Combinatorial experimental protocols for Erbicin-derived immunoagents and Herceptin

Erbicin is a human anti-ErbB2 single-chain antibody fragment with high affinity and selectivity for ErbB2-positive cancer cells. Two anti-ErbB2 immunoconjugates, called Erb-hRNase and Erb-hcAb, have been prepared and found to be selectively cytotoxic on ErbB2-positive cancer cells in vitro and vivo. In Erb-hRNase, Erbicin is linked to a human RNase and in Erb-hcAb it is linked to the key structural and functional regions of a human IgG. Herceptin is an anti-ErbB2 humanised antibody successfully used in the immunotherapy of breast cancer. We report here that the Erbicin-derived immunoagents target on breast cancer cells an ErbB2 epitope different than that of Herceptin. This finding led us to verify the effects of Herceptin on breast cancer cells when it was used in combination with the Erbicin-derived immunoagents. The results indicated that in combination experiments the antitumour action of Herceptin and that of the novel agents were significantly increased in an additive fashion. An inspection of the mechanism of action of Erb-hRNase or Erb-hcAb combined with Herceptin provided evidence that the antibody combinations engendered an increased downregulation of the ErbB2 receptor, and led to an enhanced apoptotic cell death

Epidermal growth factor receptor structural alterations in gastric cancer

<p>Abstract</p> <p>Background</p> <p>EGFR overexpression has been described in many human tumours including gastric cancer. In NSCLC patients somatic EGFR mutations, within the kinase domain of the protein, as well as gene amplification were associated with a good clinical response to EGFR inhibitors. In gastric tumours data concerning structural alterations of EGFR remains controversial. Given its possible therapeutic relevance, we aimed to determine the frequency and type of structural alterations of the <it>EGFR </it>gene in a series of primary gastric carcinomas.</p> <p>Methods</p> <p>Direct sequencing of the kinase domain of the <it>EGFR </it>gene was performed in a series of 77 primary gastric carcinomas. FISH analysis was performed in 30 cases. Association studies between <it>EGFR </it>alterations and the clinical pathological features of the tumours were performed.</p> <p>Results</p> <p>Within the 77 primary gastric carcinomas we found two <it>EGFR </it>somatic mutations and several <it>EGFR </it>polymorphisms in exon 20. Six different intronic sequence variants of <it>EGFR </it>were also found. Four gastric carcinomas showed balanced polysomy or <it>EGFR </it>gene amplification. We verified that gastric carcinoma with alterations of <it>EGFR </it>(somatic mutations or copy number variation) showed a significant increase of tumour size (<it>p </it>= 0.0094) in comparison to wild-type <it>EGFR </it>carcinomas.</p> <p>Conclusion</p> <p>We demonstrate that <it>EGFR </it>structural alterations are rare in gastric carcinoma, but whenever present, it leads to tumour growth. We considered that searching for <it>EGFR </it>alterations in gastric cancer is likely to be clinically important in order to identify patients susceptible to respond to tyrosine kinase inhibitors.</p

Preliminary study of p53 and c-erbB-2 expression in gallbladder cancer in Indian patients manuscript id: 8962091628764582

BACKGROUND: The inactivation of the tumour suppressor gene and activation of the proto-oncogene are the key steps in the development of the human cancer. The p53 and c-erbB-2 are the best examples of it. In the present study, our aim was to determine the role of these genes in the carcinogenesis of gallbladder by immunohistochemistry. METHODS: In all 78 consecutive patients of gall bladder diseases were studied for p53 and c-erbB-2 expression immunohistochemically and their expression was correlated with the age, grades and stages of the disease and presence of stone. An informed consent was obtained in each case. Chi square and z test were applied to see the association of p53 and c-erbB-2 over expression with other clinicopathological factors. RESULTS: Eight (20%) patients of gall bladder cancer were positive for p53 expression and 10 (25%) patients for c-erbB-2. The p53 positivity increased with increasing grade while cerbB-2 positivity decreased with increasing grade of gall bladder cancer. Mean age in cerbB-2 positive cases were lesser as compared to negative cases while p53 did not show such association with age. CONCLUSION: Only one case of gall bladder cancer co-expressed the p53 and c-erbB-2, thereby suggesting that p53 and c-erbB-2 may have independent role in carcinogenesis of gall bladder cancer. c-erbB-2 over expression in adenoma and younger age group indicates its role as an early event in carcinogenesis of gallbladder. However study of larger sample is required to further validate the results

