Coactosin-like protein CLP/Cotl1 suppresses breast cancer growth through activation of IL-24/PERP and inhibition of non-canonical TGFß signaling.

Coactosin-like protein (CLP, or Cotl1), is an F-actin-binding protein, whose role in cancer is largely unknown. Here we show that CLP/Cotl1 is highly expressed in a rat epithelial breast cancer cell line (FE1.3) compared with its mesenchymal counterpart (FE1.2). Knockdown of CLP/Cotl1 in FE1.3 cells increased cell proliferation, whereas its overexpression in FE1.2 cells inhibited proliferation in culture and reduced tumor growth in xenograft assays in mice. Mechanistically, we identified two major pathways through which CLP/Cotl1 exerts its suppressive effects. First, CLP/Cotl1 re-expression in FE1.2 and in human MCF7 breast cancer cells induced expression of the growth-suppressor gene interleukin-24 (IL-24), which independently of p53 upregulates the tumor-suppressor genes p53 apoptosis effector related to PMP-22 (PERP) and p21cip1. Second, overexpression of CLP/Cotl1 potentiated the growth-suppressive effect of transforming growth factor-ß1 (TGFß1), leading to downregulation of TGFß-responsive genes vascular growth factor A/B (VEGFA/VEGFB), hypoxia inducing factor 1α (HIF-1α) and trombospondin 1 (TSP1), which mediate various hallmarks of cancer progression including angiogenesis, invasion and metastasis. CLP/Cotl1 inhibited TGFß signaling via a non-canonical signaling involving IL-24-instigated inhibition of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) phosphorylation and subsequent post-transcriptional downregulation of SMAD2 and SMAD4. We also showed that CLP/COTL1 expression sensitizes breast cancer cells to chemotherapeutic drugs, and this was further enhanced by addition of exogenous TGFß1. CLP/Cotl1 expression is lost in many human malignancies including prostate, uterine and breast cancers. Thus, our results uncover a novel tumor-suppressor role for CLP/Cotl1 and identify the downstream effectors interleukin 24 (IL-24)/PERP and IL-24/MAPK/ERK/TGFß as potential targets for precision therapy.

A bipartite nuclear localization signal in the retinoblastoma gene product and its importance for biological activity.

The retinoblastoma gene product, p110RB1, appears to regulate cell growth by modulating the activities of nuclear transcription factors. The elements that specify the transport of p110RB1 into the nucleus have not yet been explored. We now report the identification of a basic region, KRSAEGGNPPKPLKKLR, in the C terminus of p110RB1, which has sequence similarity to known bipartite nuclear localization signals (NLSs). A two-amino-acid mutation introduced into this putative NLS [to give mutant NLS(NQ)] or deletion of the entire NLS (delta NLS) abrogated exclusive nuclear localization, yielding proteins which were distributed either equally throughout the cell or predominantly in the cytoplasm. A mutant protein [NLS(NQ)/delta 22] containing both the mutated NLS and a deletion of exon 22, previously shown to disrupt the interaction of p110RB1 with several cellular transcription factors and oncoproteins, accumulated only in the cytoplasm. When fused to the C terminus of Escherichia coli beta-galactosidase, the RB1 NLS directed this protein to the nucleus, indicating that the motif is not only necessary but also sufficient for nuclear transport. Neither NLS(NQ) nor delta NLS was hyperphosphorylated in vivo, but both retained their abilities to interact, in vitro, with simian virus 40 large T antigen, adenovirus E1a, and the cellular transcription factor E2F. When transfected at multiple copy number, the NLS mutant alleles displayed reduced biological activity, measured by inhibition of growth of the osteogenic sarcoma cell line Saos-2, which has no wild-type RB1. Naturally occurring mutations and deletions in exon 25 of RB1 which disrupt the NLS may lead to partial or complete inactivation of p110RB1 and may be responsible for some retinoblastoma and other tumors.

Fli-1, an Ets-related transcription factor, regulates erythropoietin-induced erythroid proliferation and differentiation: evidence for direct transcriptional repression of the Rb gene during differentiation.

