CANCER GENOMICS & PROTEOMICS 3: 369-372 (2006)
A Comparison of KGF Receptor Expression
in Various Types of Human Cancer
XIAO-PING ZANG1, MEGAN R. LERNER2,3, STEVEN J. BAHR1,
DANIEL J. BRACKETT2,3 and J. THOMAS PENTO1
Departments of 1Pharmaceutical Sciences and 2Surgery, University of Oklahoma
Health Sciences Center; 3VA Medical Center, Oklahoma City, Oklahoma 73190, OK, U.S.A.
Abstract. Background: Keratinocyte growth factor (KGF) has been observed to produce a rapid increase in the motility of breast cancer cells. KGF/KGFR (KGF receptor) signaling has also been demonstrated in the progression of many types of human cancer. The objective of the present study was to compare KGFR expression in various types of cancer. Materials and Methods: A cancer profiling array containing cDNA from 154 tumor and paired normal samples representing 19 types of human cancer was employed. Results: The results of the present study indicate that KGFR expression is enhanced in many types of human carcinomas at an early stage of cancer development, suggesting that KGFR overexpression may be an early signal in the progression of these cancers. However, the stage of cancer progression and relative level of expression varied considerably among the various types of cancer. Conclusion: These findings suggest that tumor KGFR levels may serve as a prognostic biomarker for cancer staging and/or treatment.
Correspondence to: J. Thomas Pento, Ph.D., Department of Pharmaceutical Sciences, College of Pharmacy, University of Oklahoma Health Sciences Center, 1110 N. Stonewall Ave., Oklahoma City, OK 73117, U.S.A. Tel: +405 271 6593, Fax: +405 271-7505,
Key Words: KGFR, cDNA cancer array, cancer, gene expression, progression.
Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family (also designated FGF-7) that is produced in stromal tissue and stimulates DNA synthesis, proliferation and migration of epithelial cells in the breast and other tissues (1-3). It is well established that target epithelial cells contain a high affinity KGF receptor (KGFR) (4, 5). In situ hybridization studies have confirmed the specific mesenchymal production of KGF and epithelial localization of the KGFR in mammary tissue which provides further evidence that KGF is a mesenchymally-derived mediator of epithelial cell proliferation and migration (6, 7).
The mammary glands of adult female animals are remarkably sensitive to KGF (8). Systemic administration of KGF in adult rats for three to five days was found to produce massive mammary ductal hyperplasia and an elevation of mitotic figures (8). Intraductal hyperplasia is a well-known characteristic of pre-malignant breast lesions which lead to neoplasia. Similarly, Kitsberg and Leder (9) have observed that female mice, with a constitutively up-regulated KGF transgene, developed mammary epithelial hyperplasia and eventually all animals developed metastatic mammary carcinomas. Consistent with this concept, KGFR gene up-regulation has been observed in human primary breast tumor specimens (10). Conversely, highly malignant metastatic breast cancer tissue expressed relatively little KGFR (11). It has been observed that KGF treatment induced an up-regulation of the KGFR gene in estrogen receptor (ER)-positive breast cancer cells (12). These observations suggest that KGF-mediated stimulation of breast epithelial proliferation and migration may be an early event in the molecular cascade, which leads to cancer progression and metastasis (13). It has been previously determined that KGF produces a rapid, direct motility enhancement effect in ER-positive human breast cancer cell lines (14). Furthermore, KGF enhanced the growth of breast tumors in a mouse xenograft model (15). In addition to breast cancer, there is evidence which indicates that KGF/KGFR signaling is involved in the proliferation, invasion and malignancy of prostate (16-19), cervical (20), colorectal (21), ovarian (22), lung (23), stomach (24) and endometrial (25, 26) carcinomas. Therefore, KGF/KGFR signaling may be involved in the progression of many types of cancer.
The objective of the present study was to compare the expression of KGFR in cancer tissue relative to patient-paired normal tissue in order to determine the potential value of KGFR as a predictive biomarker of cancer metastasis and as a therapeutic target for the prevention of cancer progression.
