NF-κB gene signature predicts prostate cancer progression

Abstract

In many patients with prostate cancer, the cancer will be recurrent and eventually progress to lethal metastatic disease after primary treatment, such as surgery or radiation therapy. Therefore, it would be beneficial to better predict which patients with early-stage prostate cancer would progress or recur after primary definitive treatment. In addition, many studies indicate that activation of NF-κB signaling correlates with prostate cancer progression; however, the precise underlying mechanism is not fully understood. Our studies show that activation of NF-κB signaling via deletion of one allele of its inhibitor, IκBα, did not induce prostatic tumorigenesis in our mouse model. However, activation of NF-κB signaling did increase the rate of tumor progression in the Hi-Myc mouse prostate cancer model when compared with Hi-Myc alone. Using the nonmalignant NF-κB-activated androgen-depleted mouse prostate, a NF-κB-activated recurrence predictor 21 (NARP21) gene signature was generated. The NARP21 signature successfully predicted disease-specific survival and distant metastases-free survival in patients with prostate cancer. This transgenic mouse model-derived gene signature provides a useful and unique molecular profile for human prostate cancer prognosis, which could be used on a prostatic biopsy to predict indolent versus aggressive behavior of the cancer after surgery.

Figure 1. Activation of NF-κB signaling did not induce prostatic tumorigenesis but it did increase the rate of tumor progression in the Hi-Myc mouse PCa model

A) Continuous activation of NF-κB signaling induced prostate epithelial and stromal hyper-proliferation. Prostates from IκBα+/− and wild type mice were harvested at 3 and 6 months of age. Histological analysis was performed by H&E staining. B and C) Continuous activation of NF-κB signaling promotes PCa progression in the ARR₂PB-myc-PAI transgenic mouse. The prostates from Myc alone (ARR₂PB-myc-PAI) and Myc/IκBα bigeneic mice were harvested at 3 (B) and 6 (C) months of age. Histological analysis was performed by H&E staining. (DP: Dorsal Prostate; LP: Lateral Prostate; VP: Ventral Prostate; AP: Anterior Prostate).

Figure 2. The NARP21 gene signature predicts significant difference in the overall cancer-specific survival of PCa patients

(A) KM analyses were used to examine whether there was a significant association between overall cancer-specific survival prediction and the signature generated from NF-κB activated castrated mouse prostate (NARP21) or from the wild type castrated mouse prostate (AD228) (B). Two types of overall cancer-specific survival outcomes were compared in the plot: a poor-prognosis group (black dashed line) and a favorable-prognosis group (red solid line). The disease-specific survival (DSS) time in years is displayed on the X-axis, and the Y-axis shows the probability of overall cancer-specific survival. P value is by log-rank test.

Figure 3. The NARP21 gene signature predicts significant differences in the distant metastasis-free survival of PCa patients

(A and B) KM analyses were used to examine whether there was a significant association between distant metastasis-free survival and the signature generated from NF-κB activated castrated mouse prostate (NARP21) (A) or from the wild type castrated mouse prostate (AD228) (B). Two types of distant metastasis-free survival outcomes were compared in the plot: a poor-prognosis group (black dashed line) and a favorable-prognosis group (red solid line). The distant metastases-free survival (DMFS) time in years is displayed on the X-axis, and the Y-axis shows the probability of metastasis-free survival. P value is by log-rank test. (C) 47 out of 77 PCa patients who had lymph node metastasis at the time of RRP surgery progressed to systemic metastatic PCa eventually. The time of post-surgery is displayed on the X-axis, and the Y-axis shows the percentage of systemic metastasis from poor-prognosis and favorable-prognosis groups which predicted by the NARP21 gene signature at each time point, respectively. (D) KM plot for the systemic metastasis-free survival of PCa patients who had lymph node metastasis at the time of RRP surgery. Two types of distant metastases-free survival (DMFS) outcomes were compared in the plot: a poor-prognosis group (black dashed line) and a favorable-prognosis group (red solid line) stratified by the NARP21 gene signature gene expression profile. The distant metastases-free survival (DMFS) time in years is displayed on the X-axis, and the Y-axis shows the probability of systemic metastasis-free survival. P value is by log-rank test.

Figure 4. Molecular network analysis using Ingenuity Pathway Analysis (IPA)

Network analysis showed pathways associated with the NARP21 gene signature genes (21 genes) derived from NF-κB activated androgen depleted mouse prostate. The network was generated using the NARP21 gene set. A simplified diagram from IPA shows the key molecular pathways detected by the NARP21 gene signature genes (red). A full molecular network is presented in Supplementary Fig. 5.

Figure 5. Activation of NF-κB signaling correlates with increases in JNK phosphorylation (but not in p38MAPK) and decreases E-cadherin expression in PCa cells

NF-κB signaling was activated in LNCaP PCa cells by infecting with IKK2-EE retroviral vector, in which NF-κB activity was activated with a constitutively active (EE) mutants of IKK2 (28, 43); while NF-κB signaling was inactivated in C4-2B PCa cells by infecting with IKK2-KD retroviral vector, in which NF-κB activity was inhibited with a kinase dead (KD) IKK2 mutant (28, 43). The cells infected with empty vector were used as controls. Western blotting evaluating total and phosphorylated JNK (A) and p38 (B), and E-cadherin (C) levels in NF-κB activated (LNCaP-EE and C4-2B-EV) or inactivated (LNCaP-EV and C4-2B-KD) PCa cells.