© The Author 2006. Published by Oxford University Press.
REVIEW |
Keratinocyte Growth Factor Expression and Activity in Cancer: Implications for Use in Patients With Solid Tumors
Affiliations of authors: Croton-on-Hudson, New York, NY (PWF); Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, MD (JSR)
Correspondence to: Jeffrey S. Rubin, MD, PhD, LCMB/NCI, Building 37, Room 2042, 37 Convent Drive, MSC 4256, Bethesda, MD 20892-4256 (e-mail: rubinj{at}mail.nih.gov).
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ABSTRACT |
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 TopNotes Abstract IntroductionFgf signaling in cancerExpression of kgf and...Kgf effects on tumor...Investigations of the...Kgf use in patients...References |
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Keratinocyte growth factor (KGF) is a locally acting epithelial mitogen that is produced by cells of mesenchymal origin and has an important role in protecting and repairing epithelial tissues. Use of recombinant human KGF (palifermin) in patients with hematologic malignancies reduces the incidence and duration of severe oral mucositis experienced after intensive chemoradiotherapy. These results suggest that KGF may be useful in the treatment of patients with other kinds of tumors, including those of epithelial origin. However, its application in this context raises issues that were not pertinent to its use in hematologic cancer because epithelial tumor cells, unlike blood cells, often express the KGF receptor (FGFR2b). Thus, it is important to examine whether KGF could promote the growth of epithelial tumors or protect such tumor cells from the effects of chemotherapy agents. Analyses of KGF and FGFR2b expression in tumor specimens and of KGF activity on transformed cells in vitro and in vivo do not indicate a definitive role for KGF in tumorigenesis. On the contrary, restoring FGFR2b expression to certain malignant cells can induce cell differentiation or apoptosis. However, other observations suggest that, in specific situations, KGF may contribute to epithelial tumorigenesis. Thus, further studies are warranted to examine the nature and extent of KGF involvement in these settings. In addition, clinical trials in patients with solid tumors are underway to assess the potential benefits of using KGF to protect normal tissue from the adverse effects of chemoradiotherapy and its possible impact on clinical outcome.
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INTRODUCTION |
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TopNotesAbstractIntroductionFgf signaling in cancerExpression of kgf and...Kgf effects on tumor...Investigations of the...Kgf use in patients...References |
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The fibroblast growth factors (FGFs) comprise a family of at least 22 polypeptide growth factors that have diverse roles in regulating cell proliferation, migration, and differentiation during vertebrate development, as well as homeostasis, response to injury, and tissue repair in adult animals (1). Fibroblast growth factor 7 (FGF-7), also known as keratinocyte growth factor (KGF), is produced by cells of mesenchymal origin (2,3) and acts exclusively through a subset of FGF receptor isoforms (the FGFR2b isoforms) that are expressed primarily by epithelial cells (4). The restricted pattern of FGFR2b expression and the high specificity of KGF for FGFR2b isoforms account for the predominant epithelial activity of KGF (5). The finding that KGF is expressed by mesenchymal cells and acts specifically on epithelial cells supports the hypothesis that KGF functions as a paracrine signal that mediates mesenchymal–epithelial communication (5).
Both in vitro and in vivo studies have demonstrated that KGF has potent cytoprotective and regenerative effects on epithelial tissues that are subjected to a variety of toxic exposures (6–15). These beneficial effects arise from multiple mechanisms that act collectively to strengthen the integrity of the epithelium by stimulating cell proliferation, migration, differentiation, survival, DNA repair, and induction of enzymes involved in the detoxification of reactive oxygen species (5). For this reason, efforts are underway to identify clinical applications for KGF in which the integrity of epithelial surfaces is at risk and patients would benefit from preservation or rapid restoration of these tissues. Currently, a truncated form of recombinant KGF—palifermin (brand name Kepivance)—is being evaluated in clinical trials sponsored by Amgen, Inc. (Thousand Oaks, CA) for its ability to ameliorate the severe oral mucositis that results from cancer chemoradiotherapy. In a phase III trial involving patients with hematologic malignancies who were treated with high doses of chemotherapy and radiation before autologous peripheral blood progenitor cell transplantation, palifermin treatment statistically significantly reduced both the incidence and duration of severe oral mucositis (P<.001 for both criteria) (16). Accordingly, the U.S. Food and Drug Administration approved palifermin for clinical use in this context.
The far larger population of patients who have tumors of epithelial origin may also benefit from the use of KGF to treat oral mucositis. Moreover, KGF also might decrease the acute and chronic side effects of chemoradiotherapy on epithelia beyond the oral cavity (5,7–9,13,15,17,18). However, the potential use of KGF in patients with epithelial tumors raises important questions that were not relevant to the treatment of patients with hematologic neoplasias, because epithelial cells—unlike blood cells—express the KGF receptor, FGFR2b. KGF has been shown to be mitogenic for many normal epithelial cell populations (3,5,19–24). Furthermore, it enhances the migration of normal keratinocytes (25,26) and type II pneumocytes (27–30), perhaps in association with a wound healing response (31–34). Thus, it is possible that KGF treatment might result in enhanced growth or metastasis of epithelial tumors. Furthermore, in addition to having a cytoprotective effect on normal epithelial populations, KGF might protect the malignant cells themselves, rendering them resistant to cancer treatments intended to kill them.
