Prolactin-Induced Protein (PIP) Regulates Proliferation of Luminal A Type Breast Cancer Cells in an Estrogen-Independent Manner

<div><p>Prolactin-induced Protein (PIP), an aspartyl protease unessential for normal mammalian cell function, is required for the proliferation and invasion of some breast cancer (BCa) cell types. Because PIP expression is particularly high in the Luminal A BCa subtype, we investigated the roles of PIP in the related T47D BCa cell line. Nucleic acid and antibody arrays were employed to screen effects of PIP silencing on global gene expression and activation of receptor tyrosine kinases (RTKs), respectively. Expression of PIP-stimulated genes, as defined in the T47D cell culture model, was well correlated with the expression of PIP itself across a cohort of 557 mRNA profiles of diverse BCa tumors, and bioinformatics analysis revealed cJUN and cMYC as major nodes in the PIP-dependent gene network. Among 71 RTKs tested, PIP silencing resulted in decreased phosphorylation of focal adhesion kinase (FAK), ephrin B3 (EphB3), FYN, and hemopoietic cell kinase (HCK). Ablation of PIP also abrogated serum-induced activation of the downstream serine/threonine kinases AKT, ERK1/2, and JNK1. Consistent with these results, PIP-depleted cells exhibited defects in adhesion to fibronectin, cytoskeletal stress fiber assembly and protein secretion. In addition, PIP silencing abrogated the mitogenic response of T47D BCa cells to estradiol (E2). The dependence of BCa cell proliferation was unrelated, however, to estrogen signaling because: 1) PIP silencing did not affect the transcriptional response of estrogen target genes to hormone treatment, and 2) PIP was required for the proliferation of tamoxifen-resistant BCa cells. Pharmacological inhibition of PIP may therefore serve the bases for both augmentation of existing therapies for hormone-dependent tumors and the development of novel therapeutic approaches for hormone-resistant BCa.</p></div

Identification of novel androgen receptor target genes in prostate cancer-3

<p><b>Copyright information:</b></p><p>Taken from "Identification of novel androgen receptor target genes in prostate cancer"</p><p>http://www.molecular-cancer.com/content/6/1/39</p><p>Molecular Cancer 2007;6():39-39.</p><p>Published online 6 Jun 2007</p><p>PMCID:PMC1904239.</p><p></p>wo days in CSS-supplemented medium. Gene expression was analyzed side-by-side by RT-qPCR and corrected for 18S rRNA. Bars represent the comparative ratio between the expression in C4-2B and LNCaP cells, where the expression level in LNCaP cells is defined as 1. Included in this Figure are only genes for which the expression levels were significantly different between the two cell lines in two independent experiments (n = 3; Mean ± SD). TRPV3 mRNA was detected in LNCaP cells in only one of the three measurements, and this value was used as the upper limit for TRPV3 expression in these cells. See additional file for details

Identification of novel androgen receptor target genes in prostate cancer-2

<p><b>Copyright information:</b></p><p>Taken from "Identification of novel androgen receptor target genes in prostate cancer"</p><p>http://www.molecular-cancer.com/content/6/1/39</p><p>Molecular Cancer 2007;6():39-39.</p><p>Published online 6 Jun 2007</p><p>PMCID:PMC1904239.</p><p></p>A (black bars), followed by administration of either DHT (10 nM) or Ethanol vehicle for 16 hours. Expression levels of the indicated genes were analyzed in triplicate by RT-qPCR and corrected for 18S rRNA levels. Values measured with the non-specific siRNA and ethanol were defined as 1. Results (Mean ± SD) are representative of three independent experiments

