Michael C. Ostrowski

Pten in stromal fibroblasts suppresses mammary epithelial tumours

The tumour stroma is believed to contribute to some of the most malignant characteristics of epithelial tumours. However, signalling between stromal and tumour cells is complex and remains poorly understood. Here we show that the genetic inactivation of Pten in stromal fibroblasts of mouse mammary glands accelerated the initiation, progression and malignant transformation of mammary epithelial tumours. This was associated with the massive remodelling of the extracellular matrix (ECM), innate immune cell infiltration and increased angiogenesis. Loss of Pten in stromal fibroblasts led to increased expression, phosphorylation (T72) and recruitment of Ets2 to target promoters known to be involved in these processes. Remarkably, Ets2 inactivation in Pten stroma-deleted tumours ameliorated disruption of the tumour microenvironment and was sufficient to decrease tumour growth and progression. Global gene expression profiling of mammary stromal cells identified a Pten-specific signature that was highly represented in the tumour stroma of patients with breast cancer. These findings identify the Pten–Ets2 axis as a critical stroma-specific signalling pathway that suppresses mammary epithelial tumours.

Ambient Air Pollution Exaggerates Adipose Inflammation and Insulin Resistance in a Mouse Model of Diet-Induced Obesity

Background— There is a strong link between urbanization and type 2 diabetes mellitus. Although a multitude of mechanisms have been proposed, there are no studies evaluating the impact of ambient air pollutants and the propensity to develop type 2 diabetes mellitus. We hypothesized that exposure to ambient fine particulate matter (<2.5 &mgr;m; PM2.5) exaggerates diet-induced insulin resistance, adipose inflammation, and visceral adiposity. Methods and Results— Male C57BL/6 mice were fed high-fat chow for 10 weeks and randomly assigned to concentrated ambient PM2.5 or filtered air (n=14 per group) for 24 weeks. PM2.5-exposed C57BL/6 mice exhibited marked whole-body insulin resistance, systemic inflammation, and an increase in visceral adiposity. PM2.5 exposure induced signaling abnormalities characteristic of insulin resistance, including decreased Akt and endothelial nitric oxide synthase phosphorylation in the endothelium and increased protein kinase C expression. These abnormalilties were associated with abnormalities in vascular relaxation to insulin and acetylcholine. PM2.5 increased adipose tissue macrophages (F4/80+ cells) in visceral fat expressing higher levels of tumor necrosis factor-α/interleukin-6 and lower interleukin-10/N-acetyl-galactosamine specific lectin 1. To test the impact of PM2.5 in eliciting direct monocyte infiltration into fat, we rendered FVBN mice expressing yellow fluorescent protein (YFP) under control of a monocyte-specific promoter (c-fms, c-fmsYFP) diabetic over 10 weeks and then exposed these mice to PM2.5 or saline intratracheally. PM2.5 induced YFP cell accumulation in visceral fat and potentiated YFP cell adhesion in the microcirculation. Conclusion— PM2.5 exposure exaggerates insulin resistance and visceral inflammation/adiposity. These findings provide a new link between air pollution and type 2 diabetes mellitus.

MicroRNA-451 Regulates LKB1/AMPK Signaling and Allows Adaptation to Metabolic Stress in Glioma Cells

To sustain tumor growth, cancer cells must be able to adapt to fluctuations in energy availability. We have identified a single microRNA that controls glioma cell proliferation, migration, and responsiveness to glucose deprivation. Abundant glucose allows relatively high miR-451 expression, promoting cell growth. In low glucose, miR-451 levels decrease, slowing proliferation but enhancing migration and survival. This allows cells to survive metabolic stress and seek out favorable growth conditions. In glioblastoma patients, elevated miR-451 is associated with shorter survival. The effects of miR-451 are mediated by LKB1, which it represses through targeting its binding partner, CAB39 (MO25 alpha). Overexpression of miR-451 sensitized cells to glucose deprivation, suggesting that its downregulation is necessary for robust activation of LKB1 in response to metabolic stress. Thus, miR-451 is a regulator of the LKB1/AMPK pathway, and this may represent a fundamental mechanism that contributes to cellular adaptation in response to altered energy availability.

