Xiaolan Fang

Novel 3D Co-Culture Model for Epithelial-Stromal Cells Interaction in Prostate Cancer

Paracrine function is a major mechanism of cell-cell communication within tissue microenvironment in normal development and disease. In vitro cell culture models simulating tissue or tumor microenvironment are necessary tools to delineate epithelial-stromal interactions including paracrine function, yet an ideal three-dimensional (3D) tumor model specifically studying paracrine function is currently lacking. In order to fill this void we developed a novel 3D co-culture model in double-layered alginate hydrogel microspheres, incorporating prostate cancer epithelial and stromal cells in separate compartments of the microspheres. The cells remained confined and viable within their respective spheres for over 30 days. As a proof of principle regarding paracrine function of the model, we measured shedded component of E-cadherin (sE-cad) in the conditioned media, a major membrane bound cell adhesive molecule that is highly dysregulated in cancers including prostate cancer. In addition to demonstrating that sE-cad can be reliably quantified in the conditioned media, the time course experiments also demonstrated that the amount of sE-cad is influenced by epithelial-stromal interaction. In conclusion, the study establishes a novel 3D in vitro co-culture model that can be used to study cell-cell paracrine interaction.

Novel In Vivo Model for Combinatorial Fluorescence Labeling in Mouse Prostate

The epithelial layer of prostate glands contains several types of cells, including luminal and basal cells. Yet there is paucity of animal models to study the cellular origin of normal or neoplastic development in the prostate to facilitate the treatment of heterogenous prostate diseases by targeting individual cell lineages.

Cell line modeling to study biomarker panel in prostate cancer.

African–American men with prostate cancer (PCa) present with higher‐grade and ‐stage tumors compared to Caucasians. While the disparity may result from multiple factors, a biological basis is often strongly suspected. Currently, few well‐characterized experimental model systems are available to study the biological basis of racial disparity in PCa. We report a validated in vitro cell line model system that could be used for the purpose.

Beta-catenin represses protein kinase D1 gene expression by non-canonical pathway through MYC/MAX transcription complex in prostate cancer

Down regulation of Protein Kinase D1 (PrKD1), a novel serine threonine kinase, in prostate, gastric, breast and colon cancers in humans leads to disease progression. While the down regulation of PrKD1 by DNA methylation in gastric cancer and by nuclear beta-catenin in colon cancer has been shown, the regulatory mechanisms in other cancers are unknown. Because we had demonstrated that PrKD1 is the only known kinase to phosphorylate threonine 120 (T120) of beta-catenin in prostate cancer resulting in increased nuclear beta-catenin, we explored the role of beta-catenin in gene regulation of PrKD1. An initial CHIP assay identified potential binding sites for beta-catenin in and downstream of PrKD1 promoter and sequencing confirmed recruitment of beta-catenin to a 166 base pairs sequence upstream of exon 2. Co-transfection studies with PrKD1-promoter-reporter suggested that beta-catenin represses PrKD1 promoter. Efforts to identify transcription factors that mediate the co-repressor effects of beta-catenin identified recruitment of both MYC and its obligate heterodimer MAX to the same binding site as beta-catenin on the PrKD1 promoter site. Moreover, treatment with MYC inhibitor rescued the co-repressor effect of beta-catenin on PrKD1 gene expression. Prostate specific knock out of PrKD1 in transgenic mice demonstrated increased nuclear expression of beta-catenin validating the in vitro studies. Functional studies showed that nuclear translocation of beta-catenin as a consequence of PrKD1 down regulation, increases AR transcriptional activity with attendant downstream effects on androgen responsive genes. In silico human gene expression analysis confirmed the down regulation of PrKD1 in metastatic prostate cancer correlated inversely with the expression of MAX, but not MYC, and positively with MXD1, a competing heterodimer of MAX, suggesting that the dimerization of MAX with either MYC or MXD1 regulates PrKD1 gene expression. The study has identified a novel auto-repressive loop that perpetuates PrKD1 down regulation through beta-catenin/MYC/MAX protein complex.

