LncRNA RP11-19E11 is an E2F1 target required for proliferation and survival of basal breast cancer.

Long non-coding RNAs (lncRNAs) play key roles in the regulation of breast cancer initiation and progression. LncRNAs are differentially expressed in breast cancer subtypes. Basal-like breast cancers are generally poorly differentiated tumors, are enriched in embryonic stem cell signatures, lack expression of estrogen receptor, progesterone receptor, and HER2 (triple-negative breast cancer), and show activation of proliferation-associated factors. We hypothesized that lncRNAs are key regulators of basal breast cancers. Using The Cancer Genome Atlas, we identified lncRNAs that are overexpressed in basal tumors compared to other breast cancer subtypes and expressed in at least 10% of patients. Remarkably, we identified lncRNAs whose expression correlated with patient prognosis. We then evaluated the function of a subset of lncRNA candidates in the oncogenic process in vitro. Here, we report the identification and characterization of the chromatin-associated lncRNA, RP11-19E11.1, which is upregulated in 40% of basal primary breast cancers. Gene set enrichment analysis in primary tumors and in cell lines uncovered a correlation between RP11-19E11.1 expression level and the E2F oncogenic pathway. We show that this lncRNA is chromatin-associated and an E2F1 target, and its expression is necessary for cancer cell proliferation and survival. Finally, we used lncRNA expression levels as a tool for drug discovery in vitro, identifying protein kinase C (PKC) as a potential therapeutic target for a subset of basal-like breast cancers. Our findings suggest that lncRNA overexpression is clinically relevant. Understanding deregulated lncRNA expression in basal-like breast cancer may lead to potential prognostic and therapeutic applications.

Essential versus accessory aspects of cell death: recommendations of the NCCD 2015.

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as 'accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. 'Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death.

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes.

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

Active repression and E2F inhibition by pRB are biochemically distinguishable.

To understand mechanistically how pRB represses transcription, we used a reconstituted transcription assay and compared pRB activity on naked versus chromatin templates. Surprisingly, when pRB was directly recruited to a naked template, no transcriptional repression was observed. However, we observed active repression when the same promoter was assembled into chromatin. Histone deacetylases do not appear to play a role in this observed repression. Further experiments showed repression could occur after preinitiation complex assembly, in contrast with pRB inhibition of E2F, suggesting discrete mechanisms by which pRB directly inhibits an activator such as E2F or actively represses proximally bound transcription factors.

Analysis of promoter binding by the E2F and pRB families in vivo: distinct E2F proteins mediate activation and repression.

The E2F transcription factor plays a pivotal role in the timely activation of gene expression during mammalian cell cycle progression, whereas pRB and related proteins control cell growth in part through the ability to block the action of E2F. To identify physiologically important E2F-responsive promoters and to study their occupancy and histone acetylation state in vivo, we have taken advantage of a cross-linking approach in synchronized, living cells. We find that the pattern of E2F and pRB-related polypeptides recruited to these promoters changes in a strikingly dynamic fashion as cells progress from quiescence into G(1) and S phase: Repression of each promoter in quiescent cells is associated with recruitment of E2F-4 and p130 and low levels of histone acetylation, but by late G(1), these proteins are replaced largely by E2F-1 and E2F-3, in concert with acetylation of histones H3 and H4 and gene activation. These findings suggest that repression and activation of E2F-responsive genes may occur through distinct E2F heterodimers that direct the sequential recruitment of enzymes able to deacetylate and then acetylate core histones.

Human PC4 is a substrate-specific inhibitor of RNA polymerase II phosphorylation.

The activity of cyclin-dependent protein kinases (cdks) is physiologically regulated by phosphorylation, association with the specific cyclin subunits, and repression by specific cdk inhibitors. All three physiological regulatory mechanisms are specific for one or more cdks, but none is known to be substrate specific. In contrast, synthetic cdk peptide inhibitors that specifically inhibit cdk phosphorylation of only some substrates, "aptamers," have been described. Here, we show that PC4, a naturally occurring transcriptional coactivator, competitively inhibits cdk-1, -2, and -7-mediated phosphorylation of the largest subunit of RNA polymerase II (RNAPII), but it does not inhibit phosphorylation of other substrates of the same kinases. Interestingly, the phosphorylated form of PC4 is devoid of kinase inhibitory activity. We also show that wild-type PC4 but not the kinase inhibitory-deficient mutant of PC4 represses transcription in vivo. Our results point to a novel role for PC4 as a specific inhibitor of RNAPII phosphorylation.

Mechanism of transcriptional repression of E2F by the retinoblastoma tumor suppressor protein.

