Kari Rostad

Genome-Wide Profiling of Histone H3 Lysine 4 and Lysine 27 Trimethylation Reveals an Epigenetic Signature in Prostate Carcinogenesis

Background Increasing evidence implicates the critical roles of epigenetic regulation in cancer. Very recent reports indicate that global gene silencing in cancer is associated with specific epigenetic modifications. However, the relationship between epigenetic switches and more dynamic patterns of gene activation and repression has remained largely unknown. Methodology/Principal Findings Genome-wide profiling of the trimethylation of histone H3 lysine 4 (H3K4me3) and lysine 27 (H3K27me3) was performed using chromatin immunoprecipitation coupled with whole genome promoter microarray (ChIP-chip) techniques. Comparison of the ChIP-chip data and microarray gene expression data revealed that loss and/or gain of H3K4me3 and/or H3K27me3 were strongly associated with differential gene expression, including microRNA expression, between prostate cancer and primary cells. The most common switches were gain or loss of H3K27me3 coupled with low effect on gene expression. The least prevalent switches were between H3K4me3 and H3K27me3 coupled with much higher fractions of activated and silenced genes. Promoter patterns of H3K4me3 and H3K27me3 corresponded strongly with coordinated expression changes of regulatory gene modules, such as HOX and microRNA genes, and structural gene modules, such as desmosome and gap junction genes. A number of epigenetically switched oncogenes and tumor suppressor genes were found overexpressed and underexpressed accordingly in prostate cancer cells. Conclusions/Significance This work offers a dynamic picture of epigenetic switches in carcinogenesis and contributes to an overall understanding of coordinated regulation of gene expression in cancer. Our data indicate an H3K4me3/H3K27me3 epigenetic signature of prostate carcinogenesis.

TMPRSS2:ERG fusion transcripts in urine from prostate cancer patients correlate with a less favorable prognosis.

Rostad K, Hellwinkel OJC, Haukaas SA, Halvorsen OJ, Øyan AM, Haese A, Budäus L, Albrecht H, Akslen LA, Schlomm T, Kalland K‐H. TMPRSS2:ERG fusion transcripts in urine from prostate cancer patients correlate with a less favorable prognosis. APMIS 2009; 117: 575–82.

MiR‐182 and miR‐203 induce mesenchymal to epithelial transition and self‐sufficiency of growth signals via repressing SNAI2 in prostate cells

MicroRNAs play critical roles in tumorigenesis and metastasis. Here, we report the dual functions of miR‐182 and miR‐203 in our previously described prostate cell model. MiR‐182 and miR‐203 were completely repressed during epithelial to mesenchymal transition (EMT) from prostate epithelial EP156T cells to the progeny mesenchymal nontransformed EPT1 cells. Re‐expression of miR‐182 or miR‐203 in EPT1 cells and prostate cancer PC3 cells induced mesenchymal to epithelial transition (MET) features. Simultaneously, miR‐182 and miR‐203 provided EPT1 cells with the ability to self‐sufficiency of growth signals, a well‐recognized oncogenic feature. Gene expression profiling showed high overlap of the genes affected by miR‐182 and miR‐203. SNAI2 was identified as a common target of miR‐182 and miR‐203. Knock‐down of SNAI2 in EPT1 cells phenocopied re‐expression of either miR‐182 or miR‐203 regarding both MET and self‐sufficiency of growth signals. Strikingly, considerable overlaps of changed genes were found between the re‐expression of miR‐182/203 and knock‐down of SNAI2. Finally, P‐cadherin was identified as a direct target of SNAI2. We conclude that miR‐182 and miR‐203 induce MET features and growth factor independent growth via repressing SNAI2 in prostate cells. Our findings shed new light on the roles of miR‐182/203 in cancer related processes.

