Transcriptional activation of the Axl and PDGFR-α by c-Met through a ras- and Src-independent mechanism in human bladder cancer
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A cross-talk between different receptor tyrosine kinases (RTKs) plays an important role in the pathogenesis of human cancers.
Both NIH-Met5 and T24-Met3 cell lines harboring an inducible human c-Met gene were established. C-Met-related RTKs were screened by RTK microarray analysis. The cross-talk of RTKs was demonstrated by Western blotting and confirmed by small interfering RNA (siRNA) silencing, followed by elucidation of the underlying mechanism. The impact of this cross-talk on biological function was demonstrated by Trans-well migration assay. Finally, the potential clinical importance was examined in a cohort of 65 cases of locally advanced and metastatic bladder cancer patients.
A positive association of Axl or platelet-derived growth factor receptor-alpha (PDGFR-α) with c-Met expression was demonstrated at translational level, and confirmed by specific siRNA knock-down. The transactivation of c-Met on Axl or PDGFR-α in vitro was through a ras- and Src-independent activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathway. In human bladder cancer, co-expression of these RTKs was associated with poor patient survival (p < 0.05), and overexpression of c-Met/Axl/PDGFR-α or c-Met alone showed the most significant correlation with poor survival (p < 0.01).
In addition to c-Met, the cross-talk with Axl and/or PDGFR-α also contributes to the progression of human bladder cancer. Evaluation of Axl and PDGFR-α expression status may identify a subset of c-Met-positive bladder cancer patients who may require co-targeting therapy.
KeywordsAxl PDGFR-α c-Met bladder cancer
The RTK c-Met is expressed during normal development and plays a crucial role in many cell regulatory processes . After binding to its cognate ligand-hepatocyte growth factor (HGF), activated c-Met transmits signals implicated in the cell proliferation, motility, survival, and morphogenesis [2, 3, 4]. C-Met is over-expressed and usually associated with metastatic progression of a variety of human malignant tumors, including bladder cancer [1, 5]. We have reported that c-Met is over-expressed in 32.3%, 63.2%, and 65.2% of superficial, locally advanced and metastatic bladder cancer, respectively . Over-expression of c-Met is positively associated with muscle invasion and poor long term survival (p < 0.001), while it is not related to patient outcome in the subset of superficial bladder cancer. Miyata et al. also reported the significance of c-Met in bladder cancer development and as an important predictor of metastasis and patient survival . Therefore, c-Met is emerging as a novel therapeutic target in many solid tumors [8, 9, 10].
Dimerization is generally required for activating RTKs . In addition to heterodimeric complex formation of the same subfamily [6, 11, 12, 13, 14], heterologous RTK interaction is also involved in the pathogenesis of human cancers, e.g. between EGFR and RON (a member of the c-Met family) [15, 16]. The biological significance of inhibition of both RTK signaling pathways of cancer cells was demonstrated in the context of cell proliferation, migration, anti-apoptosis and transformation in vitro. . Therefore, identification of cross-talk partners of c-Met involved in the tumorigenesis may provide important biomarkers for co-targeting therapy. In our prior RTK profiling experiment, c-Met was frequently co-expressed with Axl, platelet-derived growth factor receptor α (PDGFR-α), DDR2 and/or IGF1R in the same uroepithelial cells , suggesting the existence of yet unspecified cross-talk partners of c-Met.
Axl overexpression is detected in various human cancers, and is associated with invasiveness and/or metastasis of carcinoma of the breast , stomach , kidney , lung , and prostate . High expression of PDGFR-α is also detected in a variety of tumors, such as prostatic intraepithelial neoplasia, and carcinoma of the ovary, kidney, breast and liver [23, 24, 25, 26]. Furthermore, PDGFR-α expression provides additional predictive value related to breast cancer progression , and patient's survival in the kidney cancer  or lung cancer . The implications of these two receptor-related signaling events in the bladder carcinogenesis, however, remain unclear. This study was aimed to identify the novel interaction partners of c-Met, investigate their regulation, effect on biological activity, and the potential significance in association with patient outcome.
Cell Lines, transfection, and stable cell line establishment
NIH/3T3 mouse fibroblast cell line and bladder cancer cell line T24 were obtained commercially. The four bladder cancer cell lines UB09: stage B2; UB40: stage A, papillary; UB47: stage B1; TSGH8301: stage A were established from patients of transitional cell carcinoma of the urinary tract [6, 29]. UB47 was cultured in RPMI medium 1640 supplemented with 15% fetal bovine serum (FBS). Other cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS.