Erythropoietin (Epo) is a major regulator of erythropoiesis that alters the survival, proliferation, and differentiation of erythroid progenitor cells. The mechanism by which these events are regulated has not yet been determined. Using HB60, a newly established erythroblastic cell line, we show here that Epo-induced terminal erythroid differentiation is associated with a transient downregulation in the expression of the Ets-related transcription factor Fli-1. Constitutive expression of Fli-1 in HB60 cells, similar to retroviral insertional activation of Fli-1 observed in Friend murine leukemia virus (F-MuLV)-induced erythroleukemia, blocks Epo-induced differentiation while promoting Epo-induced proliferation. These results suggest that Fli-1 modulates the response of erythroid cells to Epo. To understand the mechanism by which Fli-1 regulates erythropoiesis, we searched for downstream target genes whose expression is regulated by this transcription factor. Here we show that the retinoblastoma (Rb) gene, which was previously shown to be involved in the development of mature erythrocytes, contains a Fli-1 consensus binding site within its promoter. Fli-1 binds to this cryptic Ets consensus site within the Rb promoter and transcriptionally represses Rb expression. Both the expression level and the phosphorylation status of Rb are consistent with the response of HB60 cells to Epo-induced terminal differentiation. We suggest that the negative regulation of Rb by Fli-1 could be one of the critical determinants in erythroid progenitor cell differentiation that is specifically deregulated during F-MuLV-induced erythroleukemia.

Transcription Factors in Breast Cancer-Lessons From Recent Genomic Analyses and Therapeutic Implications.

Multiplatform genomic analyses have identified 93 frequently altered genes in breast cancer. Of these, as many as 49 genes are directly or indirectly involved in transcription. These include constitutive and inducible DNA-binding transcription factors (DB-TFs, 13 genes), corepressors/coactivators (14 genes), epigenetic (10), and mediator/splicing/rRNA (3) factors. At least nine additional genes are immediate upstream regulators of transcriptional cofactors. G:profiler analysis reveals that these alterations affect cell cycle, development/differentiation, steroid hormone, and chromatin modification pathways. A notable observation is that DB-TFs that mediate major oncogenic signaling (e.g., WNT, receptor tyrosine kinase (RTK), NOTCH, and HIPPO), which switch from default repression (signal OFF) to transcriptional activation (signal ON), are not altered in breast cancer. Instead, corepressors (e.g., pRb for E2F1 downstream of various proliferation signals) or upstream factors (e.g., APC and AXIN for TCF, downstream of canonical WNT signaling) are lost, or coactivators (e.g., NOTCH1/2 for CSL/RBPJk) are induced. In contrast, constitutive (MYC, TBX3) and signal-induced (TP53, FOXA1) DB-TFs that do not mediate default repression are directly altered in breast cancer. Some of these TFs have been implicated in the establishment of super-enhancers and positive transcriptional elongation. In addition, oncogenic transcription is induced by mutations affecting regulatory elements or chromatin conformation that create new TF-binding sites in promoters and enhancers of oncogenic genes to promote tumorigenesis. Here we review these diverse oncogenic alterations in TFs in BC and discuss implications for therapy.

Inactivation of the retinoblastoma tumor suppressor induces apoptosis protease-activating factor-1 dependent and independent apoptotic pathways during embryogenesis.

Inactivation of the retinoblastoma (Rb) tumor suppressor in the mouse induces mid-gestational death accompanied by massive apoptosis in certain tissues. Herein, we analyzed the role of the apoptosis protease-activating factor Apaf-1, an essential component of the apoptosome, in mediating apoptosis in Rb-deficient mice. Analysis of compound mutant embryos lacking Rb and Apaf-1 revealed that Apaf-1 was absolutely required for apoptosis in the central nervous system and lens. In contrast, apoptosis in the peripheral nervous system and skeletal muscles only partly depended on Apaf-1 function. The dependency on Apaf-1 coincided with the requirement documented previously for E2F1 and p53 in the respective tissues. Loss of Apaf-1 specifically suppressed apoptosis but not the proliferation and differentiation defects in Rb-mutant embryos. We also show that the Apaf1+ but not the Rb+ allele is retained in pituitary tumors arising in Rb+/-:Apaf1+/- double heterozygous mice. Our results indicate that Apaf-1 plays a critical role in apoptosis in a subset of tissues and that both E2F1:p53:Apaf-1-dependent and -independent apoptotic pathways operate downstream of Rb.

