Previously, we isolated a series of cell lines from a human diploid fibroblast lineage as a model for multistep tumorigenesis in humans. After passaging a single LT-transfected fibroblast clone, differently progressed cell lines were obtained, including immortalized, anchorage-independent and tumorigenic cell lines. In the present paper, we analysed the gene expression profiles of these model cell lines, and observed that expression of the CapG protein was lost in the tumorigenic cell line. To examine the possibility that loss of CapG protein expression was required for tumorigenic progression, we transfected CapG cDNA into the tumorigenic cell line and tested for tumor-forming ability in nude mice. Results showed that ectopic expression of CapG suppressed tumorigenicity, but not growth in soft agar or liquid medium. We also found that certain cancer cell lines including stomach cancer, lung cancer and melanoma had also lost CapG expression. One such cancer cell line AZ521 also became non-tumorigenic after the introduction of CapG cDNA. Moreover, we showed that CapG expression was repressed in small-cell lung cancer tissues. Together, our findings indicated that CapG is a new tumor suppressor gene involved in the tumorigenic progression of certain cancers.
Carcinogenesis is thought to proceed in a stepwise fashion with the accumulation of multiple genetic abnormalities, such as activation of proto-oncogenes and inactivation of tumor suppressor genes (Hahn and Weinberg, 2002). In the case of colorectal cancer, for example, sequential alterations of a specific set of genes, APC, K-ras and p53, can account for each clinical stage of carcinogenesis (Rajogopalan et al., 2003). However, the direct correlation between such genetic abnormalities and defined clinical stages and the molecular mechanism of progression through the different clinical stages remain poorly understood.
To clarify the mechanisms of multistep carcinogenesis, we decided that it would be useful to study a series of differently transformed cell lines derived from a single line of normal cells and then analyse the differences in gene expression between these cell lines. To this end, we previously isolated a series of variously transformed cell lines (retinoblastoma (RB) cell lineage) from human skin fibroblasts (RB) from a patient with hereditary RB (Oka et al., 1999). Whereas RB cells had the normal diploid set of 46 chromosomes, one copy of chromosome 13 contained a large deletion spanning the region from q14 to q22. We introduced early genes of SV40 into RB cells and obtained several mortal clones with extended lifespan (RBSV). After repeated passages of a single RBSV cell clone, we succeeded in isolating immortalized (RBI), anchorage-independent (RBS) and tumorigenic (RBT) cell lines. Such model cell lines of multistep tumorigenesis are very rare, and to our knowledge no other similar cell lines have been previously published.
In the present study, we searched for differences in gene expression between these model cell lines and found that CapG protein expression was completely absent only in the most progressed line, the tumorigenic RBT cells. When introduced into RBT cells, the CapG gene suppressed RBT tumorigenicity in nude mice, but did not affect RBT colony formation in soft agar. This effect of CapG was not limited to our model cell lines. Although the CapG gene is ubiquitously expressed in normal tissues, expression was frequently lost in human cancer cell lines and tissues including the tumorigenic gastric cancer cell line AZ521. This cell line was also converted to a non-tumorigenic state by ectopic expression of CapG protein. These results suggested that the CapG functioned as a tumor suppressor gene and was involved in tumorigenic conversion in various human cancers.
