Histone demethylase KDM5B collaborates with TFAP2C and Myc to repress the cell cycle inhibitor p21(cip) (CDKN1A).

The TFAP2C transcription factor has been shown to downregulate transcription of the universal cell cycle inhibitor p21(cip) (CDKN1A). In examining the mechanism of TFAP2C-mediated repression, we have identified a ternary complex at the proximal promoter containing TFAP2C, the oncoprotein Myc, and the trimethylated lysine 4 of histone H3 (H3K4me3) demethylase, KDM5B. We demonstrated that while TFAP2C and Myc can downregulate the CDKN1A promoter independently, KDM5B acts as a corepressor dependent on the other two proteins. All three factors collaborate for optimal CDKN1A repression, which requires the AP-2 binding site at -111/-103 and KDM5B demethylase activity. Silencing of TFAP2C-KDM5B-Myc led to increased H3K4me3 at the endogenous promoter and full induction of CDKN1A expression. Coimmunoprecipitation assays showed that TFAP2C and Myc associate with distinct domains of KDM5B and the TFAP2C C-terminal 270 amino acids (aa) are required for Myc and KDM5B interaction. Overexpression of all three proteins resulted in forced S-phase entry and attenuation of checkpoint activation, even in the presence of chemotherapy drugs. Since each protein has been linked to poor prognosis in breast cancer, our findings suggest that the TFAP2C-Myc-KDM5B complex promotes cell cycle progression via direct CDKN1A repression, thereby contributing to tumorigenesis and therapy failure.

Alternative TFAP2A isoforms have distinct activities in breast cancer.

INTRODUCTION: AP-2α is a transcription factor implicated in the regulation of differentiation and proliferation in certain tissues, including the mammary gland. In breast tumours, continued expression of AP-2α has been correlated with a better prognosis, but this is hard to reconcile with a reported role in the upregulation of the ERBB2 oncogene. The existence of TFAP2A isoforms, deriving from alternative first exons and differing in their N-terminal sequence, has been described in some mammals, but their relative abundance and activity has not been investigated in the human breast. METHODS: Expression levels of four TFAP2A isoforms were assayed at the level of RNA and protein (via the generation of isoform-specific antibodies) in a panel of breast tumour cell lines and in tissue from normal breast and primary tumour samples. Expression constructs for each isoform were used in reporter assays with synthetic and natural promoters (cyclin D3 and ERBB2) to compare the activation and repression activity of the isoforms. RESULTS: We demonstrate that the two isoforms AP-2α 1b and AP-2α 1c, in addition to the originally cloned, AP-2α 1a, are conserved throughout evolution in vertebrates. Moreover, we show that isoform 1c in particular is expressed at levels at least on a par with the 1a isoform in breast epithelial lines and tissues and may be more highly expressed in tamoxifen resistant tumours. The isoforms share a similar transactivation mechanism involving the recruitment of the adaptors CITED2 or 4 and the transactivators p300 or CBP. However, isoform 1b and 1c are stronger transactivators of the ERBB2 promoter than isoform 1a. In contrast, AP-2α 1a is the only isoform able to act as a repressor, an activity that requires an intact sumoylation motif present within the N-terminus of the protein, and which the other two isoforms lack. CONCLUSIONS: Our findings suggest that TFAP2A isoforms may be differentially regulated during breast tumourigenesis and this, coupled with differences in their transcriptional activity, may impact on tumour responses to tamoxifen therapy. These data also have implications for the interpretation of tumour studies that seek to correlate outcomes with TFAP2A expression level.

PLU-1/JARID1B/KDM5B is required for embryonic survival and contributes to cell proliferation in the mammary gland and in ER+ breast cancer cells.

The four members of the JARID1/KDM5 family of proteins, a sub-group of the larger ARID (AT rich DNA binding domain) family, have been shown to demethylate trimethylated lysine 4 on histone 3 (H3K4me3), a chromatin mark associated with actively transcribed genes. In some lower organisms a single homologue of JARID1 is found, and functions of the four proteins found in mice and humans may be specific or overlapping. To investigate the function of the Jarid1B protein we examined the effects of deletion of the gene in mice. Systemic knock out of Jarid1b resulted in early embryonic lethality, whereas mice not expressing the related Jarid1A gene are viable and fertile. A second mouse strain expressing a Jarid1b gene with the ARID domain deleted was viable and fertile but displayed a mammary phenotype, where terminal end bud development and side branching was delayed at puberty and in early pregnancy. Since development of terminal end buds are completely dependent on signalling from the estrogen receptor (ERα), we investigated the expression of a target gene (progesterone receptor) in the ∆ARID mouse and found levels to be reduced as compared to wild-type. JARID1B is widely expressed in ER+ breast cancers and breast cancer cell lines, and interaction with ERα was demonstrated by co-immunoprecipitations in cells transfected with tagged ERα and JARID1B genes. Down-regulation of expression of JARID1B using shRNAi in MCF-7 cells resulted in a dramatic decrease in E2 stimulated tumour growth in nude mice. The data demonstrate a specific role for Jarid1B in early embryonic development, in the development and differentiation of the normal mammary gland, and in estrogen induced growth of ER+ breast cancer.