E2F1 mediates ectopic proliferation and stage-specific p53-dependent apoptosis but not aberrant differentiation in the ocular lens of Rb deficient fetuses.

The retinoblastoma tumor suppressor, Rb, is a transcription cofactor that controls cell proliferation, survival and differentiation. Mutant mouse embryos lacking Rb exhibit ectopic proliferation and apoptosis that are mediated in some tissues by E2F1, a major partner of Rb, and by the p53 tumor suppressor. Whether E2F1 and p53 also mediate the differentiation defects in Rb mutant embryos is, however, not clear. Here we show that partially rescued mgRb:Rb-/- mutant fetuses exhibit ectopic lens epithelial cell proliferation, apoptosis and severe cataract. The abnormal cell proliferation and apoptosis were significantly suppressed in the lens of compound mutant fetuses lacking both Rb and E2F1 at embryonic day (E) E15.5. Interestingly however, at E18.5, only ectopic proliferation, not apoptosis, was dramatically reduced in mgRb:Rb-/-:E2F1-/- lenses. In contrast, p53 did not exert such a stage-specific effect and apoptosis was invariably suppressed in mgRb:Rb-/-:p53-/- composite mutant lenses throughout embryogenesis. Using RT-PCR and in situ hybridization analyses, we identified a subset of lens specific genes, most notably the late differentiation marker filensin, which were not properly induced during lens development in mgRb:Rb-/-fetuses. Remarkably, despite the inhibition of cell proliferation and apoptosis, the degeneration of lens fibers and aberrant expression of filensin were only marginally corrected in mgRb:Rb-/-:E2F1-/- fetuses at E15.5 but not at all at E18.5 or in mgRb:Rb-/-:p53-/mutant fetuses. Thus, inactivation of E2F1 reduces ectopic cell proliferation and stage-specific p53-dependent apoptosis but does not rescue the differentiation defects associated with loss of Rb during lens development.

Molecular cloning and characterization of the mouse RB1 promoter.

We report the isolation and characterization of the mouse RB1 promoter and surrounding DNA sequences, and the identification of elements required for basal transcriptional activity. The mouse RB1 promoter, like the human homologue, has a high G + C content, constitutes a CpG island and is devoid of typical TATA and CAAT boxes. The first 235 base-pairs upstream of the translation initiation codon in the mouse promoter exhibit 80% sequence homology with the human sequence. This homology includes a region which contains putative binding sites for the transcription factors Sp1, ATF and E2F/DRTF1. Four major transcription initiation sites were identified downstream of this conserved region. Mutational analysis revealed that the Sp1 and ATF binding sites, but not the putative E2F/DRTF1 binding site, are critical for promoter activity. Complete disruption of the putative Sp1 and ATF sites abrogated transcription, whereas the introduction of point mutations, previously identified in the Sp1 and ATF sites in two low penetrance retinoblastoma families, reduced promoter activity in a cell type specific manner. Less reduction in activity occurred in retinoic acid induced differentiated P19 cells and NIH3T3 mouse fibroblasts than in undifferentiated embryonal carcinoma P19 cells. Activity of the RB1 promoter was found to be stimulated in retinoic-acid induced differentiated P19 cells compared to undifferentiated P19; this stimulation required intact Sp1 and ATF sites but not the putative E2F/DRTF1 binding site. Our results indicate that basal level of RB1 expression is governed by Sp1 and ATF.

The retinoblastoma gene family is differentially expressed during embryogenesis.

