Synergistic interaction of the Neu proto-oncogene product and transforming growth factor alpha in the mammary epithelium of transgenic mice.

Transgenic mice expressing either the neu proto-oncogene or transforming growth factor (TGF-alpha) in the mammary epithelium develop spontaneous focal mammary tumors that occur after a long latency. Since the epidermal growth factor receptor (EGFR) and Neu are capable of forming heterodimers that are responsive to EGFR ligands such as TGF-alpha, we examined whether coexpression of TGF-alpha and Neu in mammary epithelium could cooperate to accelerate the onset of mammary tumors. To test this hypothesis, we interbred separate transgenic strains harboring either a mouse mammary tumor virus/TGF-alpha or a mouse mammary tumor virus/neu transgene to generate bitransgenic mice that coexpress TGF-alpha and neu in the mammary epithelium. Female mice coexpressing TGF-alpha and neu developed multifocal mammary tumors which arose after a significantly shorter latency period than either parental strain alone. The development of these mammary tumors was correlated with the tyrosine phosphorylation of Neu and the recruitment of c-Src to the Neu complex. Immunoprecipitation and immunoblot analyses with EGFR- and Neu-specific antisera, however, failed to detect physical complexes of these two receptors. Taken together, these observations suggest that Neu and TGF-alpha cooperate in mammary tumorigenesis through a mechanism involving Neu and EGFR transactivation.     

Signaling through ShcA is required for transforming growth factor beta- and Neu/ErbB-2-induced breast cancer cell motility and invasion

Cooperation between the Neu/ErbB-2 and transforming growth factor beta (TGF-beta) signaling pathways enhances the invasive and metastatic capabilities of breast cancer cells; however, the underlying mechanisms mediating this synergy have yet to be fully explained. We demonstrate that TGF-beta induces the migration and invasion of mammary tumor explants expressing an activated Neu/ErbB-2 receptor, which requires signaling from autophosphorylation sites located in the C terminus. A systematic analysis of mammary tumor explants expressing Neu/ErbB-2 add-back receptors that couple to distinct signaling molecules has mapped the synergistic effect of TGF-beta-induced motility and invasion to signals emanating from tyrosine residues 1226/1227 and 1253 of Neu/ErbB-2. Given that the ShcA adaptor protein is known to interact with Neu/ErbB-2 through these residues, we investigated the importance of this signaling molecule in TGF-beta-induced cell motility and invasion. The reduction of ShcA expression rendered cells expressing activated Neu/ErbB-2, or add-back receptors signaling specifically through tyrosines 1226/1227 or 1253, unresponsive to TGF-beta-induced motility and invasion. In addition, a dominant-negative form of ShcA, lacking its three known tyrosine phosphorylation sites, completely abrogates the TGF-beta-induced migration and invasion of breast cancer cells expressing activated Neu/ErbB-2. Our results implicate signaling through the ShcA adaptor as a key component in the synergistic interaction between these pathways.     

Tumor metastasis in an orthotopic murine model of head and neck cancer: possible role of TGF-beta 1 secreted by the tumor cells

In an orthotopic murine model of head and neck cancer, combined subcutaneous and intratumoral vaccination with recombinant vaccinia virus expressing interleukin-2 (rvv-IL-2) induced significant tumor regression early on therapy. However, its efficacy was restricted by recurrent tumor growth and loco-regional metastases. In this study, we explored the mechanism of tumor metastasis. We compared the levels of expression of a number of molecules involved in tumor metastasis, which included transforming growth factor-beta1 (TGF-beta1), E-cadherin, matrix metalloproteinases (MMPs): MT1-MMP, MMP-2, MMP-9, their tissue inhibitors (TIMPs): TIMP-1/TIMP-2, and pro-angiogenic factors CD31, VEGF-R2, and iNOS between primary and metastatic tumors by real-time RT-PCR and immunohistochemistry. We detected spontaneous lymph node and tongue metastasis. Metastasis was delayed in rvv-IL-2 treated mice. Cultured tumor cells expressed negligible amount of TGF-beta1. Untreated or metastatic tumors, on the other hand, expressed high levels of TGF-beta1 and secreted TGF-beta1 in the sera of tumor-bearing mice. Levels of TGF-beta1 in the sera suddenly jumped at the time when tumor metastasis started. In the metastatic tumors, levels of MT1-MMP, MMP-2, and MMP-9 were significantly elevated (P < 0.001), while levels of TIMP-1/TIMP-2 and E-cadherin were decreased (P < 0.001) compared to control or primary tumors. Levels of CD31, VEGF-R2, and iNOS were also significantly elevated in the metastatic lesions (P < 0.001). The concurrence of high levels of TGF-beta1 in the sera, expression of proteins involved in metastasis and initiation of metastasis suggested possible role of TGF-beta1 in on setting the metastatic cascade in this model.     