Although there has been substantial interest in the potential role of KGF in epithelial cell tumorigenesis, much of the information that has been published is contradictory, making interpretation of the results difficult. Here we review these data and assess the relationship of KGF to cancer. We first provide some general information on FGF-mediated signaling in cancer. We then present a summary of the data on KGF and FGFR2b expression in human tumors, the effects of KGF and FGFR2b on tumor cell phenotypes, and the oncogenic potential of KGF in epithelial cells in vitro and in vivo. We end by considering various questions concerning KGF use in patients with tumors of epithelial origin.
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FGF SIGNALING IN CANCER |
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TopNotesAbstractIntroductionFgf signaling in cancerExpression of kgf and...Kgf effects on tumor...Investigations of the...Kgf use in patients...References |
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FGFs are expressed in most mammalian tissues, with varying patterns of spatiotemporal distribution. Some FGFs are expressed exclusively during embryonic development, whereas others are present in both embryonic and adult tissues (1). The oncogenic potential of FGFs was originally demonstrated by the identification of several FGFs as transforming genes (35–38). Subsequent studies have shown that many FGFs are capable of inducing the transformation of both epithelial cells and fibroblasts, either when supplied exogenously or expressed ectopically, and that FGF-transformed cells are tumorigenic in immunocompromised animals (39–44). The expression of FGFs in tumor cell lines and in human tumor tissues has been well established (45–49). In several cases, overexpression of FGF mRNA and/or protein occurs in conjunction with amplification of the respective FGF locus (50–53).
FGF signaling in responsive cells is mediated by a family of high-affinity transmembrane tyrosine kinase FGF receptors (FGFRs) that are encoded by four structurally related genes (FGFR1–4) (54,55). Further heterogeneity among the FGFRs is produced by alternative splicing of transcripts, which results in protein tyrosine kinases that contain either two or three immunoglobulin (Ig)-like extracellular domains and either contain or lack a highly acidic region (54,56). Specificity of FGF–FGFR binding is determined, in part, by three alternatively spliced exons that encode the carboxyl-terminal half of the Ig domain closest to the transmembrane region and approximately 20 residues of adjacent downstream sequence in FGFRs 1, 2, and 3. These alternative exons generate receptor variants that have different ligand binding properties and are expressed in a tissue-specific manner (54,56).
FGFR overexpression has been observed in many different human cancers. For example, FGFR1 is amplified and overexpressed in breast carcinoma (57). FGFR4 immunoreactivity is substantially higher in prostate cancer specimens than in benign prostatic hypertrophy specimens, and this elevated level of expression was associated with decreased survival (58). Activating mutations of FGFR3 are common in bladder and cervical carcinomas (59) and in multiple myeloma (60,61). FGFRs may also become constitutively activated in a ligand-independent fashion through truncation or fusion of the protein. In multiple myeloma, a frequent translocation involving the FGFR3 gene and the immunoglobulin heavy chain locus results in overexpression of FGFR3 (62–64). A constitutively activated form of FGFR4 has been identified in human pituitary tumors, and targeted expression of this altered receptor in transgenic mice recapitulated the human disease (65). Various FGFR1 fusion proteins have been identified in myeloproliferative disorders in which dimerization domains present in a fusion partner may induce ligand-independent dimerization and constitutive activation of the receptor (66–75).
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EXPRESSION OF KGF AND ITS RECEPTOR IN HUMAN CANCERS |
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TopNotesAbstractIntroductionFgf signaling in cancerExpression of kgf and...Kgf effects on tumor...Investigations of the...Kgf use in patients...References |
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KGF and FGFR2b Expression in Normal Cells and Tissues
Normal stromal cells from a variety of organs and tissues, including embryonic and adult lung, skin, mammary gland, stomach, bladder and prostate, as well as smooth muscle cells and microvascular endothelial cells express KGF when grown in culture (5). Alternative splicing of the FGFR2 gene results in either the FGFR2b or FGFR2c isoforms. FGFR2b isoforms, the only FGFRs to which KGF binds, are expressed primarily by epithelial cells (76), whereas FGFR2c, also known as bacterially expressed kinase (BEK), is expressed in mesenchymal lineages (76,77). KGF and FGFR2b transcripts are expressed during development (78–80) and in adult tissues (19,21,34,81–83). KGF transcripts are generally detected in mesenchymal cells adjacent to epithelia that express FGFR2b (78–80,83). These patterns of expression are consistent with the hypothesis that KGF functions as a paracrine mediator of mesenchymal–epithelial communication. However, KGF expression by a few types of normal epithelial cells has been reported (84–86), and such a pattern is suggestive of an autocrine mode of action.
A number of factors can stimulate stromal cells to express KGF. Treatment with interleukin (IL)-1 and IL-6, proinflammatory cytokines that are expressed by macrophages, polymorphonuclear leukocytes, and a few other cells, stimulated either large (IL-1) or moderate (IL-6) increases in KGF transcript and protein synthesis in fibroblasts from multiple sources (87,88). KGF expression was also induced by serum or various purified serum growth factors, including platelet-derived growth factor BB (PDGF BB), and transforming growth factor 
(TGF). These results suggest a mechanism for KGF induction during acute or chronic injury or repair (31–34,89,90), with increased expression initiated by growth factors, such as PDGF BB and TGF, that are released from platelets. Presumably, the elevated levels of KGF mRNA at later stages of the repair process are due to the release of proinflammatory cytokines, such as IL-1 and IL-6, from macrophages and polymorphonuclear leukocytes, which infiltrate the wound within 24 hours of injury (91). KGF also has been implicated as a potential paracrine mediator of steroid hormone action on epithelia in a number of organs in the male and female reproductive tracts, including the seminal vesicle, prostate, and endometrium (81,92,93).