CapG expression was lost at the tumorigenic stage of the RB cell lineage
As previously reported (Oka et al., 1999), we isolated a set of human cell lines (RB cell lineage) as a model of multistep carcinogenesis. These cell lines were derived from a single human diploid fibroblast strain (RB) that was transformed by the introduction of SV40 early genes (RBSV) and resultant lines selected for progression to the immortalized (RBI), anchorage-independent (RBS) or tumorigenic (RBT) stages (Figure 1a). To investigate for altered gene expression between the RB cell lineage stages, we performed Western blot analysis using various antibodies. When one polyclonal antibody (HD-2) was used, a protein band with apparent molecular weight of 45 kDa was found to be absent only from the RBT tumorigenic cell line (Supplementary Figure 1a). This antibody was made by us to analyse another cancer-related protein, but the antibody obtained recognized several unknown protein bands probably because of contamination of other proteins in the antigen preparation for immunization. During analyses using this antibody, we found loss of the 45 kDa protein in some cell lines including the RBT cell line and strong expression of this protein in other cell lines (Supplementary Figure 1b). We next determined a protein spot corresponding to the 45 kDa protein on two-dimensional gel (Supplementary Figure 2a). Mass spectrometric analysis identified this protein as CapG (Supplementary Figure 2b). We then cloned CapG cDNA, expressed and purified glutathione-S-transferase-fused CapG protein from Escherichia coli and generated polyclonal anti-CapG antibody (Supplementary Figure 3). Using this antibody, we confirmed that RBT cells showed no CapG protein expression (Figure 1b). To understand the mechanism by which CapG protein expression was inhibited in the RBT cell line, we performed Southern and Northern blot hybridizations. As shown in Figure 1c and d, whereas CapG mRNA was not detected in RBT cells, no gross structural alterations of the CapG gene were observed in any of the RB cell lines. This suggested that CapG expression was repressed at the transcriptional level at the stage of progression from anchorage independence to tumorigenicity in the RB cell lineage.
Ectopic CapG expression suppressed RBT cell tumorigenicity
The finding that loss of CapG expression was associated with progression from the anchorage-independent to the tumorigenic stage of the RB cell lineage led us to hypothesize that the CapG protein played a role in tumor suppression. To test this possibility, we introduced CapG cDNA into RBT cells by infection with a recombinant retroviral vector, and isolated cell clones expressing CapG protein. Levels of CapG protein in these clones were largely comparable to those observed in RBS cells, which grow anchorage independently but do not form tumors in nude mice (Figures 1a and 2a, and data not shown). The growth rate in liquid media and colony-forming ability in soft agar of RBT cell clones expressing CapG protein were similar to those of parental non-infected RBT cells (Figure 2b and c), which indicated that proliferation and anchorage-independent growth were not affected by CapG expression. However, when injected subcutaneously into nude mice, the tumor-forming capacity of CapG-expressing RBT cells was greatly reduced compared to non-infected RBT cells and RBT cells infected with a control retroviral vector (Figure 2d). These observations indicated that CapG gene suppressed RBT tumorigenicity, and that tumor suppression was independent of growth in liquid culture or soft agar.
Lack of CapG expression in various human cancer cell lines
Although ectopic CapG expression suppressed RBT cell tumorigenesis, the RB cell lineage is a series of artificially transformed cell lines rather than actual cancer cell lines. Therefore, we examined whether CapG expression was altered in human cancer cells. As shown in Figure 3a, CapG mRNA was ubiquitously expressed in normal human tissues, although expression levels were not uniform. However, some cancer cell lines, especially those derived from human stomach cancer (1/3), cutaneous melanoma (1/3), small-cell lung cancer (3/5) and lung adenocarcinoma (1/5) had completely lost CapG expression (Figure 3b). Downregulated or reduced CapG protein expression was also observed at a high frequency in various lung cancers (Figure 3b). These results suggested that loss of CapG expression may contribute to the development of human cancers.
CapG suppressed the tumorigenicity of stomach cancer cell line AZ521
To examine whether ectopic CapG expression could also suppress the tumorigenicity of an established human cancer cell line, we introduced CapG cDNA into the human stomach cancer cell line AZ521 that lacked CapG protein expression as shown in Figure 3 and isolated three cell clones that expressed CapG protein (Figure 4a). These CapG-expressing AZ521 clones exhibited similar growth rates and colony-forming ability compared to control AZ521 cells (Figure 4b and c), which again suggested that CapG protein expression did not affect cell growth in liquid media or soft agar. However, CapG-expressing AZ521 clones did not produce tumors in nude mice (Figure 4d), which suggested that CapG expression suppressed the tumorigenicity of the human cancer cells in nude mice.
