Co-expression of heparin-binding EGF-like growth factor and related peptides in human gastric carcinoma

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family of polypeptide growth factors, which includes EGF, transforming growth factor α (TGF-α), amphiregulin (AR) and betacellulin (BTC). To assess the potential role of HB-EGF in human gastric carcinomas, the expression of HB-EGF and EGF receptor (EGF-R) was examined in normal and cancerous gastric tissues and cultured gastric cancer cell lines. By Northern blot analysis, there was a 4.7-fold increase in HB-EGF mRNA levels in human gastric cancers compared with normal gastric tissues. There was a concomitant 3.9-fold increase in EGF-R mRNA levels in these cancers. Immunostaining revealed co-localization in 72% of the cancer cells of HB-EGF and EGF-R. AR and BTC moieties were not evident by Northern blot analysis. However, using PCR, both AR and BTC mRNA species were demonstrated in normal and cancerous gastric tissues. By Northern blot analysis, HB-EGF, TGF-α, AR, BTC and EGF-R mRNA moieties were co-expressed in KATO III and NCI-N87 gastric cancer cell lines. Furthermore, HB-EGF, EGF and TGF-α enhanced the growth of both cell lines in a dose-dependent manner. Our findings suggest that HB-EGF is relatively abundant in human gastric cancers and that co-expression of the EGF ligand family may lead to excessive activation of EGF-R in this disorder. © 1996 Wiley-Liss, Inc.     

Cooperation between heparin-binding EGF-like growth factor and interleukin-6 in promoting the growth of human myeloma cells

Interleukin-6 (IL-6) is a major survival and proliferation factor of human malignant plasma cells and IL-6 dependent myeloma cell lines can be obtained from patients with terminal disease. We show here that mutated diphtheria toxin, a specific inhibitor of heparin-binding epidermal growth factor-like growth factor (HB-EGF), blocked the IL-6-induced growth of two myeloma cell lines (XG-1 and XG-14) and did not significantly affect that of two other cell lines (XG-6 and XG-13). The IL-6 mediated growth of myeloma cells was also inhibited by antibodies to ErbB1, a receptor for HB-EGF. The XG-1 and XG-14 cell lines that are sensitive to HB-EGF inhibitors overexpressed HB-EGF and EGF receptor (ErbB1) genes. They also overexpressed CD9, a tetraspanin that binds to the heparin-binding domain of HB-EGF and is critical for promoting ErbB1 activation by HB-EGF. The XG-6 and XG-13 myeloma cells that were not significantly sensitive to HB-EGF antagonists, poorly expressed HB-EGF, ErbB1 and CD9 genes or proteins. We demonstrated that recombinant HB-EGF supported the long-term growth of myeloma cells, as did IL-6. The myeloma cell growth factor activity of HB-EGF was completely inhited by antibodies to ErbB1, but also by antibodies to gp130 IL-6 transducer or to IL-6. These data indicate that in the XG-1 and XG-14 IL-6-dependent myeloma cell lines, the CD9/HB-EGF/erbB1 and the IL-6/IL-6R/gp130 pathways cooperate synergistically to trigger myeloma cell growth. They suggest that inhibitors of the EGF receptor or HB-EGF may be useful for inducing myeloma cell apoptosis in patients with multiple myeloma.     

Proteolytic activation of heparin-binding EGF-like growth factor by membrane-type matrix metalloproteinase-1 in ovarian carcinoma cells

