Late stage prostate cancer (CAP) tumors are resistant to anticancer therapies that cause cell death through the induction of apoptosis. To understand how CaP cells become resistant to apoptotic stimuli, it is important to characterize the molecular pathways that control apoptosis. This proposal focuses on the requirement of the transcription factor NF-kappaB for cell survival in CaP cells in response to interleukin-1 (IL-1)beta-dependent Ca 2+ signaling and apoptosis. Preliminary data presented in this proposal demonstrates that IL-1beta-induced NF-kappaB activity will overcome growth factor induced apoptosis in CaP cells. However, IL-1beta will also induce apoptosis in CaP cells through a Ca2+ -dependent pathway, following the inactivation of NF-kappaB. These results suggest that NF-kappaB activity is required to maintain Ca 2+ homeostasis in CaP cells following IL-1beta stimulation. This is supported by the fact that IL-1beta activates NF-kappaB, in part, through Ca2+/calmodulin-dependent kinases (CaMKs), namely CaMKK and CaMKIV. IL-1beta-induced NF-kappaB activity is required to upregulate transcripts encoding Ca2+-binding proteins (CaBP), including calreticulin, calbindin-3, and DSCR1. Expression of calreticulin blocked Ca 2+ -induced apoptosis in our model system. The overall hypothesis of this proposal is that IL-1beta-induced NF-kappaB activity is required to upregulate gene products that feedback to regulate Ca 2+ levels and modulate apoptosis in CaP cells. To address this hypothesis, the following aims will be explored.
Aim 1 will establish whether CaP patients display, elevated IL-1beta. Experiments' proposed in Aim 1 will also identify whether intracellular release of Ca2+ is responsible for inducing apoptosis, and will determine if this effect impacts cytochrome c release, caspase and/or calpain activation, and subsequent cleavage of CaMK and IkappaB/NF-kappaB substrates.
Aim 2 will identify the CaMK signaling pathways responsible for upregulating NF-kappaB transcriptional activity and cell survival. Experiments outlined in Aim 2 will use dominant negative and constitutively active kinases to identify CaMK pathways required to activate NF-kappaB in response to IL-1beta, and will determine the impact of these signaling pathways on the transactivation potential of NF-kappaB.
Aim 3 will elucidate whether the calreticulin promoter is a direct target of NF-kappaB and determine if the expression of calreticulin alone or in combination with other NF-kappaB-regulated CaBPs rescues apoptosis. Experiments described in Aim 3 will determine whether NF-kappaB directly upregulates the calreticulin promoter, and will potentially identify regulated Ca2+ -binding proteins. Additional experiments proposed in Aim 3 will determine whether the expression of these proteins alone or in combination with calreticulin will rescue Ca2+ -induced apoptosis. Collectively, the experiments outlined in this proposal will offer insight into the anti-apoptotic role of NF-kappaB in Ca 2+ signaling in CaP cells, and will potentially provide a better understanding of how NF-kappaB overcomes Ca2+ -induced apoptotic cues initiatcd by growth factors and cytokines.