Amphiregulin as a Novel Target for Breast Cancer Therapy

Amphiregulin, an EGF family growth factor, binds and activates the epidermal growth factor receptor (EGFR or ErbB1). Activation of the EGFR by amphiregulin can occur through autocrine, paracrine and juxtacrine mechanisms. Amphiregulin plays a role in several biological processes including nerve regeneration, blastocyst implantation, and bone formation. Amphiregulin also plays an important role in mammary duct formation as well as the outgrowth and branching of several other human tissues such as the lung, kidney and prostate. This effect is most likely due to the induction of genes involved in invasion and migration such as cytokines and matrix metalloproteases. Clinical studies have suggested that amphiregulin also plays a role in human breast cancer progression and its expression has been associated with aggressive disease. Therefore, amphiregulin may be a novel and effective target for the treatment of breast cancer and could represent an alternative to targeting the EGFR.     

Lysophosphatidic acid-induced effects in human colon carcinoma DLD1 cells are partially dependent on transactivation of epidermal growth factor receptor

BACKGROUND: Lysophosphatidic acid (LPA) is a lipid mediator of diverse effects on various cells. LPA is well known to induce phosphorylation of the epidermal growth factor receptor (EGFR), which is termed transactivation, in some cell types. In this study, we investigated the contribution of EGFR transactivation in LPA-induced responses in colon cancer DLD1 cells. MATERIALS AND METHODS: Immunoprecipitation was performed to investigate whether LPA induced EGFR phosphorylation. Then, we investigated LPA-induced migration and IL-8 secretion in DLD1 cells. Migration was measured in a modified Boyden chamber and IL-8 secretion was measured by ELISA. In these experiments we used an EGFR inhibitor, AG1478 or matrix metalloproteinase (MMP) inhibitor, GM6001. RESULTS: Immunoprecipitation analysis revealed that LPA induced a significant level of tyrosine phosphorylation of EGFR in DLD1 cells. The LPA-induced phosphorylation of EGFR was almost completely abrogated by either AG1478 or GM6001. LPA induced significant migration and IL-8 secretion in DLD1, both of which were significantly inhibited by AG1478 or GM6001. However, the inhibitory effects were only partial (migration; 29% +/- 2%, 32 +/- 13% inhibition, IL-8 secretion; 33% +/- 1%, 26% +/- 5% inhibition, respectively). CONCLUSION: These results clearly indicate that LPA acts upstream of EGFR and compensates the EGF signal and antagonism of the EGF signal cannot completely block tumor progression in colon cancer cells. Blockade of the LPA signal may have clinical significance in the treatment of colon cancer.     

Heparin-binding epidermal growth factor-like growth factor functionally antagonizes interstitial cystitis antiproliferative factor via mitogen-activated protein kinase pathway activation

OBJECTIVE

To delineate the mechanism underlying the potential functional relationship between interstitial cystitis antiproliferative factor (APF) and heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF), as APF has previously been shown to decrease the proliferation rate of normal bladder epithelial cells and the amount of HB‐EGF produced by these cells.

MATERIALS AND METHODS

APF‐responsive T24 transitional carcinoma bladder cells were treated with high‐pressure liquid chromatography‐purified native APF with or without HB‐EGF to determine the involvement of signalling pathways and proliferation by Western blot analysis, p38 mitogen‐activated protein kinase (MAPK) and extracellular signal‐regulated kinase (Erk)/MAPK assays, and 3‐(4,5‐dimethylthiazolyl‐2)‐2,5‐diphenyltetrazolium bromide (MTT) assay.

RESULTS

Cyclic stretch induced the secretion of HB‐EGF from T24 cells overexpressing the HB‐EGF precursor, resulting in enhanced proliferation. T24 cells treated with APF had increased p38MAPK activity and suppressed cell growth, events that were both reversed by treatment with a p38MAPK‐selective inhibitor. Activation of Erk/MAPK by HB‐EGF was inhibited by APF, and APF did not stimulate p38MAPK in the presence of soluble HB‐EGF or when cells overexpressed constitutively secreted HB‐EGF. Lastly, APF inhibitory effects on cell growth were attenuated by HB‐EGF.

