Localized production of TGF-beta mRNA in tumour promoter-stimulated mouse epidermis

Tumour promoters induce a wide spectrum of morphological and biochemical alterations when applied to mouse epidermis in vivo. These include the induction of RNA, DNA and protein synthesis during discrete phases of proliferation and differentiation. This constitutes an ideal model for studying molecular events underlying the disruption of epidermal homeostasis by TPA, and its subsequent re-establishment. Transforming growth factor-beta (TGF-beta) can induce either growth stimulation, inhibition, or differentiation, depending on the target cell. A function has been proposed for TGF-beta in wound healing and in tumour promotion, but the main source of TGF-beta is generally thought to be platelets, macrophages or lymphocytes, and a direct role for this growth factor in regulating tissue homeostasis in vivo has not been demonstrated. We show here that when the tumour promoter 12-tetradecanoyl-phorbol-13-acetate (TPA) is applied to the skin of mice, very high levels of TGF-beta messenger RNA are induced in the epidermal cells. In situ hybridization techniques show that the main site of TGF-beta synthesis is in the suprabasal differentiating epidermal cells. These results suggest that TGF-beta may be a natural regulator of epidermal homeostasis which is important in tumour promotion.     

Interactions between cancer stem cells and their niche govern metastatic colonization

Metastatic growth in distant organs is the major cause of cancer mortality. The development of metastasis is a multistage process with several rate-limiting steps. Although dissemination of tumour cells seems to be an early and frequent event, the successful initiation of metastatic growth, a process termed 'metastatic colonization', is inefficient for many cancer types and is accomplished only by a minority of cancer cells that reach distant sites. Prevalent target sites are characteristic of many tumour entities, suggesting that inadequate support by distant tissues contributes to the inefficiency of the metastatic process. Here we show that a small population of cancer stem cells is critical for metastatic colonization, that is, the initial expansion of cancer cells at the secondary site, and that stromal niche signals are crucial to this expansion process. We find that periostin (POSTN), a component of the extracellular matrix, is expressed by fibroblasts in the normal tissue and in the stroma of the primary tumour. Infiltrating tumour cells need to induce stromal POSTN expression in the secondary target organ (in this case lung) to initiate colonization. POSTN is required to allow cancer stem cell maintenance, and blocking its function prevents metastasis. POSTN recruits Wnt ligands and thereby increases Wnt signalling in cancer stem cells. We suggest that the education of stromal cells by infiltrating tumour cells is an important step in metastatic colonization and that preventing de novo niche formation may be a novel strategy for the treatment of metastatic disease.     

Similarity of teleocidin B and phorbol ester tumour promoters in effects on membrane receptors

The tumour promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and structurally related phorbol esters effect changes in avian and mammalian cell cultures that mimic transformation by oncogenic viruses or chemical carcinogens and the inhibition or induction of various types of differentiation (for review see refs 1--3). Unlike initiating carcinogens, which seem to act by binding covalently to cellular DNA, the primary site of action of the phorbol ester tumour promoters seems to be the cell membrane; indeed, specific high-affinity saturable receptors for phorbol esters have been identified in cell membranes. The recently discovered class of tumour promoters, the teleocidins, are as potent as TPA in the induction of ornithine decarboxylase in mouse skin, the inhibition of differentiation of Friend erythroleukaemia cells, the induction of HL-60 cell adhesion and the promotion of tumours on mouse skin. As the teleocidins are structurally unrelated to the phorbol esters, we set out to determine their effects on cell membranes and receptors. We found that in rodent cell cultures, teleocidin B and dihydroteleocidin B have effects similar to those of TPA and that, at nanomolar concentrations, teleocidin inhibits the binding of phorbol esters to cell-surface receptors, which suggests that the action of both classes of compounds may be mediated by the same or a similar receptor system.     

An elaborate pathway required for Ras-mediated epigenetic silencing

The conversion of a normal cell to a cancer cell occurs in several steps and typically involves the activation of oncogenes and the inactivation of tumour suppressor and pro-apoptotic genes. In many instances, inactivation of genes critical for cancer development occurs by epigenetic silencing, often involving hypermethylation of CpG-rich promoter regions. It remains to be determined whether silencing occurs by random acquisition of epigenetic marks that confer a selective growth advantage or through a specific pathway initiated by an oncogene. Here we perform a genome-wide RNA interference (RNAi) screen in K-ras-transformed NIH 3T3 cells and identify 28 genes required for Ras-mediated epigenetic silencing of the pro-apoptotic Fas gene. At least nine of these RESEs (Ras epigenetic silencing effectors), including the DNA methyltransferase DNMT1, are directly associated with specific regions of the Fas promoter in K-ras-transformed NIH 3T3 cells but not in untransformed NIH 3T3 cells. RNAi-mediated knockdown of any of the 28 RESEs results in failure to recruit DNMT1 to the Fas promoter, loss of Fas promoter hypermethylation, and derepression of Fas expression. Analysis of five other epigenetically repressed genes indicates that Ras directs the silencing of multiple unrelated genes through a largely common pathway. Last, we show that nine RESEs are required for anchorage-independent growth and tumorigenicity of K-ras-transformed NIH 3T3 cells; these nine genes have not previously been implicated in transformation by Ras. Our results show that Ras-mediated epigenetic silencing occurs through a specific, complex, pathway involving components that are required for maintenance of a fully transformed phenotype.     