The plasmids pTRE-Met and pTet-Lac-Hyg were transfected into NIH/3T3 and T24 cells by Lipofectamine™ 2000 reagent according to manufacturer's protocol (Invitrogen, Carlsbad, CA, USA) . Two stable cell lines: NIH-Met5 and T24-Met3 were established.
The mRNA expression levels of eight RTK genes clustered together with c-Met in RTK c-DNA microarray analysis
NIH-Met5 day 1
NIH-Met5 day 4
NIH-Met5 day 7
Anti-phospho-tyrosine antibody was purchased from BD Transduction Laboratories (Lexington, KY, USA), and antibodies to Axl, c-Met, p-Met (phosphorylation of c-Met;Tyr 1234), PDGFR-α, and p-PDGFR-α (phosphorylation of PDGFR-α;Tyr 754) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Ras antibody was obtained from Calbiochem (Merck, Darmstadt, Germany), Sp1 from Upstate Biotechnology Inc. (Golden, CO, USA), p-Axl (phosphorylation of Axl;Tyr 702) from Cell Signaling Technology Inc. (Beverly, MA, USA) and β-actin was purchased from Sigma-Aldrich (St. Louis, MO, USA). The Src antibody was obtained from Millipore (Billerica, MA, USA) and p-Src (phosphorylation of Src;Tyr 418) purchased from Invitrogen (Carlsbad, CA, USA).
Western blot analysis
The western blot analysis was performed as previously described . Briefly, the total lysates were prepared using RIPA solution. Total protein (50 μg) was analyzed by polyacrylamide gel electrophoresis and transferred to the PVDF membrane. The membrane was probed with targeted protein antibodies and the immune complex was detected with an enhanced chemiluminescence (ECL) detection system (Perkin Elmer Life Sciences, USA).
Specific siRNA sense sequences were as follows: c-Met siRNA: 5`-AAGTGCAGTATCCTCTGACAG-3`, Axl siRNA: 5`-CGTGGAGAACAGCGAGATTTA-3`, and PDGFRα siRNA: 5`-CGAGACGATTGATGCAGGATA-3`. The cells (5 × 105) were seeded into a 6-cm cell culture dish and incubated in DMEM medium without antibiotics. Lipofectamine 2000 reagent (10 μl) was diluted in 500 μl of DMEM serum-free media and incubated for 5 min at RT. The siRNA was diluted in 500 μl of DMEM serum-free medium to the assigned concentrations. Mock transfection was conducted in parallel using distilled water as the negative control. Then cells were incubated at 37°C in the 5% CO2 incubator for 4 h. The media were replaced with normal media and cells were incubated for additional 48 h before protein extraction.
Trans-well migration assay
The effect of RTK cross-talk on cell migration was analyzed in TSGH8301 bladder cancer cells using a 24-well Transwell™ system (Corning inc., Lowell, MA). Briefly, cells were cultured in a 6-cm plate and transfected with c-Met, Axl, or PDGFR-α siRNA for 24 h, respectively. Then, cells were resuspended with serum-free medium and added into the upper chamber of the trans-well insert (2 × 105 cells/well). The 10% FBS-containing DMEM was added in the lower chamber. Cells were incubated h at 37°C for 36 h. Migrated cells were fixed with 4% formaldehyde in PBS and stained with 2% crystal violet in 2% ethanol. The non-migrated cells in the upper chamber were removed by wiping with a cotton swab. The cells on the lower surface of the filter, representing migration of the cells through the membrane, were counted under a light microscope.
Clinicopathological characteristics of study cases
Since c-Met is important in the progression of bladder cancer, both locally advanced and metastatic bladder tumors were recruited to evaluate the significance of co-expression patterns of c-Met and other RTKs. Archival material of 65 patients (44 men and 21 women; age range, 40 to 84 yr old; mean ± SD, 61.5 ± 9.4 yrs) with locally advanced or metastatic urothelial bladder cancer (21pT2, 27pT3, 17pT4) was analyzed for RTK expression. These patients were diagnosed and treated in the National Cheng Kung University Hospital, Tainan, Taiwan, between 1990 and 1999 years. The numbers with low or high grade urothelial carcinoma were 20 and 45, respectively, according to definitions described previously . Seven patients received partial cystectomy and remaining fifty-eight patients received radical cystectomy and bilateral pelvic lymph node dissection. Among them, 23 (35.4%) patients had pelvic lymph node involvement. An adjuvant systemic chemotherapy, including methotrexate, vinblastine, epirubicin, and cisplatin (M-VEC regimen), was given to 20 patients (30.8%) after radical cystectomy. The survival status was determined by outpatient clinic records and/or confirmed by interview with patients' families. Clinical follow-up ranged from 26 to 140 months (mean ± SD: 50.02 ± 6.46 months). The time of the first tumor recurrence and for disease specific survivals were counted. The time is calculated until the death of the patient due to bladder cancer. Patients who died of other causes or were still alive at the last follow-up were censored.
