Andréa Fournier

Androgens induce expression of SPAK, a STE20/SPS1-related kinase, in LNCaP human prostate cancer cells.

Genes that are regulated by androgens in the human prostate are believed to play an essential role in prostate physiology and they may also be involved in the proliferative response of prostate cancer cells to androgens. We used a cDNA subtraction approach to identify novel androgen-regulated transcripts in LNCaP cells that were exposed to 0.1 nM R1881 for 24 h. We report here that SPAK, a recently identified STE20/SPS1-related kinase that modulates p38 MAP kinase activity, exhibited increased expression in androgen-treated LNCaP cells. Androgen regulation of SPAK was both dose- and time-dependent. R1881-induced SPAK expression was completely abrogated by the antiandrogen casodex and by actinomycin D indicating that androgen induction of SPAK requires the androgen receptor and transcription. Cycloheximide caused a partial inhibition of R1881-induced SPAK expression which suggests that androgen induction of SPAK expression may require synthesis of additional proteins. Northern blot and ribonuclease protection assays demonstrated that SPAK is expressed at high levels in normal human testes and prostate, as well as in a number of breast and prostate cancer cell lines. These results identify SPAK, a member of a key cell signalling pathway, as an androgen-responsive gene in LNCaP cells. We hypothesize that SPAK may mediate androgen action in the normal and cancerous prostate gland.

Androgens differentially regulate the expression of NEDD4L transcripts in LNCaP human prostate cancer cells

The NEDD4L gene encodes a ubiquitin ligase that targets the epithelial sodium channel for degradation. A search for transcripts whose levels increase following androgen treatment of LNCaP human prostate cancer cells led to the isolation of three new NEDD4L transcripts designated NEDD4Lf, NEDD4Lg and NEDD4Lh. The three transcripts encode different forms of the NEDD4L protein, two of which contain an N-terminal C2 domain. These transcripts were detected at high levels in human prostate and mammary gland, and at lower levels in brain and skeletal muscle. We also found that the previously described NEDD4La and NEDD4Lc (KIAA0439) transcripts are also expressed in prostate and LNCaP cells. However, only NEDD4Lf, NEDD4Lg and NEDD4Lh were up-regulated by androgen in LNCaP cells. These data provide new information on the structure and expression profile of NEDD4L-derived transcripts and identify specific isoforms of the NEDD4L ubiquitin ligase as proteins with potentially important roles in androgen action and prostate physiology.

Androgen-regulated transcription factor AIbZIP in prostate cancer

Androgen-induced bZIP (AIbZIP/CREB3L4) is a transcription factor of the bZIP family that associates with the membrane of the endoplasmic reticulum (ER). In humans, AIbZIP RNA is most abundant in the prostate gland where the protein is produced in luminal cells of the glandular epithelium. AIbZIP could play an important role in prostate cancer because its expression is up-regulated by androgens in LNCaP prostate cancer cells and the protein is more abundant in cancerous than in non-cancerous prostate cells. We recently added 74 adenocarcinomas and 43 specimens of prostatic intraepithelial neoplasia (PIN) to our survey of AIbZIP expression in prostate tumours. This study showed that AIbZIP is expressed in all grades of adenocarcinoma and that it is more abundant in high-grade PIN and in adenocarcinoma than in normal prostate. The physiological function of AIbZIP remains unknown but its association with the ER and its structural homology to transcription factors such as ATF6 suggest that AIbZIP could be activated by regulated intramembrane proteolysis during the cellular response to ER stress. This review will describe the characteristics of human and mammalian AIbZIP, its relationship to prostate cancer, and our recent efforts to characterize the transcriptional properties and targets of AIbZIP.

Glucocorticoids stimulate the growth of mouse mammary carcinoma Shionogi cells in culture.

The relative potency of a series of glucocorticoids to stimulate the growth of a cloned cell line (SEM-1) derived from the androgen-sensitive Shionogi mouse mammary carcinoma is proportional to their known affinity for the glucocorticoid receptor. The stimulatory action of glucocorticoids is not inhibited by the pure antiandrogen hydroxyflutamide while the antiglucocorticoids RU25593 and RU38486 cause 100% and 80% inhibitions of the activity of triamcinolone acetonide, respectively, thus indicating that the stimulatory effect of glucocorticoids on Shionogi cell growth is mediated by the glucocorticoid receptor. Such data indicate that not only androgens but also glucocorticoids should be taken into account when assessing the endocrine control of the growth of these mammary carcinoma cells.

Androgens down-regulate myosin light chain kinase in human prostate cancer cells.

Androgens play a major role in the growth and survival of primary prostate tumors. The molecular mechanisms involved in prostate cancer progression are not fully understood but genes that are regulated by androgens clearly influence this process. We searched for new androgen-regulated genes using the Affymetrix GeneChip Human Genome U95 Set in the androgen-sensitive LNCaP prostate cancer cell line. Analysis of gene expression profiles revealed that myosin light chain kinase (MLCK) mRNA levels were markedly down-regulated by the synthetic androgen R1881. The microarray data were confirmed by ribonuclease protection assays. RNA and protein analyses revealed that LNCaP cells express both long (non-muscle) and short (smooth muscle) isoforms, and that both isoforms are down-regulated by androgens. Taken together, these data identify MLCK as a novel downstream target of the androgen signalling pathway in prostate cells.

The co-chaperone DNAJC12 binds to Hsc70 and is upregulated by endoplasmic reticulum stress

Human DNAJC12 is a J domain-containing protein whose regulation, subcellular localization, and function are currently unknown. We show here that the abundance of DNAJC12 in human LNCaP prostate cancer cells is upregulated by the stress-inducing drug A23187 and by the stress-regulated transcription factor AIbZIP/CREB3L4. The DNAJC12 gene encodes two isoforms, only one of which (isoform a) is expressed in these cells. Immunofluorescence studies showed that a recombinant DNAJC12 protein is diffusely distributed in the cytoplasm. To identify substrates of DNAJC12, we used an immunoaffinity-mass spectrometry approach in cells that express epitope-tagged DNAJC12. The list of potential DNAJC12-binding proteins that were identified in this screen includes several nucleotide-binding proteins. The most frequently identified partner of DNAJC12 in unstressed cells was Hsc70, a cognate Hsp70 chaperone, whereas in stressed cells, the ER chaperone BiP was frequently associated with DNAJC12. Immunoprecipitation experiments confirmed that the endogenous DNAJC12 and Hsc70 proteins interact in LNCaP cells. These results clarify the role of DNAJC12 in the regulation of Hsp70 function.