Correlation between trophoblast cell-surface antigen-2 (Trop-2) expression and pathological complete response in patients with HER2-positive early breast cancer treated with neoadjuvant docetaxel, carboplatin, trastuzumab, and pertuzumab.

PURPOSE: The prognostic and predictive role of trophoblast cell-surface antigen-2 (Trop-2) overexpression in human epidermal growth factor receptor 2-positive (HER2-positive) breast cancer is currently unknown. We retrospectively analyzed Trop-2 expression and its correlation with clinicopathologic features and pathological complete response (pCR) in HER2-positive early breast cancer (EBC) patients treated with neoadjuvant docetaxel, carboplatin, trastuzumab, and pertuzumab in the PHERGain study. METHODS: Trop-2 expression at baseline was determined in formalin-fixed, paraffin-embedded primary tumor biopsies by immunohistochemistry and was first classified into expressing (Trop-2-positive) or not-expressing (Trop-2-negative) tumors. Then, it was classified by histochemical score (H-score) according to its intensity into low (0-9), intermediate (10-49), and high (≥ 50). The association between clinicopathologic features, pCR, and Trop-2 expression was performed with Fisher's exact test. RESULTS: Forty-one patients with tissue evaluable for Trop-2 expression were included, with 28 (68.3%) Trop-2-positive tumors. Overall, 17 (41.46%), 14 (34.15%), and 10 (24.40%) tumors were classified as low, intermediate, and high, respectively. Trop-2 expression was significantly associated with decreased pCR rates (50.0% vs. 92.3%; odds ratio [OR] 0.05; 95% CI, 0.002-0.360]; p adjusted = 0.01) but was not correlated with any clinicopathologic features (p ≥ 0.05). Tumors with the highest Trop-2 H-score were less likely to obtain a pCR (OR 0.03; 95% CI, 0.001-0.290, p adjusted < 0.01). This association was confirmed in univariate and multivariate regression analyses. CONCLUSION: These findings suggest a potential role of Trop-2 expression as a biomarker of resistance to neoadjuvant chemotherapy plus dual HER2 blockade and may become a strategic target for future combinations in HER2-positive EBC patients.

The novel proautophagy anticancer drug ABTL0812 potentiates chemotherapy in adenocarcinoma and squamous nonsmall cell lung cancer.

Around 40% of newly diagnosed lung cancer patients are Stage IV, where the improvement of survival and reduction of disease-related adverse events is the main goal for oncologists. In this scenario, we present preclinical evidence supporting the use of ABTL0812 in combination with chemotherapy for treating advanced and metastatic Nonsmall cell lung adenocarcinomas (NSCLC) and squamous carcinomas. ABTL0812 is a new chemical entity, currently in Phase 1b/2a clinical trial for advanced squamous NSCLC in combination with paclitaxel and carboplatin (P/C), after successfully completing the first-in-human trial where it showed an excellent safety profile and signs of efficacy. We show here that ABTL0812 inhibits Akt/mTOR axis by inducing the overexpression of TRIB3 and activating autophagy in lung squamous carcinoma cell lines. Furthermore, treatment with ABTL0812 also induces AMPK activation and ROS accumulation. Moreover, combination of ABTL0812 with chemotherapy markedly increases the therapeutic effect of chemotherapy without increasing toxicity. We further show that combination of ABTL0812 and chemotherapy induces nonapoptotic cell death mediated by TRIB3 activation and autophagy induction. We also present preliminary clinical data indicating that TRIB3 could serve as a potential novel pharmacodynamic biomarker to monitor ABTL0812 activity administered alone or in combination with chemotherapy in squamous NSCLC patients. The safety profile of ABTL0812 and its good synergy with chemotherapy potentiate the therapeutic potential of current lines of treatment based on chemotherapy regimens, arising as a promising option for improving these patients therapeutic expectancy.

Glucocorticoids promote transition of ductal carcinoma in situ to invasive ductal carcinoma by inducing myoepithelial cell apoptosis.

