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PDF - 752K, Screening of genes involved in TNBC tumorigenesis.

PDF File - 117K, Supplementary Materials and Methods, Supplementary References and Supplementary Figure legends.

PDF File - 72K, Sequences of siRNA oligo used in Figure 2A and Supplementary Figure S2A.

Supplementary Fig. S2 from Heparin-binding epidermal growth factor–like growth factor promotes transcoelomic metastasis in ovarian cancer through epithelial-mesenchymal transition

Heparin-binding epidermal growth factor–like growth factor (HB-EGF) is involved in several biological processes including cell adhesion, invasion, and angiogenesis. HB-EGF also plays a pivotal role in the progression of ovarian cancer. To investigate the significance of HB-EGF in peritoneal dissemination, we examined the roles of HB-EGF in cell adhesion, invasion, and angiogenesis in ovarian cancer. Through the suppression of focal adhesion kinase and EGF receptor activation, cell adhesive properties mediated by integrin β1 were diminished by the inhibition of HB-EGF expression. The reduction of HB-EGF expression attenuated the chemotactic invasive ability and the expression of matrix metalloprotease (MMP)-2 and vascular endothelial growth factor (VEGF), leading to the inhibition of cell invasion and angiogenesis. Suppression of the Snail family, which regulates the epithelial-mesenchymal transition, blocked the cell adhesion properties on extracellular matrices, the chemotactic invasive ability, and the expression of MMP9 and VEGF through the reduction of HB-EGF expression. The volume of tumor burden in the peritoneal cavity was dependent on the expression of HB-EGF. According to these results, HB-EGF contributes to cell adhesion, invasion, and angiogenesis, which are integral to transcoelomic metastasis in ovarian cancer. CRM197, an inhibitor of HB-EGF, resulted in a significant decrease of tumor burden in peritoneal dissemination, accompanied with a reduction in both cellular spreading, when assayed on an extracellular matrix, and invasive ability, when assayed in a chemotaxis chamber, as well as decreased expression of MMP9 and VEGF. Thus, HB-EGF is a mutual validating target in the peritoneal dissemination of ovarian cancer, and CRM197 may be useful as a anticancer agent for advanced ovarian cancer. [Mol Cancer Ther 2008;7(9):3441–51]

Supplementary Fig. from Heparin-binding epidermal growth factor–like growth factor promotes transcoelomic metastasis in ovarian cancer through epithelial-mesenchymal transition

This study aimed to evaluate the learning curve and perioperative outcomes of robot-assisted hysterectomy (RAH).We retrospectively analyzed data from 45 patients who underwent RAH using the da Vinci Xi surgical system. The learning curve was evaluated using the cumulative summation method. Demographic data and various perioperative parameters, including total operative time, docking time, and console time, were obtained from the medical records.Cumulative summation analysis indicated that proficiency regarding hysterectomy time was reached after 33 cases. There were two unique phases of the learning curve for console time: the introduction phase identified by the bottom point in the curve, and the proficient phase, identified by an upward line after the bottom point in the curve. There were no significant differences between the two phases in terms of patient age and body mass index. Total operative time, docking time, and console time were significantly decreased in the proficient phase compared with those in the introduction phase. There was a significant reduction in blood loss during operation in the proficient phase. The perioperative complication rates were 12.1% in the introduction phase and 0% in the proficient phase (p=0.5606). No blood transfusion or conversion to laparotomy was required in either phase.The introduction and proficient phases identified by cumulative summation analysis demonstrated progressive improvement of surgical performance in surgeons carrying out RAH.

Endometriosis, which exhibits enigmatic pathological features such as stromal fibrosis and proliferation of ectopic epithelial cells, is known as a refractory disease. Mesenchymal stem cells modulate the fibrosis in stromal tissues through their trophic and immunomodulatory properties. To investigate the potential of stem cells in treating endometriosis, we examined the secondary morphology and molecular alterations in endometriosis-like lesions after the administration of adipose tissue-derived stem cells (ASCs) to an experimental murine model of endometriosis. The infused ASCs were found integrated in the endometriosis-like lesions. Accompanied by the suppression of stromal fibrosis and proliferation of endometriotic epithelial cells, the infusion of ASCs with stemness potential suppressed the growth of endometriosis-like lesions and inhibited the expression of pro-inflammatory and pro-fibrotic cytokines, whereas no significant attenuation of endometriosis-like lesions occurred after the infusion of ASCs without stemness potential. Accordingly, it is plausible that the trophic and immunomodulatory properties of ASCs may regulate fibrosis in endometriosis-like lesions, suggesting that regenerative medicine has strong potential as an innovative treatment for patients with endometriosis.