KGF and FGFR2b Expression in Epithelial Cancer Cell Lines
KGF expression has not been observed in established epithelial tumor cell lines derived from a variety of tissue sources (2), including prostate (94), colon (95), esophagus (96), stomach (96,97), and squamous cell carcinoma (SCC) of the head and neck (98). However, low levels of the KGF transcript have been detected in some pancreatic and breast cancer cell lines using reverse transcriptase–polymerase chain reaction (RT–PCR) analysis, even though the transcript was not detected by northern blot analysis of polyadenylated mRNA (99,100). KGF transcript and protein were induced in the human breast cancer cell line MCF-7 after treatment with vitamin D3, as determined by RT–PCR and western blot analysis (101). KGF protein has also been detected using immunohistochemistry in MCF-7 and another estrogen receptor (ER)–positive breast cancer cell line, ZR-75–1, but not in two ER-negative breast tumor cell lines (102). Finally, a low level of KGF transcript was seen in five pancreatic ductal carcinoma cell lines using northern blot analysis of total cellular RNA, whereas KGF transcript was not detected in primary cell cultures derived from normal pancreatic duct epithelium (103). These data are summarized in Table 1.
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FGFR2b mRNA is expressed in many, although not all, carcinoma cell lines derived from multiple tissues, including breast (99,104), colon (95), stomach and esophagus (97,105,106), pancreas (100), prostate (107), oral mucosa (108), and uterus (109). There is little evidence to suggest that FGFR2b expression in cancer cell lines is abnormal. Instead, its expression probably reflects the properties of the normal epithelial cells from which these tumors arise (5,19). On the other hand, in some in vitro and animal models of tumorigenesis, loss of FGFR2b expression was associated with activation of FGFR2c expression, and/or a shift to more virulent behavior (107,110,111) (Table 2).
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KGF Expression in Human Tumor Specimens
Although KGF is rarely expressed by tumor cells in vitro, KGF transcripts are readily detectable in human tumor samples by northern blot analysis or RT-PCR. However, analyses of KGF expression have not yielded consistent results (Table 1). For example, KGF transcript levels in tumor tissue specimens from patients with advanced SCC of the head and neck were lower than those in normal mucosa from the same patients (98). On the other hand, KGF mRNA levels were substantially higher in pancreatic (100) and colorectal (112) cancer specimens than in the corresponding normal tissues. Yet another study found that normal and malignant colorectal tissue specimens contained similar levels of KGF transcript and protein (95). In addition, there have been conflicting reports regarding the level of KGF expression in benign prostatic hypertrophy and prostate cancer (113,114).
One explanation for these inconsistencies may lie in the cellular composition of the tissue samples analyzed. For example, samples with a higher proportion of stromal tissue would be likely to have a stronger KGF signal, especially if factors released from tumor cells, such as TGF or PDGF BB, stimulated stromal KGF expression, or if an inflammatory reaction around the growing tumor resulted in the release of IL-1 and IL-6 that then increased stromal KGF expression. Alternatively, tumor tissue samples consisting predominantly of cancerous epithelial cells, which express little or no KGF, would have a lower signal than normal tissue. Also, because KGF expression levels vary in different organs (19), differences in KGF expression between tumors of different origins may simply reflect the variation in KGF expression normally found in these organs.
Immunohistochemical analysis offers a more sophisticated approach to the localization of KGF expression because it identifies the site of KGF protein in tissue samples. One study of KGF expression in SCC using immunohistochemistry found that only tumor stroma and submucosal areas of normal tissue stained intensely with anti-KGF antibody, whereas closely packed tumor cells were negative, consistent with the in vitro data (98). However, in benign prostatic hyperplasia and prostate cancer, KGF was detected not only in fibroblasts and smooth muscle cells (independent of disease stage) but also in the benign hyperplastic epithelial cells and in the cancer cells (115). Similarly, in breast cancer, in addition to stromal expression in lobular carcinomas and a subset of invasive ductal carcinomas, strong staining was observed in carcinoma cells (116,117). These staining patterns raise the possibility of autocrine KGF expression, although they might simply represent KGF binding to FGFR2b or low-affinity receptors, such as heparin sulfate proteoglycans (5).
Examination of KGF expression in tumor tissue using in situ hybridization has provided additional evidence that KGF is indeed expressed in an autocrine manner by cancer cells. In situ hybridization performed with digoxigenin-labeled oligonucleotides revealed weak expression of KGF in stromal cells from normal prostate as well as in benign prostatic hyperplasia and low-grade prostate carcinoma but high expression in tumor cells in high-grade prostatic tumors and in metastases to the lung and lymph nodes (118). In colorectal cancer, in situ hybridization detected KGF expression in neuroendocrine cells in close proximity to the cancer cells and in the cancer cells themselves. This signal was associated with the same cells that expressed KGF protein as detected by immunohistochemistry (112). Similarly, in immunohistochemical analysis of pancreatic cancer, KGF staining was observed in many cancer cells, whereas a stronger signal was present in the ductal and acinar cells adjacent to the tumor. This pattern of expression was paralleled by the KGF in situ hybridization signal detected with riboprobes (119). Finally, the general overlap between immunohistochemical and in situ hybridization signals suggested that KGF may be expressed in an autocrine fashion by breast (102) and lung cancer (120) cells.
