Yaping Hua

Generation of Prostate Tumor–Initiating Cells Is Associated with Elevation of Reactive Oxygen Species and IL-6/STAT3 Signaling

How prostate cancer is initiated remains a topic of debate. In an effort to establish a human model of prostate carcinogenesis, we adapted premalignant human prostate EPT2-D5 cells to protein-free medium to generate numerous tight prostate spheres (D5HS) in monolayer culture. In contrast to EPT2-D5 cells, the newly generated D5HS efficiently formed large subcutaneous tumors and subsequent metastases in vivo, showing the tumorigenicity of D5HS spheres. A striking production of interleukin (IL)-6 mRNA and protein was found in D5HS cells. The essential roles of IL-6 and the downstream STAT3 signaling in D5HS tumor sphere formation were confirmed by neutralizing antibody, chemical inhibitors, and fluorescent pathway reporter. In addition, elevated reactive oxygen species (ROS) produced upon protein depletion was required for the activation of IL-6/STAT3 in D5HS. Importantly, a positive feedback loop was found between ROS and IL-6 during tumor sphere formation. The association of ROS/IL-6/STAT3 to the carcinogenesis of human prostate cells was further examined in xenograft tumors and verified by limiting dilution implantations. Collectively, we have for the first time established human prostate tumor-initiating cells based on physiologic adaption. The intrinsic association of ROS and IL-6/STAT3 signaling in human prostate carcinogenesis shed new light on this relationship and define therapeutic targets in this setting.

Context dependent regulatory patterns of the androgen receptor and androgen receptor target genes

BackgroundExpression of the androgen receptor (AR) is associated with androgen-dependent proliferation arrest and terminal differentiation of normal prostate epithelial cells. Additionally, activation of the AR is required for survival of benign luminal epithelial cells and primary cancer cells, thus androgen deprivation therapy (ADT) leads to apoptosis in both benign and cancerous tissue. Escape from ADT is known as castration-resistant prostate cancer (CRPC). In the course of CRPC development the AR typically switches from being a cell-intrinsic inhibitor of normal prostate epithelial cell proliferation to becoming an oncogene that is critical for prostate cancer cell proliferation. A clearer understanding of the context dependent activation of the AR and its target genes is therefore desirable.MethodsImmortalized human prostate basal epithelial EP156T cells and progeny cells that underwent epithelial to mesenchymal transition (EMT), primary prostate epithelial cells (PrECs) and prostate cancer cell lines LNCaP, VCaP and 22Rv1 were used to examine context dependent restriction and activation of the AR and classical target genes, such as KLK3. Genome-wide gene expression analyses and single cell protein analyses were applied to study the effect of different contexts.ResultsA variety of growth conditions were tested and found unable to activate AR expression and transcription of classical androgen-dependent AR target genes, such as KLK3, in prostate epithelial cells with basal cell features or in mesenchymal type prostate cells. The restriction of androgen- and AR-dependent transcription of classical target genes in prostate basal epithelial cells was at the level of AR expression. Exogenous AR expression was sufficient for androgen-dependent transcription of AR target genes in prostate basal epithelial cells, but did not exert a positive feedback on endogenous AR expression. Treatment of basal prostate epithelial cells with inhibitors of epigenetic gene silencing was not efficient in inducing androgen-dependent transcription of AR target genes, suggesting the importance of missing cofactor(s).ConclusionsRegulatory mechanisms of AR and androgen-dependent AR target gene transcription are insufficiently understood and may be critical for prostate cancer initiation, progression and escape from standard therapy. The present model is useful for the study of context dependent activation of the AR and its transcriptome.

An androgen response element driven reporter assay for the detection of androgen receptor activity in prostate cells

