Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Cisplatin treatment increases survival and expansion of a highly tumorigenic side-population fraction by upregulating VEGF/Flt1 autocrine signaling

Abstract

The cellular and molecular mechanisms of tumor progression following chemotherapy are largely unknown. Here, we demonstrate that cisplatin (CDDP) treatment upregulates VEGF and Flt1 expression leading to the survival and expansion of a highly tumorigenic fraction of side-population (SP) cells in osteosarcoma (HOS), neuroblastoma (SK-N-BE2) and rhabdomyosarcoma (RH-4) cell lines. In all three lines, we show that CDDP treatment increases levels of VEGF and Flt1 expression, and induces enhanced clonogenic capacity and increased expression of the ‘stemness’-associated genes Nanog, Bmi-1 and Oct-4 in the SP fraction. In HOS, these changes are associated with the transformation of a non-tumorigenic osteosarcoma SP fraction to a highly tumorigenic phenotype. Inhibition of Flt1 led to complete reduction of tumorigenicity in the HOS SP fraction, and reduction of clonogenic capacity and expression of stemness genes in the SK-N-BE(2) and RH-4 SP fractions. Treatment with U0126, a specific inhibitor of MAPK/ERK1,2 completely downregulates CDDP-induced VEGF and Flt1 expression and induction/expansion of SP fraction in all three cell lines, indicating that these effects are mediated through MAPK/ERK1,2 signaling. In conclusion, we report a novel mechanism of CDDP-induced tumor progression, whereby the activation of VEGF/Flt1 autocrine signaling leads to the survival and expansion of a highly tumorigenic SP fraction.

Introduction

Vascular endothelial growth factor (VEGF) is a potent angiogenic agent having tumor-promoting activity. Tumor cells secrete VEGF that increases the proliferation of endothelial cells leading to tumor angiogenesis and subsequent tumor progression (Ferrara et al., 2003). In addition to its effects on endothelial cells, VEGF may also act directly on tumor cells in an autocrine manner to increase survival and proliferation (Fukumura et al., 1998; Dias et al., 2000; Bellamy et al., 2001; Soker et al., 2001; Strizzi et al., 2001; Bates et al., 2003; Qi et al., 2003; Mercurio et al., 2004; Steiner et al., 2004). We have shown that VEGF binds to its cognate receptor VEGFR-1 (Flt1 also known as fms-like tyrosine kinase-1) in neuroblastoma and rhabdomyosarcoma leading to activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase-1,2 (ERK1,2) and subsequent tumor cell survival (Das et al., 2005; Gee et al., 2005). During hypoxia-induced stress, increased expression of Flt1 rescued the tumor cells from hypoxia-induced cell death (Das et al., 2005). Our preliminary data suggested that exposure to cisplatin (cis-diammine-dichloroplatinum (II); CDDP) may increase Flt1 expression in osteosarcoma cell lines (Das B et al.). The role of VEGF autocrine signaling during oxidative stress. 7th International Symposium on Cytokines and Chemokines; 8–9 September 2005; World Congress of Gastroenterology, Montreal, Canada). Therefore, we speculated that in the fraction of tumor cells resistant to chemotherapy, treatment-induced Flt1 autocrine signaling may provide survival signaling. Recently, a tumor side-population (SP) fraction has been isolated from tumors exhibiting high resistance to chemotherapeutic treatment (Patrawala et al., 2005). Therefore, we studied the potential upregulation of Flt1 in the SP fraction and subsequent tumorigenicity.

Chemotherapeutic agents have multiple effects on tumor cells including transient modulation of growth factor signaling, drug efflux pumps and detoxification systems leading to tumor cell survival (Cara and Tannock, 2001; Cole and Tannock, 2004; Kim and Tannock, 2005). Recently, Biswas et al. (2007) showed that chemotherapeutic agents like doxorubicin may increase transforming growth factor-β signaling leading to survival and expansion of highly tumorigenic cells. Chemotherapeutic agents may transform non-tumorigenic into tumorigenic tumor cells. For example, an osteosarcoma cell line HOS, which is non-tumorigenic became tumorigenic following acute exposure to heavy metals including platinum (Lin and Costa, 1994; Miller et al., 1998, 2001; Salnikow et al., 1999). Cisplatin, a platinum drug, has been shown to activate MAPK/ERK1,2 growth signaling pathway leading to either tumor cell survival or death depending upon the cell types (Woessmann et al., 2002; Brozovic and Osmak, 2007). Our unpublished study suggested that cisplatin treatment may activate the VEGF/Flt1 autocrine signaling pathway in tumor cell lines including HOS (Das B, Tsuchida R, Yeger H, Malkin D and Baruchel S). The role of VEGF autocrine signaling during oxidative stress. 7th International Symposium on Cytokines and Chemokines; 8–9 September 2005; World Congress of Gastroenterology, Montreal, Canada).

Thus, chemotherapeutic drugs may induce cellular signaling pathways involved in tumor growth and progression leading to tumorigenicity and/or repopulation. Understanding the molecular mechanism involved in these processes may help to develop new combination therapy regimens to reduce the incidence of tumor relapse. Therefore, we investigated the potential role of VEGF/Flt1 autocrine signaling in a CDDP model of drug-induced tumorigenicity. We found that CDDP treatment activates VEGF/Flt1 autocrine signaling in the highly tumorigenic SP fraction of cell lines obtained from osteosarcoma, rhabdomyosarcoma and neuroblastoma. Subsequently, we show that the CDDP-induced SP fraction and its tumorigenic potential can be reduced by inhibiting VEGF/Flt1 autocrine signaling. Furthermore, we found that several stemness genes, including Nanog, are highly expressed in the CDDP-treated SP fraction both in vitro and in vivo. Our findings highlight the importance of VEGF/Flt1 autocrine signaling in drug-induced tumorigenicity including the expansion of an SP fraction expressing self-renewal genes including Nanog and Oct-4.