BACKGROUND: The microenvironment and stress factors like glucocorticoids have a strong influence on breast cancer progression but their role in the first stages of breast cancer and, particularly, in myoepithelial cell regulation remains unclear. Consequently, we investigated the role of glucocorticoids in ductal carcinoma in situ (DCIS) in breast cancer, focusing specially on myoepithelial cells. METHODS: To clarify the role of glucocorticoids at breast cancer onset, we evaluated the effects of cortisol and corticosterone on epithelial and myoepithelial cells using 2D and 3D in vitro and in vivo approaches and human samples. RESULTS: Glucocorticoids induce a reduction in laminin levels and favour the disruption of the basement membrane by promotion of myoepithelial cell apoptosis in vitro. In an in vivo stress murine model, increased corticosterone levels fostered the transition from DCIS to invasive ductal carcinoma (IDC) via myoepithelial cell apoptosis and disappearance of the basement membrane. RU486 is able to partially block the effects of cortisol in vitro and in vivo. We found that myoepithelial cell apoptosis is more frequent in patients with DCIS+IDC than in patients with DCIS. CONCLUSIONS: Our findings show that physiological stress, through increased glucocorticoid blood levels, promotes the transition from DCIS to IDC, particularly by inducing myoepithelial cell apoptosis. Since this would be a prerequisite for invasive features in patients with DCIS breast cancer, its clinical management could help to prevent breast cancer progression to IDC.

HER2DX Genomic Assay in HER2-positive Early Breast Cancer Treated with Trastuzumab and Pertuzumab: A Correlative Analysis from PHERGain Phase II Trial.

PURPOSE: To assess the predictive capability of HER2DX assay following (neo)adjuvant trastuzumab-pertuzumab (HP)-based therapy in HER2-positive (HER2+) early breast cancer (EBC). EXPERIMENTAL DESIGN: HER2DX was analyzed in baseline pre-treatment tumors from PHERGain trial. Patients with stage I-IIIA HER2+ EBC were randomized to group A (docetaxel, carboplatin, and HP [TCHP]) and group B (HP ± endocrine therapy). PET response was evaluated after 2 cycles. Group A received TCHP for 6 cycles regardless of PET response. Group B continued with HP ± endocrine therapy for 6 cycles (PET-responders) or with TCHP for 6 cycles (PET-non-responders). The primary objective was to associate HER2DX pCR-score with pathological complete response (pCR). The secondary objective was the association of HER2DX risk-score with 3-year invasive disease-free survival (iDFS). RESULTS: HER2DX was performed on 292 (82.0%) tumors. The overall pCR rate was 38.0%, with pCR rates of 56.4% in group A and 33.8% in group B. In multivariable analysis including treatment and clinicopathological factors, HER2DX pCR-score (continuous variable) significantly correlated with pCR (odds ratio [OR]=1.29, 95% confident interval [CI] 1.10-1.54, p<0.001). HER2DX-defined pCR-high, med, and low groups exhibited pCR rates of 50.4%, 35.8%, and 23.2%, respectively (pCR-high vs pCR-low OR=3.27, CI 1.54-7.09, p<0.001). In patients with residual disease, HER2DX high-risk group demonstrated numerically worse 3-year iDFS than the low-risk group (89.8% vs 100%; HR= 2.70, 95% CI 0.60-12.18, p=0.197). CONCLUSIONS: HER2DX predicts pCR in the context of neoadjuvant HP-based therapy, regardless of chemotherapy addition, and might identify patients at higher risk of recurrence among patients with residual disease.

The neuronal influence on tumor progression.

Nerve fibers accompany blood and lymphatic vessels all over the body. An extensive amount of knowledge has been obtained with regard to tumor angiogenesis and tumor lymphangiogenesis, yet little is known about the potential biological effects of "neoneurogenesis". Cancer cells can exploit the advantage of the factors released by the nerve fibers to generate a positive microenvironment for cell survival and proliferation. At the same time, they can stimulate the formation of neurites by secreting neurotrophic factors and axon guidance molecules. The neuronal influence on the biology of a neoplasm was initially described several decades ago. Since then, an increasing amount of experimental evidence strongly suggests the existence of reciprocal interactions between cancer cells and nerves in humans. Moreover, researchers have been able to demonstrate a crosstalk between cancer cells and nerve fibers as a strategy for survival. Despite all these evidence, a lot remains to be done in order to clarify the role of neurotransmitters, neuropeptides, and their associated receptor-initiated signaling pathways in the development and progression of cancer, and response to therapy. A global-wide characterization of the neurotransmitters or neuropeptides present in the tumor microenvironment would provide insights into the real biological influences of the neuronal tissue on tumor progression. This review is intended to discuss our current understanding of neurosignaling in cancer and its potential implications on cancer prevention and therapy. The review will focus on the soluble factors released by cancer cells and nerve endings, their biological effects and their potential relevance in the treatment of cancer.

