C-met Promoter Research Articles

Advancing Lung Cancer Treatment with Combined c-Met Promoter-Driven Oncolytic Adenovirus and Rapamycin.

Lung cancer remains a formidable health challenge due to its high mortality and morbidity rates. Non-small cell lung cancer (NSCLC) constitutes approximately 85% of all lung cancer cases, with small cell lung cancer (SCLC) accounting for the remainder. Both NSCLC and SCLC cells express receptor tyrosine kinases, which may be overexpressed or mutated in lung cancer, leading to increased activation. The c-Met receptor tyrosine kinase, crucial for cell transformation and tumor growth, invasion, and metastasis, became the focus of our study. We used an E1B55KD-deleted, replication-selective oncolytic adenovirus (Ad.What), driven by the c-Met promoter, targeting lung cancer cells with c-Met overexpression, thus sparing normal cells. Previous studies have shown the enhanced antitumor efficacy of oncolytic adenoviruses when combined with chemotherapeutic agents. We explored combining rapamycin, a selective mTOR inhibitor with promising clinical trial outcomes for various cancers, with Ad.What. This combination increased infectivity by augmenting the expression of coxsackievirus and adenovirus receptors and αV integrin on cancer cells and induced autophagy. Our findings suggest that combining a c-Met promoter-driven oncolytic adenovirus with rapamycin could be an effective lung cancer treatment strategy, offering a targeted approach to exploit lung cancer cells' vulnerabilities, potentially marking a significant advancement in managing this deadly disease.

Dnmt3b catalytic activity is critical for its tumour suppressor function in lymphomagenesis and is associated with c-Met oncogenic signalling.

BackgroundDNA methylation regulates gene transcription in many physiological processes in mammals including development and haematopoiesis. It is catalysed by several DNA methyltransferases, including Dnmt3b that mediates both methylation-dependant and independent gene repression. Dnmt3b is critical for mouse embryogenesis and functions as a tumour suppressor in haematologic malignancies in mice. However, the extent to which Dnmt3b's catalytic activity (CA) is involved in development and cancer is unclear.MethodsWe used a mouse model expressing catalytically inactive Dnmt3b (Dnmt3bCI) to study a role of Dnmt3b's CA in development and cancer. We utilized global approaches including Whole-genome Bisulfite sequencing and RNA-seq to analyse DNA methylation and gene expression to identify putative targets of Dnmt3b's CA. To analyse postnatal development and haematopoiesis, we used tissue staining, histological and FACS analysis. To determine potential involvement of selected genes in lymphomagenesis, we used overexpression and knock down approaches followed by in vitro growth assays.FindingsWe show that mice expressing Dnmt3bCI only, survive postnatal development and develop ICF (the immunodeficiency-centromeric instability-facial anomalies) -like syndrome. The lack of Dnmt3b's CA promoted fibroblasts transformation in vitro, accelerated MLL-AF9 driven Acute Myeloid Leukaemia and MYC-induced T-cell lymphomagenesis in vivo. The elimination of Dnmt3b's CA resulted in decreased methylation of c-Met promoter and its upregulation, activated oncogenic Met signalling, Stat3 phosphorylation and up-regulation of Lin28b promoting lymphomagenesis.InterpretationOur data demonstrates that Dnmt3b's CA is largely dispensable for mouse development but critical to prevent tumourigenesis by controlling events involved in cellular transformation.FundingThis study was supported by Department of Anatomy and Cell Biology and Cancer Centre at the University of Florida start-up funds, NIH/NCI grant 1R01CA188561-01A1 (R.O.).

HDAC1-Smad3-mSin3A complex is required for Smad3-induced transcriptional inhibition of hepatocyte growth factor receptor in human lung cancers.