Tyrosine phosphatase-epsilon activates Src and supports the transformed phenotype of Neu-induced mammary tumor cells

Few tyrosine phosphatases support, rather than inhibit, survival of tumor cells. We present genetic evidence that receptor-type protein-tyrosine phosphatase (RPTP)-epsilon performs such a function, as cells from mammary epithelial tumors induced by activated Neu in mice genetically lacking RPTPepsilon appeared morphologically less transformed and exhibited reduced proliferation. We show that at the molecular level, RPTPepsilon activates Src, a known collaborator of Neu in mammary tumorigenesis. Lack of RPTPepsilon reduced Src activity and altered Src phosphorylation in tumor cells; RPTPepsilon dephosphorylated and activated Src; and Src bound a substrate-trapping mutant of RPTPepsilon. The altered morphology of tumor cells lacking RPTPepsilon was corrected by exogenous Src and exogenous RPTPepsilon or RPTPalpha; exogenous activated Src corrected also the growth rate phenotype. Together, these results suggest that the altered morphology of RPTPepsilon-deficient tumor cells is caused by reduced Src activity, caused, in turn, by lack of RPTPepsilon. Unexpectedly, the phenotype of RPTPepsilon-deficient tumor cells occurs despite expression of the related RPTPalpha, indicating that endogenous RPTPalpha does not compensate for the absence of RPTPepsilon in this case. We conclude that RPTPepsilon is a physiological activator of Src in Neu-induced mammary tumors and suggest that pharmacological inhibition of phosphatases that activate Src may be useful to augment direct pharmacological inhibition of Src.     

Effects of BRCA1 transgene expression on murine mammary gland development and mutagen-induced mammary neoplasia

To characterize the role of BRCA1 in mammary gland development and tumor suppression, a transgenic mouse model of BRCA1 overexpression was developed. Using the mouse mammary tumor virus (MMTV) promoter/enhancer, transgenic mice expressing human BRCA1 or select mutant controls were generated. Transgenic animals examined during adolescence were shown to express the human transgene in their mammary glands. The mammary glands of 13-week-old virgin homozygous MMTV-BRCA1 mice presented the morphology of moderately increased lobulo-alveolar development. The mammary ductal trees of both hemizygous and homozygous MMTV-BRCA1t340 were similar to those of control non-transgenic littermates. Interestingly, both hemi- and homozygous mice expressing a splice variant of BRCA1 lacking the N-terminal RING finger domain (MMTV-BRCA1sv) exhibited marked mammary lobulo-alveolar development, particularly terminal end bud proliferation. Morphometric analyses of mammary gland whole mount preparations were used to measure epithelial staining indices of ~35% for homozygous MMTV-BRCA1 mice and ~60% for both hemizygous and homozygous MMTV-BRCA1sv mice versus ~25% for non-transgenic mice. Homozygous MMTV-BRCA1 mice showed delayed development of tumors when challenged with 7,12 dimethylbenzanthracene (DMBA), relative to non-transgenic and homozygous BRCA1t340 expressing mice. In contrast, homozygous MMTV-BRCA1sv transgenic animals were sensitized to DMBA treatment and exhibited a very rapid onset of mammary tumor development and accelerated mortality. MMTV-BRCA1 effects on mortality were restricted to DMBA-induced tumors of the mammary gland. These results demonstrate in vivo roles for BRCA1 in both mammary gland development and in tumor suppression against mutagen-induced mammary gland neoplasia.     

