Search

Hepatocyte growth factor (HGF) has an inhibitory effect on human HepG2 hepatoma cell proliferation. Previously, it was shown that HGF treatment downregulated Id1 and upregulated p16INK4a in an ERK-dependent manner, leading to the inhibition of cellular proliferation. Here, new insight suggests that Skp2, an SCF complex component and potential prognosticator in cancer, is downregulated by injection of HGF into established HepG2 xenograft tumors. The downregulation was evident at both the mRNA and protein level and in an ERK-dependent manner. Critically, high expression of Skp2 restored HGF-inhibited cell proliferation, indicating that the inhibitory effect of HGF required the downregulation of Skp2. However, downregulation was not involved in the HGF-induced upregulation of a CDK inhibitor, p27Kip1, a known SCF-Skp2 target. Instead, data revealed that Skp2 regulated Myc activity, which has oncogenic potential in the generation of hepatocellular carcinoma. Elevated expression of Skp2 or a mutant that is unable to associate with the SCF complex was capable of activating Myc, suggesting that Skp2 does not act on Myc as a component of the SCF complex, and thus functions as an activator of Myc independent of its role in ubiquitination. Furthermore, Skp2 regulated Id1 expression by regulating Myc activity, and the regulation of Skp2 is involved in the activity of p16 promoter through regulation of Id1 expression. Overall, these mechanistic findings provide the first evidence that ERK-dependent downregulation of Skp2 reduced Myc activity, leading to HGF-induced inhibition of cell proliferation through decreased Id1 expression. Implications: This study elucidates the molecular details of HGF-induced inhibition of cellular proliferation in liver cancer cells. Mol Cancer Res; 11(11); 1437–47. ©2013 AACR.

Hepatocyte growth factor (HGF) inhibits the proliferation of several tumor cell lines and tumor growth in vivo. We showed previously that HGF induces cell cycle arrest at G1 in a human hepatoma cell line, HepG2, by up-regulating the expression of p16INK4a through strong activation of extracellular signal-regulated kinase (ERK). However, although essential, the activation was not sufficient for the up-regulation of p16. In this study, we examined regulatory mechanisms of p16 expression through a transcription factor, Ets, which has been shown previously to bind to the promoter. The treatment of HepG2 cells with HGF induced ERK-dependent phosphorylation of Ets, which leads to its activation, before the up-regulation of p16, suggesting that another factor suppresses Ets activity. We found that HGF reduces the amount of Id1, which is a dominant-negative inhibitor of Ets, leading to a decrease in Ets associated with Id1. Id1 was down-regulated via transcriptional regulation not via the ubiquitin-proteasome-mediated pathway. Inhibition of the HGF-induced high-intensity ERK activity had a modest effect on the Id1 down-regulation, and inhibition of the phosphatidylinositol 3-kinase pathway had no effect, showing that Id1 is regulated by ERK-dependent and -independent pathways other than the phosphatidylinositol 3-kinase pathway. Exogenously expressed Id1 suppressed the up-regulation of p16 by HGF and the antiproliferative effect of HGF. Knockdown of Id1 significantly enhanced the activity of the p16 promoter coordinately with the activation of ERK. Our results indicated that down-regulation of Id1 plays a key role in the inhibitory effect of HGF on cell proliferation and provides a molecular basis for cancer therapy with HGF. (Mol Cancer Res 2009;7(7):1179–88)