Introduction This study was performed to determine whether the combination of maternal blood and amniotic fluid biomarkers can improve the predictive accuracy of histologic chorioamnionitis (HC). Methods This retrospective study included 80 singleton pregnant women who were suspected to have intrauterine infection and underwent measurement of two maternal blood biomarkers [maternal white blood cell count (mWBC) and maternal C-reactive protein level (mCRP)] and three amniotic fluid biomarkers [amniotic white blood cell count (aCell), amniotic glucose level (aGlucose), and amniotic lactate dehydrogenase level (aLDH)]. We divided the patients into two groups based on the presence or absence of HC and assessed the predictors of HC using logistic regression models: Model 1, combination of mWBC and mCRP; Model 2, combination of Model 1 and aGlucose; and Model 3, combination of Model 2, aCell, and aLDH. Results The multivariable analysis showed that aCell was the only significant predictor of HC [odds ratio, 1.24; 95% confidence interval (CI), 1.06–1.68] independent of mWBC, mCRP, aGlucose, and aLDH. The c-statistics were higher in Model 3 (0.803; 95% CI, 0.701–0.905) than Model 1 (0.634; 95% CI, 0.511–0.758) and Model 2 (0.785; 95% CI, 0.684–0.887). Discussion We found that the combination of maternal blood and amniotic fluid biomarkers can improve the predictive accuracy of HC. Therefore, our data provide relevant information to support counseling with regard to improving the predictive accuracy of HC in patients with suspected intrauterine infection.

Intra-amniotic infection (IAI) is a major cause of preterm birth with a poor perinatal prognosis. We aimed to determine whether analyzing vaginal microbiota can evaluate the risk of chorioamnionitis (CAM) in preterm labor cases. Vaginal discharge samples were collected from 83 pregnant women admitted for preterm labor. Based on Blanc’s classification, the participants were divided into CAM (stage ≥ II; n = 46) and non-CAM (stage ≤ I; n = 37) groups. The 16S rDNA amplicons (V1–V2) from vaginal samples were sequenced and analyzed. Using a random forest algorithm, the bacterial species associated with CAM were identified, and a predictive CAM (PCAM) scoring method was developed. The α diversity was significantly higher in the CAM than in the non-CAM group (P P = 0.022). Multivariate analysis revealed a significant difference in the frequency of developmental disorders in 3-year-old infants (PCAM, 28%, non-PCAM, 4%; P = 0.022). Analyzing vaginal microbiota can evaluate the risk of IAI. Future studies should establish appropriate interventions for IAI high-risk patients to improve perinatal prognosis.

Background: This study was performed to investigate the clinical significance of miR-4535 and miR-1915-5p in severe chorioamnionitis. Materials & methods: Amniotic fluid samples from 37 patients with severe chorioamnionitis were subjected to miRNA array analysis and ddPCR™. Diagnostic values were assessed using the receiver operating characteristic curve. The patients were separated into three groups according to Blanc’s criteria. Results: The expression of miR-4535 and miR-1915-5p was significantly correlated with the copy number of 16S rDNA, had extremely high diagnostic accuracy for severe chorioamnionitis, and was linked to maternal and fetal inflammation. Conclusion: miR-4535 and miR-1915-5p serve as promising biomarkers for the diagnosis of severe chorioamnionitis., Lay abstract Chorioamnionitis and fetal inflammatory response syndrome, which is linked to chorioamnionitis, are considered serious diseases in perinatal care. In this study, miR-4535 and miR-1915-5p are recognized as promising biomarkers for the diagnosis of chorioamnionitis before delivery. In particular, the increased expression of miR-4535 in amniotic fluid is expected to be regarded as a positive indicator for fetal inflammatory response syndrome, and the elevated expression of miR-4535 in serum is also considered to predictively diagnose intrauterine infection in pregnancy. Our results highlight that further studies should explore the underlying clinical significance of miR-4535 and miR-1915-5p.

Aim: Embryo implantation and subsequent pregnancy depends on endometrial thickness. To investigate potential fertility strategies for women with thin endometrium, we explored the efficacy of adipose tissue-derived regenerative cells (ADRCs) on thin endometrium and embryo implantation in a mouse model. Materials & methods: ADRCs isolated from mouse subcutaneous fat were characterized by flow cytometry. Endometrium thickness, endometrial fibrosis, embryo implantation and angiogenesis factors were evaluated in uterine cavities of ethanol-induced thin endometrium mice with ADRC transplantation. Results: ADRCs included adipose-derived stem cells and some blood vessel component cells. ADRCs improved endometrial thickness, endometrial fibrosis and embryo implantation and augmented vascular endothelial growth factor expression in the mouse uterine. Conclusion: ADRCs may be a useful therapeutic strategy to improve fertility of women with thin endometrium.