Comparative analysis of IFN-gamma B7.1 and antisense TGF-beta gene transfer on the tumorigenicity of a poorly immunogenic metastatic mammary carcinoma

Cancer progression is attributed in part to immune evasion strategies that include lack of co-stimulation, down-regulation of cell surface MHC molecules, and secretion of immunosuppressive factors, such as transforming growth factor-beta (TGF-beta). Gene therapy has been employed to counter these mechanisms of immune evasion by transference of B7.1, IFN-gamma or antisense TGF-beta genes into tumor cells, resulting in cell surface expression of B7.1, upregulation of MHC class I and class II molecules, or elimination of tumor-derived TGF-beta, respectively. Although each of these transgenes has been shown to alter tumorigenicity in murine models, a direct comparison of their efficacy has not been performed. In this study, we have employed a very aggressive, poorly immunogenic and highly metastatic mammary model, 4T1, to compare the efficacy of B7.1, IFN-gamma and antisense TGF-beta gene transfer in stimulating an anti-tumor response. We demonstrate that both IFN-gamma and antisense TGF-beta gene expression significantly reduced the tumorigenicity of these cells compared to mock transduced cells, with IFN-gamma having a greater effect. In contrast, B7.1 gene transfer did not affect the tumorigenicity of 4T1 cells. The anti-tumor response directed against antisense TGF-beta-expressing 4T1 tumors was mediated by CD4+ and CD8+ T cells. However, CD8+ T cells, and not CD4+ T cells, appeared to mediate the anti-tumor response against IFN-gamma-expressing tumors. Treatment of tumor-bearing animals with IFN-gamma or antisense TGF-beta gene-modified tumor cell vaccines reduced the number of clonogenic metastases to the lungs and liver compared to treatment with mock-transduced cells. Finally, in a residual disease model in which the primary tumor was excised and mice were vaccinated with irradiated tumor cells, treatment of mice with vaccinations consisting of 4T1 cells expressing both antisense TGF-beta and IFN-gamma genes was the most effective in prolonging survival.     

Mammary tumors expressing the neu proto-oncogene possess elevated c-Src tyrosine kinase activity.

Amplification and overexpression of the neu (c-erbB2) proto-oncogene has been implicated in the pathogenesis of 20 to 30% of human breast cancers. Although the activation of Neu receptor tyrosine kinase appears to be a pivotal step during mammary tumorigenesis, the mechanism by which Neu signals cell proliferation is unclear. Molecules bearing a domain shared by the c-Src proto-oncogene (Src homology 2) are thought to be involved in signal transduction from activated receptor tyrosine kinases such as Neu. To test whether c-Src was implicated in Neu-mediated signal transduction, we measured the activity of the c-Src tyrosine kinase in tissue extracts from either mammary tumors or adjacent mammary epithelium derived from transgenic mice expressing a mouse mammary tumor virus promoter/enhancer/unactivated neu fusion gene. The Neu-induced mammary tumors possessed six- to eightfold-higher c-Src kinase activity than the adjacent epithelium. The increase in c-Src tyrosine kinase activity was not due to an increase in the levels of c-Src but rather was a result of the elevation of its specific activity. Moreover, activation of c-Src was correlated with its ability to complex tyrosine-phosphorylated Neu both in vitro and in vivo. Together, these observations suggest that activation of the c-Src tyrosine kinase during mammary tumorigenesis may occur through a direct interaction with activated Neu.     