Direct Evidence for Epithelial-Mesenchymal Transitions in Breast Cancer

We developed stromal- and epithelial-specific cre-transgenic mice to directly visualize epithelial-mesenchymal transition (EMT) during cancer progression in vivo. Using three different oncogene-driven mouse mammary tumor models and cell-fate mapping strategies, we show in vivo evidence for the existence of EMT in breast cancer and show that myc can specifically elicit this process. Hierarchical cluster analysis of genome-wide loss of heterozygosity reveals that the incidence of EMT in invasive human breast carcinomas is rare, but when it occurs it is associated with the amplification of MYC. These data provide the first direct evidence for EMT in breast cancer and suggest that its development is favored by myc-initiated events.

Microphthalmia Transcription Factor Is a Target of the p38 MAPK Pathway in Response to Receptor Activator of NF-κB Ligand Signaling

Receptor activator of NF-κB ligand (RANKL) activates signaling pathways that regulate osteoclast differentiation, function, and survival. The microphthalmia transcription factor (MITF) is required for terminal differentiation of osteoclasts. To determine whether MITF could be a target of RANKL signaling, a phosphospecific MITF antibody directed against conserved residue Ser307, a potential mitogen-activated protein kinase (MAPK) site, was produced. Using this antibody, we could demonstrate that MITF was rapidly and persistently phosphorylated upon stimulation of primary osteoclasts with RANKL and that phosphorylation of Ser307 correlated with expression of the target gene tartrate-resistant acid phosphatase. MITF phosphorylation at Ser307 also correlated with persistent activation of p38 MAPK, and p38 MAPK could utilize MITF Ser307 as a substratein vitro. The phosphorylation of MITF and activation of target gene expression in osteoclasts were blocked by p38 MAPK inhibitor SB203580. In transient transfections, a constitutively active Rac1 or MKK6 gene could collaborate with MITF to activate the tartrate-resistant acid phosphatase gene promoter dependent on Ser307. Dominant negative p38 α and β could inhibit the collaboration between upstream signaling components and MITF in the transient assays. These results indicate that MITF is a target for the RANKL signaling pathway in osteoclasts and that phosphorylation of MITF leads to an increase in osteoclast-specific gene expression.

Eos Mediates Foxp3-Dependent Gene Silencing in CD4+ Regulatory T Cells

Treg Responses to Eos CD4+ regulatory T cells (Tregs) are critical for keeping our immune system in check: They prevent immune responses from getting out of hand and keep autoimmunity at bay. By activating the expression of some genes and turning off expression of others, the master regulatory transcription factor of Tregs, Foxp3, endows these cells with the appropriate gene expression program to mediate their suppressive effects. Pan et al. (p. 1142, published online 20 August) now demonstrate that the transcription factor Eos is selectively required for Foxp3-mediated gene suppression in mice. Genes normally suppressed by Foxp3 in Tregs remained “on” when Eos expression was suppressed, whereas genes activated by Foxp3 were unaffected. Treg function was also affected by Eos suppression. With half their genetic program disrupted, these cells resembled an intermediate between Tregs and conventional CD4+ T cells—unable to suppress immune responses properly and partially responsive to T cell–activating stimulation. A transcription factor required for gene suppression in regulatory T cells is identified. CD4+ regulatory T cells (Tregs) maintain immunological self-tolerance and immune homeostasis by suppressing aberrant or excessive immune responses. The core genetic program of Tregs and their ability to suppress pathologic immune responses depends on the transcription factor Foxp3. Despite progress in understanding mechanisms of Foxp3-dependent gene activation, the molecular mechanism of Foxp3-dependent gene repression remains largely unknown. We identified Eos, a zinc-finger transcription factor of the Ikaros family, as a critical mediator of Foxp3-dependent gene silencing in Tregs. Eos interacts directly with Foxp3 and induces chromatin modifications that result in gene silencing in Tregs. Silencing of Eos in Tregs abrogates their ability to suppress immune responses and endows them with partial effector function, thus demonstrating the critical role that Eos plays in Treg programming.

Transgenic Mice Overexpressing Tartrate‐Resistant Acid Phosphatase Exhibit an Increased Rate of Bone Turnover

Tartrate‐resistant acid phosphatase (TRAP) is a secreted product of osteoclasts and a lysosomal hydrolase of some tissue macrophages. To determine whether TRAP expression is rate‐limiting in bone resorption, we overexpressed TRAP in transgenic mice by introducing additional copies of the TRAP gene that contained the SV40 enhancer. In multiple independent mouse lines, the transgene gave a copy number–dependent increase in TRAP mRNA levels and TRAP activity in osteoclasts, macrophages, serum, and other sites of normal low‐level expression (notably, liver parenchymal cells, kidney mesangial cells, and pancreatic secretory acinar cells). Transgenic mice had decreased trabecular bone consistent with mild osteoporosis. Measurements of the bone formation rate suggest that the animals compensate for the increased resorption by increasing bone synthesis, which partly ameliorates the phenotype. These mice provide evidence that inclusion of an irrelevant enhancer does not necessarily override a tissue‐specific promoter.

