Amplification of the 20q Chromosomal Arm Occurs Early in Tumorigenic Transformation and May Initiate Cancer

Duplication of chromosomal arm 20q occurs in prostate, cervical, colon, gastric, bladder, melanoma, pancreas and breast cancer, suggesting that 20q amplification may play a causal role in tumorigenesis. According to an alternative view, chromosomal imbalance is mainly a common side effect of cancer progression. To test whether a specific genomic aberration might serve as a cancer initiating event, we established an in vitro system that models the evolutionary process of early stages of prostate tumor formation; normal prostate cells were immortalized by the over-expression of human telomerase catalytic subunit hTERT, and cultured for 650 days till several transformation hallmarks were observed. Gene expression patterns were measured and chromosomal aberrations were monitored by spectral karyotype analysis at different times. Several chromosomal aberrations, in particular duplication of chromosomal arm 20q, occurred early in the process and were fixed in the cell populations, while other aberrations became extinct shortly after their appearance. A wide range of bioinformatic tools, applied to our data and to data from several cancer databases, revealed that spontaneous 20q amplification can promote cancer initiation. Our computational model suggests that 20q amplification induced deregulation of several specific cancer-related pathways including the MAPK pathway, the p53 pathway and Polycomb group factors. In addition, activation of Myc, AML, B-Catenin and the ETS family transcription factors was identified as an important step in cancer development driven by 20q amplification. Finally we identified 13 "cancer initiating genes", located on 20q13, which were significantly over-expressed in many tumors, with expression levels correlated with tumor grade and outcome suggesting that these genes induce the malignant process upon 20q amplification

TMPRSS2/ERG Promotes Epithelial to Mesenchymal Transition through the ZEB1/ZEB2 Axis in a Prostate Cancer Model

Prostate cancer is the most common non-dermatologic malignancy in men in the Western world. Recently, a frequent chromosomal aberration fusing androgen regulated TMPRSS2 promoter and the ERG gene (TMPRSS2/ERG) was discovered in prostate cancer. Several studies demonstrated cooperation between TMPRSS2/ERG and other defective pathways in cancer progression. However, the unveiling of more specific pathways in which TMPRSS2/ERG takes part, requires further investigation. Using immortalized prostate epithelial cells we were able to show that TMPRSS2/ERG over-expressing cells undergo an Epithelial to Mesenchymal Transition (EMT), manifested by acquisition of mesenchymal morphology and markers as well as migration and invasion capabilities. These findings were corroborated in vivo, where the control cells gave rise to discrete nodules while the TMPRSS2/ERG-expressing cells formed malignant tumors, which expressed EMT markers. To further investigate the general transcription scheme induced by TMPRSS2/ERG, cells were subjected to a microarray analysis that revealed a distinct EMT expression program, including up-regulation of the EMT facilitators, ZEB1 and ZEB2, and down-regulation of the epithelial marker CDH1(E-Cadherin). A chromatin immunoprecipitation assay revealed direct binding of TMPRSS2/ERG to the promoter of ZEB1 but not ZEB2. However, TMPRSS2/ERG was able to bind the promoters of the ZEB2 modulators, IL1R2 and SPINT1. This set of experiments further illuminates the mechanism by which the TMPRSS2/ERG fusion affects prostate cancer progression and might assist in targeting TMPRSS2/ERG and its downstream targets in future drug design efforts

Mutant p53 Attenuates the Anti-Tumorigenic Activity of Fibroblasts-Secreted Interferon Beta

<div><p>Mutations in the p53 tumor suppressor protein are highly frequent in tumors and often endow cells with tumorigenic capacities. We sought to examine a possible role for mutant p53 in the cross-talk between cancer cells and their surrounding stroma, which is a crucial factor affecting tumor outcome. Here we present a novel model which enables individual monitoring of the response of cancer cells and stromal cells (fibroblasts) to co-culturing. We found that fibroblasts elicit the interferon beta (IFNβ) pathway when in contact with cancer cells, thereby inhibiting their migration. Mutant p53 in the tumor was able to alleviate this response via SOCS1 mediated inhibition of STAT1 phosphorylation. IFNβ on the other hand, reduced mutant p53 RNA levels by restricting its RNA stabilizer, WIG1. These data underscore mutant p53 oncogenic properties in the context of the tumor microenvironment and suggest that mutant p53 positive cancer patients might benefit from IFNβ treatment.</p></div

A schematic representation of the interaction between mutant p53 and the IFNβ pathway.

<p>Blue arrows denote positive effect while red arrows denote negative effect. Upon encounter with cancer cell CAFs activate the IFNβ pathway which limits cancer cells' migration. When mutant p53 is present in the cancer cells, this pathway is moderated via SOCS1 mediated inhibition of STAT1 phosphorylation. IFNβ is able to reduce mutant p53 RNA levels by attenuating the expression of mutant p53 RNA stabilizer WIG1.</p

IFNβ reduces mutant p53 mRNA levels by inhibiting its RNA stabiliser WIG1 (ZMAT3).