Results

CDDP treatment increases tumorigenic potential of osteosarcoma HOS cells

We used a human osteosarcoma cell line, HOS, as a model to study the role of VEGF/Flt1 autocrine signaling in CDDP-induced tumorigenicity. This cell line is non-tumorigenic when injected into nude mice (McAllister et al., 1971), and becomes tumorigenic following exposure to heavy metals (Lin and Costa, 1994; Miller et al., 2001; Miura et al., 2005). We found that exposure of HOS cells to cisplatin (CDDP) (2 μM × 3 days) increased in vitro tumorigenicity (Figures 1a and b) as measured by in vitro matrigel assay (Miller et al., 2001), and induced formation of large tumors when injected into nude mice (2 × 106 cells per injection, n=16; Figures 1c and d).We observed that the untreated HOS group (10 × 106 cells per injection) reached 0.05 cm3 size (four out of eight injection; Figure 1c). Histopathological examination of tumors at 50 days revealed that the area of inoculation was filled with fibroblasts and a few scattered tumor cells, whereas the CDDP-treated tumors showed tumor cells with surrounding blood vessels (Figure 1d). Considering that neovascularization is involved in the progression of tumor from dormancy to a state of rapid growth (Folkman, 1971), we measured the MVD (microvessel density) of the 0.05 cm3 growth using CD34 staining (Gasparini and Harris, 1995) and found that CDDP-treated xenograft showed numerous CD34 staining microvessels, whereas untreated growth did not show evidence of angiogenesis (Supplementary Figure 1).

Figure 1
figure1

Cisplatin (cis-diammine-dichloroplatinum (II); CDDP) treatment transforms non-tumorigenic HOS cells to form large, vascular tumors. (a) CDDP treatment group colonies were larger than untreated cells (× 200). To the right, histogram shows significantly higher plating efficiency following 2 μM CDDP × 3 days treatment (*P=0.0092; n=4). (b) In vitro matrigel-based tumorigenicity assay showing that treated cells formed branched networks of cells that grew and invaded the matrigel. (c) CDDP treatment group formed a large tumor mass with numerous visible vessels on its surface (50-day-old xenograft) (d) Hematoxylin and eosin staining of the tumors showed tumor cells embedded in stroma and blood vessels (× 200). An enlarged view (× 400) shows infiltrating macrophages and actively proliferating tumor cells. The untreated cells formed a small tumor mass (0.05 cm3), where a few tumor cells were seen among numerous fibroblasts (× 400). Detailed results are given in Figure 2c and the associated table in the figure.

We then investigated the potential role for SP cells in CDDP-treated HOS cell tumorigenicity. In all the following assays, 3-day exposure to 2 μM CDDP was used as a standard treatment for in vitro as well as in vivo studies.

CDDP-treated HOS SP fraction (SPcddp) is highly tumorigenic

Tumor SP cells characterized by high expression of drug efflux pumps such as BCRP1 and MDR1 have been found to contain the tumor stem cell fraction (Hirschmann-Jax et al., 2004; Kondo et al., 2004; Patrawala et al., 2005). We show that the HOS cell line contains a small fraction of SP cells, which can be maintained in serum-free media containing growth factors bFGF (basic fibroblast growth factor) and PDGF (platelet-derived growth factor; Supplementary Figure 2A). Figure 2a shows that CDDP treatment increases the SP cell fraction significantly.

Figure 2
figure2

Cisplatin (cis-diammine-dichloroplatinum (II); CDDP)-treated HOS side-population (SP) fraction (SPcddp) expresses stemness genes and is highly tumorigenic. (a) Histogram showing that CDDP treatment increased the SP cells in a dose-dependent manner (untreated: 2.6±0.2; CDDP (1 μM): 4.3±0.2; CDDP (2 μM): 6.6±0.5; *P=0.0380 and **P=0.0193). (b) Quantitative reverse transcriptase (RT)-PCR analysis showing significant increase of Bmi-1 and Nanog in SPcddp fraction compared with other fractions (*P<0.05 and **P<0.01). Human embryonic stem (hES) cell line was used as a reference control to obtain the ΔΔCt value. (c) Histogram showing the increase clonogenic activity of the SPcddp fraction (*P=0.0021). (d) Images of a representative SPcddp tumor, and its SP counterpart, the latter showing the avascular remnant of the injected matrigel plug. The associated table shows the details of the tumorigenic potential of the SPcddp fraction compared with other fractions.

We then sorted SP, non-SP, SPcddp (SP cells obtained following 3 days treatment of HOS cells with 2 μM CDDP) and non-SPcddp (non-SP cells obtained following 3 days treatment of HOS cells with 2 μM CDDP) and investigated the expression of the stemness-associated genes Nanog and Bmi-1 by quantitative reverse transcriptase (RT)-PCR (qPCR). Both these genes are highly expressed in the SPcddp fraction (Figure 2b). Furthermore, colony assay showed increased clonogenic efficiency of CDDP-treated SP fraction (SPcddp) compared with the untreated SP and treated non-SP (non-SPcddp) fractions (Figure 2c). In addition, when SPcddp cells were injected into nude mice, they formed rapidly growing tumors (Figure 2d) compared with the SP and non-SPcddp group. Furthermore, the SPcddp xenograft showed a 1.5-fold increase in MVD compared with CDDP-treated unsorted HOS cell-derived xenografts (Mann–Whitney test; Supplementary Figure 1).

