Neu Gene Expression Research Articles

Comparison of Her-2/Neu Gene Amplification or Expression between IHC and FISH in BC.

This study aimed to analyze and compare the application of FISH and IHC in BC tissue Her-2 / neu gene amplification or expression. For this purpose, 110 patients with BC were selected. FISH and IHC were performed on the BC tissues that were surgically removed, and the results were compared and analyzed. The results showed that in 110 BC tissues, the expression of HER-2 protein was (+++) 25 cases (22.73%); (++) 44 cases (40%); (+) 26 cases (23.64%); (1) 15 cases (13.64%). There were 42 cases of HER-2 gene amplification in 110 BC tissues, and 68 cases had no amplification. IHC test positive (+++) is consistent with FISH positive coincidence rate, IHC test negative (+/-) is consistent with FISH negative coincidence rate, IHC test suspicious positive (++) compared with the FISH result, the difference is statistically significant. However, the total coincidence rate between IHC test results and FISH test results was 89.29 (25/28), and the two test methods were positively correlated. Generally, IHC positive and negative expressions are in good agreement with FISH test results. The suspected positive expression of IHC is inconsistent with the FISH test result, suggesting that the IHC test is suspicious, and the positive specimens need to be tested by FISH.

Assessment of human epidermal growth factor receptor 2/neu gene amplification and expression as a biomarker for radiotherapy and hormonal-treated breast cancer patients in upper Egypt.

Breast cancer plays major public health in Egyptian women. In upper Egypt, There is an increase in the incidence of breast cancer compared to other Egyptian areas without know the reasons. In this study, we aimed to evaluate the potential of HER-2/neu status as one of the important markers to classify the women suffering from breast cancer in upper Egypt and monitoring the responsiveness to different therapies. The present study was performed on 67 female breast cancer patients in the South Egypt Cancer Institute to evaluate HER-2/neu gene amplification and expression. Tissue samples were used for immunohistological analysis of endocrine receptors, HER-2/neu, and HER-2/neu gene amplification. In addition, the blood samples were also used to determine HER-2/neu gene expression. All statistical analyses were performed using Chi-square test. The statistical difference is considered statistically significant at P < 0.05. There was a statistically significant association between HER-2/neu gene expression and the age of patients. There is decrease in the level of HER-2/neu mRNA expression in group treated with chemotherapy and group treated with chemotherapy and radiotherapy compared to each group baseline level of HER-2/neu mRNA expression before treatment. On the contrary, the group treated with chemotherapy, radiotherapy, and hormonal therapy revealed increase on the level of HER-2/neu mRNA expression when compared with their baseline for the same patients before treatment. We need further studies on the large group of upper Egypt breast cancer patients to confirm that the level of HER-2/neu mRNA expression can be used as a marker for classified them and their response to different treatment.

HER-2/neu gene amplification in gastric adenocarcinoma and its relationship with clinical and pathological findings.

Amplification of the HER-2/neu oncogene influences the progression of gastric cancer, its prognosis, and therapy. A precise examination of HER-2/neu-amplified tumor tissue is essential for managing disease and prescribing the appropriate treatment. This study aimed to investigate the status of HER-2/neu gene in the gastric cancer samples and its relationship with clinical and pathological information. In this study on 80 paraffin-embedded tissue samples from patients with gastric adenocarcinoma [2006-2011], DNA was extracted to quantify the gene expression levels of HER-2 using a polymerase chain reaction (PCR) method. Data were statistically analyzed by chi-square test using SPSS16.0 software. PCR results indicated that HER-2/neu gene amplifications occurred in 58 of the 80 samples (72.5%). HER-2/neu gene expression was not significantly related to age and sex, but the larger tumor size and the more advanced stage were significantly associated with HER-2/neu overexpression. The data show the HER-2/neu gene is more amplified in stage 4 of gastric cancer with a larger size of mass. Older age and male sex also appear to be more associated with HER-2/neu gene expression.

