Transcriptional activation of the Axl and PDGFR-α by c-Met through a ras- and Src-independent mechanism in human bladder cancer

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) [30]. Two stable cell lines: NIH-Met5 and T24-Met3 were established.

RNA was isolated using TRIzol reagent (GIBCO BRL, Gaithersburg, MD, USA), followed by mRNA purification using Oligotex™ mRNA kit (Qiagen, Valencia, CA, USA). RNA samples were reverse transcribed into cDNA fluorescently labeled either with Cy3 or with Cy5. The labeled cDNA was hybridized with a microarray cDNA chip containing 192 RTK genes [31]. Data were imported and normalized using MeV: MultiExperiment Viewer (Dana-Farber Cancer Institute, http://​www.​tm4.​org/​mev.​html) [32]. Clustering affinity search technique (CAST) was used for gene expression cluster analysis. There are 23 clusters after CAST analysis [33], the gene expression profiles of 8 genes showing the best correlation with c-Met gene were clustered as one group (table 1).

The western blot analysis was performed as previously described [6]. 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.

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.

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 [34]. 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 [6]. 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 [5]. 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 [6]. "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.

Two stable cell lines, designated as NIH-Met5 (mouse fibroblast) and T24-Met3 (human bladder cancer cell), were established to harbor the inducible c-Met gene, which was expressed only in the absence of tetracycline (Tet) (Figure 1A, lane 3 and 4; Figure 1B, lane 1). When c-Met was over-expressed, the increase of its phosphorylated form (p-Met) indicates an auto-phosphorylation (Figure 1A, lane 3; Additional file 1, lane 6). Expression of p-Met was further enhanced 10 min after treatment with hepatocyte growth factor (HGF; Sigma, St. Louis, MO, USA) (Figure 1A, lane 4; Additional file 1, lane 7). In contrast, parental NIH/3T3 cells did not express c-Met and p-Met (Figure 1A, lanes 1 and 2; Additional file 1, lane4 1-4). It is interesting to note that c-Met or p-Met was not expressed in NIH-Met5 cells when treated with Tet alone or combined with HGF treatment (Figure 1A, lanes 5 and 6; Additional file 1, lanes 8-9). Concerning T24-Met3 cells, expression of c-Met was suppressed 24 h after treatment with Tet (Figure 1B, lane 2). Together, auto-phosphorylation occurred when c-Met was over-expressed, and HGF treatment further enhanced the phosphorylation of c-Met. The results demonstrate a successful in vitro model in modulating the expression of c-Met using Tet-off system.

To identify the novel interaction partners of c-Met, NIH-Met5 cells were first treated with Tet for 24 h, and then cultured in the absence of Tet for an additional 4 and 7 days (Figure 2A), respectively. Total RNA was extracted and subjected to screening using a cDNA microarray as previously described [35]. Among 192 RTKs, a total of 8 genes were positively correlated with c-Met over-expression, including Axl, PDGFR-α, PDGFR-β, ERBB2, ERBB3, MST1R, TIE1 and TIE2 (table 1). One of these candidate genes-MST1R was recently reported in our laboratory [6]. In addition, co-expression of c-Met with Axl and/or PDGFR-α was also detected in our pilot molecular profiling of RTKs in human bladder cancer cells in vitro [16]. As a result, both Axl and PDGFR-α were chosen for subsequent analysis. The comparable expression patterns of c-Met, Axl and PDGFR-α at RNA level were shown in Figure 2A.

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.

Both UB40 and UB47 are two bladder cancer cell lines established locally from primary bladder cancer of superficial and muscle-invasive type, respectively [6]. Apparent expression of c-Met and p-Met protein was detected in these two cell lines, and both Axl and PDGFR-α also showed a comparable expression pattern (Figure 3A). To confirm their functional interaction, these cell lines were maintained under serum starvation for 12 h, and then treated with HGF (30 ng/ml) for 10 min (Figure 3A). Up-regulation of Axl and PDGFR-α was demonstrated in UB40 and UB47 cells after HGF stimulation with a corresponding increase of p-Met (Figure 3A). Level of p-Met positively correlated with the expression of Axl and PDGFR-α, suggesting a relationship among c-Met, Axl and PDGFR-α.