c-Met hyperactivity has been observed in numerous neoplasms. Several researchers have shown that the abnormal activation of c-Met is mainly caused by transcriptional activation. However, the molecular mechanism behind this transcriptional regulation is poorly understood. Here, we suggest that Smad3 negatively regulates the expression and activation of c-Met via a transcriptional mechanism. We explore the molecular mechanisms that underlie Smad3-induced c-Met transcription inhibition. We found in contrast to the high expression of c-Met, Smad3 showed low protein and mRNA levels. Smad3 and c-Met expressions were inconsistent between lung cancer tissues and cell lines. We also found that Smad3 overexpression suppresses whereas Smad3 knockdown significantly promotes Epithelial-Mesenchymal Transition and production of the angiogenic factors VEGF, CTGF and COX-2 through the ERK1/2 pathway. In addition, Smad3 overexpression decreases whereas Smad3 knockdown significantly increases protein and mRNA levels of invasion-related β-catenin and FAK through the PI3K/Akt pathway. Furthermore, using the chromatin immunoprecipitation analysis method, we demonstrate that a transcriptional regulatory complex consisting of HDAC1, Smad3 and mSin3A binds to the promoter of the c-Met gene. By either silencing endogenous mSin3A expression with siRNA or by pretreating cells with a specific HDAC1 inhibitor (MS-275), Smad3-induced transcriptional suppression of c-Met could be effectively attenuated. These results demonstrate that Smad3-induced inhibition of c-Met transcription depends on of a functional transcriptional regulatory complex that includes Smad3, mSin3A and HDAC1 at the c-Met promoter. Collectively, our findings reveal a new regulatory mechanism of c-Met signaling, and suggest a potential molecular target for the development of anticancer drugs.

MYSM1/2A-DUB is an epigenetic regulator in human melanoma and contributes to tumor cell growth.

Histone modifying enzymes, such as histone deacetylases (HDACs) and polycomb repressive complex (PRC) components, have been implicated in regulating tumor growth, epithelial-mesenchymal transition, tumor stem cell maintenance, or repression of tumor suppressor genes - and may be promising targets for combination therapies of melanoma and other cancers. According to recent findings, the histone H2A deubiquitinase 2A-DUB/Mysm1 interacts with the p53-axis in hematopoiesis and tissue differentiation in mice, in part by modulating DNA-damage responses in stem cell and progenitor compartments. Based on the identification of alterations in skin pigmentation and melanocyte specification in Mysm1-deficient mice, we hypothesized that MYSM1 may be involved in melanoma formation. In human melanoma samples, expression of MYSM1 was increased compared with normal skin melanocytes and nevi and co-localized with melanocyte markers such as Melan-A and c-KIT. Similarly, in melanoma cell lines A375 and SK-MEL-28 and in murine skin, expression of the deubiquitinase was detectable at the mRNA and protein level that was inducible by growth factor signals and UVB exposure, respectively. Upon stable silencing of MYSM1 in A375 and SK-MEL-28 melanoma cells by lentivirally-mediated shRNA expression, survival and proliferation were significantly reduced in five MYSM1 shRNA cell lines analyzed compared with control cells. In addition, MYSM1-silenced melanoma cells proliferated less well in softagar assays. In context with our finding that MYSM1 bound to the c-MET promoter region in close vicinity to PAX3 in melanoma cells, our data indicate that MYSM1 is an epigenetic regulator of melanoma growth and potentially promising new target for tumor therapy.

Abstract P6-01-20: A role of MACC1 expression and its regulation of the HGF/c-Met pathway in breast cancer

Abstract Background The newly identified gene, metastasis-associated in colon cancer 1 (MACC1), is suggested to be a transcriptional regulator of the receptor tyrosine kinase gene c-Met, leading to cancer progression and metastasis in colorectal cancer. Also in breast cancer, aberrant hepatocyte growth factor (HGF) / c-Met signaling has been shown to contribute to worse prognosis and confer resistance to endocrine therapy or trastuzumab treatment, however, little is known of the role of MACC1. Here, we report its impact on the survival for breast cancer patients and the biological function in the cell lines. Methods A total of 300 breast cancer patients who received both surgery and adjuvant treatment at Kumamoto University Hospital between 2001 and 2009 were selected. We analyzed expressions of MACC1 by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) to evaluate the associations of its expression with breast cancer survival. In an in vitro study, the expressions of MACC1 were examined by Western blotting in breast and colorectal cancer cell lines. After transfection with a MACC1-harboing plasmid, we evaluated the activities of cMet protein and cell motility and proliferation. Further, the binding ability of MACC1 to the cMet promoter was evaluated using chromatin immunoprecipitation (ChIP) assay. Results In survival analyses, reduced MACC1 expressions were associated with patient mortality. Cox proportional hazards model showed that MACC1 mRNA (HR = 0.25, P = 0.001), MACC1 protein (HR = 0.37, P = 0.016), as well as axillary nodal status and estrogen receptor status, were independent predictors of mortality. No significant correlations between MACC1 expression and other clinicopathological factors were found. We found no strong positive correlation between MACC1 protein and c-Met mRNA expression with a Spearman’s coefficient of 0.16 (P = 0.0067). In the cell lines tested, MACC1 expression was much higher in colorectal cancer cells (DLD-1) than breast cancer cells (MCF7 and MDA-MB-231). To investigate the impact of MACC1 on the biological function of the cells, we transfected with MACC1 in breast cancer and colorectal cancer cells (SW480). MACC1 overexpression did not induce cMet expression in MCF7, whereas the corresponding cMet expression was upregulated in SW480 cells. Further, SW480 cells transfected with MACC1 showed enhanced migratory ability, whereas in MDA-MB-231 cells, transfection of MACC1 had no impact on this ability. In ChIP assay, the binding of MACC1 to the cMet promoter region was suggested in SW480 cells, but not in MCF7 cells. Conclusions Our findings provide some novel insights into the role of MACC1 for breast cancer, indicating that it plays different roles in breast cancer and in several other cancers. The biological mechanism of MACC1 which underlies improvement of breast cancer prognosis remains unelucidated. There is possibility that MACC1 does not act as the exclusive master regulator of the HGF/c-Met signaling involved in disease progression in breast cancer. Further studies to validate our results are needed. Citation Format: Aiko Sueta, Yutaka Yamamoto, Mitsuhiro Hayashi, Takashi Takeshita, Mutsuko Ibusuki, Hirotaka Iwase. A role of MACC1 expression and its regulation of the HGF/c-Met pathway in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-01-20.