Increased expression of heparin-binding EGF-like growth factor (HB-EGF) and membrane-type matrix metalloproteinase-1 (MT1-MMP) is frequently associated with various types of malignant tumor. HB EGF-like growth factor has been reported to promote the malignant progression of ovarian carcinoma. Based on this finding, inhibition of HB-EGF activity with CRM197 is now under phase I clinical evaluation. On the other hand, MT1-MMP expressed in ovarian carcinoma cells is thought to promote invasion and growth of tumor cells by degrading the extracellular matrix. However, we recently demonstrated that co-expression of MT1-MMP and HB-EGF in gastric carcinoma cells leads to cleavage of HB-EGF within its N-terminal heparin-binding region, converting it into a potent heparin-independent growth factor. In this study, we evaluated the importance of regulation of HB-EGF by MT1-MMP in clinical samples of ovarian carcinoma. We detected co-expression of HB-EGF and MT1-MMP in clear cell ovarian carcinoma tissues, particularly at the invasion front and in tumor cells that had disseminated into the ascites, whereas HB-EGF alone was expressed in non-invasive borderline ovarian tumor tissue. Furthermore, a soluble HB-EGF fragment that corresponds to that processed by MT1-MMP was detected in malignant ascites obtained from patients with metastatic ovarian carcinoma. Ovarian carcinoma cells that express MT1-MMP and HB-EGF exhibited enhanced cell growth in a 3D-collagen matrix and anchorage-independent growth in suspension. These results indicate that MT1-MMP co-expressed with HB-EGF in ovarian carcinoma cells potentiates the activity of HB-EGF to promote invasive tumor growth and spreading in vivo.     

Heparin-binding EGF-like growth factor expression and biological action in human pancreatic cancer cells

The epidermal growth factor (EGF) receptor is activated by EGF and other EGF-like growth factors, including heparin-binding epidermal growth factor-like growth factor (HB-EGF). We characterized the biological actions of HB-EGF in PANC-1 and COLO-357 human pancreatic cancer cell lines, and determined whether the presence of HB-EGF in human pancreatic carcinomas correlates with patient survival. HB-EGF enhanced the growth of both cell lines in a dose-dependent manner, with a potency that was generally similar to that of EGF and transforming growth factor-alpha (TGF-alpha). HB-EGF also readily induced tyrosine phosphorylation of the EGF receptor in these cells. Immunohistochemical analysis of 47 pancreatic cancer tissues revealed the presence of HB-EGF immunoreactivity in the cancer cells in 50% of the tumors. However, the presence of HB-EGF was not associated with a statistically significant decrease in the post-operative survival period. Furthermore, coexpression of HB-EGF and the EGF receptor was not associated with shorter patient survival. These findings suggest that HB-EGF activates the EGF receptor in human pancreatic cancer cells, but that it is not involved in enhancing the biological aggressiveness of this malignancy in vivo.     

Expression of heparin-binding epidermal growth factor-like growth factor in breast carcinoma

The expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) was investigated for 76 cases of breast carcinoma. HB-EGF was expressed in 71.8% of the carcinoma cases but only slightly in normal mammary glands. Interestingly, its expression was inversely related to biological aggressiveness of the breast carcinoma. These results suggest that HB-EGF may play a crucial role in the early stage of this carcinoma.     

Amphiregulin: a new growth factor in hepatocarcinogenesis

Amphiregulin (AR) is a member of the epidermal growth factor family and a ligand of the epidermal growth factor receptor (EGFR). As other ligands of the EGFR, AR is synthesized as a precursor that is shed from the plasma membrane by metalloproteases. Hyperactive autocrine loops involving AR production have been described in a variety of tumors, and this growth factor is thought to play a non-redundant role in cancer development. AR expression is not detected in the normal liver, however it is readily induced during acute liver injury and behaves as a potent pro-regenerative and survival factor. Increased AR expression is also detected in human chronic liver injury (liver cirrhosis), which is considered a pre-neoplastic condition. Recent evidences suggest that AR can play a unique role in liver tumorigenesis and in the maintenance of the neoplastic phenotype of hepatocarcinoma cells. In this review, we summarize some aspects of AR patho-biology and the rationale behind its definition as a novel target in hepatocarcinoma therapy.     