c-erbB-2 is not a major factor in the development of colorectal cancer

We have investigated c-erbB-2 protein expression in a large cohort of well-characterized colorectal tumours, and in a subset of lymph node metastases. We have also evaluated a Val655Ile single nucleotide polymorphism, which is associated with an increased risk of breast cancer, in a subset of the colorectal cancer patients and in healthy control subjects. Immunohistochemical studies revealed that while 81.8% of tumours expressed c-erbB-2, in the majority of cases equivalent levels of c-erb-B2 were seen in adjacent normal mucosa. Colon tumours were significantly more likely to express c-erbB-2 than rectal tumours (P=0.015). Only 52.4% of the metastases displayed staining patterns concordant with their primary tumour, indicating that determination of c-erbB-2 protein in colorectal tumours cannot predict the status of lymph node metastases. PCR–RFLP analysis of the Val655Ile single nucleotide polymorphism demonstrated that allele frequencies were identical between colorectal cancer patients and a control group of Caucasian subjects (Ile=0.80 and Val=0.20 in each case), indicating that it is not related to the risk of developing colorectal cancer in this population. Furthermore, there was no relationship between c-erbB-2 protein expression and gene polymorphism (P=0.58). In terms of prognosis, no association was seen between either c-erbB-2 protein expression or the presence of the Val allele and patient survival (P>0.05 in each case), suggesting that c-erbB-2 is not a prognostic marker in colorectal cancer

Different mechanisms are implicated in ERBB2 gene overexpression in breast and in other cancers

The ERBB2 gene is overexpressed in 30% of breast cancers and this has been correlated with poor prognosis. ERBB2 is upregulated in other cancers such as prostate, pancreas, colon and ovary. In breast cancer cells, the mechanisms leading to ERBB2 gene overexpression are increased transcription and gene amplification. In these cancers, AP-2 transcription factors are involved in ERBB2 overexpression, and AP-2 levels are correlated with p185(c-erbB-2) levels. In this work, we wanted to know if the same molecular mechanisms are responsible for the ERBB2 upregulation in non-breast cancers. We compared ERBB2 gene copy number, p185(c-erbB-2) and mRNA levels with AP-2 levels in several ovary, prostate, colon and pancreas cancer cells. A moderate expression of erbB-2 mRNA and protein were observed in some cells without gene amplification. In contrast to breast cancer cells, AP-2 factors were absent or low in some non-breast cells which did express ERBB2. It is thus likely that AP-2 is not a major player in the increased levels of erbB-2 transcripts in non-breast cancer cells. The transcriptional activity of the ERBB2 promoter in colon and ovary cancer cells was estimated using reporter vectors. The results showed that the promoter regions involved in ERBB2 gene overexpression in breast cancer cells are different from those that lead to the gene upregulation in colon and ovary cancers. In conclusion, our results indicate that different transcriptional and post-transcriptional mechanisms are responsible for the increased levels of erbB-2 transcript and protein in breast and non-breast cancer cells

The combined immunodetection of AP-2α and YY1 transcription factors is associated with ERBB2 gene overexpression in primary breast tumors

INTRODUCTION: Overexpression of the ERBB2 oncogene is observed in about 20% of human breast tumors and is the consequence of increased transcription rates frequently associated with gene amplification. Several studies have shown a link between activator protein 2 (AP-2) transcription factors and ERBB2 gene expression in breast cancer cell lines. Moreover, the Yin Yang 1 (YY1) transcription factor has been shown to stimulate AP-2 transcriptional activity on the ERBB2 promoter in vitro. In this report, we examined the relationships between ERBB2, AP-2alpha, and YY1 both in breast cancer tissue specimens and in a mammary cancer cell line. METHODS: ERBB2, AP-2alpha, and YY1 protein levels were analyzed by immunohistochemistry in a panel of 55 primary breast tumors. ERBB2 gene amplification status was determined by fluorescent in situ hybridization. Correlations were evaluated by a chi2 test at a p value of less than 0.05. The functional role of AP-2alpha and YY1 on ERBB2 gene expression was analyzed by small interfering RNA (siRNA) transfection in the BT-474 mammary cancer cell line followed by real-time reverse transcription-polymerase chain reaction and Western blotting. RESULTS: We observed a statistically significant correlation between ERBB2 and AP-2alpha levels in the tumors (p < 0.01). Moreover, associations were found between ERBB2 protein level and the combined high expression of AP-2alpha and YY1 (p < 0.02) as well as between the expression of AP-2alpha and YY1 (p < 0.001). Furthermore, the levels of both AP-2alpha and YY1 proteins were inversely correlated to ERBB2 gene amplification status in the tumors (p < 0.01). Transfection of siRNAs targeting AP-2alpha and AP-2gamma mRNAs in the BT-474 breast cancer cell line repressed the expression of the endogenous ERBB2 gene at both the mRNA and protein levels. Moreover, the additional transfection of an siRNA directed against the YY1 transcript further reduced the ERBB2 protein level, suggesting that AP-2 and YY1 transcription factors cooperate to stimulate the transcription of the ERBB2 gene. CONCLUSION: This study highlights the role of both AP-2alpha and YY1 transcription factors in ERBB2 oncogene overexpression in breast tumors. Our results also suggest that high ERBB2 expression may result either from gene amplification or from increased transcription factor levels

Mutations in the epidermal growth factor receptor gene are linked to smoking-independent, lung adenocarcinoma