Epigenetic Upregulation of HGF and c-Met Drives Metastasis in Hepatocellular Carcinoma

Hepatocyte growth factor (HGF) and its receptor, c-Met, are important regulators of growth and differentiation of healthy hepatocytes. However, upregulation of HGF and c-Met have been associated with tumor progression and metastasis in hepatocellular carcinoma (HCC). Hematogenous dissemination is the most common route for cancer metastasis, but the role of HGF and c-Met in circulating tumor cells (CTCs) is unknown. We have isolated and established a circulating tumor cell line from the peripheral blood of a mouse HCC model. Our studies show that these CTCs have increased expression of HGF and c-Met in comparison to the primary tumor cells. The CTCs display phenotypic evidence of epithelial-mesenchymal transition (EMT) and the EMT appears to be inducible by HGF. Epigenetic analysis of the c-Met promoter identified significant loss of DNA methylation in CTCs which correlated with overexpression of c-Met and increased expression of HGF. Six specific CpG sites of c-Met promoter demethylation were identified. CTCs show significantly increased tumorigenicity and metastatic potential in a novel orthotopic syngeneic model of metastatic HCC. We conclude that during hematogenous dissemination in HCC, CTCs undergo EMT under the influence of increased HGF. This process also involves up regulation of c-Met via promoter demethylation at 6 CpG sites. Consequently, targeting HGF and c-Met expression by CTCs may be a novel non-invasive approach with potential clinical applications in HCC management.

Abstract SY41-02: Tumor cell effects of vascular reprogramming: Contributions of c-MET and VEGF