Assessment of HER-2/neu gene amplification and expression as a biomarker for radiotherapy and hormonal treated breast cancer patients in Upper Egypt

Assessment of HER-2/neu gene amplification and expression as a biomarker for radiotherapy and hormonal treated breast cancer patients in Upper Egypt

Detection of Her-2/neu expression in gastric cancer: Quantitative PCR versus immunohistochemistry.

The aim of this study was to compare quantitative polymerase chain reaction (qPCR) with immunohistochemistry (IHC) for the detection of Her-2 in gastric cancer, and to investigate the correlation between the expression levels of human epidermal growth factor receptor 2 (Her-2) and clinical features. Clinical data from 426 cases of gastric cancer were collected. Her-2 expression levels in cancerous tissue were detected using IHC, and the Her-2/neu gene expression levels were determined by qPCR. The correlation between the expression level of Her-2 and clinical features was investigated. The positive expression rate of Her-2 in cancerous tissue detected using qPCR and IHC was 11.17% (46/412) and 13.38% (57/426), respectively. The positive expression of the Her-2 protein/gene was significantly correlated with the depth of invasion and lymphatic metastasis, as well as the TNM stage (P<0.05). No significant correlation was identified between positive expression of the Her-2 protein/gene and tumor location, age, gender, differentiation degree and Lauren classification (P>0.05). The diagnostic consistency was good between the two methods (κ=0.828). The results indicate that the expression of Her-2/neu is closely associated with the development of gastric cancer. qPCR is a convenient, objective and efficient method, which may be used as an alternative to IHC or fluorescence in situ hybridization for the detection of Her-2/neu gene.

Distribution of breast cancer subtypes among Jordanian women and correlation with histopathological grade: molecular subclassification study

ObjectiveTo evaluate the hormone receptor status and human epidermal growth factor receptor 2 (HER2)/neu gene expression among Jordanian women with breast cancer. To classify our patients into molecular subtypes and to correlate the results with age of the patients and tumour grade.DesignEvaluation of estrogen receptor (ER), PR and HER2/neu was done by standard immunohistochemical technique and subclassification into molecular subtypes.SettingJordan University Hospital, Amman, Jordan.ParticipantsOne hundred and ninety-three cases of breast cancer diagnosed at Jordan University Hospital.Main outcome measuresMolecular subtypes of breast cancer, age and tumour grade.ResultsAll the cases were divided into two groups: the young age group less or equal 50 years of age and the older age group more than 50 years of age. The cases were subclassified into luminal A, luminal B, basal cell like (BCL) and Her2/neu+. In older age group, the most common subtype was luminal A (72%). In this age group, most of the cases (48%) were of grade II. In younger age group, 47% of the cases were of luminal A subclass. In this age group, 42% were of grade I.ConclusionsMolecular subtyping of breast cancer is an essential predicting factor of tumour response to hormonal therapy. This fact puts increased stress on the urgent need for the development of reliable and reproducible classification systems.

The transcription of theneuDgene is stronger in serotype III group B streptococci strains isolated from cerebrospinal fluid than in strains isolated from vagina

Group B streptococci (GBS) are a major cause of neonatal meningitis, and sialic acid is a determinant of the development of meningitis. The transcription level of the neuD gene, used as a marker of neu gene expression and capsular production, was significantly higher in serotype III GBS strains isolated from meningitis than from vaginal carriage. This was irrespective both of the phylogenetic position of strains and of the presence of a thymine at position 264 in the neuD gene. Differences in neuD gene transcription may explain in part why particular isolates among the GBS strains colonizing the vagina can cause meningitis.