Dual association by TFAP2A during activation of the p21cip/CDKN1A promoter.

The cyclin-dependent kinase inhibitor p21cip/CDKN1A is induced to promote growth arrest in response to a variety of stimuli in normal cells and loss of correct regulation of this gene is frequently observed in cancer. In particular, the upregulation of CDKN1A by p53 is considered to be a central mechanism of tumour suppression. Other transcription factors with tumour suppressor activity can also regulate CDKN1A, including the developmentally regulated factor, TFAP2A. Here we identify a novel AP-2 binding site within the proximal promoter of the CDKN1A gene and show this is required for optimal, p53-independent expression of p21cip/CDKN1A. We further describe a non-tumourgenic breast epithelial cell line model to study the role of endogenous TFAP2A and p53 in the control of drug-induced p21cip expression using ChIP. Maximal expression of CDKN1A requires TFAP2A which binds to two regions of the promoter: the proximal region where the AP-2 site lies and upstream near the major p53 binding site. The pattern of binding alters with time post-induction, with the proximal, p53-independent site becoming more important at later stages of p21cip induction. This pattern of promoter interaction by TFAP2A is distinct from that seen for the TFAP2C family member which represses CDKN1A expression.

AP-2gamma promotes proliferation in breast tumour cells by direct repression of the CDKN1A gene.

Overexpression of the activator protein (AP)-2gamma transcription factor in breast tumours has been identified as an independent predictor of poor outcome and failure of hormone therapy. To understand further the function of AP-2gamma in breast carcinoma, we have used an RNA interference and gene expression profiling strategy with the MCF-7 cell line as a model. Gene expression changes between control and silenced cells implicate AP-2gamma in the control of cell cycle progression and developmental signalling. A function for AP-2gamma in cell cycle control was verified using flow cytometry: AP-2gamma silencing led to a partial G1/S arrest and induction of the cyclin-dependent kinase inhibitor, p21cip/CDKN1A. Reporter and chromatin immunoprecipitation assays demonstrated a direct, functional interaction by AP-2gamma at the CDKN1A proximal promoter. AP-2gamma silencing coincided with acquisition of an active chromatin conformation at the CDKN1A locus and increased gene expression. These data provide a mechanism whereby AP-2gamma overexpression can promote breast epithelial proliferation and, coupled with previously published data, suggest how loss of oestrogen regulation of AP-2gamma may contribute to the failure of hormone therapy in patients.

Two distinct amplification events of the c-myc locus in a colorectal tumour.

Southern hybridisation of genomic DNA extracted from a human primary colorectal carcinoma revealed amplification of a fragment containing the wild-type c-myc locus. Two additional rearranged DNA fragments, lying upstream of c-myc, fused to distant non-contiguous sequences from the same chromosome, with an opposite configuration (head to head vs. head to tail), were also found to be amplified. Sequences analysis suggested that these rearrangements resulted from illegitimate recombination at two distinct points within the DNA sequence just upstream of the c-myc ORF and further that these events triggered two different amplification mechanisms, only one of which, involving a strand invasion event following DNA double strand breaks, increased the copy number of the c-myc ORF.

Functional analysis of the transcription repressor PLU-1/JARID1B.

The PLU-1/JARID1B nuclear protein, which is upregulated in breast cancers, belongs to the ARID family of DNA binding proteins and has strong transcriptional repression activity. To identify the target genes regulated by PLU-1/JARID1B, we overexpressed or silenced the human PLU-1/JARID1B gene in human mammary epithelial cells by using adenovirus and RNA interference systems, respectively, and then applied microarray analysis to identify candidate genes. A total of 100 genes showed inversely correlated differential expression in the two systems. Most of the candidate genes were downregulated by the overexpression of PLU-1/JARID1B, including the MT genes, the tumor suppressor gene BRCA1, and genes involved in the regulation of the M phase of the mitotic cell cycle. Chromatin immunoprecipitation assays confirmed that the metallothionein 1H (MT1H), -1F, and -1X genes are direct transcriptional targets of PLU-1/JARID1B in vivo. Furthermore, the level of trimethyl H3K4 of the MT1H promoter was increased following silencing of PLU-1/JARID1B. Both the PLU-1/JARID1B protein and the ARID domain selectively bound CG-rich DNA. The GCACA/C motif, which is abundant in metallothionein promoters, was identified as a consensus binding sequence of the PLU-1/JARID1B ARID domain. As expected from the microarray data, cells overexpressing PLU-1/JARID1B have an impaired G(2)/M checkpoint. Our study provides insight into the molecular function of the breast cancer-associated transcriptional repressor PLU-1/JARID1B.

