TGFβ1 inhibition increases the radiosensitivity of breast cancer cells in vitro and promotes tumor control by radiation in vivo

Abstract

Purpose: To determine whether inhibition of TGFβ signaling prior to irradiation sensitizes human and murine cancer cells in vitro and in vivo.

Experimental design: TGFβ-mediated growth and Smad phosphorylation of MCF7, Hs578T, MDA-MB-231, and T47D human breast cancer cell lines were examined and correlated with clonogenic survival following graded radiation doses with and without pretreatment with LY364947, a small molecule inhibitor of the TGFβ type I receptor kinase. The DNA damage response was assessed in irradiated MDA-MB-231 cells pretreated with LY364947 in vitro and LY2109761, a pharmacokinetically stable inhibitor of TGFβ signaling, in vivo. The in vitro response of a syngeneic murine tumor, 4T1, was tested using a TGFβ neutralizing antibody, 1D11, with single or fractionated radiation doses in vivo.

Results: Human breast cancer cell lines pretreated with TGFβ small molecule inhibitor were radiosensitized, irrespective of sensitivity to TGFβ growth inhibition. Consistent with increased clonogenic cell death, radiation-induced phosphorylation of H2AX and p53 was significantly reduced in MDA-MB-231 triple-negative breast cancer cells when pretreated in vitro or in vivo with a TGFβ type I receptor kinase inhibitor. Moreover, TGFβ neutralizing antibodies increased radiation sensitivity, blocked γH2AX foci formation, and significantly increased tumor growth delay in 4T1 murine mammary tumors in response to single and fractionated radiation exposures.

Conclusion: These results show that TGFβ inhibition prior to radiation attenuated DNA damage responses, increased clonogenic cell death, and promoted tumor growth delay, and thus may be an effective adjunct in cancer radiotherapy.

Figure 1

TGFβ growth regulation and signaling in breast epithelial cell lines. A, the bar graphs show the growth response at 24 hours or 48 hours of TGFβ treatment in 5 breast epithelial cell lines. The untreated controls are designated as “UT” and “TGFβ” refers to TGFβ-treated cells. MCF10A cells show 14% (P = 0.38) and 35% (P = 0.02) growth inhibition at 24 hours and 48 hours of TGFβ treatment. Hs578T were growth inhibited by 9% (P = 0.36) at 24 hours and 22% (P = 0.004) at 48 hours of TGFβ treatment. Proliferation of MDA-MB-231 and MCF7 cells was unaffected by TGFβ whereas proliferation of T47D cells increased slightly. B, immunoblots of phospho-Smad2, total Smad2/3 and Actin from MCF7, T47D, Hs578t, MCF10A, and MDA-MB-231 cells treated with LY364947 for 48 hours followed by TGFβ for 30 minutes or sham treated. TGFβ treatment induced phosphorylation of Smad2, which was blocked by LY364947 pretreatment. These data indicate that TGFβ signaling through the type I receptor kinase is functional and LY364947 is effective in blocking the canonical pathway of TGFβ through TGFβRI. Quantifications of the ratios of phosphorylated protein/total protein normalized to untreated control are indicated below each lane.

Figure 2

TGFβ inhibition by small molecule inhibitor LY364947 radiosensitizes epithelial breast cell lines. Cells treated for 48 hours with 400 nmol/L of LY364947 prior to exposure to graded doses of radiation were plated 3-hour postirradiation for clonogenic survival assay. Closed circles (●) represent untreated irradiated controls whereas open circles (○) represent the colony forming efficiency of LY364947 treated, irradiated cells. Mean ± SD values of triplicate determinations are shown. LY364947 treatment significantly increases radiation sensitivity (ANOVA Tukey test): MCF10A, P = 0.013, Hs578T, P = 0.005, T47D, P = 0.01, and MCF7, P = 0.01.