Background: Neoadjuvant chemotherapy (NAC) is widely performed for patients with locally advanced breast carcinomas. It is important to obtain precise information using imaging modalities about the distribution of residual carcinomas after NAC to predict the success of breast conserving surgery. However, the relationship between magnetic resonance imaging (MRI) shrinkage patterns and molecular subtypes of breast carcinomas has not yet been investigated. Methods: We analyzed the MRI shrinkage and pathological patterns of residual carcinomas after NAC in 54 consecutive breast carcinomas. The shrinkage patterns were classified into five categories: Type I and II (concentric shrinkage with or without any surrounding lesion), type III (shrinkage with residual multinodular lesions), type IV (diffuse contrast enhancement in the entire quadrant), and non-visualization. Results: The most common MRI shrinkage pattern was type I (39%) followed by non-visualization (26%). The most common pathological shrinkage pattern was type III (33%), followed by type II (28%) and non-visualization (22%). The concordance rates of the MRI patterns of non-visualization and type I were significantly lower than those of the MRI patterns of type II, III, and IV (p = 0.018). Moreover, in the luminal subtypes, the concordance rates of the MRI patterns of non-visualization and type I were significantly lower than those of the MRI patterns of type II, III, and IV (p = 0.026); however, in the non-luminal subtypes, this correlation was not significant. Conclusions: The results of the present study suggest that the cases with MRI shrinkage patterns of non-visualization and type I require caution regarding the surgical margins compared with the other types, especially the luminal subtypes. Citation Format: Tsuyoshi Mori, Kaori Tomida, Mina Kitamura, Sachiko Sakai, Yuki Kawai, Tomoharu Shimizu, Tomoko Umeda, Tomoyuki Ueki, Sachiko Kaida, Toru Miyake, Hiroya Iida, Hiroya Akabori, Naomi Kitamura, Tsuyoshi Yamaguchi, Hiromichi Sonoda, Shigeyuki Naka, Masaji Tani. Magnetic resonance imaging shrinkage patterns after neoadjuvant chemotherapy for breast carcinomas: Correlation with molecular subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3743. doi:10.1158/1538-7445.AM2017-3743

Introduction: Hepatocyte growth factor (HGF) is a multifunctional growth factor that acts as mitogen, motogen, and/or morphogen in a variety of cells. MET, a specific receptor tyrosine kinase for HGF, is up-regulated in various tumors including human head and neck squamous cell carcinoma (HNSCC) and its signal transduction induces tumor progression and invasion. However, how HGF affects the expression of downstream functional gene expression has not yet been elucidated in detail. MicroRNAs (miRNAs) are non-coding small RNAs that regulate posttranscriptional gene expression by interfering the translation of target mRNAs. Aberrant expression of miRNA is known to induce various human tumors including HNSCC, but the regulation of miRNA expression is still unknown. In this study, we examined the expression of miRNAs with or without HGF stimulation in human HNSCC cell line HSC3 and the expression of their putative target genes was evaluated. Methods: Human HNSCC cell line HSC3 was cultured with or without HGF for designated periods. Alteration of miRNA expression was examined by miRNA microarray analysis. Validation study of miRNAs showing altered expression was performed by quantitative RT-PCR (qRT-PCR). The expression of putative target mRNAs and their translated proteins were examined by qRT-PCR and western blot analysis, respectively. Results: The expression of several miRNAs including let-7a, miR-16, and miR-205, which are already known as tumor-suppressive miRNAs, was altered after HGF stimulation by miRNA microarray. Among them, we focused on miR-200c and miR-27b which were drastically down-regulated after HGF stimulation, because of their unique target mRNAs involving HGF-mediated functional molecules. The expression of ZEB1, a target mRNA for 200c, was up-regulated 3hr after HGF stimulation and that of E-cadherin, a downstream molecule of ZEB1, was up-regulated 12hr after HGF stimulation. The expression of ST14/matriptase, an activation enzyme of HGF and a target mRNA for miR-27b, was not statistically altered after HGF stimulation, but the protein level was drastically up-regulated 12hr after HGF stimulation. These changes were attenuated in part by the addition of oligo pre-mi27b or pre-miR200c into the culture medium. Conclusion: Our present results suggest that miR-200c down-regulated by HGF might play an important role for epithelial-mesenchymal transition (EMT), through up-regulation of ZEB1 followed by down-regulation of E-cadherin. In addition, it was also suggested that miR-27b down-regulated by HGF might induce auto-activation of HGF in HNSCC. These alterations of miRNA expression by HGF stimulation could be induce the enhanced progressive and invasive characteristics of HNSCC. This is a first report that HGF may directly regulate some miRNAs that affect the expression of HGF-mediated downstream functional molecules. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3972. doi:10.1158/1538-7445.AM2011-3972

