Angelo G. Scibetta

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. Immunohistochemic analysis with the antiserum α‐PLU‐1C confirmed the nuclear localisation of PLU‐1. α‐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.

Functional Analysis of the Transcription Repressor PLU-1/JARID1B

ABSTRACT 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 G2/M checkpoint. Our study provides insight into the molecular function of the breast cancer-associated transcriptional repressor PLU-1/JARID1B.

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.

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.

Regulation of MUC1 Expression in Human Mammary Cell Lines by the c-ErbB2 and Ras Signaling Pathways

The MUC1 protein is a highly O-glycosylated transmembrane molecule that is expressed at the luminal surface of most glandular epithelial cells and is upregulated in carcinomas. Here, we report the effect of the activation of the c-ErbB2 --> Ras pathway on the expression of the MUC1 gene in the nontumorigenic mammary cell lines MTSV1-7 and HB2 and in the malignant cell lines T47D and ZR75. Endogenous levels of MUC1 mRNA and protein in HB2 clones permanently overexpressing c-ErbB2 or V12-H-Ras were markedly reduced compared with levels in the parental cell lines. Furthermore, in transient transfection assays, the transcription of a CAT reporter construct driven by the MUC1 promoter was inhibited when cotransfected with a c-ErbB2 or a V12-H-ras expressing vector. Transient transfections using mutant forms of the ras oncogene, and the inhibitor chemical wortmannin, indicated that the pathway activated by c-ErbB2 proceeds via activation of Ras and that the Raf and phosphoinositide 3-kinase pathways are involved. Finally, cotransfection assays using a reporter gene driven by the MUC1 promoter carrying abolishing mutations in some of the cis-acting elements showed that a GC box at -99/-91 is crucial for responsiveness to c-ErbB2 inhibition of transcription.

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-ĸB2, 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.

PLU-1/Jarid1B contributes to estrogen-induced cell proliferation in the normal mammary gland and in breast cancer, and is required for embryonic survival

PLU-1/Jarid1B is a nuclear protein that is widely expressed in breast cancer, with higher levels being seen in ER+ cancers. Expression in normal adult issues is largely restricted to testis and the differentiating mammary gland. Through the JmjC domain the protein can demethylate H3K4me3, which correlates with its function as a transcriptional repressor. PLU-1/Jarid1B contains a DNA binding domain, and can be recruited to DNA through binding to transcription factors. We now find that the protein interacts with the ERα receptor and contributes to estrogen-induced survival of MCF-7 cells in in vitro culture and when grown as tumours in nude mice. To investigate the function of Plu-1/Jarid1B in vivo, transgenic mice expressing defective Plu-1/Jarid1B have been developed. The systemic KO is an embryonic lethal with no homozygote embryos being detected at day 7.5. Another strain expressing the protein missing the ARID AT-rich DNA binding domain (which is also required for demethylase function) shows a mammary phenotype. In these ΔARID mice, the development of the mammary tree at puberty and early pregnancy is delayed, but the gland recovers by late pregnancy. The inhibition of development of terminal end buds at puberty, which is crucially dependent on ERα signalling, suggests an involvement of Plu-1/Jarid1B in this signalling that is impaired in the ΔARID mouse. Confirming this, levels of expression of downstream targets of ERα (progesterone receptor and Wnt4) are reduced in the ΔARID mouse. The development of spontaneous mammary tumours in the ΔARID mouse is delayed compared with wild-type mice, suggesting that Plu-1/Jarid1B contributes to tumour growth, and that this action is impaired when the ARID domain is deleted. The data suggest that PLU-1/JARID1B is involved in estrogen-induced growth of normal and malignant mammary epithelial cells.