Background/aim Many anticancer agents including molecularly-targeted drugs have been developed for ovarian cancer. However, the prognosis of recurrent ovarian cancer remains extremely poor. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is reported as a rational target for ovarian cancer therapy. Moreover, serum HB-EGF expression is recognized as a biomarker in patients with primary ovarian cancer. Materials and methods We analysed serum samples with recurrent ovarian cancer at the Fukuoka University Hospital from April 2009 to March 2014. To assess the clinical significance of serum HB-EGF in recurrent ovarian cancer, the association between serum HB-EGF levels and prognosis in patients with recurrent ovarian cancer was examined using ELISA. Results Patients with high serum HB-EGF expression showed a significantly poor response to second-line chemotherapeutic agents compared with patients with low HB-EGF levels. Conclusion HB-EGF expression in serum may be a potential therapeutic indicator for novel HB-EGF-targeted therapy in recurrent ovarian cancer.

Ovarian cancer is the most lethal gynecologic malignancy. Recently, several molecularly targeted anticancer agents have been developed for ovarian cancer; however, its prognosis remains extremely poor. The development of molecularly targeted therapy, as well as companion diagnostics, is required to improve outcomes for patients with ovarian cancer. In this study, to identify microRNAs (miRNAs) involved in the progression of ovarian cancer we analyzed serum miRNAs in patients with ovarian cancer using miRNA array and quantitative RT-PCR and examined the anticancer properties of miRNA expression in ovarian cancer cells. In patients with ovarian cancer, high amount of miR-135a-3p in serum samples was significantly associated with favorable clinical prognosis. The amount of miR-135a-3p was significantly decreased in patients with ovarian cancer compared with patients with ovarian cysts or normal ovaries. In SKOV-3 and ES-2 human ovarian cancer cells, enhanced expression of miR-135a-3p induced drug sensitivity to cisplatin and paclitaxel and suppressed cell proliferation and xenograft tumor growth. These findings suggest that miR-135a-3p may be considered as a biomarker and a therapeutic agent in ovarian cancer.

Background/aim Initial treatment of endometrial cancer with surgery and platinum and taxane-based chemotherapy is often successful, but it remains unclear as to whether certain types of the disease relapse. The aim of this study was to identify the clinical features of recurrence in patients without residual tumour in endometrial cancer. Patients and methods Clinical features, histological type, and time to recurrence were analyzed in 640 endometrial cancer patients without residual tumours. Results Of 640 patients, 517 were type I and 123 were type II. For type I, early recurrent (ER) disease and late recurrent (LR) disease were noted in 80 and 8 patients, respectively, and 97.5% of ER occurred within 2 years. After recurrence, 76.2% of ER and 50% of LR patients died. In type II, ER and LR were noted in 41 and 1 patients, respectively, and 97.6% of ER occurred within 2 years, of which 75.6% died after recurrence. One LR case died of disease. Conclusion Most patients recurred within 2 years irrespective of clinical stage or type.

Background Abnormalities in the number of vessels can be found for both the umbilical artery and vein. We sometimes encounter cases of a decreased number of umbilical cord vessels, such as a single umbilical artery. In contrast, there may be an increase from three to four vessels within the umbilical cord. A supernumerary umbilical vein is particularly very rare, and it is generally found in combination with congenital anomalies. We report a case of a partial supernumerary umbilical vein. Case presentation The previous pregnancy of a 37-year-old healthy Japanese woman (gravida 2, para 1) had been uncomplicated, and the resulting child was alive and well. Prenatal examination at 36 weeks of gestation revealed the coexistence of a four-vessel part and a normal three-vessel part of the umbilical cord. A healthy female neonate weighing 2726 g was born at 38 weeks of gestation. The umbilical cord measured 40 cm in length; the four-vessel part continued to a distance of 18 cm from the surface of the infant’s body, and the remaining umbilical cord comprised three vessels. On histological examination, the fetal side of the umbilical cord had two arteries and two veins, and the placental side had two arteries and one vein. Isolated supernumerary umbilical veins tend to be overlooked. We consider that it is important to evaluate the number of umbilical cord vessels in the second trimester using ultrasound combined with color Doppler in at least three sites: the insertion sites on both the fetal abdomen and placenta, and the free loop of the umbilical cord. Conclusions Prenatal diagnosis of isolated supernumerary umbilical cord vessels tends to be overlooked. However, supernumerary vessels of the umbilical can be associated with fetal congenital anomalies. The number of vessels within the umbilical cord must be examined because the detection of such abnormalities may lead to the prenatal diagnosis of other congenital anomalies.

