Sylvie Ménard

MicroRNA Gene Expression Deregulation in Human Breast Cancer

MicroRNAs (miRNAs) are a class of small noncoding RNAs that control gene expression by targeting mRNAs and triggering either translation repression or RNA degradation. Their aberrant expression may be involved in human diseases, including cancer. Indeed, miRNA aberrant expression has been previously found in human chronic lymphocytic leukemias, where miRNA signatures were associated with specific clinicobiological features. Here, we show that, compared with normal breast tissue, miRNAs are also aberrantly expressed in human breast cancer. The overall miRNA expression could clearly separate normal versus cancer tissues, with the most significantly deregulated miRNAs being mir-125b, mir-145, mir-21, and mir-155. Results were confirmed by microarray and Northern blot analyses. We could identify miRNAs whose expression was correlated with specific breast cancer biopathologic features, such as estrogen and progesterone receptor expression, tumor stage, vascular invasion, or proliferation index.

Pilot Study of the Mechanism of Action of Preoperative Trastuzumab in Patients with Primary Operable Breast Tumors Overexpressing HER2

Purpose: To elucidate the mechanism by which trastuzumab, a humanized monoclonal antibody against HER2 with proven survival benefit in women with HER2-positive metastatic breast cancer, mediates its antitumor activity. Experimental Design: A pilot study including 11 patients with HER2-positive tumors treated in a neo-adjuvant setting with trastuzumab was performed. Trastuzumab was administered i.v. at a dose of 4 mg/kg followed by three weekly i.v. doses of 2 mg/kg. The primary tumor was surgically removed 7 days after the last treatment. Surgical samples, tumor biopsies, and lymphocytes from these patients were collected for biological studies. Result: Clinical data indicated one complete pathological remission and four partial remissions using RECIST (Response Evaluation Criteria in Solid Tumors). Trastuzumab was well tolerated and neither serious adverse events nor changes in cardiac function were observed during this short-term treatment and after surgery. The biological data showed that, independent of response, (a) all patients showed high levels of circulating trastuzumab; (b) saturating level of trastuzumab was present in all of the tumors; (c) no down-modulation of HER2 was observed in any tumors; (d) no changes in vessel diameter was observed in any tumors; (e) no changes in proliferation was observed in any tumors; and (f) a strong infiltration by lymphoid cells was observed in all cases. Patients with complete remission or partial remission were found to have a higher in situ infiltration of leukocytes and a higher capability to mediate in vitro antibody-dependent cellular cytotoxicity activity. Conclusions: The results of this pilot study argue against trastuzumab activity in patients through down-modulation of HER2 but in favor of antibody-dependent cellular cytotoxicity guiding efforts to optimize the use of trastuzumab in breast cancer patients.

High-titer HER-2/neu protein-specific antibody can be detected in patients with early-stage breast cancer.

PURPOSE To evaluate HER-2/neu-specific antibody immunity in patients with breast cancer, to determine the rate of occurrence of serum antibodies to HER-2/neu in patients with breast cancer, and to relate the presence of specific immunity to overexpression of HER-2/neu protein in primary tumor. METHODS The antibody response to HER-2/neu protein was analyzed in 107 newly diagnosed breast cancer patients. Sera was analyzed for the presence of HER-2/neu-specific antibodies with a capture enzyme-linked immunosorbent assay (ELISA) and verified by Western blot. Sera from 200 volunteer blood donors was used as a control population. RESULTS The presence of antibodies to HER-2/neu correlated with the presence of breast cancer. HER-2/neu antibodies at titers of > or = 1:100 were detected in 12 of 107 (11%) breast cancer patients versus none of 200 (0%) normal controls (P < .01). The presence of antibodies to HER-2/neu also correlated to overexpression of HER-2/neu protein in the patients primary tumor. Nine of 44 (20%) patients with HER-2/neu-positive tumors had HER-2/neu-specific antibodies, whereas three of 63 (5%) patients with HER-2/neu-negative tumors had antibodies (P = .03). The antibody responses could be substantial. Titers of greater than 1:5,000 were detected in five of 107 (5%). CONCLUSION The presence of HER-2/neu antibodies in breast cancer patients and the correlation with HER-2/neu-positive cancer implies that immunity to HER-2/neu develops as a result of exposure of patients to HER-2/neu protein expressed by their own cancer. These findings should stimulate further studies to develop the detection of immunity to oncogenic proteins as tumor markers, as well as the development and testing of vaccine strategies to induce and augment immunity to HER-2/neu for the treatment of breast cancer or prevention of recurrent disease.