Histamine signaling and metabolism identify potential biomarkers and therapies for lymphangioleiomyomatosis.

Inhibition of mTOR is the standard of care for lymphangioleiomyomatosis (LAM). However, this therapy has variable tolerability and some patients show progressive decline of lung function despite treatment. LAM diagnosis and monitoring can also be challenging due to the heterogeneity of symptoms and insufficiency of non-invasive tests. Here, we propose monoamine-derived biomarkers that provide preclinical evidence for novel therapeutic approaches. The major histamine-derived metabolite methylimidazoleacetic acid (MIAA) is relatively more abundant in LAM plasma, and MIAA values are independent of VEGF-D. Higher levels of histamine are associated with poorer lung function and greater disease burden. Molecular and cellular analyses, and metabolic profiling confirmed active histamine signaling and metabolism. LAM tumorigenesis is reduced using approved drugs targeting monoamine oxidases A/B (clorgyline and rasagiline) or histamine H1 receptor (loratadine), and loratadine synergizes with rapamycin. Depletion of Maoa or Hrh1 expression, and administration of an L-histidine analog, or a low L-histidine diet, also reduce LAM tumorigenesis. These findings extend our knowledge of LAM biology and suggest possible ways of improving disease management.

Cripto-1 is required for hypoxia to induce cardiac differentiation of mouse embryonic stem cells.

Cripto-1 is a membrane-bound protein that is highly expressed in embryonic stem cells and in human tumors. In the present study, we investigated the effect of low levels of oxygen, which occurs naturally in rapidly growing tissues, on Cripto-1 expression in mouse embryonic stem (mES) cells and in human embryonal carcinoma cells. During hypoxia, Cripto-1 expression levels were significantly elevated in mES cells and in Ntera-2 or NCCIT human embryonal carcinoma cells, as compared with cells growing with normal oxygen levels. The transcription factor hypoxia-inducible factor-1alpha directly regulated Cripto-1 expression by binding to hypoxia-responsive elements within the promoter of mouse and human Cripto-1 genes in mES and NCCIT cells, respectively. Furthermore, hypoxia modulated differentiation of mES cells by enhancing formation of beating cardiomyocytes as compared with mES cells that were differentiated under normoxia. However, hypoxia failed to induce differentiation of mES cells into cardiomyocytes in the absence of Cripto-1 expression, demonstrating that Cripto-1 is required for hypoxia to fully differentiate mES cells into cardiomyocytes. Finally, cardiac tissue samples derived from patients who had suffered ischemic heart disease showed a dramatic increase in Cripto-1 expression as compared with nonischemic heart tissue samples, suggesting that hypoxia may also regulate Cripto-1 in vivo.

Breast Mammographic Density: Stromal Implications on Breast Cancer Detection and Therapy.

Current evidences state clear that both normal development of breast tissue as well as its malignant progression need many-sided local and systemic communications between epithelial cells and stromal components. During development, the stroma, through remarkably regulated contextual signals, affects the fate of the different mammary cells regarding their specification and differentiation. Likewise, the stroma can generate tumour environments that facilitate the neoplastic growth of the breast carcinoma. Mammographic density has been described as a risk factor in the development of breast cancer and is ascribed to modifications in the composition of breast tissue, including both stromal and glandular compartments. Thus, stroma composition can dramatically affect the progression of breast cancer but also its early detection since it is mainly responsible for the differences in mammographic density among individuals. This review highlights both the pathological and biological evidences for a pivotal role of the breast stroma in mammographic density, with particular emphasis on dense and malignant stromas, their clinical meaning and potential therapeutic implications for breast cancer patients.

Tumor-Associated Fibroblasts Promote HER2-Targeted Therapy Resistance through FGFR2 Activation.