siRNA/Flt1 knockdown reduces HOS SPcddp fraction and subsequent tumorigenicity

Previously, we reported that VEGF/Flt1 autocrine signaling is involved in the survival of tumor cells during hypoxia as well as drug exposure (Das et al., 2005). Here, we first show that HOS cells express functionally active Flt1 (VEGFR1) (Figures 3a and b), but not KDR (kinase insert domain-containing receptor, VEGF receptor-2) (data not shown). Small interfering RNA (siRNA)/Flt1 knockdown completely inhibited recombinant human VEGF (rhVEGF)-mediated MAPK/ERK1,2 activity (Figure 3b), suggesting that VEGF/Flt1 autocrine signaling is mediated via the mitogenic MAPK/ERK1,2 signaling pathway. This result is consistent with previous findings on VEGF/Flt1 autocrine signaling (Das et al., 2005; Gee et al., 2005). We then investigated the potential role of VEGF/Flt1 signaling in the CDDP-induced SP fraction, and found that siRNA/Flt1 knockdown reduced the SPcddp fraction significantly, with associated reduction in Nanog and Bmi-1 expression (Figure 3c and d). Furthermore, when siRNA-treated cells were injected into nude mice, CDDP-mediated tumorigenicity was significantly reduced (Figure 4). Xenograft tissues derived from the CDDP+siRNA/Flt1-treated group showed a marked increase in necrotic cores and caspase-3-positive apoptotic cells (Figure 4b) compared with the CDDP-treated group. Most importantly, when tumor tissues were dissociated and the SP fraction sorted out, post-CDDP-treated xenografts contained a higher fraction of SP cells compared with the combination treatment (CDDP+siRNA/Flt1)-derived xenografts (P=0.013; Figure 4c). qPCR analysis of the SP fraction showed significant reduction of Nanog expression in the siRNA/Flt1-treated group. Immunohistochemical labeling revealed a significant reduction in Nanog-expressing cells in the siRNA/Flt1 knockdown group (Figure 4d). These results further suggest that the SPcddp fraction plays an important role in HOS cell tumor growth and angiogenesis. siRNA/Flt1 knockdown reduces this tumorigenic fraction (Figure 3c) leading to decreased tumor growth (Figure 4a).

Figure 3
figure3

Small interfering (siRNA)/Flt1 knockdown reduces HOS SPcddp fraction. Vascular endothelial growth factor (VEGF)/Flt autocrine signaling is involved in the survival/self-renewal of SP fraction. (a) Western blot data show Flt1 expression in HOS cells. On the right panel, immunoprecipitation result showing significant autophosphorylation of Flt1 after treatment with recombinant human VEGF165 (rhVEGF165) for 5 min. HeLa extract was used as a negative control. (b) siRNA knockdown of Flt1 expression in HOS cells. On the right panel, 5 min exposure of HOS to rhVEGF165 (100 ng ml−1) induced extracellular signal-regulated kinase-1,2 (ERK1,2) phosphorylation (44/42 kDa), whereas pretreatment with anti-Flt1 antibody (100 ng ml−1) or siRNA/Flt1 knockdown inhibited ERK1,2 phosphorylation. (c) Fluorescent-activated cell sorting profile showing increased size of SP fraction following cisplatin (cis-diammine-dichloroplatinum (II); CDDP) treatment. Histogram showing siRNA/Flt1 plus CDDP treatment reduced the SP fraction. (d) Quantitative reverse transcriptase-PCR analysis of Bmi-1 and Nanog shows corresponding decreased expression in the siRNA/Flt1+CDDP-treated group. SP, side-population. siRNA/pool is a control of siRNA. *P<0.05, **P<0.01.

Figure 4
figure4

In vitro small interfering RNA (siRNA)/Flt1 treatment of HOS reduces cisplatin (cis-diammine-dichloroplatinum (II); CDDP)-induced tumorigenicity in vivo. (a) In vivo tumorigenesis assay showing marked inhibition of HOS tumor growth in the siRNA/Flt1-treated fraction. Six to eight mice were used for each treatment group. (b) HOS xenograft (110 days after inoculation) showing marked necrosis (H&E) and increased expression of cleaved caspase-3 in the combination treatment (CDDP+siRNA/Flt1) group compared with the CDDP treatment group. (c) Both CDDP and combination treatment tumors were dissociated, and side-population (SP) fractions contained within these tumors were determined. The CDDP-treated group showed a higher percentage of SP cells compared with the combination treatment group (P=0.013). (d) Sorted SP fractions shows significant reduction of Nanog in the siRNA/Flt1 group (**P<0.01), and the immunohistochemical staining shows corresponding reduction in Nanog expression.

Interestingly, we noted that the control group (10 × 106 cells injected) showed progressive growth until it reached 0.05 cm3 but failed to ‘take off’ (Figure 4a). The injection of a large number of cells and the accumulation of fibroblasts (Figure 1d) may have allowed progressive growth for 50 days after inoculation. Failure to take off may be due to the lack of tumorigenic SPcddp cells and angiogenesis. MVD study suggests that SPcddp-derived tumor xenograft is vascular, whereas untreated dormant growth (0.05 cm3 growth) did not show evidence of angiogenesis (Supplementary Figure 1).

Inhibition of VEGF/Flt1 signaling reduces tumorigenic SP fraction in neuroblastoma and rhabdomyosarcoma cell lines

We then used two drug-resistant cell lines, SK-N-BE(2) and RH-4, to further investigate the CDDP-induced VEGF/Flt1 activity in the SP fraction. We recently described the VEGF/Flt1 autocrine loop in these two cell lines and showed that treatment with anti-Flt1 antibody (10–20 μg ml−1) completely reduces VEGF/Flt1 autocrine signaling in SK-N-BE(2) (Das et al., 2005) and RH-4 cells (Gee et al., 2005). These two cell lines contain a small fraction of SP cells (Supplementary Figure 2A) and are highly resistant to CDDP treatment (Supplementary Figure 2B). Injection of 2.5 × 105 SP cells formed rapidly progressing tumors (four tumors developed out of four injections for each cell type), whereas a similar number of non-SP (six injections for each cell type) cells failed to form tumors (data not shown). When these cell lines were exposed to CDDP, the SP fraction increased significantly and could then be reduced by anti-Flt1 antibody treatment (Figure 5a). We found that clonogenic activity and stemness gene expression of SPcddp was significantly enhanced (Figures 5b and c), which was completely downregulated following treatment with anti-Flt1 antibody.

