膀胱癌中bFGF和血管形成的研究

目的：探讨碱性成纤维细胞生长因子（ｂＦＧＦ）和血管形成与膀胱癌的发病及生物学行为之间关系。方法：采用免疫组化方法检测７２例膀胱癌组织和２１例正常膀胱组织中ｂＦＧＦ和第ＶＩＩＩ因子相关抗原表达。结果：膀胱癌组织中ｂＦＧＦ表达和微血管密度（ＭＶＤ）均显高于正常膀胱胱组织，而且两与肿瘤病理分级分期均密切相关。肿瘤复发的ｂＦＧＦ表达和ＭＶＤ均显高于复发，ｂＦＧＦ强阳性表达的ＭＶＤ值显高于阴性及弱阳性表达，结论：ｂＦＧＦ通过促进血管形成而在膀胱癌的发生发展过程中起着重要的作用，而且ｂＦＧＦ和ＭＶＤ可作为膀胱癌生物行为和预后的评估指标。     

Increased levels of urinary basic fibroblast growth factor in bladder cancer patients

Urinary basic fibroblast growth factor (bFGF) was determined by a competitive sandwich ELISA test in 26 patients with transitional bladder cancer and 26 normal volunteers or subjects with benign urological diseases. The median bFGF value in the patient group was 3.41 ng bFGF/g creatinine (range: 0-153.6), significantly higher than the median levels in the control group (1.39 ng/g creatinine; range: 0-4.5). On the basis of the optimal cut-off point of 2.64 ng bFGF/g creatinine, the sensitivity of the test for detecting bladder cancer was 61.5% and the specificity 76.9%. We also studied 17 individuals successfully treated for a previous bladder cancer and with no evidence of disease at the moment of urine collection (NED group). These subjects showed similar urinary bFGF levels as those observed in the control group (median 0.24 ng bFGF/g creatinine, range 0-5.1). Our data suggest that the dosage of urinary bFGF could be a non-invasive useful assay in the management of bladder cancer patients.     

Detection of basic fibroblast growth factor mRNA in urinary bladder cancer: correlation with local relapses.

Natural history of bladder cancer is characterized by high risk of disease progression even for patients with a clinical diagnosis of superficial disease; in these tumors, the occurrence of local relapse is known to be dependent on the angiogenesis rate. Basic fibroblast growth factor (bFGF), has been described to be elevated in urine and serum of patients with bladder cancer. We investigated the expression of bFGF at mRNA level in a panel of 32 transitional cell tumors of the urinary bladder and in normal bladder tissues used as controls. Expression of bFGF was found elevated in most tumors of high stage, where its presence was found correlated with the occurrence of early local relapses. Furthermore, bFGF was found highly expressed in the majority of tumors showing a high bcl-2 expression rate. Our data suggest that bFGF expression could contribute to the progression of disease; it may provide a prognostic indicator in the identification of patients with high risk for occurrence of local relapses.     

Quantitative analysis of basic fibroblast growth factor and vascular endothelial growth factor in human colorectal cancer.

Tumour growth is angiogenesis dependent. Some authors suggest a prognostic role of microvessel count in colorectal cancer. We tested the role of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in the switch to the angiogenic phenotype in 35 patients with colorectal cancer at different stages of disease. We evaluated the two angiogenic factors, by enzyme-linked immunosorbent assay (ELISA), in tumour, peritumoral mucosa, pathological mesenteric and peripheral blood. We used ten endoscopic intestinal biopsies and ten peripheral blood samples from healthy subjects as control. bFGF was significantly lower in tumour tissues and in peritumoral mucosas than in healthy mucosas, whereas VEGF was up-regulated in tumours but not in peritumoral mucosa. Both angiogenic factors were greatly increased in mesenteric blood. VEGF tumour and serum levels were significantly correlated with the stage of disease. bFGF tumour and serum concentration were not correlated with the stage of disease. The high levels of bFGF in mesenteric blood suggest that this growth factor might be abnormally released from tumour tissue and peritumoral mucosa and could function as an early effector in the switch to the angiogenic phenotype. In contrast, VEGF, whose levels show a significant correlation with the stage of disease, could act in a following step, supporting tumour progression.     