PURPOSE: Despite the therapeutic success of existing HER2-targeted therapies, tumors invariably relapse. This study aimed at identifying new mechanisms responsible for HER2-targeted therapy resistance. EXPERIMENTAL DESIGN: We have used a platform of HER2-targeted therapy-resistant cell lines and primary cultures of healthy and tumor-associated fibroblasts (TAF) to identify new potential targets related to tumor escape from anti-HER2 therapies. RESULTS: We have shown that TAFs promote resistance to HER2-targeted therapies. TAFs produce and secrete high levels of FGF5, which induces FGFR2 activation in the surrounding breast cancer cells. FGFR2 transactivates HER2 via c-Src, leading to resistance to HER2-targeted therapies. In vivo, coinoculating nonresistant cell lines with TAFs results in more aggressive and resistant tumors. Resistant cells activate fibroblasts and secrete FGFR ligands, creating a positive feedback loop that fuels resistance. FGFR2 inhibition not only inhibits HER2 activation, but also induces apoptosis in cells resistant to HER2-targeted therapies. In vivo, inhibitors of FGFR2 reverse resistance and resensitize resistant cells to HER2-targeted therapies. In HER2 patients' samples, α-SMA, FGF5, and FGFR2 contribute to poor outcome and correlate with c-Src activation. Importantly, expression of FGF5 and phospho-HER2 correlated with a reduced pathologic complete response rate in patients with HER2-positive breast cancer treated with neoadjuvant trastuzumab, which highlights the significant role of TAFs/FGF5 in HER2 breast cancer progression and resistance. CONCLUSIONS: We have identified the TAF/FGF5/FGFR2/c-Src/HER2 axis as an escape pathway responsible for HER2-targeted therapy resistance in breast cancer, which can be reversed by FGFR inhibitors.

Characterization of the glycosylphosphatidylinositol-anchor signal sequence of human Cryptic with a hydrophilic extension.

Epidermal Growth Factor-Cripto-1/FRL-1/Cryptic (EGF-CFC) proteins, including human Cripto-1 (hCFC2/hCR-1) and human Cryptic (hCFC1), are membrane-associated Nodal co-receptors, which have critical roles in vertebrate development. Most of the EGF-CFC proteins have been experimentally proven or predicted to be glycosylphosphatidylinositol (GPI)-anchored proteins. However, unlike other EGF-CFC proteins, hCFC1 does not exhibit a typical GPI-signal sequence, containing a 32-amino acid hydrophilic extension in its COOH-terminal end. Here we experimentally demonstrate that the COOH-terminal sequence of hCFC1 functions as a GPI-anchoring signal. Moreover, addition of a hydrophilic epitope tag of 55-amino acids (V5-His) after the GPI signal of hCR-1 interfered with generation of a GPI-anchored form of hCR-1. In contrast, addition of the same epitope tag to the end of GPI signal of hCFC1 did not affect the GPI-attachment of hCFC1. The COOH-terminal signal of hCFC1 could produce two different forms of the protein; a GPI-anchored form and an unprocessed form which was more prone to be secreted into the conditioned medium. The hydrophilic extension of hCFC1 negatively regulates the activity of hCFC1 as a Nodal co-receptor. These results demonstrate the presence of endogenous GPI-signal sequence with a hydrophilic extension, which can generate both GPI-anchored and soluble forms of the protein.

Smad2 functions as a co-activator of canonical Wnt/beta-catenin signaling pathway independent of Smad4 through histone acetyltransferase activity of p300.

Both canonical Wnt/beta-catenin and TGFbeta/Smad signaling pathways coordinately regulate pattern formation during embryogenesis as well as tumor progression. Evidence of cross-talk between these two pathways has been reported. Here we demonstrated that the Activin-like kinase 4 (Alk4)/Smad2 pathway facilitates the transcriptional activity of the oncogenic Wnt/beta-catenin/Tcf4 pathway through a novel Smad4-independent mechanism. Upon activation, Smad2 physically interacted with Tcf4, beta-catenin and the co-activator p300 to enhance transcriptional activity of beta-catenin/Tcf4 through the histone acetyltransferase activity of p300. Transactivation by Smad2 was independent of a Smad-binding element (SBE) and Smad4. Indeed, the enhancement of beta-catenin/Tcf4 transcriptional activity by activated Smad2 was negatively regulated by the presence of Smad4. Moreover, a tumor-derived missense mutant of Smad2, lacking the ability to bind to Smad4 was still able to enhance the Tcf4 transcriptional reporter in the presence of beta-catenin and Tcf4. Our findings suggest that Smad2 may function as an activator of canonical Wnt/beta-catenin/Tcf4 signaling through a SBE/Smad4-independent pathway.

