Establishment and characterization of a highly tumorigenic African American prostate cancer cell line, E006AA-hT

Abstract

Genuine racial differences in prostate cancer (PCa) biology have been considered among the potential reasons to explain PCa disparities. There is no animal model to represent all aspects of human PCa and, more specifically, to be used for PCa disparity research. The lack of a spontaneously transformed in vitro cell-based model system has been a significant impediment to investigating and understanding potential molecular mechanisms, and the hormonal, genetic, and epigenetic factors underlying the biological and clinical aggressiveness of PCa in African American (AA) men. In this study, we established and characterized the E006AA-hT cell line as a highly tumorigenic subline of the previously characterized primary AA-PCa cell line, E006AA. Extensive characterization of the E006AA-hT cell line was accomplished using cytodifferentiation and prostate-specific markers, spectral karyotyping, cell line authentication assays, cell proliferation and migration assays, and in vitro tumorigenesis assays. Spectral karyotyping of E006AA-hT showed a hypertriploid chromosome complement and shared cytogenetic changes similar to its parental cells such as diploid X, absence of Y-chromosomes, numerical gains in chromosomes 5,6,8,10,17,20,21, and marker chromosomes of unknown origin. In addition, E006AA-hT also presented numerous clonal and structural aberrations such as insertion, deletion, duplication, and translocations in chromosomes 1-5, 8, 9, 11, 13, 14, 17, and 18. The E006AA-hT cell line was shown to be highly tumorigenic and produced tumors at an accelerated growth rate in both athymic nude and triple-deficient SCID mice. Silencing the mutated androgen receptor (AR-599 Ser>Gly) did not affect proliferation (loss-of-function), but decreased migration (gain-of-function) in E006AA-hT and its parental cell type. These data support that AR-point mutations may lead simultaneously to different "loss-of-function" and "gain-of-function" phenotypes in PCa cells. E006AA-Par and its subline as the only available spontaneously transformed low- and highly-tumorigenic primary AA-PCa cell lines could be used for basic and translational research aimed in supporting prostate cancer disparity research.

Keywords: African American; Androgen Receptor; E006AA; Prostate Cancer; tumorigenic..

Figure 1

Phase contrast photomicrograph and growth of E006AA-Par and E006AA-hT cell lines. (A) E006AA-Par (40 x). (B) E006AA-hT (40 x). Incets (100 x). (C) Growth of E006AA-Par and E006AA-hT in culture medium containing normal-FBS or CS-FBS. Cells were seeded at 1.0 x 10³ per well in 96-well plates and cell proliferation was measured by MTS assay. Each data point is the average of three independent experiments; bars, Means ± SEM. Comparisons between groups were performed using Tukey-Kramer adjusted t-test. Significant differences were observed between the two cell lines cultured under normal-FBS or CS-FBS (p < 0.001).

Figure 2

Spectral karyotyping of E006AA-hT cell line. A representative metaphase spread is analyzed from twenty cells and shown as an inverted DAPI-banded image at passage 5 (A), in SKY chromosome-specific classification colors (B), and after chromosomal classification (C). Note the loss of chromosome Y, duplication of the X-chromosome, trisomy of chromosomes 1, 2, 4, 7, 9, 10, 12, 14-19, and 22, and the other additional numerical gains and aberrant chromosomes.

Figure 3

Expression of cytodifferentiation markers. Western analysis was performed on whole cell lysate using antibodies against basal cell (cytokeratin 5), epithelial (cytokeratin-8, -18, and c-Met), and stromal (desmin, HGF, and α-SM-actin) markers. DU145 used as a positive and negative control for epithelial and stromal markers, respectively. PC-3 cell line was used as negative control for p53 and PTEN expression. PrSt, normal prostate stromal cells. GAPDH was used as protein loading control.

Figure 4

Expression of prostatic markers. Western analysis was performed on whole cell lysate as described in “Materials and Methods.” LNCaP and VCaP cell lines were used as positive controls and DU145 as negative control for PSA, AR, PSMA, and Nkx3.1. GAPDH was used as protein loading control.

Figure 5

E006AA-hT cells are tumorigenic in athymic nude mice and NOG-SCID mice. Subcutaneous injection of E006AA-hT cells led to continuously growing tumors in male nude (n = 9) and NOG-SCID (n = 9) mice. E006AA-hT cells were highly tumorigenic in NOG-SCID mice as compared with nude mice. Live photographs represent E006AA-hT tumor formation in athymic and NOG-SCID mice. There was significant difference in tumor growth rate between nude and NOG-SCID mice (P < 0.001). The overall association between tumor size and time was evaluated using a linear mixed model. For each mouse strain, the overall association between tumor volume and time is evaluated using F-tests, and between times comparisons are evaluated using the appropriate least-square means t-tests. Bars, Mean ± SEM.

Figure 6

Photomicrograph of E006AA-hT tumor xenografts in athymic nude and NOD-SCID mice. Hematoxylin and eosin staining was performed on formalin-fixed and paraffin embedded tissues sections. (A and B) Diffuse sheets of poorly differentiated carcinoma with cells showing high nucleus/cytoplasmic ratio and hyperchromatic and prominent nucleoli. There is inflammatory reaction to the tumor cells around small size vessels.(C and D) Diffuse sheets of poorly differentiated carcinoma approaching the bone and invading adipose tissue and skeletal muscle. (A and C) (100 x). (B and D) (200 x). Tu, tumor cells; V, vessels; LVI, lymphovascular invasion; Inf, inflammatory cells; MI, muscle invasion; B, bone; BM, bone marrow.

Figure 7

Effect of AR-silencing on growth, migration, and invasion of E006AA-Par and E006AA-hT cell lines. (A) Androgen receptor expression in cells transiently transfected with scrambled or AR-siRNA oligos. (B) Growth of transiently-transfected E006AA-Par and E006AA-hT cells. Twenty-four hours after transfection, cells were seeded at 1.0 x 10³ per well in 96-well plates and after three days proliferation were measured by MTS assay. Significant differences were only observed between the two cell lines (p < 0.001). Each data point is the average of three independent experiments; bars, Means ± SEM. Comparisons between groups were performed using Tukey-Kramer adjusted t-test. (C) In vitro migration and invasion assay. Transfected cells were subjected to migration and invasion assays as described in “Materials and Methods”. A representative photomicrograph from control- and AR-siRNA transfected cells is presented at 100× magnification using a phase-contrast microscope. (D) Cells migrated or invaded were counted from ten random fields. Each bar represented the mean ± SEM of three independent experiments, each performed in quadruplicate. The between group comparison of cell migration and invasion was evaluated using Tukey-Kramer adjusted t-tests. There was a significant difference between control- and AR-siRNA in E006AA-Par and E006AA-hT cell lines. There was also a significant difference between the two cell lines for control-siRNA and AR-siRNA.