Ittai Ben-Porath

An embryonic stem cell–like gene expression signature in poorly differentiated aggressive human tumors

Cancer cells possess traits reminiscent of those ascribed to normal stem cells. It is unclear, however, whether these phenotypic similarities reflect the activity of common molecular pathways. Here, we analyze the enrichment patterns of gene sets associated with embryonic stem (ES) cell identity in the expression profiles of various human tumor types. We find that histologically poorly differentiated tumors show preferential overexpression of genes normally enriched in ES cells, combined with preferential repression of Polycomb-regulated genes. Moreover, activation targets of Nanog, Oct4, Sox2 and c-Myc are more frequently overexpressed in poorly differentiated tumors than in well-differentiated tumors. In breast cancers, this ES-like signature is associated with high-grade estrogen receptor (ER)-negative tumors, often of the basal-like subtype, and with poor clinical outcome. The ES signature is also present in poorly differentiated glioblastomas and bladder carcinomas. We identify a subset of ES cell-associated transcription regulators that are highly expressed in poorly differentiated tumors. Our results reveal a previously unknown link between genes associated with ES cell identity and the histopathological traits of tumors and support the possibility that these genes contribute to stem cell–like phenotypes shown by many tumors.

Autocrine TGF-β and stromal cell-derived factor-1 (SDF-1) signaling drives the evolution of tumor-promoting mammary stromal myofibroblasts

Much interest is currently focused on the emerging role of tumor-stroma interactions essential for supporting tumor progression. Carcinoma-associated fibroblasts (CAFs), frequently present in the stroma of human breast carcinomas, include a large number of myofibroblasts, a hallmark of activated fibroblasts. These fibroblasts have an ability to substantially promote tumorigenesis. However, the precise cellular origins of CAFs and the molecular mechanisms by which these cells evolve into tumor-promoting myofibroblasts remain unclear. Using a coimplantation breast tumor xenograft model, we show that resident human mammary fibroblasts progressively convert into CAF myofibroblasts during the course of tumor progression. These cells increasingly acquire two autocrine signaling loops, mediated by TGF-β and SDF-1 cytokines, which both act in autostimulatory and cross-communicating fashions. These autocrine-signaling loops initiate and maintain the differentiation of fibroblasts into myofibroblasts and the concurrent tumor-promoting phenotype. Collectively, these findings indicate that the establishment of the self-sustaining TGF-β and SDF-1 autocrine signaling gives rise to tumor-promoting CAF myofibroblasts during tumor progression. This autocrine-signaling mechanism may prove to be an attractive therapeutic target to block the evolution of tumor-promoting CAFs.

When cells get stressed: an integrative view of cellular senescence

Cells entering a state of senescence undergo a permanent cell cycle arrest, accompanied by a set of functional and morphological changes. Senescence of cells occurs following an extended period of proliferation in culture or in response to various physiologic stresses, yet little is known about the role this phenomenon plays in vivo. The study of senescence has focused largely on its hypothesized role as a barrier to extended cell division, governed by a division-counting mechanism in the form of telomere length. Here, we discuss the biological functions of cellular senescence and suggest that it should be viewed in terms of its role as a general cellular stress response program, rather than strictly as a barrier to unlimited cycles of cell growth and division. We also discuss the relative roles played by telomere shortening and telomere uncapping in the induction of senescence.

Erosion of the telomeric single-strand overhang at replicative senescence

Cultured primary human cells inevitably enter a state of replicative senescence for which the specific molecular trigger is unknown. We show that the single-strand telomeric overhang, a key component of telomere structure, is eroded at senescence. Expression of telomerase prevents overhang loss, suggesting that this enzyme prevents senescence by maintaining proper telomere structure. In contrast, progressive overhang loss occurs in cells that avoid senescence through the inactivation of p53 and Rb, indicating that overhang erosion is the result of continuous cell division and not a consequence of senescence. We thus provide evidence for a specific molecular alteration in telomere structure at senescence and suggest that this change, rather than overall telomere length, serves to trigger this state.

Mbd1 is recruited to both methylated and nonmethylated CpGs via distinct DNA binding domains

ABSTRACT MBD1 is a vertebrate methyl-CpG binding domain protein (MBD) that can bring about repression of methylated promoter DNA sequences. Like other MBD proteins, MBD1 localizes to nuclear foci that in mice are rich in methyl-CpG. In methyl-CpG-deficient mouse cells, however, Mbd1 remains localized to heterochromatic foci whereas other MBD proteins become dispersed in the nucleus. We find that Mbd1a, a major mouse isoform, contains a CXXC domain (CXXC-3) that binds specifically to nonmethylated CpG, suggesting an explanation for methylation-independent localization. Transfection studies demonstrate that the CXXC-3 domain indeed targets nonmethylated CpG sites in vivo. Repression of nonmethylated reporter genes depends on the CXXC-3 domain, whereas repression of methylated reporters requires the MBD. Our findings indicate that MBD1 can interpret the CpG dinucleotide as a repressive signal in vivo regardless of its methylation status.