In summary, whereas only very low levels of KGF appear to be present in tumor cell lines, a growing body of evidence suggests that KGF is synthesized by at least some tumor cells in vivo. Taken together, these findings may reflect the induction of KGF expression in tumor cells by specific paracrine or endocrine mechanisms. When tumor cells are isolated and established as permanent lines in culture, KGF expression may be lost in the absence of these hypothetical external signals. In this regard, it is interesting that KGF transcript and protein are detectable in early-passage SCC cells (121). Although this KGF expression may be the result of stromal cell contamination, it might also reflect tumor cell expression of KGF that persists transiently after removal of unidentified external signal(s).
FGFR2b Expression in Human Tumor Specimens
FGFR2b RNA expression has been detected in a wide range of tumor tissues (Table 2). In some cases, expression was higher in tumors than in normal tissue, including in colon (112), prostate (118), and stomach (106). However, other studies have not found differences between the expression of FGFR2b in tumor tissues or malignant epithelial cells compared with that in normal tissues and cell lines, including breast (99,104) and colon (95). In one study of 54 patients with transitional cell bladder carcinoma, FGFR2b transcript levels were lower in tumors than in normal tissue for 18 patients, whereas there was no difference between tumor and normal tissue in the remaining patients (122). As with KGF expression, measurement of FGFR2b transcripts in tumor and normal specimens will probably not yield consistent results unless the cellular composition of the tissue samples is also taken into account. In contrast to the situation with KGF expression, a higher proportion of epithelial tumor cells within a given tissue specimen may skew the result toward a more prominent FGFR2b signal than that seen in normal tissue, whereas samples consisting predominantly of stromal cells would have a weaker signal.
Efforts to determine the cellular localization of FGFR2b in tumor tissues have predominantly used immunohistochemistry. One limitation of past reports is that they typically used FGFR2 antibodies that could not distinguish between FGFR2b and FGFR2c isoforms. In one such study, immunoreactivity was seen in 20 of 38 cases of undifferentiated, advanced gastric cancer but in none of 11 cases of differentiated tumor specimens. Diffusely infiltrating lesions were also shown to be immunoreactive, as were tumor metastases in the lymph nodes and liver (105). Another study detected FGFR2 protein in islet and ductal cells of normal pancreas, and moderate to strong FGFR2 immunoreactivity in pancreatic cancer cells and in ductal and acinar cells adjacent to the cancer cells (119). Furthermore, FGFR2 protein was immunohistochemically detected in the same cells that expressed FGFR2b transcripts as determined by in situ hybridization (119).
More recent receptor localization studies have been performed using FGFR2b-specific antibodies raised against peptide sequences encoded by an exon that is restricted to that isoform (76). FGFR2b immunoreactivity was found to be higher in well-differentiated and moderately differentiated colorectal tumors than in poorly differentiated tumors and normal tissue (95). FGFR2b immunoreactivity also was observed on the luminal surfaces of epithelial cells in normal colorectal tissue and in 35 of 56 colorectal cancer specimens, predominantly those of well-differentiated histology (123). FGFR2b RNA levels also were found to be higher in colorectal cancer cells than in normal colon epithelial cells using in situ hybridization (112). FGFR2b immunoreactivity was increased in human uterine cervical cancer cells compared with that in normal squamous cervical epithelial cells (109). FGFR2b expression was not detected in normal lung tissue, but strong immunoreactivity was observed in lung tumor cells in 31 of 61 specimens. In lung adenocarcinoma, cells staining with the FGFR2b antibody were scattered throughout the tumor region, whereas in SCC, FGFR2b-positive cells often were clustered in tumor cell nests (120).
In summary, FGFR2b appears to be expressed frequently in a number of different malignancies, and, in at least some cases, increased expression seems to be associated with cell transformation and, perhaps, malignant progression. On the other hand, in some in vitro and animal models of tumorigenesis, loss of FGFR2b expression was linked to activation of FGFR2c expression and/or a shift to more virulent behavior (107,110,111,122). Thus, additional analyses of the expression and activity of FGFR2b and FGFR2c isoforms in human tumors would help to clarify the role of these FGFR2 variants in epithelial tumorigenesis.
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KGF EFFECTS ON TUMOR CELLS |
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TopNotesAbstractIntroductionFgf signaling in cancerExpression of kgf and...Kgf effects on tumor...Investigations of the...Kgf use in patients...References |
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Proliferation
Several studies have examined the effects of KGF on proliferation of tumor cells derived from a number of different histologic sources. In some cases modest stimulation of tumor cell growth was observed, but this stimulation was usually less than that seen when the corresponding normal cell types were exposed to KGF (97,99,100,117,124,125). Not all tumor cells that express FGFR2b exhibit a mitogenic response to KGF. In one report (126), cells from only five of 35 epithelial tumor cell lines showed a statistically significant increase in clonal growth after KGF treatment, even though several of the KGF-unresponsive lines expressed FGFR2b. Other studies investigating KGF modulation of tumor cell growth have yielded similar results (95,121,127,128) (Table 1).
