PTEN deficiency promotes macrophage infiltration and hypersensitivity of prostate cancer to IAP antagonist/radiation combination therapy

Abstract

PTEN loss is prognostic for patient relapse post-radiotherapy in prostate cancer (CaP). Infiltration of tumor-associated macrophages (TAMs) is associated with reduced disease-free survival following radical prostatectomy. However, the association between PTEN loss, TAM infiltration and radiotherapy response of CaP cells remains to be evaluated. Immunohistochemical and molecular analysis of surgically-resected Gleason 7 tumors confirmed that PTEN loss correlated with increased CXCL8 expression and macrophage infiltration. However PTEN status had no discernable correlation with expression of other inflammatory markers by CaP cells, including TNF-α. In vitro, exposure to conditioned media harvested from irradiated PTEN null CaP cells induced chemotaxis of macrophage-like THP-1 cells, a response partially attenuated by CXCL8 inhibition. Co-culture with THP-1 cells resulted in a modest reduction in the radio-sensitivity of DU145 cells. Cytokine profiling revealed constitutive secretion of TNF-α from CaP cells irrespective of PTEN status and IR-induced TNF-α secretion from THP-1 cells. THP-1-derived TNF-α increased NFκB pro-survival activity and elevated expression of anti-apoptotic proteins including cellular inhibitor of apoptosis protein-1 (cIAP-1) in CaP cells, which could be attenuated by pre-treatment with a TNF-α neutralizing antibody. Treatment with a novel IAP antagonist, AT-IAP, decreased basal and TNF-α-induced cIAP-1 expression in CaP cells, switched TNF-α signaling from pro-survival to pro-apoptotic and increased radiation sensitivity of CaP cells in co-culture with THP-1 cells. We conclude that targeting cIAP-1 can overcome apoptosis resistance of CaP cells and is an ideal approach to exploit high TNF-α signals within the TAM-rich microenvironment of PTEN-deficient CaP cells to enhance response to radiotherapy.

Keywords: IAP; PTEN; microenvironment; prostate cancer; radiation.

Figure 1. Comparative analysis of PTEN-status and cytokine expression in prostate cancer patient samples

(A) Scatter plot showing validation of PTEN-status profiling in prostate cancer patient samples. The data presented confirms loss of PTEN mRNA expression following cohort separation by RT-PCR. (B) Scatter plot showing CXCL8 gene expression in prostate cancer patient samples separated by PTEN mRNA status. (C) Scatter plot showing IL-6 gene expression in prostate cancer patient samples separated by PTEN mRNA status. Statistically significant differences were determined using the Spearman correlation protocol (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 2. CXCL8 induces chemotaxis of radioresistance-promoting macrophages in a PTEN-deficient setting

(A) Immunohistochemical staining of PTEN and CD68 in a prostate tissue microarray (n = 70). Presented images representative of results across all cases. (B) Bar graph demonstrating the correlation between PTEN status and CD68. Statistical analysis was performed using a Chi-squared test; p = 0.011. (C) Bar graph showing the effect of 3 nM CXCL8 in modulating cell migration of THP-1 cells. (D) Bar graph demonstrating the effect of conditioned serum-free media from irradiated PTEN-expressing DU145 NT01 and PTEN-depleted DU145 Sh11.02 cells on THP-1 cell migration. The addition of a CXCL8 neutralizing antibody represses IR-induced cell migration. Clonogenic survival curves showing the effect of THP-1 co-culture on the radiation response of (E) NT01 and (F) Sh11.02 cells. Data shown is the mean plus or minus standard error of the mean value, calculated from a minimum of three independent experiments. Statistically significant differences were determined by performing a two-tailed Students t-test for migration experiments or two-way ANOVA for clonogenic assays (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 3. Impact of ionizing radiation and PTEN-status on TNF-α signaling in prostate cancer and THP-1 cell lines

(A) Scatter plot illustrating TNF-α gene expression in prostate cancer patient samples classified by PTEN mRNA levels. (B) Bar graph showing TNF-α secretion from PTEN-depleted Sh11.02 cells following treatment with a single 3 Gy dose of IR. (C) Immunoblot demonstrating the impact of 3 Gy IR on TNFR-1 expression in Sh11.02 cells. (D) Bar graph showing TNF-α secretion levels from THP-1 cells following treatment with 3 Gy IR. (E) Bar graph showing TNF-α secretion levels from THP-1 cells following treatment with 3 nM CXCL8. Data shown is the mean plus or minus standard error of the mean value, calculated from a minimum of three independent experiments. Statistically significant differences were determined by performing a two-tailed Students t-test (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 4. Impact of radiation-induced THP-1 derived TNF-α on NFκB pro-survival signaling

(A) Bar graph illustrating luciferase reporter assay analysis of NFκB activity in Sh11.02 cells. Different experimental conditions included THP-1 co-culture, exposure to 3 Gy IR and treatment with a TNF-α neutralizing antibody (10 ng/ml). (B) Immunoblot showing basal expression of NFκB-regulated anti-apoptotic targets in PTEN-modulated DU145 populations. (C) Immunoblot showing the effect of 10 ng/ml recombinant TNF-α treatment on expression of NFκB-regulated anti-apoptotic targets in Sh11.02 cells. (D) Immunoblot illustrating expression of NFκB targets in Sh11.02 cells 4 h following 3 Gy IR in the presence or absence of THP-1 co-culture and TNF-α neutralizing antibody. Data shown is the mean plus or minus standard error of the mean value, calculated from a minimum of three independent experiments. Statistically significant differences in luciferase assay results were determined by performing a two-tailed Students t-test (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 5. Impact of AT-IAP on cIAP-1 expression and DU145 Sh11.02 cell viability

(A) Immunoblot showing cIAP-1 expression in Sh11.02 cells following treatment with 0.1, 1 or 10 μM AT-IAP. Equal protein loading was confirmed by re-probing for GAPDH. (B) Bar graph presenting MTT assay analysis of Sh11.02 cells 72 h following treatment with 10 ng/ml rhTNF-α, 1 μM AT-IAP, or a combination of both. (C) Bar graph illustrating flow cytometry data following Annexin V/PI staining of Sh11.02 cells. Different treatment conditions were similar to those mentioned above. (D) Bar graph showing caspase 3/7 activity of Sh11.02 cells following treatment with rhTNF-α, AT-IAP or both in combination for 24 h. Data shown is the mean plus or minus standard error of the mean value, calculated from a minimum of three independent experiments. Statistically significant differences were determined by performing a two-tailed Students t-test (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 6. Assessment of the radiosensitizing potential of AT-IAP in Sh11.02 cells

(A) Immunoblot showing cIAP-1 expression in Sh11.02 cells following THP-1 co-culture, 3 Gy IR and treatment with 0.1 μM AT-IAP. Equal protein loading was confirmed by re-probing for GAPDH. (B) Bar graph showing caspase 3/7 activity in Sh11.02 cells following THP-1 co-culture, 3 Gy IR and treatment with 0.1 μM AT-IAP for 6 h. (C) Clonogenic survival curve showing the radiosensitizing potential of AT-IAP on Sh11.02 cells with THP-1 co-culture. Data showing addition of a TNF-α neutralizing antibody in this system is also presented. Data shown is the mean plus or minus standard error of the mean value, calculated from a minimum of three independent experiments. Statistically significant differences were determined by performing a two-tailed Students t-test or two-way ANOVA for clonogenic assays (*p < 0.05; **p < 0.01; ***p < 0.001).