Search

Introduction: New levels of gene regulation with microRNA (miR) and gene copy number alterations (CNAs) have been identified as playing a role in various cancers. We have previously reported that sporadic breast cancer tissues exhibit significant alteration in H2AX gene copy number. However, how CNA affects gene expression and what is the role of miR, miR-24-2, known to regulate H2AX expression, in the background of the change in copy number, are not known. Further, many miRs, including miR-24-2, are implicated as playing a role in cell proliferation and apoptosis, but their specific target genes and the pathways contributing to them remain unexplored., Methods: Changes in gene copy number and mRNA/miR expression were estimated using real-time polymerase chain reaction assays in two mammalian cell lines, MCF-7 and HeLa, and in a set of sporadic breast cancer tissues. In silico analysis was performed to find the putative target for miR-24-2. MCF-7 cells were transfected with precursor miR-24-2 oligonucleotides, and the gene expression levels of BRCA1, BRCA2, ATM, MDM2, TP53, CHEK2, CYT-C, BCL-2, H2AFX and P21 were examined using TaqMan gene expression assays. Apoptosis was measured by flow cytometric detection using annexin V dye. A luciferase assay was performed to confirm BCL-2 as a valid cellular target of miR-24-2., Results: It was observed that H2AX gene expression was negatively correlated with miR-24-2 expression and not in accordance with the gene copy number status, both in cell lines and in sporadic breast tumor tissues. Further, the cells overexpressing miR-24-2 were observed to be hypersensitive to DNA damaging drugs, undergoing apoptotic cell death, suggesting the potentiating effect of mir-24-2-mediated apoptotic induction in human cancer cell lines treated with anticancer drugs. BCL-2 was identified as a novel cellular target of miR-24-2., Conclusions: mir-24-2 is capable of inducing apoptosis by modulating different apoptotic pathways and targeting BCL-2, an antiapoptotic gene. The study suggests that miR-24-2 is more effective in controlling H2AX gene expression, regardless of the change in gene copy number. Further, the study indicates that combination therapy with miR-24-2 along with an anticancer drug such as cisplatin could provide a new avenue in cancer therapy for patients with tumors otherwise resistant to drugs.

In breast cancer cells with HER2 gene amplification, HER2 receptors exist on the cell surface as monomers, homodimers, and heterodimers with EGFR/HER3. The therapeutic antibody trastuzumab, an approved therapy for HER2(+) breast cancer, cannot block ligand-induced HER2 heterodimers, suggesting it cannot effectively inhibit HER2 signaling. Hence, HER2 oligomeric states may predict the odds of a clinical response to trastuzumab in HER2-driven tumors. To test this hypothesis, we generated nontransformed human MCF10A mammary epithelial cells stably expressing a chimeric HER2-FKBP molecule that could be conditionally induced to homodimerize by adding the FKBP ligand AP1510, or instead induced to heterodimerize with EGFR or HER3 by adding the heterodimer ligands EGF/TGFα or heregulin. AP1510, EGF, and heregulin each induced growth of MCF10A cells expressing HER2-FKBP. Trastuzumab inhibited homodimer-mediated but not heterodimer-mediated cell growth. In contrast, the HER2 antibody pertuzumab, which blocks HER2 heterodimerization, inhibited growth induced by heregulin but not AP1510. Lastly, the HER2/EGFR tyrosine kinase inhibitor lapatinib blocked both homodimer- and heterodimer-induced growth. AP1510 triggered phosphorylation of Erk1/2 but not AKT, whereas trastuzumab inhibited AP1510-induced Erk1/2 phosphorylation and Shc-HER2 homodimer binding, but not TGFα-induced AKT phosphorylation. Consistent with these observations, high levels of HER2 homodimers correlated with longer time to progression following trastuzumab therapy in a cohort of patients with HER2-overexpressing breast cancer. Together, our findings confirm the notion that HER2 oligomeric states regulate HER2 signaling, also arguing that trastuzumab sensitivity of homodimers may reflect their inability to activate the PI3K (phosphoinositide 3-kinase)/AKT pathway. A clinical implication of our results is that high levels of HER2 homodimers may predict a positive response to trastuzumab., (©2011 AACR.)