CONCLUSIONS

These results indicate that HB‐EGF and APF are functionally antagonistic and signal through parallel MAPK signalling pathways in bladder cells.     

Coacervate delivery of HB‐EGF accelerates healing of type 2 diabetic wounds

Chronic wounds such as diabetic ulcers pose a significant challenge as a number of underlying deficiencies prevent natural healing. In pursuit of a regenerative wound therapy, we developed a heparin‐based coacervate delivery system that provides controlled release of heparin‐binding epidermal growth factor (EGF)‐like growth factor (HB‐EGF) within the wound bed. In this study, we used a polygenic type 2 diabetic mouse model to evaluate the capacity of HB‐EGF coacervate to overcome the deficiencies of diabetic wound healing. In full‐thickness excisional wounds on NONcNZO10 diabetic mice, HB‐EGF coacervate enhanced the proliferation and migration of epidermal keratinocytes, leading to accelerated epithelialization. Furthermore, increased collagen deposition within the wound bed led to faster wound contraction and greater wound vascularization. Additionally, in vitro assays demonstrated that HB‐EGF released from the coacervate successfully increased migration of diabetic human keratinocytes. The multifunctional role of HB‐EGF in the healing process and its enhanced efficacy when delivered by the coacervate make it a promising therapy for diabetic wounds.     

EGF-related growth factors in the pathogenesis of murine ARPKD

BACKGROUND: Epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha) and their receptor, EGFR, play key roles in polycystic kidney disease (PKD) pathogenesis. Renal expression of two related growth factors, amphiregulin and heparin-binding EGF, has not been examined previously in PKD. The aims of this study of murine autosomal-recessive polycystic kidney disease (ARPKD) were (1) to characterize amphiregulin and heparin-binding EGF expression in cystic versus normal kidneys and cells; and (2) to identify the functional effects of abnormal EGF-related growth factor expression. METHODS: Amphiregulin and heparin-binding-EGF expression were examined by immunohistology and Western blot of kidneys and conditionally-immortalized collecting tubule cells obtained from cystic bpk mice (a murine model of ARPKD) and normal littermates. EGF, TGF-alpha, amphiregulin, and heparin-binding EGF in vitro effects on cystic and control collecting tubule cells were assessed by cell proliferation, cyst fluid mitogenicity, and EGFR activation. RESULTS: By immunohistology, amphiregulin and heparin-binding EGF localized to apical and basolateral surfaces of proximal tubule cysts > normal proximal tubules. In cystic collecting tubules, heparin-binding EGF (but not amphiregulin) localized to both apical and basolateral surfaces; whereas in normal collecting tubules, amphiregulin and heparin-binding EGF localized to the basolateral surface only. Increased amphiregulin and heparin-binding EGF expression by Western blot was seen in cystic vs. normal kidneys and increased heparin-binding EGF (but not amphiregulin) expression was present in cystic collecting tubule cell lines vs. controls. EGF, TGF-alpha, amphiregulin, and heparin-binding EGF were all mitogenic to cystic > control collecting tubule cells. Immunoprecipitation of EGF and TGF-alpha reduced cyst fluid mitogenicity by almost 80%, whereas heparin-binding EGF and amphiregulin immunoprecipitations had minimal effects. Differential receptor activation was also seen: Heparin-binding EGF markedly activated EGFR (>EGF = TGF-alpha > amphiregulin), with a greater effect seen in cystic vs. control collecting tubule cells. CONCLUSION: Multiple EGF-related growth factors are abnormally expressed in murine ARPKD and may have differential roles in disease pathogenesis. In particular, newly identified abnormalities in heparin-binding EGF expression in cystic kidneys and cells may have important implications for disease pathogenesis.     

