Glucocorticoid receptor over-expression promotes human small cell lung cancer apoptosis in vivo and thereby slows tumor growth

Abstract

Small cell lung cancer (SCLC) is an aggressive tumor, associated with ectopic ACTH syndrome. We have shown that SCLC cells are glucocorticoid receptor (GR) deficient, and that restoration of GR expression confers glucocorticoid sensitivity and induces apoptosis in vitro. To determine the effects of GR expression in vivo, we characterized a mouse SCLC xenograft model that secretes ACTH precursor peptides, and so drives high circulating corticosterone concentrations (analogous to the ectopic ACTH syndrome). Infection of SCLC xenografts with GR-expressing adenovirus significantly slowed tumor growth compared with control virus infection. Time to fourfold initial tumor volume increased from a median of 9 days to 16 days (P=0.05; n=7 per group). Post-mortem analysis of GR-expressing tumors revealed a threefold increase in apoptotic (TUNEL positive) cells (P<0.01). Infection with the GR-expressing adenovirus caused a significant reduction in Bcl-2 and Bcl-xL transcripts. Furthermore, in both the GR-expressing adenovirus-infected cells and tumors, a significant number of uninfected cells underwent apoptosis, supporting a bystander cell killing effect. Therefore, GR expression is pro-apoptotic for human SCLCs in vivo, as well as in vitro, suggesting that loss of GR confers a survival advantage to SCLCs.

Figure 1

Development of SCLC xenografts in nude mice and functional testing of adenoviral vectors expressing GR-eYFP or eYFP. (A) SCLC xenografts were created by the s.c. injection of DMS 79 cells in matrigel. The SCLC xenografts displayed pathology that was similar to that of human SCLCs with large areas of necrosis (yellow arrow) and oval nuclei with scant cytoplasm (white arrow). (B) In addition, these tumors secreted ACTH precursors into the circulation at a higher level than non-SCLC human colon carcinoma xenografts (HCT116 cells; n=3, *P<0.05). (C) Infection of A549 and DMS 79 cells with Ad-GR-eYFP resulted in a significant upregulation of GR expression when compared with GR expression in cells infected with the control virus, Ad-eYFP (MOI=50; *P<0.05). (D) DMS 79 cells were spinfected with Ad-eYFP or Ad-GR-eYFP, and after 72 h, the percentage of infected (eYFP +ve) cells in each population was determined by FACS.

Figure 2

Ad-GR-eYFP infection of established SCLC xenografts resulted in apoptosis. (A) SCLC xenografts were injected with Ad-eYFP or Ad-GR-eYFP when the tumors reached 180–200 mm³ in size (n=6 per group). After 72 h, the tumors were harvested. Cryosections were stained with anti-GFP (Invitrogen) to detect the presence of virus and with TUNEL-TMR (red; Roche) to detect apoptotic cells and were examined by confocal microscopy. No GFP or TUNEL staining was seen in tumors injected with PBS. GFP staining revealed cells infected with Ad-eYFP or Ad-GR-eYFP (white arrows), while apoptotic cells were revealed by TUNEL staining (*). Co-staining with GFP and TUNEL was clearly seen in Ad-GR-eYFP-infected cells only (yellow arrows). The two Ad-GR-eYFP panels represent two different fields for the same experimental conditions. (B) The total GFP and TUNEL fluorescence of ten different sections for each treatment was quantified using Image J software and is displayed as a ratio of TUNEL/GFP. Cells infected with Ad-GR-eYFP displayed significantly more TUNEL staining than cells infected with the control virus, Ad-eYFP (*P<0.05). (C) The percentage of eYFP-positive cells remaining in the tumors after 72 h was determined by real-time quantitative PCR and compared with eYFP expression in HEK cells, where 100% of the cells express eYFP.

Figure 3

Infection of tumor cells with Ad-GR-eYFP caused growth delay. (A) When SCLC xenografts reached 180–200 mm³ in size, the tumors were injected with either Ad-eYFP or Ad-GR-eYFP, three times at 4-day intervals (n=7 per treatment). Tumor growth was measured daily using calipers and the tumors were excised when the relative tumor volume reached four times the initial injection size. (B) Restored GR expression significantly (P=0.05) slowed tumor growth to four times the initial treatment size by 1 week. (C) To determine the number of infected cells per tumor, qPCR was used to determine the number of eYFP transcripts from three tumors. The percentage of eYFP-positive cells in the tumors at harvest was compared with eYFP expression in HEK cells, where 100% of the cells express eYFP relative to GAPDH expression. Growth delay caused by Ad-GR-eYFP is mediated by increased apoptosis. (D–F) Cryosections were stained with anti-GFP (Invitrogen) to detect the presence of virus and with TUNEL-TMR (red; Roche) to detect apoptotic cells and were examined by confocal microscopy. Although Ad-eYFP was effective in infecting tumor cells (white arrows) (D), few TUNEL-positive tumor cells were seen in the representative sections shown. However, tumor cells infected with Ad-GR-eYFP also stained positive with TUNEL (E) (yellow arrows). (F) The total GFP and TUNEL fluorescence of ten different sections for each treatment was quantified using Image J software and is displayed as a ratio of TUNEL/GFP. Cells infected with Ad-GR-eYFP displayed significantly more TUNEL staining than cells infected with the control virus, Ad-eYFP (*P<0.05).

Figure 4

Apoptosis in DMS 79 cells and xenografts infected with Ad-GR-eYFP. In vitro: DMS 79 cells were infected with adenovirus. After 72 h, the cells were stained with PI and Annexin V and analyzed by flow cytometry. There is a clear shift in the Ad-GR-eYFP-infected population toward a dead or dying state (yellow arrow) (B) compared with the control virus-infected cells (A). There is also a similar shift in non-GR-eYFP-expressing cells (black arrow) (B). Ad-eYFP serves as a control where no such shift is seen (A). The proportion of uninfected (eYFP −ve) cells, drawn from the same populations described above, which are PI +ve (C) and AnnV +ve is shown (D). In C and D, a mean (±s.d.) of three separate experiments is shown (*P<0.05, compared with eYFP control). In vivo: tumor cryosections were stained with anti-GFP (Invitrogen) to detect the presence of virus and with TUNEL-TMR (red; Roche) to detect apoptotic cells and were examined by confocal microscopy. Ad-eYFP- and Ad-GR-eYFP-infected cells can be seen clearly (white arrows, E and F respectively). Little or no TUNEL staining is seen in the Ad-eYFP-injected xenograft. However, TUNEL staining is clearly visible in non-GR-eYFP-expressing cells in the Ad-GR-eYFP-infected tumor (yellow arrows, F). (G) The amount of TUNEL fluorescence in ten different sections from three different Ad-eYFP and Ad-GR-eYFP was quantified using Image J software (*P<0.05).

Figure 5

Expression levels of pro-survival and pro-apoptotic genes in tumors injected with Ad-eYFP and Ad-GR-eYFP. Seventy-two hours post infection, RNA was extracted from DMS 79, A549, and HEK 293 cells spinfected or infected with Ad-GR-eYFP or Ad-eYFP viruses as well as from tumors injected with Ad-eYFP or Ad-GR-eYFP. cDNA was synthesized and subjected to real-time quantitative PCR. The relative expression levels of Bad (A), Bim (B), Bax (C), Bcl-2 (D), and Bcl-xL (E) are shown. Data were analyzed using the 2^−ΔΔC_t method relative to GAPDH expression (*P<0.05; comparisons are shown with horizontal bars; n=3).