Her-2/neu Overexpression Due to Polysomy 17 in Breast Cancer: Molecular Testing to Guide Therapeutic Options

Background: Fluorescent in situ hybridization (FISH) equivocal results for Her-2/neu still pose a diagnostic dilemma in oncology practice. In this study, we evaluate if Her-2/neu mRNA expression is an alternative to FISH for detecting Her-2/neu positivity. Patients and Methods: Archival paraffin blocks of 54 breast cancer patients were analyzed for Her-2/neu status using immunohistochemistry (IHC), FISH, and Her-2/neu gene expression using mRNA. Results: There was a 100% positive agreement and 64.7% negative agreement of Her-2/neu mRNA expression with respect to the reference standard (FISH), with the kappa value for agreement being 0.36. mRNA levels correlated positively and strongly with FISH ratio and IHC positivity. For Her-2/neu mRNA expression, Her-2/neu copy number was a significant predictor indicating that mRNA expression is independent of polysomy status. Conclusions: Her-2/neu mRNA expression may help tide over ambiguity posed by polysomy and FISH equivocal samples.

5144 Prophylactic antibiotics are necessary to minimise the risk of febrile neutropenia in patients receiving TAC chemotherapy

Trastuzumab (Herceptin™) is a humanized antibody directed against the extracellular domain of the tyrosine kinase orphan receptor Her-2/neu (erbB-2) that has shown therapeutic efficacy against Her-2/neu-overexpressing breast tumors. However, less than 35% of patients with Her-2/neu-overexpressing metastatic breast cancer respond to trastuzumab as a single agent, whereas the remaining cases do not demonstrate tumor regression. Furthermore, the majority of patients who achieve an initial response generally acquire resistance within one year. Therefore, the identification of the potential mechanisms of resistance to trastuzumab can be very helpful for the development of new compounds, which might overcome that resistance and/or have additive/synergistic antitumor effect when given in association with trastuzumab. Recent studies in breast cancer cells have revealed a bi-directional connection between Her-2/neu and fatty acid synthase (FAS), a major lipogenic enzyme catalyzing the synthesis of long-chain saturated fatty acids from the 2-carbon donors malonyl-CoA and acetyl-CoA. Her-2/neu overexpression stimulates the FAS promoter and ultimately mediates increased endogenous fatty synthesis, and this Her-2/neu-mediated induction of breast cancer-associated FAS is inhibitable by trastuzumab. On the other hand, chemical FAS inhibitors as well as RNA interference-mediated silencing of FAS gene repress Her-2/neu gene expression at the transcriptional level. Moreover, specific FAS blockade synergistically sensitizes breast cancer cells carrying Her-2/neu-oncogene amplification and/or overexpression to trastuzumab-induced cell growth inhibition and apoptotic cell death. Strikingly, FAS inhibition synergistically interacts with trastuzumab in Her-2/neu-negative breast cancer cells engineered to overexpress Her-2/neu, thus suggesting that the molecular linkage between FAS activity and functioning of Her-2/neu cannot be explained only on the basis of a transcriptional repression of Her-2/neu gene promoter. Interestingly, while in liver and adipose tissue FAS produces fat from excess carbon consumed as carbohydrates, which is ultimately stored as triglycerides, in epithelial cancer cells, FAS activity is mainly involved in the production of phospholipids partitioning into detergent-resistant membrane microdomains (lipid raft-aggregates), which point to an active role of FAS in the deregulation of membrane functioning in tumor cells. Importantly, clusters of Her-2/neu and EGFR (erbB-1) co-localize with lipid rafts and the lipid environment in the cell membrane of breast cancer cells profoundly influences their association properties and biological functions. We hypothesize that pharmacological or small interference RNA-induced inhibition of breast cancer-associated FAS will result in major changes in the synthesis of phospholipids which, in turn, should impair a correct cellular localization of Her-2/neu at the cellular membrane of breast cancer cells. In this working model, FAS inhibition could induce a shift in the equilibrium between transport of Her-2/neu to and from the membrane favoring an increased Her-2/neu internalization followed by intracellular degradation, thus enhancing the mechanism of action of the anti-Her-2/neu antibody trastuzumab. Moreover, the inhibition of FAS-driven lipid rafts will also negatively affect EGFR-Her-2/neu cross-talk, an important mechanism of trastuzumab resistance. In summary, the specific blockade of a novel molecular linkage between FAS-regulated membrane composition and functioning of transmembrane growth factor receptors EGFR and Her-2/neu may represent a previously unrecognized therapeutic approach circumventing trastuzumab resistance in breast carcinomas.