Figure 5
figure5

Inhibition of vascular endothelial growth factor (VEGF)/Flt1 autocrine signaling reduces cisplatin (cis-diammine-dichloroplatinum (II); CDDP)-induced side-population (SP) fraction self-renewal and stemness. (a) CDDP treatment significantly increases SP fraction in SK-N-BE(2) and RH-4 fraction (n=3). Inhibition of Flt1 by anti-Flt1 antibody (10 μg ml−1) reduced CDDP-induced SP fraction significantly (n=3). Cells were treated with 2 μM CDDP plus anti-Flt1 antibody for 3 days and SP fraction analyzed by fluorescent-activated cell sorting. (b) Sorted SP fractions (treated and untreated) were analyzed for clonogenic activity and stemness gene expression. (c) Anti-Flt1 antibody treatment significantly decreased clonogenic activity (n=3) and the quantitative reverse transcriptase-PCR expression levels of Oct-4 and Nanog. *P<0.05, **P<0.01.

CDDP treatment induces VEGF/Flt1 autocrine signaling in the SPcddp fraction: role of MAPK/ERK1,2 signaling

Recently, Biswas et al. (2007) demonstrated that chemotherapeutic agents like doxorubicin may increase transforming growth factor-β signaling leading to survival and expansion of highly tumorigenic cells. Here, we found that CDDP treatment increases the SP fraction, which can be reduced by inhibiting VEGF/Flt1 autocrine signaling. Hence, it can be hypothesized that CDDP treatment may have increased VEGF and Flt1 expression in the SP fraction leading to the survival and expansion of the SPcddp fraction. Here, we first investigated the potential CDDP-induced upregulation of VEGF and Flt1 signaling in HOS cells. We found that CDDP treatment upregulates the expression of both Flt1 and VEGF expression in the HOS SP fraction (Figures 6a and c) and SK-N-BE(2)/RH-4 (Figures 7a–c). We did not observe significant upregulation of KDR and neuropilin-1 (data not shown).

Figure 6
figure6

Inhibition of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase-1,2 (ERK1,2) reduces cisplatin (cis-diammine-dichloroplatinum (II); CDDP)-induced upregulation of Flt1 expression in HOS SPcddp fraction. (a) Reverse transcriptase (RT)-PCR data show upregulation in vascular endothelial growth factor (VEGF) and Flt1 expression after CDDP treatment. Small interfering RNA (siRNA)/Flt1 knockdown reduced CDDP-mediated upregulation of VEGF and Flt1. (b) Western blot shows marked increase of phospho-ERK1,2 following CDDP treatment. Anti-Flt1 antibody treatment (100 ng ml−1 for 3 days) markedly reduced the ERK1,2 activity. Following CDDP treatment for 3 days (with or without anti-Flt1 antibody), cells were serum starved for 24 h protein extracted and western immunoblotting performed. Control cells were also subjected to 24 h serum starvation before protein extraction. HeLa was used as a positive control for MAPK/ERK1,2 activity during serum starvation. NT=not treated with CDDP. (c) Quantitative RT-PCR (qPCR) data show the increase of VEGF expression in SPcddp fraction (n=3); 20 μM of U0126 treatment reduced the VEGF secretion significantly (n=3). Note that VEGF expression was also increased in CDDP-treated non-SP fraction. Right panel: qPCR data showing the upregulation of Flt1 expression in the SPcddp fraction (n=3) and its reduction following U0126 treatment (n=3). Note that Flt1 expression was not increased in the CDDP-treated non-SP fraction. (d) Immunofluorescence data (top panel) showing marked increase of phospho-ERK1,2 in SPcddp and non-SPcddp cells. The bottom panel shows selective increase of Flt1 expression in SPcddp fraction, and its downregulation by U0126 treatment. SP, side-population. siRNA/pool is a control of siRNA. *P<0.05, **P<0.01.

Figure 7
figure7

Inhibition of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase-1,2 (ERK1,2) signaling reduces cisplatin (cis-diammine-dichloroplatinum (II); CDDP)-induced vascular endothelial growth factor (VEGF) and Flt1 upregulation in SK-N-BE(2) and RH-4. (a) ELISA (enzyme-linked immunosorbent assay) data showing the increase in VEGF165 secretion after CDDP treatment (n=4); 20 μM of U0126 treatment reduced the VEGF secretion (n=3). Note that VEGF expression was also increased in CDDP-treated non-SP fraction. (b and c) Quantitative reverse transcriptase (RT)-PCR data showing the upregulation of VEGF and Flt1 expression in the SPcddp fraction (n=3) and its reduction following U0126 treatment (n=3), respectively. Note that Flt1 expression was not increased in the CDDP-treated non-SP fraction. (d) Treatment with 20 μM of U0126 significantly reduced SK-N-BE(2) SPcddp fraction (n=3), but did not reduce RH-4 SPcddp fraction. SP, side-population. *P<0.05, **P<0.01.

When VEGF secretion was measured in HOS SP, SPcddp and non-SPcddp (CDDP-treated non-SP cells), the SPcddp cells showed significantly higher levels of secretion compared with the SP and non-SPcddp fractions (P<0.05; Supplementary Figure 2C). Interestingly, the secretion of VEGF in HOS non-SPcddp also increased significantly (P<0.05; Supplementary Figure 2C). qPCR study further revealed that the expression of VEGF increased in the non-SPcddp fraction, even though the level of expression was significantly lower than the SPcddp fraction in all the cell lines (Figures 6c and 7b). These results indicate that there was a specific increase of Flt1 in SPcddp fraction, whereas VEGF expression was increased in both SP and non-SP fraction; however, VEGF expression was higher in the SPcddp compared with the non-SPcddp fractions.