We have recently shown that OX40 (CD134) costimulation provided with GM-CSF-secreting vaccine can bypass CD8+ T cell tolerance. OX40 costimulation can also abrogate Foxp3+ regulatory T cell (Treg)-mediated suppression of antitumor immunity by reducing Foxp3 expression on Tregs. In addition, OX40 costimulation can directly augment tumor-specific CTL function without CD4+ T cell help in vivo. Here we show that pre-treatment with agonistic anti-OX40 mAb augments the antitumor immune effects of adoptive CD8+ T cell therapy in the clinically relevant wild-type model, not TCR-transgenic, mouse model. CD8+ T cells stimulated with tumor antigen (HER2/neu immunodominant peptide and T2 cells), including a small number of tumor antigen-specific effector CD8+ T cells, were transferred into tumor-bearing FVB/N mice. We could detect the response of tumor antigen-specific CD8+ T cells that were adoptively transferred into the recipient mice, however, the response was not sufficient to eradicate an established tumor. In addition, we showed that pre-treatment with OX40 costimulation in tumor-bearing recipient in order to inhibit the Treg-mediated suppressive function enhanced tumor specific immune response of adoptively transferred CD8+ T cells in the recipients, thus resulting in the eradication of an established tumor. Moreover, adoptively transferred CTLs, with OX40 costimulation in vitro before transfer, significantly enhanced a tumor-specific immune response in the tumor-bearing recipient. Furthermore, when tumor antigen-specific CTLs costimulated through OX40 in vitro were adoptively transferred into the tumor-bearing recipients that had been pre-treated with OX40 costimulation to inhibit Treg function, transferred tumor-specific CTLs were effectively maintained in this recipient 7 times more than in the recipient with CTL transfer alone without OX40 treatment, and about 3 times more than in the recipient with a single treatment of OX40 costimulation. These findings therefore suggest that both direct enhancement of adoptive transfer CTL function and pre-inhibition of Treg-mediated suppressive function in recipients with OX40 costimulation are useful strategy for antitumor adoptive CTL therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1939.

Introduction: MicroRNAs (miRNAs) are non-coding small RNAs that regulate cell proliferation and various cellular functions by interfering the translation of target mRNAs. Altered expression of miRNA is known to induce various human malignancies. Hepatoceyte growth factor (HGF) is a multifunctional growth factor that acts as mitogen, motogen, and/or morphogen in a variety of cells. MET, a specific receptor tyrosine kinase for HGF, is up-regulated in various tumors including squamous cell carcinoma of human head and neck (HNSCC) and the esophagus (ESCC), but how HGF affect the downstream gene expression have not yet been elucidated in detail. In this study, we examined the expression of miRNAs and their regulation by HGF in HNSCC and ESCC. Methods: Nine HNSCC, 8 ESCC and 2 normal squamous cell lines were used for cell culture. Total RNAs were extracted and used for microarray and following quantitative reverse transcription real-time PCR (qRT-PCR). Validation study was performed by qRT-PCR using total RNAs extracted from formalin-fixed paraffin-embedded human cancer tissues and paired normal tissues by laser-captured microdissection. For miRNA regulation study, HSC3 cells (HNSCC cell line) expressing MET was cultured with or without HGF stimulation, and their total RNAs were extracted and analyzed by miRNA microarray and following qRT-PCR. The expression of target mRNAs for miRNAs altered by HGF stimulation was also examined by qRT-PCR. Results: In HNSCC, ESCC, and normal squamous cell lines, miR-205 showed the highest expression in miRNA microarray analysis. High expression of miR-205 was observed in not only HNSCC and ESCC but also other organs’ SCC and adjacent normal squamous epithelia by qRT-PCR in validation study, although less expression was detected in cells and tissues other than squamous epithelia. MiR-21, which is an oncogenic miRNA in various malignancies, was also up-regulated in HNSCC and ESCC compared to paired normal squamous epithelia. By HGF stimulation, miR-200c and miR-27b were significantly down-regulated in HSC3 cells. Interestingly, the expression of ZEB1, a target mRNA for miR-200c, was up-regulated and E-cadherin, downstream molecule of ZEB1, was down-regulated in these cells after HGF stimulation. Conclusion: Our present results suggest that miRNA expression pattern and target mRNAs of HNSCC and ESCC are different from those of other organ's malignancies. MiR-205 might be a specific marker miRNA of both normal and malignant squamous epithelia, while miR-21 might be a putative oncogenic miRNA in HNSCC and ESCC. In addition, several miRNAs including miR-200c and miR-27b down-regulated by HGF stimulation might play an important role for epithelial-mesenchymal transition (EMT) followed by enhanced invasive characteristics of HNSCC and ESCC, through up-regulation of ZEB1 followed by down-regulation of E-cadherin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2047.