HER-2/neu expression in glioblastoma multiforme (GBM)

e13035 Background: Glioblastoma multiforme (GBM) is a disease in which very few cytotoxic chemotherapy agents have been shown to have activity. This is partly due to their inability to cross the blood brain barrier. Trials with bevacizumab, a VEGF inhibitor that disrupts tumor angiogenesis, have demonstrated activity against this otherwise chemotherapy resistant disease. This has led to interest in other biologic agents that target angiogenic pathways for the treatment of GBM. Over-expression of HER-2/neu by human tumor cells is closely associated with increased angiogenesis and expression of VEGF. Lapatinib is a recently available low molecular weight immunotherapeutic agent that targets HER-2/neu proteins. In a recent study, breast cancer patients treated with lapatinib and capcitabine had decreased brain metastases indicating that lapatinib may cross the blood brain barrier and thus have potential in the treatment of malignant gliomas. Limited studies have evaluated HER-2/neu gene expression in GBM and the results are inconsistent. We evaluated the expression of Her-2/neu protein in 41 consecutive GBM cases to explore the potential utility of targeting this pathway. Methods: Archival histopathologic sections from 41 patients (age 26–89 years) with a diagnosis of GBM from 2004–2008 were reviewed. The diagnosis was confirmed and optimal sections were selected. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue sections using the primary antibody against HER-2/neu (clone 4B5, Ventana). The results were evaluated by three independent investigators. Interpretation was performed using the semi-quantitative criteria (Score 0 to 3+) currently used for primary breast carcinomas. Results: 38 out of 41 cases showed no immunohistochemical staining with HER-2/neu antibody (score 0). Three cases demonstrated weak, incomplete membrane staining of rare tumor cells (score 1+) and were interpreted as negative. The positive and negative controls worked properly. Conclusions: Our study indicates that there is no significant immunohistochemical over-expression of HER-2/neu protein in GBM. This suggests that HER-2/neu over-expression is not a significant oncogenic pathway in GBM, and therefore may not be a potential therapeutic target in this disease. No significant financial relationships to disclose.

LED-FISH: Fluorescence microscopy based on light emitting diodes for the molecular analysis of Her-2/neu oncogene amplification

Light emitting diodes (LED), which are available as small monochromatic light sources with characteristic features such as maximum illumination power combined with minimum energy consumption and extremely long lifespan have already proved as a highly potential low-cost alternative for specific diagnostic applications in clinical medicine such as tuberculosis fluorescence microscopy. Likewise, the most reliable evaluation of Her-2/neu (c-erbB2) gene amplification, which has been established in the last few years for routine diagnosis in clinical pathology as determinant towards Herceptin-based treatment of patients with breast cancer, is based on fluorescence in situ hybridization (FISH) and corresponding high priced fluorescence equipment. In order to test the possibility to utilize the advantages of low-cost LED technology on FISH analysis of c-erbB2 gene expression for routine diagnostic purposes, the applicability of a standard bright field Carl Zeiss Axiostar Plus microscope equipped with a Fraen AFTER* LED Fluorescence Microscope Kit for the detection of Her-2/neu gene signals was compared to an advanced Nikon Eclipse 80i fluorescence microscope in combination with a conventional 100W mercury vapor lamp. Both microscopes were fitted with the same Quicam FAST CCD digital camera to unequivocally compare the quality of the captured images. C-erbB2 gene expression was analyzed in 30 different human tissue samples of primary invasive breast cancer, following formalin fixation and subsequent paraffin-embedding. The Her2/neu gene signals (green) were identifiable in the tumor cells in all cases and images of equal quality were captured under almost identical conditions by 480 nm (blue) LED module equipped standard Axiostar microscope as compared to conventional fluorescence microscopy. In this first attempt, these monochromatic LED elements proved in principle to be suitable for the detection of Her-2/neu gene expression by FISH. Thus, our own experiences emphasize the high potential of this technology to provide a serious alternative to conventional fluorescence microscopy in routine pathology; representing a sustainable technological progress, this low-cost technology will clearly give direction also to the growing field of molecular pathology.* AFTER = Amplified Fluorescence by transmitted Excitation of Radiation