Nipple fluid basic fibroblast growth factor in patients with breast cancer.

PURPOSE: It has been shown that early detection of breast cancer could save lives. Recently, there has been increasing interest in nipple fluid as a potential supplemental avenue for breast cancer diagnosis. EXPERIMENTAL DESIGN: In this study, we determined the levels of an angiogenic factor basic fibroblast growth factor (bFGF) in the nipple fluid of healthy subjects as well as patients with benign breast conditions, those at high risk for breast cancer, and patients with active breast cancer. ELISAs were used to measure bFGF. RESULTS: Nipple fluid bFGF levels were as follows (mean +/- SE): 158 +/- 17 pg/mL from benign breasts, 561 +/- 277 pg/mL from high-risk breasts, and 1,343 +/- 441 pg/mL from cancerous breasts. One-way ANOVA showed that the bFGF levels from cancerous breasts were significantly higher than those from benign and high-risk breasts (P = 0.0001 and P = 0.0193, respectively). After logarithmic transformation was applied to the data, high-risk breast bFGF levels were higher than those from benign breasts (P = 0.0028). With a cutoff level of 250 pg/mL, the sensitivity was 79.2%, specificity was 82.5%, and correct diagnosis was 66.4%. The area under the receiver operating characteristic curve was 0.86. CONCLUSIONS: We conclude that nipple fluid bFGF levels are progressively elevated in high-risk and cancerous breasts compared with benign breasts. The sensitivity and specificity of this test are promising compared with current breast cancer screening methods, and this test deserves further studies with larger clinical trials. Potential areas of usefulness include the detection of breast cancer risk or breast cancer, as well as the monitoring and/or prediction of the antiangiogenic effect of preventive therapies.     

脑膜瘤组织中bFGF及FGFR-1蛋白表达的研究

目的　探讨碱性成纤维生长因子 (basicfibroblastgrowthfactor ,bFGF)及成纤维生长因子受体 1(fibroblastgrowthfactorreceptor 1,FGFR 1)在脑膜瘤中的表达及其与脑膜瘤组织病理学和复发的关系。方法　用免疫组化技术检测bFGF、FGFR 1在不同类型的脑膜瘤组织中的蛋白表达 ,用组织病理学判断脑膜瘤的良恶性。结果　脑膜瘤细胞有不同程度的bFGF及FGFR 1表达 ,其表达阳性率与肿瘤的良恶性和复发有关。结论　bFGF和FGFR具有促进脑膜瘤细胞的增殖和生长的作用。脑膜瘤表达bFGF、FGFR的阳性率可作为鉴别肿瘤良恶性的有用指标 ,并对脑膜瘤的预后和术后复发起提示作用。     

Adenoviral-mediated gene therapy of human bladder cancer with antisense interleukin-8

We previously demonstrated the importance of interleukin-8 (IL-8) as a mediator of angiogenesis, tumorigenicity, and metastasis of transitional cell carcinoma (TCC) of the bladder. In the present study, we evaluated the feasibility of adenoviral mediated antisense IL-8 gene transfer (Ad IL-8-AS) as therapy for established TCC. In vitro, Ad IL-8-AS inhibited endothelial cell proliferation and enhanced endothelial cell apoptosis. The highly metastatic human TCC cell line 253J B-V(R) was implanted into the subcutis of athymic nude mice, and intralesional therapy with Ad IL-8-AS commenced when the tumors reached a diameter between 5 and 7 mm. Tumor growth was significantly inhibited compared with therapy in controls (saline and beta-galactosidase adenovirus). Ad IL-8-AS therapy decreased the in vivo expression of IL-8 and matrix metalloproteinase type 9 (MMP-9), reduced microvessel density, and enhanced endothelial cell apoptosis. These results indicate that Ad IL-8-AS therapy targets both tumor cells and host endothelial cells resulting in endothelial cell apoptosis and significant inhibition of tumor growth.     