Recently, we found that sustained activation of MAPK/ERK1,2 upregulates VEGF/Flt1 signaling in SK-N-BE(2) (Das et al., 2005). Since CDDP treatment may activate MAPK/ERK1,2 signaling (Woessmann et al., 2002; Brozovic and Osmak, 2007), we examined the effect of CDDP treatment on the level of phosphorylated forms of MAPK/ERK1,2 and found that the latter is markedly increased following CDDP treatment (Figure 6b). We then investigated the effect of U0126, a specific inhibitor of MAPK/ERK1,2 on the expression of VEGF and Flt1 following CDDP treatment. We used a dose of 20 μM U0126, which downregulates rhVEGF-mediated MAPK/ERK1,2 in HOS as well as RH-4 (Gee et al., 2005) and SK-N-BE(2) (Das et al., 2005). We found that U0126 treatment reduced CDDP-induced VEGF and Flt1 expression in HOS (Figure 6c) and RH-4/SK-N-BE(2) (Figures 7a–c). Immunofluorescence studies showed that phospho-ERK1,2 level was increased in both SPcddp and non-SPcddp compared with SP cells. However, Flt1 expression was mainly increased in the SPcddp fraction (Figure 6d). U0126 treatment reduced Flt1 expression in the SP fraction (Figure 6c) and reduced CDDP-induced SPcddp fraction significantly in HOS (data not shown) as well as SK-N-BE(2) (Figure 7d).

Discussion

Vascular endothelial growth factor/Flt1 autocrine signaling plays an important role in the survival of drug-resistant tumor cells (Das et al., 2005; Gee et al., 2005). Environmental stress such as hypoxia upregulates VEGF/Flt1 signaling in the tumor cells leading to enhanced tumorigenic potential including angiogenic potential (Das et al., 2005). Here, we investigated the role of chemotherapeutic-induced stress in the upregulation of VEGF/Flt1 autocrine signaling and subsequent tumorigenic potential in several tumor cell lines. We particularly investigated the role of CDDP, a commonly used chemotherapeutic agent in the upregulation of VEGF/Flt1 autocrine signaling in tumor SP fraction. We found that (1) CDDP treatment increases the SP fraction of three different cell lines; (2) CDDP treatment increases VEGF and Flt1 expression in the SP fraction by activating MAPK/ERK1,2 signaling; and (3) inhibition of Flt1 reduces the tumorigenic fraction.

Recently, tumor SP fractions have been shown to contain tumorigenic cells in many tumor types. The SP fraction is highly tumorigenic, drug-resistant and expresses ‘stemness’ genes such as Nanog, Oct-4 and Bmi-1 (Setoguchi et al., 2004; Hirschmann-Jax et al., 2005; Patrawala et al., 2005, 2007; Ho et al., 2007). It has been shown that less than 1000 SP cells are sufficient to form rapidly progressing xenografts (Ho et al., 2007; Patrawala et al., 2007). However, here we found that not all SP cells are tumorigenic since 1 × 105–0.5 × 106 SP cells were required to obtain SK-N-BE(2) and RH-4 xenografts. The HOS cell line contains a 2–3% fraction of SP cells, even though it is non-tumorigenic. CDDP-treatment increased the tumorigenic capacity including the expression of Nanog in the HOS SPcddp fraction both in vitro and in vivo. For the first time, we have demonstrated that drug exposure may actually enhance the tumorigenic potential of the SP fraction.

We found that CDDP treatment led to a significant increase in the SP fraction including specific upregulation of Flt1 and stemness gene expression in the SP fraction. In our experimental model, cells were first treated with CDDP, and then SP and non-SP fractions were sorted and qPCR analysis of Flt1 expression was performed. Hence, it can be argued that an apparent increase of Flt1/stemness gene expression in the SP fraction may be due to either survival (selection) and/or expansion (induction) of SP cells having Flt1/stemness gene expression. While selection may explain the increase of Flt1/stemness gene expressing HOS SPcddp cells, the RH-4 and SK-N-BE(2) are highly resistant to CDDP treatment (Supplementary Figure 1B), and therefore, apparent selection of Flt1-expressing SPcddp cells is unlikely. Instead, CDDP-mediated stress may expand SPcddp fraction. We found that hypoxia-induced oxidative stress expands highly tumorigenic SP cells (Das et al., 2007) (Das B. The role of VEGF autocrine signaling in hypoxia and oxidative stress driven ‘stemness switch’: implications in solid tumor progression and metastasis. PhD thesis, Institute of Medical Sciences, University of Toronto, Canada). Earlier, it was reported that chemotherapy-induced stress led to the expansion of normal bone marrow stem cells (Richman et al., 1976; Cottler-Fox et al., 2003). Hence, CDDP-induced expansion of a highly tumorigenic SPcddp fraction is a likely possibility. Such a stress-induced expansion of tumorigenic cells would involve autocrine signaling pathways such as VEGF/Flt1 signaling. Our findings that siRNA/Flt1 inhibition significantly decreased the SPcddp fraction support this possibility. It is possible that only tumorigenic SP cells are capable of activating the signaling pathway leading to expansion, which would explain why the Flt1/stemness gene expression did not change in the non-tumorigenic non-SPcddp fraction.