Anti‐tumor effect of SLPI on mammary but not colon tumor growth

Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor that was related to cancer development and metastasis dissemination on several types of tumors. However, it is not known the effect of SLPI on mammary and colon tumors. The aim of this study was to examine the effect of SLPI on mammary and colon tumor growth. The effect of SLPI was tested on in vitro cell apoptosis and in vivo tumor growth experiments. SLPI over‐expressing human and murine mammary and colon tumor cells were generated by gene transfection. The administration of murine mammary tumor cells over‐expressing high levels of SLPI did not develop tumors in mice. On the contrary, the administration of murine colon tumor cells over‐expressing SLPI, developed faster tumors than control cells. Intratumoral, but not intraperitoneal administration of SLPI, delayed the growth of tumors and increased the survival of mammary but not colon tumor bearing mice. In vitro culture of mammary tumor cell lines treated with SLPI, and SLPI producer clones were more prone to apoptosis than control cells, mainly under serum deprivation culture conditions. Herein we demonstrated that SLPI induces the apoptosis of mammary tumor cells in vitro and decreases the mammary but not colon tumor growth in vivo. Therefore, SLPI may be a new potential therapeutic tool for certain tumors, such as mammary tumors. J. Cell. Physiol. 228: 469–475, 2013. © 2012 Wiley Periodicals, Inc.     

Perforin is a major contributor to NK cell control of tumor metastasis.

We provide the first demonstration, using experimental and spontaneous models of metastasis in C57BL/6 (B6) (RM-1 prostate carcinoma) and BALB/c (DA3 mammary carcinoma) mice, that tumor metastasis is primarily controlled by perforin-dependent cytotoxicity mediated by NK1.1+ cells. MHC class Ilow RM-1 and DA3 tumor cells were sensitive in vitro to Fas-mediated lysis or spleen NK cells in a perforin-dependent fashion. Perforin-deficient NK cells did not lyse these tumors, and perforin-deficient mice were 10-100-fold less proficient than wild-type mice in rejecting the metastasis of tumor cells to the lung. Fas ligand mutant gld mice displayed uncompromised protection against tumor metastasis. Depletion of NK subsets resulted in greater numbers of metastases than observed in perforin-deficient mice, suggesting that perforin-independent effector functions of NK cells may also contribute to protection from tumor metastasis.     

Cell-type specific interaction of Neu differentiation factor (NDF/heregulin) with Neu/HER-2 suggests complex ligand-receptor relationships.

The Neu/HER-2 receptor tyrosine kinase is overexpressed in some types of human adenocarcinomas, including tumors of the breast and the ovary. A 44 kDa glycoprotein that elevates tyrosine phosphorylation of Neu has been isolated and named Neu differentiation factor (NDF), or heregulin. Here we show that NDF affects tyrosine phosphorylation of Neu in human tumor cells of breast, colon and neuronal origin, but not in ovarian cells that overexpress the receptor. By using monoclonal antibodies (mAbs) to Neu, we found that the ovarian receptor is immunologically and biochemically similar to the mammary p185neu. Nevertheless, unlike breast-derived Neu, the ovarian protein did not display covalent cross-linking to radiolabeled NDF, and was devoid of ligand-induced association with phosphatidylinositol 3'-kinase. Direct binding analysis showed that NDF binds with high affinity (Kd approximately 10(-9) M) to mammary cells, but its weak association with ovarian cells is probably mediated by heparin-like molecules. Similar to the endogenous receptor, the ectopically overexpressed Neu of mammary cells, but not of ovarian and fibroblastic cells, exhibited elevated levels of NDF-induced phosphorylation and covalent cross-linking of the radiolabeled factor. Taken together, our results imply that NDF binding to cells requires both Neu and an additional cellular component, whose identity is still unknown, but its tissue distribution is more restricted than the expression of the neu gene.     

Epithelial protein-tyrosine phosphatase 1B contributes to the induction of mammary tumors by HER2/Neu but is not essential for tumor maintenance

