PCSC Asymmetric Cell Division
MicroRNAs (miRNAs) are 20-22 nt, non-coding small RNAs that play fundamental roles in development and normal stem cell functions. Recent evidence suggests that miRNAs also play essential functions in regulating various CSCs, tumor development, and metastasis. With the emerging roles of miRNAs in regulating the properties of normal SCs, we rationalized that miRNAs may also be important regulators of PCSCs and PCa development/metastasis. We started testing this possibility early 2007 and we have recently generated strong preliminary evidence that certain miRNAs such as miR-34a indeed plays a critical role in regulating PCSC properties and PCa metastasis (Liu et al., 2011). We first purified human PCa cells from the LAPC9 (a bone marrow metastasis), LAPC4 (a lymph node metastasis), and Du145 (a brain metastasis) xenografts and determined the expression levels of a library of 324 sequence-validated human miRNAs using quantitative RT-PCR (qPCR). The library screening in unsorted cells revealed 137 miRNAs to be expressed at reliably detectable levels in the three PCa types. We then compared the expression levels of these 137 miRNAs in 6 purified marker-positive cell populations, i.e., the CD44+ cells from LAPC9, LAPC4, and Du145; the SP cells from LAPC9; the CD133+ cells from LAPC4; and the a 2b1+ cells from Du145, and compared with those in the corresponding marker-negative cell populations. The comparative qPCR analysis revealed miR-34a to be under-expressed in all marker-positive PCa cells. Subsequent work (Liu et al., 2011) demonstrated:
- In normal prostate epithelial and PCa cells, miR-34a expression levels correlate with the p53 status (i.e., cells with wt-p53 express higher levels of miR-34a than cells with mutant/null p53);
- miR-34a in bulk PCa cells inhibits proliferation and induces cell-cycle arrest and senescence;
- miR-34a overexpression in bulk PCa cells via either transfection of synthetic oligonucleotides (oligos) or infection with retroviral or lentiviral vectors encoding miR-34a, inhibits tumor regeneration;
- miR-34a re-expression in purified CD44+ PCa cells that express low levels of endogenous miR-34a abolishes tumor regeneration;
- In contrast, reducing miR-34a by an antagonist (i.e., anti-34a) in purified CD44- PCa cells promotes PCa regeneration and metastasis;
- Of clinical relevance, systemic delivery of miR-34a inhibits tumor regeneration and metastasis and extends the survival of PCa-bearing animals;
- miR-34a inhibits CSC properties such as clonal/clonogenic growth and serial prostasphere formation and tumor transplantation;
- Critically, miR-34a directly targets CD44 via two conserved miR-34a binding sites at its 3’-UTR;
- CD44 is a functional target of miR-34a and CD44 knockdown phenocopies the miR-34a effect in inhibiting tumor development & metastasis; and
- Finally, ‘rescue’ assays indicate that CD44, as a functional target of miR-34a, mainly regulates PCa cell motility and invasion.
Altogether, our studies (Liu et al., 2011) establish miR-34a as a critical negative regulator of PCSCs. At the same time, we are addressing whether prostate CSCs can undergo asymmetric cell division.