Overall, our findings in HOS, SK-N-BE(2) and RH-4 cells suggest that CDDP-induced SP fraction propagation is mediated by upregulation of VEGF and its receptor Flt1. VEGF/Flt1 autocrine signaling is mediated by MAPK/ERK1,2 signaling, the latter being found to be upregulated during stress including following CDDP treatment (Woessmann et al., 2002; Brozovic and Osmak, 2007). Numerous studies have reported the activation of the MAPK superfamily including ERK1,2 in tumor cells following stress (Brozovic and Osmak, 2007). The activation of these signaling pathways may be involved in cell survival as well as cell death depending upon the specific stimulus and cell type involved. We found that ERK1,2 activation following hypoxia reperfusion injury leads to cell survival in neuroblastoma cell lines (Das et al., 2005). On the other hand, hypoxia reperfusion-activated MAPK/ERK1,2 in cardiac tissue may lead to protection as well as damage to cardiac tissue depending on the degree of stimulation and cell type involved (Behrends et al., 2000). Ranganathan et al. (2006) reported that upregulation of MAPK/ERK1,2 signaling may activate dormant tumor cells leading to the expansion of tumorigenic cells and subsequent tumor progression. However, no molecular mechanism was identified that accounted for the activation of the dormant tumors. Here, our findings on MAPK/ERK1,2-mediated activation of VEGF/Flt1 signaling in the SP fraction suggest a molecular mechanism by which the mitogenic signaling of MAPK/ERK1,2 may activate dormant tumor stem cell-like SP cells. In the clinical setting, it may be necessary to first assess the effect of CDDP on the specific tumor cell type to determine whether the drug induces VEGF and MAPK/ERK1,2 signaling activity. If it does, combination therapy might be considered beneficial. Therefore, use of such preclinical data may help to design effective and innovative combination therapies.

Our findings on the CDDP-induced activation of VEGF/Flt1 signaling in a subset of tumor SP cells warrant further investigation including whether other chemotherapeutic agents may have similar effect. Our preliminary investigation suggests that doxorubicin and methotrexate may also increase SP fraction and Flt1 expression in the tumor cells (Supplementary Figures 3A and B).

In summary, we report on a novel mechanism of CDDP-induced tumorigenicity and demonstrate that even brief exposure of tumor cells to drugs may lead to the upregulation of VEGF autocrine signaling. One of the important and direct implications of our result is that primary dormant tumors may become highly tumorigenic following exposure to drugs by activating a signaling loop between MAPK/ERK1,2 and VEGF/Flt1. Hence, combination therapies that include either anti-Flt1 or MAPK/ERK1,2 inhibitors have the potential benefit to reduce the incidence of drug-induced tumor cell repopulation and relapse.

Methods

Cell lines and culture

The human osteosarcoma cell line HOS and neuroblastoma cell line SK-N-BE(2) were obtained from the American Tissue Culture Collection (ATCC, Rockville, MD, USA). The RH-4 rhabdomyosarcoma cell line was kindly provided by Dr Thomas Look (Dana-Farber Cancer Institute, Boston, MA, USA). All cell lines were maintained in the culture media as recommended by ATCC.

Reagents and drugs

Cisplatin (CDDP), doxorubicin and methotrexate were obtained from Sigma (St Louis, MO, USA). Recombinant human VEGF165 (rhVEGF165), nonspecific goat IgG and a neutralizing antibody against VEGF165 (anti-human mouse monoclonal antibody) were purchased from R&D Systems (Minneapolis, MN, USA). Source of other reagents, antibodies and kits are noted with assay descriptions.

Side-population analysis

The protocol was based on that described by Montanaro et al. (2004). Details are given in the Supplementary Methods section.

Western blot analysis and immunoprecipitation

The overall method has been previously described in detail (Das et al., 2005). Details are given in the Supplementary Methods section.

Methylcellulose-based clonogenic assay

The assay was performed as previously described (Das et al., 2003). Tumor cells were washed three times with phosphate-buffered saline and then 2 × 103 cells were plated in methylcellulose media (Stem Cell Inc., Vancouver, BC, USA), and colonies were counted after 2 weeks using an inverted microscope.

Reverse transcriptase-PCR

Details are given in the Supplementary Methods section.

Real-time quantitative RT-PCR

Real-time qPCR was performed using TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA). Details are provided in the Supplementary Methods section.

Small interfering RNA

Small interfering RNA knockdown of genes was performed according manufacturer's instructions (details are provided in the Supplementary Methods section).

ELISA analysis

The measurements of both VEGF were carried out using an ELISA kit (R&D Systems) according to the manufacturer's protocols. Samples consisted of conditioned media harvested after treatment with or without CDDP (2 μM) for 3 days in serum-free media. Fluorescence activity was converted to actual concentration by a standard curve and normalized by cell number.

Immunofluorescence and immunohistochemistry

Standard protocol was used as described in the Supplementary Methods section.

In vitro matrigel tumorigenicity assay

Tumor cells were incubated for 8 h on top of a matrigel layer. Cells with tumorigenic potential form branched networks of cells that grow and invade the matrigel structures, whereas non-tumorigenic cells either form colonies or undergo cell death (Miller et al., 2001). We modified the assay, where cells were treated with serum-free medium containing 200 ng ml−1 SDF1α (stromal cell-derived factor-1α), and then plated in growth factor-free matrigel (BD Bioscience, San Jose, CA, USA).

In vivo tumorigenicity assay

Tumorigenicity of the HOS cells was measured by tumor incidence (number of tumors/number of injection of 2–10 × 106 cells), and latency was measured as time required to form palpable tumors of more than 0.05 cm3. Details are given in the Supplementary Methods section.

Statistical analysis

The data are presented as mean±s.d. The statistical calculations were performed with GraphPad Prism 4.0 (Hearne Scientific Software, Chicago, IL, USA) using Student's t-test for cell survival assays. A value of P0.05 was considered statistically significant.

References

  1. Bates RC, Goldsmith JD, Bachelder RE, Brown C, Shibuya M, Oettgen P et al. (2003). Flt-1-dependent survival characterizes the epithelial–mesenchymal transition of colonic organoids. Curr Biol 13: 1721–1727.

