Characterization of Keratinocyte Growth Factor and Receptor Expression in Human Pancreatic Cancer

Keratinocyte growth factor (KGF) is an angiogenic and mitogenic polypeptide that has been implicated in cancer growth and tissue development and repair. Its actions are dependent on its binding to a specific cell-surface KGF receptor (KGFR), which is encoded by the fibroblast growth factor (FGF) receptor type II (FGFR-2) gene. In the present study, we compared the immunohistochemical localization of KGF and KGFR/FGFR-2 in the normal and cancerous pancreas using specific antibodies that recognize KGF and KGFR/FGFR-2 and examined the expression of KGF, KGFR, and FGFR-2 in human pancreatic cancer by in situ hybridization with the corresponding riboprobes. In the normal pancreas, KGF immunoreactivity was present principally in the islet cells, whereas KGFR/FGFR-2 immunoreactivity was present both in the islet and ductal cells. In the pancreatic cancers, moderate KGF and moderate to strong KGFR/FGFR-2 immunoreactivity was present in many of the cancer cells. Furthermore, the ductal and acinar cells adjacent to the cancer cells exhibited moderate to strong KGF and KGFR/FGFR-2 immunoreactivity. By in situ hybridization, KGF, KGFR, and FGFR-2 were overexpressed and co-localized in the cancer cells within the pancreatic tumor mass but were even more abundant in the acinar and ductal cells adjacent to the cancer cells. These findings indicate that KGF, KGFR, and FGFR-2 are overexpressed in both the cancer cells and the adjacent pancreatic parenchyma and raise the possibility that KGF may act in an autocrine and paracrine manner to enhance pancreatic cancer cell growth in vivo.     

Estrogen receptor-associated expression of keratinocyte growth factor and its possible role in the inhibition of apoptosis in human breast cancer

Although estrogen is known to play a crucial role in the pathogenesis of breast cancer, the molecular mechanisms underlying the action of estrogen remain elusive. In the present study, we focused on keratinocyte growth factor (KGF) and its receptor (KGFR) in the pathogenesis of breast cancer, as a growth factor mediating estrogen action, since significant roles of KGF were demonstrated in various steroid hormone-dependent tissues. First, using paraffin-embedded specimens from 42 breast cancer patients, we examined expression patterns of KGF and KGFR by both immunohistochemistry using newly generated antibodies and nonradioactive in situ hybridization with T-T dimerized synthetic oligonucleotide probes. We next compared the results with the expression of estrogen receptor (ER) alpha and beta, proliferative activity and apoptotic frequency (TUNEL staining). Also, the similar approaches were taken to analyze the expression and role of KGF in ER-positive (MCF7, ZR-75-1) and ER-negative (SK-BR-3, MDA-MB-231) human breast cancer cell lines in vitro. In the surgical specimens, KGF was expressed in cancer cells as well as stromal cells in 19/42 cases (45%), while KGFR was found in cancer cells in 24/42 cases (57%). The distribution of protein and mRNA in the analysis of both KGF and KGFR expression generally coincided. Moreover, KGF expression was closely associated with the expression of ER alpha, and the coexpression of KGF and KGFR significantly correlated with lower TUNEL index, but not with proliferative activity. In accordance with the in vivo findings, KGF expression was detected only in ER alpha-positive MCF7 and ZR-75-1 cells in vitro. And more importantly, we found the inhibitory effect of KGF upon the induction of apoptosis by anticancer drugs in MCF7 cells. Collectively, our results indicate that ER alpha may be involved in KGF expression, and that KGF may play antiapoptotic roles, rather than mitogenic, in human breast cancer.     