MicroRNA-195 inhibits human gastric cancer by directly targeting basic fibroblast growth factor

Purpose

Gastric cancer (GC) is one of the fatal malignancies worldwide with high occurrences but poor outcomes. bFGF has been shown to play significant roles in GC. Yet, whether bFGF affects the development of GC is less studied.

Methods

MicroRNA assays, real-time PCR, and western blot were conducted for expression analysis of miR-195-5p and basic fibroblast growth factor (bFGF). Luciferase activity was measured with mutated bFGF 3′-UTR sequence at the 3′ end of the luciferase gene. Two GC cell lines, SNU-1 and KATO-3 overexpressing miR-195-5p and bFGF were subjected to wound healing assay and transwell invasion assay. Mouse GC xenograft model was established and subjected to tumor size analysis.

Results

Expression levels of miR-195-5p and bFGF showed negative correlation in human GC tissues. MiR-195-5p directly targeted bFGF 3′-UTR as demonstrated by luciferase activity assay. MiR-195-5p, through downregulating bFGF, inhibited the migration and invasion of SNU-1 and KATO-3 cells, as well as tumorigenesis in a xenograft mouse model, which could be restored by re-introduction of bFGF.

Conclusions

MiR-195-5p inhibits tumorigenesis of GC through suppressing bFGF, which supports both miR-195-5p and bFGF as potential therapeutic targets in the treatment of GC.

     

肿瘤治疗新靶点:碱性成纤维细胞生长因子及其受体信号

碱性成纤维细胞生长因子（bFGF）是一个促细胞分裂、增殖及血管生成的多肽生长因子,肿瘤组织中bFGF及其受体过度表达为抗肿瘤治疗提供潜在靶目标,研制靶向性抗bFGF抗体、FGFR抑制剂等抑制肿瘤细胞增殖和新血管形成,阻断肿瘤生长的营养和转移途径,成为抗肿瘤热点。本文就bFGF及其受体信号在肿瘤的发生、发展和转移中的作用及临床转化研究进行综述。      

Role of transforming growth factor beta in the growth inhibition of human breast cancer cells by basic fibroblast growth factor

Recent studies from our laboratory have revealed that basic fibroblast growth factor (bFGF) selectively inhibits the proliferation of human MCF-7 breast cancer cells. It has also been shown to enhance cis-platinum-induced apoptosis, decrease levels of the anti-apoptotic gene product bcl-2, and increase levels of the cyclin-dependent protein kinase inhibitor p21/WAF1/Cip1. Transforming growth factor beta-1 (TGF₁), a cell growth regulator has been found to have an inhibitory effect on breast cancer cells. The aim of the present study was to evaluate the possible role of TGF₁ in the antiproliferative effects of bFGF in MCF-7 breast cancer cells. We found that exogenous, as well as endogenous (overexpressed) bFGF increased TGF₁ mRNA expression in the cells and enhanced the secretion of TGF₁ into culture medium. However, exogenous addition of TGF₁ neither led to a decrease in bcl-2 nor induced an increase in the levels of p21/WAF1/Cip1 and neutralizing antibodies to TGF₁, did not reverse bFGF-induced G₁ arrest nor the increase in p21/WAF1/Cip1 level. In contrast, antisense oligonucleotides to TGF₁ abrogated the antiproliferative effects and inhibited the induction of p21/WAF1/Cip1 by bFGF in MCF-7 cells. These data suggest that the anti-proliferative effects of bFGF in human MCF-7 breast cancer cells are mediated by endogenous TGF₁, while exogenous TGF₁ does not mimic all the effects of bFGF on these breast cancer cells. These findings provide an important basis for further investigations into the autocrine and paracrine processes that control the growth of breast cancer cells.     

