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The role of HGF/MET and FGF/FGFR in fibroblast-derived growth stimulation and lapatinib-resistance of esophageal squamous cell carcinoma
© Saito et al.; licensee BioMed Central. 2015
Received: 24 June 2014
Accepted: 2 February 2015
Published: 25 February 2015
Although advanced esophageal squamous-cell carcinoma (ESCC) is treated using a multidisciplinary approach, outcomes remain unsatisfactory. The microenvironment of cancer cells has recently been shown to strongly influence the biologic properties of malignancies. We explored the effect of supernatant from esophageal fibroblasts on the cell growth and chemo-resistance of ESCC cell lines.
We used 22 ESCC cell lines, isolated primary human esophageal fibroblasts and immortalized fibroblasts. We first examined cell proliferation induced by fibroblast supernatant. The effect of supernatant was evaluated to determine whether paracrine signaling induced by fibroblasts can influence the proliferation of cancer cells. Next, we examined the effects of adding growth factors HGF, FGF1, FGF7, and FGF10, to the culture medium of cancer cells. These growth factors are assumed to be present in the culture supernatants of fibroblasts and may exert a paracrine effect on the proliferation of cancer cells. We also examined the intrinsic role of HGF/MET and FGFs/FGFR in ESCC proliferation. In addition, we examined the inhibitory effect of lapatinib on ESCC cell lines and studied whether the fibroblast supernatants affect the inhibitory effect of lapatinib on ESCC cell proliferation. Finally, we tested whether the FGFR inhibitor PD-173074 could eliminate the rescue effect against lapatinib that was induced by fibroblast supernatants.
The addition of fibroblast supernatant induces cell proliferation in the majority of cell lines tested. The results of experiments to evaluate the effects of adding growth factors and kinase inhibitors suggests that the stimulating effect of fibroblasts was attributable in part to HGF/MET or FGF/FGFR. The results also indicate diversity in the degree of dependence on HGF/MET and FGF/FGFR among the cell lines. Though lapanitib at 1 μM inhibits cell proliferation by more than 50% in the majority of the ESCC cell lines, fibroblast supernatant can rescue the growth inhibition of ESCC cells. However, the rescue effect is abrogated by co-treatment with FGFR inhibitor.
These results demonstrate that cell growth of ESCC depends on diverse receptor tyrosine kinase signaling, in both cell-autonomous and cell-non-autonomous manners. The combined inhibition of these signals may hold promise for the treatment of ESCC.
Currently, esophageal cancer is the eighth most common cancer in the world [1,2]. Esophageal cancer remains one of the least studied and most lethal malignancies . Squamous-cell carcinoma accounts for 92.5% of all primary esophageal tumors in Japan and other Asian countries [4-6], while adenocarcinoma is the most prevalent histologic type of esophageal cancer in western countries . Since the overall incidence and mortality of esophageal squamous-cell carcinoma (ESCC) is lower than other cancers such as breast cancer, colorectal cancer and lung cancer in western countries , biological studies of ESCC have been lagging behind. Advanced ESCC is treated using a multidisciplinary approach, including surgery, chemotherapy, and radiotherapy, but outcomes remain unsatisfactory [9-12].
Cancers are the end-product of accumulated effects of many genetic alterations, and the specific combination of changes is reflected in the unique characteristics of each tumor. The microenvironment of cancer cells has recently been shown to strongly influence the biologic properties of cancer .
A tumor consists of a dynamic mixture of tumor cells, fibroblasts, endothelial cells, immune cells and extracellular matrix. In many solid tumors, the stroma has been recognized to be important in promoting tumor proliferation, invasion, metastasis, and chemo-resistance [14,15]. The proliferation of fibroblasts is frequently seen in the invasive portion of a malignant tumor and tumors with significant proliferation of those cells are associated with a poor prognosis in colorectal cancers, breast cancers and lung cancers [16-18]. In ESCC, previous reports described that stromal fibroblasts have an important role in angiogenesis  and tumor differentiation . Fibroblasts are associated with cancer cells at all stages of cancer progression, and their production of growth factors, chemokines and extracellular matrix facilitates the angiogenic recruitment of endothelial cells and pericytes .
Hepatocyte growth factor (HGF) regulates cell growth, cell motility, and morphogenesis by activating a tyrosine kinase signaling cascade after binding to the c-Met receptor . HGF is secreted by mesenchymal cells including fibroblasts and promotes invasion of ESCC cells .
