Androgen upregulates the palmitoylation of eIF3L in human prostate LNCaP cells

Abstract

Background: Prostate cancer is the second leading cause of cancer-related deaths in Western countries. Most patients diagnosed with advanced prostate cancer can be treated with the main treatment: androgen deprivation therapy (ADT). The androgen receptor (AR) signaling axis plays a pivotal role in the progression of prostate cancer. However, most patients can ultimately progress to the castration-resistant prostate cancer (CRPC) stage within 2 years. At this stage, drugs targeting the AR signaling axis, including enzalutamide and abiraterone acetate, cannot prevent the progression of prostate cancer, thus predicting a poor prognosis. The molecular mechanism lies in the aberrant AR reactivation, which exhibits an adaptive response to ADT, such as the presence of AR splice variants. Thus, CRPC treatment remains a challenge. Purpose: In addition to the AR axis, a mechanism leading to this progression should be determined. The present study mainly compared palmitoylated proteins between androgen-treated LNCaP cells and non-treated LNCaP cells by palmitoylome profiling, to illustrate the changes at proteomic levels. Materials and methods: To screen the androgen-induced palmitoylated proteins, we conducted proteomic experiments using clickable palmitate probe (Alk-C16) between three individual pairs of androgen-treated and non-treated LNCaP cells. Results: We identified 4351 unique peptides corresponding to 835 proteins, among them a number of these identified proteins were palmitoylated proteins, particularly eIF3L. Androgen treatment significantly increased the palmitoylation level of eIF3L, an individual subunit of eIF3. As an initiation factor, eIF3L plays a pivotal role in the translation of mRNAs encoding growth-promoting proteins by enhancing translation rates, thus controlling cell proliferation. Conclusion: In this study, we demonstrated that the regulation of eIF3L palmitoylation may provide new directions for the therapy of prostate cancer. Moreover, the increased level of androgen-induced eIF3L may be used as a biomarker for the diagnosis of early-stage prostate cancer.

Keywords: androgen; biomarker; eIF3L; palmitoylation; prostate cancer.

Figure 1

(A) Structure of common protein fatty acylation. S-palmitoylation is the addition of the 16-carbon palmitate to a cysteine residue via a reversible thioester linkage. (B) Reversible S-palmitoylation. Palmitate is transferred from palmitoyl-CoA, which is produced by acyl-CoA synthetase, to a protein by protein acyl transferase. By contrast, palmitate on proteins is cleaved by palmitoyl-protein thioesterase.

Figure 2

Process of labeling cellular lipid-modified proteins with fatty acid analogs. Synthetic ω-alkynyl fatty acids are added to cultured cells and metabolically incorporated into acylated proteins. After workup, the alkynyl group is chemoselectively ligated to azide-tagged biotin or fluorophore by a Cu1-catalyzed alkyne-azide [3+2] cycloaddition reaction.

Figure 3

The mass spectrum results of eIF3L palmitoylation.

Figure 4

The amino acid sequences of eIF3L of Homosapiens (human). The partial highlighted red sequences of eIF3L are consistent with the mass spectrum results by comparing Figure 3 with Figure 4.

Figure 5

Regulation of androgen on the palmitoylation level of eIF3 subunits in LNCaP cells. The LNCaP cells labeled by Alk-C16 were treated with R1881 or DMSO as a control. The palmitoylated proteins were accumulated by click chemistry. They were identified by MS and quantified by label-free strategy. Among the six eIF3 subunits, the androgen-induced candidates were identified as LFQ activities were significantly up-regulated (fold changes>1.5, P<0.05) in triplicate samples of androgen-treated vs non-treated LNCaP cells. The P-values were analyzed using two-tailed Student’s-t distribution function. *P<0.05.

Figure 6

Androgen promotion of eIF3L palmitoylation by S-palmitoylation assay. The LNCaP cells were treated with R1881 or DMSO for 24 hrs and then the cells were harvested and performed by S-palmitoylated protein assay to test the palmitoylation level of eIF3L. Abbreviatons: HAM, hydroxylamine; Palm, palmitoylation; IB, immunoblot; IP, immunoprecipitation.

Figure 7

The palmitoylation level of eIF3L was downregulated with the increase of the concentration of abiraterone. The cultured LNCaP cells were treated with androgen R1881 and different concentrations of abiraterone (0.1 µmol/L or 0.5 mol/L), then the S-palmitoylated protein assay revealed that abiraterone could downregulate eIF3L palmitoylation level by inhibiting androgen.