Loss of CapG protein expression was also observed in primary tumors
As most cancer cell lines are cultured in vitro over long periods of time, it can be argued that cancer cell lines do not necessarily accurately reflect primary tumor cells. To examine whether primary tumors also showed loss of CapG protein expression, we performed Western blot analysis using human tumor tissues isolated from cases of stomach cancer (four cases), small-cell lung cancer (four cases) and melanoma (two cases). The results showed that although downregulation of CapG protein was not observed in the stomach cancer and melanoma samples, CapG protein levels were greatly reduced in three of the four small-cell lung cancer samples (Figure 3c). Although some CapG protein was still present, tumor tissues can also contain normal cells such as stromal cells or infiltrating lymphocytes that would express normal levels of CapG. Therefore, our results strongly suggested that the development of certain human primary cancers could also involve the loss of the CapG gene function.
Taken together, our findings indicated that CapG may act as a tumor suppressor protein involved in tumorigenicity in nude mice but not in cell growth in liquid media or soft agar.
Effect of ectopic CapG expression on microfilament organization and cell motility
Dynamic reorganization of the actin cytoskeleton is an underlying factor in the tumor-related processes of invasion and metastasis. Because the CapG protein is known to possess actin-modulating activity (Silacci et al., 2004), it is possible that an alteration of microfilaments contributes to the acquisition or loss of tumorigenicity. Therefore, microfilament organization was examined in the RB cell lineage. Although the expression level of actin protein was comparable in all of the RB cell lines regardless of the presence or absence of CapG (Figure 5a), actin filament organization was greatly altered with malignant progression of the RB cell lineage (Figure 5b). Microfilaments were well organized and stretched long in diploid RB cells, and were maintained, though much finer and shorter, even in the anchorage-independent cell line RBS (Figure 5b). In contrast, the actin stress fibers seemed to disappear in the tumorigenic RBT cell line, but then reassembled to form a thick bundle at the perinuclear region (Figure 5b). These findings led us to hypothesize that the tumorigenicity of the RBT cell line might be caused, at least in part, by alteration of microfilament organization. One caveat, however, is that the ectopic CapG expression that resulted in the suppression of tumorigenicity in RBT cells did not affect formation of the actin bundle (Figure 5b).
We next examined the effect of ectopic CapG expression on cell motility that is believed to have some relation to actin cytoskeleton. The same RBT transfectants that were used for the tumorigenicity test and microfilament staining were subjected to a migration assay. As shown in Figure 5c, ectopic expression of CapG enhanced migration activity of the RBT cell line. This finding implies that cell motility and tumorigenicity may be controlled by different functions of the CapG protein.
These results suggest that the actin-modulation function of CapG may not be involved in the suppression of tumorigenicity. Clarification of the mechanism of tumor suppression by CapG will necessitate the isolation and characterization of CapG-interacting proteins.
In this paper, we analysed the RB cell lineage, a human model of multistep tumorigenesis, to identify genes involved in malignant progression. As the different RB cell lines were derived from a single line of human diploid fibroblasts, it is likely that they shared a uniform genetic background with alterations limited to the expression of genes associated with malignant progression. Therefore, the RB cell lineage represented an effective tool for the identification of oncogenes or tumor suppressor genes responsible for each step of malignant progression. The cell lineage was not transfected with exogenous genes for malignant progression from immortalized RBI cell lines, such that the malignant progression observed in this cell lineage was likely to be due, at least in part, to the inactivation of endogenous tumor suppressor genes (Oka et al., 1999). In this way, the process of malignant progression in the RB cell lineage may mimic the process that occurs in naturally occurring cancer. Therefore, identification of genes with altered expression in response to malignant progression using the RB cell lineage may be a useful tool to help elucidate the mechanisms of the multistep tumorigenic process.