Enhanced expression of keratinocyte growth factor and its receptor correlates with venous invasion in pancreatic cancer

Keratinocyte growth factor (KGF) and KGF receptor (KGFR) have been implicated in cancer growth as well as tissue development and repair. In this study, we examined whether KGF and KGFR have a role in human pancreatic ductal adenocarcinoma (PDAC). KGFR mRNA was expressed in eight pancreatic cancer cell lines, whereas the KGF mRNA was detected in seven of the cell lines and was absent in MIA PaCa-2 cells. KGFR and KGF immunoreactivity were localized in the cancer cells in 41.5 and 34.0% of patients, respectively. There was a significant correlation between KGFR or KGF immunoreactivity and venous invasion and a significant correlation between the presence of both markers and venous invasion, vascular endothelial growth factor (VEGF)-A expression, and poor prognosis. Exogenous KGF increased VEGF-A expression and release in MIA PaCa-2 cells, and PANC-1 cells stably transfected to overexpress KGF-exhibited increased VEGF-A expression. Moreover, short hairpin-KGFR transfection in MIA PaCa-2 cells reduced the stimulatory effect of exogenous KGF on VEGF-A expression. Short hairpin-KGF transfection in KLM-1 cells reduced VEGF-A expression in the cells. KGFR and KGF may act to promote venous invasion and tumor angiogenesis in PDAC, raising the possibility that they may serve as novel therapeutic targets in anti-angiogenic strategies in PDAC.     

Possible involvement of keratinocyte growth factor and its receptor in enhanced epithelial-cell proliferation and acquired recurrence of middle-ear cholesteatoma

Middle-ear cholesteatoma is characterized by enhanced proliferation of epithelial cells and granular tissue formation. However, the molecular mechanism underlying these pathological changes is largely unknown. Keratinocyte growth factor (KGF) is a mesenchymal cell-derived paracrine growth factor that specifically stimulates epithelial cell proliferation. In the present study, we investigated the possible involvement of KGF and its receptor, KGFR, in the pathogenesis of cholesteatoma using in situ hybridization and immunohistochemistry, respectively. We examined 56 cholesteatoma specimens, and 8 normal skin areas as control. KGF and KGFR expression was examined by immunohistochemistry using rabbit anti-human KGF and anti-human KGFR polyclonal antisera raised in our laboratories against synthetic peptides corresponding to parts of human KGF and KGFR, respectively. KGF protein and mRNA were detected exclusively in stromal fibroblasts and infiltrating T lymphocytes in 80% of cholesteatoma cases, whereas KGFR protein and mRNA were localized in the epithelium in 72% of cases. Assessment of the proliferative activity of cholesteatoma using the labeling index for Ki-67 showed a significantly higher Ki-67 labeling index (66%) in KGF+/KGFR+ cases than other cases. There was a significant correlation between KGF+/KGFR+ expression and recurrence. Our results indicate the possible involvement of both KGF and KGFR in enhanced epithelial cell proliferative activity and recurrence of cholesteatoma.     

Keratinocyte growth factor-induced motility of breast cancer cells

Endogenous growth factors and cytokines are known to have a major influence on the progression, motility and invasiveness of tumor cells. We have reported previously that conditioned media from mouse fibroblasts increases the motility of breast cancer cells. Further, we determined that keratinocyte growth factor (KGF) was an active factor from mouse fibroblasts responsible for most of the motility response in breast cancer cells. The present study examined the effect of human KGF on the motility of estrogen receptor (ER)-positive and ER-negative human breast cancer cell lines in culture using time-lapse videomicroscopy to quantify cell motility. In the present study we observed that recombinant human KGF enhanced several parameters of cellular motility in ER-positive cells but not in ER-negative cell lines. Further, we observed that the level of KGF receptor (KGFR) expression in ER-positive cells was much greater than in the ER-negative cell lines. The motility response to KGF was found to be both dose-and time-dependent. Of the three ER-positive breast cancer cell lines tested, MCF-7 cells were the most responsive to KGF stimulation. Finally, MCF-7 cells grown in estrogen-depleted media did not respond to KGF. These results suggest that KGF from stromal tissue surrounding a primary tumor mass can enhance tumor cell motility and may be an early signal in the progression of breast cancer cells to a more motile and metastatic phenotype. Thus, KGF, KGFR and/or the KGF signaling pathway may be important therapeutic targets for the treatment or prevention of breast cancer metastasis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Expression of keratinocyte growth factor and its receptor in human breast cancer: its inhibitory role in the induction of apoptosis possibly through the overexpression of Bcl-2