Nuclear basic fibroblast growth factor regulates triple-negative breast cancer chemo-resistance

Introduction

Chemotherapy remains the only available treatment for triple-negative (TN) breast cancer, and most patients exhibit an incomplete pathologic response. Half of patients exhibiting an incomplete pathologic response die within five years of treatment due to chemo-resistant, recurrent tumor growth. Defining molecules responsible for TN breast cancer chemo-resistance is crucial for developing effective combination therapies blocking tumor recurrence. Historically, chemo-resistance studies have relied on long-term chemotherapy selection models that drive genetic mutations conferring cell survival. Other models suggest that tumors are heterogeneous, being composed of both chemo-sensitive and chemo-resistant tumor cell populations. We previously described a short-term chemotherapy treatment model that enriches for chemo-residual TN tumor cells. In the current work, we use this enrichment strategy to identify a novel determinant of TN breast cancer chemotherapy resistance [a nuclear isoform of basic fibroblast growth factor (bFGF)].

Methods

Studies are conducted using our in vitro model of chemotherapy resistance. Short-term chemotherapy treatment enriches for a chemo-residual TN subpopulation that over time resumes proliferation. By western blotting and real-time polymerase chain reaction, we show that this chemotherapy-enriched tumor cell subpopulation expresses nuclear bFGF. The importance of bFGF for survival of these chemo-residual cells is interrogated using short hairpin knockdown strategies. DNA repair capability is assessed by comet assay. Immunohistochemistry (IHC) is used to determine nuclear bFGF expression in TN breast cancer cases pre- and post- neoadjuvant chemotherapy.

Results

TN tumor cells surviving short-term chemotherapy treatment express increased nuclear bFGF. bFGF knockdown reduces the number of chemo-residual TN tumor cells. Adding back a nuclear bFGF construct to bFGF knockdown cells restores their chemo-resistance. Nuclear bFGF-mediated chemo-resistance is associated with increased DNA-dependent protein kinase (DNA-PK) expression and accelerated DNA repair. In fifty-six percent of matched TN breast cancer cases, percent nuclear bFGF-positive tumor cells either increases or remains the same post- neoadjuvant chemotherapy treatment (compared to pre-treatment). These data indicate that in a subset of TN breast cancers, chemotherapy enriches for nuclear bFGF-expressing tumor cells.

Conclusion

These studies identify nuclear bFGF as a protein in a subset of TN breast cancers that likely contributes to drug resistance following standard chemotherapy treatment.     

Basic fibroblast growth factor in human prostate cancer cells.

To increase our understanding of the potential role of basic fibroblast growth factor (bFGF) in malignant progression of prostate cancer, we determined the production of bFGF, the expression of FGF receptor (flg), and the response to exogenous bFGF in LNCaP, DU 145, and PC 3 cells. We observed that these three prostate cancer cell lines, which differed in their dependence on androgens for growth in vitro and in their in vivo behavior in nude mice, could be distinguished as follows: (a) androgen-sensitive LNCaP cells, which do not metastasize in nude mice, did not produce measurable amounts of bFGF, expressed small but measurable amounts of FGF receptor mRNA, and did respond to exogeneous bFGF; (b) androgen-insensitive, moderately metastatic DU 145 cells did produce measurable amounts of biologically active bFGF, expressed large amounts of FGF receptor mRNA, and responded to exogeneous bFGF and the heparin-binding fractions from DU 145 cell extracts; (c) androgen-insensitive and highly metastatic PC3 cells also produced measurable amounts of bFGF but did not demonstrate a growth response to either the heparin-binding fractions from PC3 cell extracts or exogenous bFGF, even though large amounts of FGF receptor mRNA were expressed in PC 3 cells. These results suggest the possibility that differences in production of, and response to, bFGF may be associated with different biological behavior.     