To observe age-related changes in the anterior lower uterine segment (LUS) thickness in normal pregnancy from 20 to 35 weeks’ gestation. Subjects were 235 uncomplicated singleton cases that underwent single ultrasound examination at 4-week intervals during 20–35 weeks’ gestation. Sagittal LUS sections were evaluated with transvaginal ultrasonography. Anterior LUS thickness (Th) was measured every centimeter from the lowest bladder point (Th0cm) to 4 cm from that point (Th4cm). Th values were standardized by dividing by the Th0cm value. Intragroup comparisons of standardized Th values and intergroup comparisons of actual Th values were made according to gestational age. Statistical analyses were performed with the Kruskal–Wallis and Tukey honest standard deviation tests; significance was set at p

The NG2 proteoglycan promotes tumor growth as a component of both tumor and stromal cells. Using intracranial, NG2-negative B16F10 melanomas, we have investigated the importance of pericyte and macrophage NG2 in brain tumor progression. Reduced melanoma growth in myeloid-specific NG2 null (Mac-NG2ko) and pericyte-specific NG2 null (PC-NG2ko) mice demonstrates the importance of NG2 in both stromal compartments. In each genotype, loss of pericyte-endothelial cell interaction diminishes formation of endothelial junctions and assembly of the basal lamina. Tumor vessels in Mac-NG2ko mice have smaller diameters, reduced patency, and increased leakiness compared to PC-NG2ko mice, thus decreasing tumor blood supply and increasing hypoxia. While reduced pericyte interaction with endothelial cells in PC-NG2ko mice results from loss of pericyte activation of β1 integrin signaling in endothelial cells, reduced pericyte-endothelial cell interaction in Mac-NG2ko mice results from 90% reduced macrophage recruitment. The absence of macrophage-derived signals in Mac-NG2ko mice causes loss of pericyte association with endothelial cells. Reduced macrophage recruitment may be due to diminished activation of integrins in the absence of NG2, causing decreased macrophage interaction with endothelial adhesion molecules that are needed for extravasation. These results reflect the complex interplay that occurs between macrophages, pericytes, and endothelial cells during tumor vascularization.

Colorectal cancer (CRC) is one of the most frequently occurring cancers with high morbidity and mortality worldwide. Amphiregulin (AREG), a member of the epidermal growth factor family and a rational target for CRC therapy, is essential for the three-dimensional structure of tumor formation. To clone the genes associated with increased AREG expression, we performed a cDNA microarray analysis in two CRC cell lines undergoing two-dimensional (2DC) and three-dimensional culture (3DC). Upregulated (>2.0-fold) and downregulated (

Chorioamnionitis (CAM), an inflammation of the foetal membranes due to infection, is associated with preterm birth and poor perinatal prognosis. The present study aimed to determine whether CAM can be diagnosed prior to delivery based on the bacterial composition of the amniotic fluid (AF). AF samples from 79 patients were classified according to placental inflammation: Stage III (n = 32), CAM; Stage II (n = 27), chorionitis; Stage 0-I (n = 20), sub-chorionitis or no neutrophil infiltration; and normal AF in early pregnancy (n = 18). Absolute quantification and sequencing of 16S rDNA showed that in Stage III, the 16S rDNA copy number was significantly higher and the α-diversity index lower than those in the other groups. In principal coordinate analysis, Stage III formed a separate cluster from Stage 0-I, normal AF, and blank. Forty samples were classified as positive for microbiomic CAM (miCAM) defined by the presence of 11 bacterial species that were found to be significantly associated with CAM and some parameters of perinatal prognosis. The diagnostic accuracy for CAM according to miCAM was: sensitivity, approximately 94%, and specificity, 79–87%. Our findings indicate the possibility of predicting CAM prior to delivery based on the AF microbiome profile.

Advanced lung cancer is one of the most lethal malignancies. Many anticancer agents have been developed for lung cancer with epidermal growth factor receptor (EGFR) mutations, but its prognosis remains extremely poor. The development of molecularly-targeted therapies is required for patients with lung cancer with secondary mutation of the EGFR gene. In this study, in order to assess the validity of heparin-binding EGF-like growth factor (HB-EGF) as a therapeutic target for lung cancer with EGFR mutation, we examined the antitumor effects of a specific inhibitor (cross-reacting material 197; CRM197) on lung cancer cells with EGFR mutation. HB-EGF was the most predominantly expressed EGFR ligand in lung cancer cells with EGFR mutation. CRM197 induced significant cell apoptosis and marked suppression of tumorigenicity in lung cancer cells with single or double mutation of EGFR. These results suggest that HB-EGF is a rational target for the treatment of lung cancer with EGFR mutation.

Background Patients with ovarian cancer with high levels of heparin-binding epidermal growth factor-like growth factor have a poor prognosis. Here we assessed the pharmacokinetics and tumour-inhibiting effects of cross-reacting material 197, produced commercially as BK-UM, and examined the efficacy and safety of its intravenous (i.v.) administration. Materials and methods BK-UM was administered to rats, and its serum levels were measured. Ovarian cancer cell lines were either intraperitoneally (i.p.) or subcutaneously administered into mice, to establish a mouse model of ovarian cancer. BK-UM was then administered i.p. or i.v., and its tumour-inhibiting effects were examined. Results Higher maximum serum concentration (Cmax) values resulted from i.v. administration, whereas longer time to maximum serum (Tmax) values resulted from i.p. administration. In the peritoneal dissemination model, i.p. administration inhibited tumour growth and increased survival rate, whereas in the subcutaneous model, i.v. administration significantly inhibited tumour growth compared to i.p. administration. Conclusion Administration of BK-UM by i.v. is both efficacious and safe.