Protein-tyrosine phosphatase 1B (PTP1B), a well-established metabolic regulator, plays an important role in breast cancer. Using whole-body PTP1B knockout mice, recent studies have shown that PTP1B ablation delays HER2/Neu-induced mammary cancer. Whether PTP1B plays a cell-autonomous or a noncell-autonomous role in HER2/Neu-evoked tumorigenesis and whether it is involved in tumor maintenance was unknown. We generated mice expressing HER2/Neu and lacking PTP1B specifically in the mammary epithelium. We found that mammary-specific deletion of PTP1B delays the onset of HER2/Neu-evoked mammary tumors, establishing a cell autonomous role for PTP1B in such neoplasms. We also deleted PTP1B in established mouse mammary tumors or depleted PTP1B in human breast cancer cell lines grown as xenografts. PTP1B inhibition did not affect tumor growth in either model showing that neither epithelial nor stromal PTP1B is necessary for tumor maintenance. Taken together, our data show that despite the PTP1B contribution to tumor onset, it is not essential for tumor maintenance. This suggests that PTP1B inhibition could be effective in breast tumor prevention.     

Epithelial and fibroblast cell lines derived from a spontaneous mammary carcinoma in a MMTV/neu transgenic mouse

Female murine mammary tumor virus (MMTV)/neu transgenic mice, expressing a wild-type rat neu oncogene driven by an MMTV promoter, develop focal mammary adenocarcinomas that are pathologically very similar to human breast tumors. Two new cell lines were established from a mammary tumor that arose in a female MMTV/neu transgenic mouse. One of these lines, mammary carcinoma from Neu transgenic mouse A (MCNeuA), has an epithelial morphology, is cytokeratin positive, and expresses high levels of the neu transgene. Karyotyping and comparative genomic hybridization analyses demonstrated genomic alterations in the MCNeuA cell line. The other line, N202Fb3, has a fibroblast morphology, is cytokeratin negative, and expresses the neu transgene at a very low level. This cell line also expresses smooth muscle alpha-actin, suggesting that it is a myofibroblast line. The MCNeuA cell line is tumorigenic when injected into syngeneic MMTV/neu transgenic mice, with an in vivo doubling time of about 14 d. The rationale for establishing this tumor cell line was to provide a tumor transplantation system for rapidly assessing immunotherapeutic interventions before testing in the more cumbersome model of spontaneous tumor development in the MMTV/neu transgenic mice. Mice immunized with a Neu extracellular domain protein vaccine were protected against a subsequent inoculation of MCNeuA cells, indicating that this cell line will be useful for evaluating cancer vaccine strategies. This tumor cell line may also prove useful in studying the biological properties of the neu oncogene and its role in the malignant process. In addition, the tumor-derived fibroblast line may be useful for studying tumor-stromal cell interactions.     

Behavior of metastatic and nonmetastatic breast tumors in old mice

Breast cancer incidence and mortality increase with age. A better understanding of the biological behavior of metastatic and nonmetastatic breast tumors in older subjects may help to develop improved breast cancer therapies. In this study, we used syngeneic metastatic (4TO7cg) and nonmetastatic (64pT) mouse breast tumor models at three age levels to evaluate various characteristics that are considered to be important for effective anti-breast cancer immunotherapy. These included tumor size and growth, metastases, vascularization, gene expression levels of the tumor-associated antigen (TAA) Mage-b (homologous to human MAGE-B) in primary breast tumors and metastases, and the presence of CD4(+) and CD8(+) T cells in the inguinal lymph nodes at the site of the tumor. The primary breast tumors and metastases were generated by injection of mouse mammary tumor cell lines 4TO7cg or 64pT into a mammary fat pad of normal 3-, 9-, or 21/24-month old BALB/c mice. In the nonmetastatic breast tumor model, significantly smaller tumors were observed in old compared with young mice. This was associated with a significant increase in the percentage of CD8(+) T cells in inguinal lymph nodes and significantly higher Mage-b expression levels in the primary tumors at old age. In the metastatic (4TO7cg) breast tumor model, a less pronounced, not statistically significant, smaller tumor size was found in the old mice, without a difference in the percentage of CD8(+) T cells or Mage-b expression levels. However, in this mouse model almost all metastases showed high levels of Mage-b expression (2- to 3-fold higher than the primary tumors in the same animals) regardless of age. These results indicate that the metastatic and nonmetastatic breast tumor models could be useful model systems to analyze how breast cancer vaccines for humans can be tailored to old age.     

