Vivek Mittal

Role of transposable elements in heterochromatin and epigenetic control

Heterochromatin has been defined as deeply staining chromosomal material that remains condensed in interphase, whereas euchromatin undergoes de-condensation. Heterochromatin is found near centromeres and telomeres, but interstitial sites of heterochromatin (knobs) are common in plant genomes and were first described in maize. These regions are repetitive and late-replicating. In Drosophila, heterochromatin influences gene expression, a heterochromatin phenomenon called position effect variegation. Similarities between position effect variegation in Drosophila and gene silencing in maize mediated by “controlling elements” (that is, transposable elements) led in part to the proposal that heterochromatin is composed of transposable elements, and that such elements scattered throughout the genome might regulate development. Using microarray analysis, we show that heterochromatin in Arabidopsis is determined by transposable elements and related tandem repeats, under the control of the chromatin remodelling ATPase DDM1 (Decrease in DNA Methylation 1). Small interfering RNAs (siRNAs) correspond to these sequences, suggesting a role in guiding DDM1. We also show that transposable elements can regulate genes epigenetically, but only when inserted within or very close to them. This probably accounts for the regulation by DDM1 and the DNA methyltransferase MET1 of the euchromatic, imprinted gene FWA, as its promoter is provided by transposable-element-derived tandem repeats that are associated with siRNAs.

Endothelial Progenitor Cells Control the Angiogenic Switch in Mouse Lung Metastasis

Angiogenesis-mediated progression of micrometastasis to lethal macrometastasis is the major cause of death in cancer patients. Here, using mouse models of pulmonary metastasis, we identify bone marrow (BM)–derived endothelial progenitor cells (EPCs) as critical regulators of this angiogenic switch. We show that tumors induce expression of the transcription factor Id1 in the EPCs and that suppression of Id1 after metastatic colonization blocked EPC mobilization, caused angiogenesis inhibition, impaired pulmonary macrometastases, and increased survival of tumor-bearing animals. These findings establish the role of EPCs in metastatic progression in preclinical models and suggest that selective targeting of EPCs may merit investigation as a therapy for cancer patients with lung metastases.

Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance.

The role of epithelial-to-mesenchymal transition (EMT) in metastasis is a longstanding source of debate, largely owing to an inability to monitor transient and reversible EMT phenotypes in vivo. Here we establish an EMT lineage-tracing system to monitor this process in mice, using a mesenchymal-specific Cre-mediated fluorescent marker switch system in spontaneous breast-to-lung metastasis models. We show that within a predominantly epithelial primary tumour, a small proportion of tumour cells undergo EMT. Notably, lung metastases mainly consist of non-EMT tumour cells that maintain their epithelial phenotype. Inhibiting EMT by overexpressing the microRNA miR-200 does not affect lung metastasis development. However, EMT cells significantly contribute to recurrent lung metastasis formation after chemotherapy. These cells survived cyclophosphamide treatment owing to reduced proliferation, apoptotic tolerance and increased expression of chemoresistance-related genes. Overexpression of miR-200 abrogated this resistance. This study suggests the potential of an EMT-targeting strategy, in conjunction with conventional chemotherapies, for breast cancer treatment.The role of epithelial to mesenchymal transition (EMT) in metastasis is a longstanding source of controversy, largely due to an inability to monitor transient and reversible EMT phenotypes in vivo. We established an EMT lineage tracing system to monitor this process, using a mesenchymal-specific Cre-mediated fluorescent marker switch system in spontaneous breast-to-lung metastasis models. We confirmed that within a predominantly epithelial primary tumor, a small portion of tumor cells undergo EMT. Strikingly, lung metastases mainly consisted of non-EMT tumor cells maintaining their epithelial phenotype. Inhibiting EMT by overexpressing miR-200 did not impact lung metastasis development. However, EMT cells significantly contribute to recurrent lung metastasis formation after chemotherapy. These cells survived cyclophosphamide treatment due to reduced proliferation, apoptotic tolerance, and elevated expression of chemoresistance-related genes. Overexpression of miR-200 abrogated this resistance. This study suggests the potential of an EMT-targeting strategy, in conjunction with conventional chemotherapies, for breast cancer treatment.

