Reprogramming murine telomerase rapidly inhibits the growth of mouse cancer cells in vitro and in vivo

Abstract

Telomerase plays a critical role in cancer, prompting the pursuit of various telomerase-based therapeutic strategies. One such strategy, telomerase interference, exploits the high telomerase activity in cancer cells and reprograms telomerase to encode "toxic" telomeres. To date, telomerase interference has been tested in human cancer cells xenografted into mice, an approach that does not recapitulate spontaneous malignancy and offers few insights about host toxicities, because human telomerase is targeted in a mouse host. To address these limitations, we designed and validated two new gene constructs specifically targeting mouse telomerase: mutant template mouse telomerase RNA (MT-mTer) and small interfering RNA against wild-type mouse telomerase RNA (α-mTer-siRNA). Using lentiviral delivery in mouse prostate cancer cells, we achieved α-mTer-siRNA-mediated knockdown of wild-type mTer (80% depletion) and concurrent overexpression of MT-mTer (50-fold). We showed that the two constructs effectively synergize to reprogram murine telomerase to add mutant instead of wild-type telomeric repeats, resulting in rapid telomeric uncapping (5-fold increase in DNA damage foci). This, in turn, led to rapid and significant apoptosis (>90% of cells) and growth inhibition in vitro (90% reduction in viable cell mass) and in vivo (75% reduction in tumor allograft wet weight). In summary, we have shown that mouse cancer cells are vulnerable to direct telomerase interference using novel murine telomerase-targeting constructs; this approach can now be used to study the true therapeutic potential of telomerase interference in mouse spontaneous cancer models.

Figure 1. α-mTer-siRNA knocks down WT-mTer and inhibits telomerase activity in E4 mouse prostate cancer cells

(A) α-mTer-siRNA structure and cloning into lentiviral vector system (target sequence is boxed, template region is underlined). (B) α-mTer-siRNA knocks down WT-mTer by 80% three days after expression in E4 cells, as quantified by real-time PCR (RT-PCR). (C) α-mTer-siRNA inhibits telomerase activity by 95% three days after expression in E4 cells, as quantified by real-time PCR telomeric repeat amplification protocol.

Figure 2. Mutant template telomerase RNA (MT-mTer) incorporates into active telomerase enzyme and reprograms it to add mutated telomeric repeats

(A) MT-mTer and MT-mTer/siRNA structure and cloning into lentiviral vector system. (B) Ectopic over-expression (50 fold) of MT-mTer in E4 mouse prostate cancer cells three days after lentiviral infection, as quantified by RT-PCR. (C) MT-mTer levels by PCR are unaffected by co-expresseed α-mTer-siRNA, which is designed to knock down only the endogenous wild type mTer. (D) Experiment scheme of modified RT-PCR TRAP assay designed to specifically detect addition of wild-type TTAGGG vs. mutated TTTGGG nucleotide repeats.

Figure 3. MT-mTer and MT-mTer/siRNA inhibit E4 mouse prostate cancer proliferation in vitro

Cell growth by MTS colorimetric cell viability assay is significantly inhibited by day 7 after expression of MT-mTer or MT-mTer/siRNA constructs (50% and 90% reduction, respectively).

Figure 4. MT-mTer/siRNA inhibits E4 tumor growth

(A) Left: Representative samples of MT-mTer/siRNA or vector control tumors. E4 cells were infected in vitro with lentivirus expressing active or control constructs and then allografted subcutaneously into NOD/SCID mice. Middle: Growth of tumors was inhibited by E4 cells infected by MT-mTer/siRNA lentivirus. Right: Wet weights of tumors resected 4 weeks after inoculation. (B) E4 cells (Lin-Sca-1+CD49f+) were sorted by FACSAria from freshly-resected, disaggregated and digested tumors (left). MT-mTer RNA levels from sorted E4 cells were quantified by RT-PCR (right). (C) Genomic mTer DNA levels from sorted E4 cells were quantified by PCR using the single copy gene 36B4 to control for DNA loading. (bottom). Representative bands from three different samples are shown.

Figure 5. MT-mTer/siRNA induces rapid apoptosis and DNA damage in E4 cells without altering bulk telomere length

(A) Left: TUNEL assay performed 4 days after lentiviral expression of MT-mTer/siRNA demonstrates brisk apoptotic cell death, 90.7% versus 1.1% with vector control by FACS analysis; shown are representative plots of three different assays with statistical significance (p < 0.01). Right: Caspase assay performed at the same day 4 time point reveals significantly increased caspase activity consistent with apoptosis in cells expressing MT-mTer/siRNA versus control. (B) Left: Quantitation of p53BP1 DNA damage foci in MT-mTer/siRNA or vector control infected cells. Cells were grown on glass cover slips, fixed and stained on day 4 post lentiviral infection, and foci were counted under 63X magnification using a Zeiss Imager.Z1. Right: Representative fluorescence micrographs depicting p53BP1 foci, TRF2, DAPI, and merge. Cells were grown, infected, fixed, and stained as before, then photographed at 63x magnification using a Zeiss LSM 510 confocal scope. MT-mTer/siRNA-infected cells appear to have more p53BP1 DNA damage foci than vector control-infected cells and more co-localization of p53BP1 DNA damage foci with telomeres (TRF2) than irradiated cells (co-localization indicated by arrows, right). (C) Cells infected with lentivirus expressing the various constructs were harvested 3 days after infection and assayed for bulk telomere lengths using RT-PCR (mean of triplicate experiments).