Studies On Tumorigenicity Of Drug-Tolerant Du145 Cells And Functional Roles Of CD44 And CD44~+ Cells In Tumor Development

Background:Cancer stem cells (CSCs) or tumor-initiating cells have been reported to be resistant to therapeutics and therapy-resistant cancer cells have been shown to manifest phenotypic properties of CSCs. We have shown that CD44+(α2β1hi) prostate cancer cell population harbors highly tumorigenic and metastatic stem/progenitor cells, but whether these stem-like cancer cells are more resistant to chemotherapy drugs and are functionally involved in prostate cancer development remains unanswered. To address these important questions, we conducted the study.Objectives:1. To understand whether stem-like prostate cancer cells are more resistant to chemotherapy drugs or whether chronic drugs treatment enrich stem-like prostate cancer cells.2. To determine the functional roles of CD44 and/or CD44+cells in prostate cancer development.3. To provide new sight on efficiently treating prostate cancer in clinical.Methods:1. We chronically treated Du145 prostate cancer cells, which have a subset of CD44+ cells, with two clinically relevant drugs, etoposide and paclitaxel, and three experimental drugs, i.e., staurosporine and two novel paclitaxel analogs WP1102 and WP1103. IC50 assay was used to determine whether the drug-tolerant Du145 cells were actually drug-resistant to selecting drugs and also cross-resistant to non-selecting drugs.2. The limiting-dilution tumor experiments in NOD/SCID mice by injecting subcutaneously (s.c) or orthotopically in the dorsal prostate (DP) were carried out to evaluate the tumorigenicity and metastaticity of drug-tolerant cells.3. Proliferation assay and BrdU incorporation assay were used to measure cell proliferation. The clonal assay was carried out to measure the ability of cells, at the single-cell level, to founder a self-renewing clone. Western blotting and immunofluorescence staining experiments were carried out to measure the protein expressions including P27, P21, BCL-2, CD44, etc.4. To prospectively determine whether CD44 is causally involved in prostate cancer cell tumorigenicity, we infected parental Du145 cells with a lentiviral vector encoding CD44 shRNA (CD44-shRNA) or a non-silencing shRNA control vector (NS-shRNA). Then these cells were implanted either subcutaneously or orthotopically in the DP.Results:Chronic sublethal drug treatment led to generation of drug-tolerant and cross-resistant cancer cells. Surprisingly, when the drug-tolerant cells were implanted, either subcutaneously or orthotopically, into NOD/SCID mice, they showed much reduced tumorigenicity or were even non-tumorigenic. Drug-tolerant DLD1 colon cancer cells and some UC14 bladder cancer cells selected by this protocol also displayed reduced tumorigenicity.Drug-tolerant Du145 cells demonstrated low proliferative and clonogenic potential. Consistent with reduced cell proliferation, drug-tolerant Du145 cells showed increased levels of two important cyclin-dependent kinase inhibitors, p21 and p27, especially in Dul45-Paclitaxel and Du145-WP1103 cells, the two cell lines that completely lacked tumorigenicity. The p27 levels were also elevated in all three other drug-tolerant Du145 cell lines. In contrast to p21 and p27, Bcl-2, an anti-apoptotic protein, showed less again in the two non-tumorigenic Du145 lines, i.e., Du145-Paclitaxel and Du145-WP1103.Western blotting and immunofluorescence staining experiments showed that both Du145-Paclitaxel and Du145-WP1103 cultures completely lacked CD44 and/or CD44+ cells. Du145-VP16 and Du145-STS cultures also showed reduced CD44 protein levels and/or numbers of CD44+ cells. By contrast, Du145-WP1102 cells, which retained some tumor-initiating capacity, showed similar levels of CD44 protein expression to and slightly more CD44+ (many faintly positive) cells than parental Du145 cultures. The extent to which CD44 protein and/or CD44+ cells were ablated in drug-tolerant Du145 cultures appeared to correlate well with the level of reduction in their tumorigenic potential. Prospective knockdown of CD44 in Du145 cells inhibited tumor regeneration. Conclusions:Altogether, our studies reveal that:1. Some drug-resistant cancer cells are not necessarily, and may actually be depleted of, CSCs;2. CD44 and/or CD44+ cancer cells play causal roles in tumor regeneration;3. In exploring the new drugs to treat prostate cancer, it is possible to choose CD44+ prostate cancer cells as therapeutic target, and especially, efficient delivery of drugs in vivo should be considered to improve anti-tumor effects. V...