Abstract 4342: Differential expression of novel PKD1 related biomarker panel in African American men with prostate cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Prostate Cancer (PCa) exhibits striking racial disparity and is presented more aggressively in African Americans (AAs) than Caucasians (CAs). We have previously reported that the dysregulation of PKD1-centered signaling in primary AA prostate cancer cell line E006AA is akin to more aggressive Caucasian cell line C4-2. As PKD1 plays a central role in several cellular mechanisms that contribute in aggressive malignant phenotype, we defined a PKD1 related biomarker panel consisting of 11 biomarkers and explored whether PKD1 centered signaling would be differentially regulated in AA compared to CA PCa. Methods: PKD1 related biomarker panel consisted of PKD1, AR, HSP27, N-Cadherin, Snail, Vimentin, MMP-2, MMP-9, MT-1, E-cadherin, beta-Catenin. The validity of the PKD1 related biomarker panel in PCa was evaluated on available microarray databases (cbioportal.org). The differential expression of the biomarker panel was quantified on PCa tissue samples from15 AA and 15 CA men with matching Gleason score obtained from tumor tissue bank of Comprehensive Cancer Center of Wake Forest University. Results: The cbioportal consisted of six different PCa databases with approximately 662 human PCa samples in total. In average, PKD1 related biomarker panel was altered 47.7% in available cases. Quantification of the PKD1 related biomarker panel regarding gene expression demonstrated a trend toward higher expression of majority of biomarkers in AA samples in compare to CAs. However, only variances in expression of MMP2 (P<0.0001) and MT1A (P = 0.03) were significantly different between AAs and CAs. The results obtained from human PCa samples showed a moderate correlation with previously reported cell line data which showed significantly higher expression of EMT markers N-Cadherin, Vimentin and Snail in AA cell line in compare to CA cell lines. Conclusion: The differential expression of PKD1 related biomarker panel in AAs in compare to CAs suggests a biological basis for disparity in PCa. Besides, relatively high altered PKD1 related biomarker panel in available microarray databases suggests the potential of the panel as a tool in diagnosis of aggressive phenotype in PCa. Citation Format: Bita Nickkholgh, Xiaolan Fang, Shira M. Winters, Nora Fino, KC. Balaji. Differential expression of novel PKD1 related biomarker panel in African American men with prostate cancer. [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 4342. doi:10.1158/1538-7445.AM2015-4342

Abstract LB-190: Soluble E-Cadherin promotes cell invasion but not proliferation in prostate cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Metastasis is responsible for the majority of prostate cancer-related deaths. Despite progress in early detection and management, there remains no effective, long-term cure for metastatic prostate cancer, partially due to the unclear molecular mechanism of cancer cell invasion and metastasis. Clinical reports discovered that soluble E-cadherin (sE-Cad, the cleaved ectodomain of human E-Cadherin protein) was elevated in body fluids and serum of patients with a variety of cancers, and its serum levels have been shown to correlate positively with metastatic cancers and disease invasiveness. However, little is known about the role of sE-Cad in metastasis, or the molecular mechanism of its abnormal accumulation in cancerous microenvironment. Our lab generated C4-2 prostate cancer cell lines which would secret synthetic sE-Cad by Doxycycline induction. We found that increased sE-Cad in the conditioned medium promotes cell invasion, but not cell proliferation. We analyzed the protein subcellular localizations in different cell compartments (membrane, cytoskeleton, nucleus and cytoplasm) and observed increased binding of synthetic sE-Cad to membrane E-Cadherin, indicating that sE-Cad could promote cell motility by interacting with membrane proteins and possibly disrupting the cell-cell adhesion. This work is supported by Wake Forest University institutional funds to KC Balaji and by NIH grant CA079448 to X Fang. Citation Format: Xiaolan Fang, K. C. Balaji, Bita NickKholgh, Kenneth Gyabaah. Soluble E-Cadherin promotes cell invasion but not proliferation in prostate cancer. [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 LB-190. doi:10.1158/1538-7445.AM2014-LB-190

