary pattern (MPP) are associated with frequent nodal metastasis. However, little is known about the mechanisms that underlie MPP-associated nodal metastasis. We have previously reported that pT1 lung adenocarcinomas with MPP are significantly associated with small cluster invasion (SCI) and lymphatic involvement. SCI is defined as markedly resolved acinar-papillary tumor structures with single or small clusters of carcinoma cells invading stroma within fibrotic foci. In this study, we hypothesized that c-Met activation may be involved in the MPP-SCI sequence, given that the c-Met tyrosine-kinase receptor and its ligand hepatocyte growth factor (HGF), play important roles in tumor cell motility and invasion. We analyzed 125 pT1-size lung adenocarcinomas for immunohistochemical expression of phosphorylated c-Met and its correlation with MPP, SCI, lymphatic involvement and prognosis. SCI was significantly more frequent in the MPP-positive group (P<0.0001) and associated with lymphatic involvement (P<0.0001) and nodal metastasis (P=0.021). c-Met protein was detected in all tumors by immunohistochemistry as membranous and cytoplasmic staining. Phospho-c-Met (pc-Met) was positive in 119/125 tumors (95%) and expressed at high levels in 27 cases (22%). A high level of pc-Met expression was significantly associated with MPP (P=0.01) and SCI (P=0.0059). Moreover, in tumors with MPP or SCI, those expressing high levels of pc-Met were significantly more associated with lymphatic involvement. In p-Stage IA lung adenocarcinomas (n=99), patients in the high pc-Met expression group showed significantly worse survival than patient in the low expression group (P=0.0313). These results suggest that activation of c-Met through phosphorylation may be involved in MPP and SCI.

Synthetic emmprin peptides inhibit tumor cell-fibroblast interaction-stimulated upregulation of MMP-2 and tumor cell invasion.

Stromal cells are the main source of matrix metalloproteinases (MMPs) in human carcinoma tissues. Emmprin is a glycosylated transmembrane protein containing two immunoglobulin (Ig) domains that is expressed in carcinoma cells and stimulates MMP production by adjacent stromal cells. The first Ig domain (ECI) of emmprin contains the biologically active site. We investigated whether synthetic peptides carrying a partial ECI sequence could inhibit emmprin activity. Only the second peptide (emp#2), which contains a putative N-glycosylation site sequence, inhibited emmprin-stimulated production of MMP-2 in co-cultures of fibroblasts and several different human tumor cells types, including carcinoma, sarcoma, melanoma, leukemia and glioma cells. Moreover, emp#2 significantly inhibited the invasive activity of glioblastoma cells promoted by interaction with fibroblasts. Perturbation of emmprin activity by this peptide may have potential therapeutic uses in the prevention of MMP-2-dependent cancer invasion.

Imatinib mesylate inhibits cell growth of malignant peripheral nerve sheath tumors in vitro and in vivo through suppression of PDGFR-β

BackgroundMalignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive and associated with poor prognosis. Basic research to develop new treatment regimens is critically needed.MethodsThe effects of imatinib mesylate on MPNSTs were examined in six human MPNST cell lines and in a xenograft mouse model.ResultsThe results showed expression of platelet-derived growth factor receptor-β and suppression of its phosphorylation by imatinib mesylate in all six cell lines. Imatinib mesylate effectively suppressed MPNST cell growth in vitro at concentrations similar to those used clinically (1.46 − 4.6 μM) in three of six cell lines. Knockdown of PDGFR-β by transfection with a specific siRNA also caused significant reduction in cell proliferation in the sensitive cell lines, but not in the resistant cell lines. Furthermore, imatinib mesylate also significantly suppressed colony formation within soft agar and tumor growth in xenograft models using two of the three sensitive MPNST cell lines. There was excellent agreement between in vitro and in vivo sensitivity to imatinib mesylate, suggesting possible selection of imatinib-sensitive tumors by in vitro analysis.ConclusionsThe results suggest that imatinib mesylate may be useful in the treatment of MPNST patients and in vitro studies may help select cells that are sensitive to imatinib mesylate in vivo.

Tumor budding in colorectal carcinoma assessed by cytokeratin immunostaining and budding areas: Possible involvement of c-Met

Tumor budding/sprouting has been shown to be an independent adverse prognostic factor in T1 and T3N0 colorectal carcinomas, however, its assessment could be improved by more accurate identification of budding carcinoma cells and consideration of budding areas. Moreover, tumor budding mechanisms are yet to be defined. In this study, we evaluated the identification of budding tumor cells by either H&E staining alone or H&E with immunohistochemistry and developed a scoring system based on budding grades and areas. We examined whether the budding score correlated with clinicopathologic features and prognosis and the association between tumor budding/sprouting and c‐Met protein expression and phosphorylation and MET gene copy numbers because c‐Met is known to play an important role in colorectal carcinoma tumorigenesis. Cytokeratin immunohistochemistry could identify tumors with shorter disease‐free survival (DFS) from the low‐grade budding group assessed with H&E alone. High budding scores based on budding grade and area were more significantly correlated with DFS than scores obtained using the budding grade alone. In tumors with a high budding score, c‐Met expression and phosphorylation levels and MET gene copy numbers were significantly increased at the invasive front compared with those in superficial tumor portions. This study showed for the first time that high levels of phospho‐c‐Met at the invasive front were significantly associated with a high budding score and shorter DFS. In conclusion, a budding score assessed by budding grades and budding‐positive areas correlates highly with clinicopathologic aggressive features of colorectal carcinoma.