Keratinocyte growth factor (KGF), a mesenchymal cell derived paracrine growth factor that regulates normal epithelial cell proliferation, appears to be an essential mediator of steroids in various reproductive organs. The present study was designed to determine the expression and role of KGF and its receptor (KGFR) in human breast carcinoma tissues by immunohistochemistry. We also compared the results with the expression of estrogen receptor alpha(ERalpha), ERbeta, the proliferative activity assessed by the labeling index (LI) for the Ki-67 antigen, apoptotic frequency assessed by terminal dUTP nick end-labeling (TUNEL) index, and the expression of Bcl-2. All of KGF-positive cases were ERalpha- positive (p<0.05), but not that of ERbeta, while all of KGFR-positive cases were ERbeta-positive (p<0.05), but not that of ERalpha. The specimens with the coexpression of KGF and KGFR significantly correlated with a lower TUNEL index (p<0.05), but not with Ki-67 LI in breast cancer tissues. Further analysis at the cellular level revealed that Bcl-2 was colocalized in KGFR-positive cells, and these cells were almost negative for TUNEL staining. Bcl-2-positive cells were also associated with ERbeta, as expected. Therefore, the results indicate that ERalpha may be involved in KGF expression, and that the coexpression of KGF and KGFR may play an inhibitory role in the induction of apoptosis possibly through the up-regulation of Bcl-2 expression in human breast cancer.     

Expression of keratinocyte growth factor/fibroblast growth factor-7 and its receptor in human lung cancer: correlation with tumour proliferative activity and patient prognosis

Keratinocyte growth factor (KGF)/fibroblast growth factor-7 (FGF-7), a mesenchymal cell-derived paracrine growth factor that specifically stimulates epithelial cell proliferation, has been implicated in the repair of lung tissue. The present study was designed to determine the expression and role of KGF and its receptor (KGFR) in human lung cancer tissues, particularly in relation to cancer cell kinetics and prognosis. Thirty-one adenocarcinomas and 30 squamous cell carcinomas, and ten normal lung tissues as a control, were examined. The expression of KGF and KGFR proteins was examined using rabbit polyclonal anti-human KGF and anti-human KGFR antisera raised in the authors' laboratories against synthetic peptides corresponding to parts of human KGF KGFR, respectively. Their specificity was confirmed in lung tumour tissues by western blotting various controls. Proliferative activity was assessed by determining the labelling index (LI) for Ki-67 antigen. Immunohistochemistry revealed that tissue co-expression of KGF KGFR correlated significantly with higher differentiation grades in squamous cell carcinoma. Conversely, in adenocarcinoma, co-expression correlated with lower differentiation grades high Ki-67 LI, was significantly associated with lymph node metastasis shorter 5-year survival. Therefore, the results indicate that co-expression of KGF KGFR correlates significantly with poor prognosis in adenocarcinoma, but not in squamous cell carcinoma, of the lung.     

Keratinocyte growth factor-transfection-stimulated adhesion of colorectal cancer cells to extracellular matrices

The keratinocyte growth factor (KGF) regulates cell growth and behavior in an autocrine or paracrine manner. In colorectal cancer tissues, KGF is expressed in tumor cells and adjacent stromal fibroblasts. We have constructed a KGF-gene-transfected cell line (HCT15-KGF) from a colorectal cancer cell line, HCT-15, that expresses the KGF receptor, and studied the effects of KGF on cell behavior, particularly growth and adhesion to extracellular matrices (ECMs). The amount of KGF secreted from HCT15-KGF was significantly higher than that from a mock-transfected cell line (HCT15-MOCK). The modes of growth of these cell lines were similar. The degree of adhesion of HCT15-KGF to ECMs, including type-IV collagen and fibronectin was higher than that of HCT15-MOCK. The expressions of integrins in both cell lines were not significantly different. However, extracellular-regulated kinase-1 and -2 (ERK1/2) phosphorylation and focal adhesion kinase (FAK) expression that regulate the adhesive functions of integrin families were enhanced in HCT15-KGF. U0126, an inhibitor of the ERK upstream regulator MEK, attenuated the adhesion and spreading of HCT15-KGF cells to type-IV collagen. These results indicate that KGF enhances the adhesion of colorectal cancer cells to type-IV collagen through ERK and FAK signaling pathways.     

