Establishment of prostate cancer spheres from a prostate cancer cell line after phenethyl isothiocyanate treatment and discovery of androgen-dependent reversible differentiation between sphere and neuroendocrine cells

Abstract

Prostate cancer can transform from androgen-responsive to an androgen-independent phenotype. The mechanism responsible for the transformation remains unclear. We studied the effects of an epigenetic modulator, phenethyl isothiocyanate (PEITC), on the androgen-responsive LNCaP cells. After treatment with PEITC, floating spheres were formed with characteristics of prostate cancer stem cells (PCSC). These spheres were capable of self-renewal in media with and without androgen. They have been maintained in both types of media as long term cultures. Upon androgen deprivation, the adherent spheres differentiated to neuroendocrine cells (NEC) with decreased proliferation, expression of androgen receptor, and PSA. NEC reverse differentiated to spheres when androgen was replenished. The sphere cells expressed surface marker CD44 and had enhanced histone H3K4 acetylation, DNMT1 down-regulation and GSTP1 activation. We hypothesize that PEITC-mediated alteration in epigenomics of LNCaP cells may give rise to sphere cells, whereas reversible androgenomic alterations govern the shuttling between sphere PCSC and progeny NEC. Our findings identify unrecognized properties of prostate cancer sphere cells with multi-potential plasticity. This system will facilitate development of novel therapeutic agents and allow further exploration into epigenomics and androgenomics governing the transformation to hormone refractory prostate cancer.

Keywords: androgen; epigenome; prostate cancer; sphere; stem cells.

Figure 1. Structure of LNCaP prostate cancer spheres

A. Representative bright-field images of LNCaP cell spheres cultured in RPMI-1640 medium with 10% FBS. From left to right are floating 3D-sphere, 400x magnifications; fluorescent image of a GFP-labeled LNCaP cell sphere, 400x magnifications; and an adherent sphere, 200x magnifications. B. The left is an image of H & E stained paraffin embedded LNCaP cells; the center is a cross section of a sphere, paraffin embedded and H& E stained; and the right is cross section of a sphere, paraffin embedded and immuno-stained for Ki67 expression. The dark spots are the Ki67 positive cells. Images are 400x magnifications.

Figure 2. Growth and differentiation of LNCaP-derived sphere cells

A. Cell cycle analysis of sphere cells. LNCaP sphere cultures maintained in RPMI-1640 medium with 10% regular FBS (solid column) or with10% charcoal-stripped FBS (CSF) (open column) were analyzed for cell cycle phase distribution by flow cytometry. Vertical bars are mean ± SD of 3 separate experiments. B. Neuroendocrine cells developed from adherent sphere cells in medium with 10% CSF on chamber slides, Pap stained, 400 × magnifications. C. Western blots showing the expression of androgen receptor (AR), Sp1, and PSA in LNCaP cells (maintained in regular FBS medium), spheres (maintained in regular FBS medium), and neuroendocrine cells (maintained in CSF medium). β-actin was used as a loading control. D. Immunohistochemical staining of AR in paraffin embedded preparations. From left to right are LNCaP cells, spheres (regular FBS medium), and neuroendocrine cells (CSF medium), 400x magnifications.

Figure 3. Reversible differentiation between LNCaP spheres and neuroendocrine cells

A. Immunohistochemical staining of Ki67 expression in LNCaP cells (maintained in regular FBS medium), spheres (regular FBS medium), neuroendocrine cells (CSF medium), and spheres reverse differentiated from the neuroendocrine cells (maintained in regular FBS medium). Columns and vertical bars represent means ± SD of 4 independent experiments. B. Western blots for androgen receptor (AR) expression in the neuroendocrine cells (CSF medium), spheres reverse differentiated from the neuroendocrine cells (maintained in regular FBS medium), and spheres (maintained in regular FBS medium). β-actin was used as a loading control.

Figure 4. Phenotype and invasive activity of sphere and neuroendocrine cells

A. CD44 expression. This figure shows the proportion of CD44 positive cells among LNCaP cells maintained in regular FBS medium, sphere cells maintained in regular FBS medium, sphere cells maintained in CSF medium, and neuroendocrine cells maintained in CSF medium, as determined by a flow cytometric method. Columns and vertical bars represent the means ± SD of four independent experiments. B. Soft-agar gel colony formation assay showing number of colonies per plate of LNCaP cells (prepared in regular FBS medium), spheres (prepared in regular FBS medium), and neuroendocrine cells (in CSF medium). Columns and vertical bars are means ± SD of 4 separate experiments. * indicates statistically significant difference in colony numbers between LNCaP and neuroendocrine cells, P<0.05. C. Matrigel migration assay. This figure shows the number of invasive cells per membrane of LNCaP cells (maintained in regular FBS medium), spheres (regular FBS medium), and neuroendocrine cells (CSF medium) from the Matrigel chamber invasion assay. Chemoattractant used in the lower chamber: solid bar (■) indicates regular FBS; open bar (o) indicates charcoal-stripped FBS (CSF). Columns and vertical bars indicate the means ± SD of four separate experiments.

Figure 5. Epigenetic marks of LNCaP sphere cells

A. Immunoblotting showing the expression levels of DNMT1 (DNA methytransferase 1), GSTP1 (π-class glutathione S-transferase), and acetylated histone H3 lysine 4 (H3K4) in LNCaP cells and sphere cells maintained in regular FBS medium. β-actin was used as a loading control. B. Immunohistochemical staining of GSTP1 on paraffin embedded preparations of LNCaP cells (left), and sphere cells (right) maintained in regular FBS medium.

Figure 6. Hypothesis of a prostate cancer sphere model system

Androgen-dependent prostate cancer cells form spheres, with characteristics of prostate cancer stem cells (PCSC), due to epigenomic alterations mediated by an epigenetic modulator PEITC. This step is irreversible. PCSC retain tumorigenic potential and have dual proliferation capabilities allowing self-renewal in the presence and absence of androgen. In the absence of androgen (such as complete androgen blockade therapy/castration), PCSC cells are attenuated in proliferation and initiated differentiation process to neuroendocrine cells (NEC). The androgenome in the NEC cells are reprogramed in the presence of androgen, thereby the NEC cells can de-differentiate to spheres. Since LNCaP cells are epithelial in origin, this step may represent a new phenomenon, epithelial-to-neuroendorine and neuroendocrine-to-epithelial transition.