Fibroblast Growth Factors (FGFs) have been implicated in the regulation of cell differentiation, proliferation, migration and survival in many different cell types . The biological activities of FGFs are mediated by FGF receptors (FGFR). FGFR2 has two different isoforms that are designated FGFR2 IIIb and FGFR2 IIIc; the former is particularly localized in epithelial cells with growth induced by FGF-1, 3, 7, 10 and the latter binds FGF-1, 2, 4, 6, 9 and is expressed mainly in mesenchymal cells [25,26]. FGFR2 positive tumor fibroblasts may provide cancer cells with a suitable microenvironment to promote cancer development and progression .
Lapatinib is a dual tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR) and human EGFR-2 (HER2) tyrosine kinase domains [28,29]. Recently lapatinib has been evaluated for the treatment of not only breast cancer [30,31] but also gastric cancer  and ESCC .
To date, however, few studies have evaluated the role of stromal fibroblasts in ESCC. In this study, we focused on the relationship between ESCC cells and fibroblasts, the main component of cancer stroma. We prepared in vitro experimental systems to evaluate the interactions between ESCC and fibroblasts and clarify the mechanisms by which fibroblasts control proliferation of ESCC cells at the molecular level. We also studied the effects of various tyrosine kinase inhibitors to gain insight into new treatment strategies for ESCC.
PHA-665752 and PD-173074 were obtained from Tocris Bioscience (Bristol, UK). Lapatinib was obtained from Bio Vision (Milpitas, USA). Recombinant human HGF, recombinant human FGF1, 7, and 10 were obtained from R & D System (Minneapolis, USA). Stock solutions of PHA-665752, PD-173074 and lapatinib were prepared in dimethyl sulfoxide and stored at −80°C until use. Stock solutions of HGF and FGFs were prepared in phosphate-buffered saline (PBS) and stored at −80°C until use.
Esophageal squamous cell lines
We used 22 esophageal squamous cell lines: TE-1, TE-4, TE-5, TE-6, TE-8 TE-9, TE-10, TE-11, TE-14, TE-15, and EC-GI-10 were obtained from RIKEN Cell Bank (Tsukuba, Japan), while KYSE30, KYSE50, KYSE70, KYSE140, KYSE150, KYSE170, KYSE180, KYSE220, KYSE270, T.T, and TTN were obtained from Health Science Research Resources Bank (Osaka, Japan). All cancer cell lines were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS, Autogen Bioclear), glutamine, 100units/ml penicillin, and 100 μg/ml streptomycin, in a humidified atmosphere with 5% CO2.
Primary human esophageal fibroblasts designated as HEF75 , HEF2111, HEF1173 and cancer associated fibroblasts designated as HECAF2111 were isolated from human esophagus tissues which were resected in the Department of Surgery, Jichi Medical University Hospital. The patient from whom the tissue was obtained had not received neoadjuvant chemotherapy or radiation therapy. The study was approved by the Jichi Medical University Ethics Committee and written informed consent was obtained from the patient.
To isolate fibroblasts [35,36], epithelial tissue was washed twice in PBS and cut into 1–2 mm3 pieces. Several pieces were placed in a six-well plate, and the explants cultured for 48 hours in DMEM (Dulbecco’s modified Eagle’s medium; Invitrogen) supplemented with 10% FBS, antibiotics, and glutamine at 37°C in a humidified atmosphere with 5% CO2. After removing the explants and non-adherent cells, the remaining cells were incubated for 1–2 weeks. The adherent cells were then trypsinized and passaged into a new culture flask at a ratio of 1:3 for further expansion. The cells were used for subsequent experimental study after the third passage.
Human lung-derived fibroblasts, HFL-III, obtained from RIKEN Cell Bank, were also used in some experiments.
Immortalization of esophageal fibroblasts
Immortalized fibroblasts, designated as HFE75-hTERT (human telomerase reverse transcriptase), were described previously . In brief, in accordance with the protocol of Lipofectamin 2000 Reagent (Invitrogen Co., Ltd.), plasmid DNA encoding hTERT (pCLXSN-hTERT), a kind gift from Dr. T. Kiyono, National Cancer Research Institute, Japan , and pVSV-G were co-transfected into the GP2-293 cell line. After 48 hours, the culture supernatant including the retrovirus was added to fibroblasts to induce transduction. Selection was performed with G418 (Geneticin®, Invitrogen) 48 hours after transduction. In this study, cells between the 20th and 25th passage after viral transduction were used. Immortalized human esophageal fibroblasts were designated as human esophageal fibroblast (HEF) 75-human telomerase reverse transcriptase (HEF75-hTERT).