We report differential expression of the RB1 tumor suppressor gene and the homologous genes p107 and p130 during embryogenesis. Abundant RB1 transcripts were detected during neurogenesis, hematopoiesis, myogenesis, lens development and in the ganglion cell layer of the embryonic retina, prior to and during differentiation. The expression pattern of RB1 mirrored the defects in RB1 mutant mice (RB-/-). In the heart, lung, kidney and intestine, p107, but not RB1, was expressed. In the liver and the central nervous system p107 and RB1 were co-expressed, consistent with the accelerated cell death observed in RB-/-; p107-/- double knock-out mice. In the central nervous system, p107 expression was restricted to proliferating cells surrounding the ventricles, while RB1 was expressed in areas of both proliferating and differentiating cells. In contrast to RB1 and p107, expression of p130 was low throughout embryogenesis. In situ hybridization and Western blot analyses showed that the expression of p107 and p130 was not markedly altered in RB-/- embryos compared to control littermates. Our results suggest that members of RB1 gene family have distinct, but overlapping roles in embryogenesis, with p107 and RB1 possibly having redundant functions in the central nervous system and liver.

Nuclear localization conferred by the pocket domain of the retinoblastoma gene product.

The tumor suppressor Rb is a nuclear phosphoprotein that controls cell growth and differentiation by modulating the activity of certain transcription factors. Transport of Rb to the nucleus is affected by both a bipartite nuclear localization signal (NLS) in the C-terminus of the protein and a central domain, termed A/B or pocket, through which Rb interacts with transcription factors and viral oncoproteins. Mutations in either the A or B subdomains of the pocket render a NLS-deficient Rb completely cytoplasmic. Fusing the A/B domain of Rb to the Escherichia coli beta-galactosidase, to create betagal-A/B, confers nuclear localization upon this bacterial protein. Moreover, co-expression with the adenovirus oncoprotein, E1A, further augments nuclear localization of betagal-A/B. These findings provide direct evidence that the pocket domain of Rb is not only required but also sufficient to induce nuclear transport by a 'piggyback' mechanism. Thus, nuclear localization of Rb is dictated by two independent and autonomous domains: (i) the bipartite NLS and (ii) the pocket domain. We suggest that via these domains, Rb chaperons and co-compartmentalizes with its associated factors and preempts their activity prior to nuclear transport.

The ets transcription factor Fli-1 in development, cancer and disease.

Friend leukemia virus-induced erythroleukemia-1 (Fli-1), an E26 transformation specific (ETS) transcription factor, was isolated a quarter century ago through a retrovirus mutagenesis screen. Fli-1 has since been recognized to play critical roles in normal development and homeostasis. For example, it transcriptionally regulates genes that drive normal hematopoiesis and vasculogenesis. Indeed, Fli-1 is one of 10 key regulators of hematopoietic stem/progenitor cell maintenance and differentiation. Aberrant expression of Fli-1 also underlies a number of virally induced leukemias, including Friend virus-induced erythroleukemia and various types of human cancers, and it is the target of chromosomal translocations in childhood Ewing's sarcoma. Abnormal expression of Fli-1 is important in the etiology of autoimmune diseases such as systemic lupus erythematosus and systemic sclerosis. These studies establish Fli-1 as a strong candidate for drug development. Despite difficulties in targeting transcription factors, recent studies identified small-molecule inhibitors for Fli-1. Here we review past and ongoing research on Fli-1 with emphasis on its mechanistic function in autoimmune disease and malignant transformation. The significance of identifying Fli-1 inhibitors and their clinical applications for treatment of disease and cancer with deregulated Fli-1 expression are discussed.

Effect of glycogen synthase kinase-3 inactivation on mouse mammary gland development and oncogenesis.

Many components of the Wnt/ß-catenin signaling pathway have critical functions in mammary gland development and tumor formation, yet the contribution of glycogen synthase kinase-3 (GSK-3α and GSK-3ß) to mammopoiesis and oncogenesis is unclear. Here, we report that WAP-Cre-mediated deletion of GSK-3 in the mammary epithelium results in activation of Wnt/ß-catenin signaling and induces mammary intraepithelial neoplasia that progresses to squamous transdifferentiation and development of adenosquamous carcinomas at 6 months. To uncover possible ß-catenin-independent activities of GSK-3, we generated mammary-specific knockouts of GSK-3 and ß-catenin. Squamous transdifferentiation of the mammary epithelium was largely attenuated, however, mammary epithelial cells lost the ability to form mammospheres suggesting perturbation of stem cell properties unrelated to loss of ß-catenin alone. At 10 months, adenocarcinomas that developed in glands lacking GSK-3 and ß-catenin displayed elevated levels of γ-catenin/plakoglobin as well as activation of the Hedgehog and Notch pathways. Collectively, these results establish the two isoforms of GSK-3 as essential integrators of multiple developmental signals that act to maintain normal mammary gland function and suppress tumorigenesis.