The stem cell factor-c-kit system and mast cells in human pancreatic cancer

Stem cell factor (SCF) and its receptor c-kit take part in the regulation of developmental processes of mast cells, hematopoietic stem cells, and melanocytes, as well as in the growth control of human malignancies. To explore the possible role of the SCF-c-kit system and of mast cells in pancreatic cancer, the concomitant expression and distribution of the two molecules were examined in 17 normal and 26 cancerous human pancreatic tissues and in 6 cultured pancreatic cancer cell lines. Mast cell distribution was also evaluated in the same tissue samples. In addition, the effects of SCF and of the c-kit tyrosine-kinase inhibitor STI571 on the growth of the cancer cell lines and of the normal pancreatic ductal cell line TAKA-1 were assessed. SCF immunoreactivity was absent in acinar, ductal, and islet cells of the normal pancreas and faint in pancreatic cancer tissues and cell lines. In contrast, c-kit was clearly present in some normal and hyperplastic ducts of the normal pancreas, in the cancer cells of 73% of the tumor samples, and in all the cell lines tested. Mast cells, identified by tryptase and chymase immunostaining on consecutive tissue sections, showed immunoreactivity for SCF and c-kit in both normal and cancerous specimens and their number was significantly increased (p = 0.03) in pancreatic cancer compared with the normal pancreas. SCF showed a dose-dependent growth inhibitory effect on TAKA-1 cells (p < 0.001), whereas pancreatic cancer cells were resistant to the SCF-induced growth inhibition. Nonetheless, the growth of TAKA-1 cells and pancreatic cancer cells was inhibited by the c-kit tyrosine kinase inhibitor STI571. In conclusion, the SCF-c-kit system, possibly with the contribution of mast cells, may have a growth-regulating role in the normal pancreas, which is altered during malignant transformation.     

PXMP4促进结直肠癌细胞增殖、迁移和侵袭

目的 探讨过氧化物酶体膜蛋白4(peroxisomal membrane protein 4,PXMP4)对结直肠癌细胞增殖、迁移和侵袭能力的影响.方法 采用生物信息学分析65例配对结直肠癌组织中PXMP4 mRNA的表达.应用免疫组化检测112例配对结直肠癌组织中PXMP4的表达,并进行临床病理相关分析;利用RT-P...     

Differential distribution of fibroblast growth factor (FGF)-7 and FGF-10 in L-arginine-induced acute pancreatitis

The regenerative process of the pancreas after acute pancreatitis (AP) is characterized by acinar and ductal cell proliferation with synthesis and transient deposition of extracellular matrices. Various growth factors were reported to be highly expressed in AP, but their regulation has not yet been clarified. Fibroblast growth factor (FGF)-7, also known as keratinocyte growth factor (KGF), and FGF-10 are members of the FGF family and show high structural homology and similar biological characteristics. Both are mainly synthesized by mesenchymal cells and stimulate epithelial cells via KGF receptor (KGFR) which is a splice variant of FGFR-2. In the present study, we attempted to immunohistochemically determine the localization of FGF-7 and FGF-10 in pancreatic tissues of an L-arginine-induced rat pancreatitis model. Furthermore, highly specific KGFR antibodies were prepared and used for Western blot analysis and immunohistochemistry. In the normal pancreas, FGF-7 was localized in alpha cells of islets, but FGF-10 was not detected. KGFR was also localized in islet cells, ductal cells, and centroacinar cells in the normal pancreas. In the pancreatic tissues of rats with L-arginine-induced pancreatitis, FGF-7 was localized in alpha cells, whereas FGF-10 was expressed in vascular smooth muscle cells (VSMCs). KGFR was not expressed in centroacinar cells and its level decreased after L-arginine treatment. However, KGFR was detected instead in some acinar cells and VSMCs in addition to islet cells. These findings suggest that FGF-7 and FGF-10 contribute to the regeneration and differentiation of acinar cells and angiogenesis in AP through KGFR.     

Changes in survivin messenger RNA level during cisplatin treatment in gastric cancer.