Immunostaining procedures were described in detail previously . Briefly, tissue sections were incubated at RT for 2 h with monoclonal anti-cMet (1:100 dilution; Santa Cruz), anti-AXL (1:10 dilution; Santa Cruz) and anti-PDGFR-α (1:200 dilution; Santa Cruz) antibodies raised against the membrane protein. The optimal dilution was determined by using human kidney as a positive control . Then StrAviGen Super Sensitive MultiLink kit (BioGenex Laboratories, Inc., San Ramon, California) was used to detect the resulting immune complex. Peroxidase activity was visualized using an aminoethyl carbazole substrate kit (Zymed Laboratories, Inc., San Francisco, Califonia).
Because no apparent difference of staining intensity was detected, only the proportion of tumor cells stained for c-Met, Axl or PDGFR-α was considered in classification . "High level of expression" indicates > 50% of the tumor cells were immunostained, "low level of expression" indicates 10%-50% reactivity; and "negative" indicates < 10% staining for RTK protein.
The association between tumor staging or gross characteristics with expression status of c-Met, Axl, and PDGFR-α was analyzed by Chi-square test as appropriate. The correlation between co-expression patterns of RTKs and disease-specific survival of cancer patients was constructed according to Kaplan-Meier method by Log rank test.
Establishment of stable cell lines harboring inducible c-Met gene
Expression and functional association of c-Met with Axl and PDGFR-α in vitro
The regulation was then examined at protein level in NIH-Met5 cells. As shown in figure 2B (lane 3), c-Met was overexpressed in the absence of Tet, while suppressed c-Met expression was demonstrated after treatment of Tet for 24 h, as with that of figure 1. A reversion of c-Met expression gradually appeared after removal of Tet for 4 and 7 days. Expression of c-Met became visible by day 4 and almost completely reversed by day 7 after removal of Tet (Figure 2B, lanes 4 and 5). The parental NIH/3T3 cells were used as a control (Figure 2B, lanes 1 and 2).
Using total c-Met and p-Met as the reference, expression of Axl and PDGFR-α showed a comparable trend to that of c-Met at day 4 and day 7 (Figure 2B, lanes 4 - 5), respectively. This positive association of Axl or PDGFR-α with c-Met expression was also demonstrated in T24-Met3 human bladder cancer cell line (Figure 1B). However, no difference of Axl and PDGFR-α expression was detected in NIH3T3 cells (Figure 2B, lanes 1 and 2). Taken together, expression patterns of total c-Met and p-Met were positively correlated with Axl and PDGFR-α expression, suggesting a functional relationship between Axl/PDGFR-α and c-Met.
Correlation of c-Met expression with Axl and PDGFR-α status in human bladder cancer cells
To clarify the interaction among c-Met, Axl and PDGFR-α, UB40 cancer cells were transfected with c-Met, Axl and PDGFR-α specific siRNAs at the optimal concentrations for 48 h. When expression of each receptor protein was suppressed by their specific siRNA, expression levels of the other two proteins showed a trend of down-regulation, with a higher correlation between c-Met and Axl (Figure 3B). However, co-immunoprecipitation assay did not reveal evidence of direct interaction among these three RTK proteins at cell membrane level (data not shown). Taken together, the above data demonstrate a cross-talk among c-Met, Axl and PDGFR-α in a protein-protein interaction independent manner in human bladder cancer cells.
The involvement of MEK/ERK signaling pathway in the transactivation of Axl and PDGFR-α by c-Met
It is well known that MEK/ERK 1/2 is one of the most important transducer proteins when HGF binds to c-Met . Both FTI-277 (a Ras farnesylation inhibitor; Calbiochem, San Diego, CA, USA) and PD98059 (a MEK 1 inhibitor; Cashmere Biotech Co., Taipei, Taiwan) were used to verify the involvement of MEK/ERK 1/2 signaling in c-Met-mediated activation of Axl and PDGFR-α. We showed that ERK phosphorylation was abrogated by PD98059 after HGF treatment for 24 h (Figure 4B, lanes 4 and 8) compared to FTI-277 (Figure 4B, lanes 3 and 7), suggesting the existence of a ras-independent phosphorylation of ERK mediated by HGF. The HGF-up-regulated Axl and PDGFR-α could be inhibited by PD98059 (Figure 4B, lanes 4 and 8), supporting the involvement of MEK/ERK 1/2 signaling in this transactivation event. In summary, MEK/ERK 1/2 signaling is involved in the transactivation of Axl and PDGFR-α by HGF/c-Met pathway in human bladder cancer cell lines, but is independent of ras or Src activity.