Genomic analysis of metastasis reveals an essential role for RhoC

The most damaging change during cancer progression is the switch from a locally growing tumour to a metastatic killer. This switch is believed to involve numerous alterations that allow tumour cells to complete the complex series of events needed for metastasis. Relatively few genes have been implicated in these events. Here we use an in vivo selection scheme to select highly metastatic melanoma cells. By analysing these cells on DNA arrays, we define a pattern of gene expression that correlates with progression to a metastatic phenotype. In particular, we show enhanced expression of several genes involved in extracellular matrix assembly and of a second set of genes that regulate, either directly or indirectly, the actin-based cytoskeleton. One of these, the small GTPase RhoC, enhances metastasis when overexpressed, whereas a dominant-negative Rho inhibits metastasis. Analysis of the phenotype of cells expressing dominant-negative Rho or RhoC indicates that RhoC is important in tumour cell invasion. The genomic approach allows us to identify families of genes involved in a process, not just single genes, and can indicate which molecular and cellular events might be important in complex biological processes such as metastasis.     

Rae1 and H60 ligands of the NKG2D receptor stimulate tumour immunity

Natural killer (NK) cells attack many tumour cell lines, and are thought to have a critical role in anti-tumour immunity; however, the interaction between NK cells and tumour targets is poorly understood. The stimulatory lectin-like NKG2D receptor is expressed by NK cells, activated CD8+ T cells and by activated macrophages in mice. Several distinct cell-surface ligands that are related to class I major histocompatibility complex molecules have been identified, some of which are expressed at high levels by tumour cells but not by normal cells in adults. However, no direct evidence links the expression of these 'induced self' ligands with tumour cell rejection. Here we demonstrate that ectopic expression of the murine NKG2D ligands Rae1beta or H60 in several tumour cell lines results in potent rejection of the tumour cells by syngeneic mice. Rejection is mediated by NK cells and/or CD8+ T cells. The ligand-expressing tumour cells induce potent priming of cytotoxic T cells and sensitization of NK cells in vivo. Mice that are exposed to live or irradiated tumour cells expressing Rae1 or H60 are specifically immune to subsequent challenge with tumour cells that lack NKG2D ligands, suggesting application of the ligands in the design of tumour vaccines.     

Keratinocytes blocked in phorbol ester-responsive early stage of terminal differentiation by sarcoma viruses

It has been suggested that the initiation step in mouse skin carcinogenesis involves an alteration in epidermal-differentiation, as mouse basal keratinocytes exposed to initiators resist the arrest of cell growth that is normally associated with the induction of terminal differentiation by calcium ions. The growth of epidermal basal cells infected by Kirsten (Ki) or Harvey (Ha) sarcoma viruses is, however, arrested in response to calcium ions, although the cells do not progress through their entire maturation programme when a functioning ras gene of those viruses is expressed. If continuous proliferation in the differentiating cell layers is a requirement for tumour formation in skin, the response of sarcoma virus-infected cells seems inconsistent with the suggestion that an activated ras gene is sufficient to initiate skin carcinogenesis. We now show that sarcoma virus-infected keratinocytes, when induced to differentiate, are blocked at an early, reversible stage of maturation. Furthermore, the cells respond to phorbol ester tumour promoters by undergoing a phenotypic reversion to a less mature stage. These results suggest that activation of a ras gene can produce conditionally initiated cells, in which the full expression of tumorigenicity depends on exposure to tumour promoters.     

人间充质干细胞成骨分化早期HOX家族基因转录的表观遗传调控

人间充质干细胞(human mesenchymal stem cells,hMSCs)是一类具有多分化潜能的成体干细胞,在体内外可以被人工定向诱导分化成多种不同的细胞.有报道表明,在干细胞分化的过程中,细胞核内染色质发生重塑.HOX家族基因作为一类转录因子,在胚胎发育以及细胞分化过程中发挥着十分重要作用.通过体外定向诱导骨髓间充质干细胞向成骨细胞分化,对比分化前后细胞中HOX家族基因的表达状况,发现HOX家族基因的表达水平在hMSCs早期成骨分化过程中显著下降.进一步的研究发现,HOX家族基因的这种表达变化是由其启动子区的组蛋白H3-Lys9乙酰化和二甲基化水平发生变化而导致的.一系列实验证据表明,在间充质干细胞的成骨分化过程中,HOX家族基因表达受到抑制,而这种抑制作用是与其分化过程中发生的染色质重塑事件密切相关的.