While majority of the patients with metastatic CRPC benefit from enzalutamide treatment, the responders inevitably develop resistance. Hence, studies have been directed to investigate the potential mechanisms associated with the development of enzalutamide resistance. In the present study, we demonstrate that the downregulation of ISL1 serves as an important alternative therapy for CRPC treatment through the targeting of EMT via the negative regulation of the AKT/NF-κB signaling pathway.
ISL1 serves a major role in multiple tissue types, such as heart, kidneys, skeletal muscle, nervous system, and endocrine organs, and its upregulation is associated with cancer progression and poor prognosis14–17. Furthermore, ISL1 can influence the expression of genes related with EMT such as ZEB1 and N-cadherin15. We found that ISL1 was the most highly expressed EMT factor in enzalutamide-resistant cells (Fig. 4a). We analyzed the function of ISL1 in AR signaling in PCa cells and found that cell proliferation decreased and AR signaling was downregulated in ISL1 siRNA-expressing hormone-sensitive PCa cells (Fig. 5).
As epithelial plasticity driver, Snail (a master EMT-TF) is also known to promote the development of resistance against enzalutamide through the regulation of AR activity in PCa27. The loss of epithelial phenotypes, including spindle morphology and intercellular adhesion, and the acquisition of mesenchymal characteristics such as high migration and invasion capacities and reduced cell-extracellular adhesion are the two major events observed during EMT28. The expression of EMT marker genes was downregulated in ISL1-knockdown cells (Fig. 6b). Studies have demonstrated that EMT is associated with CRPC29,30. Sun et al.31 found that castration may induce EMT, as is evident from the decreased expression of epithelial markers (including E-cadherin) and increased levels of mesenchymal markers (including N-cadherin, Slug, Zeb1, and Twist1) in human LuCaP35 PCa xenograft tumors as well as in the normal mouse prostate tissue following androgen deprivation. Similar changes have also been reported in samples from individuals undergoing ADT31. EMT is driven by EMT inducing transcription factors (including Snail, Slug, Zeb1, Zeb2, and Twist), some of which have been known to be involved in the development of CRPC28. Shiota et al.32 found that castration-induced oxidative stress may promote AR overexpression through Twist1 overexpression, thereby possibly developing castration resistance. Furthermore, facilitation of castration resistance by Slug in PCa has been reported by Wu et al.33. Slug, another transcription factor driver of EMT, not only augments the expression of AR but also enhances AR transcriptional activity with or without androgen and acts as a novel coactivator for AR33. Overall, these aforementioned studies suggest that EMT is responsible for PCa progression and treatment resistance. Accordingly, treatment regimens that could reverse EMT phenotypes may become a viable alternative for CRPC therapy.
Aberrant activation of NF-κB signaling in PCa has been associated with metastatic progression34,35. In addition, NF-κB signaling plays an important role in EMT36. The knockdown of ISL1 resulted in reduced p65 phosphorylation (Fig. 6D), which is imperative for the nuclear translocation of NF-κB/p65. The NF-κB family, an important class of transcriptional regulators, comprises five members, including RelA (p65), RelB, c-Rel, p50/p105 (NF-κB1), and p52/p100 (NF-κB2). NF-κB binds to the inhibitor κB (IκB) protein in the cytoplasm in an inactive state. The IκB kinase (IKK) complex is activated under pathological conditions and subsequently induces the phosphorylation of IκB, leading to the degradation of IκB and translocation of NF-κB to the nucleus37. Increasing results indicate that the NF-κB transcription factor family is a crucial mediator of EMT38. Certain studies have shown that NF-κB binds to the promoters of genes associated with EMT, including those encoding Snail, Slug, and Twist, and increases their transcription38,39. Ozes et al.40 reported the involvement of AKT in the activation of NF-κB by mediating the phosphorylation of IKKA which is responsible for the activation of its downstream target IκB. In the current study, the knockdown of ISL1 resulted in the inhibition of the phosphorylation of both AKT and p65. These results show that ISL1 knockdown suppresses EMT by negatively regulating the AKT/NF-κB pathway. Furthermore, the AKT/NF-κB signaling pathway may drive the progression of CRPC by mechanisms other than EMT induction. Activation of NF-κB mediated by PI3K/AKT increases the expression of AR via NF-κB binding to the AR promoter41. CRPC, previously defined as hormone-refractory PCa, is thought to be androgen dependent42, indicating that targeting AR may serve as an effective strategy for CRPC treatment. The present data demonstrate that ISL1 suppressed the phosphorylation of AKT and p65; however, whether the effect of ISL1 on the AR signaling axis occurs through the regulation of the AKT/NF-κB pathway is unclear and warrants further examination. In addition, a subsequent study is needed to determine the genomic distribution of a critical EMT regulator in CRPC using chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq).
In conclusion, the present study identified that aberrant expression of ISL1 influenced enzalutamide resistance through EMT pathway. The strategy of inhibition of ISL1 holds great promise as a sensitizing strategy to restore the antitumor effects in enzalutamide-resistant cells. Targeting ISL1 may serve as an effective treatment strategy for patients resistant to enzalutamide.