Regulation of human Cripto-1 gene expression by TGF-beta1 and BMP-4 in embryonal and colon cancer cells.

Human Cripto-1 (CR-1) is a cell membrane protein that is overexpressed in several different types of human carcinomas. In the present study we investigated the mechanisms that regulate the expression of CR-1 gene in cancer cells. We cloned a 2,481 bp 5'-flanking region of the human CR-1 gene into a luciferase reporter vector and transfected NTERA-2 human embryonal carcinoma cells and LS174-T colon cancer cells to test for promoter activity. Activity of CR-1 promoter in both cell lines was modulated by two TGF-beta family members, TGF-beta1 and BMP-4. In particular, TGF-beta1 significantly up-regulated CR-1 promoter activity, whereas a dramatic reduction in CR-1 promoter activity was observed with BMP-4 in NTERA-2 and LS174-T cells. Changes in the CR-1 promoter activity following TGF-beta1 and BMP-4 treatments correlated with changes in CR-1 mRNA and protein expression in NTERA-2 and LS174-T cells. We also identified three Smad binding elements (SBEs) within the CR-1 promoter and point mutation of SBE1 (-2,197/-2,189) significantly reduced response of the CR-1 promoter to both TGF-beta1 and BMP-4 in NTERA-2 and LS174-T cells. Chromatin immunoprecipitation assay also demonstrated binding of Smad-4 to a CR-1 promoter DNA sequence containing SBE1 in LS174-T cells. Finally, BMP-4 inhibited migration of LS174-T cells and F9 mouse embryonal carcinoma cells by downregulation of CR-1 protein. In conclusion, these results suggest a differential modulation of CR-1 gene expression in embryonal and colon cancer cells by two different members of the TGF-beta family.

Netrin-1 can affect morphogenesis and differentiation of the mouse mammary gland.

Netrin-1 has been shown to regulate the function of the EGF-like protein Cripto-1 (Cr-1) and affect mammary gland development. Since Cr-1 is a target gene of Nanog and Oct4, we investigated the relationship between Netrin-1 and Cr-1, Nanog and Oct4 during different stages of development in the mouse mammary gland. Results from histological analysis show that exogenous Netrin-1 was able to induce formation of alveolar-like structures within the mammary gland terminal end buds of virgin transgenic Cripto-1 mice and enhance mammary gland alveologenesis in early pregnant FVB/N mice. Results from immunostaining and Western blot analysis show that Netrin-1, Nanog and Oct4 are expressed in the mouse embryonic mammary anlage epithelium while Cripto-1 is predominantly expressed outside this structure in the surrounding mesenchyme. We find that in lactating mammary glands of postnatal FVB/N mice, Netrin-1 expression is highest while Cripto-1 and Nanog levels are lowest indicating that Netrin-1 may perform a role in the mammary gland during lactation. HC-11 mouse mammary epithelial cells stimulated with lactogenic hormones and exogenous soluble Netrin-1 showed increased beta-casein expression as compared to control thus supporting the potential role for Netrin-1 during functional differentiation of mouse mammary epithelial cells. Finally, mouse ES cells treated with exogenous soluble Netrin-1 showed reduced levels of Nanog and Cripto-1 and higher levels of beta-III tubulin during differentiation. These results suggest that Netrin-1 may facilitate functional differentiation of mammary epithelial cells and possibly affect the expression of Nanog and/or Cripto-1 in multipotent cells that may reside in the mammary gland.

Activation of a Nodal-independent signaling pathway by Cripto-1 mutants with impaired activation of a Nodal-dependent signaling pathway.

Cripto-1, a co-receptor for Nodal, can activate Nodal-dependent and Nodal-independent signaling pathways. In this study we have investigated whether Cripto-1 mutants, that fail to activate a Nodal-dependent signaling pathway, are capable to activate a Nodal-independent signaling pathway in mammary epithelial cells. Cripto-1 mutants expressed in EpH4 mouse mammary epithelial cells are fully functional in regard to activation of a Nodal-independent signaling pathway, leading to phosphorylation of mitogen-activated protein kinase (MAPK) and Akt and to enhanced proliferation and motility of these cells, suggesting that Cripto-1 mutants with impaired Nodal signaling are still active in a Nodal-independent signaling pathway.

Histamine receptor 1 inhibition enhances antitumor therapeutic responses through extracellular signal-regulated kinase (ERK) activation in breast cancer.