HER2 is gene amplified or over-expressed in 20-25% of breast cancers resulting in elevated HER2 activation. Trastuzumab (Herceptin), a humanized monoclonal antibody, targets activated HER2 and is clinically effective in HER2-over-expressing breast cancers. However, despite prolonged survival, treated breast cancer patients develop resistance. Resistance to trastuzumab occurs upon inactivation of HER2 regulatory proteins or upon up-regulation of alternative receptors. In particular, elevated levels of EGFR, present in estrogen receptor (ER) positive, trastuzumab-resistant BT-474 xenografts caused, a trastuzumab-resistant phenotype (Ritter et al. Clin Cancer Res 13:4909-4919, 2007). However, the role of EGFR in acquired trastuzumab resistance in ER negative cell models is not well defined. In this study, SKBR3 cell line clones expressing EGFR were generated to examine the role of EGFR over-expression on trastuzumab sensitivity in an, ER-negative breast carcinoma cell line. A stable clone, SKBR3/EGFR (clone 4) expressing moderate levels of EGFR remained sensitive to trastuzumab, whereas a stable clone, SKBR3/EGFR (clone 5) expressing high levels of EGFR, became resistant to trastuzumab. Depletion of EGFR by EGFR small-interfering RNAs in the SKBR3/EGFR (clone 5) reversed trastuzumab resistance. However, the SKBR3/EGFR (clone 5) cell line remained sensitive to lapatinib, an EGFR/HER2 inhibitor. Biochemical analysis using co-immunoprecipitation and proximity-based quantitative VeraTag assays demonstrated that high levels of EGFR phosphorylation, EGFR/EGFR homo-dimerization, and EGFR/HER2 hetero-dimerization were present in the trastuzumab-resistant cells. We conclude that EGFR over-expression can mediate trastuzumab resistance in both ER positive and ER negative cells and hypothesize that a threshold level of EGFR, in the absence of autocrine ligand production, is required to induce the resistant phenotype.

The availability of drugs targeting the EGFR/HER/erbB signaling pathway has created a need for diagnostics that accurately predict treatment responses. We have developed and characterized a novel assay to provide sensitive and quantitative measures of HER proteins and homodimers in formalin-fixed, paraffin-embedded (FFPE) cell lines and breast tumor tissues, to test these variables. In the VeraTag assay, HER proteins and homodimers are detected through the release of fluorescent tags conjugated to specific HER antibodies, requiring proximity to a second HER antibody. HER2 protein quantification was normalized to tumor area, and compared to receptor numbers in 12 human tumor cell lines determined by fluorescence-activated cell sorting (FACS), and with HER immunohistochemistry (IHC) test categories and histoscores in cell lines and 170 breast tumors. HER1 and HER2 expression levels determined by the VeraTag assay are proportional to receptor number over more than a 2 log10 range, and HER homodimer levels are consistent with crosslinking and immunoprecipitation results. VeraTag HER2 measurements of breast tumor tissue and cell lines correlate with standard IHC test categories (P<0.001). VeraTag HER2 levels also agree with IHC histoscores at lower HER2 protein levels, but are continuous and overlapping between IHC test categories, extending the dynamic range 5-fold to 10-fold at higher HER2 levels. The VeraTag assay specifically and reproducibly measures HER1 and HER2 protein and homodimers in FFPE tissues. The continuous measure of HER2 protein levels over a broad dynamic range, and the novel HER2 homodimer measure, are presently being assessed as predictive markers for responses to targeted HER2 therapy.

This pilot study examines the feasibility of nipple aspiration to distinguish women with breast cancer from healthy women using surface-enhanced laser desorption ionisation time-of-flight mass spectrometry (SELDI-TOF/MS). Nipple aspiration fluid (NAF) was collected from each breast in 21 women newly diagnosed with unilateral breast cancer and 44 healthy women. No differences were found when proteomic profiles of NAF from the cancer-bearing breast and the contralateral non-cancerous breast were compared. In contrast, 9 protein peaks were significantly different between the cancer-bearing breast compared with healthy women and 10 peaks were significantly different between the contralateral healthy breast and healthy women (P<0.05). These data suggest that invasive breast cancer may result in a field change across both breasts and that proteomic profiling of NAF may have more value in breast cancer risk assessment than as a diagnostic or screening tool.