<p>Cells were treated with IFNβ for the designated time points, fixed and sorted by an “Image stream” FACS. (A) Representative images from each condition and (B) a graph representing the mean pixel intensity of mutant p53 positive cells for the entire population. (C) Cells were treated with the designated IFNs for 24 h and mutant p53 and GAPDH levels were measured by western blot. Cells were treated with IFNβ for 24 h and mutant p53 (D) and WIG1 (E) RNA levels were determined by QRT-PCR. * P<0.05. (F) HepG2 cells were introduced with mut175 plasmid and treated with IFNβ. RNA levels were determined by QRT-PCR. * P<0.05. (G) H1299 cell harbouring a TS form of mutant p53 (Wild type form at 32°C and mutant form at 37°C) were treated with IFNβ for 30 h. p53 and GAPDH levels were measured by western blot, shown in the lower panel a is normalized quantification of the bands.</p

Expression patterns of H1299 in response to co-cultivation with CAFs.

<p>(A) Three principal expression patterns of H1299 cultivated with HK3-T. (B) A heat-map depicting genes that exhibit Pearson correlation of at least 0.9 to representative bait(s) from each pattern shown in A. Beneath is the percentage of IFN targets in each list based on the ‘Interferome database’ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061353#pone.0061353-Samarajiwa1" target="_blank">[30]</a>. (C) QRT-PCR analysis of a representative gene from each expression pattern. (D) The H1299 panel was treated with the designated IFNs for 24 h. Shown is a QRT-PCR analysis of MX1 expression.</p

Mutant p53 counteracts IFNβ by SOCS1-mediated attenuation of STAT1 phosphorylation.

<p>(A) Cells were treated with IFNβ for 16 h, fixed and sorted by an “Image stream” FACS. The upper panel depicts representative images from each condition and the graph represents the mean pixel intensity of STAT1 positive cells for the entire population. (B) Same as in A, here the graph shows the similarity between p-STAT and DAPI staining, thereby quantifying both the expression and localization of pSTAT1. (C) Cells were treated with IFNβ for the designated durations, shown is a graph depicting nuclear p-STAT1. (D) The cells were also collected for RNA analysis and a QRT-PCR for SOCS1 expression was performed. (E) H1299¹⁷⁵ cells were introduced with RNAi against LacZ as a control or against SOCS1. p = 0.002 (F) Cells were then treated with IFNβ for 24 h. Shown is a QRT-PCR analysis of MX1 expression. p<0.05. (G) Cells were seeded in trans-wells in serum-free media and treated with IFNβ for 24 h. Migrating cells were collected and counted.</p

An in vitro model to study the tumor-stroma encounter in lung cancer.

<p>(A). p53-null lung carcinoma cells (H1299) were introduced with the designated mutations. p53 levels were determined by Western blot analysis (A) and by QRT-PCR (B). A fluorescent microscope image of co-cultured dsRed-labeled H1299 with GFP-labeled HK3 (C, upper panel). Representative FACS analysis depicting dsRed- and GFP-labeled sub-populations following a sorting procedure (C, middle panel). Each sub-population was then re-sorted using the same sorting gates (C, lower panel).</p

The interferon beta pathway is up regulated in fibroblasts after co-cultivation with mutp53-bearing carcinoma cells.

<p>(A) Following the described sorting procedure, HK3-T samples were subjected to a microarray analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061353#s4" target="_blank">materials and methods</a>). The presented cluster was obtained by the ‘CLICK’ algorithm from the ‘Expander’ package using default homogeneity (version 5.2) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061353#pone.0061353-Sharan2" target="_blank">[27]</a>. The log 2 ratios were standardized to have zero mean and unit standard deviation for each gene. (B) ‘HK3-T cluster’ which contains 414 genes was compared to a previously reported ‘IFN signature’ that was induced by co-cultures of cancer and stromal cells. (C) ‘HK3-T cluster’ was compared with the ‘Interferome database’ containing 1196 IFN targets. A Fisher's exact test was utilized to compare these overlaps with those of all other clusters. * = P<0.0001 (D) To assess the significance of the physical interaction between HK3-T and H1299¹⁷⁵ in eliciting the IFN pathway, HK3-T were introduced to conditioned media from each cell type grown alone or from a co-culture plate. mRNA levels of two known IFN targets were measured by QRT-PCR. (E) Interferon α and β mRNA levels. (F) CAFs and H1299 were grown either alone or in a co-culture. The co-cultured cells were incubated with the designated antibodies. Shown is the mRNA level of the designated IFN targets. * P<0.05. (G) HK3-T cells were subjected to conditioned media of HK3-T or that of HK3-T cultured with H1299¹⁷⁵. Shown is a western blot of GAPDH, STAT1, pSTAT1 and STAT2. (H) The same experimental setup was used. Shown in the upper panel are microscope images of the cells. Cells were then collected and stained with Propidium Iodide (PI) and apoptotic cells (PI positive) were detected by FACS sorting and their percentage is depicted in the lower panel.</p