Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters.

miR-17 approximately 92, miR-106b approximately 25, and miR-106a approximately 363 belong to a family of highly conserved miRNA clusters. Amplification and overexpression of miR-1792 is observed in human cancers, and its oncogenic properties have been confirmed in a mouse model of B cell lymphoma. Here we show that mice deficient for miR-17 approximately 92 die shortly after birth with lung hypoplasia and a ventricular septal defect. The miR-17 approximately 92 cluster is also essential for B cell development. Absence of miR-17 approximately 92 leads to increased levels of the proapoptotic protein Bim and inhibits B cell development at the pro-B to pre-B transition. Furthermore, while ablation of miR-106b approximately 25 or miR-106a approximately 363 has no obvious phenotypic consequences, compound mutant embryos lacking both miR-106b approximately 25 and miR-17 approximately 92 die at midgestation. These results provide key insights into the physiologic functions of this family of microRNAs and suggest a link between the oncogenic properties of miR-17 approximately 92 and its functions during B lymphopoiesis and lung development.

PTEN action in leukaemia dictated by the tissue microenvironment.

PTEN encodes a lipid phosphatase that is underexpressed in many cancers owing to deletions, mutations or gene silencing. PTEN dephosphorylates phosphatidylinositol (3,4,5)-triphosphate, thereby opposing the activity of class I phosphatidylinositol 3-kinases that mediate growth- and survival-factor signalling through phosphatidylinositol 3-kinase effectors such as AKT and mTOR. To determine whether continued PTEN inactivation is required to maintain malignancy, here we generate an RNA interference-based transgenic mouse model that allows tetracycline-dependent regulation of PTEN in a time- and tissue-specific manner. Postnatal Pten knockdown in the haematopoietic compartment produced highly disseminated T-cell acute lymphoblastic leukaemia. Notably, reactivation of PTEN mainly reduced T-cell leukaemia dissemination but had little effect on tumour load in haematopoietic organs. Leukaemia infiltration into the intestine was dependent on CCR9 G-protein-coupled receptor signalling, which was amplified by PTEN loss. Our results suggest that in the absence of PTEN, G-protein-coupled receptors may have an unanticipated role in driving tumour growth and invasion in an unsupportive environment. They further reveal that the role of PTEN loss in tumour maintenance is not invariant and can be influenced by the tissue microenvironment, thereby producing a form of intratumoral heterogeneity that is independent of cancer genotype.

p53 loss promotes acute myeloid leukemia by enabling aberrant self-renewal.

The p53 tumor suppressor limits proliferation in response to cellular stress through several mechanisms. Here, we test whether the recently described ability of p53 to limit stem cell self-renewal suppresses tumorigenesis in acute myeloid leukemia (AML), an aggressive cancer in which p53 mutations are associated with drug resistance and adverse outcome. Our approach combined mosaic mouse models, Cre-lox technology, and in vivo RNAi to disable p53 and simultaneously activate endogenous Kras(G12D)-a common AML lesion that promotes proliferation but not self-renewal. We show that p53 inactivation strongly cooperates with oncogenic Kras(G12D) to induce aggressive AML, while both lesions on their own induce T-cell malignancies with long latency. This synergy is based on a pivotal role of p53 in limiting aberrant self-renewal of myeloid progenitor cells, such that loss of p53 counters the deleterious effects of oncogenic Kras on these cells and enables them to self-renew indefinitely. Consequently, myeloid progenitor cells expressing oncogenic Kras and lacking p53 become leukemia-initiating cells, resembling cancer stem cells capable of maintaining AML in vivo. Our results establish an efficient new strategy for interrogating oncogene cooperation, and provide strong evidence that the ability of p53 to limit aberrant self-renewal contributes to its tumor suppressor activity.

Restoration of p53 function leads to tumour regression in vivo.

Tumorigenesis is a multi-step process that requires activation of oncogenes and inactivation of tumour suppressor genes. Mouse models of human cancers have recently demonstrated that continuous expression of a dominantly acting oncogene (for example, Hras, Kras and Myc) is often required for tumour maintenance; this phenotype is referred to as oncogene addiction. This concept has received clinical validation by the development of active anticancer drugs that specifically inhibit the function of oncoproteins such as BCR-ABL, c-KIT and EGFR. Identifying additional gene mutations that are required for tumour maintenance may therefore yield clinically useful targets for new cancer therapies. Although loss of p53 function is a common feature of human cancers, it is not known whether sustained inactivation of this or other tumour suppressor pathways is required for tumour maintenance. To explore this issue, we developed a Cre-loxP-based strategy to temporally control tumour suppressor gene expression in vivo. Here we show that restoring endogenous p53 expression leads to regression of autochthonous lymphomas and sarcomas in mice without affecting normal tissues. The mechanism responsible for tumour regression is dependent on the tumour type, with the main consequence of p53 restoration being apoptosis in lymphomas and suppression of cell growth with features of cellular senescence in sarcomas. These results support efforts to treat human cancers by way of pharmacological reactivation of p53.