There is evidence that KGF promotes the growth of certain gastric cancer cells. Gastric fibroblasts stimulated the growth of gastric carcinoma cells derived from scirrhous tumors but not those from well-differentiated adenocarcinomas (129). Subsequent experiments demonstrated that this effect could be inhibited by adding a KGF-neutralizing antibody or mimicked by substituting KGF for fibroblast-conditioned medium (97). Similarly, scirrhous carcinoma cell lines expressed higher levels of FGFR2b than cell lines displaying characteristics of well-differentiated gastric cancers. No KGF protein was detected in conditioned medium collected from gastric carcinoma cell lines (97). These results suggest that paracrine sources of KGF may enhance the proliferation of gastric carcinoma cells in scirrhous tumors (Table 1).
Motility and Invasion
Consistent with its function in promoting wound healing, KGF enhances the migration of normal human keratinocytes (26) and the motility of type II lung alveolar cells, as shown in a variety of in vitro models (27–30). KGF increased several parameters of cellular motility in ER-positive breast tumor cells but not in ER-negative cells. The stimulation was dose and time dependent and was postulated to be related to the levels of FGFR2b expression, which were greater in ER-positive than in ER-negative cells (130,131). KGF-induced motility could be inhibited using either specific reagents (e.g., antisense FGFR2b oligonucleotides or a soluble fusion protein containing an extracellular FGFR2b domain) or nonspecific reagents (e.g., heparin) (132,133). KGF also has been shown to enhance the invasive potential of SNU-16 gastric carcinoma cells (106). Transfection of KGF cDNA into nontumorigenic prostate epithelial cells resulted in overexpression of matrix metalloproteinase (MMP) 1 and urokinase plasminogen activator (uPA) and the acquisition of invasive properties (134) (Table 1). A possible association between KGF expression and increased metastatic behavior was also suggested by comparing gene expression in the highly metastatic C-100 breast cancer cell line with that in C-100 cells transfected with the metastasis suppressor gene nm23, which display low metastatic potential. Expression of KGF and of a number of other genes associated with cell motility was reduced in the nm23-transfected cells (135). However, whether the decreased expression of KGF in these breast cancer cells contributed to their reduced metastatic activity was not determined.
Cytoprotection Against Radiation and Chemotherapy
Evidence that KGF can protect against oxidant-induced lung injury comes from several studies demonstrating that KGF lowers the levels of DNA breaks in alveolar cells exposed to radiation (136), hydrogen peroxide (137), or hyperoxia (138). KGF also had cytoprotective effects on epithelial cells in the gastrointestinal and upper aerodigestive tracts in mice after treatment with chemotherapy agents or radiation (8,9). Moreover, KGF decreased apoptosis in hepatocytes after treatment with actinomycin and tumor necrosis factor in vitro or with lipopolysaccharide and D-galactosamine in vivo (139). KGF also reduced apoptosis in the intestinal epithelial cells of mice treated with total parenteral nutrition (140) and in lung alveolar cells after hyperoxia, both in vitro (138) and in vivo (6).
When one contemplates the use of KGF in patients with tumors of epithelial origin, the question arises whether KGF would have a cytoprotective effect on the cancer cells themselves, increasing their resistance to the cytoablative treatments intended to kill them (Table 1). An initial study of 10 SCC cell lines, seven of which expressed FGFR2b, demonstrated no difference in the radiation survival of any of the cell lines in the presence or absence of KGF (128). The ability of KGF to protect early-passage cultures of primary tumor cells from radiation (121) or tumor cell lines from 5-fluorouracil (5-FU) (126) has been tested in anchorage-independent growth assays. Addition of KGF did not alter radiation-induced impairment of proliferation or clonogenic survival (121) or diminish the sensitivity of tumor cells to 5-FU (126). Furthermore, KGF provided no growth advantage for tumor cell xenografts established in nude mice with FGFR2b-expressing human SCC cell lines when it was administered after irradiation (2.5 Gy/day for 5 consecutive days) (128). Similarly, HT-29 human intestinal adenocarcinoma cell xenografts in nude mice that were treated with KGF for 3 days before receiving 5-FU did not display any reduction in sensitivity to the growth-inhibiting effects of 5-FU compared with HT-29 xenografts in nude mice that did not receive KGF before 5-FU (8). Finally, in rats bearing breast cancer xenografts, KGF treatment was found to act synergistically with methotrexate to increase apoptosis in the breast cancer cells, resulting in a decrease in tumor size (141).
However, other experiments have suggested that KGF may have antiapoptotic activity on certain cancer cells (Table 1). KGF expression in breast tumor specimens (as determined by immunohistochemistry) was closely associated with the expression of ER and a lower level of apoptosis, as shown by a decrease in DNA fragmentation (102,142). KGF also inhibited the induction of apoptosis by 5-FU or cyclophosphamide in the ER-positive cell lines MCF-7 and ZR-75-1 (102). In another study, KGF stimulated the activity of the prosurvival mediator Akt and inhibited Fas-mediated apoptosis in A549 lung tumor cells (143). Thus, although KGF may not protect many tumor cells from the effects of radiation or chemotherapy, the results from these studies underline the need for further exploration of this subject.