The androgen receptor (AR) transcription factor plays a key role in the development and progression of prostate cancer, as is evident from the efficacy of androgen-deprivation therapy, AR is also the most frequently mutated gene, in castration resistant prostate cancer (CRPC). AR has therefore become an even more attractive therapeutic target in aggressive and disseminated prostate cancer. To investigate mechanisms of AR and AR target gene activation in different subpopulations of prostate cancer cells, a toolkit of AR expressor and androgen response element (ARE) reporter vectors were developed. Three ARE reporter vectors were constructed with different ARE consensus sequences in promoters linked to either fluorescence or luciferase reporter genes in lentiviral vector backbones. Cell lines transduced with the different vectors expressed the reporters in an androgen-dependent way according to fluorescence microscopy, flow cytometry and multi-well fluorescent and luminescence assays. Interestingly, the background reporter activity in androgen-depleted medium was significantly higher in LNCaP cells compared to the prostate transit amplifying epithelial cell lines, EP156T-AR and 957E/hTERT-AR with exogenous AR. The androgen-induced signal to background was much higher in the latter benign prostate cells than in LNCaP cells. Androgen-independent nuclear localization of AR was seen in LNCaP cells and reduced ARE-signaling was seen following treatment with abiraterone, an androgen synthesis inhibitor. The ARE reporter activity was significantly stronger when stimulated by androgens than by β-estradiol, progesterone and dexamethasone in all tested cell types. Finally, no androgen-induced ARE reporter activity was observed in tumorigenic mesenchymal progeny cells of EP156T cells following epithelial to mesenchymal transition. This underscores the observation that expression of the classical luminal differentiation transcriptome is restricted in mesenchymal type cells with or without AR expression, and presence of androgen.

Models of Tumor Progression in Prostate Cancer

Human prostate cancer is initiated in a benign prostate epithelial cell which gains the potential to progress to metastatic disease. The exact cell of origin of prostate cancer has been debated in recent years based upon different models. Primary prostate epithelial cells have restricted life-spans in culture, but can be immortalized. Prostate cancer cell lines have been difficult to establish and new ones are desirable. Attempts to transform benign prostate epithelial cells in vitro have proved difficult without the use of strong carcinogens or oncogenes in processes not likely to mimic closely carcinogenesis in the aging human prostate. Models of epithelial-to-mesenchymal transition (EMT) and cancer stem cells in prostate carcinogenesis have become available, and advances in three-dimensional organoid culture technology represent a breakthrough in prostate cancer research. Organoids may recapitulate multiple features of prostate cancer and have the potential to replace costly and laborious animal experiments. Still, animal models are needed to investigate and validate molecular mechanisms and to develop therapeutic principles in the pipeline between in vitro experiments and clinical applications. Although mice represent the most common experimental animal in prostate cancer research, species like rat, dog, and zebrafish may have advantages depending upon the hypothesis or question. Animal models can generally be categorized into spontaneous or induced development of cancer, immunodeficient animals with xenografts, and genetically engineered animals. In prostate cancer, neuroendocrine differentiation and bone metastases are prevalent in the final stages of cancer progression and animal models that recapitulate these processes are available.

Abstract 2902: Assays for androgen receptor activity using cell based ARE reporter systems

The androgen receptor (AR) transcription factor plays a key role in the development and progression of prostate cancer. Inhibition of its ligand by androgen deprivation therapy (ADT) is consequently the main medical treatment of invasive prostate cancer. AR is activated and maintained throughout prostate cancer progression even in castration resistant prostate cancer (CRPC). Prostate cancer cells escape from ADT using a variety of mechanisms. The AR and target genes have therefore become even more focused therapeutic targets in aggressive and disseminated prostate cancer. AR and its classical target genes, such as KLK3 (PSA) are, however, efficiently shut off in basal epithelial prostate cells and possibly in prostate cancer stem cells (CSCs). To investigate mechanisms of AR and AR target gene activation in different subpopulations of prostate cancer cells, several androgen response elements (ARE) reporter vectors were developed. Three ARE reporter vectors were constructed with different ARE consensus sequences in promoters linked to either fluorescence or luciferase reporter genes in lentiviral vector backbones. Cell lines transduced with the different vectors expressed the reporters in an androgen-dependent way according to fluorescence microscopy, flow cytometry and multi-well fluorescent and luminescence recording. The 241B promoter sequence reporter was selected among the constructed ARE reporters on the basis of higher activity in initial screenings. The AR positive and androgen responsive prostate cancer cell line, LNCaP, and the prostate transit amplifying epithelial cell line, EP156T-AR with exogenous AR, were transduced with the lentiviral 241B-mCherry fluorescence reporter. Flow cytometry, multi-well reader and fluorescence microscopy results corresponded when reporter cells were treated with androgen and with the AR antagonist, enzalutamide, and with the anti-androgen, abiraterone. An SV40-promoter GFP reporter element was next cloned into the 241B-mCherry reporter. Constitutive expression of GFP facilitated normalization of ARE driven mCherry fluorescent signals. Furthermore, AR expression vectors were also constructed with the AR open reading frames cloned into lentiviral expression vectors and used in co-transfection and co-transduction experiments in AR negative cell types. The developed ARE reporter system will help us to investigate the role of the AR in differentiation and proliferation of prostate cells and to study the AR activity in two and three dimensional cell cultures. This system can also be useful in screening for drugs with activity against the AR. Citation Format: Waqas Azeem, Margrete R. Hellem, Jan R. Olsen, Yaping Hua, Kristo Marvyin, Lisha Li, Yi Qu, Biaoyang Lin, Xisong Ke, Anne M. Oyan, Karl-Henning Kalland. Assays for androgen receptor activity using cell based ARE reporter systems. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2902.