Advanced lung cancer is one of the most lethal malignancies. Many anticancer agents have been developed for lung cancer with epidermal growth factor receptor (EGFR) mutations, but its prognosis remains extremely poor. The development of molecularly-targeted therapies is required for patients with lung cancer with secondary mutation of the EGFR gene. In this study, in order to assess the validity of heparin-binding EGF-like growth factor (HB-EGF) as a therapeutic target for lung cancer with EGFR mutation, we examined the antitumor effects of a specific inhibitor (cross-reacting material 197; CRM197) on lung cancer cells with EGFR mutation. HB-EGF was the most predominantly expressed EGFR ligand in lung cancer cells with EGFR mutation. CRM197 induced significant cell apoptosis and marked suppression of tumorigenicity in lung cancer cells with single or double mutation of EGFR. These results suggest that HB-EGF is a rational target for the treatment of lung cancer with EGFR mutation.

Background BK-UM (CRM197) is a mutant form of diphtheria toxin and a specific inhibitor of heparin-binding epidermal growth factor-like growth factor (HB-EGF). We assessed the safety, pharmacokinetics, recommended dose, and efficacy of BK-UM in patients with recurrent ovarian cancer (OC) or peritoneal cancer (PC), and measured HB-EGF levels in serum and abdominal fluid after BK-UM administration. Methods Eleven patients with advanced or recurrent OC or PC were enrolled and treated with BK-UM via the intraperitoneal route. The dose was escalated (1.0, 2.0, 3.3, and 5.0 mg/m2) using a 3 + 3 design. Results Eight of 11 patients completed treatment. No dose-limiting toxicity (DLT) was experienced at dose levels 1 (1.0 mg/m2) and 2 (2.0 mg/m2). Grade 3 transient hypotension as an adverse event (defined as a DLT in the present study) was observed in two of four patients at dose level 3 (3.3 mg/m2). Treatment with BK-UM was associated with decreases in HB-EGF levels in serum and abdominal fluid in seven of 11 patients and five of eight patients, respectively. Clinical outcomes included a partial response in one patient, stable disease in five patients, and progressive disease in five patients. Conclusions BK-UM was well tolerated at doses of 1.0 and 2.0 mg/m2, with evidence for clinical efficacy in patients with recurrent OC or PC. A dose of 2.0 mg/m2 BK-UM is recommended for subsequent clinical trials. Trial registration This trial was prospectively performed as an investigator-initiated clinical trial. The trial numbers are UMIN000001002 and UMIN000001001, with registration dates of 1/30/2008 and 2/4/2008, respectively. UMIN000001001 was registered as a trial for the continuous administration of BK-UM after UMIN000001002. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3071-5) contains supplementary material, which is available to authorized users.

Table S1. Pharmacokinetics of BK-UM. (PDF 48 kb)

Heparin-binding EGF-like growth factor (HB-EGF) is one of several proangiogenic factors and represents a possible therapeutic target for patients with triple-negative breast cancer (TNBC). However, the role of HB-EGF in promoting tumor aggressiveness in TNBC remains unclear. To investigate specific genes and pathways involved in TNBC tumorigenesis, we profiled gene expression changes in two TNBC cell lines under two-dimensional culture (2DC) and three-dimensional culture (3DC) and in a tumor xenograft model. We identified simultaneous upregulation of HB-EGF, VEGFA, and angiopoietin-like 4 (ANGPTL4) in 3DC and tumor xenografts, compared with 2DC. We show that HB-EGF regulates the expression of VEGFA or ANGPTL4 via transcriptional regulation of hypoxia-inducible factor-1α and NF-κB. Furthermore, suppression of VEGFA or ANGPTL4 expression enhanced HB-EGF expression, highlighting a unique regulatory loop underlying this angiogenesis network. Targeted knockdown of HB-EGF significantly suppressed tumor formation in a TNBC xenograft model, compared with individual knockdown of either VEGFA or ANGPTL4, by reducing the expression of both VEGFA and ANGPTL4. In patients with TNBC, VEGFA or ANGPTL4 expression was also significantly correlated with HB-EGF expression. Low concentrations of exogenously added HB-EGF strongly activated the proliferation of endothelial cells, tube formation, and vascular permeability in blood vessels, in a similar fashion to high doses of VEGFA and ANGPTL4. Taken together, these results suggest that HB-EGF plays a pivotal role in the acquisition of tumor aggressiveness in TNBC by orchestrating a molecular hierarchy regulating tumor angiogenesis. Mol Cancer Res; 11(5); 506–17. ©2013 AACR.