Role of DNA Methylation in Stable Gene Repression

A large fraction of the animal genome is maintained in a transcriptionally repressed state throughout development. By generating viable Dnmt1-/- mouse cells we have been able to study the effect of DNA methylation on both gene expression and chromatin structure. Our results confirm that the underlying methylation pattern has a profound effect on histone acetylation and is the major effector of me-H3(K4) in the animal genome. We demonstrate that many methylated genes are subject to additional repression mechanisms that also impact on histone acetylation, and the data suggest that late replication timing may play an important role in this process.

Directed elimination of senescent cells by inhibition of BCL-W and BCL-XL

Senescent cells, formed in response to physiological and oncogenic stresses, facilitate protection from tumourigenesis and aid in tissue repair. However, accumulation of such cells in tissues contributes to age-related pathologies. Resistance of senescent cells to apoptotic stimuli may contribute to their accumulation, yet the molecular mechanisms allowing their prolonged viability are poorly characterized. Here we show that senescent cells upregulate the anti-apoptotic proteins BCL-W and BCL-XL. Joint inhibition of BCL-W and BCL-XL by siRNAs or the small-molecule ABT-737 specifically induces apoptosis in senescent cells. Notably, treatment of mice with ABT-737 efficiently eliminates senescent cells induced by DNA damage in the lungs as well as senescent cells formed in the epidermis by activation of p53 through transgenic p14ARF. Elimination of senescent cells from the epidermis leads to an increase in hair-follicle stem cell proliferation. The finding that senescent cells can be eliminated pharmacologically paves the way to new strategies for the treatment of age-related pathologies.

Characterization of a tumor-associated gene, a member of a novel family of genes encoding membrane glycoproteins.

To isolate genes involved in tumor formation and in embryogenesis, a subtracted cDNA library was constructed from a c-myc-induced mouse brain tumor. A gene isolated in this screen, named TMP (tumor-associated membrane protein), codes for a putative glycoprotein with four transmembrane domains. The TMP gene was found to be highly expressed in brain tumor cells but not in normal brain. It is also expressed at high levels in undifferentiated embryonic stem cells, but markedly down-regulated in these cells after their differentiation into embryoid bodies. The TMP amino acid sequence bears high homology to the growth arrest specific protein PMP22/GAS-3, which is involved in several human peripheral neuropathies. The expression patterns of the TMP and PMP22 genes in NIH-3T3 fibroblasts were compared at different proliferation states. The results suggest an inverse pattern of expression for the two homologs, TMP expression being high during cell proliferation and PMP22 expression being high during growth arrest. To further characterize the TMP gene we have isolated its human homolog and examined its expression in embryonic and adult tissues. In our search for human sequences homologous to TMP and PMP22, we identified two new genes which we have named XMP and YMP. Thus, we present a novel family of membrane glycoproteins, one member of which is closely associated with proliferation and another with growth arrest.

p16Ink4a-induced senescence of pancreatic beta cells enhances insulin secretion

Cellular senescence is thought to contribute to age-associated deterioration of tissue physiology. The senescence effector p16Ink4a is expressed in pancreatic beta cells during aging and limits their proliferative potential; however, its effects on beta cell function are poorly characterized. We found that beta cell–specific activation of p16Ink4a in transgenic mice enhances glucose-stimulated insulin secretion (GSIS). In mice with diabetes, this leads to improved glucose homeostasis, providing an unexpected functional benefit. Expression of p16Ink4a in beta cells induces hallmarks of senescence—including cell enlargement, and greater glucose uptake and mitochondrial activity—which promote increased insulin secretion. GSIS increases during the normal aging of mice and is driven by elevated p16Ink4a activity. We found that islets from human adults contain p16Ink4a-expressing senescent beta cells and that senescence induced by p16Ink4a in a human beta cell line increases insulin secretion in a manner dependent, in part, on the activity of the mechanistic target of rapamycin (mTOR) and the peroxisome proliferator-activated receptor (PPAR)-γ proteins. Our findings reveal a novel role for p16Ink4a and cellular senescence in promoting insulin secretion by beta cells and in regulating normal functional tissue maturation with age.

The Tmp Gene, Encoding a Membrane Protein, Is a c-Myc Target with a Tumorigenic Activity

ABSTRACT The c-Myc oncoprotein induces cell proliferation and transformation through its activity as a transcription factor. Uncovering the genes regulated by c-Myc is an essential step for understanding these processes. We recently isolated the tumor-associated membrane protein gene, Tmp, from a c-myc-induced mouse brain tumor. Here we show that Tmp is specifically highly expressed in mammary tumors and T-cell lymphomas which develop in c-myc transgenic mice, suggesting that Tmpexpression is a general characteristic of c-Myc-induced tumors. In addition, Tmp expression is induced upon serum stimulation of fibroblasts as shown in a time course closely correlated with c-myc expression. We have isolated the Tmppromoter region and identified a putative c-Myc binding element, CACGTG, located in the first intron of the gene. We show here that constructs containing the Tmp regulatory region fused to a reporter gene are activated by c-Myc through this CACGTG element and that the c-Myc–Max protein complex can bind to this element. Moreover, an inducible form of c-Myc, the MycER fusion protein, can activate the endogenous Tmp gene. We also show that Tmp-overexpressing fibroblasts induce rapidly growing tumors when injected into nude mice, suggesting thatTmp may possess a tumorigenic activity. Thus, TMP, a member of a novel family of membrane glycoproteins with a suggested role in cellular contact, is a c-Myc target and is possibly involved in c-Myc-induced transformation.