The effect of cross-talk of c-Met, Axl, and PDGFR-α on cell migration
Clinical implication of c-Met, Axl, and PDGFR-α co-expression patterns in human bladder cancer patients
Correlation of c-Met, Axl, and PDGFR-α protein expression with clinicopathologic parameters of patients with locally advanced and metastatic bladder cancers
c-Met & Axl
c-Met & PDGFR-α
Axl & PDGFR-α
c-Met & Axl & PDGFR-α
Discussion and conclusions
In this study, we showed that both Axl and PDGFR-α have a functional interaction with c-Met in vitro and in vivo. This is the first report showing their potential clinical importance in human bladder cancer. The results concur with co-expression of c-Met/PDGFR-α in all of 9 human bladder cancer cell lines reported by Black and his colleagues . The interaction between c-Met and Axl or PDGFR-α was further corroborated by HGF stimulation and siRNA silencing experiments in vitro. The interaction among these three RTKs may be initiated by protein-protein interaction or signaling transduction. The former possibility was excluded by co-immunoprecipitation assay (data not shown). In terms of signal regulation, the successful inhibition of c-Met activation by PD98059, but not by FTI-277 (ras inhibitor) or PP2 (Src inhibitor), suggests a ras- and Src-independent MEK/ERK 1/2 signaling in the transactivation of Axl and PDGFR-α. Our results seem to imply the existence of a novel mechanism by which c-Met transactivates the expression of Axl and PDGFR-α. Additional experiments are required to clarify whether protein kinase C is involved in this cross-talk in vivo.
Further support for our hypothesis of regulation at transcriptional level comes from several prior reports. The Sp1/Sp3 cis-acting elements were demonstrated to activate the promoter of Axl in various cancer cell lines . Moreover, Sp1 response elements are detected in PDGFR-α promoter region [42, 43]. Given that c-Met induces the phosphorylation of Sp1 and enhances downstream gene expression through MEK/ERK signaling pathway [44, 45], c-Met might up-regulate the expression of Axl and PDGFR-α through Sp1. The dose-dependent suppression of Sp1, Axl and PDGFR-α by c-Met siRNA supports our speculation (Additional file 2).
It has been reported that HGF is expressed in fibroblast-like cells, smooth muscle cells, and endothelial cells of the bladder . Expression of c-Met on the cancer cell surface thus may enable the paracrine activation in vivo, irrespective of their capability to synthesize HGF. The correlation of co-expression of two or three of the RTKs with patient survival supports the "invasive growth" program in carcinomas with multiple RTK over-expression [13, 15, 47]. The prognostic significance of c-Met, whether alone or co-expressed with Axl/PDGFR-α, supports the clinical relevance of c-Met-directed therapy (e.g. PHA665752) for human bladder cancer. Since the importance of co-targeting therapy for human bladder cancer having co-expressed RTKs has been demonstrated [15, 47], a prospective study is imperative to clarify the significance of Axl and/or PDGFR-α as an additional biomarker or implementation of MEK1/2 inhibitor in the design of c-Met-targeting therapy for human bladder cancer patients.
It is interesting to note that induction of Axl via ''kinase switching'' confers the Gleevec resistance in relapsed patients with c-Kit- or PDGFR-α-driven tumors of the gastrointestinal tract . Therefore, evaluation of RTK expression profile in human cancer may provide signaling network information and help in prediction of potential drug resistance . Olaussen et al. showed that combinations of tyrosine kinase inhibitors could induce a synergistic antitumor effect and thus improve the therapeutic efficacy . When more highly selective or multi-target tyrosin kinase inhibitors become available, the discovery of co-expression of RTKs in cancer cells highlights the necessity for individualized therapies in the future.
This project was supported by grants of Landmark Project Grant A25 of the National Cheng Kung University funded by the Ministry of Education in Taiwan, 96-2628-B-006-003-MY3 from the National Science Council; and NHRI-EX99-9930BI from the National Health Research Institutes, Taiwan. We thank Dr. R. Zuchini for critical reviewing this manuscript.
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