Forced expression of hepatocyte-specific fibroblast growth factor 21 delays initiation of chemically induced hepatocarcinogenesis

Inappropriate fibroblast growth factor (FGF) signaling is involved in most tissue-specific pathologies including cancer. Previously we showed that inappropriate expression and chronic activity of FGF receptor (FGFR) 1 in hepatocytes accelerated diethylnitrosamine (DEN)-initiated hepatocarcinogenesis. Here we showed that although widely expressed FGF1 and FGF2 are frequently upregulated in hepatocellular carcinoma (HCC), germline deletion of both FGF1 and FGF2 had no effect on DEN-initiated hepatocarcinogenesis. Thus overexpression of FGF1 or FGF2 may be a consequence rather than contributor to hepatoma progression. FGF21 is the first of 22 homologues whose expression has been reported to be preferentially in the liver. We showed that similar to FGF1 and FGF2, FGF21 mRNA was upregulated in neoplastic and regenerating liver after partial hepatectomy (PH) and CCl4 administration. In situ hybridization analysis confirmed that in contrast to FGF1 and FGF2, expression of FGF21 mRNA was limited to hepatocytes. Forced overexpression of FGF21 in hepatocytes by gene targeting had no apparent impact on normal liver development and compensatory response to injury. Surprisingly, overexpression of FGF21 delayed the appearance of DEN-induced liver tumors. At 8 and 10 mo, only 10% and 30% of transgenic mice, respectively, developed adenomas compared to 50% (all adenomas) and 80% (60% adenoma/20% HCC) in the wild-type (WT) mice. However, the incidence and burden of HCC at 10 mo and later was equal in the FGF21 transgenic and WT mice. We propose that FGF21 may delay development of adenomas through activation of resident hepatocyte FGFR4 at early times, but counteracts the delay by acceleration of progression to HCC through interaction with ectopic FGFR1 once it appears in hepatoma cells. This indicates a dual function of FGF21 that may reflect changes in FGFR isotype during progression of differentiated hepatoma cells.     

hst-1 transforming protein: expression in silkworm cells and characterization as a novel heparin-binding growth factor

A protein encoded by an hst-1 transforming gene was expressed in silkworm-derived BmN cells and secreted into the culture medium using a recombinant baculovirus vector. The strong affinity for heparin of the secreted protein made it possible to purify the hst-1 protein to homogeneity in a two-step procedure. The purified hst-1 protein has a molecular weight of 18,000 and stimulates both DNA synthesis in NIH3T3 cells and human umbilical vein endothelial cell proliferation. In addition, morphological changes and anchorage-independent growth of NIH3T3 cells are induced by this product. These results show that the hst-1 transforming protein is a novel heparin-binding growth factor as predicted by nucleotide sequence analysis.     

Clinical significance of heparin-binding epidermal growth factor-like growth factor and a disintegrin and metalloprotease 17 expression in human ovarian cancer.

PURPOSE: Lysophosphatidic acid, which is enriched in the peritoneal fluid of ovarian cancer patients, plays a key role in the progression of ovarian cancer. Lysophosphatidic acid can generate epidermal growth factor receptor (EGFR) signal transactivation involving processing of EGFR ligands by ADAM (a disintegrin and metalloprotease) family metalloproteases. We aimed to investigate the clinical significance of EGFR ligands and ADAM family in the lysophosphatidic acid-induced pathogenesis of ovarian cancer. EXPERIMENTAL DESIGN: We examined the expression of EGFR ligands and ADAM family members in 108 patients with normal ovaries or ovarian cancer, using real-time PCR, immunohistochemistry, and in situ hybridization, and analyzed the clinical roles of these molecules. Statistical analyses of these data were done using the Mann-Whitney test, Kaplan-Meier method, or Spearman's correlation analysis. RESULTS: Large differences in expression were found for heparin-binding EGF-like growth factor (HB-EGF) and other EGFR ligands and for ADAM 17 and other ADAM family members. HB-EGF expression was significantly increased in advanced ovarian cancer compared with that in normal ovaries (P < 0.01). HB-EGF expression was significantly associated with the clinical outcome (P < 0.01). ADAM 17 expression was significantly enhanced in both early and advanced ovarian cancer compared with that in normal ovaries (both P < 0.01), although it had no clinical significance in the progression-free survival. HB-EGF expression was significantly correlated with ADAM 17 expression (gamma = 0.437, P < 0.01). CONCLUSIONS: Our findings suggest that HB-EGF and ADAM 17 contribute to the progression of ovarian cancer and that HB-EGF plays a pivotal role in the aggressive behavior of a tumor in ovarian cancer.     