Macrophage Colony-stimulating Factor Promotes Cell Survival through Akt/Protein Kinase B*

The signaling pathways activated by the macrophage colony-stimulating factor (M-CSF) to promote survival of monocyte and macrophage lineage cells are not well established. In an effort to elucidate these pathways, we have used two cell types responsive to M-CSF: NIH 3T3 fibroblasts genetically engineered to express human M-CSF receptors (3T3-FMS cells) and human monocytes. M-CSF treatment induced M-CSF receptor tyrosine phosphorylation and recruitment of the p85 subunit of phosphatidylinositol 3-kinase (PI3K) to these receptors. These M-CSF receptor events correlated with activation of the serine/threonine kinase Akt. To clarify that PI3K products activate Akt in response to M-CSF, NIH 3T3 fibroblasts expressing mutant human M-CSF receptors (3T3-FMS(Y809F)) that fail to activate Ras in response to M-CSF also exhibit increased Akt kinase activity in response to M-CSF challenge. Furthermore, Akt appears to be the primary regulator of survival in 3T3-FMS cells, as transfection of genes encoding dominant-negative Akt isoforms into these fibroblasts blocked M-CSF-induced survival. In normal human monocytes, M-CSF increased the levels of tyrosine-phosphorylated proteins and induced Akt activation in a PI3K-dependent manner. The PI3K inhibitor LY294002 blocked M-CSF-mediated monocyte survival, an effect that was partially restored by caspase-9 inhibitors. These data suggest that M-CSF may induce cell survival through Akt-induced suppression of caspase-9 activation.

Trisomy represses Apc(Min)-mediated tumours in mouse models of Down's syndrome.

Epidemiological studies spanning more than 50 yr reach conflicting conclusions as to whether there is a lower incidence of solid tumours in people with trisomy 21 (Down’s syndrome). We used mouse models of Down’s syndrome and of cancer in a biological approach to investigate the relationship between trisomy and the incidence of intestinal tumours. ApcMin-mediated tumour number was determined in aneuploid mouse models Ts65Dn, Ts1Rhr and Ms1Rhr. Trisomy for orthologues of about half of the genes on chromosome 21 (Hsa21) in Ts65Dn mice or just 33 of these genes in Ts1Rhr mice resulted in a significant reduction in the number of intestinal tumours. In Ms1Rhr, segmental monosomy for the same 33 genes that are triplicated in Ts1Rhr resulted in an increased number of tumours. Further studies demonstrated that the Ets2 gene contributed most of the dosage-sensitive effect on intestinal tumour number. The action of Ets2 as a repressor when it is overexpressed differs from tumour suppression, which requires normal gene function to prevent cellular transformation. Upregulation of Ets2 and, potentially, other genes involved in this kind of protective effect may provide a prophylactic effect in all individuals, regardless of ploidy.

MITF and PU.1 Recruit p38 MAPK and NFATc1 to Target Genes during Osteoclast Differentiation

Transcription factors NFATc1, PU.1, and MITF collaborate to regulate specific genes in response to colony-stimulating factor-1 (CSF-1) and receptor activator of NF-κB ligand (RANKL) signaling during osteoclast differentiation. However, molecular details concerning timing and mechanism of specific events remain ill-defined. In bone marrow-derived precursors, CSF-1 alone promoted assembly of MITF-PU.1 complexes at osteoclast target gene promoters like cathepsin K and acid 5 phosphatase without increasing gene expression. The combination of RANKL and CSF-1 concurrently increased the levels of MAPK-phosphorylated forms of MITF, p38 MAPK, and SWI/SNF chromatin-remodeling complexes bound to these target promoters and markedly increased expression of the genes. NFATc1 was subsequently recruited to complexes at the promoters during terminal stages of osteoclast differentiation. Genetic analysis of Mitf and Pu.1 in mouse models supported the critical interaction of these genes in osteoclast differentiation. The results define MITF and PU.1 as nuclear effectors that integrate CSF-1/RANKL signals during osteoclast differentiation to initiate expression of target genes, whereas a complex that includes NFATc1 may act to maintain target gene expression in differentiated cells.