Nuclear accumulation of basic fibroblast growth factor in human astrocytic tumors

BACKGROUND: The authors recently reported that nuclear accumulation of basic fibroblast growth factor (bFGF) demonstrated a significant correlation with recurrence of pituitary adenomas. The current study sought to determine whether nuclear bFGF accumulation was a predictor of survival in patients with astrocytic tumors. METHODS: The authors examined 52 patients with primary astrocytic tumors. Immunohistochemical assays for bFGF, fibroblast growth factor receptor 1 (FGFR1), and proliferating cell nuclear antigen were performed. Immunoreactivity of bFGF in nuclei was recorded in terms of the bFGF nuclear index (NI), which was calculated as the percentage of tumor cells with nuclear immunoreactivity. Western blot analysis of bFGF in nuclear fractions was performed. RESULTS: The bFGF NI had a mean value of 35.1% and was < 30% (low NI) in 27 patients and >or= 30% (high NI) in 25 patients. In all cases, FGFR1 immunoreactivity was observed in the cytoplasm but not in the nucleus. Western blot analysis indicated that the nuclear fractions from tumor specimens with high NI contained high-molecular-weight bFGF. Univariate analyses showed that age, tumor histology, gender, and bFGF NI were significantly correlated with patient survival. Multivariate analyses demonstrated that NI had the greatest influence (P = 0.0073) on survival rate, compared with age (P = 0.0083) and gender (P = 0.0492). Compared with low NI, high NI was associated with a relative risk of 3.292. CONCLUSIONS: The findings of the current study suggest that bFGF NI may be a useful predictor of survival in patients with astrocytic tumors.     

The future of bladder cancer therapy: Optimizing the inhibition of the fibroblast growth factor receptor

Therapeutic options for metastatic bladder cancer (BC) have seen minimal evolution over the past 30 years, with platinum-based chemotherapy remaining the mainstay of standard of care for metastatic BC. Recently, five immune checkpoint inhibitors (ICIs) have been approved by the FDA as second-line therapy, and two ICIs are approved as first-line treatment in selected patients. Molecular alterations of muscle-invasive bladder cancer (MIBC) have been reported by The Cancer Genome Atlas. About 15% of patients with MIBC have molecular alterations in the fibroblast growth factor (FGF) axis. Several ongoing trials are testing novel FGF receptor (FGFR) inhibitors in patients with FGFR genomic aberrations. Recently, erdafitinib, a pan-FGFR inhibitor, was approved by the FDA in patients with metastatic BC who have progressed on platinum-based chemotherapy. We reviewed the literature over the last decade and provide a summary of current knowledge of FGF signaling, and the prognosis associated with FGFR mutations in BC. We cover the role of FGFR inhibition with non-selective and selective tyrosine kinase inhibitors as well as novel agents in metastatic BC. Efficacy and safety data including insights from mechanism-based toxicity are reported for selected populations of metastatic BC with FGFR aberrations. Current strategies to managing resistance to anti-FGFR agents is addressed, and the importance of developing reliable biomarkers as the therapeutic landscape moves towards an individualized therapeutic approach.     

Immunolocalization of basic fibroblast growth factor to the microvasculature of human brain tumors.

BACKGROUND. Microvascular proliferation, a prominent feature of tumors of the central nervous system, is a prime target for anti-cancer therapy. METHODS. Because basic fibroblast growth factor (bFGF) plays a key role in the regulation of angiogenesis, surgical specimens from 52 human brain tumors were examined by immunocytochemical studies with a murine monoclonal antibody to bFGF. Sections from these tumors also were incubated with Ki-67 monoclonal antibody to measure the growth fraction. RESULTS. Immunostaining for bFGF was observed in 45 of 52 (87%) neoplasms, reacting with 97% of the malignant brain tumors and 67% of benign tumors (P < 0.01). The nonreactive tumors were a medulloblastoma and 7 of 21 (33%) benign, noninvasive, slow-growing neoplasms (1 acoustic schwannoma, 3 meningiomas, 2 pituitary adenomas, and 1 cholesteatoma). The indices of proliferation (Ki-67 labeling) were lower for the 21 benign tumors (1.2 +/- 1.1%) than the 31 malignant tumors (10.3 +/- 10.5%; P < 0.001). The bFGF was immunolocalized in the tumor cell nuclei in 23 of 52 tumors (44%) and in the cytoplasm of 8 of 52 (15%) tumors. Immunostaining to bFGF was prominent in the microvascular endothelial compartment in 84% of the malignant tumors and only 52% of benign tumors (P < 0.01). Immunostaining was not present after preabsorption of the antibody with pure human recombinant bFGF. CONCLUSIONS. The presence of bFGF predominantly within the tumor microvasculature indicates a cellular depot for this potent growth factor that mediates angiogenesis and tumorigenesis. These data support a role for bFGF in the transition from the benign to the malignant phenotype.     