Abstract 1824: Context dependent regulatory patterns of the androgen receptor (AR) and androgen receptor target genes

Background: Expression of the androgen receptor (AR) is associated with androgen-dependent proliferation arrest and terminal differentiation of normal prostate epithelial cells. Additionally, activation of the AR is required for survival of benign luminal epithelial cells and primary cancer cells. Androgen deprivation therapy (ADT) leads to apoptosis in both benign and cancerous tissue. Cancer cell escape from ADT is known as castration-resistant prostate cancer (CRPC). In the course of CRPC development the AR typically switches from being a cell-intrinsic inhibitor of normal prostate epithelial cell proliferation to becoming an oncogene that is critical for prostate cancer cell proliferation. Methods: Immortalized human prostate basal epithelial EP156T cells and progeny cells that underwent epithelial to mesenchymal transition (EMT), primary prostate epithelial cells (PrECs) and prostate cancer cell lines LNCaP, VCaP and 22Rv1 were used to examine context dependent restriction and activation of the AR and classical target genes, such as KLK3. Genome-wide gene expression analyses and single cell protein analyses were applied to study the effect of different contexts. Results: A variety of growth conditions were tested and found unable to activate AR expression and transcription of classical androgen-dependent AR target genes, such as KLK3, in prostate epithelial cells with basal cell features or in mesenchymal type prostate cells. The restriction of androgen and AR dependent transcription of classical target genes in prostate basal epithelial cells was at the level of AR expression. Exogenous AR expression was sufficient for androgen-dependent transcription of AR target genes in prostate basal epithelial EP156T-AR cells, but did not exert a positive feedback on endogenous AR expression. Mesenchymal type prostate EPT3-AR cells with exogenous AR expression, in contrast to epithelial type EP156T-AR cells, were androgen non-responsive and were unable to produce detectable PSA in the culture supernatants even with higher levels of exogenous AR protein than in EP156T-AR and LNCaP cells for up to 2 weeks in androgen containing growth medium. The restricted PSA expression in the mesenchymal context suggests that if ADT increases the pool of mesenchymal type prostate cancer cells, then this might go undetected during PSA monitoring of disease progression. Citation Format: Jan Roger Olsen, Waqas Azeem, Margrete R. Hellem, Kristo Marvyin, Yaping Hua, Yi Qu, Lisha Li, Biaoyang Lin, XiSong Ke, Anne Margrete Oyan, Karl-Henning Kalland. Context dependent regulatory patterns of the androgen receptor (AR) and androgen receptor target genes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1824.

Abstract 79: An androgen response element-based reporter assay for the detection of androgen receptor activity in prostate cells

Background: The androgen receptor (AR) transcription factor plays pivotal roles in the development and progression of prostate cancer. AR is activated and maintained throughout prostate cancer progression even in castration-resistant prostate cancer (CRPC). AR and its classical target genes such as PSA (KLK3) are, however, efficiently shut off in basal epithelial prostate cells and possibly in prostate cancer stem cells (CSCs). There is limited information on the mechanisms that keep the AR OFF in these cells, and the conditions that turn the AR and target genes ON. Results: Fluorescent and luminescent reporters were constructed to develop a functional assay for AR activity. These reporters contain the repeated AR binding promoter sequences and were designed in a way that the AR can activate these AR element (ARE) reporters. The 241B reporter was selected among the constructed ARE reporters on the basis of higher activity. The AR positive prostate cancer cell line, LNCaP, was transduced with the lentiviral 241B-mCherry fluorescence reporter. FACS analysis quantitated the ARE reporter activity when reporter cells were treated with androgen and with the AR antagonist, enzalutamide, or the anti-androgen, abiraterone. AR expression vectors were also constructed with the AR open reading frames cloned into lentiviral expression vectors. All constructs were sequenced to verify the correct AR DNA sequence and their activity were tested using ARE reporter system. Conclusion: The developed ARE reporter system is useful to understand the mechanisms of AR and AR target gene activation in different subpopulations of prostate cancer cells. It will help us to investigate the role of the AR in differentiation and proliferation of prostate cells and to study the AR activity in two and three dimensional cell cultures. This system can also be useful in screening for drugs with activity against the AR. Citation Format: Waqas Azeem, Margrete R. Hellem, Jan R. Olsen, Yaping Hua, Kristo Marvyin, Lisha Li, Yi Qu, Biaoyang Lin, Xisong Ke, Anne M. Oyan, Karl-Henning Kalland. An androgen response element-based reporter assay for the detection of androgen receptor activity in prostate cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 79. doi:10.1158/1538-7445.AM2015-79

