CCL2 Signaling Research Articles

Abstract A063: Targeting the CCL2 chemokine pathway in breast tumors through intratumoral delivery of calcium cross linked TAT peptide: siRNA complexes

Abstract Increased expression of CCL2 correlates with poor patient survival and development of invasive breast cancer. CCL2 regulates mammary tumor progression by recruitment of tumor promoting macrophages. Our laboratory and others have shown that CCL2 signaling mediates stromal: tumor interactions to regulate breast cancer cell survival, invasion and metastasis. While the CCL2 signaling represents a novel and potentially effective therapeutic target in invasive breast cancer, it is necessary to develop new tools to specifically inhibit CCL2 signaling in vivo. siRNA knockdown could specifically target CCL2, but is unstable and toxic when delivered in vivo. The HIV-1 derived TAT peptide penetrates cell plasma membranes at high efficiency and contains nuclear translocation signals to enhance siRNA delivery to the nucleus. Recent studies from our research team have shown that CaCl2 condenses TAT/nucleic acids to nanoparticles facilitating cellular uptake. The goals of these studies are to design TAT nanoparticle formulas to target CCL2 protein expression in breast tumors. Using an in vitro luciferase reporter system, we identified specific N/P (TAT:siRNA) ratios and CaCl2 concentrations that would efficiently deliver siRNAs to PyVmT mammary tumor cells. Utilizing a novel ex vivo mammary tumor system, TAT/CaCl2 nanocomplexes carrying GFP plasmid or Cy3 labeled siRNAs were then injected into PyVmT mammary tumors. We observed visible GFP expression and Cy3 labeled siRNA uptake in over 50% of the PyVmT mammary tumor. Flow cytometry analysis showed that low N/P ratio resulted in efficient TAT/CaCl2/siRNA nanoparticle uptake in tumor epithelial cells. Normal mammary tissues did not show significant uptake of TAT nanoparticles. CCL2 knockdown was observed in tumor epithelial cells, but not stromal cells as determined by flow cytometry. Similar results were observed in cultured cells. At low N/P ratio, PyVmT tumor cells showed a 50% reduction in CCL2 expression upon delivery of TAT/CaCl2 nanoparticles carrying CCL2 siRNAs, while there was no significant effect on stromal cells as determined by ELISA. Downregulation of CCL2 in the tumor cells significantly decreased proliferation as determined by flow cytometry analysis. Importantly, treatment with TAT/siRNA nanoparticle did not significantly affect viability of normal cells. These data indicate this nanoparticle strategy represents a novel, feasible approach to target the CCL2 signaling in breast cancer, with important implications on therapeutic targeting. Citation Format: Wei Bin Fang, Nabil Alhakamy, Cory Berkland, Nikki Cheng. Targeting the CCL2 chemokine pathway in breast tumors through intratumoral delivery of calcium cross linked TAT peptide: siRNA complexes. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A063.

A role for CCL2 in both tumor progression and immunosurveillance

The chemokine CCL2, which is best known for its chemotactic functions, is expressed not only by immune cells, but also by several types of malignant and stromal cells. CCL2 has been shown to exert both pro- and anti-tumor effects. However, recent results demonstrate a main role for CCL2 in tumor progression and metastasis, suggesting that this chemokine may constitute a therapeutic target for anticancer drugs. Mammary carcinoma models, including models of implantable, transgenic, and chemically-induced tumors, were employed in the setting of Ccl2 or Ccr2 knockout mice or CCL2 neutralization with a monoclonal antibody to further investigate the role of the CCL2/CCR2 signaling axis in tumor progression and metastatic spread. In our implantable tumor models, an anti-CCL2 monoclonal antibody inhibited the growth of primary malignant lesions in a biphasic manner and reduced the number of metastases. However, in Ccl2−/− or Ccr2−/− mice developing implanted or transgenic tumors, the number of pulmonary metastases was increased despite a reduction in the growth rate of primary neoplasms. Transgenic Mtag.Ccl2−/− or Mtag.Ccr2−/− mice also exhibited a significantly earlier of disease onset. In a chemical carcinogenesis model, anti-CCL2 monoclonal antibody inhibited the growth of established lesions but was ineffective in the tumor induction phase. In contrast to previous studies indicating a role for CCL2 in the establishment of metastases, we have demonstrated that the absence of CCL2/CCR2-signaling results in increased metastatic disease. Thus, the CCL2/CCR2 signaling axis appears to play a dual role in mediating early tumor immunosurveillance and sustaining the growth and progression of established neoplasms. Our findings support the use of anti-CCL2 therapies for the treatment of established breast carcinoma, although the complete abrogation of the CCL2 signaling cascade may also limit immunosurveillance and support metastatic spread.

