Androgen receptor acetylation governs trans activation and MEKK1-induced apoptosis without affecting in vitro sumoylation and trans-repression function

This work was supported by grants from the NIH (R01CA86072 to R.G.P. and R01CA72038-01 to S.A.W.F.) and The Susan Komen Breast Cancer Foundation (to R.G.P.). R.T.H. and E.J. were supported by the Medical Research Council. Y.-G.Y. is supported by grant CA26504 to E. R. Stanley. Work conducted at the Albert Einstein College of Medicine was supported by Cancer Center Core National Institutes of Health grant 5-P30-CA13330-26.The androgen receptor (AR) is a nuclear hormone receptor superfamily member that conveys both traits repression and ligand-dependent trans-activation function. Activation of the AR by dihydrotestosterone (DHT) regulates diverse physiological functions including secondary sexual differentiation in the male and the induction of apoptosis by the JNK kinase, MEKK1. The AR is posttranslationally modified on lysine residues by acetylation and sumoylation. The histone acetylases p300 and P/CAF directly acetylate the AR in vitro at a conserved KLKK motif. To determine the functional properties governed by AR acetylation, point mutations of the KLKK motif that abrogated acetylation were engineered and examined in vitro and in vivo. The AR acetylation site point mutants showed wild-type trans repression of NF-kappaS, AP-1, and Sp1 activity; wild-type sumoylation in vitro; wild-type ligand binding; and ligand-induced conformational changes. However, acetylation-deficient AR mutants were selectively defective in DHT-induced trans activation of androgen-responsive reporter genes and coactivation by SRC1, Ubc9, TIP60, and p300. The AR acetylation site mutant showed 10-fold increased binding of the N-CoR corepressor compared with the AR wild type in the presence of ligand. Furthermore, histone deacetylase 1 (HDAC1) bound the AR both in vivo and in cultured cells and HDAC1 binding to the AR was disengaged in a DHT-dependent manner. MEKK1 induced AR-dependent apoptosis in prostate cancer cells. The AR acetylation mutant was defective in MEKK1-induced apoptosis, suggesting that the conserved AR acetylation site contributes to a pathway governing prostate cancer cellular survival. As AR lysine residue mutations that abrogate acetylation correlate with enhanced binding of the N-CoR repressor in cultured cells, the conserved AR motif may directly or indirectly regulate ligand-dependent corepressor disengagement and, thereby, ligand-dependent trans activation.Publisher PDFPeer reviewe

Evidence for Diversity in Transcriptional Profiles of Single Hematopoietic Stem Cells

Hematopoietic stem cells replenish all the cells of the blood throughout the lifetime of an animal. Although thousands of stem cells reside in the bone marrow, only a few contribute to blood production at any given time. Nothing is known about the differences between individual stem cells that dictate their particular state of activation readiness. To examine such differences between individual stem cells, we determined the global gene expression profile of 12 single stem cells using microarrays. We showed that at least half of the genetic expression variability between 12 single cells profiled was due to biological variation in 44% of the genes analyzed. We also identified specific genes with high biological variance that are candidates for influencing the state of readiness of individual hematopoietic stem cells, and confirmed the variability of a subset of these genes using single-cell real-time PCR. Because apparent variation of some genes is likely due to technical factors, we estimated the degree of biological versus technical variation for each gene using identical RNA samples containing an RNA amount equivalent to that of single cells. This enabled us to identify a large cohort of genes with low technical variability whose expression can be reliably measured on the arrays at the single-cell level. These data have established that gene expression of individual stem cells varies widely, despite extremely high phenotypic homogeneity. Some of this variation is in key regulators of stem cell activity, which could account for the differential responses of particular stem cells to exogenous stimuli. The capacity to accurately interrogate individual cells for global gene expression will facilitate a systems approach to biological processes at a single-cell level

Elevated Expression of MAPK Phosphatase 3 in Breast Tumors—A Mechanism of Tamoxifen Resistance