Effect of the Silybin-Phosphatidylcholine Complex (IdB 1016) on the Development of Mammary Tumors in HER-2/neu Transgenic Mice

Silybin, a main component of the milk thistle of Silybum marianum, has been reported to possess anticancer activity. We investigated the effects of IdB 1016, a complex of silybin with phosphatidylcholine, on the development of mammary tumors appearing spontaneously in HER-2/neu transgenic mice. The mechanisms involved in the antitumor effect of IdB 1016 were evaluated by studying the apoptosis, senescent-like growth arrest, intratumoral leukocyte infiltrate, and the expression of HER-2/neu and p53 in tumoral mammary glands from transgenic mice and in human breast SKBR3 tumor cells. The administration of IdB 1016 delayed the development of spontaneous mammary tumors, reduced the number and size of mammary tumor masses, and diminished lung metastasization in HER-2/neu transgenic mice. In tumoral mammary glands from IdB 1016-treated mice, a down-regulation of HER-2/neu gene expression was associated with an increased senescent-like growth arrest of tumor cells, and an increased infiltrate of neutrophils, CD4, and CD8 T cells. Both senescent-like growth arrest and apoptosis were significantly increased and were associated with a reduced p185(HER-2/neu) protein and an increased p53 mRNA in SKBR3 in vitro treated with IdB 1016 in comparison with control cells. The results show the antitumor effect of IdB 1016 in the development of spontaneous mammary tumors in HER-2/neu transgenic mice. The effect of IdB 1016 might be related to the down-regulation of HER-2/neu expression and the induction of senescent-like growth arrest and apoptosis through a p53-mediated pathway in tumor cells.

Amplification of HER-2/neu (erbB2) gene expression in gallbladder (GBC) and in bile duct cancer (BDC) (biliary tract cancer, BTC)

4011 Background: GBC and BDC (BTC) are uncommon in the United States, but are endemic in parts of South America and Asia. BTC are aggressive tumors with poor survival rates. Chemotherapy RR is &lt;30%, and no survival benefit has been demonstrated. Improved understanding of the molecular carcinogenic mechanisms of BTC may lead to improved therapeutic regimens. Activation of HER-2/neu play a prominent role in the development of breast cancer. In this study, BTC patient (pt) specimens from the U.S., Chile, and Japan were examined the HER-2/neu immunohistochemical (IHC) expression and its gene amplification by fluorescence in situ hybridization (FISH). Methods: Specimens from 26 GBC patients (Chile, US), 12 BDC (USA), 6 cholecystitis (Chole.) (Japan) and 7 normal GB (Chile) were examined. pStage (AJCC) of the pt was: IA: 2, IB: 9, IIA: 7, IIB: 9, III: 3, IV: 4, unknown: 4. Histologic grade was G1 and G2 adenocarcinoma (adca): 22, G3 adca: 13, squamous cell carcinoma: 1, unknown: 2. All cases were screened for HER-2/neu by IHC (0–3+). HER-2/neu gene amplification (FISH (+)) (defined as a HER-2/CEP17 ratio of 2.0 or greater) was performed by double color Her-2/neu gene/chromosome 17 centromere (CEP17) FISH assays using PathVysion HER-2/neu DNA probe kit. Results: In BTC, HER-2/neu IHC 0, 1+, 2+ and 3+ scores were found in 0 (0%), 20 (53%), 11 (29%) and 7 (18%), respectively. In Chole., 3 of 6 (50%) showed 1+ while the remaining and normal GB were all negative for IHC. FISH on paraffin sections was considered successful on 36 (95%) of the 38 cases. HER-2/neu FISH (+) was detected in 7 of 36 (19%) (6 from 3+ cases and 1 from 2+ case) and none in Chole. and normal GB. There was a significant correlation between overexpression by IHC (2+ and 3+) and FISH (+) (Fisher’s exact test; P=0.0123), but no significant correlation between pStage, histologic grade and FISH (+). Conclusions: The HER-2/neu amplification was identified in 19% of BTC in comparison with the other organ cancer (breast cancer:20–25%, lung cancer: 4.5%, colorectal cancer; 2.4%, prostate cancer: 9.3%). There was a strong correlation between IHC overexpression and FISH (+). These data support therapeutic approaches using HER-2/neu targeted agents in BTC pt. HER-2/neu IHC may be a useful surrogate for its gene amplification. No significant financial relationships to disclose.