Identification of human gene complementing ts AlS9 mouse L-cell defect in DNA replication following DNA-mediated gene transfer.

The temperature-sensitive (ts) mouse L-cell, ts AlS9, is defective in a gene required for nuclear DNA replication early in the S phase of the cell cycle. Human DNA sequences were introduced into ts AlS9 cells together with the plasmid pSV2neo, which can confer resistance to the drug geneticin. Cotransformants, expressing both the plasmid-derived neomycin gene and the transferred human AlS9 gene, were selected for growth in the presence of the drug at the nonpermissive temperature (npt). The resulting transformants retained a common set of human-specific Alu repetitive DNA sequences. These are likely to be accommodated within, or in proximity to, the transferred human AlS9 gene. The results obtained provide the basis for cloning human genes required for DNA replication.

Molecular cloning of human A1S9 locus: an X-linked gene essential for progression through S phase of the cell cycle.

The temperature-sensitive (ts) A1S9 mouse L-cell mutant is defective in an X-linked gene essential for the progression of cells through the S phase of the cell duplication cycle. We recently reported the complementation of the ts A1S9 cell defect with total human DNA and the isolation of independent temperature-resistant transformants that retained a common set of human specific Alu-containing fragments. Here we describe the molecular cloning of these human DNA sequences from one of the secondary transformants. ST-1-0. A genomic library prepared from ST-1-0 was screened with a total human DNA probe, and two recombinant bacteriophages carrying overlapping segments were isolated. The cloned region was extended in both directions using a human X-chromosome specific library. In total, a human region spanning 42 kb in length, and containing all the Alu-specific DNA sequences found in ST-1-0, was isolated in five overlapping recombinant phages. The A1S9 gene appeared to be larger than the DNA recovered in individual phage isolates, as was assessed by transfection experiments. A single-copy probe derived from the phage DNA was shown to be conserved in independent primary, secondary, and tertiary transformants of ts A1S9 cells and mapped to the X chromosome by molecular hybridization. Northern blot hybridization of this probe with poly(A)+ mRNA derived from ST-1-0 cells identified a transcript of about 3.6 kb.

Molecular cloning, primary structure and expression of the human X linked A1S9 gene cDNA which complements the ts A1S9 mouse L cell defect in DNA replication.

The temperature-sensitive ts A1S9 mutation of mouse L cells was previously shown to affect nuclear DNA replication and to be complemented by active and inactive human X chromosomes in human-ts A1S9 somatic cell hybrids. We report the isolation of cDNA clones which correct the ts A1S9 lesion, using as a probe a genomic fragment derived from the human A1S9 locus. The nucleotide sequence of the A1S9 cDNA encompasses a single open reading frame of 2409 bp which could encode a heretofore unreported protein of 90 393 daltons. Southern blot hybridization of the A1S9 cDNA probe with DNA from various species revealed homologous sequences in vertebrates but not in yeast. Northern blot analysis of serum-starved, synchronized cells demonstrated that the A1S9 gene was expressed at a relatively low level in quiescent cells and at a higher and constant level throughout the cell cycle. Human cell lines harbouring increasing numbers of inactive X chromosomes (47, XXX, 49, XXXXX) were found to express the A1S9 gene at the same level as control cells (45, X), suggesting that the gene does not escape X chromosome inactivation.

Localization of the human A1S9 gene complementing the ts A1S9 mouse L-cell defect in DNA replication and cell cycle progression to Xp11.2----p11.4.

The temperature-sensitive ts A1S9 mouse L-cell mutant is defective in an X-linked gene essential for progression of cells through the S phase of the cell division cycle. A single copy fragment derived from the complementing human A1S9 gene was used as a probe to localize the gene on the X chromosome. Southern blot analysis of human x rodent hybrids and in situ hybridization to human metaphase chromosomes allowed the regional assignment of the human A1S9 gene to Xp11.2----p11.4.