By comparing gene expression profiles of the RB cell lineage, we found that CapG expression was lost at the transcriptional level at the stage of progression from the non-tumorigenic to the tumorigenic state. Similar downregulation of CapG expression was also observed in various human cancer cell lines and cancer tissues. These findings allowed us to hypothesize that the CapG protein may be involved in the tumorigenic progression of cancer cells as a tumor suppressor gene. To examine this possibility, we introduced CapG cDNA into the tumorigenic line of the RB cell lineage, RBT, as well as the gastric cancer cell line AZ521, both of which showed no endogenous CapG protein expression. We then tested the ability of these cell lines to form tumors in nude mice and to form colonies in soft agar. Our results indicated that ectopic CapG gene expression suppressed tumorigenicity, but did not affect the anchorage-independent growth of these cell lines.
CapG, also known as gCap39, Mbh1 or MCP, is a 348-amino-acid protein that is ubiquitously expressed in normal tissues, being particularly abundant in macrophages (Yu et al., 1990; Prendergast and Ziff, 1991; Dabiri et al., 1992). CapG is a member of the gelsolin family of actin filament modulating proteins that also includes gelsolin, villin, adseverin, advellin, supervillin and flightless I (Silacci et al., 2004). However, CapG has features that distinguish it from other gelsolin family proteins. CapG has only three repeated gelsolin-like domains, in contrast to the usual six domains present in other gelsolin family proteins, and it lacks the actin-severing activity exhibited by the other family members. Another unique characteristic of CapG is its subcellular localization. Whereas CapG localizes to both the cytoplasm and nucleus, the other gelsolin family members are present only in the cytoplasm. Therefore, CapG may have a function in addition to actin or cytoskeleton modulation. Indeed, it has been reported that CapG represses transcriptional activation (De Corte et al., 2004), although it was not clear which gene was trans-repressed by CapG.
Among these CapG properties, the cytoskeleton-modulating function may be dispensable for the suppression of tumorigenicity, as ectopic CapG expression in the tumorigenic RBT cell line did not affect cytoskeletal appearance (Figure 5b) but did suppress tumorigenicity. CapG expression and nuclear localization patterns were also unchanged between the non-tumorigenic cell line RBS and the non-tumorigenic transfectants, RBT/CapG cells and AZ521/CapG cells, which expressed ectopic CapG protein (Supplementary Figure 5). This persistent expression suggests that tumor suppression does not result from abnormal expression or localization of CapG protein.
On the other hand, CapG reportedly possesses an oncogenic function involved in the control of cell migration or invasion. When cells were transfected with CapG expression vector, they acquired stimulated migration/invasion activity (Pellieux et al., 2003; De Corte et al., 2004). Enhanced migration of RBT cells was also observed in this study after ectopic expression of CapG (Figure 5c). This latter experiment utilized the same transfected cell clones that were examined for tumorigenicity (Figures 2d and 5c). Consequently, the indication is that CapG overexpression leads to the simultaneous suppression of tumorigenicity and activation of migration/invasion. Thus, the implication is that activated migration/invasion does not hamper tumor suppression. In this regard, it is noteworthy that CapG protein overexpression in ocular melanoma and glioblastoma has been reported (Van Ginkel et al., 1998; Lal et al., 1999). Although the effect of the overexpression on tumorigenesis has not been clarified, it may be interesting to examine whether these cancers contain cells with CapG gene mutations.
Given that CapG function can be activated via the Ras/MAPK signaling pathway (De Corte et al., 2004), it is possible that there may be a relationship between loss of CapG expression and activation of the Ras/MAPK signaling pathway. It is well known that the K-Ras, N-Ras and BRAF genes are frequently mutated and activated in melanoma and stomach cancer (Davies et al., 2002; Rajagopalan et al., 2002). Furthermore, our results showed that some melanoma and stomach cancer cell lines had lost CapG protein expression, which suggests that Ras/MAPK mutations may affect CapG protein expression levels. Therefore, we examined the melanoma and stomach cancer cell lines used in this paper for the presence of K-Ras, N-Ras and BRAF mutations by RT–PCR and sequencing. However, we found no relationship between mutation of these genes and CapG protein levels (data not shown). Further study is necessary to clarify the mechanism of tumor suppression by CapG.
