PTTG1 attenuates drug-induced cellular senescence

Abstract

As PTTG1 (pituitary tumor transforming gene) abundance correlates with adverse outcomes in cancer treatment, we determined mechanisms underlying this observation by assessing the role of PTTG1 in regulating cell response to anti-neoplastic drugs. HCT116 cells devoid of PTTG1 (PTTG1(-/-)) exhibited enhanced drug sensitivity as assessed by measuring BrdU incorporation in vitro. Apoptosis, mitosis catastrophe or DNA damage were not detected, but features of senescence were observed using low doses of doxorubicin and TSA. The number of drug-induced PTTG1(-/-) senescent cells increased ∼4 fold as compared to WT PTTG1-replete cells (p<0.001). p21, an important regulator of cell senescence, was induced ∼3 fold in HCT116 PTTG1(-/-) cells upon doxorubicin or Trichostatin A treatment. Binding of Sp1, p53 and p300 to the p21 promoter was enhanced in PTTG1(-/-) cells after treatment, suggesting transcriptional regulation of p21. p21 knock down abrogated the observed senescent effects of these drugs, indicating that PTTG1 likely suppresses p21 to regulate drug-induced senescence. PTTG1 also regulated SW620 colon cancer cells response to doxorubicin and TSA mediated by p21. Subcutaneously xenografted PTTG1(-/-) HCT116 cells developed smaller tumors and exhibited enhanced responses to doxorubicin. PTTG1(-/-) tumor tissue derived from excised tumors exhibited increased doxorubicin-induced senescence. As senescence is a determinant of cell responses to anti-neoplastic treatments, these findings suggest PTTG1 as a tumor cell marker to predict anti-neoplastic treatment outcomes.

Figure 1. HCT116 PTTG1^−/− cells are more sensitive to doxorubicin (Dox).

a) HCT116 WT and PTTG1^−/− cells expressing an empty vector (WT+EV and PTTG1^−/− +EV), and PTTG1 re-introduced PTTG1^−/− (PTTG1^−/− +PTTG1) cells were plated in 96-well plates and DNA synthesis assessed at different time points (time 0 represents 0 hours after cell plating); b) HCT116 WT and PTTG1^−/− cells expressing an empty vector (WT+EV and PTTG1^−/− +EV), and PTTG1 re-introduced PTTG1^−/− (PTTG1^−/− +PTTG1) cells were plated in 96-well plates for 24 hours and treated with control vehicle (C) or different doxorubicin doses for another 48 hours. DNA synthesis was assessed by measuring BrdU incorporation; c) HCT116 WT and PTTG1^−/− cells were plated for 24 hours and treated with control vehicle (C) or doxorubicin (0.02 uM) for another 48 hours, fixed, stained for β-gal activity and cells counted; d) HCT116 WT and PTTG1^−/− cells were plated for 24 hours and treated with control vehicle (C) or different doses of doxorubicin for another 48 hours, Tunel assay performed and positive cells counted. Each experiment was repeated three times. Results expressed as mean±SD, n = 3, *p<0.05, **p<0.001.

Figure 2. HCT116 PTTG1^−/− and SW620 PTTG1 knock down (PTTG1-kd) cells exhibit enhanced drug-induced senescence.

a) HCT116 WT, PTTG1^−/− and PTTG1 re-introduced PTTG1^−/− (PTTG1^−/− +PTTG1) cells were plated in 96-well plates for 24 hours and treated with control vehicle (C) or TSA for another 48 hours. DNA synthesis was assessed by measuring BrdU incorporation; b) HCT116 WT and PTTG1^−/− cells were plated for 24 hours and treated with control vehicle (C) or TSA (0.005 uM) for another 48 hours, fixed, stained for β-gal activity and cell number counted; c) SW620 WT, PTTG1 knock down (PTTG1-kd) cells and PTTG1-transfected (PTTG1-tf) cells were plated in 96-well plates for 24 hours and treated with control vehicle (C) or doxorubicin (Dox) for another 48 hours, DNA synthesis was assessed by measuring BrdU incorporation; d) SW620 WT, PTTG1 knock down (PTTG1-kd) cells and PTTG1 transfected (PTTG1-tf) cells were plated in 24-well plates for 24 hours, treated with control vehicle (C) or doxorubicin (0.02 uM) for another 48 hours, β-gal staining performed and positive cells counted. Each experiment was repeated three times. Results are expressed as mean±SD, n = 3, *p<0.05, **p<0.001.

