Osteoclast differentiation factor RANKL controls development of progestin-driven mammary cancer

Breast cancer is one of the most common cancers in humans and will on average affect up to one in eight women in their lifetime in the United States and Europe. The Women's Health Initiative and the Million Women Study have shown that hormone replacement therapy is associated with an increased risk of incident and fatal breast cancer. In particular, synthetic progesterone derivatives (progestins) such as medroxyprogesterone acetate (MPA), used in millions of women for hormone replacement therapy and contraceptives, markedly increase the risk of developing breast cancer. Here we show that the in vivo administration of MPA triggers massive induction of the key osteoclast differentiation factor RANKL (receptor activator of NF-κB ligand) in mammary-gland epithelial cells. Genetic inactivation of the RANKL receptor RANK in mammary-gland epithelial cells prevents MPA-induced epithelial proliferation, impairs expansion of the CD49f(hi) stem-cell-enriched population, and sensitizes these cells to DNA-damage-induced cell death. Deletion of RANK from the mammary epithelium results in a markedly decreased incidence and delayed onset of MPA-driven mammary cancer. These data show that the RANKL/RANK system controls the incidence and onset of progestin-driven breast cancer.     

Androgen regulation of cell proliferation and expression of viral sequences in mouse mammary tumour cells

The role of steroids in promoting cell proliferation is well established but the molecular mechanisms are not clear. The S115 mouse mammary tumour cell line provides a model system for molecular studies in vitro in that it exhibits in tissue culture both a positive proliferative response to androgens and a change from a transformed phenotype in the presence of androgen to a normal phenotype when androgen is removed. We have considered here the possible involvement of mouse mammary tumour virus (MMTV) in these processes. We have demonstrated the presence in S115 cells of MMTV-related sequences which are transcribed into RNA only in the long-term presence of androgen. Prolonged culture in the absence of androgen, which results in loss of proliferative response to androgen, is accompanied by loss of MMTV-related RNA and increased methylation of MMTV-related sequences.     

Regulation of cancer cell migration and bone metastasis by RANKL

Bone metastases are a frequent complication of many cancers that result in severe disease burden and pain. Since the late nineteenth century, it has been thought that the microenvironment of the local host tissue actively participates in the propensity of certain cancers to metastasize to specific organs, and that bone provides an especially fertile 'soil'. In the case of breast cancers, the local chemokine milieu is now emerging as an explanation for why these tumours preferentially metastasize to certain organs. However, as the inhibition of chemokine receptors in vivo only partially blocks metastatic behaviour, other factors must exist that regulate the preferential metastasis of breast cancer cells. Here we show that the cytokine RANKL (receptor activator of NF-kappaB ligand) triggers migration of human epithelial cancer cells and melanoma cells that express the receptor RANK. RANK is expressed on cancer cell lines and breast cancer cells in patients. In a mouse model of melanoma metastasis, in vivo neutralization of RANKL by osteoprotegerin results in complete protection from paralysis and a marked reduction in tumour burden in bones but not in other organs. Our data show that local differentiation factors such as RANKL have an important role in cell migration and the tissue-specific metastatic behaviour of cancer cells.     

Pygo2转基因小鼠模型的建立及表型的初步分析

主要通过建立Pygo2转基因小鼠的模型对其表型进行初步分析.首先构建K14-2×flag-Pygo的转基因构件,经酶切、纯化后构建Pygo2转基因小鼠.出生后的仔鼠用PCR和Western方法检测基因型,并通过进一步的免疫组化验证Pygo2基因的表达.PCR检测获得7只转基因阳性鼠,6只Western检测为阳性.对转基因小鼠子代的胚胎和成体进行免疫组化证明,Pygo2基因在皮肤和乳腺组织中有过量表达.转基因小鼠的皮肤、乳腺以及鼠尾椎骨等组织出现了异常的表型.乳腺中有肿瘤组织的形成,且Pygo2在肿瘤中有大量表达.该模型的成功建立为进一步研究Pygo2的功能奠定了基础.     