Abstract Intratumoral hypoxia is associated with greater risk of metastasis and less favorable prognosis. Hypoxia increases the expression in tumor cells of c-MET, the tyrosine kinase receptor for hepatocyte growth factor, through HIF-1α binding sites on the c-MET promoter. Activation of c-MET promotes tumor cell proliferation, motility, and invasion and is associated with tumor aggressiveness. Intratumoral hypoxia is increased by vascular pruning produced by angiogenesis inhibitors that block VEGF signaling. We asked whether selective VEGF inhibition, which exaggerates intratumoral hypoxia and activates c-MET, is sufficient to increase tumor invasiveness and metastasis and whether selective inhibition c-MET is sufficient to block these effects. We found that treatment of pancreatic neuroendocrine tumors in RIP-Tag2 mice with a neutralizing anti-VEGF antibody or with the tyrosine kinase inhibitor sunitinib reduced tumor burden. Both treatments also increased intratumoral hypoxia, expression and activation of c-MET, tumor invasion into the exocrine pancreas, and metastasis to the liver. Increased intratumoral hypoxia was documented by increased HIF-1α and staining for pimonidazole, carbonic anhydrase-IX, and Glut1 in tumor cells. Treatment with either agent for only a week was accompanied by increased expression of c-MET mRNA and corresponding increases in c-MET protein and c-MET phosphorylation in tumor cells. Exposure to hypoxia also increased c-MET phosphorylation in freshly isolated RIP-Tag2 tumor cells in vitro. With these findings, the selective anti-VEGF antibody #AF-493-NA was added to the list, which already includes sunitinib, anti-VEGFR-2 antibody (DC101), and genetic deletion of VEGF, of interventions reported to promote tumor invasion and metastasis in preclinical models. To test the involvement of c-MET in the exaggerated tumor aggressiveness, we used three complementary approaches to block c-MET signaling. The first used the tyrosine kinase inhibitor PF-04217903 because of its high selectivity for c-MET. The second used PF-02341066 (crizotinib), a tyrosine kinase inhibitor that has high potency against c-MET but also blocks anaplastic lymphoma kinase (ALK). The third used the tyrosine kinase inhibitor XL184 (cabozantinib), which simultaneously blocks c-MET and VEGFR among other receptor tyrosine kinases. We found that RIP-Tag2 tumors treated with either PF-04217903 or PF-02341066 from age 14 to 17 weeks were about the same size as vehicle-treated controls but were less invasive and had fewer metastases. After this treatment, the tumors had a smoother contour, a distinctive ball-like shape, and less invasion into the exocrine pancreas. Liver metastases were significantly smaller and less numerous. Inhibition of c-MET and VEGF together, by administration of PF-04217903 or PF-02341066 together with anti-VEGF antibody or sunitinib, slowed tumor growth and had even more potent effects on invasion and metastasis than found with either c-MET inhibitor used alone. These findings in RIP-Tag2 tumors were corroborated by parallel experiments on orthotopic Panc-1 pancreatic adenocarcinomas. Expression of c-MET and invasiveness of Panc-1 tumors were increased by treatment with sunitinib, and the increase was blocked by concurrent administration of PF-04217903. XL184 given as a single agent reduced tumor invasion and metastasis in RIP-Tag2 mice as much as the drug combinations. The similarity of effects of XL184 on tumor invasion and metastasis to those of selective inhibitors given together argues for c-MET and VEGFR being relevant targets of XL184, although additional involvement of inhibition of AXL, KIT, RET or other targets cannot be excluded for this multi-targeted agent. To test whether concurrent inhibition of c-MET and VEGF signaling can reverse tumor invasion, we compared tumors in RIP-Tag2 mice treated for 3 weeks with tumors in onset controls at age 14 weeks. The amount of invasion found after treatment with PF-042107903 plus sunitinib or with XL184 given alone was less than in onset controls. Similarly, the metastatic burden in the liver was less after XL184 for 3 weeks than in onset controls. Overall, the experiments revealed that, consistent with published reports, inhibition of VEGF signaling by function-blocking antibody or sunitinib causes vascular pruning and slowing of tumor growth but also increases invasion and metastasis in these preclinical models. The findings fit with a mechanism involving vascular pruning, intratumoral hypoxia, HIF-1α accumulation, and activation of c-MET in tumor cells. Invasion and metastasis that accompany VEGF inhibition can be blocked by concurrent inhibition of c-MET. PF-04217903 and PF-02341066 have similar efficacy in this regard when either is given together with anti-VEGF antibody or sunitinib. Inhibition of both c-MET and VEGF signaling by XL184 has potent anti-angiogenic activity and reduces tumor growth while also reducing invasion and metastasis. The findings indicate that tumor invasion and metastasis can be reduced along with tumor growth by blocking c-MET and VEGF signaling together to offset the consequences of intratumoral hypoxia resulting from angiogenesis inhibition and vascular pruning. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY41-02. doi:1538-7445.AM2012-SY41-02

Overexpression of hepatocyte growth factor receptor in scleroderma dermal fibroblasts is caused by autocrine transforming growth factor β signaling