Suppression of solid tumor growth by immunoneutralizing monoclonal antibody against human basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is a potent angiogenic mitogen. To elucidate the effect of bFGF inhibitors in vivo, anti-bFGF immunoneutralizing monoclonal antibody was prepared. One monoclonal antibody against human bFGF, obtained by cell fusion and designated 3H3, completely inhibited bFGF-induced proliferation of human umbilical vein endothelial cells at a concentration of 100 ng/ml. 3H3 did not bind to acidic fibroblast growth factor or HST1 protein, indicating high specificity for bFGF. Furthermore, the immunoneutralizing activity of 3H3 was examined in vivo. K1000 cells (a BALB/c 3T3 transformant in which the leader sequence-fused bFGF gene was transfected) were transplanted s.c. into BALB/c nude mice. Growth of the tumor cells was inhibited by i.v. treatment with 3H3 at a concentration of 200 micrograms/mouse. Histological observation showed that the antitumor effect of 3H3 was due to the inhibition of bFGF-induced angiogenesis. This experiment provides direct causal evidence for the hypothesis that tumor growth is angiogenesis dependent. This finding could also have implications for the development of novel therapeutic approaches to angiogenic solid tumors.     

乙酰肝素酶反义寡核苷酸对人胃癌细胞株SGC 7901表达bFGF的抑制作用

目的探讨乙酰肝素酶反义寡核苷酸对人胃癌细胞株SGC 7901细胞中表达碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)的影响.方法用脂质体介导乙酰肝素酶反义寡核苷酸转染人胃癌细胞株SGC 7901细胞,采用免疫细胞化学观察细胞内bFGF的变化.结果脂质体介导转染乙酰肝素酶反义寡核苷酸前后,SGC 7901胃癌细胞内乙酰肝素酶mRNA积分光密度值(integra optical densit,IOD)分别为256.32±3.21、146.89±2.35、85.36±3.58和63.78±1.42.bFGF阳性表达率转染前72.23%,转染0.2、0.4和0.8μmol/L的AS-ODN后分别为57.15%、51.11%和42.36%.与转染前比较,转染后细胞bFGF表达明显减少,P值分别为0.007、0.003和0.001.结论转染乙酰肝素酶反义寡核苷酸可以有效抑制胃癌细胞bFGF的表达.     

Expression of basic fibroblast growth factor and its receptor in human pancreatic carcinomas.

We examined the expression of basic fibroblast growth factor (FGF) and FGF receptor by immunohistochemistry in 32 human pancreatic ductal adenocarcinomas. Mild to marked basic FGF immunoreactivity was noted in 19 (59.4%) of the 32 tumours examined, and 30 (93.3%) of the tumours exhibited a cytoplasmic staining pattern against FGF receptor. The tumours were divided into two groups according to the proportion of positively stained tumour cells: a low expression group (positive cells < 25%) and a high expression group (positive cells > or = 25%). No statistically significant difference in tumour size, differentiation, metastases or stage was found between the low and high basic FGF expression groups. However, a significant correlation was found between FGF receptor expression level and the presence of retroperitoneal invasion, lymph node metastasis, and tumour stage. In addition, low FGF receptor expression was significantly associated with a longer post-operative survival as compared with high FGF receptor expression, whereas there was no significant difference in post-operative survival between the low and high basic FGF expression groups. Increased expression of FGF receptor is correlated with the extent of malignancy and post-operative survival in human pancreatic ductal adenocarcinomas. Thus, overexpression of FGF receptor may prove to be a more useful prognostic marker than basic FGF expression level in pancreatic cancer patients.