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INVESTIGATIONS OF THE TUMORIGENICITY OF KGF |
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TopNotesAbstractIntroductionFgf signaling in cancerExpression of kgf and...Kgf effects on tumor...Investigations of the...Kgf use in patients...References |
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Autocrine signaling by many growth factor–receptor combinations is a major stimulus of cell transformation. Although there are many examples of elevated KGF and/or FGFR2b expression in various human cancers and a few examples of low KGF expression in tumor cell lines, the functional consequences of autocrine KGF/FGFR2b expression in these settings are largely undefined. However, a number of experimental models have been used to investigate the effects of autocrine KGF expression on cell growth.
Creation of KGF/FGFR2b Autocrine Loops in Nontumorigenic Cells
Many fibroblasts synthesize and secrete KGF but lack the specific high-affinity receptors for KGF-induced mitogenesis, whereas most epithelial cells express FGFR2b but not KGF (2,3). However, when mouse NIH/3T3 fibroblasts, which synthesize KGF, were transfected with full-length FGFR2b cDNA an autocrine loop was created, and these cells formed foci in monolayer culture (4) (Table 2). Conversely, human PNT1A prostatic epithelial cells transfected with KGF cDNA acquired invasive potential in extracellular matrix migration assays, an effect that was specifically inhibited by a KGF neutralizing antibody (134). Furthermore, these cells overexpressed MMP1 and uPA, which have been implicated in cell migration, and displayed anchorage-independent growth, growth in serum-free medium, and increased proliferation (134). These studies suggest that the creation of a KGF autocrine loop, by the introduction either of FGFR2b into fibroblasts or of KGF into epithelial cells, can induce at least some features of the transformed phenotype. However, KGF-transfected PNT1A epithelial cells were not tumorigenic in nude mice (134), suggesting that the presence of a KGF autocrine loop is not sufficient for neoplasia (Table 1).
This conclusion is reinforced by the results of a number of studies in transgenic animals in which the consequences of targeting KGF expression to various epithelial cell populations has been examined. Only one instance of an artificially engineered autocrine expression system has been reported to result in neoplasia (144). In that case, mice that expressed KGF as a transgene under the control of the mouse mammary tumor virus long terminal repeat (LTR) were initially characterized by mammary epithelial cell hyperplasia; however, after multiple pregnancies, which presumably induced high levels of transgenic KGF expression, the mice developed mammary adenocarcinomas (144) (Table 1). It could be argued that the long lag period and the requirement for multiple pregnancies before neoplasia developed indicate that the KGF autocrine loop is not sufficient for neoplasia but rather provides conditions in which subsequent changes can occur that culminate in malignancy. Furthermore, the incidence of cancer in this model was specific for breast tissue, with no tumors arising in the salivary gland even though this tissue expressed the KGF transgene from the mouse mammary tumor virus LTR. Male mice carrying the same transgene developed hyperplasia of the genital tract, including the seminal vesicle and prostate, but no tumors (144). This result is consistent with that of a study in which KGF expression was targeted to the prostate using the probasin promoter, which also resulted in prostatic hyperplasia but not neoplasia (145). In other transgenic studies, targeted KGF expression in the epidermis (146), lung (147), pancreas (148), and corneal epithelium (149) elicited widespread histologic changes, including increased cell proliferation, but no reported instances of neoplasia. Taken together, these findings indicate that persistent production of KGF in transgenic animals caused permanent histologic abnormalities, such as hyperplasia, in targeted epithelial tissues, although these changes rarely resulted in malignancy (Table 1).
Restoration of FGFR2b Expression in Malignant Epithelial Cells
An alternative strategy to examine the oncogenic potential of KGF/FGFR2b signaling involves the introduction of FGFR2b to malignant epithelial cells that do not express this receptor. This approach was suggested by the finding that FGFR2b expression is lost during the malignant progression of various tumor types, including bladder (122), prostate (107), and salivary gland adenocarcinoma (110) (Table 2). Furthermore, in a rat Dunning model of prostate cancer, FGFR2b expression was associated with a well-differentiated phenotype, whereas its loss was associated with a shift to more virulent behavior (111). Transfection of FGFR2b cDNA into malignant prostate cells resulted in epithelial cell differentiation (150) and KGF-dependent inhibition of proliferation (151). A malignant bladder cancer cell line transfected with FGFR2b showed reduced growth in vitro, and these cells formed fewer and slower growing tumors in nude mice than those formed by the parent cell line (152). Finally, expression of transfected FGFR2b cDNA in the HSY human salivary adenocarcinoma cell line inhibited its growth by inducing differentiation and apoptosis, both in vitro and in vivo (153). These results indicate that, in particular settings, signaling through FGFR2b might serve to maintain epithelial differentiation, inhibit tumor growth, and constrain tumor invasion.
Identification of FGFR2b Variants Associated With Increased Transforming Activity
Several FGFR2b variants, all carboxyl-terminal truncations that are generated by alternative splicing, have increased transforming activity (Fig. 1). K-sam-IIC3, which was identified as an amplified gene in the stomach cancer cell line KATO-III (154,155), exhibits higher transforming activity in NIH/3T3 cells than does prototypical FGFR2b (also designated KGFR-WT) (155). The KGFR-PA variant was isolated as a transforming gene from parathyroid adenoma tissue after its introduction into NIH/3T3 cells using an expression cDNA library (156). A natural isoform of mouse FGFR2b (designated KGFR-ET, for early termination) containing a partially truncated carboxyl terminus that is identical to the carboxyl terminus of the human FGFR2/BEK isoform, TK25 (157,158), is also associated with increased transforming potential (156).