Gelsolin, the prototype gelsolin family protein, also exhibits tumor suppressor activity in certain human cancer cell lines. Downregulation of gelsolin has been observed at a high frequency in various cancer cell lines, and ectopic expression of gelsolin suppressed tumorigenicity of bladder and lung cancer cell lines (Tanaka et al., 1995; Sagawa et al., 2003). However, in these cases, gelsolin suppressed not only tumorigenicity but also anchorage-independent cell growth, which suggests that the mechanisms of tumor suppression by CapG and gelsolin are different.
In this study, based on RB cell lineage cell lines, we attempted to investigate the mechanisms that underlie multistep malignant progression and identified a candidate tumor suppressor gene, CapG. Loss of CapG protein and mRNA expression was observed at the progression from the non-tumorigenic to tumorigenic state, and ectopic CapG expression in tumorigenic RBT cells resulted in the inhibition of tumorigenicity, but not anchorage-independent cell growth. CapG protein levels in the RB cell lineage coincided well with the activity of the CapG gene, which indicated that our cell lines were a useful model to clarify the mechanisms of malignant progression. Although the molecular mechanism of CapG-induced tumor suppression remains unclear, we expect our model cell lines will contribute greatly to the understanding of tumorigenesis.
Materials and methods
RB cell lines were cultured in Dulbecco's modified minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS) as described previously (Oka et al., 1999). The gastric cancer cell line AZ521 was cultured in minimal essential medium (MEM) supplemented with 10% FBS. Cancer cell lines were obtained from the Japanese Cancer Research Resources Bank, Health Science Research Resources Bank or Riken Cell Bank.
Western blot analysis
Cells were lyzed with RIPA buffer (0.15 M NaCl, 50 mM Tris-HCl (pH 7.4), 1 mM ethylenediaminetetraacetic acid, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 100 μg/ml phenylmethylsulfonyl fluoride and 0.25 TIU/ml aprotinin). The cell lysates were run on 5–20% SDS–polyacrylamide gel and electroblotted onto polyvinylidene difluoride membrane. The membranes were incubated successively with rabbit polyclonal anti-CapG (TransGenic, Kumamoto, Japan), mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Chemicon International, Temecula, CA, USA) or α-tubulin (Sigma, St Louis, MO, USA) antibody and then with horseradish peroxidase-conjugated anti-rabbit or mouse IgG antibody (Cell Signaling Technology, Beverly, MA, USA). Protein bands were detected using enhanced chemiluminescence reagent (Amersham, Piscataway, NJ, USA).
Southern blot hybridization
DNA was digested with restriction enzymes, subjected to 1% agarose gel electrophoresis and transferred onto nylon membranes. Membranes were then hybridized with 32P-labeled probe, washed and autoradiographed.
Northern blot hybridization
RNA isolated using an SV total RNA isolation kit (Promega, Madison, WI, USA) was subjected to formaldehyde-1.2% agarose gel electrophoresis, blotted onto nylon membranes, hybridized with 32P-labeled probe, washed and autoradiographed.
Construction of retrovirus vector and virus production
pCX4bsr, a Moloney murine leukemia virus-based retrovirus vector containing the blasticidin S resistance gene (bsr) as a selectable marker, was constructed by Akagi et al. (2000). pCX4-CapG was constructed by inserting full-length human CapG cDNA into the multi-cloning site of the pCX4bsr vector.
Phoenix-A cells were transfected with pCX4bsr or pCX4-CapG constructs using FuGene 6 (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's protocols. Two days after transfection, the culture supernatants were collected and stored at −70°C until use.