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.

There are several reports of signaling regulation about RTK transactivation. For example, a HGF-independent activation of c-Met by fibronectin was reported to promote the tumor invasion/metastasis [36]. Through binding to α₅β₁-integrin, fibronectin directly associates with c-Met and activates both Src and focal adhesion kinase activity. To clarify the potential involvement of this c-Met/Src-related signaling event, the Src inhibitor PP2 (Calbiochem, Merck, Darmstadt, Germany) was used to treat serum starved UB40 cells for 24 h. As shown in Figure 4A, suppression of Src phosphorylation did not alter the levels of c-Met and Axl, indicating that Src is not involved in the cross-talk of the three RTKs.

It is well known that MEK/ERK 1/2 is one of the most important transducer proteins when HGF binds to c-Met [1]. 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.

Upon HGF stimulation, c-Met induces several biological responses that collectively give rise to a program known as "invasive growth". To clarify the biological relevance of cross-talk among c-Met, Axl and PDGFR-α, cell migration assay was conducted. The transwell experiment showed that migration of TSGH8301 bladder cancer cells was considerably suppressed by c-Met siRNA knock-down (p < 0.001). In addition, apparent inhibition was also demonstrated when shRNA for Axl or PDGFR-α alone was treated (p < 0.01) (Figure 5A and 5B). Figure 5C shows that the siRNA for c-Met and shRNAs for Axl and PDGFR-α,TSGH8301, indeed suppressed their target gene expression in TSGH8301 cells. This result is consistent with the reports on Axl in the breast [37] and liver cancers [38], and on the PDGFR-α in liver cancer [39], respectively. Our result suggests that c-Met, Axl and PDGFR-α may induce comparable biological functions, possibly through the same signaling pathway or inter-connecting signal network.

To clarify the clinical implication of the above-mentioned findings in vitro, expression levels of c-Met, Axl and PDGFR-α were examined by immunohistochemistry in a total of 65 cases of locally advanced and metastatic bladder tumors. Co-expression of c-Met/Axl/PDGFR-α in a case of a bladder cancer tissue was demonstrated in Figure 6. Collectively, overexpression of c-Met, Axl, and PDGFR-α was found in 30 (46.2%), 52 (80%), and 40 (61.5%) cases, respectively. Co-expression of two receptors was revealed in 22 (33.8%, c-Met/Axl), 27 (41.5%, c-Met/PDGFR-α), and 17 (26.2%, Axl/PDGFR-α) cases. Fourteen cases (21.5%) showed co-expression of three receptors (c-Met/Axl/PDGFR-α). In these human bladder tumors, over-expression of PDGFR-α was correlated with nodal metastasis and overexpression of c-Met or c-Met/Axl/PDGFR-α showed the most significant correlation with poor patient survival (p < 0.01) followed by c-Met/PDGFR-α, PDGFR-α, c-Met/Axl, and Axl/PDGFR-α (p < 0.05) (table 2). Kaplan Meier survival analysis showed that cumulative survival of patients with high expression of c-Met/Axl and c-Met/PDGFR-α was significantly lower than those with lower expression (Log rank test, p < 0.05) (Figure 7A and 7B). After adjusting for nodal status, multivariate analysis using log rank test revealed that indicators associated with poor long term survival were over-expression of c-Met and co-expression of c-Met/Axl/PDGFR-α (p = 0.015) (data not shown). We next used a Cox proportional hazards models to determine the relative risk (RR) of overall survival with 95% confidence interval (CI). The RR of poor long term survival was 3.340 for over-expression of c-Met, and 3.860 for co-expression of c-Met/Axl/PDGFR-α. Taken together, our results indicate that, in addition to c-Met, both Axl and PDGFR-α play a positive role in the progression of human bladder cancer.