    CAS  Article  Google Scholar 

  2. Behrends M, Schulz R, Post H, Alexandrov A, Belosjorow S, Michel MC et al. (2000). Inconsistent relation of MAPK activation to infarct size reduction by ischemic preconditioning in pigs. Am J Physiol Heart Circ Physiol 279: H1111–H1119.

    CAS  Article  Google Scholar 

  3. Bellamy WT, Richter L, Sirjani D, Roxas C, Glinsmann-Gibson B, Frutiger Y et al. (2001). Vascular endothelial cell growth factor is an autocrine promoter of abnormal localized immature myeloid precursors and leukemia progenitor formation in myelodysplastic syndromes. Blood 97: 1427–1434.

    CAS  Article  Google Scholar 

  4. Biswas S, Guix M, Rinehart C, Dugger TC, Chytil A, Moses HL et al. (2007). Inhibition of TGF-beta with neutralizing antibodies prevents radiation-induced acceleration of metastatic cancer progression. J Clin Invest 117: 1305–1313.

    CAS  Article  Google Scholar 

  5. Brozovic A, Osmak M . (2007). Activation of mitogen-activated protein kinases by cisplatin and their role in cisplatin-resistance. Cancer Lett 251: 1–16.

    CAS  Article  Google Scholar 

  6. Cara S, Tannock IF . (2001). Retreatment of patients with the same chemotherapy: implications for clinical mechanisms of drug resistance. Ann Oncol 12: 23–27.

    CAS  Article  Google Scholar 

  7. Cole S, Tannock IF . (2004). Drug Resistance, 4th edn. McGraw-Hill: Toronto, 390 pp.

    Google Scholar 

  8. Cottler-Fox MH, Lapidot T, Petit I, Kollet O, DiPersio JF, Link D et al. (2003). Stem cell mobilization. Hematology Am Soc Hematol Educ Program, 419–437.

    Article  Google Scholar 

  9. Das B, Tsuchida R, Malkin D, Baruchel S, Yeger H . (2007). Hypoxia enhances tumor stemness by increasing the invasive and tumorigenic side-population fraction. Stem Cells (under review).

  10. Das B, Yeger H, Baruchel H, Freedman M, Koren G, Baruchel S . (2003). In vitro cytoprotective activity of squalene on a bone marrow versus neuroblastoma model of cisplatin-induced toxicity. Implications in cancer chemotherapy. Eur J Cancer 39: 2556–2565.

    CAS  Article  Google Scholar 

  11. Das B, Yeger H, Tsuchida R, Torkin R, Gee MF, Thorner PS et al. (2005). A hypoxia-driven vascular endothelial growth factor/Flt1 autocrine loop interacts with hypoxia-inducible factor-1alpha through mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 pathway in neuroblastoma. Cancer Res 65: 7267–7275.

    CAS  Article  Google Scholar 

  12. Dias S, Hattori K, Zhu Z, Heissig B, Choy M, Lane W et al. (2000). Autocrine stimulation of VEGFR-2 activates human leukemic cell growth and migration. J Clin Invest 106: 511–521.

    CAS  Article  Google Scholar 

  13. Ferrara N, Gerber HP, LeCouter J . (2003). The biology of VEGF and its receptors. Nat Med 9: 669–676.

    CAS  Article  Google Scholar 

  14. Folkman J . (1971). Tumor angiogenesis: therapeutic implications. N Engl J Med 285: 1182–1186.

    CAS  Article  Google Scholar 

  15. Fukumura D, Xavier R, Sugiura T, Chen Y, Park EC, Lu N et al. (1998). Tumor induction of VEGF promoter activity in stromal cells. Cell 94: 715–725.

    CAS  Article  Google Scholar 

  16. Gasparini G, Harris AL . (1995). Clinical importance of the determination of tumor angiogenesis in breast carcinoma: much more than a new prognostic tool. J Clin Oncol 13: 765–782.

    CAS  Article  Google Scholar 

  17. Gee MF, Tsuchida R, Eichler-Jonsson C, Das B, Baruchel S, Malkin D . (2005). Vascular endothelial growth factor acts in an autocrine manner in rhabdomyosarcoma cell lines and can be inhibited with all-trans-retinoic acid. Oncogene 24: 8025–8037.

    CAS  Article  Google Scholar 

  18. Hirschmann-Jax C, Foster AE, Wulf GG, Goodell MA, Brenner MK . (2005). A distinct ‘side population’ of cells in human tumor cells: implications for tumor biology and therapy. Cell Cycle 4: 203–205.

    CAS  Article  Google Scholar 

  19. Hirschmann-Jax C, Foster AE, Wulf GG, Nuchtern JG, Jax TW, Gobel U et al. (2004). A distinct ‘side population’ of cells with high drug efflux capacity in human tumor cells. Proc Natl Acad Sci USA 101: 14228–14233.

    CAS  Article  Google Scholar 

  20. Ho MM, Ng AV, Lam S, Hung JY . (2007). Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells. Cancer Res 67: 4827–4833.

    CAS  Article  Google Scholar 

  21. Kim JJ, Tannock IF . (2005). Repopulation of cancer cells during therapy: an important cause of treatment failure. Nat Rev Cancer 5: 516–525.

    CAS  Article  Google Scholar 

  22. Kondo T, Setoguchi T, Taga T . (2004). Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line. Proc Natl Acad Sci USA 101: 781–786.

    CAS  Article  Google Scholar 

  23. Lin X, Costa M . (1994). Transformation of human osteoblasts to anchorage-independent growth by insoluble nickel particles. Environ Health Perspect 102 (Suppl 3): 289–292.

    CAS  Article  Google Scholar 

  24. McAllister RM, Gardner MB, Greene AE, Bradt C, Nichols WW, Landing BH . (1971). Cultivation in vitro of cells derived from a human osteosarcoma. Cancer 27: 397–402.