Cutaneous fibrosis seen in systemic sclerosis (SSc) is caused by fibroblast activation and abnormal collagen accumulation due to 'autocrine transforming growth factor (TGF)-β/Smad signaling'. Hepatocyte growth factor (HGF) may have therapeutic value against SSc, because of its inducible effect on the expression of matrix metalloproteinase (MMP)-1. Previous studies indicated SSc dermal fibroblasts overexpress HGF receptor c-met, which suggest specific and effective induction of MMP-1 in SSc fibroblasts caused by HGF treatment. However, the exact mechanism of c-met overexpression in SSc cells was hardly investigated. We hypothesized that such c-met overexpression is also caused by autocrine TGF-β/Smad signaling. Expression of c-met protein in cultured SSc dermal fibroblasts was significantly up-regulated compared with that in normal fibroblasts. Ectopic TGF-β stimulation induced c-met synthesis in normal fibroblasts, while a TGF-β knockdown normalized the up-regulated c-met levels in SSc fibroblasts. Furthermore, we found the c-met promoter contains a putative binding site for Smads, and the binding activity of Smad2/3 to the c-met promoter was constitutively up-regulated in SSc fibroblasts as well as in normal fibroblasts treated with exogenous TGF-β1. Taken together, c-met may be overexpressed due to autocrine TGF-β/Smad signaling in SSc. Considering that HGF has an antifibrotic effect, such c-met overexpression in SSc fibroblasts may be a negative feedback against cutaneous fibrosis. Clarifying the mechanisms of c-met overexpression and controlling the HGF/c-met pathway may lead to a new therapeutic approach for this disease.

Filamin a mediates HGF/c‐MET signaling in tumor cell migration

Deregulated hepatocyte growth factor (HGF)/c-MET axis has been correlated with poor clinical outcome and drug resistance in many human cancers. Identification of novel regulatory mechanisms influencing HGF/c-MET signaling may therefore be necessary to develop more effective cancer therapies. In our study, we show that multiple human cancer tissues and cells express filamin A (FLNA), a large cytoskeletal actin-binding protein, and expression of c-MET is significantly reduced in human tumor cells deficient for FLNA. The FLNA-deficient tumor cells exhibited poor migrative and invasive ability in response to HGF. On the other hand, the anchorage-dependent and independent tumor cell proliferation was not altered by HGF. The FLNA-deficiency specifically attenuated the activation of the c-MET downstream signaling molecule AKT in response to HGF stimulation. Furthermore, FLNA enhanced c-MET promoter activity by its binding to SMAD2. The impact of FLNA deficiency on c-MET expression and HGF-mediated cell migration in human tumor cells was confirmed in primary mouse embryonic fibroblasts deficient for Flna. These data suggest that FLNA is one of the important regulators of c-MET signaling and HGF-induced tumor cell migration.

The effect of hepatitis B virus X protein on the c-met promoter activity in HepG2 cells

To confirm the effect of hepatitis B virus X (HBx) protein on the c-met promoter activity in HepG2 cells. The expression of c-met protein was detected by western blot in HBx-transfected HepG2 cells, the human c-met promote activity was checked by luciferase assay using five different constructs with deletion or point mutation. HBx protein stimulated the expression of the c-met in HepG2 cells. The enhanced expression of c-met in HBx-transfected cells was mediated by the activation of AP-2 and SP-1 transcriptional activity at the c-met promoter region (-183bp--100bp), HBx increased the invasiveness of HepG2 cells as determined by Matrigel invasion assay. These results suggests that HBx induces the expression of c-met through the activation of AP-2 and SP-1 activity at the promoter region; in addition, our data indicate that HBx stimulates the invasive potential of HepG2 cells.

Interaction of Actinomycin D with Promoter Element of c-met and Its Inhibitory Effect on the Expression of c-Met

c-Met, the tyrosine kinase receptor for hepatocyte growth factor plays a pivotal role in normal cellular signaling and overexpression of c-Met protein is reported in several human cancers. Thus, transcriptional regulation of c-met appears to be an attractive target for chemotherapy. Therefore, we selected a 24mer GC rich sequence (24R) from the c-met promoter located at −142 to −119 from transcription start site and studied its interaction with anticancer drug, Actinomycin D. Spectroscopic analysis demonstrated a strong complexation between ActD and 24RY as shown by: (i) a high binding constant, K of 4–5 × 105 M−1 with ΔΔG of −47 ± 1.5 Kcalmol−1; (ii) marked increase by + 10 °C in melting temperature of 24RY; and (iii) significant changes in circular dichroic spectra of both ActD and 24RY. Molecular modeling revealed the preference of ActD to the Spl binding site, GGCGGG, in 24RY. Expression of the c-Met was checked in HepG2 cells, a human hepatocellular carcinoma cell line by using western blotting and immunocytochemistry. Downregulation of c-Met expression by as much as 50% was observed in the presence of 20ng/ml (IC50) of ActD. Taking into account of the binding studies also, we feel that the down regulation of c-Met perhaps involves binding of ActD to the promoter site of c-met. Therefore, c-met could be a challenging and promising target for therapeutic strategies in combating cancer.