Biologic and therapeutic role of HER2 in cancer

Overexpression of the human epidermal growth factor-2 (HER2) oncogene in human breast carcinomas has been associated with a more aggressive course of disease. The reason for this association is still unclear, although it has been suggested to rest in increased proliferation, vessel formation, and/or invasiveness. Alternatively, prognosis may not be directly related to the presence of the oncoprotein on the cell membrane, but instead to the breast carcinoma subset identified by HER2 overexpression and characterized by a peculiar gene expression profile. HER2 has also been associated with sensitivity to anthracyclins and resistance to endocrine therapy, suggesting that tyrosine kinase receptor and hormone receptor pathways represent two major proliferation pathways exclusively active in breast carcinomas, one sensitive to chemotherapeutic drugs and the other to antiestrogens. HER2 currently represents one of the most appropriate targets for specific therapy. Indeed, trastuzumab, a monoclonal antibody directed against the extracellular domain of HER2, is therapeutically active in HER2-positive breast carcinomas. However, a consistent number of HER2-positive tumors is not responsive to HER2-driven therapy, indicating the need for a better understanding of the mechanism of action of this new biological drug in vivo. While preclinical studies suggest antibody-dependent cell cytotoxicity as the major mechanism, determination of NK activity at the time of treatment remains mandatory, especially in patients treated with immunosuppressive drugs. The efficacy of prophylactic vaccination has been fully demonstrated in preclinical models, whereas ongoing studies of active immunotherapy using a variety of vaccination regimens against HER2 in tumor-bearing mice and patients have met with only moderate success.

microRNA-205 Regulates HER3 in Human Breast Cancer

An increasing amount of experimental evidence shows that microRNAs can have a causal role in breast cancer tumorigenesis as a novel class of oncogenes or tumor suppressor genes, depending on the targets they regulate. HER2 overexpression is a hallmark of a particularly aggressive subset of breast tumors, and its activation is strictly dependent on the trans-interaction with other members of HER family; in particular, the activation of the PI3K/Akt survival pathway, so critically important in tumorigenesis, is predominantly driven through phosphorylation of the kinase-inactive member HER3. Here, we show that miR-205, down-modulated in breast tumors compared with normal breast tissue, directly targets HER3 receptor, and inhibits the activation of the downstream mediator Akt. The reintroduction of miR-205 in SKBr3 cells inhibits their clonogenic potential and increases the responsiveness to tyrosine-kinase inhibitors Gefitinib and Lapatinib, abrogating the HER3-mediated resistance and restoring a potent proapoptotic activity. Our data describe miR-205 as a new oncosuppressor gene in breast cancer, able to interfere with the proliferative pathway mediated by HER receptor family. Our study also provides experimental evidence suggesting that miR-205 can improve the responsiveness to specific anticancer therapies.

New insights into the role of extracellular matrix during tumor onset and progression

Recently, a view of the tumor as a functional tissue interconnected with the microenvironment has recently been described. For many years, the stroma has been studied in the context of the malignant lesion, and only rarely has its role been considered before carcinogenic lesions appear. Recent studies have provided evidence that stromal cells and their products can cause the transformation of adjacent cells through transient signaling that leads to the disruption of homeostatic regulation, including control of tissue architecture, adhesion, cell death, and proliferation. It is now well established that tumor progression requires a continually evolving network of interactions between neoplastic cells and extracellular matrix. A relevant step of this process is the remodeling of microenvironment which surrounds tumors leading to the release of ECM‐associated growth factors which can then stimulate tumor and/or endothelial cells. Finally, tumor cells reorganizing the extracellular matrix to facilitate communications and escape the homeostatic control exerted by the microenvironment modify response to cytotoxic treatments.

Role of HER2 gene overexpression in breast carcinoma

The HER2 proto‐oncogene encodes a transmembrane glycoprotein of 185 kDa (p185HER2) with intrinsic tyrosine kinase activity. Amplification of the HER2 gene and overexpression of its product induce cell transformation. Numerous studies have demonstrated the prognostic relevance of p185HER2, which is overexpressed in 10% to 40% of human breast tumors. Recent data suggest that p185HER2 is a ligand orphan receptor that amplifies the signal provided by other receptors of the HER family by heterodimerizing with them. Ligand‐dependent activation of HER1, HER3, and HER4 by EGF or heregulin results in heterodimerization and, thereby, HER2 activation. HER2 overexpression is associated with breast cancer patient responsiveness to doxorubicin, to cyclophosphamide, methotrexate, and fluorouracil (CMF), and to paclitaxel, whereas tamoxifen was found to be ineffective and even detrimental in patients with HER2‐positive tumors. In vitro analyses have shown that the role of HER2 overexpression in determining the sensitivity of cancer cells to drugs is complex, and molecules involved in its signaling pathway are probably the actual protagonists of the sensitivity to drugs. The association of HER2 overexpression with human tumors, its extracellular accessibility, as well as its involvement in tumor aggressiveness are all factors that make this receptor an appropriate target for tumor‐specific therapies. A number of approaches are being investigated as possible therapeutic strategies that target HER2: (1) growth inhibitory antibodies, which can be used alone or in combination with standard chemotherapeutics; (2) tyrosine kinase inhibitors (TKI), which have been developed in an effort to block receptor activity because phosphorylation is the key event leading to activation and initiation of the signaling pathway; and (3) active immunotherapy, because the HER2 oncoprotein is immunogenic in some breast carcinoma patients. J. Cell. Physiol. 182:150–162, 2000.