Antiestrogen resistance is a major clinical problem in the treatment of breast cancer. Altered growth factor signaling with estrogen receptor (ER) α has been shown to be associated with the development of resistance. Gene expression profiling was utilized to identify MAPK phosphatase 3 (MKP3) whose expression was correlated with response to the antiestrogen tamoxifen in both patients and in vitro derived cell line models. Overexpression of MKP3 rendered ERα-positive breast cancer cells resistant to the growth inhibitory effects of tamoxifen, and enhanced tamoxifen agonist activity in endometrial cells. MKP3 overexpression was associated with lower levels of activated ERK1,2 phosphorylation in the presence of estrogen, but that estrogen deprivation and tamoxifen treatment decreased MKP3 phosphatase activity, leading to an up-regulation of pERK1,2 MAPK, phosphoserine 118 of ERα, and cyclin D1. The MEK inhibitor PD98059 blocked tamoxifen-resistant growth. Accumulation of reactive oxygen species was observed with tamoxifen treatment of MKP3 overexpressing cells, and antioxidant treatment increased MKP3 phosphatase activity, thereby blocking resistance. Furthermore, PD98059 increased the levels of phospho-JNK in tamoxifen-treated MKP3 overexpressing cells, suggesting an interaction between MKP3 levels, activation of ERK1,2 MAPK, and JNK signaling in human breast cancer cells. MKP3 represents a novel mechanism of resistance which may be a potential biomarker for the use of ERK1,2 and/or JNK inhibitors in combination with tamoxifen treatment

RNA sequencing of cancer reveals novel splicing alterations

Breast cancer transcriptome acquires a myriad of regulation changes, and splicing is critical for the cell to “tailor-make” specific functional transcripts. We systematically revealed splicing signatures of the three most common types of breast tumors using RNA sequencing: TNBC, non-TNBC and HER2-positive breast cancer. We discovered subtype specific differentially spliced genes and splice isoforms not previously recognized in human transcriptome. Further, we showed that exon skip and intron retention are predominant splice events in breast cancer. In addition, we found that differential expression of primary transcripts and promoter switching are significantly deregulated in breast cancer compared to normal breast. We validated the presence of novel hybrid isoforms of critical molecules like CDK4, LARP1, ADD3, and PHLPP2. Our study provides the first comprehensive portrait of transcriptional and splicing signatures specific to breast cancer sub-types, as well as previously unknown transcripts that prompt the need for complete annotation of tissue and disease specific transcriptome

A Renewable Tissue Resource of Phenotypically Stable, Biologically and Ethnically Diverse, Patient-Derived Human Breast Cancer Xenograft Models

Breast cancer research is hampered by difficulties in obtaining and studying primary human breast tissue, and by the lack of in vivo preclinical models that reflect patient tumor biology accurately. To overcome these limitations, we propagated a cohort of human breast tumors grown in the epithelium-free mammary fat pad of SCID/Beige and NOD/SCID/IL2γ-receptor null (NSG) mice, under a series of transplant conditions. Both models yielded stably transplantable xenografts at comparably high rates (~21% and ~19%, respectively). Of the conditions tested, xenograft take rate was highest in the presence of a low-dose estradiol pellet. Overall, 32 stably transplantable xenograft lines were established, representing 25 unique patients. Most tumors yielding xenografts were “triple-negative” (ER-PR-HER2+) (n=19). However, we established lines from three ER-PR-HER2+ tumors, one ER+PR-HER2−, one ER+PR+HER2− and one “triple-positive” (ER+PR+HER2+) tumor. Serially passaged xenografts show biological consistency with the tumor of origin, are phenotypically stable across multiple transplant generations at the histologic, transcriptomic, proteomic, and genomic levels, and show comparable treatment responses as those observed clinically. Xenografts representing 12 patients, including two ER+ lines, showed metastasis to the mouse lung. These models thus serve as a renewable, quality-controlled tissue resource for preclinical studies investigating treatment response and metastasis