Linda L. Xu

Quantitative expression profile of androgen-regulated genes in prostate cancer cells and identification of prostate-specific genes.

Quantitative expression profile of androgen‐regulated genes (ARGs) was evaluated in the hormone‐responsive prostate cancer cell line LNCaP by serial analysis of gene expression (SAGE). A total of 83,489 SAGE tags representing 23,448 known genes or expressed sequence tags (ESTs) and 1,655 potentially novel sequences have unraveled the transcriptome of LNCaP cells, the most common cell line used in prostate cancer research. Comparison of transcripts between control and R1881‐treated LNCaP cells revealed the induction of 136 genes and repression of 215 genes in response to androgen (p < 0.05). Strikingly, a high fraction (∼90%) of ARGs identified in our study has not been described as ARGs previously. A number of prostate‐specific transcription factors were among the ARGs identified here. Classification of the ARGs on the basis of biochemical functions revealed that a great majority of ARGs identified in our experimental system appear to be involved in regulation of transcription, splicing, ribosomal biogenesis, mitogenesis, bioenergetics and redox processes. One of the novel aspects of androgen signaling included androgen regulation of genes involved in DNA repair/recombination process. By comparing our LNCaP‐C and LNCaP‐T SAGE libraries with SAGE tag libraries available at the NCBI‐SAGE website, we have identified >200 potential prostate specific/abundant transcripts. The discovery of new prostate‐specific genes and ARGs provides a unique opportunity to determine the role of these genes in prostate cell growth, differentiation and tumorigenesis.

Androgen-induced expression of endoplasmic reticulum (ER) stress response genes in prostate cancer cells

Evaluations of androgen regulated gene (ARG) repertoire provide new insights into the androgen receptor (AR) mediated signaling at the transcriptional level. Definition of ARGs having critical functions in the biology of normal and malignant prostate should aid in identifying new bio-markers and therapeutic targets for prostate cancer (CaP). Using Affymetrix HuGene FL oligonucleotide arrays, temporal expression profiles of ARGs in widely used hormone responsive LNCaP cells, were analysed by hierarchical clustering methods and functional classification. ARGs in response to different androgen concentrations showed temporal co-regulation of genes involved in specific biochemical pathways. This study focuses on our new observations of the coordinated androgen induction of genes (NDRG1, PDIR, HERPUD1, ORP150) involved in the endoplasmic reticulum (ER) stress response pathway. Expression analysis of the two selected ER stress responsive genes, NDRG1 and HERPUD1 in primary CaPs revealed a significantly reduced tumor associated expression. Intriguing linkage of the androgen signaling to ER stress responsive genes, a protective response to protein unfolding or protein damage resulting from cellular stress signals, suggests that androgens may induce such stress signals in CaP cells. Decreased CaP associated expression of two ER stress responsive genes also suggests that possible abrogation of this pathway in prostate tumorigenesis.

Expression profile of an androgen regulated prostate specific homeobox gene NKX3.1 in primary prostate cancer

PURPOSEnNKX3.1, a member of the family of homeobox genes, exhibits prostate tissue specific expression and appears to play a role in mouse prostate development. Rapid induction of NKX3.1 gene expression in response to androgens has also been described. On the basis of the established role of androgens in prostatic growth and differentiation and studies showing an association of aberrant homeobox gene expression with the neoplastic process, we hypothesize that alterations of NKX3.1 gene expression play a role in prostate tumorigenesis.nnnMATERIALS AND METHODSnNKX3.1 expression was analyzed in matched, microdissected normal and tumor tissues from 52 primary prostate cancer specimens from radical prostatectomy by semiquantitative RT-PCR and in situ hybridization and correlated with the clinicopathologic features. NKX3.1 expression was quantified as differential expression between matched tumor and normal tissues and was grouped as overexpression in tumor tissue, reduced expression in tumor tissue and no change between tumor and normal tissues. Androgen regulation of NKX3.1 expression was also studied in LNCaP cells. Androgen receptor (AR) expression in prostate tumor and normal tissue was correlated with NKX3.1 expression.nnnRESULTSnComparison of NKX3.1 expression between normal and tumor tissues revealed overexpression in 31% tumor specimens (16 of 52), decreased expression in 21% tumor specimens (11 of 52) and no change in 48% specimens (25 of 52). When these expression patterns were stratified by organ confined and non-organ-confined tumor, a higher percentage of patients exhibited NKX3.1 overexpression in non-organ confined tumor (40%) versus organ confined tumor (22%). Elevated NKX3.1 expression significantly correlated with tumor volume and serum prostate specific antigen (PSA) level in the NKX3.1 overexpression group (p<0.05). Metastatic prostate cancer cell lines did not exhibit mutations in the protein coding sequence of NKX3.1. Additionally, the NKX3.1 expression correlated with AR expression (p<0.01) in vivo in human prostate tissues. Comparison of PSA and NKX3.1 expression in response to androgen revealed a rapid androgen mediated induction of NKX3.1 expression in LNCaP cells. In situ hybridization analysis of representative specimens confirmed RT-PCR observations.nnnCONCLUSIONSnThese results suggest an association of NKX3.1 with a more aggressive phenotype of carcinoma of the prostate. Correlation of AR expression with NKX3.1 in human prostate tissues underscores the androgen regulation of NKX3.1 in the physiologic context of human prostate tissues.

