Glenn Merlino

TGFα overexpression in transgenic mice induces liver neoplasia and abnormal development of the mammary gland and pancreas

To define the role of TGF alpha in normal tissue function and in pathogenesis, transgenic mice have been generated bearing a fusion gene consisting of the mouse metallothionein 1 promoter and a human TGF alpha cDNA. In these mice, human TGF alpha RNA and protein are abundant in many tissues and TGF alpha is detectable in blood and urine. The effects of TGF alpha overproduction in transgenic mice are pleiotropic and tissue specific. The liver frequently contains multifocal, well-differentiated hepatocellular carcinomas that express enhanced levels of human TGF alpha RNA. The mammary gland exhibits impeded morphogenetic penetration of epithelial duct cells into the stromal fat pad. The pancreas shows progressive interstitial fibrosis and a florid acinoductular metaplasia, during which acinar cells appear to degranulate, dedifferentiate, and assume characteristics of intercalated or centroacinar duct cells. TGF alpha therefore plays an important role in cellular proliferation, organogenesis, and neoplastic transformation.

Expression profiling identifies the cytoskeletal organizer ezrin and the developmental homeoprotein Six-1 as key metastatic regulators

Patients presenting with metastatic rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children, have a very poor clinical prognosis. This is due, in large part, to our rudimentary knowledge of the molecular events that dictate metastatic potential. We used cDNA microarray analysis of RMS cell lines, derived from Ink4a/Arf-deficient mice transgenic for hepatocyte growth factor/scatter factor (HGF/SF), to identify a set of genes whose expression was significantly different between highly and poorly metastatic cells. Subsequent in vivo functional studies revealed that the actin filament–plasma membrane linker ezrin (encoded by Vil2) and the homeodomain-containing transcription factor Six-1 (sine oculis–related homeobox-1 homolog) had essential roles in determining the metastatic fate of RMS cells. VIL2 and SIX1 expression was enhanced in human RMS tissue, significantly correlating with clinical stage. The identification of ezrin and Six-1 as critical regulators of metastasis in RMS provides new mechanistic and therapeutic insights into this pediatric cancer.

Lifetime exposure to a soluble TGF-β antagonist protects mice against metastasis without adverse side effects

TGF-betas play diverse and complex roles in many biological processes. In tumorigenesis, they can function either as tumor suppressors or as pro-oncogenic factors, depending on the stage of the disease. We have developed transgenic mice expressing a TGF-beta antagonist of the soluble type II TGF-beta receptor:Fc fusion protein class, under the regulation of the mammary-selective MMTV-LTR promoter/enhancer. Biologically significant levels of antagonist were detectable in the serum and most tissues of this mouse line. The mice were resistant to the development of metastases at multiple organ sites when compared with wild-type controls, both in a tail vein metastasis assay using isogenic melanoma cells and in crosses with the MMTV-neu transgenic mouse model of metastatic breast cancer. Importantly, metastasis from endogenous mammary tumors was suppressed without any enhancement of primary tumorigenesis. Furthermore, aged transgenic mice did not exhibit the severe pathology characteristic of TGF-beta null mice, despite lifetime exposure to the antagonist. The data suggest that in vivo the antagonist may selectively neutralize the undesirable TGF-beta associated with metastasis, while sparing the regulatory roles of TGF-betas in normal tissues. Thus this soluble TGF-beta antagonist has potential for long-term clinical use in the prevention of metastasis.

Soluble dominant-negative receptor uncovers essential roles for fibroblast growth factors in multi-organ induction and patterning

Despite a wealth of experimental data implicating fibroblast growth factor (FGF) signaling in various developmental processes, genetic inactivation of individual genes encoding specific FGFs or their receptors (FGFRs) has generally failed to demonstrate their role in vertebrate organogenesis due to early embryonic lethality or functional redundancy. Here we show that broad mid‐gestational expression of a novel secreted kinase‐deficient receptor, specific for a defined subset of the FGF superfamily, caused agenesis or severe dysgenesis of kidney, lung, specific cutaneous structures, exocrine and endocrine glands, and craniofacial and limb abnormalities reminiscent of human skeletal disorders associated with FGFR mutations. Analysis of diagnostic molecular markers revealed that this soluble dominant‐negative mutant disrupted early inductive signaling in affected tissues, indicating that FGF signaling is required for growth and patterning in a broad array of organs and in limbs. In contrast, transgenic mice expressing a membrane‐tethered kinase‐deficient FGFR were viable. Our results demonstrate that secreted FGFR mutants are uniquely effective as dominant‐negative agents in vivo, and suggest that related soluble receptor isoforms expressed in wild‐type mouse embryos may help regulate FGF activity during normal development.