Inductive angiocrine signals from sinusoidal endothelium are required for liver regeneration

During embryogenesis, endothelial cells induce organogenesis before the development of circulation. These findings suggest that endothelial cells not only form passive conduits to deliver nutrients and oxygen, but also establish an instructive vascular niche, which through elaboration of paracrine trophogens stimulates organ regeneration, in a manner similar to endothelial-cell-derived angiocrine factors that support haematopoiesis. However, the precise mechanism by which tissue-specific subsets of endothelial cells promote organogenesis in adults is unknown. Here we demonstrate that liver sinusoidal endothelial cells (LSECs) constitute a unique population of phenotypically and functionally defined VEGFR3+CD34−VEGFR2+VE-cadherin+FactorVIII+CD45− endothelial cells, which through the release of angiocrine trophogens initiate and sustain liver regeneration induced by 70% partial hepatectomy. After partial hepatectomy, residual liver vasculature remains intact without experiencing hypoxia or structural damage, which allows study of physiological liver regeneration. Using this model, we show that inducible genetic ablation of vascular endothelial growth factor (VEGF)-A receptor-2 (VEGFR2) in the LSECs impairs the initial burst of hepatocyte proliferation (days 1–3 after partial hepatectomy) and subsequent reconstitution of the hepatovascular mass (days 4–8 after partial hepatectomy) by inhibiting upregulation of the endothelial-cell-specific transcription factor Id1. Accordingly, Id1-deficient mice also manifest defects throughout liver regeneration, owing to diminished expression of LSEC-derived angiocrine factors, including hepatocyte growth factor (HGF) and Wnt2. Notably, in in vitro co-cultures, VEGFR2-Id1 activation in LSECs stimulates hepatocyte proliferation. Indeed, intrasplenic transplantation of Id1+/+ or Id1−/− LSECs transduced with Wnt2 and HGF (Id1−/−Wnt2+HGF+ LSECs) re-establishes an inductive vascular niche in the liver sinusoids of the Id1−/− mice, initiating and restoring hepatovascular regeneration. Therefore, in the early phases of physiological liver regeneration, VEGFR2-Id1-mediated inductive angiogenesis in LSECs through release of angiocrine factors Wnt2 and HGF provokes hepatic proliferation. Subsequently, VEGFR2-Id1-dependent proliferative angiogenesis reconstitutes liver mass. Therapeutic co-transplantation of inductive VEGFR2+Id1+Wnt2+HGF+ LSECs with hepatocytes provides an effective strategy to achieve durable liver regeneration.

Engraftment and Reconstitution of Hematopoiesis Is Dependent on VEGFR2-Mediated Regeneration of Sinusoidal Endothelial Cells

Myelosuppression damages the bone marrow (BM) vascular niche, but it is unclear how regeneration of bone marrow vessels contributes to engraftment of transplanted hematopoietic stem and progenitor cells (HSPCs) and restoration of hematopoiesis. We found that chemotherapy and sublethal irradiation induced minor regression of BM sinusoidal endothelial cells (SECs), while lethal irradiation induced severe regression of SECs and required BM transplantation (BMT) for regeneration. Within the BM, VEGFR2 expression specifically demarcated a continuous network of arterioles and SECs, with arterioles uniquely expressing Sca1 and SECs uniquely expressing VEGFR3. Conditional deletion of VEGFR2 in adult mice blocked regeneration of SECs in sublethally irradiated animals and prevented hematopoietic reconstitution. Similarly, inhibition of VEGFR2 signaling in lethally irradiated wild-type mice rescued with BMT severely impaired SEC reconstruction and prevented engraftment and reconstitution of HSPCs. Therefore, regeneration of SECs via VEGFR2 signaling is essential for engraftment of HSPCs and restoration of hematopoiesis.

Improving the efficiency of RNA interference in mammals

RNA interference (RNAi) has been very successfully applied as a gene-silencing technology in both plants and invertebrates, but many practical obstacles need to be overcome before it becomes viable in mammalian systems. Greater specificity and efficiency of RNAi in mammals is being achieved by improving the design and selection of small interfering RNAs (siRNAs), by increasing the efficacy of their delivery to cells and organisms, and by engineering their conditional expression. Genome-wide functional RNAi screens, which are predominantly done in worms and flies, have now begun to revolutionize large-scale loss-of-function studies in mammals.

Effect of angiogenesis inhibition by Id loss and the contribution of bone-marrow-derived endothelial cells in spontaneous murine tumors

Angiogenic defects in Id mutant mice inhibit the growth of tumor xenografts, providing a genetic model for antiangiogenic stress. Our work tests the consequences of such stress on progression of more physiological Pten+/- tumors. While tumor growth occurs despite impaired angiogenesis, disruption of vasculature by Id loss causes tumor cells to experience hypoxia and necrosis, the extent of which is tumor dependent. We show that bone-marrow-derived endothelial precursors contribute functionally to neovasculature of some but not all Pten+/- tumors, partially rescuing Id mutant phenotype. We demonstrate that loss of Id1 in tumor endothelial cells results in downregulation of several proangiogenic genes, including alpha6 and beta4 integrins, matrix metalloprotease-2, and fibroblast growth factor receptor-1. Inhibition of these factors phenocopies loss of Id in in vivo angiogenesis assays.