Endometrial cancer (EC) has a poor prognosis due to drug resistance.We evaluated the safety and efficacy of adjuvant combination chemotherapy with docetaxel plus cisplatin ((DP) docetaxel, 70 mg/m(2); cisplatin, 60 mg/m(2); every 28 days) in EC patients at intermediate-risk (IR) or high-risk (HR) for recurrence.Sixty-four patients diagnosed with EC were enrolled. Stage-I, -II, -III and -IV disease was noted in 23, 7, 28 and 6 patients, respectively. Histopathological analyses revealed that 56, 3, 1 and 4 patients had endometrioid, serous, clear-cell or "other" types of carcinoma. Grade-3/4 hematologic toxicities were found at 80% and 95% in patients in IR and HR groups, respectively. In IR and HR groups, mean progression-free (PFS) survival was 69.5 and 29.5, while overall survival (OS) was 59.6 and 47.5 months, respectively.DP may be clinically safe and useful treatment for EC.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), which belongs to the epidermal growth factor family, is a rational therapeutic target for triple-negative breast cancer (TNBC). This study aimed to assess the anti-tumor efficacy of intravenous (i.v.) HB-EGF-specific inhibitor (CRM197) for TNBC.NOD/SCID mice were subcutaneously injected withTNBC cells, MDA-MB-231, and, then, treated with i.v. CRM197 in either dose- or frequency-dependent manners, using an advanced cancer model and an adjuvant therapy model. Tumor volume and mouse body weight were calculated weekly. Statistical significance was assessed by the Mann-Whitney U-test.Mice that received i.v. CRM197 showed a significant anti-tumor effect in dose- and frequency-dependent manners in both models. However, their body weight did not differ significantly among groups.These results suggest that i.v. CRM197 is an effective treatment for TNBC.

Colostrum contains a wide variety of crucial nutritional elements including growth factors for newborn infants to adapt to the extrauterine environment.To investigate the clinical significance of epidermal growth factor receptor ligands in milk during the first month of lactation.The concentrations of epidermal growth factor (EGF), amphiregulin (AR) and transforming growth factor-α (TGF-α) in milk sampled from a total of 31 normal mothers at days 1-3, 5, and 30 postpartum were examined using ELISA.At days 1-3, the concentration of EGF was extremely high [131.6 ± 20.4 (mean ± SEM) ng/ml] compared to that of AR (4,197.2 ± 1,055.2 pg/ml) or TGF-α (261.7 ± 33.6 pg/ml), while the concentration of AR was significantly elevated compared to that of TGF-α. At days 5 and 30, the concentration of EGF was significantly elevated compared to that of AR or TGF-α. In 16 mothers among the same 31 subjects, samples were longitudinally obtained on days 1, 2, 5, and 30 postpartum. Concentrations of AR were higher on days 1 and 2 and rapidly declined to below 1 ng/ml on day 5, and were maintained at lower levels on day 30. Concentrations of EGF were high on day 1 (greater than 10 ng/ml) but gradually declined by days 2, 5, and 30. Concentrations of TGF-α remained at lower levels of below 1 ng/ml throughout the lactation period from days 1 to 30.These results suggested that EGF and amphiregulin in colostrum might contribute to the early stage of development of neonatal gastrointestinal function.

Purpose: Heparin-binding epidermal growth factor (EGF)–like growth factor (HB-EGF) is a member of the EGF family and plays a pivotal role in tumor progression in ovarian cancer. We developed an anti-HB-EGF monoclonal antibody (mAb) and investigated its antitumor activities in vitro and in vivo to evaluate its potential as a therapeutic antibody against ovarian cancer. Experimental Design: We prepared mAbs from HB-EGF null mice immunized with recombinant human soluble HB-EGF and evaluated their binding and neutralizing activity against HB-EGF. Next, we generated a mouse–human chimeric antibody and examined its in vitro and in vivo antitumor activities. Results: Two murine anti-HB-EGF mAbs were developed, and one of them, KM3566, was revealed to have a high binding reactivity for membrane-anchored HB-EGF (pro-HB-EGF) expressed on the cell surface, as well as neutralizing activity against growth promoting activity of soluble HB-EGF. The mouse–human chimeric counterpart for KM3566 (cKM3566) induced dose-dependent antibody-dependent cellular cytotoxicity (ADCC) against cancer cells expressing HB-EGF in vitro, and significantly inhibited tumor growth in severe combined immunodeficient mice inoculated with MCAS or ES-2 human ovarian cancer cells. Conclusions: A novel anti-HB-EGF chimeric antibody, cKM3566, with two antitumor mechanisms, neutralization and ADCC, exhibits potent in vivo antitumor activity. These results indicate that cKM3566 is a promising antiovarian cancer therapeutic antibody. Clin Cancer Res; 17(21); 6733–41. ©2011 AACR.