Genistein inhibits expression of vascular endothelial growth factor in HER-2/neu transfected human breast cancer MCF-7 cells

Objectiveour previous studies have demonstrated that HER-2/neu gene expression in human breast cancer MCF-7 cells promotes angiogenesis in MCF-7 cells xenograft tumors, and genistein inhibits angiogenesis in MCF-7 cells with HER-2/neu expression xenograft tumors. Here, the effects of genistein on the expression of vascular endothelial growth factor (VEGF) in MCR-7 cells with HER-2/neu expression were further studied for exploring the molecular mechanism of anti-angiogenesis in HER-2/neu-overexpressing breast cancer by genistein.MethodsHER-2/neu-overexpressing MCF-7 cells (MCF-7/HER-2) were established by transfecting HER-2/neu gene into HER-2/neu negative expression breast cancer MCF-7 cells. Immunocytochemical staining, western blot and reverse transcription-polymerase chain reaction (RT-PCR) were adopted to measure the expression of VEGF in MCF-7/HER-2 cells treated by genistein for 24, 48 and 72h.ResultsHER-2/neu expression up-regulated VEGF mRNA and protein in MCF-7 cells, genistein decreased VEGF mRNA and protein level in MCF-7/HER-2 cells in a time-dependent manner.ConclusionThese results suggest that VEGF plays an important role in HER-2/neu gene expression promoted antiogenesis in breast cancer and genistein induced down-regulation of the expression of VEGF may be one of the molecular mechanisms of its anti-angiogenesis in HER-2/neu-overexpressing breast cancer.

The effect of RNAi-mediated gene silencing on her-2/neu gene expression in lung adenocarcinoma cells

Objective: Her-2/neu protein overexpression has been demonstrated in Non-small cell lung cancer, especially in lung adenocarcinoma. Its overexpression often indicates a poor prognosis. Therapeutic agents against her-2/neu have been intensively sought over the past decades. The objective of this paper is to investigate the effect of chemically synthesized siRNA targeting her-2/neu on her-2/neu dysregulated human lung adenocarcinoma cells. Methods: Calu-3 cells were transfected with siRNAs formulated LipofectAMINE 2000. The her-2/neu mRNA and protein levels were detected by RT-PCR and flow cytometry (FCM). The biological morphology and growth inhibition of calu-3 cells were observed with light microscopy and MTT assay, respectively. The cell cycle and apoptosis rate were analyzed by FCM. Results: siRNA targeting Her-2/neu down-regulated the transcription of her-2/neu oncogene and protein level. Slowed cell proliferation and cell cycle arrest at G0/1 stage could be also observed, accompanied with enhanced cell apoptosis. Conclusion: Specific siRNA targeting her-2/neu can effectively inhibit her-2/neu expression in her-2/neu overexpressing calu-3 cell lines. siRNA-mediated gene silencing may be a useful therapeutic strategy for cancer.