转基因成纤维细胞向创面持续投递新型分泌型表皮细胞生长因子

目的 探索通过转基因成纤维细胞向创面持续投递表皮细胞生长因子（ＥＧＦ）促进创面愈合的新方法。方法 构建新型分泌型人ＥＧＦｃＤＮＡ并转染人成纤维细胞株ＫＭＳＴ６。将经照射的转基因细胞移植于裸鼠全厚创面。结果 稳定转染的细胞克隆可分泌有活性的ＥＧＦ。移植后可在伤口组织中检测到人ＥＧＦ,其含量缓慢下降,但至少可持续７天。结论 一次性移植少量转基因细胞可在创面愈合早期这一关键阶段持续向创面释放ＥＧＦ。     

Upregulation of heparin‐binding epidermal growth factor‐like growth factor and osteopontin in experimental hydronephrosis

SUMMARY This study examined the expression of heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) and osteopontin in unilateral ureteral obstruction (UUO) in the rat, a model of obstructive uropathy. HB‐EGF mRNA was upregulated 5.5‐fold at 4 h post‐obstruction (P < 0.05) and 4.5‐fold after 12 h (P < 0.05). Immunohistochemical staining for HB‐EGF demonstrated an increase in protein in the distended tubules. To determine what effects increased HB‐EGF might have in the obstructed kidney, we attempted to determine whether HB‐EGF upregulates osteopontin and α‐smooth muscle actin (α‐SMA) in the tubular line NRK‐52E. Both of these molecules are increased in UUO. Osteopontin mRNA was upregulated in NRK‐52E cells after 24, 48 and 72 h HB‐EGF stimulation. In contrast, HB‐EGF caused a downregulation of α‐SMA protein by Western blot in NRK‐52E cells. When a blocking mAb against secreted HB‐EGF was administered, however, there was no effect on osteopontin mRNA levels or immunohistochemical staining for α‐smooth muscle actin. These data suggest that the action of HB‐EGF in UUO may be to increase osteopontin and reduce α‐smooth muscle actin expression by tubular epithelial cells by an autocrine or intracrine mechanism. By reducing α‐SMA expression, HB‐EGF may also act to maintain epithelial cell morphology in this model.     

Potential molecular mechanisms of negative pressure in promoting wound healing

Negative pressure wound therapy (NPWT) has been widely used in various lesions. This study aimed to explore the biological effects of negative pressure on the polymorphonuclear neutrophils (PMNs), macrophages, and epidermal keratinocyte cells involved in wound healing. PMNs differentiated from HL‐60, macrophages were derived from THP‐1 monocytes, and keratinocytes were cultured in vitro, and they were treated with 0, ?0.03 mp, and ?0.05 mp, respectively. Cell ultrastructure; viability; apoptosis; and protein factors such as tumour necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ), epidermal growth factor (EGF), epidermal growth factor receptor (EGFR), interleukin‐17 (IL‐17), and cell division cycle 42 (Cdc42) were determined by transmission electron microscopy (TEM), CCK8, flow cytometry (FCM), ELISA, and simple Western assays, respectively. After negative pressure stimulation, the cell ultrastructure of PMNs and macrophages cells was presented with a marked increase of lysosomes and a relative decrease of mitochondria. In addition, the cell viability was enhanced in PMNs and macrophages in a pressure‐dependent manner and apoptosis ratios were significantly reduced in PMNs and macrophages. In addition, under ?0.05 negative pressure, IFN‐γ and IL‐17 were significantly increased in PMNs or macrophages. Moreover, increased EGF and EGFR and Cdc42 levels in keratinocytes induced by the ?0.05 mpa were detected, indicating that the migration chemotaxis of keratinocyte cells was enhanced. Negative pressure might promote cell proliferation, accelerate inflammatory responses, and promote epithelialisation during wound healing by increasing IFN‐γ, IL‐17, Cdc42, EGF, and EGFR in PMNs, macrophages, or keratinocytes under different negative pressures.     

High Cortical Bone Mass Phenotype in Betacellulin Transgenic Mice Is EGFR Dependent