Therapeutics targeting the ErbB protein family receptors have not always yielded favorable or successful results in present cancer therapy. This review discusses the possibility of the clinical adaptation of targeting against heparin-binding epidermal growth factor-like growth factor (HB-EGF), one of the ligands of the ErbB system, in ovarian cancer therapy.We have previously described the results of studies concerning roles of HB-EGF in tumor formation in ovarian cancer. In brief, lisophosphatidic acid (LPA) and HB-EGF are predominantly expressed in advanced ovarian cancer, and LPA-induced, a disintegrin and metalloprotease-mediated ectodomain shedding of HB-EGF was found to be critical to tumor formation. We also noted that exogenous expression of HB-EGF enhanced tumor formation but inhibition blocked both extracellular signal-related kinase and serine/threonine protein kinase activation. Finally we investigated the antitumor effects of CRM197 - a specific HB-EGF inhibitor - on ovarian cancer cells by evaluating human ovarian cancer cell proliferation.We discuss alternative strategies to develop the chemotherapeutic agent based on targeting ErbB family ligands rather than their receptors. A phase I study of CRM197 for advanced ovarian cancer has already begun, which is the first approved trial of ErbB-ligand-targeted therapy. We also discuss clinical adaptations based on combination of CRM197 with other conventional chemotherapeutic agents.

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.

Pancreatic cancer is one of the most lethal malignancies. Epidermal growth factor receptor (EGFR), HER3, Akt, and amphiregulin have been recognized as targets for pancreatic cancer therapy. Although gemcitabine + erlotinib has been the recommended chemotherapy for pancreatic cancer, the prognosis is extremely poor. The development of molecularly targeted therapies has been required for patients with pancreatic cancer. To assess the validation of amphiregulin as a target for pancreatic cancer therapy, we examined its expression in pancreatic cancer using real-time PCR analyses and ELISA. We also measured the apoptotic cell rate using TUNEL assays. In addition, alterations in signaling pathways were detected by immunoblotting analyses. Treatment with gemcitabine, which reduced the cell viability and augmented the cell apoptotic rate, activated and subsequently attenuated ERK and EGFR signals. However, gemcitabine, paclitaxel, or cisplatin treatment enhanced the Akt activation, heterodimer formation of EGFR with HER3, and secretion of amphiregulin, indicating that the presence of gemcitabine promoted the activity of targeted molecules including amphiregulin, Akt, and HER3 for pancreatic cancer therapy. Combined treatment with an inhibitor for amphiregulin and gemcitabine, paclitaxel, or cisplatin induced synergistic antitumor effects, accompanied by the suppression of Akt and ERK activation. Blockade of amphiregulin suppressed the activities of EGFR, HER3, and Akt and the expression of amphiregulin itself. According to this evidence, combination chemotherapy of conventional anticancer drugs plus an inhibitor for amphiregulin would allow us to provide more favorable clinical outcomes for patients with pancreatic cancer.

Aberrant expression levels of epidermal growth factor receptor (EGFR) and its cognate ligands have been recognized as one of the causes of cancer progression. To investigate the validity of EGFR ligands as targets for cancer therapy, we examined the expression of EGFR ligands and in vitro anti-tumor effects of small interference RNA (siRNA) for EGFR ligands in various cancer cells. HB-EGF expression was dominantly elevated in ovarian, gastric, and breast cancer, melanoma and glioblastoma cells, whereas amphiregulin was primarily expressed in pancreatic, colon, and prostate cancer, renal cell carcinoma and cholangiocarcinoma cells. Transfection of siRNAs for HB-EGF or amphiregulin into these cells significantly increased the numbers of apoptotic cells with attenuation of EGFR and ERK activation. In lung cancer cells, any EGFR ligand was not recognized as a validated target for cancer therapy. These results suggest that HB-EGF and amphiregulin are promising targets for cancer therapy.

Neuroblastoma (NB) is the most common and lethal extracranial solid tumor in children. The present study aimed to verify that the heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a rational target in NB therapy.We examined expression of EGFR ligands in four NB cell lines using 2-dimensional culture (DC) and 3DC conditions. To assess the anti-tumor effect of cross-reacting material 197 (CRM197), which is a specific inhibitor of HB-EGF, on NB cells, we also performed terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay to detect apoptotic cells.HB-EGF was predominantly expressed in two out of four NB cell lines under 2DC and 3DC conditions. CRM197 significantly induced apoptosis of NB cells with high HB-EGF expression.HB-EGF plays an important role in neuroblastoma tumorigenesis and CRM197 showed an effective antitumor effect in neuroblastoma cells.