Neonatal sunburn and melanoma in mice.

Retrospective epidemiological data have indicated that cutaneous malignant melanoma may arise as a consequence of intense, intermittent exposure of the skin to ultraviolet radiation, particularly in children, rather than from the cumulative lifetime exposure that is associated with other forms of skin cancer. Here we use a genetically engineered mouse model to show that a single dose of burning ultraviolet radiation to neonates, but not adults, is necessary and sufficient to induce tumours with high penetrance which are reminiscent of human melanoma. Our results provide experimental support for epidemiological evidence that childhood sunburn poses a significant risk of developing this potentially fatal disease.

The Two Faces of Interferon-γ in cancer.

Interferon-γ is a cytokine whose biological activity is conventionally associated with cytostatic/cytotoxic and antitumor mechanisms during cell-mediated adaptive immune response. It has been used clinically to treat a variety of malignancies, albeit with mixed results and side effects that can be severe. Despite ample evidence implicating a role for IFN-γ in tumor immune surveillance, a steady flow of reports has suggested that it may also have protumorigenic effects under certain circumstances. We propose that, in fact, IFN-γ treatment is a double-edged sword whose anti- and protumorigenic activities are dependent on the cellular, microenvironmental, and/or molecular context. As such, inhibition of the IFN-γ/IFN-γ receptor pathway may prove to be a viable new therapeutic target for a subset of malignancies. Clin Cancer Res; 17(19); 6118–24. ©2011 AACR.

Expression of a dominant-negative mutant TGF-β type II receptor in transgenic mice reveals essential roles for TGF-β in regulation of growth and differentiation in the exocrine pancreas

Using a dominant‐negative mutant receptor (DNR) approach in transgenic mice, we have functionally inactivated transforming growth factor‐β (TGF‐β) signaling in select epithelial cells. The dominant‐negative mutant type II TGF‐β receptor blocked signaling by all three TGF‐β isoforms in primary hepatocyte and pancreatic acinar cell cultures generated from transgenic mice, as demonstrated by the loss of growth inhibitory and gene induction responses. However, it had no effect on signaling by activin, the closest TGF‐β family member. DNR transgenic mice showed increased proliferation of pancreatic acinar cells and severely perturbed acinar differentiation. These results indicate that TGF‐β negatively controls growth of acinar cells and is essential for the maintenance of a differentiated acinar phenotype in the exocrine pancreas in vivo. In contrast, such abnormalities were not observed in the liver. Additional abnormalities in the pancreas included fibrosis, neoangiogenesis and mild macrophage infiltration, and these were associated with a marked up‐regulation of TGF‐β expression in transgenic acinar cells. This transgenic model of targeted functional inactivation of TGF‐β signaling provides insights into mechanisms whereby loss of TGF‐β responsiveness might promote the carcinogenic process, both through direct effects on cell proliferation, and indirectly through up‐regulation of TGF‐βs with associated paracrine effects on stromal compartments.