Molecular signatures suggest a major role for stromal cells in development of invasive breast cancer.

BACKGROUND: Breast cancer invasion and metastasis involves both epithelial and stromal changes. Our objective was to delineate the pivotal role stroma plays in invasion by comparing transcriptomes among stromal and epithelial cells in normal tissue and invasive breast cancer. METHODS: Total RNA was isolated from epithelial and stromal cells that were laser captured from normal breast tissue (n = 5) and invasive breast cancer (n = 28). Gene expression was measured using Affymetrix U133A 2.0 GeneChips. Differential gene expression was evaluated and compared within a model that accounted for cell type (epithelial [E] versus stromal [S]), diagnosis (cancer [C] versus normal [N]) as well as cell type-diagnosis interactions. RESULTS: Compared to NE, the CE transcriptome was highly enriched with genes in proliferative, motility and ECM ontologies. Differences in CS and NS transcriptomes suggested that the ECM was being remodeled in invasive breast cancer, as genes were over-represented in ECM and proteolytic ontologies. Genes more highly expressed in CS compared to CE were primarily ECM components or were involved in the remodeling of ECM, suggesting that ECM biosynthesis and remodeling were initiated in the tumor stroma. CONCLUSION: Based on identified molecular cross-talk between the two contiguous cell populations, a mechanistic model that spurs invasion is proposed, that shows breast cancer invasion proceeds through the acquisition of a motile phenotype in tumor epithelial cells and a reactive phenotype in cancer associated fibroblasts.

In a hypergravity environment neonatal survival is adversely affected by alterations in dam tissue metabolism rather than reduced food intake.

Exposure of rat dams to hypergravity during pregnancy is associated with increased pup mortality, reduced food intake, and decreased rates of glucose oxidation and lipogenesis in mammary tissue. We hypothesized that increased pup mortality is due to changes in maternal metabolism and not to reduced food intake of dams. Effects of hypergravity on rate of glucose oxidation and lipogenesis in mammary, liver, and adipose tissue were measured in rat dams centrifuged at 2.0 G [hypergravity (HG)], kept at 1.0 G (control), or fed to match the intake of HG rats (pair fed) from gestation day 11 (G11) until G21 or postpartum day 3 (P3). Body weight, percent body fat, metabolizable energy, and nitrogen balance were significantly less in HG dams compared with controls (P<0.05); however, these factors were not different between HG and pair-fed dams. By P3, 100% of control and pair-fed pups survived, while only 49% of HG pups survived. At G21, rates of glucose oxidation and lipogenesis in mammary and adipose tissue were less in HG than in control and pair-fed dams (P<0.1 and P<0.05). In liver, at G21, the rate of lipogenesis was greater in HG than control and pair-fed dams (P<0.01); at P3, lipogenesis was greater in control than HG and pair-fed dams (P<0.05). Gene expression of ATP citrate lyase, acetyl-CoA carboxylase, and fatty acid synthase increased in liver from pregnancy to lactation in control and pair-fed dams but not HG dams. Thus reduced food intake and body mass due to hypergravity exposure cannot explain the dramatic decrease in HG pup survival.

Hypergravity disruption of homeorhetic adaptations to lactation in rat dams include changes in circadian clocks.

Altered gravity load induced by spaceflight (microgravity) and centrifugation (hypergravity) is associated with changes in circadian, metabolic, and reproductive systems. Exposure to 2-g hypergravity (HG) during pregnancy and lactation decreased rate of mammary metabolic activity and increased pup mortality. We hypothesize HG disrupted maternal homeorhetic responses to pregnancy and lactation are due to changes in maternal metabolism, hormone concentrations, and maternal behavior related to gravity induced alterations in circadian clocks. Effect of HG exposure on mammary, liver and adipose tissue metabolism, plasma hormones and maternal behavior were analyzed in rat dams from mid-pregnancy (Gestational day [G]11) through early lactation (Postnatal day [P]3); comparisons were made across five time-points: G20, G21, P0 (labor and delivery), P1 and P3. Blood, mammary, liver, and adipose tissue were collected for analyzing plasma hormones, glucose oxidation to CO(2) and incorporation into lipids, or gene expression. Maternal behavioral phenotyping was conducted using time-lapse videographic analyses. Dam and fetal-pup body mass were significantly reduced in HG in all age groups. HG did not affect labor and delivery; however, HG pups experienced a greater rate of mortality. PRL, corticosterone, and insulin levels and receptor genes were altered by HG. Mammary, liver and adipose tissue metabolism and expression of genes that regulate lipid metabolism were altered by HG exposure. Exposure to HG significantly changed expression of core clock genes in mammary and liver and circadian rhythms of maternal behavior. Gravity load alterations in dam's circadian system may have impacted homeorhetic adaptations needed for a successful lactation.