Early stage growth of intracranial B16F10 tumors is reduced by 87% in myeloid-specific NG2 null (Mac-NG2ko) mice and by 77% in pericyte-specific NG2 null (PC-NG2ko) mice, demonstrating the importance of the NG2 proteoglycan in each of these stromal compartments. In both genotypes, loss of pericyte-endothelial cell interaction results in numerous structural defects in tumor blood vessels, including decreased formation of endothelial cell junctions and decreased assembly of the vascular basal lamina. All vascular deficits are larger in Mac-NG2ko mice than in PC-NG2ko mice, correlating with the greater decrease in pericyte-endothelial cell interaction in Mac-NG2ko animals. Accordingly, tumor vessels in Mac-NG2ko mice have a smaller diameter, lower degree of patency, and higher degree of leakiness than tumor vessels in PC-NG2ko mice, leading to less efficient tumor blood flow and to increased intratumoral hypoxia. While reduced pericyte interaction with endothelial cells in PC-NG2ko mice is caused by loss of NG2-dependent pericyte activation of β1 integrin signaling in endothelial cells, reduced pericyte-endothelial cell interaction in Mac-NG2ko mice is due to a 90% reduction in NG2-dependent macrophage recruitment to tumors. The absence of a macrophage-derived signal(s) in Mac-NG2ko mice results in the loss of pericyte ability to associate with endothelial cells, possibly due to reduced expression of N-cadherin by both pericytes and endothelial cells.

Objectives. RCAS1 expression is significantly associated with clinical prognosis in various human cancers, which suggests that RCAS1 may be involved in acquisition of malignant phenotypes. To investigate the relationship between RCAS1 and one such characteristic, tumor invasiveness, we examined RCAS1 expression in cervical neoplasms ranging from the precancerous state to invasive cancer. Methods. RCAS1 expression was studied retrospectively via immunohistochemical methods. Samples consisted of biopsy tissue from 90 patients with intraepithelial neoplasia and resected tumor tissue from 154 patients with invasive cancer. Statistical analysis was done to correlate RCAS1 expression and clinicopathologic variables in patients with a depth of cancer cell invasion into stromal tissue of >5 mm. Results. RCAS1 expression was detected in patients with intraepithelial cancer and invasive cancer but not in patients with dysplasia. The occurrence and degree of RCAS1 expression increased with the depth of invasion. In patients with invasive cancer, RCAS1 overexpression was significantly correlated with invasion of the lymph-vascular space, lymph node metastasis in two or more sites, and tumor volume; RCAS1 expression was not associated with histologic subtype. Overall survival rates for patients with RCAS1 overexpression were significantly shorter than those for patients without RCAS1 overexpression. In connective tissue surrounding tumor cells, the number of cells expressing vimentin significantly decreased in relation to RCAS1 expression level. Moreover, significant associations between expression levels of RCAS1 and those of MMP-1 and laminin-5 were found. Conclusion. RCAS1 may contribute to acquisition of malignant uterine cervical phenotypic characteristics including invasion, metastasis, and tumor growth via connective tissue remodeling.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the epidermal growth factor family, is a target for ovarian cancer therapy. The present study investigated the administration schedule of BK-UM, an anticancer agent targeting HB-EGF.The ovarian cancer cell line, RMG-I, was injected subcutaneously into five-week-old female nude mice. The BK-UM was administered intraperitoneally, using three administration schedules with different doses. The tumor volume was calculated every week. Statistical significance was assessed using the Mann-Whitney U-test.At doses0.1 mg/kg, BK-UM displayed significant antitumor effects, although the antitumor effects and body weights of mice did not significantly differ by dose or by three different administration schedules. At a dose0.1 mg/kg, however, BK-UM had little inhibitory effect on tumor growth.Daily administration of BK-UM, which has a potentially dose-dependent antitumor effect, may be the optimal schedule for clinical application.

Ovarian clear cell carcinoma (OCCC) is a worst histological subtype than other ovarian malignant tumor. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a promising target for ovarian cancer therapy. The aims of this study were to validate the efficacy of HB-EGF–targeted therapy for OCCC and to identify the transcription factor that contributed to the induction of HB-EGF by SN38 treatment in OCCC cells. HB-EGF was highly expressed in OCCC cells, and an increase of HB-EGF was induced by SN38 which had only antitumor effect among conventional anticancer agents on OCCC. A specific inhibitor of HB-EGF, a cross-reacting material 197 (CRM197), led to a synergistic increase in the number of apoptotic OCCC cells with the treatment of SN38. The luciferase assay with 5′-deletion promoter constructs identified a GC-rich element between −125 and −178 (the distal transcription start site was denoted +1) as a cis-regulatory region, and the treatment of SN38 induced luciferase activity in this region. An in silico and chromatin immunoprecipitation analysis estimated that SP1 bound to the cis-regulatory region of HB-EGF in OCCC cells. Real-time PCR and cell viability assays showed that the transfection of a small interfering RNA targeting SP1 suppressed the expression of HB-EGF induced by SN38, resulting in the enhanced sensitivity of SN38. Taken together, these results indicate that induction of HB-EGF expression contributed to defense mechanism against treatment of SN38 through the transcriptional activity of SP1 in OCCC cells.