Rabbit polyclonal anti-phospho-Akt, anti-phospho-Erk antibodies (Cell Signaling Technology, Danvers, MA) and goat polyclonal anti-beta actin (Santa Cruz Biotechnology, Santa Cruz, CA) were used in this study.
Gene expression profile
A comprehensive gene expression analysis was performed using an oligonucleotide microarray (GeneChip Human Genome U133A, Affymetrix, Santa Clara, CA) as described previously . The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus  and are accessible through GEO Series accession number GSE63941 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE63941).
Western blot analysis
Cells were lysed in a lysis buffer consisting of 20 mmol/L Tris–HCl (pH7.4), 150 mmol/L NaCl, 50 mmol/L NaF, and 1 mmol/L Na3VO4 with a cocktail of proteinase inhibitors. After sonication, lysates were immersed in water at 98°C for five minutes and cleared by centrifugation. Protein concentrations were determined using the DC Protein Assay kit (BioRad). For Western blot analysis, equal amounts of protein samples were size-separated on 8% polyacrylamide gels and electroblotted onto a nitrocellulose membrane. Nonspecific binding was blocked by immersion of the membranes for 20 minutes in 5% skim milk in Tris-buffer saline at room temperature.
Membranes were washed with Tris-buffer saline buffer containing 0.1% Tween 20, incubated for one hour at room temperature with primary antibodies, washed, and then reacted with peroxidase-conjugated secondary antibodies. The antigen was detected using ECL Western Blotting Detection Reagents (Amersham) following the manufacturer’s instructions.
Cell proliferation assay
Cell viability was measured using the Cell Counting Kit (CCK) 8 assay (Dojindo, Tokyo, Japan) according to the manufacturer’s instructions. ESCC cells (4-10×103 cells) were plated in 96-well microtiter plates. After 24 hours, a supernatant of fibroblasts or growth factors (HGF, FGF1, FGF7, and FGF10), was added to the wells as appropriate.
The fibroblast supernatant was prepared by incubating HEF75 or HEF75-hTERT in DMEM containing 0.1%FBS for 12 hrs. The prepared supernatant was diluted at 1×, 1/2×, 1/10×, 1/100×, and 1/1000× with 0.1%FBS + DMEM and added to the ESCC cells. The final concentrations of the growth factors were: 20 ng/ml, 10 ng/ml, 2 ng/ml, 0.2 ng/ml and 0.02 ng/ml for HGF; 10 ng/ml, 1 ng/ml, 0.1 ng/ml, 0.01 ng/ml and 0.001 ng/ml for FGF1; 40 ng/ml, 20 ng/ml, 2 ng/ml, 0.2 ng/ml, 0.02 ng/ml for FGF7; and 20 ng/ml, 10 ng/ml, 1 ng/ml, 0.1 ng/ml and 0.01 ng/ml for FGF10.
After the addition of fibroblast supernatant or growth factors, cells were incubated for four days at 37°C. For inhibition experiments, cells were incubated in the presence of 1 μM of lapatinib (a dual inhibitor of epidermal growth factor receptor and human epidermal growth factor receptor 2), PHA-665752 (MET inhibitor), PD-173074 (FGFR inhibitor), or 0.1% of dimethyl sulfoxide (DMSO, negative control).
At the end of the four-day incubation, the absorbance of each well at 450 nm was measured with a reference at 630 nm using a BIO-RAD model 680XR microplate reader (Bio-Rad, Hercules, CA).
The percentage of cell viability was calculated by following formula:%cell viability = (mean absorbance in test wells)/ (mean absorbance in control well) ×100. Results were plotted as cell viability versus log10 (concentration of reagents).
To describe the blocking effect of PHA-665752 and PD-173074 on the growth promoting effect of fibroblast supernatant, a blunting index was calculated by the formula: blunting index (%) = 100-(fold change in the presence of PHA-665752 (or PD-173074) and fibroblast supernatant/fold change in the presence fibroblast supernatant alone) × 100.
Enzyme-linked immunosorbent assay (ELISA)
Human HEF75, HEF75-hTERT, and some cancer cells in a confluent state were cultured for 24 hours in RPMI/DMEM containing 0.1%FBS, and the supernatant concentration was measured by Quantikine® human FGF 7 immunoassay or human HGF immunoassay (R&D Systems). Measurements were performed in accordance with the manufacturer’s instructions.