Materials and Methods
The CLONTECH Cancer Profiling Array II (BD Biosciences, Palo Alto, CA, USA) was employed in this study. This nylon membrane-based array was spotted with total cDNA extracted from 19 different cancer types and adjacent normal tissue taken from 154 individual cancer patients, according to the manufacturer's instructions. The membrane was also spotted with total cDNA from the following human cancer cells lines: HeLa, cervical carcinoma; Daudi and Raji, Burkitt's lymphoma; K562, chronic myelogenous leukemia; HL60, acute promyelocytic leukemia; G361, malignant melanoma; A549, lung carcinoma; MOLT4, acute lymphoblastic leukemia and SW480, colorectal adenocarcinoma. The array cDNA samples were normalized with ubiquitin.
The KGFR (FGFR2-IIIb) probe used in this study was created by isolating total RNA from an MCF-7 cell line using the RNeasy Mini-Kit (Quiagen, Valencia, CA, USA). The KGFR cDNA was directly prepared from 5 µg of total RNA by a SuperScript First-Strand Synthesis System for RT-PCR Kit (Invitrogen, Carlsbad, CA, USA). The polymerase chain reaction (PCR) for the probe was carried out using gene-specific primers: FGFR2-IIIb Reverse 5'-CAC TCG GGG ATA AAT AGT T-3' and FGFR2-IIIb Forward 5'-ACT CGG AGA CCC CTG CCA3'. Gel electrophoresis of the final PCR product produced a single band with a size of 461 bp. An epidermal growth factor receptor (EGFR) Probe was created using the same methodology. The KGFR and EGFR cDNA probes were hybridized to the cancer array, as described in the manufacturer's protocol. Following hybridization, the arrays were placed on a phospho-imager (model 820 Storm Phospho-imaging System) overnight to create a digitized image of the array. The images were then analyzed using Array Vision software (Imaging Research Inc.) which calculates the pixel density of each of the elements (cancer and normal) and the background density. The element density is corrected for background density and the ratio of the corrected density of each cancer elements is divided by the corrected density of the patient-paired normal elements to obtain the hybridization ratios.
The KGFR cDNA probe did not hybridize to the negative controls on the array which included total yeast RNA, E. Coli DNA, Poly A and a human DNA tandem repeat. The KGFR probe did hybridize to human genomic DNA used as a positive control. The ratio of the expression of KGFR in the tumor samples, relative to that present in the patient-paired normal tissue, is presented in Table I.
Zang et al: KGFR Expression in Cancer
A ratio of greater than 1 indicates overexpression of KGFR in the cancer sample. In summary, the percentage of cancer samples which were observed to have an enhanced level of KGFR expression are as follows: breast 60%; ovary 100%; uterus 70%; cervix 30%; vulva 20%; prostate 75%; testes 80%; thyroid 10%; skin 0%; stomach 60%; small intestine 71%; colon 10%; rectum 40%; pancreas 29%; liver 0%; trachea 67%; lung 60%; kidney 10%; and bladder 40%. Breast tumor specimens with a higher KGFR expression were observed to be in an early stage of cancer development (stage I). This was found to be true in the other types of cancer except for ovary, stomach, rectum, bladder, trachea, small intestine and pancreas cancer samples, which were found to be in stages III or IV. All cDNA samples from the human cancer cell lines on the array were found to hybridize with KGFR and the density hybridization was similar among the cancer cell lines.
In this study the cancer array was also hybridized with an EGFR probe. It was observed that hybridization with the EGFR probe produced a pattern of tumor and tissue expression of EGFR which was completely different than that observed with the KGFR probe (data not shown). Thus, suggesting that the pattern of KGFR expression observed in this study was unique to KGFR.
KGF binds to the KGFR (also known as FGFR2-IIIb) found in epithelial cells, which is a splice variant of FGFR-2 encoded by the FGFR-2 gene (27). KGFR is a member of the fibroblast growth factor receptor (FGFR) family which are membrane-spanning tyrosine kinase receptors consisting of four known peptides whose sequences are highly conserved (7). It has been shown that there is a transition in the KGFR from the IIIb isoform in primary tumors, which is KGF-responsive, to the IIIIc isoform in advanced prostate cancer, which is unresponsive to KGF (19). This observation also supports the concept that KGF is an early signal that is involved in the initiation of cancer cell migration and progression to a metastatic phenotype.