Abstract 4000: Biological basis of aggressive phenotype in African-American prostate cancer cell line

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: It is well established that African American men (AAM) with prostate cancer (PC) present more frequently with higher grade tumors compared to Caucasians. While the disparity is likely multifactorial, the biological basis is perhaps the strongest contender. Protein kinase D1 (PKD1) is a tumor and metastasis suppressor in PC. Down regulation of PKD1 was shown to increased cell proliferation, migration and metastasis in PC. Aim and experimental procedure: In order to address the fundamental question whether PC in AAM is inherently aggressive, we compared the proliferation, invasion and the expression of PKD1-centered signaling based 13-biomarker panel in primary and metastatic AAM cell lines (E006AA and MDApca2b) to benign (RWPE) and metastatic Caucasian cell lines (LNCaP and C4-2) using Western blot within different cell compartments. The results were further validated by RT-PCR and immunofluoroscent staining. Results: The proliferation, viability and invasion assays showed that E006AA cells proliferate and invade matrigel significantly more rapidly than metastatic LNCaP and MDAPca2b cells. The 13-biomarker panel analysis showed PKD1-centered dysregulaiton in AAM prostate cancer cell lines. Although E006AA is a primary PC cell line, PKD1 was down regulated similar to more aggressive C4-2 cells. Nuclear androgen receptor (AR), which shows active AR, was higher in LNCaP and MD2Pca2b; Vimentin showed higher expression in C4-2 and E006AA. Localization of E-Cadherin, N-Cadherin, T120 phosphorylated s- Catenin, MMP-2 and S11 phosphorylated snail in MD2Pca2b was similar to LNCaP cells but different to C4-2 and E006AA. Conclusion: Our Data demonstrates that the dysregulaiton of PKD1-centered signaling occurs in primary AAM prostate cancer cell line E006AA is akin to other metastatic PC cell lines suggesting an inherently aggressive nature of PC in AAM. Acknowledgment: Authors would like to thank Dr. Shahriar Koochekpour (Roswell park institute, University at Buffalo,State University of NewYork) for his kind gift E006AA cell line. Citation Format: Bita Nickkholgh, Xiaolan Fang, Kenneth Gyabaah, K.C. Balaji. Biological basis of aggressive phenotype in African-American prostate cancer cell line. [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 4000. doi:10.1158/1538-7445.AM2014-4000

Abstract 4078: Novel 3D co-cultural model for epithelial-stromal cells interaction in prostate cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Paracrine function is a major mechanism of cell-cell communication within tissue microenvironment in both normal development and disease. In vitro cell culture models simulating tissue or tumor microenvironment are necessary tools to delineate epithelial stromal interactions including paracrine function. Yet an ideal 3-dimensional (3D) tumor model specifically studying paracrine function is currently lacking. In order to fill this void we developed a novel 3D co-culture model, using double layered alginate hydrogel microspheres to incorporate prostate cancer epithelial and stromal cells. The cells remained confined and viable within their respective spheres for over 30 days. As a proof of principle regarding paracrine function of the model, we measured shedded component of E-cadherin (sE-cad) in the conditioned media, a major membrane bound cell adhesive molecule that is highly dysregulated in cancers including prostate. In addition to demonstrating that sE-cad can be reliably quantified in the conditioned media, the time course experiments also proved that the amount of sE-cad is influenced by epithelial stromal interaction. In conclusion, the study establishes a novel 3D in vitro co-culture model that can be used to study cell-cell paracrine interaction. Citation Format: Xiaolan Fang, Sittadjody Sivanandane, Kenneth Gyabaah, K.C. Balaji. Novel 3D co-cultural model for epithelial-stromal cells interaction in prostate cancer. [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 4078. doi:10.1158/1538-7445.AM2013-4078