David M. Feldser

Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals

There is growing recognition that mammalian cells produce many thousands of large intergenic transcripts. However, the functional significance of these transcripts has been particularly controversial. Although there are some well-characterized examples, most (>95%) show little evidence of evolutionary conservation and have been suggested to represent transcriptional noise. Here we report a new approach to identifying large non-coding RNAs using chromatin-state maps to discover discrete transcriptional units intervening known protein-coding loci. Our approach identified ∼1,600 large multi-exonic RNAs across four mouse cell types. In sharp contrast to previous collections, these large intervening non-coding RNAs (lincRNAs) show strong purifying selection in their genomic loci, exonic sequences and promoter regions, with greater than 95% showing clear evolutionary conservation. We also developed a functional genomics approach that assigns putative functions to each lincRNA, demonstrating a diverse range of roles for lincRNAs in processes from embryonic stem cell pluripotency to cell proliferation. We obtained independent functional validation for the predictions for over 100 lincRNAs, using cell-based assays. In particular, we demonstrate that specific lincRNAs are transcriptionally regulated by key transcription factors in these processes such as p53, NFκB, Sox2, Oct4 (also known as Pou5f1) and Nanog. Together, these results define a unique collection of functional lincRNAs that are highly conserved and implicated in diverse biological processes.

Requirement for NF-kappaB signalling in a mouse model of lung adenocarcinoma

NF-κB transcription factors function as crucial regulators of inflammatory and immune responses as well as of cell survival. They have also been implicated in cellular transformation and tumorigenesis. However, despite extensive biochemical characterization of NF-κB signalling during the past twenty years, the requirement for NF-κB in tumour development in vivo, particularly in solid tumours, is not completely understood. Here we show that the NF-κB pathway is required for the development of tumours in a mouse model of lung adenocarcinoma. Concomitant loss of p53 (also known as Trp53) and expression of oncogenic Kras(G12D) resulted in NF-κB activation in primary mouse embryonic fibroblasts. Conversely, in lung tumour cell lines expressing Kras(G12D) and lacking p53, p53 restoration led to NF-κB inhibition. Furthermore, the inhibition of NF-κB signalling induced apoptosis in p53-null lung cancer cell lines. Inhibition of the pathway in lung tumours in vivo, from the time of tumour initiation or after tumour progression, resulted in significantly reduced tumour development. Together, these results indicate a critical function for NF-κB signalling in lung tumour development and, further, that this requirement depends on p53 status. These findings also provide support for the development of NF-κB inhibitory drugs as targeted therapies for the treatment of patients with defined mutations in Kras and p53.

Telomere Dysfunction Increases Mutation Rate and Genomic Instability

The increased tumor incidence in telomerase null mice suggests that telomere dysfunction induces genetic instability. To test this directly, we examined mutation rate in the absence of telomerase in S. cerevisiae. The mutation rate in the CAN1 gene increased 10- to 100-fold in est1Delta strains as telomeres became dysfunctional. This increased mutation rate resulted from an increased frequency of terminal deletions. Chromosome fusions were recovered from est1Delta strains, suggesting that the terminal deletions may occur by a breakage-fusion-bridge type mechanism. At one locus, chromosomes with terminal deletions gained a new telomere through a Rad52p-dependent, Rad51p-independent process consistent with break-induced replication. At a second locus, more complicated rearrangements involving multiple chromosomes were seen. These data suggest that telomerase can inhibit chromosomal instability.

Growth Inhibitory and Tumor- Suppressive Functions of p53 Depend on its Repression of CD44 Expression

The p53 tumor suppressor is a key mediator of cellular responses to various stresses. Here, we show that under conditions of basal physiologic and cell-culture stress, p53 inhibits expression of the CD44 cell-surface molecule via binding to a noncanonical p53-binding sequence in the CD44 promoter. This interaction enables an untransformed cell to respond to stress-induced, p53-dependent cytostatic and apoptotic signals that would otherwise be blocked by the actions of CD44. In the absence of p53 function, the resulting derepressed CD44 expression is essential for the growth and tumor-initiating ability of highly tumorigenic mammary epithelial cells. In both tumorigenic and nontumorigenic cells, CD44s expression is positively regulated by p63, a paralogue of p53. Our data indicate that CD44 is a key tumor-promoting agent in transformed tumor cells lacking p53 function. They also suggest that the derepression of CD44 resulting from inactivation of p53 can potentially aid the survival of immortalized, premalignant cells.

Short Telomeres, even in the Presence of Telomerase, Limit Tissue Renewal Capacity

Autosomal-dominant dyskeratosis congenita is associated with heterozygous mutations in telomerase. To examine the dosage effect of telomerase, we generated a line of mTR+/- mice on the CAST/EiJ background, which has short telomeres. Interbreeding of heterozygotes resulted in progressive telomere shortening, indicating that limiting telomerase compromises telomere maintenance. In later-generation heterozygotes, we observed a decrease in tissue renewal capacity in the bone marrow, intestines, and testes that resembled defects seen in dyskeratosis congenita patients. The progressive worsening of disease with decreasing telomere length suggests that short telomeres, not telomerase level, cause stem cell failure. Further, wild-type mice derived from the late-generation heterozygous parents, termed wt*, also had short telomeres and displayed a germ cell defect, indicating that telomere length determines these phenotypes. We propose that short telomeres in mice that have normal telomerase levels can cause an occult form of genetic disease.

