Senescence-secreted factors activate Myc and sensitize pretransformed cells to TRAIL-induced apoptosis

Abstract

Senescent cells secrete a plethora of factors with potent paracrine signaling capacity. Strikingly, senescence, which acts as defense against cell transformation, exerts pro-tumorigenic activities through its secretome by promoting tumor-specific features, such as cellular proliferation, epithelial-mesenchymal transition and invasiveness. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has the unique activity of activating cell death exclusively in tumor cells. Given that the senescence-associated secretome (SAS) supports cell transformation, we asked whether SAS factor(s) would establish a program required for the acquisition of TRAIL sensitivity. We found that conditioned media from several types of senescent cells (CMS) efficiently sensitized pretransformed cells to TRAIL, while the same was not observed with normal or immortalized cells. Dynamic transcription profiling of CMS-exposed pretransformed cells indicated a paracrine autoregulatory loop of SAS factors and a dominant role of CMS-induced MYC. Sensitization to TRAIL coincided with and depended on MYC upregulation and massive changes in gene regulation. Senescent cell-induced MYC silenced its target gene CFLAR, encoding the apoptosis inhibitor FLIPL , thus leading to the acquisition of TRAIL sensitivity. Altogether, our results reveal that senescent cell-secreted factors exert a TRAIL-sensitizing effect on pretransformed cells by modulating the expression of MYC and CFLAR. Notably, CMS dose-dependent sensitization to TRAIL was observed with TRAIL-insensitive cancer cells and confirmed in co-culture experiments. Dissection and characterization of TRAIL-sensitizing CMS factors and the associated signaling pathway(s) will not only provide a mechanistic insight into the acquisition of TRAIL sensitivity but may lead to novel concepts for apoptogenic therapies of premalignant and TRAIL-resistant tumors.

Keywords: Myc; TRAIL; apoptosis; secretome; senescence; tumor.

Figure 1

Conditioned medium from senescent cells (CMS) specifically sensitizes pretransformed cells to TRAIL-induced apoptosis. (A) The stepwise tumorigenesis model systems comprise (top) normal human foreskin fibroblasts (BJ), immortalized (BJEH), pretransformed (BJEL), and transformed (BJELM) cells, obtained through sequential overexpression of h-TERT, SV40-ER, and Myc ER, and (bottom) normal human embryonic epithelial kidney cells (HEK), pretransformed (HA1E), and H-RasV12-transformed (HA1ER) cells. (B) TRAIL-induced apoptosis assays, cells of BJ system pretreated (+) or not (−) with conditioned medium from senescent cells (CMS) for 20 h, and then with 200 ng mL⁻¹ of TRAIL for 24 h. Apoptosis measured by FACS analysis, using 7A6 (APO2.7) antibody (C) TRAIL-induced apoptosis of BJEL cells treated with two types of CMS (oncogene-induced senescence-OIS and replicative senescence-RS) and their nonsenescent counterparts. OIS cells obtained by stably overexpressing Ras in WI-38 fibroblasts (‘WI-38 CMS-OIS’), nontransfected WI-38 cells (‘WI-38 CMY’) were used as their nonsenescent equivalent. Replicative senescence cells were obtained through serial passaging of BJ cells (‘BJ CMS-RS’), young proliferating BJ fibroblasts used as their nonsenescent counterpart (‘BJ CMY’). (D) TRAIL-induced apoptosis in pretransformed and transformed cells of the HEK stepwise tumorigenesis system. Pretransformed HA1E cells were pretreated (+) or not (−) with conditioned medium from senescent cells (CMS) and then with TRAIL as in (B).

Figure 2

Dynamic regulatory map of CMS-induced sensitization to TRAIL sensitivity in BJEL cells and its analysis. (A) DREM generated dynamic regulatory map, with six bifurcation points (BPs) in green and paths stemming from them. Microarray gene expression data are represented in log2 values, Myc association to a specific path is noted. (B) Heatmap representing hierarchical clustering, Euclidean distance, complete linkage. (C) Western blot analysis of protein extracts from BJEL cells preincubated with CMS for 0, 8, or 16 h and treated with TRAIL. Immunoblots carried using apoptosis antibodies: anti-PARP, anticaspase-8, anticaspase-9, and actin antibody as a loading control.

