CD44-negative prostate cancer cells give rise to CD44 high cells that display phenotypical and functional stem-like traits

Hormonal therapy is effective for advanced prostate cancer (PCa) but the disease often recurs and becomes hormone-refractory. Neuroendocrine (NE) cells, expressing selectively the prostate cancer stem cell (CSC) marker CD44, may be resistant to androgen ablation and promote tumor recurrence. Our group demonstrated that the Toll-Like receptor (TLR) 3 agonist poly (I:C) induces apoptosis in the androgen-dependent prostate tumor cell line LNCaP, negative for NE and CD44 markers, while the androgen-independent PC3 and DU-145 cell lines are partially resistant and are positive for NE and CD44 markers. In order to investigate if the apoptosis resistance to poly (I:C) can be correlated to CD44 expression, the modulation of membrane-CD44 protein in PC3 cells after poly (I:C) treatment was assessed by flow cytometry. We found very small CD44-negative cells, resistant to poly (I:C), that gave rise to a CD44-positive population in PC3 cells after sorting. The same rate of conversion was observed in the 2-4% of sorted CD44-negative very small in size cells contained in parental PC3 and DU-145 cells. Moreover, clonogenic assay indicated that CD44-negative-sorted PC3 cells converted in CD44high cells, expanded in vitro culture, displayed an higher clonogenic potential than sorted CD44high -derived population producing mainly holoclones described to be enriched in stem-like cells, whereas CD44high population generated mainly meroclones, a mixture of cells of different proliferative potential. In addition, all the clones produced by CD44-negative cells became positive for CD44. Each individual clone was subjected to secondary clonogenic assay that showed a greater clonogenic potential in CD44-negative-derived clones compared to those generated by CD44high -sorted population. The cells generated by CD44-negative-sorted population were indistinguishable from the CD44high -derived cells in terms of CD44 expression, but CD44-negative population re-expressed specifically CD44 v8-v10 splice variant isoform in both the androgen-independent cell lines PC3 and DU-145. Because CD44v isoforms are more specifically expressed in CSCs than CD44s, the presence of a specific CD44v isoform might be better CSC marker than CD44s isoform. These results show that CD44high cells, described to be stem-like and more tumorigenic in prostate cancer, may arise from a CD44-negative population. Moreover CD44high -derived population contain a smaller proportion of self-renewing cells than CD44-negative-derived population.