Breast cancer associated transcriptional repressor PLU-1/JARID1B interacts directly with histone deacetylases.

The PLU-1/JARID1B nuclear protein, which is expressed in a high proportion of breast cancers, but shows restricted expression elsewhere, belongs to the ARID family of proteins, known to play important roles in development, differentiation, transcriptional regulation and chromatin remodeling. PLU-1/JARID1B is a strong transcriptional repressor, and here we show that the protein localizes in MAD bodies when cotransfected with class IIa histone deacetylases (HDACs) or N-CoR. Direct binding to class I and class IIa HDACs is demonstrated, while the interaction with N-CoR appears to be indirect. The domains involved in the HDAC4-PLU-1/JARID1B interaction were investigated in detail, and the data show that 2 PHD domains in PLU-1/JARID1B, which are involved in transcriptional repression, are also crucial for binding to a domain in the 5' region of HDAC4, overlapping the MEF-2 binding region. Physiological relevance of this interaction in the mammary gland is suggested from the observation that HDAC4 and PLU-1/JARID1B are coexpressed in the pregnant and involuting mouse mammary gland and are both silenced at lactation. Significantly, the expression of both proteins is seen in breast cancers.

Gene expression changes induced by a recombinant E1-/E3- adenovirus type 5 vector in human mammary epithelial cells.

OBJECTIVES: Adenoviral vectors are used in transferring exogenous genes to a variety of cells and tissue types both in vitro and in vivo. Gene expression changes induced by an E1/E3-defective adenovirus vector have been studied in human mammary epithelial cells by comparing the gene expression profile in infected and uninfected cells. METHODS: The human mammary epithelial cell line HB2 was infected with an E1/E3-defective adenovirus type 5 vector. Total RNA was extracted from infected and uninfected cells 24 and 72 h after infection and subjected to microarray analysis using the Affymetrix U133A genomic chip system. Semiquantitative RT-PCR confirmed the regulation of genes observed by microarray analysis. RESULTS: The microarray analysis showed 24 and 95 transcripts to be regulated 24 and 72 h after infection, respectively. A relatively high number of genes involved in innate and inflammatory host immune responses, including interleukin-8, interleukin-6, NF-kappaB(2), RELB and fos, were induced. As expected from an E1-defective virus, changes in the expression of genes involved in the G1-S transition and in the activation of cell proliferation were not detected. CONCLUSION: Our study provides insight into the host transcriptional response following transduction of an adenoviral vector into mammary epithelial cells.

Heterogeneity within and between primary colorectal carcinomas and matched metastases as revealed by analysis of Ki-ras and p53 mutations.

Analysis of the genetic status of Ki-ras and p53 in primary colorectal carcinomas and matched colorectal liver metastasis from 30 patients reveals an overall heterogeneity both within and between the two tumoral tissues. Both genes were found mutated with a similar frequency in both tissues; however, identical mutations in primary tumor and matched metastasis were found less frequently in the case of the Ki-ras than the p53 gene. Only in three cases the same p53 and Ki-ras mutations found in the primary tumor were found also in the metastasis. In several metastatic specimens the DNA bearing a mutation detected also in the primary tumor appears significantly less abundant than the wild-type DNA. These data are discussed in the light of current models of primary tumor/metastasis relationships.

PLU-1 nuclear protein, which is upregulated in breast cancer, shows restricted expression in normal human adult tissues: a new cancer/testis antigen?

The PLU-1 gene is expressed at the level of message in breast cancers and breast cancer cell lines and shows restricted expression in normal adult tissues with the exception of testis. The predicted protein sequence contains several domains, including the PLU domain, which is shared by other proteins involved in transcription and/or development. We have developed a polyclonal antiserum to a C-terminal fragment of the PLU-1 protein, which shows little homology to other family members. Immunohistochemical analysis with the antiserum alpha-PLU-1C confirmed the nuclear localisation of PLU-1. alpha-PLU-1C also reacted with the mouse homologue of PLU-1 (mPlu-1) but not with the closest family member, RBP2. Using Western blot analysis, PLU-1 was shown to be well expressed in breast cancers and breast cancer cell lines, while it was not detected in a range of normal adult tissues. Our results suggest that the PLU-1 protein may belong to the class of testis/cancer antigens.