Suppression of lung adenocarcinoma progression by Nkx2-1

Despite the high prevalence and poor outcome of patients with metastatic lung cancer the mechanisms of tumour progression and metastasis remain largely uncharacterized. Here we modelled human lung adenocarcinoma, which frequently harbours activating point mutations in KRAS and inactivation of the p53 pathway, using conditional alleles in mice. Lentiviral-mediated somatic activation of oncogenic Kras and deletion of p53 in the lung epithelial cells of KrasLSL-G12D/+;p53flox/flox mice initiates lung adenocarcinoma development. Although tumours are initiated synchronously by defined genetic alterations, only a subset becomes malignant, indicating that disease progression requires additional alterations. Identification of the lentiviral integration sites allowed us to distinguish metastatic from non-metastatic tumours and determine the gene expression alterations that distinguish these tumour types. Cross-species analysis identified the NK2-related homeobox transcription factor Nkx2-1 (also called Ttf-1 or Titf1) as a candidate suppressor of malignant progression. In this mouse model, Nkx2-1 negativity is pathognomonic of high-grade poorly differentiated tumours. Gain- and loss-of-function experiments in cells derived from metastatic and non-metastatic tumours demonstrated that Nkx2-1 controls tumour differentiation and limits metastatic potential in vivo. Interrogation of Nkx2-1-regulated genes, analysis of tumours at defined developmental stages, and functional complementation experiments indicate that Nkx2-1 constrains tumours in part by repressing the embryonically restricted chromatin regulator Hmga2. Whereas focal amplification of NKX2-1 in a fraction of human lung adenocarcinomas has focused attention on its oncogenic function, our data specifically link Nkx2-1 downregulation to loss of differentiation, enhanced tumour seeding ability and increased metastatic proclivity. Thus, the oncogenic and suppressive functions of Nkx2-1 in the same tumour type substantiate its role as a dual function lineage factor.

Hypoxia, HIF-1, and the pathophysiology of common human diseases

Hypoxia plays a fundamental role in the pathophysiology of common causes of mortality, including ischemic heart disease, stroke, cancer, chronic lung disease, and congestive heart failure. In these disease states, hypoxia induces changes in gene expression in target organs that either fail to result in adequate adaptation or directly contribute to disease pathogenesis. Hypoxia-inducible factor 1 (HIF-1) is a transcriptional activator that is expressed in response to cellular hypoxia and mediates multiple cellular and systemic homeostatic responses to hypoxia. Recent studies have provided evidence that important pathophysiological responses to hypoxia in pulmonary hypertension, myocardial ischemia, and cancer are mediated by HIF-1. Pharmacologic and gene therapy strategies designed to modulate HIF-1 activity may represent a novel and effective therapeutic approach to these common disorders.

Telomere dysfunction and the initiation of genome instability

Tumour growth is an evolutionary process that is characterized by the selection of clonal populations of cells that acquire distinct genetic changes. Many cancer therapies aim to exploit the specific changes that occur in cancer cells, but understanding the underlying mechanisms of genomic instability that cause these mutations could lead to more effective therapies. If common mechanisms exist for initiating genomic instability in tumours, selection could explain the differences in specific gene mutations that accumulate in different tumour types. The cause of genomic instability in human tumours is unclear, although there is evidence to indicate that telomere dysfunction could make an important contribution.

Stage-specific sensitivity to p53 restoration during lung cancer progression

Tumorigenesis is a multistep process that results from the sequential accumulation of mutations in key oncogene and tumour suppressor pathways. Personalized cancer therapy that is based on targeting these underlying genetic abnormalities presupposes that sustained inactivation of tumour suppressors and activation of oncogenes is essential in advanced cancers. Mutations in the p53 tumour-suppressor pathway are common in human cancer and significant efforts towards pharmaceutical reactivation of defective p53 pathways are underway. Here we show that restoration of p53 in established murine lung tumours leads to significant but incomplete tumour cell loss specifically in malignant adenocarcinomas, but not in adenomas. We define amplification of MAPK signalling as a critical determinant of malignant progression and also a stimulator of Arf tumour-suppressor expression. The response to p53 restoration in this context is critically dependent on the expression of Arf. We propose that p53 not only limits malignant progression by suppressing the acquisition of alterations that lead to tumour progression, but also, in the context of p53 restoration, responds to increased oncogenic signalling to mediate tumour regression. Our observations also underscore that the p53 pathway is not engaged by low levels of oncogene activity that are sufficient for early stages of lung tumour development. These data suggest that restoration of pathways important in tumour progression, as opposed to initiation, may lead to incomplete tumour regression due to the stage-heterogeneity of tumour cell populations.

Response and Resistance to NF-κB Inhibitors in Mouse Models of Lung Adenocarcinoma

UNLABELLED Lung adenocarcinoma is a leading cause of cancer death worldwide. We recently showed that genetic inhibition of the NF-κB pathway affects both the initiation and the maintenance of lung cancer, identifying this pathway as a promising therapeutic target. In this investigation, we tested the efficacy of small-molecule NF-κB inhibitors in mouse models of lung cancer. In murine lung adenocarcinoma cell lines with high NF-κB activity, the proteasome inhibitor bortezomib efficiently reduced nuclear p65, repressed NF-κB target genes, and rapidly induced apoptosis. Bortezomib also induced lung tumor regression and prolonged survival in tumor-bearing Kras(LSL-G12D/wt);p53(flox/flox) mice but not in Kras(LSL-G12D/wt) mice. After repeated treatment, initially sensitive lung tumors became resistant to bortezomib. A second NF-κB inhibitor, Bay-117082, showed similar therapeutic efficacy and acquired resistance in mice. Our results using preclinical mouse models support the NF-κB pathway as a potential therapeutic target for a defined subset of lung adenocarcinoma. SIGNIFICANCE Using small-molecule compounds that inhibit NF-κB activity, we provide evidence that NF-κB inhibition has therapeutic efficacy in the treatment of lung cancer. Our results also illustrate the value of mouse models in validating new drug targets in vivo and indicate that acquired chemoresistance may later influence bortezomib treatment in lung cancer.