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.

Cell plasticity regulated by the balance between the mesenchymal to epithelial transition (MET) and the opposite program, EMT, is critical in the metastatic cascade. Several transcription factors (TFs) are known to regulate EMT, though the mechanisms of MET remain unclear. We demonstrate a novel function of two TFs, OVOL1 and OVOL2, as critical inducers of MET in human cancers. Our findings indicate that the OVOL-TFs control MET through a regulatory feedback loop with EMT-inducing TF ZEB1, and the regulation of mRNA splicing by inducing Epithelial Splicing Regulatory Protein 1 (ESRP1). Using mouse prostate tumor models we show that expression of OVOL-TFs in mesenchymal prostate cancer cells attenuates their metastatic potential. The role of OVOL-TFs as inducers of MET is further supported by expression analyses in 917 cancer cell lines, suggesting their role as crucial regulators of epithelial-mesenchymal cell plasticity in cancer.

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.

During tumor progression, immune system phagocytes continually clear apoptotic cancer cells in a process known as efferocytosis. However, the impact of efferocytosis in metastatic tumor growth is unknown. In this study, we observed that macrophage-driven efferocytosis of prostate cancer cells in vitro induced the expression of proinflammatory cytokines such as CXCL5 by activating Stat3 and NF-κB(p65) signaling. Administration of a dimerizer ligand (AP20187) triggered apoptosis in 2 in vivo syngeneic models of bone tumor growth in which apoptosis-inducible prostate cancer cells were either coimplanted with vertebral bodies, or inoculated in the tibiae of immunocompetent mice. Induction of 2 pulses of apoptosis correlated with increased infiltration of inflammatory cells and accelerated tumor growth in the bone. Apoptosis-induced tumors displayed elevated expression of the proinflammatory cytokine CXCL5. Likewise, CXCL5-deficient mice had reduced tumor progression. Peripheral blood monocytes isolated from patients with bone metastasis of prostate cancer were more efferocytic compared with normal controls, and CXCL5 serum levels were higher in metastatic prostate cancer patients relative to patients with localized prostate cancer or controls. Altogether, these findings suggest that the myeloid phagocytic clearance of apoptotic cancer cells accelerates CXCL5-mediated inflammation and tumor growth in bone, pointing to CXCL5 as a potential target for cancer therapeutics.

Apoptosis is crucial for tissue homeostasis and organ development. In bone, apoptosis is recognized to be a main fate of osteoblasts, yet the relevance of this process remains underexplored. Using our murine model with inducible Caspase 9, the enzyme that initiates intrinsic apoptosis, we triggered apoptosis in a proportion of mature osteocalcin (OCN⁺) osteoblasts and investigated the impact on postnatal bone development. Osteoblast apoptosis stimulated efferocytosis by osteal macrophages. A five-week stimulation of OCN⁺ osteoblast apoptosis in 3-week-old male and female mice significantly enhanced vertebral bone formation while increasing osteoblast precursors. A similar treatment regimen to stimulate osterix⁺ cell apoptosis had no impact on bone volume or density. The vertebral bone accrual following stimulation of OCN⁺ osteoblast apoptosis did not translate in improved mechanical strength due to disruption of the lacunocanalicular network. The observed bone phenotype was not influenced by changes in osteoclasts but was associated with stimulation of macrophage efferocytosis and vasculature formation. Phenotyping of efferocytic macrophages revealed a unique transcriptomic signature and expression of factors including VEGFA. To examine whether macrophages participated in the osteoblast precursor increase following osteoblast apoptosis, macrophage depletion models were employed. Depletion of macrophages via clodronate-liposomes and the CD169-diphtheria toxin receptor mouse model resulted in marked reduction in leptin receptor⁺ and osterix⁺ osteoblast precursors. Collectively, this work demonstrates the significance of osteoblast turnover via apoptosis and efferocytosis in postnatal bone formation. Importantly, it exposes the potential of targeting this mechanism to promote bone anabolism in the clinical setting.