    CAS  Article  Google Scholar 

  25. Mercurio AM, Bachelder RE, Bates RC, Chung J . (2004). Autocrine signaling in carcinoma: VEGF and the alpha6beta4 integrin. Semin Cancer Biol 14: 115–122.

    CAS  Article  Google Scholar 

  26. Miller AC, Blakely WF, Livengood D, Whittaker T, Xu J, Ejnik JW et al. (1998). Transformation of human osteoblast cells to the tumorigenic phenotype by depleted uranium-uranyl chloride. Environ Health Perspect 106: 465–471.

    CAS  Article  Google Scholar 

  27. Miller AC, Mog S, McKinney L, Luo L, Allen J, Xu J et al. (2001). Neoplastic transformation of human osteoblast cells to the tumorigenic phenotype by heavy metal-tungsten alloy particles: induction of genotoxic effects. Carcinogenesis 22: 115–125.

    CAS  Article  Google Scholar 

  28. Miura K, Uniyal S, Leabu M, Oravecz T, Chakrabarti S, Morris VL et al. (2005). Chemokine receptor CXCR4-beta1 integrin axis mediates tumorigenesis of osteosarcoma HOS cells. Biochem Cell Biol 83: 36–48.

    CAS  Article  Google Scholar 

  29. Montanaro F, Liadaki K, Schienda J, Flint A, Gussoni E, Kunkel LM . (2004). Demystifying SP cell purification: viability, yield, and phenotype are defined by isolation parameters. Exp Cell Res 298: 144–154.

    CAS  Article  Google Scholar 

  30. Patrawala L, Calhoun T, Schneider-Broussard R, Zhou J, Claypool K, Tang DG . (2005). Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2− cancer cells are similarly tumorigenic. Cancer Res 65: 6207–6219.

    CAS  Article  Google Scholar 

  31. Patrawala L, Calhoun-Davis T, Schneider-Broussard R, Tang DG . (2007). Hierarchical organization of prostate cancer cells in xenograft tumors: the CD44+{alpha}2{beta}1+ cell population is enriched in tumor-initiating cells. Cancer Res 67: 6796–6805.

    CAS  Article  Google Scholar 

  32. Qi L, Robinson WA, Brady BM, Glode LM . (2003). Migration and invasion of human prostate cancer cells is related to expression of VEGF and its receptors. Anticancer Res 23: 3917–3922.

    CAS  PubMed  Google Scholar 

  33. Ranganathan AC, Adam AP, Aguirre-Ghiso JA . (2006). Opposing roles of mitogenic and stress signaling pathways in the induction of cancer dormancy. Cell Cycle 5: 1799–1807.

    CAS  Article  Google Scholar 

  34. Richman CM, Weiner RS, Yankee RA . (1976). Increase in circulating stem cells following chemotherapy in man. Blood 47: 1031–1039.

    CAS  PubMed  Google Scholar 

  35. Salnikow K, An WG, Melillo G, Blagosklonny MV, Costa M . (1999). Nickel-induced transformation shifts the balance between HIF-1 and p53 transcription factors. Carcinogenesis 20: 1819–1823.

    CAS  Article  Google Scholar 

  36. Setoguchi T, Taga T, Kondo T . (2004). Cancer stem cells persist in many cancer cell lines. Cell Cycle 3: 414–415.

    CAS  Article  Google Scholar 

  37. Soker S, Kaefer M, Johnson M, Klagsbrun M, Atala A, Freeman MR . (2001). Vascular endothelial growth factor-mediated autocrine stimulation of prostate tumor cells coincides with progression to a malignant phenotype. Am J Pathol 159: 651–659.

    CAS  Article  Google Scholar 

  38. Steiner HH, Karcher S, Mueller MM, Nalbantis E, Kunze S, Herold-Mende C . (2004). Autocrine pathways of the vascular endothelial growth factor (VEGF) in glioblastoma multiforme: clinical relevance of radiation-induced increase of VEGF levels. J Neurooncol 66: 129–138.

    Article  Google Scholar 

  39. Strizzi L, Catalano A, Vianale G, Orecchia S, Casalini A, Tassi G et al. (2001). Vascular endothelial growth factor is an autocrine growth factor in human malignant mesothelioma. J Pathol 193: 468–475.

    CAS  Article  Google Scholar 

  40. Woessmann W, Chen X, Borkhardt A . (2002). Ras-mediated activation of ERK by cisplatin induces cell death independently of p53 in osteosarcoma and neuroblastoma cell lines. Cancer Chemother Pharmacol 50: 397–404.

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work is supported by the National Cancer Institute of Canada, the Andrew Mizzoni Cancer Research Fund and the Harry and Hannah Fisher Research Fund. RT and BD are supported in part by awards of the Hospital for Sick Children's Research Training Centre and the National Cancer Institute of Canada Fellowship with funds from the Terry Fox Foundation. We thank Dr Meredith Irwin for critical review of the manuscript; Sherry Zhao, Shamim Lotif, Micky Tsui, Reza Mokhtari, Michael Ho, Suzanne McGovern and Ana Novokmet for technical assistance; and the staff of the animal facility of the Hospital for Sick Children for their technical support.

Author information

Affiliations

Authors

Corresponding author

Correspondence to D Malkin.

Additional information

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Tsuchida, R., Das, B., Yeger, H. et al. Cisplatin treatment increases survival and expansion of a highly tumorigenic side-population fraction by upregulating VEGF/Flt1 autocrine signaling. Oncogene 27, 3923–3934 (2008). https://doi.org/10.1038/onc.2008.38

Download citation

Keywords

  • osteosarcoma
  • cisplatin
  • tumorigenic SP
  • VEGF/Flt1 autocrine

Further reading

Search

Quick links