Selective targeting of radiation-resistant tumor-initiating cells

Abstract

Tumor-initiating cells (TICs) have been shown both experimentally and clinically to be resistant to radiation and chemotherapy, potentially resulting in residual disease that can lead to recurrence. In this study, we demonstrate that TICs isolated from p53 null mouse mammary tumors repair DNA damage following in vivo ionizing radiation more efficiently than the bulk of the tumor cells. Down-regulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was observed both in fluorescence activated cell sorting (FACS)-isolated TICs as compared to non-TICs and in TIC-enriched mammospheres as compared to primary tumor cells depleted of TICs. This effect was accompanied by increased Akt signaling, as well as by the direct activation of the canonical Wnt/beta-catenin signaling pathway specifically within the TIC subpopulation by phosphorylation of beta-catenin on serine 552. Using limiting dilution transplantation performed on p53 null tumor cells transduced with Wnt reporter lentivirus, we demonstrated that FACS sorting of cells expressing TOP-eGFP resulted in a marked enrichment for TICs. Furthermore, FACS analysis demonstrated that cells with active Wnt signaling overlapped with the TIC subpopulation characterized previously using cell surface markers. Finally, pharmacological inhibition of the Akt pathway in both mammospheres and syngeneic mice bearing tumors was shown to inhibit canonical Wnt signaling as well as the repair of DNA damage selectively in TICs, sensitizing them to ionizing radiation treatment. Thus, these results suggest that pretreatment with Akt inhibitors before ionizing radiation treatment may be of potential therapeutic benefit to patients.

Fig. 1.

Altered DNA damage repair in TICs. (A) γ-H2AX and 53BP1 foci of individual subpopulations isolated by FACS immediately following IR (Insets, ≈5 h posttreatment) and isolated by FACS 48 h later (≈53 h posttreatment). γ-H2AX, red; 53BP1, green; and DAPI, blue. (Scale bar, 5 μm.) (B) Similar numbers of cells exhibit DNA damage following 6 Gy (□, ≈5 h posttreatment), whereas DNA damage was significantly reduced 48 h later (■, ≈53 h posttreatment) in TICs as compared to the majority of tumor cells (*, P < 0.03; **, P = 0.05). Two hundred to ∼500 cells from each subpopulation of cells were counted. Tumors T1, T6, and T7 were included in the study.

Fig. 2.

Akt and Wnt signaling pathways were activated in TICs. (A) qPCR analysis of PTEN, using tumors T6 and T7. The relative expression of TIC was designated as “1” to calculate the fold change. Three biological replicates were included in each sample (*, P < 0.01). , Lin⁻CD29^HCD24^H; ▪, Lin⁻CD29^HCD24^L; □, Lin⁻CD29^LCD24^H; ■, Lin⁻CD29^LCD24^L. (B) Western blot demonstrates activation of AKT and increased p-β-catenin^Ser552, a direct target of Akt activation, in freshly FACS isolated TICs, as well as in MSs enriched for TICs, as compared to non-TICs and the total tumor cells cultured on plastic (2D), which are depleted in TICs, respectively.

Fig. 3.

Canonical Wnt signaling marks TICs in the p53 null mammary tumors. (A) Totals of 6.1% (T1) and 2.7% (T6) of tumor cells show GFP expression following transduction with lentiviral TOP-eGFP compared to little to no expression in the FOP-eGFP-transduced control. (B) FACS analysis shows that the majority (90% in T1 and 70–75% in T6) of eGFP-positive cells are also CD29^HCD24^H (Left) and reciprocally most CD29^HCD24^Hcells are also eGFP positive (Right).

Fig. 4.

Akt inhibitor perifosine-sensitized radiation-resistant mammary tumorspheres enriched for TICs in vitro as well as in vivo. (A) Western blot showing decreased active β-catenin and active Akt during perifosine treatment of MSs enriched for TICs. Proteins were isolated from perifosine-treated secondary MSs from T1. (B) Treatment of secondary MSs with perifosine inhibited their self-renewal and sensitized them to IR treatment (*, P < 0.03; **, P < 0.01) from tumors T1 and T7. Three replicates from each tumor were included. (C) Perifosine alone and perifosine plus 6 Gy IR treatment reduce the percentage of the TICs as shown by FACS analysis using tumor T7, tumor T6, and TOP-eGFP-transduced tumor T1. Five independent tumors from both untreated and IR alone groups were included for each tumor type. Ten tumors were included for perifosine alone and perifosine plus IR groups for each tumor type (***, P < 0.002; Δ P < 0.05). (D) A similar level of DNA damage foci observed from various subpopulations isolated by FACS 48 h following irradiation (≈53 h posttreatment) in perifosine plus IR treated groups. After perifosine plus IR treatment and FACS sorting, cells were cytospun and stained for DNA damage foci. Fifty to ∼170 cells of each subpopulation were counted, and the percentage of cells with γ-H2AX or 53BP1 foci was calculated for each subpopulation. Tumors T1 and T7 were included in the study, and three replicates from each tumor were included. No significant differences were observed between TICs and the three subpopulations of cells (P > 0.05).