It has been previously observed that KGF enhances the scattering motility of human breast cancer cells and that ER-positive, KGF-responsive cancer cells express KGFR while ER-negative, KGF-unresponsive, cells do not (14). Others have reported KGFR overexpression of in situ but not metastatic breast cancer (10, 11). The present study demonstrated that KGFR is up-regulated in breast and other types of cancer tissue at an early stage of cancer development (i.e., uterus, cervix, vulva, prostate, testes and lung), suggesting that KGFR up-regulation may be an early signal in the progression of these cancers, and thus, may be a useful prognostic biomarker.
On the other hand, it was found that KGFR was up-regulated in more advanced tumors in other types of cancer (i.e., ovary, stomach, small intestine, rectum, bladder, trachea and pancreas), while it was actually down-regulated in some other types of cancer on the array (i.e., skin, liver, colon and kidney). These findings support the concept that KGFR tumor levels can serve as a prognostic biomarker for the staging and/or treatment of many types of cancer. Further, KGF, KGFR and associated signaling pathways may serve as therapeutic targets for the development of important new therapeutic agents to inhibit the metastatic progression of KGF-responsive cancers.
This study was supported in part by grants from NCI (CA-89740) and DOD (DAMD17-01-1-0591).
1. Baird A and Klagsbrun M: The fibroblast growth factor family; Nomenclature meeting report and recommendations. Ann NY Acad Sci 638: 13-16, 1991.
2. Rubin JS, Bottaro DP, Chedid M, Mikki T, Ron D, Cunha G and Finch PW (eds.). Epithelial-Mesenchymal Interactions in Cancer. Verlag Basel, pp. 191-214, 1995.
3. Rubin JS, Osada H, Finch PW, Taylor WG, Rudikoff S and Aaronson SA: Purification and characterization of a newly identified growth factor specific for epithelial cells. Proc Natl Acad Sci USA 86: 802-806, 1989.
4. Aaronson SA, Bottaro DP, Miki T, Ron D, Finch PW, Fleming TP, Ahn J, Taylor WG and Rubin JS: Keratinocyte growth factor. A fibroblast growth factor family member with unusual target cell specificity. Ann N Y Acad Sci USA 638: 62-77, 1991.
5. Bottaro DP, Rubin JS, Ron D, Finch PW, Florio C and Aaronson SA: Characterization of the receptor for keratinocyte growth factor. Evidence for multiple fibroblast growth factor receptors. J Biol Chem 265: 12767-12770, 1990.
6. Mason IJ, Fuller-Pace F, Smith R and Dickson C: FGF-7 (keratinocyte growth factor) expression during mouse development suggests roles in myogenesis, forebrain regionalisation and epithelial-mesenchymal interactions. Mech Dev 45: 15-30, 1994.
7. Miki T, Fleming TP, Bottaro DP, Rubin JS, Ron D and Aaronson SA: Expression cDNA cloning of the KGF receptor by creation of a transforming autocrine loop. Science 251: 72-75, 1991.
8. Ulich TR, Yi ES, Cardiff R, Yin S, Bikhazi N, Biltz R, Morris CF and Pierce GF: Keratinocyte growth factor is a growth factor for mammary epithelium in vivo. The mammary epithelium of lactating rats is resistant to the proliferative action of keratinocyte growth factor. Am J Pathol 144: 862-868, 1994.
9. Kitsberg DI and Leder P: Keratinocyte growth factor induces mammary and prostatic hyperplasia and mammary adenocarcinoma in transgenic mice. Oncogene 13: 2507-2515, 1996.
10. Koos RD, Banks PK, Inkster SE, Yue W and Brodie AM: Detection of aromatase and keratinocyte growth factor expression in breast tumors using reverse transcription-polymerase chain reaction. J Steroid Biochem Mol Biol 45: 217-225, 1993.
11. Bansal GS, Cox HC, Marsh S, Gomm JJ, Yiangou C, Luqmani Y, Coombes RC and Johnston CL: Expression of keratinocyte growth factor and its receptor in human breast cancer. Br J Cancer 75: 1567-1574, 1997.