Epithelial to Mesenchymal Transition of a Primary Prostate Cell Line with Switches of Cell Adhesion Modules but without Malignant Transformation

Background Epithelial to mesenchymal transition (EMT) has been connected with cancer progression in vivo and the generation of more aggressive cancer cell lines in vitro. EMT has been induced in prostate cancer cell lines, but has previously not been shown in primary prostate cells. The role of EMT in malignant transformation has not been clarified. Methodology/Principal Findings In a transformation experiment when selecting for cells with loss of contact inhibition, the immortalized prostate primary epithelial cell line, EP156T, was observed to undergo EMT accompanied by loss of contact inhibition after about 12 weeks in continuous culture. The changed new cells were named EPT1. EMT of EPT1 was characterized by striking morphological changes and increased invasion and migration compared with the original EP156T cells. Gene expression profiling showed extensively decreased epithelial markers and increased mesenchymal markers in EPT1 cells, as well as pronounced switches of gene expression modules involved in cell adhesion and attachment. Transformation assays showed that EPT1 cells were sensitive to serum or growth factor withdrawal. Most importantly, EPT1 cells were not able to grow in an anchorage-independent way in soft agar, which is considered a critical feature of malignant transformation. Conclusions/Significance This work for the first time established an EMT model from primary prostate cells. The results show that EMT can be activated as a coordinated gene expression program in association with early steps of transformation. The model allows a clearer identification of the molecular mechanisms of EMT and its potential role in malignant transformation.

Increased Expression of SIM2-s Protein Is a Novel Marker of Aggressive Prostate Cancer

Purpose: The human SIM2 gene is located within the Downs syndrome critical region of chromosome 21 and encodes transcription factors involved in brain development and neuronal differentiation. SIM2 has been assigned a possible role in the pathogenesis of solid tumors, and the SIM2-short isoform (SIM2-s) was recently proposed as a molecular target for cancer therapy. We previously reported SIM2 among the highly up-regulated genes in 29 prostate cancers, and the purpose of our present study was to examine the expression status of SIM2 at the transcriptional and protein level as related to outcome in prostate cancer. Experimental Design: By quantitative PCR, mRNA in situ hybridization, and immunohistochemistry, we evaluated the expression and significance of SIM2 isoforms in 39 patients with clinically localized prostate cancer and validated the expression of SIM2-s protein in an independent cohort of 103 radical prostatectomies from patients with long and complete follow-up. Results: The SIM2 isoforms (SIM2-s and SIM2-l) were significantly coexpressed and increased in prostate cancer. Tumor cell expression of SIM2-s protein was associated with adverse clinicopathologic factors like increased preoperative serum prostate-specific antigen, high histologic grade, invasive tumor growth with extra-prostatic extension, and increased tumor cell proliferation by Ki-67 expression. SIM2-s protein expression was significantly associated with reduced cancer-specific survival in multivariate analyses. Conclusions: These novel findings indicate for the first time that SIM2 expression might be important for clinical progress of human cancer and support the recent proposal of SIM2-s as a candidate for targeted therapy in prostate cancer.

p63 attenuates epithelial to mesenchymal potential in an experimental prostate cell model.

The transcription factor p63 is central for epithelial homeostasis and development. In our model of epithelial to mesenchymal transition (EMT) in human prostate cells, p63 was one of the most down-regulated transcription factors during EMT. We therefore investigated the role of p63 in EMT. Over-expression of the predominant epithelial isoform ΔNp63α in mesenchymal type cells of the model led to gain of several epithelial characteristics without resulting in a complete mesenchymal to epithelial transition (MET). This was corroborated by a reciprocal effect when p63 was knocked down in epithelial EP156T cells. Global gene expression analyses showed that ΔNp63α induced gene modules involved in both cell-to-cell and cell-to-extracellular-matrix junctions in mesenchymal type cells. Genome-wide analysis of p63 binding sites using ChIP-seq analyses confirmed binding of p63 to regulatory areas of genes associated with cell adhesion in prostate epithelial cells. DH1 and ZEB1 are two elemental factors in the control of EMT. Over-expression and knock-down of these factors, respectively, were not sufficient alone or in combination with ΔNp63α to reverse completely the mesenchymal phenotype. The partial reversion of epithelial to mesenchymal transition might reflect the ability of ΔNp63α, as a key co-ordinator of several epithelial gene expression modules, to reduce epithelial to mesenchymal plasticity (EMP). The utility of ΔNp63α expression and the potential of reduced EMP in order to counteract metastasis warrant further investigation.

