Zachary S. Varsos

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 cFLIPL (cellular caspase-8 (FLICE)-like inhibitory protein), Bcl-2, and Bcl-XL 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 protects prostate cancer PC3 cells from autophagic death via phosphatidylinositol 3-kinase/AKT-dependent survivin Up-regulation

Resistance to cell death is a hallmark of cancer. Autophagy is a survival mechanism activated in response to nutrient deprivation; however, excessive autophagy will ultimately induce cell death in a nonapoptotic manner. The present study demonstrates that CCL2 protects prostate cancer PC3 cells from autophagic death, allowing prolonged survival in serum-free conditions. Upon serum starvation, CCL2 induced survivin up-regulation in PC3, DU 145, and C4-2B prostate cancer cells. Both cell survival and survivin expression were stunted in CCL2-stimulated PC3 cells when treated either with the phosphatidylinositol 3-kinase inhibitor LY294002 (2 μm) or the Akt-specific inhibitor-X (Akti-X; 2.5 μm). Furthermore, CCL2 significantly reduced light chain 3-II (LC3-II) in serum-starved PC3; in contrast, treatment with LY294002 or Akti-X reversed the effect of CCL2 on LC3-II levels, suggesting that CCL2 signaling limits autophagy in these cells. Upon serum deprivation, the analysis of LC3 localization by immunofluorescence revealed a remarkable reduction in LC3 punctate after CCL2 stimulation. CCL2 treatment also resulted in a higher sustained mTORC1 activity as measured by an increase in phospho-p70S6 kinase (Thr389). Rapamycin, an inducer of autophagy, both down-regulated survivin and decreased PC3 cell viability in serum-deprived conditions. Treatment with CCL2, however, allowed cells to partially resist rapamycin-induced death, which correlated with survivin protein levels. In two stable transfectants expressing survivin-specific short hairpin RNA, generated from PC3, survivin protein levels controlled both cell viability and LC3 localization in response to CCL2 treatment. Altogether, these findings indicate that CCL2 protects prostate cancer PC3 cells from autophagic death via the phosphatidylinositol 3-kinase/Akt/survivin pathway and reveal survivin as a critical molecule in this survival mechanism.

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. J. Cell. Biochem. 113: 1569–1580, 2012.

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 up-regulation is essential to 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. Addendum to: Roca H, Varsos Z, Pienta KJ. CCL2 protects prostate cancer PC3 cells from autophagic death via PI3K/AKT-dependent survivin up-regulation. J Biol Chem 2008; In press.

Abstract 166: MicroRNA profiling analysis identify new signature of tumor-promoting M2-type macrophages

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL M2-type macrophages are a major inflammatory component in prostate cancer and are associated with cancer cell growth, survival, and metastasis. Since recent findings have demonstrated the importance of microRNAs in the differentiation of human cells, we hypothesize that microRNAs are involved in regulating monocytic differentiation into M2-type macrophages and we aim to identify the microRNAs involved. Methods: Human CD14+ PBMCs were isolated from healthy donors and stimulated with cytokines known to promote differentiation into the M2-type macrophages (IL4, IL13, IL6, and MCSF). As controls, we also treated cells with media only and with INFγ, a cytokine known to promote M1-type polarization. Cells were collected at 48 hours post-stimulation and sent for microRNA profiling by microarray. Microarray findings were then confirmed by qRT-PCR. Results: Microarray profiling of cytokine-stimulated M2-type macrophages demonstrated an altered miRNA expression compared to M1-type macrophages and untreated monocytic cells. Specifically, of the 858 microRNAs that were profiled, miR-135a, miR-193b, miR-203, miR-302, miR-375, miR-452, miR-511, miR-603, miR-622, miR-654, miR-938 and miR-1238 were found to be up-regulated by greater than 2 folds. Furthermore, qRT-PCR analysis confirmed the microarray results. When the qRT-PCR analysis was repeated for cells treated with cytokines for 6 hours, 18 hours and 24 hours, we noticed that the overexpression in these microRNAs could already be observed at 6 hours post-treatment, although the expression levels were noticeably greater at later timepoints. Flow cytometric analysis showed the surface expression of CD206 (mannose receptor: the well characterized surface marker of M2-type macrophages) in both MCSF and IL-4-treated cells but only after at least 18 hours of stimulation. Conclusions: MicroRNA expression in PBMCs is altered when they are stimulated by M2-type promoting cytokines and the change in microRNA expression preceded the expression of the M2-specific CD206 surface marker. This change in microRNA expression can therefore be used as a marker for M2-polarized macrophages and may play critical roles in the regulation of macrophage differentiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 166. doi:10.1158/1538-7445.AM2011-166