Transgenic mice with mammary gland targeted expression of human cortactin do not develop (pre-malignant) breast tumors: studies in MMTV-cortactin and MMTV-cortactin/-cyclin D1 bitransgenic mice

BACKGROUND: In human breast cancers, amplification of chromosome 11q13 correlates with lymph node metastasis and increased mortality. To date, two genes located within this amplicon, CCND1 and EMS1, were considered to act as oncogenes, because overexpression of both proteins, respectively cyclin D1 and cortactin, correlated well with 11q13 amplification. Cyclin D1 is involved in cell cycle regulation and the F-actin-binding protein cortactin in cytoskeletal dynamics and cell migration. To study the role of cortactin in mammary gland tumorigenesis, we examined mouse mammary tumor virus (MMTV)-cortactin transgenic mice and MMTV-cortactin/-MMTV-cyclin D1 bitransgenic mice. METHODS: MMTV-cortactin transgenic mice were generated and intercrossed with previously described MMTV-cyclin D1 transgenic mice. Immunohistochemical, Northern and Western blot analyses were performed to study the expression of human transgene cortactin during mammary gland development and in mammary tumors. For tumor incidence studies, forced-bred, multiparous mice were used to enhance transgene expression in the mammary gland. Microscopical examination was performed using haematoxylin and eosin staining. RESULTS: Mammary gland tumors arose stochastically (incidence 21%) with a mean age of onset at 100 weeks. This incidence, however, did not exceed that of aged-matched control FVB/N mice (38%), which unexpectedly, also developed spontaneous mammary gland tumors. We mimicked 11q13 amplification by generating MMTV-cortactin/-MMTV-cyclin D1 bitransgenic mice but did not observe any synergistic effect of cortactin on cyclin D1-induced mammary hyperplasias or carcinomas, nor development of distant metastasis. CONCLUSION: From this study, we conclude that development of (pre-malignant) breast tumors in either wild type or MMTV-cyclin D1 mice was not augmented due to mammary gland targeted overexpression of human cortactin

Cortactin overexpression results in sustained epidermal growth factor receptor signaling by preventing ligand-induced receptor degradation in human carcinoma cells

The chromosome 11q13 region is frequently amplified in human carcinomas and results in an increased expression of various genes including cortactin, and is also associated with an increased invasive potential. Cortactin acts as an important regulator of the actin cytoskeleton. It is therefore very tempting to speculate that cortactin is the crucial gene within the 11q13 amplicon that mediates the invasive potential of these carcinomas. Cortactin also participates in receptor-mediated endocytosis, and recent findings have shown that, during receptor internalization, cortactin overexpression inhibits the ubiquitylation-mediated degradation of the epidermal growth factor receptor, resulting in a sustained ligand-induced epidermal growth factor receptor activity

Comparative genome analysis of cortactin and HS1: the significance of the F-actin binding repeat domain

Abstract Background In human carcinomas, overexpression of cortactin correlates with poor prognosis. Cortactin is an F-actin-binding protein involved in cytoskeletal rearrangements and cell migration by promoting actin-related protein (Arp)2/3 mediated actin polymerization. It shares a high amino acid sequence and structural similarity to hematopoietic lineage cell-specific protein 1 (HS1) although their functions differ considerable. In this manuscript we describe the genomic organization of these two genes in a variety of species by a combination of cloning and database searches. Based on our analysis, we predict the genesis of the actin-binding repeat domain during evolution. Results Cortactin homologues exist in sponges, worms, shrimps, insects, urochordates, fishes, amphibians, birds and mammalians, whereas HS1 exists in vertebrates only, suggesting that both genes have been derived from an ancestor cortactin gene by duplication. In agreement with this, comparative genome analysis revealed very similar exon-intron structures and sequence homologies, especially over the regions that encode the characteristic highly conserved F-actin-binding repeat domain. Cortactin splice variants affecting this F-actin-binding domain were identified not only in mammalians, but also in amphibians, fishes and birds. In mammalians, cortactin is ubiquitously expressed except in hematopoietic cells, whereas HS1 is mainly expressed in hematopoietic cells. In accordance with their distinct tissue specificity, the putative promoter region of cortactin is different from HS1. Conclusions Comparative analysis of the genomic organization and amino acid sequences of cortactin and HS1 provides inside into their origin and evolution. Our analysis shows that both genes originated from a gene duplication event and subsequently HS1 lost two repeats, whereas cortactin gained one repeat. Our analysis genetically underscores the significance of the F-actin binding domain in cytoskeletal remodeling, which is of importance for the major role of HS1 in apoptosis and for cortactin in cell migration.</p

Transgenic mice with mammary gland targeted expression of human cortactin do not develop (pre-malignant) breast tumors: studies in MMTV-cortactin and MMTV-cortactin/-cyclin D1 bitransgenic mice-4

<p><b>Copyright information:</b></p><p>Taken from "Transgenic mice with mammary gland targeted expression of human cortactin do not develop (pre-malignant) breast tumors: studies in MMTV-cortactin and MMTV-cortactin/-cyclin D1 bitransgenic mice"</p><p>BMC Cancer 2006;6():58-58.</p><p>Published online 14 Mar 2006</p><p>PMCID:PMC1450299.</p><p>Copyright © 2006 van Rossum et al; licensee BioMed Central Ltd.</p>plastic alveolar nodules (HAN) or adenosquamous adenoma; adenosquamous carcinoma; papillary carcinoma; cribriform carcinoma; adeno carcinoma; and spindle cell carcinoma. All pictures are taken with the same magnification except (H), which is taken with a two times higher magnification

Transgenic mice with mammary gland targeted expression of human cortactin do not develop (pre-malignant) breast tumors: studies in MMTV-cortactin and MMTV-cortactin/-cyclin D1 bitransgenic mice-2

