Prostate tumor cells with cancer progenitor properties have high telomerase activity and are rapidly killed by telomerase interference

Abstract

Background: Cancer progenitor cells (CPCs) have been postulated to promote treatment resistance and disease progression in prostate and other malignancies. We investigated whether the enzyme telomerase, which is active in cancer cells and in normal stem cells, plays an important role in CPC which can be exploited to neutralize these cells.

Methods: We used flow cytometry and assays of gene expression, clonogenicity, and invasiveness to isolate and characterize a putative CPC subpopulation from freshly resected human prostatectomy specimens. Telomerase activity was measured by qPCR-based Telomeric Repeat Amplification Protocol (TRAP). Telomerase interference was achieved by ectopic expression of a mutated telomerase RNA construct which reprograms telomerase to generate "toxic" uncapped telomeres. Treated cells were assayed for apoptosis, proliferation in culture, and xenograft tumor formation.

Results: CPC in prostate tumors expressed elevated levels of genes associated with a progenitor phenotype and were highly clonogenic and invasive. Significantly, CPC telomerase activity was 20- to 200-fold higher than in non-CPC from the same tumors, and CPC were exquisitely sensitive to telomerase interference which induced rapid apoptosis and growth inhibition. Similarly, induction of telomerase interference in highly tumorigenic CPC isolated from a prostate cancer cell line abrogated their ability to form tumor xenografts.

Conclusions: Human prostate tumors contain a CPC subpopulation with markedly elevated telomerase activity which renders them acutely susceptible to telomerase interference. These findings offer the first tumor-derived and in vivo evidence that telomerase may constitute a CPC "Achilles heel" which may ultimately form the basis for more effective new CPC-targeting therapies.

Figure 1. CPC phenotypes of cell subpopulations isolated from human prostate tumors

(A) Left: Gene expression (by RT-PCR) of cell subpopulations from 3 individual tumors (“+” = CPC, and “−” = non-CPC). Right: Semi-quantitative densitometric analysis of gel at left (mean values from 3 tumors). (B) Relative colony formation and (C) matrigel invasion of cell subpopulations from 8 individual tumors with sample micrographs (arrows on matrigel micrograph indicate cells that migrated across the membrane).

Figure 2. Telomerase and telomere characterization of CPC and non-CPC cell subpopulations isolated from human prostate tumors

(A) Telomerase activity of cell subpopulations from 6 individual tumors by qPCR-TRAP, normalized to a standard control telomerase activity from DU145 cancer cells (p=0.03). (B) Relative hTERT mRNA levels of cell subpopulations in 4 individual tumors (p=0.008). (C) Telomere lengths of cell subpopulations in 3 individual tumors, respectively. In 2A-B, “ND” = not detectable. In all panels, 1000 cells were used for each experiment, and assays were conducted in triplicate and compared using a two-sided Wilcoxon matched-pairs signed rank test of statistical significance.

Figure 3. Induction of telomerase interference (reprogramming of telomerase) in tumor-derived CPC

(A) MT-hTer expression by qPCR and (B) MT-telomerase activity levels by MT-specific qPCR-TRAP 48 hours after infection with lentivirus expressing MT-hTer/siRNA. (C) Percent apoptosis by TUNEL assay at day 4 after treatment with MT-hTer/siRNA or vector control. (D) Proliferation after treatment with MT-hTer/siRNA or vector control. Data in A-D reflects biological triplicate experiments conducted using CPC cells isolated from 1 patient tumor. All experiments were also repeated in triplicate using 2 additional patient tumors with similar results (data not shown).

Figure 4. CPC phenotypes of cell subpopulations isolated from DU145 human prostate cancer cell line

(A) Relative gene expression and (B) Colony formation (left), and invasiveness (right) of CPC and non-CPC subpopulations. (C) Relative telomerase activity (left) and hTERT mRNA levels (right), and bulk telomere lengths (D) of CPC and non-CPC subpopulations normalized to control standard (LNCaP cell line).

Figure 5. Effect of telomerase interference on in vivo tumor formation by DU145-derived CPC

(A) Relative growth inhibition of CPC and non-CPC subpopulations 6 days after treatment with MT-hTer/siRNA. (B) CPC were more tumorigenic than non-CPC, and tumor formation by CPC was abrogated by telomerase interference. (C) Mean tumor weights at excision on day 90 post inoculation (NT = no tumors).