Studies of the cytokine-induced regulation and the biological functions of Lipocalin-2 in human prostate cancer cell lines

  • Untersuchungen zur zytokin-induzierten Regulierung und den biologischen Funktionen von Lipocalin-2 in humanen Prostatakrebs-Zelllinien

Schröder, Sarah Katharina; Pradel, Gabriele (Thesis advisor); Weiskirchen, Ralf (Thesis advisor); Müller, Frank (Thesis advisor)

Aachen : RWTH Aachen University (2021)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021


Prostate cancer (PCa) is one of the most common and deadly cancers in men worldwide. An important factor for initiation and progression of PCa is a proinflammatory tumor microenvironment (TME). The tumor and the surrounding cells secrete diverse cytokines and other soluble mediators that affect tumorigenesis. One soluble mediator in the TME is the secreted transporter protein Lipocalin-2 (LCN2). In PCa patients, LCN2 levels were found to be increased and a correlation with unfavorable clinicopathological features has been described. Besides, LCN2 is known to affect different stages of tumor development in other cancers. Nevertheless, the precise mechanisms of LCN2 regulation and its exact role in PCa tumorigenesis are still poorly understood. The aim of the present thesis was to investigate whether LCN2 is regulated by proinflammatory cytokines in PCa to identify possible novel target structures for therapy. First, LCN2 expression was studied in the human metastatic PCa cell lines LNCaP and PC-3 via RT-qPCR and Western blot analysis. It was found that PC-3 cells express and secrete high quantities of LCN2, whereas LNCaP cells exhibit a significantly lower LCN2 expression. The cell lines were stimulated with proinflammatory cytokines to investigate LCN2 induction and to unravel activated signaling pathways. It was shown for the first time that LCN2 mRNA and its protein expression are strongly inducible by TNF-α in the highly metastatic PC-3, but not altered in LNCaP cells. Contrarily, stimulation with IL-1β significantly increased LCN2 levels in LNCaP cells only. p38, NF-κB or JNK pathways were activated shortly after cytokine treatment in both cell lines. Specific pharmacological inhibitors revealed NF-κB and JNK signaling axis to be responsible for TNF-α-mediated LCN2 induction in PC-3 cells. On the other hand, in LNCaP cells, IL-1β-induced upregulation of LCN2 was identified to be mediated by IκBζ stabilization and NF-κB induction. The second part of this thesis examined the function of LCN2 in metastatic PCa. For this purpose, LCN2 was transiently downregulated or stably depleted in PC-3 cells using siRNA or CRISPR/Cas9-mediated knockout strategy. Knockdown of LCN2 led to a distinct decrease of IL-1β. Interestingly, different stable single-cell derived LCN2-knockout (KO) cell lines exhibited a general reduction in cytokine expression. Conversely, re-expression or supplementation of LCN2 restored IL-1β expression in these cells. Moreover, LCN2-deficient cells showed further characteristics such as reduced proliferation and adhesion, as well as an increased unfolded protein response (UPR). Various UPR markers were activated in PC-3 and LCN2-KO cells by treatment with tunicamycin. However, LCN2-KO cells showed increased and lastingly UPR as they exhibited higher levels of p-NF κB and p-EIF2α compared to parenteral PC-3 cells. Collectively, the present thesis gained new insights into the interplay between cytokines and LCN2 in PCa. The data indicate that besides NF κB, JNK signaling axis is an additional target for approaches in therapy in PCa. The results of the generated LCN2 deficient cells hint towards various functions of LCN2 in PCa tumorigenesis, including a role in inflammation, adhesion and UPR. In addition, IL-1β was identified as a novel downstream target of LCN2 in PC-3 cells. The findings of the present thesis contribute to the understanding of the regulation and role of LCN2 in vitro and pave the way for developing animal models to study the tumorigenesis of PCa in vivo.