Dual Blockade of PKA and NF–κB Inhibits H2 Relaxin-Mediated Castrate-Resistant Growth of Prostate Cancer Sublines and Induces Apoptosis

Abstract

We previously demonstrated H2 relaxin (RLN2) facilitates castrate resistant (CR) growth of prostate cancer (CaP) cells through PI3K/Akt/β-catenin-mediated activation of the androgen receptor (AR) pathway. As inhibition of this pathway caused only ~50% reduction in CR growth, the goal of the current study was to identify additional RLN2-activated pathways that contribute to CR growth. Next-generation sequencing (NGS)-based transcriptome and gene ontology (GO) analyses comparing LNCaP stably transfected with RLN2 (LNCaP-RLN2) versus LNCaP-vector identified differential expression of genes associated with cell proliferation (12.7% of differentially expressed genes), including genes associated with the cAMP/PKA and NFκB pathways. Subsequent molecular analyses confirmed that the cAMP/PKA and NFκB pathways play a role in facilitating H2 relaxin-mediated CR growth of CaP cells. Inhibition of PKA attenuated RLN2-mediated AR activity, inhibited proliferation and caused a small but significant increase in apoptosis. Combined inhibition of the PKA and NFκB signaling pathways via inhibition of PKA and Akt induced significant apoptosis and dramatically reduced clonogenic potential, outperforming docetaxel, the standard of care treatment for CR CaP. Immunohistochemical (IHC) analysis of tissue microarrays (TMA) in combination with multispectral quantitative imaging comparing RLN2 levels in patients with BPH, PIN and CaP determined that RLN2 is significantly upregulated in CaP vs BPH (p=0.002). The combined data indicate RLN2 overexpression is frequent in CaP patients and provides a growth advantage to CaP cells. A near complete inhibition of RLN2-induced CR growth can be achieved by simultaneous blockade of both pathways.

Interaction between angiotensin II and relaxin 2 in the progress of growth and spread of prostate cancer cells

“…Furthermore PI3K/Akt, factors of the wnt pathway, can facilitate H2-relaxin-mediated activation of the AR pathway (23). Vinall et al (24) report that the cAMP/PKA and NF-κB pathways play an important role in facilitating the H2 relaxin-mediated castration resistant growth of prostate cancer cells.…”

A novel role and mechanism of cystic fibrosis transmembrane conductance regulator in bisphenol A‐induced prostate cancer

“…16 However, increased cAMP combined with inhibited androgen receptor could decrease the activity of PKA and then inhibit the apoptosis of prostate cancer cells. 17 AMPK is an important kinase maintaining cellular energy metabolism by sensing AMP/ ATP ratio. 11 In the presence of excessive loss of ATP, the production of cAMP from ATP depyrophosphate was inhibited, and then AMPK is activated by cytochrome C to regulate the expression of apoptosis-related factor Bax, Bim, and Bcl-2, and finally, caspase 9 and caspase 3 were increased to induce cell apoptosis.…”

Targeting the relaxin hormonal pathway in prostate cancer

“…Despite the immense body of research on relaxin's role in a variety of physiological functions and demonstrations of relaxin's effects on various tumor cell lines, the role of relaxin and relaxin‐like factors in human cancers has only started to emerge relatively recently . Initial observations revealed over‐expression of relaxin in neoplastic tissues of the mammary gland and the prostate . Relaxin has been implicated in the progression of a number of cancers, however, to date, a direct role for relaxin has been characterized only in the prostate.…”

“…H2 relaxin signalling in prostate cancer can drive androgen receptor signalling pathway activation via an intracellular mechanism ( via PI3K/Akt and Wnt pathways through the stabilization of cytoplasmic β‐catenin) that is independent of external stimulation by androgens . The dual inhibition of signaling downstream of RXFP1, specifically the cAMP/PKA/β‐catenin and the PI3K/Akt/NFκB pathways, was shown to inhibit the growth of castrate‐resistant tumors that is promoted by relaxin . Thus, the relaxin pathway is a critical signalling cascade, facilitating the growth of both androgen‐sensitive and androgen‐independent prostate cancer tumors, and blocking the relaxin pathway could counter tumor growth by a dual mechanism: ( i ) it would counteract the tumor growth promoting effects of relaxin, and ( ii ) prevent the crosstalk and activation of the AR pathway.…”