Abstract 1290: The ability of the transcription factor p63 to induce selected gene expression modules associated with mesenchymal to epithelial transition of prostate cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Genome-wide gene expression analysis identified p63 as one of the most consistently under-expressed transcription factors in prostate cancer samples compared to matched benign prostate tissue. Two alternative p63 promoters generate two different N-terminal variants, TA or ΔN which contains and lacks the transactivating domain, respectively. Differential splicing generates multiple additional isoforms. The activity and interactions of the different isoforms remain unresolved. Isoform-specific real-time quantitative PCR assays revealed that ΔNp63α is the predominant isoform in epithelial prostate tissues, but other isoforms are detectable. Our group has established an experimental cell culture model based upon primary, immortalized prostate epithelial cells (EP156T cells), where p63 was shut down when EP156T cells underwent epithelial-to-mesenchymal transition (EMT) to become EPT1 cells. Subsequently, several additional mesenchymal like cell subtypes with additional malignant features, including anchorage independent growth in soft agar and ability to grow at much higher density in monolayers, were derived from EPT1 cells. All of these subtypes exhibited very low p63 expression. To examine the potential of p63 to induce mesenchymal to epithelial transition (MET) in this EMT model, we have stably re-expressed the ΔNp63α and TAp63α isoforms in different mesenchymal like cells of the model using retroviral/lentiviral vector transductions. Genome-wide gene expression analyses and phenotypic cellular assays revealed that the ΔNp63α isoform induced re-expression of multiple genes involved in cell adhesion. But this was not associated with complete MET induction. Cells re-expressing ΔNp63α obtained an intermediate morphology with both epithelial and mesenchymal traits, and with actin filaments organized in stress fibers. ΔNp63α re-expression significantly compromised migratory but not invasive ability of the cells. Based upon these findings we want to investigate whether p63 may compromise epithelial to mesenchymal plasticity in an animal model of tumorigenic cells and if there is differential effect on primary tumor growth compared to metastatic growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1290. doi:1538-7445.AM2012-1290

Abstract 2329: Restriction of androgen receptor and target gene expression

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: The androgen receptor (AR) is activated and pathogenic in castration resistant prostate cancer (CRPC) due to a variety of escape mechanisms from anti-androgen treatment. The “cell of origin” of prostate cancer is not finally defined, but in different models prostate epithelial basal cells have been shown to develop into cancer with authentic prostate cancer features. The AR and target genes are effectively silenced in prostate epithelial basal cells. Given the importance of activated AR in prostate cancer we performed a reassessment of the basis of its silencing in prostate epithelial basal cells. Results: EP156T cells are derived from primary prostate epithelial basal cells and were propagated in monolayer cultures with features of transit amplifying cells in medium with low calcium concentration. The AR was not detectable in these cells and neither the AR nor target genes such as KLK3 (PSA), TMPRSS2 and NKX3-1 were induced following treatment with synthetic androgen (R1881) and a variety of growth factors and combinations as determined using sensitive Taqman real-time quantitative PCR assays, gene expression microarrays of cell lysates and PSA assays of cell supernatants. This was in contrast to LNCaP control cells where KLK3 was 12-fold and TMPRSS2 was 8-fold increased following R1881 induction. EP156T cells underwent epithelial to mesenchymal transition (EMT), and a series of progeny cells with an accumulating number of malignant hallmarks were derived. In these cells a spontaneous, but limited increase in AR mRNA was found. But the cells were neither androgen responsive nor androgen dependent as determined using R1881 stimulation and bicalutamide inhibitor, respectively. Shift to high calcium concentration in the medium rapidly induced morphological differentiation of basal cells, but still the AR and AR target gene expression was restricted. In 3-dimensional cultures in Matrigel structures with a distinct outer cell layer versus different inner cells were observed. A variety of methods was used to explore the molecular basis of restricted AR and target gene expression in these different culture conditions, including chromatin immunoprecipitation, indirect immunofluorescence, fluorescent promoter reporter constructs, gene expression and Western blot assays. Conclusion: In human prostate epithelial cells with basal cell traits we found strong restrictions to expression of the AR and AR target genes in monolayer cultures with a large variety of growth factors and combinations, including androgens. High calcium concentration induced morphological changes associated with epithelial differentiation, but with restricted AR and target gene expression. Growth of epithelial cells in Matrigel was associated with evidence of morphological differentiation. This model is useful for further identification of the molecular basis of restricted AR and target gene expression in prostate cells in comparison with malignant prostate cells. Citation Format: Margrete R. Hellem, Jan Roger Olsen, Yaping Hua, Yi Qu, Kristo Marvyin, Kari Rostad, Jie Liu, Lisha Li, Varda Rotter, Biaoyang Lin, Xisong Ke, Anne Margrete Oyan, Karl-Henning Kalland. Restriction of androgen receptor and target gene expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2329. doi:10.1158/1538-7445.AM2014-2329