12. Zang XP, Lerner ML, Brackett DJ and Pento JT: Keratinocyte growth factor-mediated pattern of gene expression in breast cancer cells. Cancer Genomics Proteomics 1: 339-344, 2004.
13. Aznavoorian S, Murphy AN, Stetler-Stevenson WG and Liotta LA: Molecular aspects of tumor cell invasion and metastasis. Cancer 71: 1368-1383, 1993.
14. Zang XP and Pento JT: Keratinocyte growth factor-induced motility of breast cancer cells. Clin Exp Metastasis 18: 573-580, 2001.
15. Zang XP, Howard EW, Jupe ER, Manjeshwar S and Pento JT: Enhanced growth of KGF-transfected human breast cancer cells in a mouse xenograft model. Breast Cancer Res Treat 82: 190, 2005.
16. Leung HY, Mehta P, Gray LB, Collins AT, Robson CN and Neal DE: Keratinocyte growth factor expression in hormone insensitive prostate cancer. Oncogene 15: 1115-1120, 1997.
17. McGarvey TW and Stearns ME: Keratinocyte growth factor and receptor mRNA expression in benign and malignant human prostate. Exp Mol Pathol 63: 52-62 ,1995.
18. Planz B, Oltean H, Deix T, Kirley SD, Wang QF, McDougal WS and Marberger M: Effect of keratinocyte growth factor and activin on cell growth in the human prostatic cancer cell line LNCaP. World J Urol 22: 140-144, 2004.
19. Yan G, Fukabori Y, McBride G, Nikolaropolous S and McKeehan WL: Exon switching and activation of stromal and embryonic fibroblast growth factor (FGF)-FGF receptor genes in prostate epithelial cells accompany stromal independence and malignancy. Mol Cell Biol 13: 4513-4522, 1993.
20. Kurban G, Ishiwata T, Kudo M, Yokoyama M, Sugisaki Y and Naito Z: Expression of keratinocyte growth factor receptor (KGFR/FGFR2 IIIb) in human uterine cervical cancer. Oncol Rep 11: 987-991, 2004.
21. Otte JM, Schmitz F, Banasiewicz T, Drews M, Folsch UR and Herzig KH: Expression of keratinocyte growth factor and its receptor in colorectal cancer. Eur J Clin Invest 30: 222-229, 2000.
22. Parrott JA, Kim G, Mosher R and Skinner MK: Expression and action of keratinocyte growth factor (KGF) in normal ovarian surface epithelium and ovarian cancer. Mol Cell Endocrinol 167: 77-87, 2000.
23. Yamayoshi T, Nagayasu T, Matsumoto K, Abo T, Hishikawa Y and Koji T: Expression of keratinocyte growth factor/fibroblast growth factor-7 and its receptor in human lung cancer: correlation with tumour proliferative activity and patient prognosis. J Pathol 204: 110-118, 2004.
24. Nakazawa K, Yashiro M and Hirakawa K: Keratinocyte growth factor produced by gastric fibroblasts specifically stimulates proliferation of cancer cells from scirrhous gastric carcinoma. Cancer Res 63: 8848-8852, 2003.
25. Taniguchi F, Harada T, Sakamoto Y, Yamauchi N, Yoshida S, Iwabe T and Terakawa N: Activation of mitogen-activated protein kinase pathway by keratinocyte growth factor or fibroblast growth factor-10 promotes cell proliferation in human endometrial carcinoma cells. J Clin Endocrinol Metab 88: 773-780, 2003.
26. Visco V, Carico E, Marchese C, Torrisi MR, Frati L, Vecchione A and Muraro R: Expression of keratinocyte growth factor receptor compared with that of epidermal growth factor receptor and erbB-2 in endometrial adenocarcinoma. Int J Oncol 15: 431-435, 1999.
27. Ornitz DM, Xu J, Colvin JS, McEwen DG, MacArthur CA, Coulier F, Gao G and Goldfarb M: Receptor specificity of the fibroblast growth factor family. J Biol Chem 271: 15292-15297, 1996.
Received September 25, 2006
Accepted October 27, 2006