We provide a comprehensive data to establish the significant contribution of CXCR1, in prostate cancer cell growth, survival, and tumorigenic potential. The CXCR1 was active in AIPC cells where the cells produce its ligand, IL-8. It was not active in an IL-8 deficient androgen-responsive cell line, such as LNCaP. Using both positive (over expression) and negative (shRNA-mediated knockdown) expression strategies, we demonstrated the distinct role of CXCR1, and the lack of such function by CXCR2 in cell proliferation and survival (inhibition of apoptosis) in CaP cells.
Growth inhibition induced by CXCR1 depletion was causally associated with alteration in the expression of critical cell cycle regulatory proteins. Cell proliferation involves activation and suppression of a large repertoire of both cytoplasmic and nuclear proteins. However, we could demonstrate a link between IL-8 – CXCR1 signaling and cell-cycle progression. A depletion of CXCR1 led to both cell cycle arrest and decreased expression of molecules that promote the transit of cells in G0/G1 phase to S-phase. This included decreased expression of Cyclin D1, increased expression of P27 but reduction in the levels of phosphorylated ERK1/2 and Rb proteins (). These results demonstrate that IL-8-CXCR1 signaling is upstream in the mitogenic pathway, control several critical molecules during cell cycle progression. These finding increase the potential to inhibit critical cell proliferation pathways using inhibitors of IL-8 and/or CXCR1.
MacManus et al (2007) [25
] reported that external addition of IL-8 can also regulate the expression of Cyclin D1 in AIPC cells, such as PC-3 and DU145. We demonstrate here that blocking one of its receptor by mRNA depletion is sufficient to produce not only cell proliferation inhibition, cyclinD1 synthesis but also induction of apoptosis and angiogenic molecules. Since IL-8 is constitutively produced by AIPC cells, an autocrine growth stimulation is likely to be present in these cells, and probably in patients with AIPC, as increased IL-8 levels have been reported in patients with AIPC [[22
]. Considering multiple publications have reported the differential expression of IL-8Rs (CXCR1 and CXCR2), in aggressive prostate tumors and the lack of them in benign tissues or normal epithelial cells [23
], our results clearly establish the critical and functional role of such increased expression of IL-8Rs in aggressive tumors. The present finding in concert with earlier reports establishes that external and internal stimulation of CXCR1 by IL-8 mediate autocrine growth stimulation. Furthermore, our experiments show active growth signaling by autocrine mechanism, by IL-8 produced by the tumor cells alone, not the stromal cells, a condition necessitated by the earlier results by MacManus et al., [25
]. However, our observation that external IL-8 (paracrine stimulation) can induce mitogenic response in LAPC-4 cells, that lack autocrine IL-8 stimulation (Fig. S2C
) suggests that controlling CXCR1 may have a greater consequence than that produced by controlling IL-8 levels for tumor growth inhibition.
Although we observed an increase in Cyclin D1 expression by addition of other growth factor, such as EGF in PC-3V cells, we found a significantly diminished response to EGF signaling in CXCR1-depleted cells (e.g., PLK2) () indicating CXCR1-IL8 interaction may mediate EGF-EGFR mitogenic signaling, as shown in lung cancer cells [42
The depletion of CXCR1 in PC-3 cells, thus depletion of IL-8-CXCR1 signaling pathway, caused decrease in cell survival via increase in apoptosis () and it was through mitochondria-mediated (intrinsic) mechanism. To our knowledge, this is the first report of control of mitochondrial integrity by IL-8-CXCR1 signaling. Lack of IL-8-CXCR1 signaling resulted in not only depolarization of mitochondria but also significant decreases in anti-apoptotic proteins, BCL2 and BCLxL
. Furthermore, CXCR1 depletion caused an increase in pro-apoptotic proteins, BAX and BAD and caspase-9 activation. It was reported previously, that increased levels of IL-8 in the serum of cancer patients, might increase tissue BCL2, and thus increase the survival of tumor cells [43
]. More over, Nor et al [44
] noted that increased IL-8 is associated with increased endothelial cell survival in tumor vasculature, that may cause increased tumor cell survival. This is also evident in PLK2 tumors in vivo, as a steep increase in apoptosis was observed in the tumor tissue, demonstrating potential clinical application of CXCR1 (and therefore, CXCR2 down modulation) antagonists for clinical application.
Our results demonstrate a co-regulation of CXCR1 and CXCR2 at the transcriptional level. We show that () a concordant down regulation of CXCR2 mRNA when we down regulated CXCR1 by shRNA, and an up regulation of CXCR2 when shRNA knockdown is rescued by shRNA incompatible mutant CXCR1 cDNA. However, down regulation of CXCR2 by siRNA did not alter CXCR1 mRNA, indicating a hierarchical regulation, most likely in the post-transcriptional level, of CXCR2 mRNA. Although this may provide a unique opportunity to regulate CXCR2 function, we reserve this investigation for a later study. However, our observation of a hierarchical regulation of CXCR1 and CXCR2 does bear significance in the regulation of tumor angiogenesis, largely controlled by tumor cells’ angiogenic activity largely mediated by CXCR2 [24
The expression of angiogenic CXC chemokines is high in AIPC cells [7
]. CXCR2 activation does not contribute to tumorigenesis but it stimulates angiogenesis [24
]. We corroborate this finding with our results, as we found no change in Cyclin D1 level by depleting CXCR2 mRNA () and further, a CXCR2 specific inhibitor did not inhibit cell proliferation (Supplement Fig. 3S
). Our finding of decreased VEGF in PLK2 cells may be due to the down regulation of both CXCRs, as noted above.
In summary, the data presented in this report clearly establish that IL-8-CXCR1 autocrine activity is essential for AIPC growth and survival. Since, AIPC is the most difficult form of CaP to treat; patients typically survive less than two years at this stage, our study provides a novel avenue to control, potentially, the growth of AIPC, by using inhibitors of IL-8-CXCR1 signaling, or by direct antagonists of IL-8 or CXCR1. Overall, these studies establish a novel strategy to control AIPC.