Epidermal growth factor receptor (EGFR) mutations are a potential predictor of the effectiveness of EGFR inhibitors for the treatment of lung cancer. Although EGFR mutations were reported to occur with high frequency in nonsmoking Japanese adenocarcinoma patients, the exact nature has not been fully elucidated. We examined EGFR gene mutations within exons 18–21 and their correlations to clinico-pathological factors and other genetic alterations in tumour specimens from 154 patients who underwent resection for lung cancer at Kyoto University Hospital. Epidermal growth factor receptor mutations were observed in 60 tumours (39.0%), all of which were adenocarcinoma. Among the patients with adenocarcinoma (n=108), EGFR mutations were more frequently observed in nonsmokers than former smokers or current smokers (83.0, 50.0, 15.2%, respectively), in women than men (76.3 vs 34.0%), in tumours with bronchio-alveolar component than those without bronchio-alveolar component (78.9 vs 42.9%), and in well or moderately differentiated tumours than poorly differentiated tumours (72.0, 64.4, 34.2%). No tumours with EGFR mutations had any K-ras codon 12 mutations, which were well-known smoking-related gene mutations. In conclusion, adenocarcinomas with EGFR mutation had a distinctive clinico-pathological feature unrelated to smoking. Epidermal growth factor receptor mutations may play a key role in the development of smoking-independent adenocarcinoma

Designing a HER2/neu promoter to drive α1,3galactosyltransferase expression for targeted anti-αGal antibody-mediated tumor cell killing

INTRODUCTION: Our goal was to specifically render tumor cells susceptible to natural cytolytic anti-αGal antibodies by using a murine α1,3galactosyltransferase (mαGalT) transgene driven by a designed form of HER2/neu promoter (pNeu), the transcription of which is frequently observed to be above basal in breast tumors. Indeed, the αGalT activity that promotes Galα1,3Galβ1,4GlcNAc-R (αGal) epitope expression has been mutationally disrupted during the course of evolution, starting from Old World primates, and this has led to the counter-production of large amounts of cytotoxic anti-αGal antibodies in recent primates, including man. METHOD: Expression of the endogenous c-erbB-2 gene was investigated in various cell lines by northern blotting. A mαGalT cDNA was constructed into pcDNA3 vector downstream of the original CMV promoter (pCMV/mαGalT) and various forms of pNeu were prepared by PCR amplification and inserted in the pCMV/mαGalT construct upstream of the mαGalT cDNA, in the place of the CMV promoter. These constructs were transferred into HEK-293 control and breast tumor cell lines. Stably transfected cells were analyzed by northern blotting for their expression of αGalT and c-erbB-2, and by flow cytometry for their binding with fluorescein isothiocyanate-conjugated Griffonia simplicifolia/isolectin B4. RESULTS: We show that expression of the mαGalT was up- or down-modulated according to the level of endogenous pNeu activity and the particular form of constructed pNeu. Among several constructs, two particular forms of the promoter, pNeu250 containing the CCAAT box and the PEA3 motif adjacent to the TATAA box, and pNeu664, which has three additional PEA3 motifs upstream of the CCAAT box, were found to promote differential αGalT expression. CONCLUSION: Our results strengthen current concepts about the crucial role played by the proximal PEA3 motif of pNeu, and may represent a novel therapeutic approach for the development of targeted transgene expression

The Cooperation between hMena Overexpression and HER2 Signalling in Breast Cancer

hMena and the epithelial specific isoform hMena11a are actin cytoskeleton regulatory proteins belonging to the Ena/VASP family. EGF treatment of breast cancer cell lines upregulates hMena/hMena11a expression and phosphorylates hMena11a, suggesting cross-talk between the ErbB receptor family and hMena/hMena11a in breast cancer. The aim of this study was to determine whether the hMena/hMena11a overexpression cooperates with HER-2 signalling, thereby affecting the HER2 mitogenic activity in breast cancer. In a cohort of breast cancer tissue samples a significant correlation among hMena, HER2 overexpression, the proliferation index (high Ki67), and phosphorylated MAPK and AKT was found and among the molecular subtypes the highest frequency of hMena overexpressing tumors was found in the HER2 subtype. From a clinical viewpoint, concomitant overexpression of HER2 and hMena identifies a subgroup of breast cancer patients showing the worst prognosis, indicating that hMena overexpression adds prognostic information to HER2 overexpressing tumors. To identify a functional link between HER2 and hMena, we show here that HER2 transfection in MCF7 cells increased hMena/hMena11a expression and hMena11a phosphorylation. On the other hand, hMena/hMena11a knock-down reduced HER3, AKT and p44/42 MAPK phosphorylation and inhibited the EGF and NRG1-dependent HER2 phosphorylation and cell proliferation. Of functional significance, hMena/hMena11a knock-down reduced the mitogenic activity of EGF and NRG1. Collectively these data provide new insights into the relevance of hMena and hMena11a as downstream effectors of the ErbB receptor family which may represent a novel prognostic indicator in breast cancer progression, helping to stratify patients