Abstract 1638: Effect of the phytochemical crinumaquine on prostate mesenchymal type cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In a prostate carcinogenesis cell culture model benign EP156T prostate epithelial cells underwent epithelial to mesenchymal transition (EMT), and a series of progeny EPT mesenchymal type cells accumulated malignant features in a stepwise fashion. Through the screening of phytochemicals using this model, we have found that crinumaquine inhibited proliferation of mesenchymal type EPT1 and EPT2 cells more efficiently than the compound inhibited epithelial type EP156T cells. Genome-wide gene expression analysis revealed a pattern where crinumaquine induced patterns associated with cell cycle arrest at both G1-S and G2-M phase checkpoints and strong up-regulation of genes involved in DNA repair and CDKN1A and CDKN1C and chromatin rearrangement. Differentially expressed genes were uploaded to the Connectivity Map tools (The Broad Institute, MA, USA) and the highest score was associated with histone deacetylase inhibitors. ChemMap analysis based on its chemical structure indicated that crinumaquine targets ER-β and cyclines. Crinumaquine was also found to induce up-regulation of CDH1 and other genes associated with an incomplete mesenchymal to epithelial transition (MET) of EPT1 and EPT2 cells, and this was associated with increased apoptosis markers in gene expression and Western blot analyses. Due to the selective inhibition of mesenchymal type and more malignant cells of the model, crinumaquine will be further explored concerning its anti-cancer leading compound potential. Citation Format: Kristo Marvyin, Run-hui Liu, Huizi Jin, Yaping Hua, Yi Qu, Xisong Ke, Karl-Henning Kalland, Wei-dong Zhang, Anne Margrete Oyan. Effect of the phytochemical crinumaquine on prostate mesenchymal type cells. [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 1638. doi:10.1158/1538-7445.AM2014-1638

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 304: Reactive oxygen species induce prostate cancer cells via activation of the IL6/STAT3 pathway.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Previously we have established a stepwise prostate carcinogenesis model including human primary prostate epithelial EP156T cells, non-malignant mesenchymal EPT1 cells and pre-malignant EPT2-D5 cells. Aims and methods: To further achieve malignant cells, EPT2-D5 cells were grown in protein-free medium and the tumorigenesis was tested in vivo. Results: 1) Lots of spheres were generated in monolayer culture of D5 cells in protein free medium. The sphere forming cells were named D5HS. 2) Different from EPT2-D5 cells, D5HS formed large subcutaneous tumors (EPT3) and metastasis (EPT3M) in SCID mice. 3) Gene expression profiling showed inflammation signatures in D5HS. 4) Cellular Reactive Oxygen Species Detection Assay (DCFDA) showed high reactive oxygen species (ROS) in D5HS, and ELISA and Western blotting showed high secreted IL6 and high STAT3 expression in D5HS. 5) Blocking of ROS or STAT3 signaling reduced the sphere formation. 6) Immunohistochemistry staining showed high pSTAT3 level in the EPT3 tumor. Conclusions: Reactive oxygen species induce prostate cancer cells via activation of the IL6/STAT3 pathway. Citation Format: Yi Qu, Runhui Liu, Jigang Zhang, Mihaela Popa, Yaping Hua, Biaoyang Lin, Emmet McCormack, Weidong Zhang, Anne Margrete Oyan, Karl-Henning Kalland, Xisong Ke. Reactive oxygen species induce prostate cancer cells via activation of the IL6/STAT3 pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 304. doi:10.1158/1538-7445.AM2013-304