Identification of novel androgen receptor target genes in prostate cancer-0

<p><b>Copyright information:</b></p><p>Taken from "Identification of novel androgen receptor target genes in prostate cancer"</p><p>http://www.molecular-cancer.com/content/6/1/39</p><p>Molecular Cancer 2007;6():39-39.</p><p>Published online 6 Jun 2007</p><p>PMCID:PMC1904239.</p><p></p>ation with target loci. Two independent AR ChIPs, and IgG control ChIPs were subjected to the CD procedure as described in Methods. In the example shown here, PCRs were performed with the 'AC' and the 'TG' PCR primers (see Methods and Additional file ) with the annealing temperature set at either 70°C or 71°C as indicated. Amplified products were resolved using 8% PAGE and visualized by EtBr staining. The arrowheads point at bands amplified more prominently in the AR compared to the Control (IgG) lanes. , marker DNA; numbers above bands indicate size in bps. . The two bands indicated in panel A by arrowheads were excised, purified and re-amplified with the same 'AC' and 'TG' primers used for CD. The products were subjected to secondary digestion with the indicated enzymes, followed by agarose gel electrophoresis. Arrowheads point at similar III sub-fragments obtained from the two AR ChIPs. – , no template control, , uncut, , marker DNA. . The III subfragments from B were excised, purified and sequenced. By blasting against the human genome using Ensembl [65], both sequences mapped to chromosome 17q25.3, ~1.5-kb upstream of the MAFG gene and ~2.5-kb downstream of the PYCR1 gene as shown in the diagram. The two genes are transcribed in the same direction as indicated by the horizontal arrows. , polyadenylation signal. The AR binding region discovered through CD ('''') abuts a CpG island (), but does not overlap with any repetitive elements (). Several AREs () were identified in this region using Consite [66]. . AR occupancy at the PYCR1/MAFG locus was tested by conventional ChIP assay. The PSA enhancer serves as positive control. A non-target locus serves as the negative control. Genomic DNA was used to demonstrate that the ChIP amplification was performed within a dynamic range. – , no template control. , marker DNA

Identification of novel androgen receptor target genes in prostate cancer-4

<p><b>Copyright information:</b></p><p>Taken from "Identification of novel androgen receptor target genes in prostate cancer"</p><p>http://www.molecular-cancer.com/content/6/1/39</p><p>Molecular Cancer 2007;6():39-39.</p><p>Published online 6 Jun 2007</p><p>PMCID:PMC1904239.</p><p></p>roarray sets (21) and results are mined for all probesets (rows) interrogating each of the 32 CD-disclosed genes (Table 1). Heat map shows relative expression for each of the indicated probesets, where darker shades represent higher mRNA levels. Tumors included 23 prostate cancers from patients not receiving therapy (), 17 primary prostate cancers following 3-month neoadjuvant androgen ablation therapy (), and 7 AR-positive metastatic lesions (). All Grade A probesets interrogating each gene are shown, except for probesets 59776_at (WBSCR28) and 36904_at (KIF1A), which did not detect significant expression in any sample. Samples are grouped and ranked as follows. – probesets for the known AR-stimulated genes KLK3/PSA and TMPRSS2. – probesets exhibiting statistically greater mean expression in untreated compared to AAT-treated primary PCa samples (< 0.05), thereby representing putative AR-stimulated genes. – probesets exhibiting statistically lower mean expression in untreated compared to AAT-treated PCa samples (p < 0.05), thereby representing putative AR-repressed genes. – probesets exhibiting no statistical difference between samples without or with AAT. Probesets in Groups II-IV are ranked by -value in descending order

Identification of novel androgen receptor target genes in prostate cancer-1

<p><b>Copyright information:</b></p><p>Taken from "Identification of novel androgen receptor target genes in prostate cancer"</p><p>http://www.molecular-cancer.com/content/6/1/39</p><p>Molecular Cancer 2007;6():39-39.</p><p>Published online 6 Jun 2007</p><p>PMCID:PMC1904239.</p><p></p>SS-containing medium for three days, and then re-fed (time 0) with the same medium supplemented with either 10 nM DHT or ethanol vehicle. RNA was extracted at the indicated time points during the time course and expression of the specified genes was measured by RT-qPCR. Expression levels relative to 18S rRNA (which itself stayed stable throughout the time course) are shown with the 0 time values defined as 1 for each cell line. Representative data is shown from one of two independent experiments with n = 3, except for panels 2L, O, U, Y and ZC, where the C4-2B data is derived from 6 measurements (see Additional file for the complete set of raw data). Error bars are SEM. Genes are roughly ordered based on the DHT-responsiveness in C4-2B cells, with stimulated genes first (panels A-J) to repressed genes last (panels ZC-ZF). TRVP3 mRNA was barely detectable in LNCaP cells