Prostate cancer (PCa) is the most common male malignancy and the second most common cause of cancer-related deaths in the United Kingdom and the United States. It is a heterogeneous disease caused by different genetic mutations where the majority of prostate cancers are indolent and slow growing. However, some tumours are aggressive with the ability to invade local structures and metastasise, leading to poor clinical outcome. Radiation therapy (XRT) can be used to treat localized PCa, but 30-50% of patients undergoing XRT will experience disease recurrence within 10 years. Androgen Deprivation Therapy (ADT) has also demonstrated improved clinical success with further anti-tumour efficacy when combined with XRT, however half of patients develop a resistance to ADT caused by failure to completely block androgen receptor (AR) signaling by a number of mechanisms.
A novel, FDA-approved therapeutic to treat metastatic castration-resistant prostate cancer (mCRPC), Enzalutamide (ENZA) has recently been shown to enhance the effect of radiation therapy by impairing the DNA damage repair process. ENZA, an AR antagonist, blocks AR signaling at a number of locations in the pathway including androgen binding, nuclear AR translocation and DNA binding.
Ghashghaei et al., have previously demonstrated that ENZA with or without ADT enhances the effect of XRT in both LNCaP (hormone-sensitive) and C4‑2 (hormone-resistant) cell lines. To better understand the mechanism of action of ENZA on PCa cells, the authors sought to identify a radiation sensitive gene signature induced by ENZA in both LNCaP (AR-positive, hormone-sensitive PCa cells) and C4‑2 (AR-positive, hormone-resistant PCa cells).
Cells were treated with ENZA alone and in combination with ADT and XRT and profiled for changes in gene expression using the Affymetrix Human Clariom S arrays. Microarray data was background-corrected and normalized using “RMA” and differential expression (DE) analysed using the Bioconductor package “Limma”. They compared groups ENZA + XRT vs. XRT only, ADT + XRT vs. XRT, ENZA + ADT + XRT vs. XRT.
In non-irradiated experiments 5 genes were DE for all treatment groups (ENZA, ADT, ENZA + ADT) vs. controls in the LNCaP cells and 7 in the C4‑2 cell lines. Only 2 DE genes, CYP1A1 and CYP1A2 were common to both cell types.
In LNCaP cells, where radiation was included with the treatments, microarray analysis identified 13 DE genes vs. radiation only controls. LAT, CDH10, SLC39A5, PTPRN2 and OSBPL10 were upregulated and NKX3‑1, ZMIZ1, INSL6, LRFN1, SPDEF, TEX44, PDE9A and MMEL1 downregulated.
ZMIZ1 is a co-factor for the androgen receptor and has been demonstrated to enhance AR-mediated transcription. Previous studies have shown that ZMIZ1 inhibition resulted in reduced growth of a human PCa cell lines.
KEGG pathway analysis determined that LAT is involved in immune and inflammatory pathways and PTPRN2 and PDE9A genes are associated with diabetes pathways and purine metabolism respectively.
In C4‑2 cells 11 DE genes were identified in combined radiation treatments compared to radiation alone. CYP1A1, CYP1A2, FBXL5, SEMA6A and TIPARP were upregulated and TRIB2, CYP1B1, NKX3‑1, PFKFB3, PMEPA1 and C1orf116, however as CYP1A1, CYP1A2, and FBXL5 were identified in the DE without radiation treatment, these genes are unlikely to be involved in radiation sensitivity.
KEGG pathway analysis only identified pathways involving CYP1A1, CYP1A2 and CYP1B1, associated with metabolic pathway regulation.
Potentiation of radiation by ENZA or ENZA in combination with ADT is potentially mediated through immune/inflammatory pathways in LNCaP cells or metabolic pathways in C4‑2 cells.
To validate the microarray data authors performed qRT PCR analysis on PCa cultures treated with XRT and ENZA+/-ADT in combination with XRT. Quantification confirmed the differential expression of set of nine (9) genes that showed increased irradiation sensitivity induced by ENZA. The gene set included LAT, PTPRN2, NKX3‑1, PDE9A, ZMZI1, CYP1A1, CYPA2, TANK, and TRAF5.
Analysis of clinical significance was assessed using the publicly available Cancer Genome Atlas Prostate Adenocarcinoma (TCGA PRAD). A univariate Cox proportional hazards survival model suggested association between expression of the identified genes and time to recurrence. Patients with low expression of all the candidate genes, except for PTPRN2, were associated with lower disease free survival times whereas the 10-year survival rate was higher in patients with high expression levels.
In summary the present study revealed potential prognostic biomarkers for response to combined ENZA, ADT and XRT therapy. The biomarker signature predicted an enhanced radiation response to the combination therapy and genes identified were shown to be associated with improvement in recurrence-free survival in a cohort of prostate cancer patients. Further validation of the predictive potential of the identified gene markers in clinical trials is required to assess the long term survival and efficacy of combined ENZA, ADT and XTR treatment.