Peripheral neuropathies, hepatocellular carcinomas and islet cell adenomas in transgenic mice

The ability to introduce foreign DNA into the genome of mice offers unique opportunities to produce new models of disease process. Recent experiments have shown that integration and expression of simian virus 40 (SV40) T antigen genes and the murine mammary tumour virus (MMTV)-myc genes in transgenic mice can lead to the development of neoplasia in a remarkably tissue-specific manner. In the case of SV40-bearing mice, tumours consistently develop in the choroid plexus. In the accompanying paper, we show that the 72-base pair (bp) enhancer in the SV40 genome is instrumental in directing tumorigenesis to the choroid plexus. However, when the enhancer is deleted from a construction also containing the metallothionein-human growth hormone fusion gene (SV delta e-MGH), an entirely new pattern of pathology results. The present report focuses on transgenic mice carrying this construct; they develop demyelinating peripheral neuropathies, hepatocellular carcinomas and islet cell adenomas.     

Deletion of vascular endothelial growth factor in myeloid cells accelerates tumorigenesis

Angiogenesis and the development of a vascular network are required for tumour progression, and they involve the release of angiogenic factors, including vascular endothelial growth factor (VEGF-A), from both malignant and stromal cell types. Infiltration by cells of the myeloid lineage is a hallmark of many tumours, and in many cases the macrophages in these infiltrates express VEGF-A. Here we show that the deletion of inflammatory-cell-derived VEGF-A attenuates the formation of a typical high-density vessel network, thus blocking the angiogenic switch in solid tumours in mice. Vasculature in tumours lacking myeloid-cell-derived VEGF-A was less tortuous, with increased pericyte coverage and decreased vessel length, indicating vascular normalization. In addition, loss of myeloid-derived VEGF-A decreases the phosphorylation of VEGF receptor 2 (VEGFR2) in tumours, even though overall VEGF-A levels in the tumours are unaffected. However, deletion of myeloid-cell VEGF-A resulted in an accelerated tumour progression in multiple subcutaneous isograft models and an autochthonous transgenic model of mammary tumorigenesis, with less overall tumour cell death and decreased tumour hypoxia. Furthermore, loss of myeloid-cell VEGF-A increased the susceptibility of tumours to chemotherapeutic cytotoxicity. This shows that myeloid-derived VEGF-A is essential for the tumorigenic alteration of vasculature and signalling to VEGFR2, and that these changes act to retard, not promote, tumour progression.     

Bone metastasis: can osteoclasts be excluded?

The RANK/RANKL signalling mechanism is the final common pathway of osteoclast formation and activity. Inhibitors of RANK ligand (RANKL) that bind to RANK (for 'receptor activator of NF-kappaB'), such as osteoprotegerin (OPG), neutralizing antibodies against RANKL and soluble RANK antagonists, are well described inhibitors of bone metastasis in preclinical and clinical models, presumably because of their effects on osteoclasts. Jones et al. show that OPG inhibits bone metastasis after intracardiac injection of B16F10 murine melanoma cells, but claim that bone metastases are entirely independent of osteoclast formation and bone resorption: rather, they are caused by an effect on cell migration through RANK. However, we question whether these surprising conclusions are rigorously supported by their data.     

Cytokinesis failure generating tetraploids promotes tumorigenesis in p53-null cells

A long-standing hypothesis on tumorigenesis is that cell division failure, generating genetically unstable tetraploid cells, facilitates the development of aneuploid malignancies. Here we test this idea by transiently blocking cytokinesis in p53-null (p53-/-) mouse mammary epithelial cells (MMECs), enabling the isolation of diploid and tetraploid cultures. The tetraploid cells had an increase in the frequency of whole-chromosome mis-segregation and chromosomal rearrangements. Only the tetraploid cells were transformed in vitro after exposure to a carcinogen. Furthermore, in the absence of carcinogen, only the tetraploid cells gave rise to malignant mammary epithelial cancers when transplanted subcutaneously into nude mice. These tumours all contained numerous non-reciprocal translocations and an 8-30-fold amplification of a chromosomal region containing a cluster of matrix metalloproteinase (MMP) genes. MMP overexpression is linked to mammary tumours in humans and animal models. Thus, tetraploidy enhances the frequency of chromosomal alterations and promotes tumour development in p53-/- MMECs.     