Targeting and Regulation of the HER-2/neu Oncogene Promoter with Bis-Peptide Nucleic Acids

Antigene oligonucleotides have the potential to regulate gene expression through site-specific DNA binding. However, in vivo applications have been hindered by inefficient cellular uptake, degradation, and strand displacement. Peptide nucleic acids (PNAs) address several of these problems, as they are resistant to degradation and bind DNA with high affinity. We designed two cationic pyrimidine bis-PNAs (cpy-PNAs) to target the polypurine tract of the HER-2/neu promoter and compared them to an unmodified phosphodiester triplex-forming oligonucleotide (TFO1) and a TFO-nitrogen mustard conjugate (TFO2). PNA1 contains a + 2 charge and bound two adjacent 9-bp target sequences with high affinity and specificity, but only at low pH. PNA2 contains a +5 charge and bound one 11-bp target with high affinity up to pH 7.4, but with lower specificity. The PNA:DNA:PNA triplex formed by these cpy-bis-PNAs presented a stable barrier to DNA polymerase extension. The cpy-bis-PNAs and the TFO-alkylator conjugate prevented HER-2/neu transcription in a reporter gene assay (TFO2 = PNA1 > PNA2 >> TFO1). Both PNAs and TFOs were effective at binding the target sequence in naked genomic DNA, but only the TFO-alkylator (TFO2) and the more cationic PNA (PNA2) were detected at the endogenous HER-2/neu promoter in permeabilized cells. This work demonstrates the potential for preventing HER-2/neu gene expression with cpy-bis-PNAs in tumor cells.

Effect of resveratrol on the development of spontaneous mammary tumors in HER‐2/neu transgenic mice

Resveratrol (Res) has been reported to possess cancer chemopreventive activity on the basis of its in vitro effects on tumor cells and in vivo experimental models of rodents transplanted with parental tumors or treated with carcinogens. We investigated the effects of Res on the development of mammary tumors appearing spontaneously in HER-2/neu transgenic mice at an early age. The mechanisms involved in the Res antitumor effect were evaluated by studying the immune effectiveness, tumor apoptosis and expression of mRNA and protein for HER-2/neu in tumoral mammary glands from Res-treated mice and in tumor cell lines. Res supplementation delayed the development of spontaneous mammary tumors (p < 0.001), reduced the mean number and size of mammary tumors (p < 0.0001) and diminished the number of lung metastases in HER-2/neu transgenic mice. The effects of Res were associated with downregulation of HER-2/neu gene expression and increased apoptosis both in tumoral mammary glands and in murine (N202) and human (SKBr3) tumor cell lines. Neither the basal, the IL-2-induced NK activity nor the lymphocyte number and proliferation was modified in Res-supplemented compared to control mice. Our results demonstrate that Res supplementation delays the development and reduces the metastasizing capacity of spontaneous mammary tumors in HER-2/neu transgenic mice. The antitumor effect of Res might be related to the downregulation of HER-2/neu expression and the induction of apoptosis in tumor cells.

Targeting fatty acid synthase-driven lipid rafts: a novel strategy to overcome trastuzumab resistance in breast cancer cells