Androgen receptor mutation (T877A) promotes prostate cancer cell growth and cell survival.

Alteration of the AR functions due to amplification, overexpression and somatic mutation of the AR itself or altered interaction of AR with other cell growth regulatory proteins, may contribute to a significant subset of advanced prostate cancer (CaP). Very little is known about the pathways impacted by AR dysfunctions, although CaP associated AR alterations suggest the biological role of the AR dysfunction in disease progression. Comparative evaluations of wild type (wt) AR and mutant (mt) ARs in appropriate experimental models should provide a better understanding of the functional impact of AR alterations in CaP. Here, we provide direct evidence showing cell growth/cell survival promoting effects of the widely studied CaP associated AR mutation (T877A). In contrast to Ad-wtAR or Ad-control infected LNCaP or LAPC4 cells, Ad-mtAR (T877A) infected LNCaP or LAPC4 cells continued to grow in the androgen-deprived medium and exhibited an androgen independent AR-transcription factor activity. Further, Ad-mtAR (T877A) infected LNCaP or LAPC4 cells exhibited enhanced cell growth in the presence of lower concentrations of the synthetic androgen, R1881. Of note, Ad-mtAR (T877A) infected LNCaP cells showed striking resistance to cell growth inhibition/apoptosis mediated by the wt p53. Taken together, these findings provide novel insights into the AR dysfunctions resulting from the T877A mutation and functionally similar AR alterations may provide selective cell growth/survival advantage for CaP progression. These observations have important implications for developing biology-based prognostic biomarkers and therapeutic strategies for CaP showing such AR dysfunctions.

A Feedback Loop between the Androgen Receptor and a NEDD4-binding Protein, PMEPA1, in Prostate Cancer Cells

PMEPA1 was identified originally as a highly androgen-inducible gene with prostate-abundant expression that was restricted to prostatic epithelial cells. PMEPA1 protein is a NEDD4 (ubiquitin-protein isopeptide ligase)-binding protein, which negatively regulates prostate cancer cell growth. In this study we establish that PMEPA1 is a direct transcriptional target of the androgen receptor (AR). We also demonstrate that PMEPA1 negatively regulates AR protein levels in different cell culture models. Transient expression of PMEPA1 down-regulates AR protein levels and AR transcriptional targets in prostate cancer cells. Conversely, knockdown of PMEPA1 leads to elevated levels of AR protein, AR transcriptional targets (prostate-specific antigen), and increased cell cycle S phase. We define that the PMEPA1-dependent down-regulation of AR is because of AR ubiquitination and proteasome-mediated degradation. The mutant PMEPA1 (PY1/2 motif mutation) that is impaired in NEDD4 recruitment shows attenuated AR ubiquitination and AR protein down-regulation. These data support the hypothesis that PMEPA1 negatively regulates the stability of AR protein by enhancing AR ubiquitination and proteasome-mediated degradation through NEDD4. The effect of PMEPA1 on AR ubiquitination and degradation appears to be MDM2-independent. Thus, the PMEPA1-AR degradation pathway may represent a new androgen-dependent mechanism for regulating AR levels in prostate epithelial cells. These findings underscore that the decreased PMEPA1 expression frequently noted in prostate cancers may lead to increased AR functions and strengthen the biological role of PMEPA1 in prostate cancers.