<p><b>Copyright information:</b></p><p>Taken from "Transgenic mice with mammary gland targeted expression of human cortactin do not develop (pre-malignant) breast tumors: studies in MMTV-cortactin and MMTV-cortactin/-cyclin D1 bitransgenic mice"</p><p>BMC Cancer 2006;6():58-58.</p><p>Published online 14 Mar 2006</p><p>PMCID:PMC1450299.</p><p>Copyright © 2006 van Rossum et al; licensee BioMed Central Ltd.</p>ce as indicated by % mammary tumor free. No significant differences were seen. The age at which 50% of the animals were found to have mammary gland tumors, the T, is for each mouse line more than 100 weeks. n, number of mice examined; +, indicates those mice that died without a MG tumor (censored). Kaplan-Meier analysis determining the survival ratio of wild type and transgenic mice. MP1 and T16*MP1 were significantly different from WT (p = 0,034 and p = 0,017 respectively, log-rank test)

Transgenic mice with mammary gland targeted expression of human cortactin do not develop (pre-malignant) breast tumors: studies in MMTV-cortactin and MMTV-cortactin/-cyclin D1 bitransgenic mice-1

<p><b>Copyright information:</b></p><p>Taken from "Transgenic mice with mammary gland targeted expression of human cortactin do not develop (pre-malignant) breast tumors: studies in MMTV-cortactin and MMTV-cortactin/-cyclin D1 bitransgenic mice"</p><p>BMC Cancer 2006;6():58-58.</p><p>Published online 14 Mar 2006</p><p>PMCID:PMC1450299.</p><p>Copyright © 2006 van Rossum et al; licensee BioMed Central Ltd.</p>om the abdominal mammary gland of non-transgenic (W) and transgenic mouse line 16 (T) of virgin female mice (V), mice at day 11 and 17 of pregnancy (P11, P17), day 4, 10 and 17 of lactation (L4, L10, L17) and day 4 and 8 of involution (I4, I8). Monoclonal antibody Esab157 was used to recognize specific human transgene expression, whereas monoclonal antibody 4F11 recognizes both human and mouse cortactin. Actin was used as a loading control. Immunohistochemical detection of cortactin transgene in a 10-days lactating mammary gland (L10). WT, wild type; T16, transgenic mouse line 16. About 50% of epithelial cells from the transgenic section stained positive for cortactin. Arrows indicate cortactin localization at cell-cell contacts. Sections were counterstained with haematoxylin

Transgenic mice with mammary gland targeted expression of human cortactin do not develop (pre-malignant) breast tumors: studies in MMTV-cortactin and MMTV-cortactin/-cyclin D1 bitransgenic mice-0

<p><b>Copyright information:</b></p><p>Taken from "Transgenic mice with mammary gland targeted expression of human cortactin do not develop (pre-malignant) breast tumors: studies in MMTV-cortactin and MMTV-cortactin/-cyclin D1 bitransgenic mice"</p><p>BMC Cancer 2006;6():58-58.</p><p>Published online 14 Mar 2006</p><p>PMCID:PMC1450299.</p><p>Copyright © 2006 van Rossum et al; licensee BioMed Central Ltd.</p>rate a stable transgene mRNA; black bar, 1.8 kb human EMS1 cDNA [29]; hatched bar, transcriptional processing sequences derived from SV40. Relevant restriction sites are indicated. Southern blot analysis of tail DNA of three cortactin transgenic founders and one wild type (WT) mouse. The intensity of the bands corresponds to the increased copy number of the integrated transgenic cortactin cDNA. T6 contains one cDNA copy. Northern blot analysis of 10-days lactating mammary glands. Expression of human transgenic cortactin mRNA (3,6 kb) was detected in T16 only. Full-length human cortactin cDNA was used as a probe. Western blot analysis of 10-days lactating mammary glands to detect transgenic cortactin protein expression. Polyclonal antibody RA444 was used to recognize specific human transgene expression, whereas polyclonal antibody RA23 recognized both human and endogenous mouse cortactin. Virgin T16 transgenic (T) and non-transgenic (W) female mice were treated with glucocorticosteroid dexamethasone to stimulate transgene expression from the MMTV promoter. Lysates from liver (Li), salivary gland (SG), kidney (Ki), spleen (Sp), pancreas (Pa), lung (Lu) and 10-days lactating mammary gland as a positive control (MG) were subjected to Western blotting. Polyclonal antibody RA444 was used to detect specific human transgene expression, whereas monoclonal 4F11 antibody recognizes both human and mouse cortactin. Transgene expression was significantly induced in the pancreas, liver and kidney

Transgenic mice with mammary gland targeted expression of human cortactin do not develop (pre-malignant) breast tumors: studies in MMTV-cortactin and MMTV-cortactin/-cyclin D1 bitransgenic mice-3

<p><b>Copyright information:</b></p><p>Taken from "Transgenic mice with mammary gland targeted expression of human cortactin do not develop (pre-malignant) breast tumors: studies in MMTV-cortactin and MMTV-cortactin/-cyclin D1 bitransgenic mice"</p><p>BMC Cancer 2006;6():58-58.</p><p>Published online 14 Mar 2006</p><p>PMCID:PMC1450299.</p><p>Copyright © 2006 van Rossum et al; licensee BioMed Central Ltd.</p> 20 weeks of age. The dark spot in the middle represents a lymph node. Whole mounts of the abdominal mammary gland of post-weaned multiparous mice with mammary intraepithelial neoplasia (MIN) and hyperplastic alveolar nodules (HAN, arrows). The age in weeks and the number of pregnancies that the mouse went through, are indicated between brackets