Disseminated Breast Cancer Cells Acquire a Highly Malignant and Aggressive Metastatic Phenotype during Metastatic Latency in the Bone

Background

Disseminated tumor cells (DTCs) in the bone marrow may exist in a dormant state for extended periods of time, maintaining the ability to proliferate upon activation, engraft at new sites, and form detectable metastases. However, understanding of the behavior and biology of dormant breast cancer cells in the bone marrow niche remains limited, as well as their potential involvement in tumor recurrence and metastasis. Therefore, the purpose of this study was to investigate the tumorigenicity and metastatic potential of dormant disseminated breast cancer cells (prior to activation) in the bone marrow.

Methodology/Principal Findings

Total bone marrow, isolated from mice previously injected with tumorspheres into the mammary fat pad, was injected into the mammary fat pad of NUDE mice. As a negative control, bone marrow isolated from non-injected mice was injected into the mammary fat pad of NUDE mice. The resultant tumors were analyzed by immunohistochemistry for expression of epithelial and mesenchymal markers. Mouse lungs, livers, and kidneys were analyzed by H+E staining to detect metastases. The injection of bone marrow isolated from mice previously injected with tumorspheres into the mammary fat pad, resulted in large tumor formation in the mammary fat pad 2 months post-injection. However, the injection of bone marrow isolated from non-injected mice did not result in tumor formation in the mammary fat pad. The DTC-derived tumors exhibited accelerated development of metastatic lesions within the lung, liver and kidney. The resultant tumors and the majority of metastatic lesions within the lung and liver exhibited a mesenchymal-like phenotype.

Conclusions/Significance

Dormant DTCs within the bone marrow are highly malignant upon injection into the mammary fat pad, with the accelerated development of metastatic lesions within the lung, liver and kidney. These results suggest the acquisition of a more aggressive phenotype of DTCs during metastatic latency within the bone marrow microenvironment.     

Involvement of insulin receptor substrate 2 in mammary tumor metastasis

The insulin receptor substrate (IRS) proteins are adaptor molecules that integrate signals generated by receptors that are implicated in human breast cancer. We investigated the specific contribution of IRS-2 to mammary tumor progression using transgenic mice that express the polyoma virus middle T antigen (PyV-MT) in the mammary gland and IRS-2-null (IRS-2(-/-)) mice. PyV-MT-induced tumor initiation and growth were similar in wild-type (WT) and IRS-2(-/-) mice. However, the latency and incidence of metastasis were significantly decreased in the absence of IRS-2 expression. The contribution of IRS-2 to metastasis is intrinsic to the tumor cells, because IRS-2(-/-) mammary tumor cells did not metastasize when grown orthotopically in the mammary fat pads of WT mice. WT and IRS-2(-/-) tumors contained similar numbers of mitotic cells, but IRS-2(-/-) tumors had a higher incidence of apoptosis than did WT tumors. In vitro, IRS-2(-/-) mammary tumor cells were less invasive and more apoptotic in response to growth factor deprivation than their WT counterparts. In contrast, IRS-1(-/-) tumor cells, which express only IRS-2, were highly invasive and were resistant to apoptotic stimuli. Collectively, our findings reveal an important contribution of IRS-2 to breast cancer metastasis.     

Metastasis-associated protein 1 deregulation causes inappropriate mammary gland development and tumorigenesis