The retinoblastoma tumor suppressor protein (pRB) is a transcriptional repressor, critical for normal cell cycle progression. We have undertaken studies using a highly purified reconstituted in vitro transcription system to demonstrate how pRB can repress transcriptional activation mediated by the E2F transcription factor. Remarkably, E2F activation became resistant to pRB-mediated repression after the establishment of a partial (TFIIA/TFIID) preinitiation complex (PIC). DNase I footprinting studies suggest that E2F recruits TFIID to the promoter in a step that also requires TFIIA and confirm that recruitment of the PIC by E2F is blocked by pRB. These studies suggest a detailed mechanism by which E2F activates and pRB represses transcription without the requirement of histone-modifying enzymes.

Activity and nature of p21(WAF1) complexes during the cell cycle.

Elevated levels of the p21(WAF1) (p21) cyclin-dependent kinase inhibitor induce growth arrest. We have characterized a panel of monoclonal antibodies against human p21 in an effort to understand the dynamic regulatory interactions between this and other cellular proteins during the cell cycle. The use of these reagents has allowed us to address several important, yet unresolved, issues concerning the biological activity of p21, including the potential kinase activity of complexes that associate with this cyclin-dependent kinase inhibitor. We have found that the kinase activity of cyclin A/Cdk2 associated with p21 is significantly lower than that of cyclin A/Cdk2 free of p21, suggesting that p21 abolishes its activity in vivo, and the use of multiple antibodies has enabled us to begin the study of the molecular architecture of p21 complexes in vivo. In addition, we found that human fibroblasts released from a quiescent state display abundant amounts of p21 devoid of associated proteins ("free" p21), the levels of which decrease as cells approach S phase. Cyclin A levels increase as the amount of monomeric p21 decreases, resulting in an excess of cyclin A/Cdk2 complexes that are not bound to, or inactivated by, p21. Our data strengthen the notion that the G1-to-S phase transition in human fibroblasts occurs when the concentration of cyclin A/Cdk2 surpasses that of p21.

Dual cyclin-binding domains are required for p107 to function as a kinase inhibitor.

The retinoblastoma (pRB) family of proteins includes three proteins known to suppress growth of mammalian cells. Previously we had found that growth suppression by two of these proteins, p107 and p130, could result from the inhibition of associated cyclin-dependent kinases (cdks). One important unresolved issue, however, is the mechanism through which inhibition occurs. Here we present in vivo and in vitro evidence to suggest that p107 is a bona fide inhibitor of both cyclin A-cdk2 and cyclin E-cdk2 that exhibits an inhibitory constant (Ki) comparable to that of the cdk inhibitor p21/WAF1. In contrast, pRB is unable to inhibit cdks. Further reminiscent of p21, a second cyclin-binding site was mapped to the amino-terminal portions of p107 and p130. This amino-terminal domain is capable of inhibiting cyclin-cdk2 complexes, although it is not a potent substrate for these kinases. In contrast, a carboxy-terminal fragment of p107 that contains the previously identified cyclin-binding domain serves as an excellent kinase substrate although it is unable to inhibit either kinase. Clustered point mutations suggest that the amino-terminal domain is functionally important for cyclin binding and growth suppression. Moreover, peptides spanning the cyclin-binding region are capable of interfering with p107 binding to cyclin-cdk2 complexes and kinase inhibition. Our ability to distinguish between p107 and p130 as inhibitors rather than simple substrates suggests that these proteins may represent true inhibitors of cdks.

Regulation of transcription by proteins that control the cell cycle.

In eukaryotes, progression of a cell through the cell cycle is partly controlled at the level of transcriptional regulation. Yeast and mammalian cells use similar mechanisms to achieve this regulation. Although gaps still remain, progress has been made recently in connecting the links between the cell's cycle and its transcriptional machinery.

p130 and p107 use a conserved domain to inhibit cellular cyclin-dependent kinase activity.

