IL-4 induces proliferation in prostate cancer PC3 cells under nutrient-depletion stress through the activation of the JNK-pathway and survivin up-regulation.

Interleukin (IL)-4 plays a critical role in the regulation of immune responses and has been detected at high levels in the tumor microenvironment of cancer patients where it correlates with the grade of malignancy. The direct effect of IL-4 on cancer cells has been associated with increased cell survival; however, its role in cancer cell proliferation and related mechanisms is still unclear. Here it was shown that in a nutrient-depleted environment, IL-4 induces proliferation in prostate cancer PC3 cells. In these cells, under nutrient-depletion stress, IL-4 activates mitogen-activated protein kinases (MAPKs), including Erk, p38, and JNK. Using MAP-signaling-specific inhibitors, it was shown that IL-4-induced proliferation is mediated by JNK activation. In fact, JNK-inhibitor-V (JNKi-V) stunted IL-4-mediated cell proliferation. Furthermore, it was found that IL-4 induces survivin up-regulation in nutrient-depleted cancer cells. Using survivin-short-hairpin-RNAs (shRNAs), it was demonstrated that in this milieu survivin expression above a threshold limit is critical to the mechanism of IL-4-mediated proliferation. In addition, the significance of survivin up-regulation in a stressed environment was assessed in prostate cancer mouse xenografts. It was found that survivin knockdown decreases tumor progression in correlation with cancer cell proliferation. Furthermore, under nutrient depletion stress, IL -4 could induce proliferation in cancer cells from multiple origins: MDA-MB-231 (breast), A253 (head and neck), and SKOV-3 (ovarian). Overall, these findings suggest that in a tumor microenvironment under stress conditions, IL-4 triggers a simultaneous activation of the JNK-pathway and the up-regulation of survivin turning on a cancer proliferation mechanism.

CCL2 and interleukin-6 promote survival of human CD11b+ peripheral blood mononuclear cells and induce M2-type macrophage polarization.

CCL2 and interleukin (IL)-6 are among the most prevalent cytokines in the tumor microenvironment, with expression generally correlating with tumor progression and metastasis. CCL2 and IL-6 induced expression of each other in CD11b(+) cells isolated from human peripheral blood. It was demonstrated that both cytokines induce up-regulation of the antiapoptotic proteins cFLIP(L) (cellular caspase-8 (FLICE)-like inhibitory protein), Bcl-2, and Bcl-X(L) and inhibit the cleavage of caspase-8 and subsequent activation of the caspase-cascade, thus protecting cells from apoptosis under serum deprivation stress. Furthermore, both cytokines induced hyperactivation of autophagy in these cells. Upon CCL2 or IL-6 stimulation, CD11b(+) cells demonstrated a significant increase in the mannose receptor (CD206) and the CD14(+)/CD206(+) double-positive cells, suggesting a polarization of macrophages toward the CD206(+) M2-type phenotype. Caspase-8 inhibitors mimicked the cytokine-induced up-regulation of autophagy and M2 polarization. Furthermore, E64D and leupeptin, which are able to function as inhibitors of autophagic degradation, reversed the effect of caspase-8 inhibitors in the M2-macrophage polarization, indicating a role of autophagy in this mechanism. Additionally, in patients with advanced castrate-resistant prostate cancer, metastatic lesions exhibited an increased CD14(+)/CD206(+) double-positive cell population compared with normal tissues. Altogether, these findings suggest a role for CCL2 and IL-6 in the survival of myeloid monocytes recruited to the tumor microenvironment and their differentiation toward tumor-promoting M2-type macrophages via inhibition of caspase-8 cleavage and enhanced autophagy.

CCL2 is a negative regulator of AMP-activated protein kinase to sustain mTOR complex-1 activation, survivin expression, and cell survival in human prostate cancer PC3 cells.