Figure 3

Myc and FLIP-L play a key role in sensitization to TRAIL-induced apoptosis by CMS. (A) Quantification of TRAIL-induced apoptosis in BJEL cells pretreated with siRNAs against Myc or siGFP as control for 24 h and then with CMS and TRAIL as usual. (B) Western blot analysis of total protein extracts from BJEL cells treated with CMS as specified using anti-Myc antibody and actin antibody as a loading control. (C) Western blot analysis of total protein extracts from BJEL cells treated with siRNAs against Myc or siGFP as a control for 24 h. Immunoblotting performed using anti-Myc antibody, anti-FLIP antibody, and actin antibody as a loading control. (D) Measurement of luciferase activity in BJEL cells transfected with FLIP-luc and Myc ER or FLIP-luc and pBabe. Cells were seeded in triplicates and treated with 4-OHT (Myc ER transfected cells, 10⁻⁶ m) or CMS for 5 and 20 h as indicated (pBabe-transfected cells). Luciferase activity, normalized to β-gal activity, is represented relative to pBabe at 0 h, which was given a value of 1. Statistically significant differences are shown (*P = 0.005 and **P < 0.0001). (E) Western blot analysis of total protein extracts from BJEL cells incubated with CMS for 20 h using anti-FLIP antibody and actin antibody as a loading control. (F) Western blot analysis of total protein extracts from BJEL cells transfected as in (C) and then treated with CMS for 20 h. All samples were collected together and immunoblotting was performed using anti-Myc antibody, anti-FLIP antibody, and actin antibody as a loading control. (G) TRAIL-induced apoptosis of BJEL cells transfected with siFLIP or siGFP as a control for 24 h and then treated with TRAIL as usual. (left panel). Western blot analysis of total protein extracts of siRNA-transfected BJEL cells at the moment of TRAIL treatment (right panel) using anti-FLIP antibody and actin as a loading control.

Figure 4

FLIP-L inhibits sensitivity of cells to TRAIL by blocking full cleavage of caspase-8 and formation of p-18 fragment. (A) Western blot analysis of total protein extracts from BJEL and BJELM cells treated with CMS as indicated and then with TRAIL for 3 h. (B) BJEL cells were pretreated with siMyc and siGFP as a control (left panel) or siFLAR and siGFP as a control and then treated with CMS as in A (right panel). Immunoblots were performed using anticaspase 8 antibody and actin antibody as a loading control. (C) Comparison of FLIP_L and MYC RNA and protein levels. Reverse transcription–PCR was performed on total RNA extracted from cells of the BJ system. CFLAR mRNA was normalized to GAPDH (upper left), MYC RNA to 36B4 (upper right). Western blot analysis of total protein extracts was performed with anti-FLIP_L or anti-MYC antibodies using actin as loading control (lower panel).

Figure 5

Sensitization to TRAIL in cancer cells and in co-culture conditions. (A) Generation of senescent and pretransformed cells from BJ cells, through genetic changes. Pretransformed (BJEL) cell, obtained by Hahn and Weinberg after introduction of hTERT and SV40EReg and senescent (BJ-S) cells are formed through H-Ras overexpression in BJ cells. Senescent (T47D-S) cells were obtained after doxorubicin treatment of TRAIL-unresponsive T47D cancer cells. (B) Comparison of young and senescent cells. Senescence-associated β-gal (SA β-Gal) staining of young (BJ, T47D) and their derivative senescent (BJ-S, T47D-S) cells (20×, upper left panel). Immunoblots with protein extracts from BJ and BJ-S cells with anti-H-Ras and actin antibodies (lower left panel). Reverse transcription–PCR was performed on RNA extracted from T47D and T47D-S cells. IL6 and IL8 mRNA were normalized to 36B4 (right panel). (C) Quantification of TRAIL-induced apoptosis in BJEL or T47D cells grown alone (/) or incubated with conditioned medium from appropriate young (BJ, T47D) or senescent (BJ-S, T47D-S) cell. (D) Co-culture of BJEL or T47D cells with BJ/T47D or BJ-S/T47D-S cells at increasing confluences, mean, and SEM of triplicates;**P < 0.005 and *P < 0.03.