The pRB-related proteins p107 and p130 are thought to suppress growth in part through their associations with two important cell cycle kinases, cyclin A-cdk2 and cyclin E-cdk2, and transcription factor E2F. Although each protein plays a critical role in cell proliferation, the functional consequences of the association among growth suppressor, cyclin-dependent kinase, and transcription factor have remained elusive. In an attempt to understand the biochemical properties of such complexes, we reconstituted each of the p130-cyclin-cdk2 and p107-cyclin-cdk2 complexes found in vivo with purified, recombinant proteins. Strikingly, stoichiometric association of p107 or p130 with either cyclin E-cdk2 or cyclin A-cdk2 negated the activities of these kinases. The results of our experiments suggest that inhibition does not result from substrate competition or loss of cdk2 activation. Kinase inhibitory activity was dependent upon an amino-terminal region of p107 that is highly conserved with p130. Further, a role for this amino-terminal region in growth suppression was uncovered by using p107 mutants unable to bind E2F. To determine whether cellular complexes might display similar regulatory properties, we purified p130-cyclin A-cdk2 complexes from human cells and found that such complexes exist in two forms, one that contains E2F-4-DP-1 and one that lacks the heterodimer. These endogenous complexes behaved like the in vitro-reconstituted complexes, exhibiting low levels of associated kinase activity that could be significantly augmented by dissociation of p130. The results of these experiments suggest a mechanism whereby p130 and p107 suppress growth by inhibiting important cell cycle kinases.

Specific regulation of E2F family members by cyclin-dependent kinases.

The transcription factor E2F-1 interacts stably with cyclin A via a small domain near its amino terminus and is negatively regulated by the cyclin A-dependent kinases. Thus, the activities of E2F, a family of transcription factors involved in cell proliferation, are regulated by at least two types of cell growth regulators: the retinoblastoma protein family and the cyclin-dependent kinase family. To investigate further the regulation of E2F by cyclin-dependent kinases, we have extended our studies to include additional cyclins and E2F family members. Using purified components in an in vitro system, we show that the E2F-1-DP-1 heterodimer, the functionally active form of the E2F activity, is not a substrate for the active cyclin D-dependent kinases but is efficiently phosphorylated by the cyclin B-dependent kinases, which do not form stable complexes with the E2F-1-DP-1 heterodimer. Phosphorylation of the E2F-1-DP-1 heterodimer by cyclin B-dependent kinases, however, did not result in down-regulation of its DNA-binding activity, as is readily seen after phosphorylation by cyclin A-dependent kinases, suggesting that phosphorylation per se is not sufficient to regulate E2F DNA-binding activity. Furthermore, heterodimers containing E2F-4, a family member lacking the cyclin A binding domain found in E2F-1, are not efficiently phosphorylated or functionally down-regulated by cyclin A-dependent kinases. However, addition of the E2F-1 cyclin A binding domain to E2F-4 conferred cyclin A-dependent kinase-mediated down-regulation of the E2F-4-DP-1 heterodimer. Thus, both enzymatic phosphorylation and stable physical interaction are necessary for the specific regulation of E2F family members by cyclin-dependent kinases.

Cyclin-binding motifs are essential for the function of p21CIP1.

The cyclin-dependent kinase (Cdk) inhibitor p21 is induced by the tumor suppressor p53 and is required for the G1-S block in cells with DNA damage. We report that there are two copies of a cyclin-binding motif in p21, Cy1 and Cy2, which interact with the cyclins independently of Cdk2. The cyclin-binding motifs of p21 are required for optimum inhibition of cyclin-Cdk kinases in vitro and for growth suppression in vivo. Peptides containing only the Cy1 or Cy2 motif partially inhibit cyclin-Cdk kinase activity in vitro and DNA replication in Xenopus egg extracts. A monoclonal antibody which recognizes the Cy1 site of p21 specifically disrupts the association of p21 with cyclin E-Cdk2 and with cyclin D1-Cdk4 in cell extracts. Taken together, these observations suggest that the cyclin-binding motif of p21 is important for kinase inhibition and for formation of p21-cyclin-Cdk complexes in the cell. Finally, we show that the cyclin-Cdk complex is partially active if associated with only the cyclin-binding motif of p21, providing an explanation for how p21 is found associated with active cyclin-Cdk complexes in vivo. The Cy sequences may be general motifs used by Cdk inhibitors or substrates to interact with the cyclin in a cyclin-Cdk complex.

Transcriptional control of the cell cycle.

Although a significant amount of evidence has demonstrated that there are intimate connections between transcriptional controls and cell cycle regulation, the precise mechanisms underlying these connections remain largely obscure. A number of recent advances have helped to define how critical cell cycle regulators, such as the retinoblastoma family of tumor suppressor proteins and the cyclin-dependent kinases, might function on a biochemical level and how such mechanisms of action have been conserved not only in the regulation of transcription by all three RNA polymerases but also across species lines. In addition, the use of in vivo techniques has begun to explain how the activity of the E2F transcription factor family is tied to the cell cycle dependent expression of target genes.

The anti-proliferative effect of sulindac and sulindac sulfide on HT-29 colon cancer cells: alterations in tumor suppressor and cell cycle-regulatory proteins.