FOXP3 Expression and Overall Survival in Breast Cancer

PURPOSE The transcription factor forkhead box P3 (FOXP3) up- or downregulates a large number of genes and has been recently reported to be expressed in tumor cells. We investigated the prognostic importance of FOXP3 expression in patients with breast cancer. PATIENTS AND METHODS The expression patterns of FOXP3 were characterized by immunohistochemistry in primary breast carcinoma specimens from patients of the Milan 3 and 1 trials. Kaplan-Meier analysis and Cox proportional hazards regression modeling were used to assess the overall survival, distant metastasis-free survival, and local relapse cumulative incidence, according to the presence or absence of FOXP3 expression. RESULTS FOXP3 expression in tumors was associated with worse overall survival probability and the risk increased with increasing FOXP3 immunostaining intensity. FOXP3 was also a strong prognostic factor for distant metastases-free survival but not for local recurrence risk. In multivariate analysis FOXP3 resulted an independent prognostic factor and the hazard ratio of FOXP3 expression and of lymph node positivity were similar. In the Milan 3 trial, the probability of 10-year survival in node-negative subgroup was 100% for FOXP3-negative and 82% for FOXP3-positive patients; in node-positive subgroup 82% for FOXP3-negative and 41% for FOXP3-positive patients. Even in the Milan 1 trial the lack of FOXP3 expression in node-positive subgroup was related to a significantly better prognosis than in FOXP3-positive patients (10-year survival probability, 89% v 59%). CONCLUSION The data identify FOXP3 expression as a new independent prognostic factor in breast carcinoma, which might help to improve the selection of patients for appropriate therapy.

Tumor-Initiating Cells of HER2-Positive Carcinoma Cell Lines Express the Highest Oncoprotein Levels and Are Sensitive to Trastuzumab

Purpose: The existence of tumor-initiating cells in breast cancer has profound implications for cancer therapy. In this study, we investigated the sensitivity of tumor-initiating cells isolated from human epidermal growth factor receptor type 2 (HER2)-overexpressing carcinoma cell lines to trastuzumab, a compound used for the targeted therapy of breast cancer. Experimental Design: Spheres were analyzed by indirect immunofluorescence for HER2 cell surface expression and by real-time PCR for HER2 mRNA expression in the presence or absence of the Notch1 signaling inhibitor (GSI) or Notch1 small interfering RNA. Xenografts of HER2-overexpressing breast tumor cells were treated with trastuzumab or doxorubicin. The sphere-forming efficiency (SFE) and serial transplantability of tumors were assessed. Results: In HER2-overexpressing carcinoma cell lines, cells with tumor-initiating cell properties presented increased HER2 levels compared with the bulk cell population without modification in HER2 gene amplification. HER2 levels were controlled by Notch1 signaling, as shown by the reduction of HER2 cell surface expression and lower SFE following γ-secretase inhibition or Notch1 specific silencing. We also show that trastuzumab was able to effectively target tumor-initiating cells of HER2-positive carcinoma cell lines, as indicated by the significant decrease in SFE and the loss of serial transplantability, following treatment of HER2-overexpressing xenotransplants. Conclusions: Here, we provide evidence for the therapeutic efficacy of trastuzumab in debulking and in targeting tumor-initiating cells of HER2-overexpressing tumors. We also propose that Notch signaling regulates HER2 expression, thereby representing a critical survival pathway of tumor-initiating cells.

Role of HER receptors family in development and differentiation

Members of the epidermal growth factor receptor family of receptor tyrosine kinases play a critical role in both development and oncogenesis. The latter is suggested by the frequent overexpression of HER‐2, EGFR, and HER‐3 in some human carcinomas, primarily breast and squamous cancer. The biological activities of the EGFR family are exerted through various ligand–receptor and receptor–receptor interactions. One receptor that plays a central role in this signaling network is HER‐2/Neu, which is considered the preferred heterodimerization partner for other members of the EGFR family. The role of these receptors and their ligands in development is discussed, with particular emphasis on their ability to mediate a variety of pathways and cellular responses, including proliferation, differentiation, and apoptosis.