CCL2 is a cytokine prevalent in the prostate cancer tumor microenvironment. Recently, we reported that CCL2 induces the mammalian target of rapamycin (mTOR) pathway to promote prostate cancer PC3 cell survival; however, the mechanism used by CCL2 to maintain mTOR complex-1 (mTORC1) activation requires clarification. This study demonstrates that upon serum starvation, CCL2 functions as a negative regulator of AMP-activated protein kinase (AMPK) by decreasing phosphorylation at its major regulatory site (Thr(172)) in PC3, DU145, and C4-2B prostate cancer cells. The CCL2-mediated AMPK regulation decreased raptor phosphorylation (Ser(792)) resulting in hyperactivation of mTORC1. D942, a pharmacological activator of AMPK, stunted CCL2-induced mTORC1 activity, survivin expression, and cell survival without significantly affecting Akt activity. CCL2, however, conferred some resistance to the lethal effect of D942 compared with untreated cells. By using Akt-specific inhibitor X, it was shown that Akt inactivation did not cause an increase in AMPK phosphorylation in CCL2-stimulated cells, suggesting that CCL2-mediated negative regulation of AMPK is independent of Akt. Furthermore, bisindolylmaleimide-V, a specific inhibitor of p70(S6K), stunted survivin expression and induced cell death in CCL2-treated PC3. Altogether, these findings suggest that CCL2 hyperactivates mTORC1 through simultaneous regulation of both AMPK and Akt pathways and reveals a new network that promotes prostate cancer: CCL2-AMPK-mTORC1-survivin.

The chemokine CCL2 increases prostate tumor growth and bone metastasis through macrophage and osteoclast recruitment.

CC chemokine ligand 2 (CCL2, also known as monocyte chemoattractant protein-1) has been demonstrated to recruit monocytes to tumor sites. Monocytes are capable of being differentiated into tumor-associated macrophages (TAMs) and osteoclasts (OCs). TAMs have been shown to promote tumor growth in several cancer types. Osteoclasts have also been known to play an important role in cancer bone metastasis. To investigate the effects of CCL2 on tumorigenesis and its potential effects on bone metastasis of human prostate cancer, CCL2 was overexpressed into a luciferase-tagged human prostate cancer cell line PC-3. In vitro, the conditioned medium of CCL2 overexpressing PC-3luc cells (PC-3(lucCCL2)) was a potent chemoattractant for mouse monocytes in comparison to a conditioned medium from PC-3(lucMock). In addition, CCL2 overexpression increased the growth of transplanted xenografts and increased the accumulation of macrophages in vivo. In a tumor dissemination model, PC-3(lucCCL2) enhanced the growth of bone metastasis, which was associated with more functional OCs. Neutralizing antibodies targeting both human and mouse CCL2 inhibited the growth of PC-3(luc), which was accompanied by a decrease in macrophage recruitment to the tumor. These findings suggest that CCL2 increases tumor growth and bone metastasis through recruitment of macrophages and OCs to the tumor site.

CCL2, survivin and autophagy: new links with implications in human cancer.

Recent data strongly support the idea that the orchestrated interaction between cancer and other cells in the tumor microenvironment is a vital component in the neoplastic process. Thus, tumor cells take advantage of the signaling molecules of the immune system to proliferate, survive, and invade other tissues. CCL2 (Chemokine (C-C motif) ligand 2, Monocyte chemoattractant protein-1 (MCP-1) has been demonstrated to play a significant role in prostate cancer neoplasia and invasion, and is highly expressed in the tumor microenvironment. We recently reported that CCL2 elicits a strong survival advantage in prostate cancer PC3 cells through PI3K/Akt-dependent regulation of autophagy via the mammalian target of rapamycin (mTOR) pathway and importantly, survivin upregulation is essential in this survival mechanism. Autophagy protects cells from nutrient depletion stress, but, paradoxically, excessive autophagy will result in cell death. How these life or death decisions are regulated remains unclear. Here we discuss the function of survivin in the control of autophagy and the interaction between CCL2, survivin and autophagy in the complex program of tumor progression.