Induction of angiogenesis during the transition from hyperplasia to neoplasia

It is now well established that unrestricted growth of tumours is dependent upon angiogenesis. Previous studies on tumour growth, however, have not revealed when or how the transition to an angiogenic state occurs during early tumour development. The advent of transgenic mice carrying oncogenes that reproducibly elicit tumours of specific cell types is providing a new format for studying multi-step tumorigenesis. In one of these models, transgenic mice expressing an oncogene in the beta-cells of the pancreatic islets heritably recapitulate a progression from normality to hyperplasia to neoplasia. We report here that angiogenic activity first appears in a subset of hyperplastic islets before the onset of tumour formation. A novel in vitro assay confirms that hyperplasia per se does not obligate angiogenesis. Rather, a few hyperplastic islets become angiogenic in vitro at a time when such islets are neovascularized in vivo and at a frequency that correlates closely with subsequent tumour incidence. These findings suggest that induction of angiogenesis is an important step in carcinogenesis.     

High expression of human serum albumin in milk of transgenic mice directed by the goat β-casein gene promoter region

We have constructed a mammary gland expression vector that contained the goat β-casein gene promoter, 5′upstream regulatory region, exons 1,2, intron 1 as well as the human serum albumin (hALB) mini-gene (including the full-long sequences of hALB cDNA and its intron 1). Injection of the vector into mouse tail veins showed that the recombinant construct was expressed only in mammary glands. The vector was microinjected into the mouse fertilized eggs, followed by transferring the eggs into the foster mice. 33 F₀ mice were obtained. Of the 33, 8 mice (5♀, 3 ♂) were transgenic with hALB gene integration identified by PCR as well as Southern blot hybridization. The integration rate was 24.2% (8/33). Western blot analysis showed that 3 female transgenic mice had hALB expression in their milk. The hALB contents in milk reached 3.54, 0.21 and 3.03 g/L, respectively.     

A paracrine requirement for hedgehog signalling in cancer

Ligand-dependent activation of the hedgehog (Hh) signalling pathway has been associated with tumorigenesis in a number of human tissues. Here we show that, although previous reports have described a cell-autonomous role for Hh signalling in these tumours, Hh ligands fail to activate signalling in tumour epithelial cells. In contrast, our data support ligand-dependent activation of the Hh pathway in the stromal microenvironment. Specific inhibition of Hh signalling using small molecule inhibitors, a neutralizing anti-Hh antibody or genetic deletion of smoothened (Smo) in the mouse stroma results in growth inhibition in xenograft tumour models. Taken together, these studies demonstrate a paracrine requirement for Hh ligand signalling in the tumorigenesis of Hh-expressing cancers and have important implications for the development of Hh pathway antagonists in cancer.     

High expression of human serum albumin in milk of trans- genie mice directed by the goat p-casein gene promoter region

We have constructed a mammary gland expression vector that contained the goat β-casein gene pro-moter, 5'upstream regulatory region, exons 1, 2, intron 1 as well as the human serum albumin (hALB) mini-gene (including the full-long sequences of hALB cDNA and its intron 1). Injection of the vector into mouse tail veins showed that the recombinant construct was expressed only in mammary glands. The vector was microinjected into the mouse fertilized eggs, followed by transferring the eggs into the foster mice. 33 F0 mice were obtained. Of the 33, 8 mice (5 , 3 ) were transgenic with hALB gene integration identified by PCR as well as Southern blot hybridization. The integration rate was 24.2% (8/33). Western blot analysis showed that 3 female transgenic mice had hALB expression in their milk. The hALB contents in milk reached 3.54, 0.21 and 3.03 g/L, respectively.     

Expression of recombinant human lysozyme in the milk of transgenic mice

Human lysozyme is a 130-aa (amino acid) alkaline polypeptide, and has both anti-bacterial and anti-viral properties which make it an important component of human natural immunity system. As a first step toward the ultimate goal of improving the anti-bacterial properties of bovine and ovine milk, a transgenic mouse that contains the genomic DNA sequence of the human lysozme gene has been generated for the first time. From 83 mice generated by microinjection, a total of 6 positive transgenic mice were identified by PCR and Southern blot. F1 mice positive for transgene in lines were also detected by PCR. This shows that transgene could be transmitted from founder transgenic mice to their offspring. Recombinant human lysozyme (rHlys) was found in the whey of 3 female positive transgenic mice by Western blot. The highest concentration of rHlys for transgenic mice was 0.2mg/mL. The antibacterial activity of the whey for transgenic mice was highly enhanced up to 0.4 times as much as that of human, while that of non-transgenic mouse was very low. Although the lysozyme activity of transgenic mice is still lower than that of human, the rHlys exhibits the same specific activity as that of human lysozyme. It provides a strong basis for further studies into the possible application of rHlys express in mammary gland.