Tumorigenicity in nude mice
Female 4-week-old BALB/cJ (nu/nu) mice were injected subcutaneously in the back with CapG- or empty virus-infected cells in 0.2 ml of DMEM (without serum). After 7 weeks, tumor size was measured in two dimensions using hand calipers, and tumor volume calculated by the formula 0.5 × L × W2, where L and W are the length and width of a tumor, respectively (Sagawa et al, 2003).
Anchorage-independent growth assay
Anchorage-independent growth was assessed by colony-forming ability in soft agar. Ten thousand cells were inoculated into 0.35% agarose in DMEM supplemented with 10% FBS per 60 mm dish. After 2 weeks’ incubation, the number of colonies (>0.125 mm in diameter) was scored.
Cell migration assay
Cells (4 × 104) were suspended in 200 μl of serum-free DMEM containing 0.1% bovine serum albumin, and plated in upper chamber of Chemotaxicell (Kurabo, Osaka, Japan). The lower chamber contained 600 μl of phosphate-buffered saline supplemented with 6 μg/ml fibronectin. After incubation for 6 h at 37°C, cells were fixed with 80% methanol, and stained with hematoxylin. The number of cells migrated through membrane was counted under microscope.
Akagi T, Shishido T, Murata K, Hanafusa H . (2000). Proc Natl Acad Sci USA 97: 7290–7295.
Dabiri GA, Young CL, Rosenbloom J, Southwick FS . (1992). J Biol Chem 267: 16545–16552.
Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S et al (2002). Nature 417: 949–954.
De Corte V, Van Impe K, Bruyneel E, Boucherie C, Mareel M, Vandekerckhove J et al. (2004). J Cell Sci 117: 5283–5292.
Hahn WC, Weinberg RA . (2002). Nat Rev Cancer 2: 331–341.
Lal A, Lash AE, Altschul SF, Velculescu V, Zhang L, McLendon RE et al. (1999). Cancer Res 59: 5403–5407.
Oka K, Tomonaga Y, Nakazawa T, Ge H-Y, Bengtsson U, Stanbridge EJ et al. (1999). Genes, Chromosome Cancer 26: 47–53.
Pellieux C, Desgeorges A, Pigeon CH, Chambaz C, Yin H, Hayoz D et al. (2003). J Biol Chem 278: 29136–29144.
Prendergast GC, Ziff EB . (1991). EMBO J 10: 757–766.
Rajagopalan H, Bardelli A, Lengauer C, Kinzler KW, Vogelstein B, Velculescu VE . (2002). Nature 418: 934.
Rajogopalan H, Nowak MA, Vogelstein B, Lengauer C . (2003). Nat Rev Cancer 3: 695–701.
Sagawa N, Fujita H, Banno Y, Nozawa Y, Katoh H, Kuzumaki N . (2003). Br J Cancer 88: 606–612.
Silacci P, Mazzolai L, Gauci C, Stergiopulos N, Yin HL, Hayoz D . (2004). Cell Mol Life Sci 61: 2614–2623.
Tanaka M, Mullauer L, Ogiso Y, Fujita H, Moriya S, Furuuchi K et al. (1995). Cancer Res 55: 3228–3232.
Van Ginkel PR, Gee RL, Walker TM, Hu D-N, Heizmann CW, Polans AS . (1998). Biochim Biophys Acta 1448: 290–297.
Yu F-X, Johnston PA, Sudhof TC, Yin HL . (1990). Science 250: 1413–1415.
We thank Dr Akagi for providing the pCX4 retrovirus vector. SH is a recipient of a grant from the Charitable Trust Osaka Cancer Researcher-Fund.
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Cite this article
Watari, A., Takaki, K., Higashiyama, S. et al. Suppression of tumorigenicity, but not anchorage independence, of human cancer cells by new candidate tumor suppressor gene CapG. Oncogene 25, 7373–7380 (2006) doi:10.1038/sj.onc.1209732
- tumor suppressor gene
- multistep tumorigenesis
- gastric cancer
- lung cancer
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