Signaling through the epidermal growth factor receptor (EGFR) by ligands such as epidermal growth factor (EGF), transforming growth factor α (TGFA), and amphiregulin (AREG) has been reported to have effects on skeletal growth. The role of betacellulin (BTC), another EGFR ligand, in skeletal development and bone metabolism is unknown. In previous experiments, transgenic mice overexpressing BTC ubiquitously under the control of the chicken β‐actin promoter (BTC‐tg) exhibited stunted growth and disproportionately sized long bones. In this study, we performed a detailed phenotypic analysis of BTC‐tg mice at 3, 6, and 9 wk of age. Osteoblastic cells from transgenic mice showed strong expression of BTC as determined by Western blots and by immunohistochemistry on bone sections. In femurs of male and female BTC‐tg mice, we found reduced longitudinal bone growth and a pronounced increase in total volumetric BMD. The increased femoral BMD was mainly caused by augmented endocortical bone apposition and subsequent cortical bone thickening. In contrast, vertebral BMD was reduced in BTC‐tg mice of both sexes. An overall similar phenotype was found in 6‐mo‐old BTC‐tg mice. The increase in cortical bone mass in the appendicular skeleton of BTC‐tg mice was largely blocked when they were crossed into the Egfr^Wa5 background characterized by a dominant negative EGFR. Our study showed that overexpression of BTC results in an EGFR‐dependent upregulation of cortical bone mass in the appendicular skeleton of mice, uncovering a potential novel anabolic pathway for cortical bone.     

Possible autocrine loop of the epidermal growth factor system in patients with benign prostatic hyperplasia treated with finasteride: a placebo-controlled randomized study

OBJECTIVE: To analyse the expression of the epidermal growth factor (EGF) system in prostate tissue and secretions obtained from patients with benign prostatic hyperplasia (BPH) treated with or without finasteride (which primarily targets the androgen-sensitive secretory epithelial cells in the prostate, with little effect on basal epithelial and stromal cells). PATIENTS AND METHODS: The expression of the EGF system was evaluated by enzyme-linked immunosorbent assay and immunohistochemistry in samples of prostate tissue and secretions from patients with BPH randomized for treatment with finasteride or placebo for 3 months before surgery. RESULTS: Prostate tissue expressed the EGF receptor (HER1) and HER2, and the ligands EGF, transforming growth factor alpha (TGFalpha), heparin-binding (HB) EGF, betacellulin and amphiregulin. Treatment with finasteride produced greater concentrations of amphiregulin (P < 0.05) than did placebo, did not change the level of TGFalpha, HER1 and HER2, and tended to decrease the concentration of EGF, betacellulin and HB-EGF in prostate tissue. Using immunohistochemistry, HER1 and TGFalpha were both localized to the basal epithelial cells, and there was a strong positive correlation among the tissue concentrations of HER1, HER2 and TGFalpha. Amphiregulin localized to the luminal secretory epithelium. Prostate secretions contained only EGF, which was at levels approximately 150 times higher than in prostate tissue; treatment with finasteride did not affect the concentration of EGF in prostate secretion. CONCLUSIONS: There were only minor changes in the expression of TGFalpha, HER1 and HER2 after finasteride treatment. This may represent an important system for the continuous growth and homeostasis of the androgen-independent basal epithelial cells in the prostate.     

Changes in epidermal growth factor receptor expression in human bladder cancer cell lines following interferon-alpha treatment

PURPOSE: We examined the regulation of epidermal growth factor (EGF) receptor (EGFR) expression in human bladder cancer cell lines by interferon-alpha (IFN-alpha), the ability of IFN-alpha to inhibit cell proliferation and the sensitivity of IFN-alpha pretreated cells to EGF. MATERIALS AND METHODS: Cell proliferation was determined using crystal violet colorimetric and clonogenic assays. EGFR expression was measured by flow cytometry using specific antibody or ligand binding approaches. RESULTS: After IFN-alpha (100 IU/ml) treatment cell surface EGFR expression was upregulated in 6 of 11 and down-regulated in 2 of 11 bladder cancer cell lines. The over expression of cell surface EGFR peaked within 48 to 96 hours and increased by 35% to 241% in individual cell lines. High level cell surface EGFR correlated with intracellular EGFR expression. Cell growth inhibition by IFN-alpha coexisted with EGFR over expression in the 6 lines. IFN-alpha treated cells remained sensitive to EGF treatment. CONCLUSIONS: IFN-alpha transiently up-regulates EGFR expression and inhibits in vitro growth in some human bladder cancer cells. IFN-alpha does not prevent EGFR from binding EGF or signal transduction via the EGF-EGFR pathway. This may have clinical implications for improving treatment based on EGFR targeting in select patients with bladder cancer.     