Nonsteroidal anti-inflammatory drugs lower the incidence of and mortality from colon cancer. Sulindac reduces the number and size of polyps in patients with familial adenomatous polyposis. We have shown that sulindac and sulindac sulfide reversibly reduce the proliferation rate of HT-29 colon cancer cells, alter their morphology, induce them to accumulate in the G0/G1 phase of the cell cycle, and sulindac sulfide induces cell death by apoptosis. In this study we confirmed that sulindac and sulindac sulfide prevent HT-29 cells from progressing from the G0/G1 into the S phase. This block in cell cycle progression is associated with an initial rise, then an abrupt decrease in the levels of p34cdc2 protein. Sulindac and sulindac sulfide decrease the levels of mitotic cyclins, induce the levels of p21WAF-1/cip1, and reduce the total levels of pRB, with a relative increase in the amount of the underphosphorylated form of pRB in a time- and concentration-dependent manner. In addition, these compounds reduce the levels of mutant p53. These responses are not associated with intestinal cell differentiation and occur independent of the ability of these compounds to induce apoptosis. We conclude that sulindac and sulindac sulfide reduce the levels of major components of the molecular cell cycle machinery and alter the levels of several tumor suppressor proteins in a manner consistent with cell cycle quiescence. These mechanisms may be operative in vivo to account, in part, for the anti-neoplastic effects of these compounds.

p107 uses a p21CIP1-related domain to bind cyclin/cdk2 and regulate interactions with E2F.

The kinase activities of the cyclin/cdk complexes can be regulated in a number of ways. The most recently discovered mechanism of regulation is the association of cdk inhibitors (CKIs), such as p21, p27, and p57, with these complexes. In this report we demonstrate that the pRB-related protein p107, like the p21 family of cdk inhibitors, can inhibit the phosphorylation of target substrates by cyclin A/cdk2 and cyclin E/cdk2 complexes, and the associations of p107 and p21 with cyclin/cdk2 rely on a structurally and functionally related interaction domain. Furthermore, interactions between p107 or p21 with cyclin/cdk2 complexes are mutually exclusive. In cells treated with DNA-damaging agents elevated levels of p21 cause a dissociation of p107/cyclin/cdk2 complexes to yield p21/cyclin/cdk2 complexes. Finally, the consequences of cyclin/cdk2 interactions with p107 have been examined. The activation of the p107-bound cyclin/cdk kinases leads to dissociation of p107 from the transcription factor E2F. Together, these results suggest that cyclin/cdk complexes can be regulated by protein molecules from different families in a mutually exclusive manner in response to certain signals and that these inhibitory proteins may have a potential role in regulating macromolecular assembly.

Tumour-derived p16 alleles encoding proteins defective in cell-cycle inhibition.

The cyclin-dependent kinase inhibitor p16 is a candidate tumour-suppressor protein that maps to a genomic locus strongly associated with familial melanoma and other tumour types. Screening of primary tumours and linkage analysis of familial melanoma pedigrees have identified many potential mutations in p16, but the functional significance of these sequence variants has remained unclear. We report here that p16 can act as a potent and specific inhibitor of progression through the G1 phase of the cell cycle, and we demonstrate that several tumour-derived alleles of p16 encode functionally compromised proteins. The ability of p16 to arrest cell-cycle progression generally correlates with inhibition of cyclin D1/Cdk4 kinase activity in vitro, with two exceptions among the alleles tested. In vivo, the presence of functional retinoblastoma protein appears to be necessary but may not be sufficient to confer full sensitivity to p16-mediated growth arrest. Our results provide support for the notion that p16 is an important cell-cycle regulator whose inactivation contributes to the outgrowth of human tumours.

Differential regulation of E2F transactivation by cyclin/cdk2 complexes.

The mammalian transcription factor E2F plays a critical role in the expression of genes required for cellular proliferation. To understand how E2F is regulated, we have developed a reconstituted in vitro transcription assay. Using this E2F-responsive assay, we can demonstrate that E2F-mediated transcription can be directly repressed by the tumor suppressor protein pRB. This inhibition is abolished by phosphorylation of pRB with either cyclin A/cdk2 or cyclin E/cdk2. However, these cyclin/kinase complexes exhibit differences in the ability to phosphorylate E2F. Only cyclin A/cdk2 can phosphorylate E2F effectively, and this phosphorylation abolishes its ability to bind DNA and mediate trans-activation. Thus, this in vitro transcriptional assay allows activation and inactivation of E2F transcription, and our findings demonstrate how transcriptional regulation of E2F can be linked to cell cycle-dependent activation of kinases.