Trastuzumab (Herceptin) is a humanized antibody directed against the extracellular domain of the tyrosine kinase orphan receptor Her-2/neu (erbB-2) that has shown therapeutic efficacy against Her-2/neu-overexpressing breast tumors. However, less than 35% of patients with Her-2/neu-overexpressing metastatic breast cancer respond to trastuzumab as a single agent, whereas the remaining cases do not demonstrate tumor regression. Furthermore, the majority of patients who achieve an initial response generally acquire resistance within one year. Therefore, the identification of the potential mechanisms of resistance to trastuzumab can be very helpful for the development of new compounds, which might overcome that resistance and/or have additive/synergistic antitumor effect when given in association with trastuzumab. Recent studies in breast cancer cells have revealed a bi-directional connection between Her-2/neu and fatty acid synthase (FAS), a major lipogenic enzyme catalyzing the synthesis of long-chain saturated fatty acids from the 2-carbon donors malonyl-CoA and acetyl-CoA. Her-2/neu overexpression stimulates the FAS promoter and ultimately mediates increased endogenous fatty synthesis, and this Her-2/neu-mediated induction of breast cancer-associated FAS is inhibitable by trastuzumab. On the other hand, chemical FAS inhibitors as well as RNA interference-mediated silencing of FAS gene repress Her-2/neu gene expression at the transcriptional level. Moreover, specific FAS blockade synergistically sensitizes breast cancer cells carrying Her-2/neu-oncogene amplification and/or overexpression to trastuzumab-induced cell growth inhibition and apoptotic cell death. Strikingly, FAS inhibition synergistically interacts with trastuzumab in Her-2/neu-negative breast cancer cells engineered to overexpress Her-2/neu, thus suggesting that the molecular linkage between FAS activity and functioning of Her-2/neu cannot be explained only on the basis of a transcriptional repression of Her-2/neu gene promoter. Interestingly, while in liver and adipose tissue FAS produces fat from excess carbon consumed as carbohydrates, which is ultimately stored as triglycerides, in epithelial cancer cells, FAS activity is mainly involved in the production of phospholipids partitioning into detergent-resistant membrane microdomains (lipid raft-aggregates), which point to an active role of FAS in the deregulation of membrane functioning in tumor cells. Importantly, clusters of Her-2/neu and EGFR (erbB-1) co-localize with lipid rafts and the lipid environment in the cell membrane of breast cancer cells profoundly influences their association properties and biological functions. We hypothesize that pharmacological or small interference RNA-induced inhibition of breast cancer-associated FAS will result in major changes in the synthesis of phospholipids which, in turn, should impair a correct cellular localization of Her-2/neu at the cellular membrane of breast cancer cells. In this working model, FAS inhibition could induce a shift in the equilibrium between transport of Her-2/neu to and from the membrane favoring an increased Her-2/neu internalization followed by intracellular degradation, thus enhancing the mechanism of action of the anti-Her-2/neu antibody trastuzumab. Moreover, the inhibition of FAS-driven lipid rafts will also negatively affect EGFR-Her-2/neu cross-talk, an important mechanism of trastuzumab resistance. In summary, the specific blockade of a novel molecular linkage between FAS-regulated membrane composition and functioning of transmembrane growth factor receptors EGFR and Her-2/neu may represent a previously unrecognized therapeutic approach circumventing trastuzumab resistance in breast carcinomas.

Role of androgen deprivation in EGFR and Her-2/neu gene expression patterns in prostate cancer

9574 BACKGROUND: Her2/neu and epidermal growth factor receptor (EGFR) signaling pathways have been implicated in prostate cancer progression. Androgenic regulation of these epidermal growth factors (EGF) has been previously reported in prostate cancer. To evaluate the effect of androgen deprivation, a standard treatment for advanced stage on EGF members, we compared HER2/neu and EGFR gene expression in freshly resected tissue from advanced, hormone sensitive, prostate cancer patients undergoing androgen deprivation (AD) and tissue from patients who had not received androgen deprivation. METHODS: Tissue from 9 early stage cancer patients (prostatectomy) and 6 advanced stage patients undergoing trans-urethral resection of prostate were analyzed. Gene expression was quantitated at mRNA level by real–time polymerase chain reaction. Transcript abundance was calculated relative to standard curves generated using a cDNA pool of all samples and was normalized to 18s (control). T–test for independent samples was used for detecting differences between means for the two groups. RESULTS: All specimens (n=15) expressed HER2/neu and EGFR. Mean expression and comparisons between groups are shown in Table 1. A correlation between EGFR and Her2/neu was also performed using Pearson's coefficient; r = 0.69 (95% CI: 0.27–0.89; 2 tailed p–value 0.0048). CONCLUSIONS: Stimulation of prostate cancer proliferation by androgens and EGFs has also been reported in cell culture (1). We hypothesize that AD decreased gene expression of EGF family members when patients are initially responding to this maneuver as seen in our study population. Additional inhibition of EGFR and/or Her2/neu during hormone sensitive disease may further prolong duration of response to AD and delay emergence of hormone refractoriness and should be tested in clinical trials. Ref 1 Ye, D. et al: Clin Can Research vol 5, 2171–77, 1999. No significant financial relationships to disclose.

Role of androgen deprivation in EGFR and Her-2/neu gene expression patterns in prostate cancer

Role of androgen deprivation in EGFR and Her-2/neu gene expression patterns in prostate cancer