Emerging data suggest that metastasis-associated protein 1 (MTA1) represses ligand-dependent transactivation functions of estrogen receptor-alpha in cultured breast cancer cells and that MTA1 is upregulated in human breast tumors. However, the role of MTA1 in tumorigenesis in a physiologically relevant animal system remains unknown. To reveal the role of MTA1 in mammary gland development, transgenic mice expressing MTA1 under the control of the mouse mammary tumor virus promoter long terminal repeat were generated. Unexpectedly, we found that mammary glands of these virgin transgenic mice exhibited extensive side branching and precocious differentiation because of increased proliferation of ductal and alveolar epithelial cells. Mammary glands of virgin transgenic mice resemble those from wild-type mice in mid-pregnancy and inappropriately express beta-casein, cyclin D1 and beta-catenin protein. Increased ductal growth was also observed in the glands of ovariectomized female mice, as well as of transgenic male mice. MTA1 dysregulation in mammary epithelium and cancer cells triggered downregulation of the progesterone receptor-B isoform and upregulation of the progesterone receptor-A isoform, resulting in an imbalance in the native ratio of progesterone receptor A and B isoforms. MTA1 transgene also increased the expression of progesterone receptor-A target genes Bcl-XL (Bcl2l1) and cyclin D1 in mammary gland of virgin mice, and, subsequently, produced a delayed involution. Remarkably, 30% of MTA1 transgenic females developed focal hyperplastic nodules, and about 7% exhibited mammary tumors within 18 months. These studies establish, for the first time, a potential role of MTA1 in mammary gland development and tumorigenesis. The underlying mechanism involves the upregulation of progesterone receptor A and its targets, Bcl-XL and cyclin D1.     

Transforming growth factor-beta signaling helps specify tumor type in DMBA and hormone-induced mammary cancers

To determine the role of transforming growth factor-beta (TGF-beta) signaling in mammary development and tumor formation, we previously generated transgenic mice that expressed a dominant-negative form of the TGF-beta type II receptor (DNIIR) under the control of DNA regulatory elements from the metallothionein promoter (MT-DNIIR-28). In this report, we tested the hypothesis that loss of TGF-beta signaling in the mammary gland alters the development of chemically or hormonally induced tumors in mice. Four groups of mice were used in the study: wild-type and MT-DNIIR-28 mice on zinc with pituitary isograft, and wild-type and MT-DNIIR-28 mice on zinc with pituitary isograft treated with the carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA). Tumor-free survival over time, tumor growth rate, and tumor pathology were measured. Statistically significant differences in tumor free survival over time or tumor growth rate were not detected in wild-type versus transgenic mice treated with DMBA. In contrast, tumor-free survival was significantly altered in transgenic mice that were treated with the pituitary isograft alone with MT-DNIIR mice developing tumors more quickly. Alterations in the types of tumors that formed in wild-type versus MT-DNIIR DMBA-treated mice were detected. In wild-type mice, tumors with squamous differentiation or bicellular adenomyoepitheliomas were most common. Adenomyoepitheliomas were not detected in transgenic mice. Furthermore, there was reduced staining for alpha smooth muscle actin and keratin 14, markers for myoepithelial cells, in the glandular portion of tumors in transgenic mice. The pathology of tumors induced by pituitary isograft alone was also markedly different in wild-type and transgenic mice. All the tumors classified from wild-type mice demonstrated some form of squamous differentiation, whereas squamous differentiation was not detected in the pituitary-induced transgenic tumors. The results suggest that TGF-beta acts as a tumor suppressor for hormone-induced cancers and that TGF-beta has a role in determining tumor pathology by regulating myoepithelial or squamous differentiation, maintenance, or transformation.     

Transduction of a mesenchyme-specific gene periostin into 293T cells induces cell invasive activity through epithelial-mesenchymal transformation

Tumor metastasis is a multistep pathological process involved in the final phase of tumor development. During this process, epithelium-derived tumor cells undergo fibroblast-like transformation, referred to as epithelial-mesenchymal transition (EMT), which contributes to aggressive behavior of tumors. We identify periostin, a mesenchyme-specific gene product, as a contributor to EMT and metastatic potential. Stable expression of a periostin transgene in tumorigenic but non-metastatic 293T cells caused cells to undergo fibroblast-like transformation accompanied by increased expressions of vimentin, epidermal growth factor receptor (EGFR), and matrix metalloproteinase-9. The cells expressing ectopic periostin increased cell migration, invasion, and adhesion by 2-9-fold. Invasive characteristics required signaling through integrin alpha(v)beta5 and EGFR. In addition, periostin-engineered 293T cells formed metastases in immunodeficient mice following either cardiac inoculation or injection into mammary fat pad. These data demonstrate an active role for periostin in EMT and metastasis that requires cross-talk between integrin and EGFR signaling pathways.