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The intrinsic transforming activity of these carboxyl-terminal truncations of FGFR2b has been difficult to assess in NIH/3T3 cells because these cells express KGF and are transformed by the ectopic expression of the prototypical FGFR2b receptor (4). However, replacement of the FGFR2b extracellular domain of the KGFR-PA and KGFR-ET variants with that of FGFR2c, which does not bind to KGF (4,76), resulted in chimeric receptor molecules that, unlike FGFR2c itself, displayed potent transforming activity when transfected into NIH/3T3 cells (156). This result implies that the FGFR2b carboxyl-terminal truncations have inherent transforming capability. The finding that the transforming activity of K-sam-IIC3 is higher than that of prototypical FGFR2b (155) is consistent with this interpretation.
The mechanism that underlies the increased transforming activity of the FGFR2b carboxyl-terminally truncated variants is not well understood. In the untruncated prototypical FGFR2b receptor, tyrosine 770 is required for the binding and subsequent tyrosine phosphorylation of phospholipase C
, as well as for mitogen-activated protein kinase activation of cell proliferation (159). Furthermore, this amino acid residue has been reported to have a negative effect on cell transformation in other FGFRs (160–162). Although K-sam-IIC3 and KGFR-PA lack this residue, its presence in KGFR-ET (Fig. 1) suggests that it is unlikely to have a pivotal role in determining the transforming activity of these molecules. K-sam-IIC3 has been reported to exhibit less autophosphorylation than FGFR2b in NIH/3T3 cells and to mediate a stronger mitogenic response to KGF (163). The higher transforming activity of KGFR-PA is not associated with increased receptor dimerization or autophosphorylation (156). However, tyrosine phosphorylation of downstream substrates, including SH2-containing protein (SHC) isoforms and possibly FGFR substrate 2 (FRS2), was increased in transfectants expressing KGFR-PA (156).
On a cellular level, the carboxyl-terminally truncated variants are less able to promote differentiation or inhibit various growth promoting pathways in transformed cells than prototypical FGFR2b. For example, when FGFR2b was introduced into L6 myoblasts that were cultured in differentiation-inducing medium, it conferred a differentiated phenotype and reduced the growth rate of these cells, whereas the introduction of K-sam-IIC3 had little effect on them (163). Furthermore, transfection of FGFR2b into T24 bladder carcinoma cells resulted in growth inhibition as well as in decreased expression and secretion of insulin-like growth factor II (IGF-II). Addition of exogenous IGF-II restored growth rates to original levels, suggesting that the decreased expression of IGF-II was responsible for the impaired proliferation (164). This growth inhibitory effect was not observed with the K-sam-IIC3 variant, indicating that the increased transforming activity associated with this receptor variant may involve its inability to downregulate IGF-II expression and that this property is associated with the carboxyl-terminal domain of FGFR2b (164).
Taken together, these observations suggest that the carboxyl terminus of prototypical FGFR2b/KGFR-WT possesses some negative regulatory activity that may play a role in muting the proliferative response while also promoting differentiation. However, there has been no systematic analysis of the prevalence of carboxyl-terminally truncated FGFR2b variants in epithelial tumors. Therefore, it is unclear whether the expression of these FGFR2b variants occurs in only a few specific tumor types or whether it is a common phenomenon, and their contribution to epithelial tumorigenesis in general remains uncertain (Table 2).
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KGF USE IN PATIENTS WITH SOLID TUMORS |
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In light of the experimental findings with regard to KGF and FGFR2b expression and activity in cancer cells, a number of issues related to the exogenous administration of KGF in patients with solid tumors merit consideration.
Safety
Does KGF/FGFR2b signaling drive tumorigenesis? As noted above, there are many examples of FGFs and FGFRs that participate in neoplasia through a variety of mechanisms, including gene amplification and/or translocation and activating mutations. Although such genetic changes have not been described for KGF in cancer cells, paracrine or autocrine stimulation of KGF/FGFR2b signaling probably occurs in many epithelial malignancies. However, there is little evidence that KGF promotes tumorigenesis. Nevertheless, one report (120) suggested that coexpression of KGF and FGFR2b by lung adenocarcinoma cells is associated with a worse prognosis. In contrast, a high level of FGFR2b expression in colorectal cancer was associated with a minimally invasive, well-differentiated phenotype (123), and the absence of FGFR2b expression in bladder carcinoma was characteristic of aggressive tumors (122). Gene amplification of a truncated FGFR2b isoform (K-sam-IIC3) has been observed in scirrhous gastric carcinomas, but amplification does not appear to be associated with prognosis (105). Thus, although it is possible that KGF/FGFR2b signaling may contribute to tumorigenesis in specific situations, it may not do so generally. Indeed, in vitro studies and animal models have indicated that this pathway might actually constrain malignant behavior in some instances (150–153,164). Additional studies with FGFR2b-specific reagents and prognostic information are needed to confirm that these findings are applicable to human tumors in vivo. Combined with the results from transgenic animal models, in which KGF overexpression typically caused hyperplasia but rarely resulted in neoplasia (144–149), it appears that the KGF/FGFR2b pathway has, at most, a limited role in tumorigenesis.