Local expression of epidermal growth factor-like growth factors in human testis and its role in spermatogenesis

Microdissection testicular sperm extraction (micro-TESE) has revealed that spermatogenesis in nonobstructive azoospermia (NOA) patients is heterogeneous, even in the same testis, but there is no information regarding growth factors to support spermatogenesis. We investigated the involvement of epidermal growth factor (EGF)-like growth factors, which play important roles in cell proliferation and differentiation in NOA patients who underwent micro-TESE. Testicular samples were obtained from 5 fertile men (15 samples), 5 prostate cancer patients receiving maximum androgen blockade (10 samples), and 13 NOA patients who underwent micro-TESE (50 samples). The expression of the mRNA for EGF, heparin binding (HB)-EGF, amphiregulin, epiregulin, betacellulin, and transforming growth factor (TGF)-α were analyzed by real-time polymerase chain reaction analysis and adjusted using the expression of glyceraldehyde-3-phosphate dehydrogenase. Heterogeneous expression of these EGF-like growth factors were observed even in the same testis. The expression of HB-EGF, amphiregulin and TGF-α in NOA and prostate cancer patients was significantly lower than observed in fertile controls. In NOA patients, expression in the testicular sample comprising mature sperm was significantly higher than those without mature sperm, indicating that HB-EGF, amphiregulin, and TGF-α are considered to participate in creating a suitable niche for spermatogenesis. Considering the findings that ablation of gonadotropin inhibited and human chorionic gonadotropin stimulation increased these EGF-like growth factors, the expressions are presumably under gonadotropin regulation.     

Inhibition of the epidermal growth factor receptor preserves podocytes and attenuates albuminuria in experimental diabetic nephropathy

Aim: Early renal enlargement may predict the future development of nephropathy in patients with diabetes. The epidermal growth factor (EGF)‐EGF receptor (EGFR) system plays a pivotal role in mediating renal hypertrophy, where it may act to regulate cell growth and proliferation and also to mediate the actions of angiotensin II through transactivation of the EGFR. In the present study we sought to investigate the effects of long‐term inhibition of the EGFR tyrosine kinase in an experimental model of diabetes that is characterized by angiotensin II dependent hypertension. Methods: Female heterozygous streptozotocin‐diabetic TGR(mRen‐2)27 rats were treated with the EGFR inhibitor PKI 166 by daily oral dosing for 16 weeks. Results: Treatment of TGR(mRen‐2)27 rats with PKI 166 attenuated the increase in kidney size, glomerular hypertrophy and albuminuria that occurred with diabetes. The reduction in albuminuria, with EGFR inhibition in diabetic TGR(mRen‐2)27 rats, was associated with preservation of the number of glomerular cells staining positively for the podocyte nuclear marker, WT1. Immunostaining for WT1 inversely correlated with glomerular volume in diabetic rats. In contrast to agents that block the renin‐angiotensin system (RAS), EGFR inhibition had no effect on either the quantity of mesangial matrix or the magnitude of tubular injury in diabetic animals. Conclusion: These observations indicate that inhibition of the tyrosine kinase activity of the EGFR attenuates kidney and glomerular enlargement in association with podocyte preservation and reduction in albuminuria in diabetes. Accordingly, targeting the EGF‐EGFR pathway may represent a therapeutic strategy for patients who continue to progress despite RAS‐blockade.     

蛋白酪氨酸磷酸酶-3对结肠癌细胞表皮生长因子相关信号通路的调节

【摘要】〓目的〓观察蛋白酪氨酸磷酸酶-3(PRL-3)在结肠癌LoVo细胞中对表皮生长因子(EGFR)相关信号通路的调节作用。方法〓转染PRL-3的LoVo细胞(以下简称LoVo-P)及对照组LoVo细胞(以下简称LoVo-C)48 h后,通过Western blot检测LoVo细胞PRL-3蛋白表达,并检测EGFR及相关信号MEK-ERK通路蛋白p-MEK1/2,p-Erk1/2,p-Msk1/2的表达。结果〓Western-Blot检测发现高表达PRL-3的LoVo细胞能够激活EGFR磷酸化水平,蛋白灰度分析显示其表达增多72.3%(P<0.01)。并且转染PRL-3后LoVo细胞的MEK-ERK通道被激活。结论〓PRL-3通过激活细胞表皮生长因子磷酸化参与表皮生长因子相关信号通路的调节。     