Preferential binding of anti-cancer drug adriamycin to the Sp1 binding site in c-met promoter region: A spectroscopic and molecular modeling study

Abstract The c-met gene encodes a transmembrane glycoprotein receptor with tyrosine kinase activity and overexpression of MET receptor is found in a number of common human malignancies. Regulation of c-met oncogene expression in general can be controlled by several DNA binding anti-cancer drugs. Interaction of adriamycin with a short oligonucleotide (24RY), which is part of the positive regulatory element (−233 to −68) in c-met gene was studied using UV–Vis absorption and fluorescence spectroscopy, UV-thermal melting, and molecular modeling. Strong binding of adriamycin to 24RY (overall binding constant K, 1− 3 × 105 M−1) is thermodynamically favored and is accompanied by the following: a marked increase in the melting temperature of 24RY by +15 °C and ∼60% decrease in absorption at 480 nm, ∼80% quenching of fluorescence at 555 nm along with a blue shift of the λ emi max to 522 nm of adriamycin. Present data reveals that adriamycin binds to ∼ 5 bp (GCGGG) of the Sp1 binding site in 24RY and thus competes with Sp1 binding to the promoter site which results in down-regulation of kinase. Therefore, targeting c-met is a promising approach as it is an attractive novel oncogene for cancer therapeutics.

Regulation of c-met expression by transcription repressor Daxx

The protooncogene c-met encodes the tyrosine kinase receptor for the hepatocyte growth factor/scatter factor (HGF/SF). While overexpression of c-met is documented in many types of tumors, the mechanism of c-met regulation remains elusive. Here, we demonstrate Daxx as a repressor of c-met transcription. The expression of c-met is elevated in Daxx knockout mouse cells and is reversed by Daxx reconstitution. C-met promoter analysis of Daxx-/- cells reveled changes in chromatin acetylation, but not in DNA methylation. Daxx binds to the mouse c-met promoter and Daxx-binding region is sufficient for transcription repression, while HDAC2 is associated with c-met promoter mostly in Daxx+/+ cells, pointing to Daxx-dependent HDAC2 recruitment as a potential mechanism of c-met repression. HGF-induced cell mobility and invasion confirmed augmented activity of c-Met/HGF pathway in Daxx-/- cells. Finally, inverse correlation between Daxx and c-Met in cancer cell lines and in metastatic breast cancer specimens suggests potential function of Daxx as a c-met repressor during cancer progression.

Hypoxia can induce c‐Met expression in glioma cells and enhance SF/HGF‐induced cell migration

The c-Met receptor and its ligand scatter factor/hepatocyte growth factor (SF/HGF) are strongly overexpressed in malignant gliomas. Signaling through c-Met as well as exposure to hypoxia can stimulate glioma cell migration and invasion. In several cancer cell types, hypoxia was shown to activate the c-met promoter, which contains hypoxia inducible factor-1 (HIF-1) binding sites. We hypothesized that hypoxia might upregulate c-Met also in glioma cells. Analyzing 18 different glioblastoma cell lines and 10 glioblastoma primary cultures, we found that in 50% of both the cell lines and the primary cultures c-Met protein levels were increased following exposure to hypoxia. Upregulation of c-met in response to hypoxia was also detected at the transcriptional level. In all primary cultures and in 16 of the 18 cell lines (89%), HIF-1 alpha levels were increased by hypoxia. Transfection of siRNA against HIF-1 alpha abgrogated the hypoxic induction of c-Met, suggesting that c-Met expression is upregulated by a HIF-1 alpha-dependent mechanism. Hypoxia sensitized glioblastoma cell lines which showed hypoxic induction of c-Met to the motogenic effects of SF/HGF. These findings suggest that approximately half of all human glioblastomas respond to hypoxia with an induction of c-Met, which can enhance the stimulating effect of SF/HGF on tumor cell migration.