Histamine receptor 1 (HRH1) belongs to the rhodopsin-like G-protein-coupled receptor family. Its activation by histamine triggers cell proliferation, embryonic development, and tumor growth. We recently established that HRH1 is up-regulated in basal and human epidermal growth factor receptor 2 (HER2)-enriched human breast tumors and that its expression correlates with a worse prognosis. Nevertheless, the functional role of HRH1 in basal and HER2-targeted therapy-resistant breast cancer (BC) progression has not yet been addressed. Using terfenadine, a selective chemical inhibitor of HRH1, we showed that the inhibition of HRH1 activity in basal BC cells leads to sub-G0 cell accumulation, suppresses proliferation, promotes cell motility and triggers the activation of extracellular signal-regulated kinase (ERK) signaling, initiating the mitochondrial apoptotic pathway. Furthermore, HER2-targeted therapy-resistant cells express higher levels of HRH1 and are more sensitive to terfenadine treatment. Moreover, in vivo experiments showed that terfenadine therapy reduced the tumor growth of basal and trastuzumab-resistant BC cells. In conclusion, our results suggest that targeting HRH1 is a promising new clinical approach to consider that could enhance the effectiveness of current therapeutic treatment in patients with basal and BC tumors resistant to HER2-targeted therapies.

Identification of cripto-1 as a novel serologic marker for breast and colon cancer.

PURPOSE: Human Cripto-1 (CR-1), a cell membrane glycosylphosphatidylinositol-anchored glycoprotein that can also be cleaved from the membrane, is expressed at high levels in several different types of human tumors. We evaluated whether CR-1 is present in the plasma of patients with breast and colon cancer, and if it can represent a new biomarker for these malignancies. EXPERIMENTAL DESIGN: We determined CR-1 plasma levels using a sandwich-type ELISA in 21 healthy volunteers, 54 patients with breast cancer, 33 patients with colon carcinoma, and 21 patients with benign breast lesions. Immunohistochemical analysis was also used to assess CR-1 expression in cancerous tissues. RESULTS: Very low levels of CR-1 (mean+/-SD) were detected in the plasma of healthy volunteers (0.32+/-0.19 ng/mL). A statistically significant increase in the levels of plasma CR-1 was found in patients with colon carcinoma (4.68+/-3.5 ng/mL) and in patients with breast carcinoma (2.97+/-1.48 ng/mL; P<0.001). Although moderate levels of plasma CR-1 were found in women with benign lesions of the breast (1.7+/-0.99 ng/mL), these levels were significantly lower than in patients with breast cancer (P<0.001). Finally, immunohistochemical analysis and real-time reverse transcription-PCR confirmed strong positivity for CR-1 in colon and/or breast tumor tissues. CONCLUSION: This study suggests that plasma CR-1 might represent a novel biomarker for the detection of breast and colon carcinomas.

Epidermal growth factor receptor (EGFR) signaling in cancer.

The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases (RTK). These trans-membrane proteins are activated following binding with peptide growth factors of the EGF-family of proteins. Evidence suggests that the EGFR is involved in the pathogenesis and progression of different carcinoma types. The EGFR and EGF-like peptides are often over-expressed in human carcinomas, and in vivo and in vitro studies have shown that these proteins are able to induce cell transformation. Amplification of the EGFR gene and mutations of the EGFR tyrosine kinase domain have been recently demonstrated to occur in carcinoma patients. Interestingly, both these genetic alterations of the EGFR are correlated with high probability to respond to anti-EGFR agents. However, ErbB proteins and their ligands form a complex system in which the interactions occurring between receptors and ligands affect the type and the duration of the intracellular signals that derive from receptor activation. In fact, proteins of the ErbB family form either homo- or hetero-dimers following ligand binding, each dimer showing different affinity for ligands and different signaling properties. In this regard, evidence suggests that cooperation of multiple ErbB receptors and cognate ligands is necessary to induce cell transformation. In particular, the growth and the survival of carcinoma cells appear to be sustained by a network of receptors/ligands of the ErbB family. This phenomenon is also important for therapeutic approaches, since the response to anti-EGFR agents might depend on the total level of expression of ErbB receptors and ligands in tumor cells.

Differential expression of neurogenes among breast cancer subtypes identifies high risk patients.