Analysis of variance was used to compare mean values of continuous variables among three or more groups, and P-values were calculated. When the P-value was less than 0.05 and the null hypothesis was rejected, Dunnett’s test, a method for post hoc comparison, was used to compare the groups. The Jmp9 (SAS Institute Inc., Cary, North Carolina, USA) statistical software package was used for analysis.
Effect of fibroblast supernatant on ESCC cell proliferation
When proliferation assays were performed using culture supernatant obtained from fibroblasts established from normal esophagus (HEF 2111) and purchased lung-derived fibroblasts (HFL-III), generally similar trends are observed (data not shown). These findings imply that growth factors or cytokines secreted by fibroblasts play an important role in cancer cell proliferation.
Extrinsic and intrinsic roles of HGF/MET and FGFs/FGFR in ESCC cell proliferation
These results indicate that cell proliferation of many ESCC cell lines is dependent on HGF/MET and FGF/FGFR either in an extrinsic or intrinsic manner. These experiments also indicate diversity in the degree of dependence on HGF/MET and FGF/FGFR among the cell lines tested, with more cell lines exogenously dependent on FGF/FGFR than on HGF/MET.
Western blot analysis
Both Akt and ERK1/2 participate in the biological effects of growth factors [41-43]. The ERK signaling cascade is reported to control the proliferation of multiple cell types in response to growth factor treatment [44,45]. In contrast, Akt signaling is best known for mediating cell survival .
Effects of PHA-665752 (MET inhibitor) and PD-173074 (FGFR inhibitor) on cell proliferation induced by fibroblast supernatant
Testing all ESCC cell lines for the relative contribution of HGF/MET and FGF/FGFR in a similar experiment, demonstrates that four cell lines (KYSE220, TTN, TE-6, and TE-15) are dependent on HGF/MET, while eight cell lines (KYSE30, KYSE50, TT, TE-6, TE-9, TE-11, TE-14, and TE-15) are dependent on FGF/FGFR. These results indicate that paracrine stimulation by fibroblast is dependent, at least in part, on HGF/MET or FGF/FGFR. These results also show that there is diversity among ESCC cell lines with regard to dependence on HGF/MET and FGF/FGFR.
Inhibitory effects of lapatinib on ESCC cell proliferation and rescue by fibroblast supernatant
Cancer treatment strategies are currently shifting to targeted therapies. Several drugs are target members of the human epidermal growth factor receptor (EGFR/HER) family . This family includes four membrane receptor tyrosine kinases, EGFR (HER1/erbB1), HER2 (erbB2), HER3 (erbB3), and HER4 (erbB4), which activate key cell signaling pathways controlling cell growth, proliferation, migration, apoptosis, and resistance to cytotoxic agents . Wang et al. reported that supernatants of human fibroblast cell lines induce gefinitib resistance in lung cancer cell lines .
FGFR inhibitor (PD-173074) blocks the rescue effect of fibroblast supernatants against lapatinib
Recent studies indicate that paracrine stimuli from stromal fibroblasts may confer resistance to various molecular targeted agents including lapatinib [49-51]. We reasoned that blockade of paracrine stimuli may attenuate the rescue effect of fibroblast supernatant in lapatinib-sensitive ESCC cell lines. We chose TE-11 and TE-14 cell lines in which the paracrine effect of fibroblasts are dependent on FGFRs, and tested the effect of FGFR inhibitor on the rescue effect of fibroblast supernatant.
The concentrations of HGF in culture supernatants of the fibroblasts HEF75, HEF75-hTERT, and HFL-III were 4049 pg/mL, 9390 pg/mL, and 9381 pg/mL, respectively, and the concentrations of FGF7 were 249 pg/mL, 1087 pg/mL, and 17 pg/mL, respectively. Culture supernatants of HFL-III, a lung-derived fibroblast line, contained high concentrations of HGF, but very low concentrations of FGF7. The culture supernatants of measured cancer cells (KYSE30, KYSE50, KYSE150, KYSE220, T.TN, TE-8, TE-11, TE-14, and TE-15) contained virtually no HGF or FGF7, suggesting that the autocrine activity of these cells was minimal.
The importance of the tumor microenvironment for tumor growth has been recognized for many years . Cancers develop in a complex and dynamic microenvironment. Stromal fibroblasts are one of the major stromal components and it is becoming increasingly clear that fibroblasts are also prominent modifiers of cancer progression .