表皮细胞生长因子及其受体在胎儿和成人肠道组织中的表达特征

目的探讨表皮细胞生长因子(EGF)及其受体(EGFR)在不同发育阶段人小肠组织中的表达特征及其可能的生物学意义。方法24例被测标本中包括胎儿(胎龄13～31周)的小肠组织18例，成人(16～54岁)的小肠组织6例。用病理学技术和免疫组织化学方法确定EGF及EGFR两种蛋白在胎儿、成人小肠组织中的定位以及表达量的变化规律．结果EGF及EGFR在不同胎龄的胎儿和成人小肠组织中均有阳性表达。随着胎儿生长发育，小肠组织中EGF和EGFR的蛋白含量逐渐增加，这一变化趋势一直保持到成人小肠组织中。其中EGF主要存在于小肠黏膜上皮细胞、黏膜下层的血管内皮细胞内和浆膜上皮细胞的胞浆和胞外基质中，EGFR则分布于这些细胞的细胞膜上。结论EGF及EGFR在不同发育阶段的小肠组织中呈阳性表达，这种细胞因子可能以自分泌或旁分泌方式调节人肠道的生长发育、结构和功能的维持，并可能在小肠损伤后的修复中起重要作用。     

Augmentation of 5-fluorouracil cytotoxicity by epidermal growth factor in a newly established human signet-ring cell carcinoma of the stomach in culture

A cell line designated TSG6 was established from a signet-ring cell gastric carcinoma developed in a 57-year-old female patient. The TSG6 cells had well preserved the features of signet-ring cell carcinoma based on morphology. The cells exhibited both epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) immunoreactivities, and also secreted EGF. Moreover, the growth of TSG6 cells was stimulated in the presence of exogenous EGF. These results suggest that the possible presence of an EGF/EGFR autocrine growth mechanism is expressed in the TSG6 cells. The simultaneous treatment with EGF and 5-fluorouracil (5-FU) produced a nearly 2.4-fold enhancement of 5-FU cytotoxicity against TSG6 cells. A bromodeoxyuridine/DNA How cytometry analysis revealed that EGF augmented 5-FU cytotoxicity by inducing the accumulation of S phase cells which might be more susceptible to 5-FU. Moreover, we found that the incorporation of 5-FU into the TSG6 cells was increased with the addition of EGF. These data indicate that EGF may be a potent agent as a biological response modifier for 5-FU against the tumors which express the EGF/EGFR autocrine mechanism, and that the TSG6 cell line is useful in furthering our understanding of the interaction between anticancer drugs and EGF.     

Keloid fibroblast responsiveness to epidermal growth factor and activation of downstream intracellular signaling pathways.

Keloids, which overgrow the boundaries of the original injury, represent aberrations in the fundamental process of wound healing that include over-abundant cell in-migration, cell proliferation, and inflammation, as well as increased extracellular matrix synthesis and defective remodeling. To understand the key events that result in the formation of these abnormal scars would open new avenues for better understanding of excessive repair, and might provide new therapeutic options. We examined epidermal growth factor receptor (EGFR)-induced cell motility in keloid fibroblasts, as this receptor initiates cell migration during normal wound repair. We show that keloid fibroblasts respond to EGF-induced cell migration but the response is somewhat diminished compared to normal adult fibroblasts (approximately 30% reduced); the mitogenic response was similarly blunted (approximately 5% reduced). Keloid fibroblasts express near normal levels of EGFR (82%), but show a much more attenuated activation of EGFR itself and the motility-associated phospholipase C-gamma. This was reflected in part by rapid loss of EGFR upon exposure to EGF. Interestingly, while extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) activation was relatively robust in keloid fibroblasts, the downstream triggering of the motility-associated calpain activity was blunted. This was reflected by high cell-substratum adhesiveness in the keloid fibroblasts. Thus, the blunted migratory response to EGF noted in keloid fibroblasts appears due to limited activation of two important biochemical switches for cell motility.