The nervous system is now recognized to be a relevant component of the tumor microenvironment. Receptors for neuropeptides and neurotransmitters have been identified in breast cancer. However, very little is known about the role of neurogenes in regulating breast cancer progression. Our purpose was to identify neurogenes associated with breast cancer tumorigenesis with a potential to be used as biomarker and/or targets for treatment. We used three databases of human genes: GeneGo, GeneCards and Eugenes to generate a list of 1266 relevant neurogenes. Then we used bioinformatics tools to interrogate two published breast cancer databases SAGE and MicMa (n=96) and generated a list of 7 neurogenes that are differentially express among breast cancer subtypes. The clinical potential was further investigated using the GOBO database (n=1881). We identified 6 neurogenes that are differentially expressed among breast cancer subtypes and whose expression correlates with prognosis. Histamine receptor1 (HRH1), neuropilin2 (NRP2), ephrin-B1 (EFNB1), neural growth factor receptor (NGFR) and amyloid precursor protein (APP) were differentially overexpressed in basal and HER2-enriched tumor samples and syntaxin 1A (STX1A) was overexpressed in HER2-enriched and luminal B tumors. Analysis of HRH1, NRP2, and STX1A expression using the GOBO database showed that their expression significantly correlated with a shorter overall survival (p < 0.0001) and distant metastasis-free survival (p < 0.0001). In contrast, elevated co-expression of NGFR, EFNB1 and APP was associated with longer overall (p < 0.0001) and metastasis-free survival (p < 0.0001). We propose that HRH1, NRP2, and STX1A can be used as prognostic biomarkers and therapeutic targets for basal and HER2-enriched breast cancer subtypes.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in breast cancer: current status and future development.

Evidence suggests that the epidermal growth factor receptor (EGFR) and its ligands are involved in the pathogenesis of different human carcinomas, including breast cancer. Results of phase II clinical trials of EGFR tyrosine kinase inhibitors (TKIs) have shown that these compounds have little activity in breast cancer patients when used as single agents. The potential pitfalls of these clinical trials, and the molecular mechanisms that might be involved in regulating the sensitivity/resistance of breast cancer cells to EGFR TKIs are discussed in this brief article. In particular, preclinical findings clearly demonstrate that breast cancer cells are able to activate different mechanisms to escape the anti-tumor effects of drugs directed against growth factor-driven pathways. Therefore, it is conceivable that significant blockade of tumor growth might be obtained only through contemporary blockade of different growth promoting pathways, at least in advanced disease. In addition, preclinical and clinical findings support the use of EGFR TKIs in specific subgroups of breast cancer patients, such as estrogen receptor positive (ER+), tamoxifen resistant patients. In this regard, we describe potential future applications of these compounds in combination with other agents in the treatment of breast carcinoma.

Comparison of methods for the isolation of human breast epithelial and myoepithelial cells.

Two lineages, epithelial, and myoepithelial cells are the main cell populations in the normal mammary gland and in breast cancer. Traditionally, cancer research has been performed using commercial cell lines, but primary cell cultures obtained from fresh breast tissue are a powerful tool to study more reliably new aspects of mammary gland biology, including normal and pathological conditions. Nevertheless, the methods described to date have some technical problems in terms of cell viability and yield, which hamper work with primary mammary cells. Therefore, there is a need to optimize technology for the proper isolation of epithelial and myoepithelial cells. For this reason, we compared four methods in an effort to improve the isolation and primary cell culture of different cell populations of human mammary epithelium. The samples were obtained from healthy tissue of patients who had undergone mammoplasty or mastectomy surgery. We based our approaches on previously described methods, and incorporated additional steps to ameliorate technical efficiency and increase cell survival. We determined cell growth and viability by phase-contrast images, growth curve analysis and cell yield, and identified cell-lineage specific markers by flow cytometry and immunofluorescence in 3D cell cultures. These techniques allowed us to better evaluate the functional capabilities of these two main mammary lineages, using CD227/K19 (epithelial cells) and CD10/K14 (myoepithelial cells) antigens. Our results show that slow digestion at low enzymatic concentration combined with the differential centrifugation technique is the method that best fits the main goal of the present study: protocol efficiency and cell survival yield. In summary, we propose some guidelines to establish primary mammary epithelial cell lines more efficiently and to provide us with a strong research instrument to better understand the role of different epithelial cell types in the origin of breast cancer.