The Androgen Receptor Negatively Regulates the Expression of c-Met: Implications for a Novel Mechanism of Prostate Cancer Progression

The precise molecular mechanisms by which prostate cancer cells progress from androgen-sensitive to androgen-insensitive status still remain largely unclear. The hepatocyte growth factor/scatter factor (HGF/SF) plays a critical role in the regulation of cell growth, cell motility, morphogenesis, and angiogenesis. The aberrant expression of HGF/SF and its receptor, c-Met, often correlates with poor prognosis in a variety of human malignancies, including prostate cancer. Here, we investigate a potential link between androgen signaling and c-Met expression in prostate cancer cells. First, we showed that the androgen receptor (AR) represses the expression of c-Met in a ligand-dependent manner. Using different c-Met promoter/reporter constructs, we identified that Sp1 induces the transcription of c-Met and that AR can repress the Sp1-induced transcription in prostate cancer cells. Moreover, the data from electrophoretic mobility shift assay showed that AR interferes with the interaction between Sp1 and the functional Sp1 binding site within the c-Met promoter. Furthermore, we tested the effect of AR on c-Met expression in an androgen-insensitive prostate cancer cell line, CWR22Rv1. Finally, the repressive role of androgen signaling on c-Met expression was confirmed in prostate cancer xenografts. The above data indicate a dual role of AR in transcriptional regulation. Although the current androgen ablation therapy can repress the expression of growth-promoting genes that are activated by the AR, it may also attenuate the repressive role of AR on c-Met expression. Therefore, the therapeutic strategies to inhibit the activation of the HGF/c-Met pathway may be of benefit when combined with current androgen ablation treatment.

C-Met Expression Is Regulated by Mitf in the Melanocyte Lineage

Hepatocyte growth factor (HGF)/c-Met signaling is thought to be a key pathway in both melanocyte development and melanoma metastasis. Here, HGF stimulation of melanocytes was seen to up-regulate c-Met expression. In an effort to decipher the mechanism by which HGF up-regulates its receptor, we found that c-Met is a direct transcriptional target of Mitf. This was confirmed with chromatin immunoprecipitation experiments of the human c-Met promoter, as well as by the ability of adenovirally expressed Mitf to modulate endogenous c-Met protein levels in melanocytes. Disruption of Mitf blocked HGF-dependent increases in endogenous c-Met message and protein levels, indicating that HGF regulates its own receptor levels via Mitf. Finally, dominant-negative inhibition of Mitf resulted in profound resistance of melanocytes and melanoma cells to HGF-dependent matrix invasion, suggesting a physiologic role for this pathway in melanocytic development and melanoma.

Both Sp1 and Smad participate in mediating TGF-beta1-induced HGF receptor expression in renal epithelial cells.

Hepatocyte growth factor (HGF) receptor is a transmembrane receptor tyrosine kinase encoded by the c-met protooncogene. In this study, we demonstrated that c-met expression was upregulated in the kidney after obstructive injury in mice. Because the pattern of c-met induction was closely correlated with transforming growth factor-beta1 (TGF-beta1) expression in vivo, we further investigated the regulation of c-met expression in renal tubular epithelial (HKC) cells by TGF-beta1 in vitro. Real-time RT-PCR and Northern and Western blot analyses revealed that TGF-beta1 significantly induced c-met expression in HKC cells, which primarily took place at the gene transcriptional level. Overexpression of inhibitory Smad7 completely abolished c-met induction, indicating its dependence on Smad signaling. Interestingly, TGF-beta1-induced c-met expression was also contingent on a functional Sp1, as ablation of Sp1 binding with mithramycin A abrogated c-met induction in HKC cells. Transfection and sequence analysis identified a cis-acting TGF-beta1-responsive region in the c-met promoter, in which resided a putative Smad-binding element (SBE) and an adjacent Sp1 site. TGF-beta1 not only induced Smad binding to the SBE/Sp1 sites in the c-met promoter, but also enhanced the binding of Sp proteins. Furthermore, Sp1 could form a complex with Smads in a TGF-beta1-dependent fashion. These results suggest a novel regulatory mechanism controlling c-met expression by TGF-beta1 in renal epithelial cells, in which both Smad and Sp proteins participate and cooperate in activating c-met gene transcription.