Retinoblastoma gene promoter directs transgene expression exclusively to the nervous system.

In human, germ line mutations in the tumor suppressor retinoblastoma (Rb) predispose individuals to retinoblastoma, whereas somatic inactivation of Rb contributes to the progression of a large spectrum of cancers. In mice, Rb is highly expressed in restricted cell lineages including the neurogenic, myogenic, and hematopoietic systems, and disruption of the gene leads to specific embryonic defects in these tissues. The symmetry between Rb expression and the defects in mutant mice suggest that transcriptional control of Rb during embryogenesis is pivotal for normal development. We have initiated studies to dissect the mechanisms of transcriptional regulation of Rb during development by promoter lacZ transgenic analysis. DNA sequences up to 6 kilobase pairs upstream of the mouse Rb promoter, isolated from two different genomic libraries, directed transgene expression exclusively to the developing nervous system, excluding skeletal muscles and liver. Expression of the transgene in the central and peripheral nervous systems, including the retina, recapitulated the expression of endogenous Rb during embryogenesis. A promoter region spanning approximately 250 base pairs upstream of the transcriptional starting site was sufficient to confer expression in the central and peripheral nervous systems. To determine whether this expression pattern was conserved, we isolated the human Rb 5' genomic region and generated transgenic mice expressing lacZ under control of a 1.6-kilobase pair human Rb promoter. The human Rb promoter lacZ mice also expressed the transgene primarily in the nervous system in several independent lines. Thus, transgene expression directed by both the human and mouse Rb promoters is restricted to a subset of tissues in which Rb is normally expressed during embryogenesis. Our findings demonstrate that regulatory elements directing Rb expression to the nervous system are delineated within a well defined core promoter and are regionally separated from elements, yet to be identified, that are required for expression of Rb in the developing hematopoietic and skeletal muscle systems.

Dual mechanisms of repression of E2F1 activity by the retinoblastoma gene product.

The retinoblastoma gene product, pRb, negatively regulates cell proliferation by modulating the activity of the transcription factor E2F1 that controls expression of S-phase genes. To dissect transcriptional regulation of E2F1 by pRb, we developed a means to control the subcellular localization of pRb by exchanging its constitutive nuclear localization signal (NLS) with an inducible nuclear targeting domain from the glucocorticoid receptor (GR). In co-transfection experiments in hormone-free media, pRb delta NLS-GR sequestered E2F1 in the cytoplasm; addition of steroid hormones induced co-translocation of pRb delta NLS-GR and E2F1 to the nucleus. A pRb allele lacking a NLS, pRb delta NLS, also sequestered E2F1 in the cytoplasm. Both nuclear and cytoplasmic pRb delta NLS-GR repressed transcription from a simple, E2F1-activated, promoter equally well. pRb delta NLS-GR exerted differential effects on complex promoters containing an activator and E2F sites that acted as either positive or negative elements. We propose a dual mechanism of transcriptional repression by pRb which allows tight control of E2F1-responsive genes: a pRb-E2F1 repressor unit is assembled off DNA to pre-empt transcriptional activation by E2F1; recruitment of this repressor unit to cognate binding sites on promoters allows silencing of adjacent promoter elements.

Characterization of the human RB1 promoter and of elements involved in transcriptional regulation.

The retinoblastoma gene (RB1) is a recessive oncogene implicated in a number of human tumors. Although the RB1 gene is expressed in most proliferating cells, there is considerable evidence for the transcriptional regulation of this gene. Therefore, we have performed a detailed analysis of the regulatory elements in the promoter of the human RB1 gene. Deletion analysis of the 5' upstream region determined the location of the basal promoter to be between -208 and -179 nucleotides relative to the translational start. This region contains essential binding sites for the transcription elements ATF and SP1 and potentially important sites for E2F and steroid hormone responsiveness but no TATA or CAAT boxes. Primer extension and RNase protection analysis identified two initiation sites at -176 and -128 base pairs, both downstream of the promoter. Cotransfection experiments revealed repression of the RB1 promoter by its protein product p110RB1. This repression has been mapped to the core promoter region containing the E2F-binding site; however, this site is not required for autorepression.