Abstract 4128: miR203 induces mesenchymal to epithelial transition via repressing SNAI2 and activating P-cadherin in prostate cells

MicroRNAs play critical roles in tumorigenesis and metastasis. Here we report the functions of miR203 in prostate cancer based upon an experimental cell culture model. While highly expressed in prostate epithelial cells, miR203 became completely repressed following their epithelial to mesenchymal transition (EMT). Forced re-expression of miR203 in the mesenchymal cells induced many epithelial features, including morphological change in 2D and 3D culture, decreased abilities of invasion and migration, as well as increased epithelial markers and decreased mesenchymal markers. By computer prediction combined with microarray profiling and luciferase reporter target assays, we identified SNAI2 as target of miR203. Knock-down of SNAI2 can mimic the epithelial phenotypes induced by miR203. Most of the affected genes by miR203 were also found to be changed with the same pattern by knock-down of SNAI2. Furthermore, P-cadherin (CDH3), an important epithelial adhesion component, was identified as the downstream target of SNAI2. CDH3-promoter-Luc reporter assay demonstrated that 3 E-boxes in the CDH3 promoter mediated direct targeting by SNAI2. Decreased and increased expression of CDH3 were found in culture cells with overexpression and knock-down of SNAI2, respectively. We concluded that miR203 represses SNAI2, thus activating P-cadherin in induction of epithelial features. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4128. doi:1538-7445.AM2012-4128

Abstract 3089: The role of p63 regulation during epithelial to mesenchymal transition (EMT) and subsequent accumulation of malignant features of primary immortalized prostate cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL A family of transcription factors is constituted by p53, p63 and p73. Unlike p53, which is expressed in response to environmental stress, p63 (TP73L) is constitutively expressed at high levels in a variety of epithelial tissues including prostate. Our genome-wide gene expression analysis identified p63 as one of the most consistently under-expressed genes in prostate cancer samples compared to matched benign prostate tissue. All benign prostate samples including benign prostate hyperplasias (BPH) exhibited high p63 expression. Two alternative p63 promoters generate two different N-terminal variants, TA or ΔN which contains and lacks the transactivating domain, respectively. Differential splicing generates multiple additional isoforms. The activity and interactions of the different isoforms remain unresolved. Using isoform-specific TaqMan real-time quantitative PCR assays, we have found that ΔNp63α is the predominant isoform in prostate tissues, but the other isoforms are detectable. Our group has established a new experimental cell culture model based upon primary, immortalized prostate epithelial cells (EP156T cells), where p63 was found to be shut-down as EP156T cells underwent epithelial-to-mesenchymal transition (EMT) to become EPT1 cells. Subsequently, EPT2 cells were derived from EPT1 cells and p63 stayed shut-down. EPT2 cells had acquired several malignant features in contrast to the progenitors, including anchorage independent growth in soft agar and ability to grow at much higher density in monolayers. To dissect the role of p63 in EMT and in gene expression modules associated with carcinogenesis, we have stably re-expressed the predominant isoform ΔNp63α in both EPT1 and EPT2 cells using retroviral vector transductions. Microarray analysis shows that p63 re-expression leads to re-expression of genes participating in several cell-junction components, such as hemidesmosomes, focal adhesions and gap junctions. These genes were down-regulated during EMT. There were also morphological changes and a decreased ability to migrate following p63 re-expression. This indicates that ΔNp63α re-expression might result in a partial mesenchymal-to-epithelial transition (MET). The present focus is to identify co-factors that may act complementary or synergetically with p63 to induce a complete MET in EPT1 cells and to identify p63 target genes and regulatory cross-talk with relevance for anti-cancer strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3089. doi:10.1158/1538-7445.AM2011-3089