The potential role of the heparin-binding growth factor pleiotrophin in breast cancer

Summary We propose that the secreted protein pleiotrophin (PTN) is a major factor in the malignant progression of breast cancer. This hypothesis is based on the growth-stimulatory effects of PTN on cellsin vitro andin vivo and on its high levels of expression in 60% of tumor samples from breast cancer patients. The stimulation of proliferation and tube formation of endothelial cells by PTN suggests that it can serve as an angiogenesis factor during tumor growth. We hypothesize that PTN has the potential to support growth of breast cancer at its primary site and to enhance the ability of tumor cells to metastasize. Furthermore, we suggest that specific endocrine signals interact to regulate the expression of PTNin vitro andin vivo. Finally, we propose that understanding the functions of PTN and its hormonal regulation can lead to the development of novel therapeutic strategies for breast cancer.     

Resident hepatocyte fibroblast growth factor receptor 4 limits hepatocarcinogenesis

Fibroblast growth factor (FGF) family signaling mediates cell‐to‐cell communication in development and organ homeostasis in adults. Of the FGF receptor (FGFR) isotypes, FGFR4 is the sole resident isotype present in mature parenchymal hepatocytes. FGFR1 that is normally associated with activated nonparenchymal cells appears ectopically in hepatoma cells. Ectopic expression and chronic activity of FGFR1 in hepatocytes accelerates diethylnitrosamine (DEN)‐initiated hepatocarcinogenesis by driving unrestrained cell proliferation and tumor angiogenesis. Hepatocyte FGFR4 mediates liver's role in systemic cholesterol/bile acid and lipid metabolism and affects proper hepatolobular restoration after damage without effect on cell proliferation. Here we ask whether FGFR4 plays a role in progression of hepatocellular carcinoma (HCC). We report that although spontaneous HCC was not detected in livers of FGFR4‐deficient mice, the ablation of FGFR4 accelerated DEN‐induced hepatocarcinogenesis. In contrast to FGFR1 that induced a strong mitogenic response and depressed rate of cell death in hepatoma cells, FGFR4 failed to induce a mitogenic response and increased the rate of cell death. FGFR1 but not FGFR4 induced cyclin D1 and repressed p27 expression. Analysis of activation of Erk, JNK, and PI3K‐related AKT signaling pathways indicated that in contrast to FGFR1, FGFR4 failed to sustain Erk activation and did not activate AKT. These differences may underlie the opposing effects of FGFR1 and FGFR4. These results suggest that in contrast to ectopic FGFR1 that is a strong promoter of hepatoma, resident FGFR4 that mediates differentiated hepatocyte metabolic functions also serves to suppress hepatoma progression. © 2008 Wiley‐Liss, Inc.     

Clinical significance of heparin-binding epidermal growth factor-like growth factor in peritoneal fluid of ovarian cancer

Epidermal growth factor receptor (EGFR) has been implicated in tumour growth and extension of ovarian cancer. Peritoneal fluid in ovarian cancer patients contains various growth factors that can promote tumour growth and extension. In order to investigate the clinical significance of EGFR ligands as activating factors of ovarian cancer, we examined the cell proliferation-promoting activity and the level of EGFR ligands in peritoneal fluid obtained from 99 patients. Proliferation-promoting activity in peritoneal fluid from 63 ovarian cancer patients (OVCA) was much higher than peritoneal fluid from 18 ovarian cyst patients (OVC) and 18 normal ovary patients (NO), and the activity was suppressed only by antibodies against EGFR or heparin-binding epidermal growth factor (HB-EGF). A large difference was observed in the level of EGFR ligands between HB-EGF and TGF-alpha or amphiregulin. The concentration of HB-EGF in OVCA significantly increased compared to that in OVC or NO (P<0.01). No significant difference in the concentration of TGF-alpha and amphiregulin was found between the OVCA and NO or OVC groups. In peritoneal fluid, HB-EGF is sufficiently elevated to activate cancer cells even at an early stage of OVCA. These results suggested that HB-EGF in peritoneal fluid might play a key role in cell survival and in the proliferation of OVCA.