TGF-β IL-6 axis mediates selective and adaptive mechanisms of resistance to molecular targeted therapy in lung cancer

The epidermal growth-factor receptor (EGFR) tyrosine kinase inhibitor erlotinib has been proven to be highly effective in the treatment of nonsmall cell lung cancer (NSCLC) harboring oncogenic EGFR mutations. The majority of patients, however, will eventually develop resistance and succumb to the disease. Recent studies have identified secondary mutations in the EGFR (EGFR T790M) and amplification of the N-Methyl-N′-nitro-N-nitroso-guanidine (MNNG) HOS transforming gene (MET) oncogene as two principal mechanisms of acquired resistance. Although they can account for approximately 50% of acquired resistance cases together, in the remaining 50%, the mechanism remains unknown. In NSCLC-derived cell lines and early-stage tumors before erlotinib treatment, we have uncovered the existence of a subpopulation of cells that are intrinsically resistant to erlotinib and display features suggestive of epithelial-to-mesenchymal transition (EMT). We showed that activation of TGF-β–mediated signaling was sufficient to induce these phenotypes. In particular, we determined that an increased TGF-β–dependent IL-6 secretion unleashed previously addicted lung tumor cells from their EGFR dependency. Because IL-6 and TGF-β are prominently produced during inflammatory response, we used a mouse model system to determine whether inflammation might impair erlotinib sensitivity. Indeed, induction of inflammation not only stimulated IL-6 secretion but was sufficient to decrease the tumor response to erlotinib. Our data, thus, argue that both tumor cell-autonomous mechanisms and/or activation of the tumor microenvironment could contribute to primary and acquired erlotinib resistance, and as such, treatments based on EGFR inhibition may not be sufficient for the effective treatment of lung-cancer patients harboring mutant EGFR.

Inducible, reversible, and stable RNA interference in mammalian cells.

RNA interference is a powerful genetic approach for efficiently silencing target genes. The existing method of gene suppression by the constitutive expression of short hairpin RNAs (shRNAs) allows analysis of the consequences of stably silencing genes but limits the analysis of genes essential for cell survival, cell cycle regulation, and cell development. We have developed an inducible U6 promoter for synthesis of shRNAs in both human and murine cells. Cells containing stably integrated shRNA expression constructs demonstrate stringent dosage- and time-dependent kinetics of induction with undetectable background expression in the absence of the inducer ecdysone. Inducible suppression of human p53 in glioblastoma cells shows striking morphological changes and defects in cell cycle arrest caused by DNA damage, as expected. Remarkably, the inducibility is reversible after withdrawal of the inducer, as observed by reappearance of the protein and a restoration of the original cell phenotype. Inducible and reversible regulation of RNA interference has broad applications in the areas of mammalian genetics and molecular therapeutics.

Adipogenesis is inhibited by brief, daily exposure to high-frequency, extremely low-magnitude mechanical signals

Obesity, a global pandemic that debilitates millions of people and burdens society with tens of billions of dollars in health care costs, is deterred by exercise. Although it is presumed that the more strenuous a physical challenge the more effective it will be in the suppression of adiposity, here it is shown that 15 weeks of brief, daily exposure to high-frequency mechanical signals, induced at a magnitude well below that which would arise during walking, inhibited adipogenesis by 27% in C57BL/6J mice. The mechanical signal also reduced key risk factors in the onset of type II diabetes, nonesterified free fatty acid and triglyceride content in the liver, by 43% and 39%, respectively. Over 9 weeks, these same signals suppressed fat production by 22% in the C3H.B6–6T congenic mouse strain that exhibits accelerated age-related changes in body composition. In an effort to understand the means by which fat production was inhibited, irradiated mice receiving bone marrow transplants from heterozygous GFP+ mice revealed that 6 weeks of these low-magnitude mechanical signals reduced the commitment of mesenchymal stem cell differentiation into adipocytes by 19%, indicating that formation of adipose tissue in these models was deterred by a marked reduction in stem cell adipogenesis. Translated to the human, this may represent the basis for the nonpharmacologic prevention of obesity and its sequelae, achieved through developmental, rather than metabolic, pathways.