Suppression of HGF receptor gene expression by oxidative stress is mediated through the interplay between Sp1 and Egr-1

Hepatocyte growth factor (HGF) receptor, the product of the c-met protooncogene, is transcriptionally regulated by a wide variety of cytokines as well as extracellular environmental cues. In this report, we demonstrate that c-met expression was significantly suppressed by oxidative stress. Treatment of mouse renal inner medullary collecting duct epithelial cells with 0.5 mM H(2)O(2) inhibited c-met mRNA and protein expression, which was concomitant with induction of Egr-1 transcription factor. Ectopic expression of Egr-1 in renal epithelial cells markedly inhibited endogenous c-met expression in a dose-dependent fashion, suggesting a causative effect of Egr-1 in mediating c-met suppression. The cis-acting element responsible for H(2)O(2)-induced c-met inhibition was localized at nucleotide position -223 to -68 of c-met promoter, in which reside an imperfect Egr-1 and three Sp1-binding sites. Egr-1 markedly suppressed c-met promoter activity but did not directly bind to its cis-acting element in the c-met gene. Induction of Egr-1 by oxidative stress attenuated the binding of Sp1 to its cognate sites, but it did not affect Sp1 abundance in renal epithelial cells. Immunoprecipitation uncovered that Egr-1 physically interacted with Sp1 by forming the Sp1/Egr-1 complex, which presumably resulted in a decreased availability of unbound Sp1 as a transcriptional activator for the c-met gene. Thus it appears that inhibition of c-met expression by oxidative stress is mediated by the interplay between Sp1 and Egr-1 transcription factors. Our findings reveal a novel transcriptional regulatory mechanism by which Egr-1 sequesters Sp1 as a transcriptional activator of c-met via physical interaction.

Sp1 and Sp3 transcription factors synergistically regulate HGF receptor gene expression in kidney.

We investigated the expression pattern and underlying mechanism that controls hepatocyte growth factor (HGF) receptor (c-met) expression in normal kidney and a variety of kidney cells. Immunohistochemical staining showed widespread expression of c-met in mouse kidney, a pattern closely correlated with renal expression of Sp1 and Sp3 transcription factors. In vitro, all types of kidney cells tested expressed different levels of c-met, which was tightly proportional to the cellular abundances of Sp1 and Sp3. Both Sp1 and Sp3 bound to the multiple GC boxes in the promoter region of the c-met gene. Coimmunoprecipitation suggested a physical interaction between Sp1 and Sp3. Functionally, Sp1 markedly stimulated c-met promoter activity. Although Sp3 only weakly activated the c-met promoter, its combination with Sp1 synergistically stimulated c-met transcription. Conversely, deprivation of Sp proteins by transfection of decoy Sp1 oligonucleotide or blockade of Sp1 binding with mithramycin A inhibited c-met expression. The c-met receptor in all types of kidney cells was functional and induced protein kinase B/Akt phosphorylation in a distinctly dynamic pattern after HGF stimulation. These results indicate that members of the Sp family of transcription factors play an important role in regulating constitutive expression of the c-met gene in all types of renal cells. Our findings suggest that HGF may have a broader spectrum of target cells and possess wider implications in kidney structure and function than originally thought.

Interferon-α activates multiple STAT signals and down-regulates c-Met in primary human hepatocytes

Interferon (IFN)-alpha therapy is currently the primary choice for viral hepatitis and a promising treatment for hepatocellular carcinoma (HCC). Primary mouse and rat hepatocytes respond poorly to IFN-alpha stimulation. Thus, it is very important to examine the IFN-alpha signal pathway in primary human hepatocytes. The IFN-alpha-activated signals and genes in primary human hepatocytes and hepatoma cells were examined by Western blotting and microarray analyses. Primary human hepatocytes respond very well to IFN-alpha stimulation as shown by activation of multiple signal transducer and activator of transcription factor (STAT) 1, 2, 3, 5, and multiple genes. The differential response to IFN-alpha stimulation in primary human and mouse hepatocytes may be caused by expression of predominant functional IFN-alpha receptor 2c (IFNAR2c) in primary human hepatocytes vs. expression of predominant inhibitory IFNAR2a in mouse hepatocytes. Microarray analyses of primary human hepatocytes show that IFN-alpha up-regulates about 44 genes by over 2-fold and down-regulates about 9 genes by 50%. The up-regulated genes include a variety of antiviral and tumor suppressors/proapoptotic genes. The down-regulated genes include c-myc and c-Met, the hepatocyte growth factor (HGF) receptor. Down-regulation of c-Met is caused by IFN-alpha suppression of the c-Met promoter through down-regulation of Sp1 binding and results in attenuation of HGF-induced signals and cell proliferation. IFN-alpha directly targets human hepatocytes, followed by activation of multiple STATs and regulation of a wide variety of genes, which may contribute to the antiviral and antitumor activities of IFN-alpha in human liver.