Figure 3. PTTG1 down regulates p21 in colon cancer cells.

a) HCT116 WT and PTTG1^−/− cells were treated with control vehicle (C), doxorubicin (Dox, 0.02 uM) or TSA (0.005 uM), and proteins analyzed by Western blotting to assess histone H3 and H4 acetylation; b) PTTG1^−/− cells were transfected with scrambled siRNA (C), or siRNA targeting p21, p53 or Sp1 respectively and treated with doxorubicin (0.02 uM) for 48 hours. Protein levels were evaluated by Western blotting and senescence evaluated with β-gal staining; c) HCT116 or SW620 cells were transfected with empty vector or scrambled siRNA (C) or PTTG1 plasmids or siRNA as indicated, proteins were extracted and PTTG1 and p21 levels evaluated by Western blotting; d) WT and PTTG1^−/− cells were treated with control vehicle (C) or different doses of doxorubicin (0.005, 0.05, 0.5 and 5 uM) for 48 hours Sp1 and p53 levels analyzed by Western blotting. Each experiment was repeated three times. Results are expressed as mean±SD, n = 3, *p<0.05, **p<0.001.

Figure 4. Sp1 and p53 regulate p21 levels and promoter activities in HCT116 WT and PTTG1^−/− cells.

a) Primer pair 1 was designed to detect protein binding close to p53 motifs on p21 promoter. Primer pair 2 was used to detect protein binding close to Sp1 binding site on p21 promoter; b) WT and PTTG1^−/− cells treated with control vehicle (C), doxorubicin (Dox, 0.02 uM) or TSA (0.005 uM) for 48 hours and chromatin immunoprecipitation performed using p53 antibody. Enriched chromatin was analyzed by qPCR using primer pair 1; c) WT and PTTG1^−/− cells were treated with control vehicle (C), Dox (0.02 uM) or TSA (0.005 uM) for 48 hours and chromatin immunoprecipitation performed using Sp1 antibody. Enriched chromatins were analyzed by qPCR using primer pair 2; d) luciferase plasmids containing p21 promoters (with/out p53 binding motifs) were transfected into drug-treated HCT116 WT and PTTG1^−/− cells. Cells were harvested and luciferase activity analyzed. Each experiment was repeated three times. Results are expressed as mean±SD, n = 3, *p<0.05, **p<0.001.

Figure 5. Protein levels on p21 promoter after drug treatment.

a) WT and PTTG1^−/− cells were treated with control vehicle (C), 0.02 uM doxorubicin (Dox) or 0.005 uM TSA for 48 hours. Cells were harvested and chromatin immunoprecipitations performed using the indicated antibodies. Enriched chromatins were evaluated using qPCR and primer pair 1. Levels of p21 promoter binding were normalized to these of WT control HCT116 cells after doxorubicin or TSA treatment and are depicted as a) histone H3 acetylation; b) histone H4 acetylation; c) P300 binding; d) HDAC1 binding; e) PTTG1^−/− or p53^−/− cells were transfected with scramble (C) or p300 siRNA, and p21 promoter containing or devoid of the p53 binding site. Results are expressed as mean±SD, n = 3, *p<0.05, **p<0.001.

Figure 6. Xenografted PTTG1^−/− tumors are more sensitive to doxorubicin (Dox).

a) HCT116 WT and PTTG1^−/− HCT116 cells were implanted subcutaneously into nude mice and treated with control vehicle (C) or doxorubicin. Tumor dimensions were measured twice a week; b) twenty one days after tumor cell xenografts, mice were sacrificed, tumors separated and weighed; c) harvested tumor tissues were sectioned and stained with β-gal; d) tumor tissue was stained for p21 immunoreactivity; e) harvested tumor tissues were homogenized and Western blot performed. Results are expressed as mean±SD, n = 10, *p<0.05, **p<0.001.