To date, few studies have comprehensively evaluated the interactions between ESCC and stromal fibroblasts. We studied 22 ESCC cell lines, the maximum available commercially, because many previous studies used few cell lines that were appropriate for the experiments. Furthermore, we isolated esophageal stromal fibroblasts from resected specimens and immortalized a portion of them. These studies clarify the roles of fibroblasts in the proliferation of ESCC and in resistance to lapatinib, given the diversity of cancer.
We first examined cell proliferation induced by fibroblasts. The addition of fibroblast supernatant induced cell proliferation in the majority of ESCC cell lines. In particular, supernatants from immortalized fibroblasts had a stronger impact on cancer cell lines. HEF75-hTERT, immortalized fibroblasts, increased cell proliferation by 1.25 fold or more in 21 of 22 cell lines, while normal fibroblasts increased proliferation in 12 of 22 cell lines (Figure 1). These results imply that immortalized fibroblasts may develop properties similar to the so-called cancer associated fibroblasts.
Stromal fibroblasts promote tumor progression in several ways such as secretion of multiple factors and matrix metalloproteinases . We hypothesized that some growth factors, such as HGF or FGFs secreted by stromal fibroblasts may be responsible for the proliferation effect of ESCC cells. In fact, previous studies have suggested that HGF promotes invasion of ESCC cells  and FGF7 (keratinocyte growth factor: KGF) increases the growth rate of esophageal cancer cell lines (TE-8 and TE-11) . We next examined the effects of adding growth factors to the culture medium of cancer cells and the effects of FGFR inhibitor and MET inhibitor treatment on cell proliferation induced by fibroblast supernatant. The results of experiments to evaluate the effects of adding growth factors and kinase inhibitors suggest that the stimulating effect of fibroblasts was attributable in part to HGF/MET or FGF/FGFR (Figure 4). The results indicate diversity in the degree of dependence on HGF/MET and FGF/FGFR among the cell lines. The exogenous proliferation of more ESCC cell lines may depend on FGF/FGFR signaling rather than on HGF/MET signaling.
The development of targeted therapies is a major advance in the treatment of cancer , but data for esophageal cancer are still lacking . Previous reports imply that targeted therapy has potential for the treatment of patients with ESCC . Stromal fibroblasts also might correlate with resistance to targeted therapy [49,51]. Lapatinib, which is a dual tyrosine kinase inhibitor, is considered a promising candidate . When we added 1 μM of lapatinib to the 22 ESCC cell lines tested, proliferation was strongly inhibited in a majority of the cells lines (Figure 7). However, lapatinib-induced inhibition was abrogated by fibroblast supernatants in many cell lines, and proliferation in 10 of the 22 cell lines was rescued more than 25% (Figure 7). These findings suggest that lapatinib inhibits the proliferation of ESCC cells in an intrinsic manner, while fibroblasts might antagonize the effect of lapatinib. Moreover, these results imply that the effect of targeted therapies can be attenuated by stromal fibroblasts. Fibroblasts might thus have a role in drug resistance, even in ESCC cell lines.
Finally, we tested whether the FGFR inhibitor PD-173074 could eliminate the rescue effect against lapatinib that was induced by fibroblast supernatants. In two representative cell lines (TE-11 and TE-14), fibroblast supernatant restored the growth of cancer cells inhibited by lapatinib, but the subsequent addition of PD-173074 abrogated the rescue effect of fibroblast supernatant against lapatinib (Figure 8). These findings imply that combination therapy with lapatinib and an FGFR inhibitor might be effective in overcoming therapeutic resistance against to lapatinib caused by stromal fibroblasts.
We studied 22 ESCC cell lines taking into account the diversity of malignancies. In more than half of the cancer cell lines tested, proliferation was induced by culture supernatants of immortalized fibroblasts. Growth factors, such as HGF or FGFs secreted by fibroblasts influence cell proliferation and resistance to lapatinib in some cancer cell lines, although the degree of dependence differs among the cell lines. The addition of fibroblast supernatant attenuates the effect of lapatinib. However, the rescue effect of fibroblast supernatant is abrogated by combining an FGFR inhibitor with lapatinib. Although this study was performed in vitro, these results may lead to the development of new treatment strategies.
This study was supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan, Grants for Research on Human Genome Tailor-made from the Ministry of Health, Labor, and Welfare of Japan, and the Foundation for Development of Community, Japan.
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