Would administration of KGF enhance tumor growth? The data we have summarized indicate that tumor cells exhibit little or no mitogenic response to KGF in vitro or in vivo, even when they express FGFR2b. The lack of increased proliferation reflects their high basal level of mitogenic activity, which presumably is a consequence of aberrant oncogene expression and loss of tumor suppressor functions. Given the small proliferative response of malignant cells to KGF and the fact that endogenous KGF is already available from paracrine or autocrine sources, it is unlikely that additional exposure from pharmacologic doses of KGF would have much impact on tumor growth, especially because the duration of KGF treatment is typically short and the effects of exogenous KGF are transient (19).
Would KGF inhibit the cytotoxicity of cancer treatment regimens? This possibility is of greater theoretical concern than KGF stimulation of tumor growth because the timing of KGF administration would coincide with chemoradiotherapy treatments. However, results from several in vitro and in vivo studies suggest that KGF does not typically block the lethal activity of chemotherapy or radiation on tumor cells. For example, whereas KGF inhibited radiation and peroxide induction of DNA strand breaks and Fas-mediated apoptosis in well-differentiated A549 lung carcinoma cells (136,137), the KGF-treated cells still succumbed to cytotoxic doses of radiation (136). Nonetheless, a hypothetical effect of KGF on tumor cell survival warrants attention and careful monitoring of patients enrolled in palifermin clinical trials.
Would KGF promote development of secondary tumors? As the number of cancer survivors increases, the incidence of secondary tumors also has risen (165). Although some of these malignancies may have occurred spontaneously in a cancer-prone population, it is likely that exposure to cancer treatment regimens that cause DNA damage also contributed to the neoplastic process. Presumably, mutations resulting from cytotoxic therapy predispose patients to the development of subsequent cancer. Insofar as KGF can inhibit apoptosis in cells that normally would die after accumulating DNA damage (6,138), it is possible that KGF treatment would foster the development of secondary malignancies. However, the opposite outcome is also possible: by promoting DNA repair, KGF might reduce the number of cells that have mutations and, consequently, limit the likelihood of subsequent cancers. Again, careful monitoring of patients who receive KGF should help to resolve this issue.
Are any solid tumor types especially well or poorly suited for KGF treatment? Currently, definitive evidence concerning the preferential use or avoidance of KGF in a particular solid tumor setting is lacking. Preliminary indications suggest that KGF might be beneficial for the treatment of bladder, salivary, and perhaps prostate cancer, based on data showing that the introduction of FGFR2b into malignant cells from these tumor types induced cell differentiation or apoptosis (150–153). On the other hand, KGF has been found to inhibit apoptosis in ER-positive breast tumor cells (102,142) and to promote their motility (130,131), and prolonged expression of a KGF transgene in mouse mammary epithelium ultimately resulted in neoplasia (144). In addition, KGF specifically stimulated the growth of gastric carcinoma cells derived from scirrhous tumors, but not those from well-differentiated adenocarcinomas (97,129). Furthermore, the FGFR2b isoform K-sam-IIC3, which exhibits elevated transforming activity (155), is amplified in stomach cancer cell lines (154,155). Thus, patients with breast or stomach cancers probably should not be treated with KGF pending further information about its activity in these contexts. Considering the uncertainties summarized here, KGF use in patients with epithelial tumors should be confined to clinical trials where its effects would be closely monitored.
Little information is available about KGF/FGFR2b expression or activity in sarcomas. Given their mesenchymal origin, it seems likely that many of these tumors would express KGF but not FGFR2b, although some might express both, a pattern that has been documented in a few mesenchymal cell types (5). Thus, aside from the latter possibilities, it is unlikely that sarcomas would be directly affected by KGF treatment.
Potential Therapeutic Benefits
Currently, the primary objective of KGF use in cancer patients is a reduction of chemoradiotherapy-induced oral mucositis. Palifermin (Kepivance) has been approved by the Food and Drug Administration to decrease the incidence and duration of severe oral mucositis in patients with hematologic malignancies who receive high-dose cytotoxic therapy followed by hematopoietic stem cell transplantation. The safety and efficacy of palifermin in nonhematologic malignancies has not been established. However, in a randomized phase II clinical trial in patients with advanced colorectal cancer who were treated with 5-FU and leucovorin, patients who received palifermin had a lower incidence of ulcerative oral mucositis and were more likely to receive the full dose of chemotherapy on time than patients who did not receive palifermin (166). The ability of KGF to help patients endure current treatment regimens suggests that its use might enable the adoption of more aggressive therapies that could have a greater likelihood of a favorable outcome, if patients were able to tolerate toxicities associated with increased doses of chemoradiotherapy. It is possible that KGF could facilitate the development of more effective chemoradiotherapy by increasing the therapeutic window of available agents and treatment methods.
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NOTES |
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TopNotesAbstractIntroductionFgf signaling in cancerExpression of kgf and...Kgf effects on tumor...Investigations of the...Kgf use in patients...References |
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Support for the writing of this review was provided by the Intramural Research Program of the National Institutes of Health, National Cancer Institute.
As coinventors on patents pertaining to KGF, both P. W. Finch and J. S. Rubin have a financial interest in its commercial development. P. W. Finch holds stock in Amgen, the maker of Kepivance.
We express our appreciation to Dr. Stuart A. Aaronson for his critical role in initiating KGF research and his ongoing interest.
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REFERENCES |
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