Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3. Loss or injury to these networks is associated with a number of motility disorders4. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6. Importantly, normal KIT activity is required for ICC development5,6. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations

Modulators of Prostate Cancer Cell Proliferation and Viability Identified by Short-Hairpin RNA Library Screening

There is significant need to identify novel prostate cancer drug targets because current hormone therapies eventually fail, leading to a drug-resistant and fatal disease termed castration-resistant prostate cancer. To functionally identify genes that, when silenced, decrease prostate cancer cell proliferation or induce cell death in combination with antiandrogens, we employed an RNA interference-based short hairpin RNA barcode screen in LNCaP human prostate cancer cells. We identified and validated four candidate genes (AKT1, PSMC1, STRADA, and TTK) that impaired growth when silenced in androgen receptor positive prostate cancer cells and enhanced the antiproliferative effects of antiandrogens. Inhibition of AKT with a pharmacologic inhibitor also induced apoptosis when combined with antiandrogens, consistent with recent evidence for PI3K and AR pathway crosstalk in prostate cancer cells. Recovery of hairpins targeting a known prostate cancer pathway validates the utility of shRNA library screening in prostate cancer as a broad strategy to identify new candidate drug targets

Silencing of a subset of genes inhibited VCaP proliferation and induced apoptosis.

<p>Candidate target genes from LNCaP screen probes that were depleted in the presence of bicalutamide were selectively targeted using siRNAs. (A, left panel), VCaP cells were transfected with siRNAs that scored in the LNCaP drug screen and were incubated with 1 uM MDV3100 or vehicle, and the number of viable cells was measured 6 days post-treatment. (A, right panel), VCaP cells were transfected and treated as in (A, left panel) except Caspase 3/7 activity was measured after 3 days of treatment. <i>PLK1</i> siRNA transfection was used as a positive control. Dashed lines indicate the level of growth inhibition or apoptosis induced by MDV3100, for comparison. NT, non-targeting siRNA. (B) Gene silencing (10–50%) was confirmed by RT-qPCR 6 days post-transfection of VCaP cells with the siRNA SMARTpools. Reactions were done in triplicate and normalized to RPL27 for each cDNA and then normalized to vehicle-treated NT. Standard error of the mean was calculated. Bic, bicalutamide.</p

Candidate gene expression and alterations in human prostate tumors.

*<p>4 of the samples with upregulated AR mRNA also had <i>AR</i> amplification.</p

Probes depleted in bicalutamide-treated LNCaP cells.

*<p>Number of probes that had a log2 value ≤−0.58.</p>**<p>Bic, bicalutamide; Veh, vehicle.</p

shRNA probes depleted or enriched in bicalutamide-treated LNCaP cells.

<p>(A) Schematic of shRNA screen. Details can be found in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034414#s4" target="_blank">Materials and Methods</a> section. (B) A heatmap was generated by clustering based on probes that were depleted or enriched (log2 bicalutamide/vehicle ≤+/−0.58 and a p-value≤0.01) in the bicalutamide (0.4 uM and 1.0 uM)-treated LNCaP cells at T = 1 and T = 2 compared to the vehicle-treated cells at the same timepoints. The shRNA target gene associated with each probe is indicated to the right of the heatmap. Target genes that appear more than one time on the heatmap indicate that more than one probe scored for that gene.</p

Higher <i>TTK</i> expression is associated with biochemical recurrence.

<p>Kaplan Meier plot of the risk of biochemical recurrence in patients (n = 131) with TTK overexpressing prostate tumors (n = 20; green line) versus those without TTK overexpressing (n = 111; blue line) tumors (p = 0.003, log-rank test).</p

ETV1 is a lineage survival factor that cooperates with KIT in gastrointestinal stromal tumours

Gastrointestinal stromal tumour (GIST) is the most common human sarcoma and is primarily defined by activating mutations in the KIT or PDGFRA receptor tyrosine kinases. KIT is highly expressed in interstitial cells of Cajal (ICCs)-the presumed cell of origin for GIST-as well as in haematopoietic stem cells, melanocytes, mast cells and germ cells. Yet, families harbouring germline activating KIT mutations and mice with knock-in Kit mutations almost exclusively develop ICC hyperplasia and GIST, suggesting that the cellular context is important for KIT to mediate oncogenesis. Here we show that the ETS family member ETV1 is highly expressed in the subtypes of ICCs sensitive to oncogenic KIT mediated transformation, and is required for their development. In addition, ETV1 is universally highly expressed in GISTs and is required for growth of imatinib-sensitive and resistant GIST cell lines. Transcriptome profiling and global analyses of ETV1-binding sites suggest that ETV1 is a master regulator of an ICC-GIST-specific transcription network mainly through enhancer binding. The ETV1 transcriptional program is further regulated by activated KIT, which prolongs ETV1 protein stability and cooperates with ETV1 to promote tumorigenesis. We propose that GIST arises from ICCs with high levels of endogenous ETV1 expression that, when coupled with an activating KIT mutation, drives an oncogenic ETS transcriptional program. This differs from other ETS-dependent tumours such as prostate cancer, melanoma and Ewing sarcoma where genomic translocation or amplification drives aberrant ETS expression. It also represents a novel mechanism of oncogenic transcription factor activation