Figure 3

TGFβ inhibition by LY364947 small molecule inhibitor radiosensitizes and attenuates DDR in MDA-MB-231 breast cancer cells in vitro. A, MDA-MB-231 cells treated for 48 hours with 400 nmol/L of LY364947 prior to exposure to graded doses of radiation were plated 3-hour postirradiation for clonogenic survival assay. Closed circles (●) represent untreated irradiated controls whereas open circles (○) represent the colony forming efficiency of LY364947 treated, irradiated cells. Mean ± SD values of triplicate determinations are shown for an assay representative of 3 experiments. LY364947 treatment significantly increases radiation sensitivity (ANOVA Tukey test, P = 0.023). B, MDA-MB-231 cells treated with LY364947 for 24 hours prior to irradiation with 5 Gy were subjected to immunoblot analysis to examine the effect of inhibiting TGFβ signaling on p53 phosphorylated at serine 15 and at serine 20. TGFβ inhibition prior to irradiation reduces p53 phosphorylation at both sites. The ratio of phosphorylated protein/total protein relative to untreated controls is indicated below each. C, induction of γH2AX foci in cultured MDA-MB-231 cells irradiated with 2 Gy was decreased by pretreatment with LY364947. Cells exhibit nuclear immunostaining of γH2AXfoci (green); nuclei are counterstained with DAPI (blue). D, γH2AXfoci (green) were reduced in MDA-MB-231 xenograft tumors from mice-treated LY2109761 (bottom) and irradiated with 3 Gy (right). Nuclei are counterstained with DAPI (blue).

Figure 4

TGFβ neutralizing antibody 1D11 radiosensitized murine mammary cancer cells in vitro. A, colony forming efficiency of cells treated for 48 hours with LY364947, prior to irradiation. Closed circles (●) represent control cells whereas open circles (○) represent the colony forming efficiency of LY364947 treated irradiated cells irradiated to the indicated dose. Radiation sensitivity was increased by LY364947 treatment (P = 0.024, ANOVA Tukey test). A representative experiment of 3 is shown. B, immunoblots of phospho-Smad2 and total Smad2/3 from 4T1 cells treated with LY364947 for 24 hours followed by TGFβ for 30 minutes or sham treated. The ratio of phosphorylated protein/total protein normalized to control is indicated below each. C, colony forming efficiency of cells treated for 24 hours with 1D11 or 13C4 antibody control, prior to irradiation. Closed circles (●) represent control antibody-treated cells whereas open circles (○) represent the colony forming efficiency of 1D11 TGFβ neutralizing antibody-treated irradiated cells irradiated to the indicated dose. Radiation sensitivity was increased by 1D11 treatment (P = 0.03, ANOVA Tukey test). A representative experiment of 3 is shown. D, immunoblots of phospho-Smad2 and total Smad2/3 from 4T1 cells treated with 1D11 for 24 hours followed by TGFβ for 30 minutes or sham treated. The ratio phosphorylated protein/total protein normalized to control is indicated below each. E, 4T1 cells treated with LY364947 for 48 hours prior to irradiation with 2 Gy were immunostained for γH2AX (green) and DAPI (blue). Radiation-induced γH2AX foci were decreased by LY364947TGFβ inhibition.

Figure 5

TGFβ neutralizing antibody 1D11 increases radiation-induced tumor delay growth in vivo. A, 4T1 tumors treated i.p. with 5 mg/kg 1D11 or 13C4 control antibody and irradiated at 8 Gy and harvested 1 hour later were immunostained for γH2AX (green) and DAPI (blue). γH2AX foci were decreased in tumors from mice treated with 1D11 (bottom) compared with the tumor treated with 13C4 (top). Low (left) and high (right) magnifications are shown. B, 4T1 tumor volumes from mice treated as above on day 13 with 13C4 control antibody (circles) or 1D11 TGFβ neutralizing antibody (squares) with (open symbols) and without (closed symbols) radiation (8 Gy) at day 14. 1D11 treatment increased the response to radiation (P = 0.01) but did not itself affect tumor growth rate (P = 0.77). C, individual tumor growth curves according to treatment. D, tumor growth delay compared with control was significantly increased by radiation and combination at day 23. n = 13 mice/group; *, <0.05; **, <0.01; ***, <0.001; NS, not significant.

Figure 6

TGFβ neutralizing antibody 1D11 treatment increases the response to multiple fractions of radiation in vivo. A, 4T1 tumor volume growth curves from mice treated i.p. on day 13 with 50 mg/kg 13C4 control antibody (circles) or 1D11 TGFβ neutralizing antibody (squares). Tumors were treated with 3 fractions of 12 Gy (open symbols) initiated at day 14 for 3 consecutive days. 1D11 and radiation decreased tumor growth rate compared with radiation and 13C4 (P < 0.05). 1D11 treatment alone did not significantly affect tumor growth rate (P = 0.16). B, individual tumor growth curves according to treatment. C, tumor weight at experiment termination was significantly decreased by the treatment combination. n = 5 mice/group; *, <0.05; **, <0.01; ***, <0.001; NS, not significant.