Rhabdomyosarcoma – working out the pathways

Rhabdomyosarcomas constitute a collection of childhood malignancies thought to arise as a consequence of regulatory disruption of skeletal muscle progenitor cell growth and differentiation. Our understanding of the pathogenesis of this neoplasm has recently benefited from the study of normal and malignant myogenic cells in vitro, facilitating the identification of diagnostic cytogenetic markers and the elucidation of mechanisms by which myogenesis is regulated. It is now appreciated that the delicate balance between proliferation and differentiation, mutually exclusive yet intimately associated processes, is normally controlled in large part through the action of a multitude of growth factors, whose signals are interpreted by members of the MyoD family of helix – loop – helix proteins, and key regulatory cell cycle factors. The latter have proven to be frequent targets of mutational events that subvert myogenesis and promote the development of rhabdomyosarcoma. Although significant progress has been made in the treatment of rhabdomyosarcoma, patients presenting with metastatic disease or certain high risk features are still faced with a dismal prognosis. Only now are genetically engineered mouse models becoming available that are certain to provide fresh insights into the molecular/genetic pathways by which rhabdomyosarcomas arise and progress, and to suggest novel avenues of therapeutic opportunity.

Ultraviolet B but not Ultraviolet A Radiation Initiates Melanoma

Cutaneous malignant melanoma is one of the fastest increasing cancers with an incidence that has more than doubled in the last 25 years. Sunlight exposure is strongly implicated in the etiology of cutaneous malignant melanoma and the UV portion of the sunlight spectrum is considered responsible. Data are, however, conflicting on the roles of ultraviolet B [UVB; 280–320 nanometers (nm)] and ultraviolet A (UVA; 320–400 nm), which differ in their ability to initiate DNA damage, cell signaling pathways and immune alterations. To address this issue, we have used specialized optical sources, emitting isolated or combined UVB or UVA wavebands or solar simulating radiation, together with our hepatocyte growth factor/scatter factor-transgenic mouse model of UV-induced melanoma that uniquely recapitulates human disease. Only UVB-containing sources initiated melanoma. These were the isolated UVB waveband (>96% 280–320 nm), the unfiltered F40 sunlamp (250–800 nm) and the solar simulator (290–800 nm). Kaplan-Meier survival analysis indicated that the isolated UVB waveband was more effective in initiating melanoma than either the F40 sunlamp or the solar simulator (modified log rank P < 0.02). The latter two sources showed similar melanoma effectiveness (P = 0.38). In contrast, transgenic mice irradiated with either the isolated UVA waveband (>99.9% 320–400 nm, 150 kJ/m2), or an F40 sunlamp filtered to remove > 96% of the UVB, responded like unirradiated control animals. We conclude that, within the constraints of this animal model, UVB is responsible for the induction of mammalian cutaneous malignant melanoma whereas UVA is ineffective even at doses considered physiologically relevant. This finding may have major implications with respect both to risk assessment from exposure to solar and artificial UVB, and to development of effective protection strategies against melanoma induction by UVB. Moreover, these differences in wavelength effectiveness can now be exploited to identify UV pathways relevant to melanomagenesis.

Targeting expression of a transforming growth factor beta 1 transgene to the pregnant mammary gland inhibits alveolar development and lactation.

Transforming growth factor‐beta 1 (TGF‐beta 1) possesses highly potent, diverse and often opposing cell‐specific activities, and has been implicated in the regulation of a variety of physiologic and developmental processes. To determine the effects of in vivo overexpression of TGF‐beta 1 on mammary gland function, transgenic mice were generated harboring a fusion gene consisting of the porcine TGF‐beta 1 cDNA placed under the control of regulatory elements of the pregnancy‐responsive mouse whey‐acidic protein (WAP) gene. Females from two of four transgenic lines were unable to lactate due to inhibition of the formation of lobuloalveolar structures and suppression of production of endogenous milk protein. In contrast, ductal development of the mammary glands was not overtly impaired. There was a complete concordance in transgenic mice between manifestation of the lactation‐deficient phenotype and expression of RNA from the WAP/TGF‐beta 1 transgene, which was present at low levels in the virgin gland, but was greatly induced at mid‐pregnancy. TGF‐beta 1 was localized to numerous alveoli and to the periductal extracellular matrix in the mammary gland of transgenic females late in pregnancy by immunohistochemical analysis. Glands reconstituted from cultured transgenic mammary epithelial cells duplicated the inhibition of lobuloalveolar development observed in situ in the mammary glands of pregnant transgenic mice. Results from this transgenic model strongly support the hypothesis that TGF‐beta 1 plays an important in vivo role in regulating the development and function of the mammary gland.