Survivin is a member of the inhibitor of apoptosis protein (IAP) family. The expression of survivin has not been reported in differentiated normal tissues, but it has been observed in many cancerous tissues. Recent studies have revealed that survivin may correlate with the chemo-radio resistance of certain malignant cells. In the present study, the correlation between the occurrence of apoptosis and the level of expression of survivin messenger RNA (mRNA) was investigated in a gastric cancer cell line (MKN-45) and in patients with advanced gastric cancer during cisplatin (CDDP) treatment. In the gastric cancer cell line, the percentage of apoptotic cells (apoptotic index: AI) did not change after 48 h incubation with low-dose CDDP (1 microg/ml), whereas the AI explosively increased between 12 and 24 h treatment with high-dose CDDP (10 microg/ml). Relative levels of expression of survivin mRNA and survivin protein increased after low- and high-dose CDDP treatment. Survivin mRNA was not detected in normal gastric mucosas. Also, in 13 patients with advanced gastric cancer who underwent CDDP-based preoperative chemotherapy, survivin mRNA was detected in only 2 cases (15.4%). Survivin mRNA was observed in the resected tumor specimens of two cases. No significant correlation between survivin mRNA expression and the occurrence of apoptosis in resected tumors or between survivin mRNA expression and patient survival was observed. These findings indicate that survivin may play an important role for the chemoresistance of this cancer cell line. However, the clinical importance of survivin expression remains unclear in patients with gastric cancer.     

维甲酸对高氧暴露新生大鼠肺组织角化细胞生长因子及其受体表达的影响

目的: 探讨高氧暴露新生大鼠肺组织结构的变化和角化细胞生长因子(KGF)及其受体(KGFR)的表达情况及维甲酸(RA)对其的影响。方法: 将出生24 h内SD大鼠90只随机分为3组,Ⅰ组:空气+生理盐水(NS);Ⅱ组:高氧+NS;Ⅲ组:高氧+RA。Ⅱ、Ⅲ组持续暴露于85% O₂中,Ⅰ组置于空气中;Ⅲ组每天腹腔注射RA,Ⅰ、Ⅱ组每天腹腔注射NS。分别于生后3、7、14 d取肺标本,用HE染色法观察肺组织结构变化及辐射状肺泡计数(RAC);RT-PCR检测KGF和KGFR mRNA表达强度和免疫组织化学法检测KGF蛋白表达水平。结果: (1)生后第14 d,Ⅱ、Ⅲ组较Ⅰ组RAC值显著减少(P<0.05),但Ⅲ组较Ⅱ组明显增高(P<0.05)。(2)与Ⅰ组相比,Ⅱ组3 d时,KGF mRNA表达明显增强(P<0.05);其后开始下降,7 d仍高于Ⅰ组(P<0.05);14 d时较Ⅰ组有所降低(P<0.05);Ⅲ组各时点表达量均高于同期Ⅱ组(P<0.05)。3 d时,各组KGFR mRNA表达量无明显差异;7 d、14 d时,Ⅱ、Ⅲ组表达量明显低于Ⅰ组(均P<0.05),且Ⅱ组和Ⅲ组之间无显著差异(P>0.05)。(3)KGF阳性细胞主要分布在部分肺泡壁及肺泡周围血管内皮细胞和间质细胞。KGF蛋白表达强度与其mRNA表达变化相似。结论: RA可促进肺组织KGF表达,改善高氧所致肺发育受阻。对未成熟肺高氧损伤有一定的保护作用。     

Overexpression of leucine‐rich repeat‐containing G protein‐coupled receptor 5 in gastric cancer

Gastric cancer is one of the most common malignancies worldwide and patients with advanced gastric cancer still have poor clinical outcomes. The overexpression of leucine‐rich repeat‐containing G protein‐coupled receptor 5 (LGR5) mRNA in colorectal cancer and its correlation with clinicopathological factors were recently reported by us. In this study, we show LGR5 mRNA overexpression in human gastric cancer specimens by quantitative RT‐PCR and in situ hybridization and assess a correlation with clinicopathological factors. The mean expression of LGR5 mRNA in cancerous tissues was five times higher than that in normal tissue (P = 0.0002). Furthermore, LGR5 mRNA expression show marked variation among cases and significantly increased in cases where lymphatic invasion was present compared with those where it was absent (P = 0.0056). Although the mean expression level of LGR5 was observed to be higher in nodal metastasis and venous invasion positive cases compared to negative cases, a significant difference was not observed. These results suggest that LGR5 can be a biomarker for malignancy in gastric cancer.