A proteomic approach to nipple aspiration fluid (NAF) has been used in a number of studies comparing women with breast cancer and healthy women. However, to make useful comparisons between women with breast cancer and healthy women it is necessary to establish whether there is physiological variation in the proteomic profiles of NAF. The purpose of this study was, for the first time, to examine how the proteomic profile of NAF using surface-enhanced laser desorption ionisation time-of-flight mass spectrometry varies across the menstrual cycle in healthy pre-menopausal women. Twelve women were recruited and nipple aspiration was carried out weekly from both breasts of each subject for two menstrual cycles. Matching serum samples for luteinising hormone, follicle stimulating hormone and oestradiol were obtained at each aspiration attempt. Statistically significant peaks were found for three healthy volunteers (p < 0.05). However, the peaks that varied across the menstrual cycle were different from one healthy volunteer to another and the differences were small compared with the large variation in proteomic profiles between healthy volunteers. This study provides proof of concept that the NAF proteomic profile does not vary substantially during the menstrual cycle and that therefore it is valid to compare NAF profiles from pre-menopausal women that have been taken at different stages in the menstrual cycle.

Established methods of breast cancer detection have well-described limitations, and new diagnostic techniques are evolving continually to improve diagnostic accuracy. The intraductal approach encompasses the modalities of nipple aspiration, ductal lavage, and duct endoscopy, and is a means of directly accessing the microenvironment of the breast and either sampling or visualizing this intraductal milieu. The aim of sampling this mammary microenvironment is to obtain samples from the physical surroundings of cells that are undergoing malignant transformation, thereby providing a new method of detection before the development of a clinically or radiologically discernible mass. A literature review was conducted to investigate the evolution of the intraductal approach and its particular application in the field of biomarker discovery, primarily using the intraductal technique of nipple aspiration, in combination with emerging protein profiling techniques.

Background: Capsular contracture occurs more frequently when immediate breast reconstruction (IBR) is associated with radiotherapy (RT) in a post-mastectomy field. The aim of this study was to investigate the impact of RT on surgical outcome after IBR using a single implant type., Methods: One hundred and thirty-six breast reconstructions were studied in 114 patients: 62 reconstructions were performed using submuscular implants alone and 74 had an implant-assisted latissimus dorsi myocutaneous flap using a McGhan 150 biodimensional permanent expander implant. Data were prospectively collected on capsule contracture, geometric measurements, photographic assessments and pain scores. The median follow-up was 4 (range, 2-5) years., Results: The mean age of the 114 patients studied was 45 (range, 20-77) years. Forty-four reconstructed breasts received RT. Capsule formation was detected in 13/92 (14.1%) reconstructed breasts with no RT and in 17/44 (38.6%) reconstructed breasts with RT. On univariate analysis, RT was the only variable related to capsule formation (p<0.001). Significant differences in geometric measurements of symmetry were identified in patients with capsules compared with those without capsules. Photographic assessments were worse in the capsule group: mean photo score 8 (95% CI 8, 8.5) compared with the no capsule group 6.5 (95% CI 5, 7.5), p<0.001. Persistent pain two years or more after surgery was present in 8/30 patients with capsules and 1/106 with no capsule group, p<0.01. Capsule formation is three times more likely to occur after IBR in association with an RT field. However, as more than 60% of patients do not get capsules despite RT at four years, implant-assisted tissue expansion techniques using a biodimensional device is a viable breast reconstructive option in selected cases.

Aims: It is well recognised that intravasation of tumour cells into the vasculature and/or lymphatics is a key stage in the metastatic process. It is also clear that very little is known about the mechanisms underlying this event. In this review, we will focus on cell surface molecules that may be instrumental in mediating the attachment of tumour cells, and in particular breast carcinoma cells, to the lymphatic and microvascular endothelia and discuss the therapeutic and prognostic value in targeting these receptors in metastatic disease., Methods: A literature search was carried out from PubMed for indexed articles and reviews. Websites containing information on gene expression profiles were located using standard web browser search functions. For articles containing gene expression data, relevant information was frequently located in supplementary tables or in associated websites., Findings: The search yielded a very large number of indexed published articles and websites. Important major reports and studies were reviewed, screened and tracked for other relevant publications. The most important articles were analysed and discussed., Conclusions: The lack of knowledge as to the mechanism by which tumour cells intra-vasate into the vasculature and/or lymphatics is perhaps not surprising given the lack of suitable models with which to investigate tumour cell intravasation. However, recent advances in the identification of molecular markers of angiogenic and lymphangiogenic endothelium, the development of techniques to image tumour cells in vivo and a better understanding of the architecture of these vessels is beginning to offer hope that this least well understood event in the metastatic process is becoming more amenable to study.