Recruitment of a Myeloid Cell Subset (CD11b/Gr1mid) via CCL2/CCR2 Promotes the Development of Colorectal Cancer Liver Metastasis

Liver metastasis from colorectal cancer is a leading cause of cancer mortality. Myeloid cells play pivotal roles in the metastatic process, but their prometastatic functions in liver metastasis remain incompletely understood. To investigate their role, we simulated liver metastasis in C57BL/6 mice through intrasplenic inoculation of MC38 colon carcinoma cells. Among the heterogeneous myeloid infiltrate, we identified a distinct population of CD11b/Gr1(mid) cells different from other myeloid populations previously associated with liver metastasis. These cells increased in number dramatically during establishment of liver metastases and were recruited from bone marrow by tumor-derived CCL2. Liver metastasis of Lewis lung carcinoma cells followed this pattern but this mechanism is not universal as liver colonization by B16F1 melanoma cells did not recruit similar subsets. Inhibition of CCL2 signaling and absence of its cognate receptor CCR2 reduced CD11b/Gr1(mid) recruitment and decreased tumor burden. Depletion of the CD11b/Gr1(mid) subset in a transgenic CD11b-diphtheria toxin receptor mouse model markedly reduced tumor cell proliferation. There was no evidence for involvement of an adaptive immune response in the prometastatic effects of CD11b/Gr1(mid) cells. Additionally, an analogous myeloid subset was found in liver metastases of some colorectal cancer patients. Collectively, our findings highlight the importance of myeloid cells--in this case a selective CD11b/Gr1(mid) subset--in sustaining development of colorectal cancer liver metastasis and identify a potential target for antimetastatic therapy.

Role of glutaminyl cyclases in thyroid carcinomas

CCL2 is a chemokine known to recruit monocytes/macrophages to sites of inflammation. CCL2 is also associated with tumor progression in several cancer types. Recently, we showed that the N-terminus of CCL2 is modified to a pyroglutamate (pE)-residue by both glutaminyl cyclases (QC (QPCT)) and its isoenzyme (isoQC (QPCTL)). The pE-residue increases stability against N-terminal degradation by aminopeptidases. Here, we report an upregulation of QPCT expression in tissues of patients with thyroid carcinomas compared with goiter tissues, whereas QPCTL was not regulated. In thyroid carcinoma cell lines, QPCT gene expression correlates with the mRNA levels of its substrate CCL2. Both QPCT and CCL2 are regulated in a NF-κB-dependent pathway shown by stimulation with TNFa and IL1b as well as by inhibition with the IKK2 inhibitor and RNAi of p50. In the culture supernatant of thyroid carcinoma cells, equal amounts of pECCL2 and total CCL2 were detected by two ELISAs discriminating between total CCL2 and pECCL2, concluding that all CCL2 is secreted as pECCL2. Activation of the CCL2/CCR2 pathway by recombinant CCL2 increased tumor cell migration of FTC238 cells in scratch assays as well as thyroid carcinoma cell-derived CCL2-induced migration of monocytic THP1 cells. Suppression of CCL2 signaling by CCR2 antagonist, IKK2 inhibitor, and QPCT RNAi reduced FTC238 cell growth measured by WST8 proliferation assays. Our results reveal new evidence for a novel role of QC in thyroid carcinomas and provide an intriguing rationale for the use of QC inhibitors as a means of blocking pECCL2 formation and preventing thyroid cancer metastasis.

Absence of CCL2 is sufficient to restore hippocampal neurogenesis following cranial irradiation

Cranial irradiation for the treatment of brain tumors causes a delayed and progressive cognitive decline that is pronounced in young patients. Dysregulation of neural stem and progenitor cells is thought to contribute to these effects by altering early childhood brain development. Earlier work has shown that irradiation creates a chronic neuroinflammatory state that severely and selectively impairs postnatal and adult neurogenesis. Here we show that irradiation induces a transient non-classical cytokine response with selective upregulation of CCL2/monocyte chemoattractant protein-1 (MCP-1). Absence of CCL2 signaling in the hours after irradiation is alone sufficient to attenuate chronic microglia activation and allow the recovery of neurogenesis in the weeks following irradiation. This identifies CCL2 signaling as a potential clinical target for moderating the long-term defects in neural stem cell function following cranial radiation in children.

Expansion of a subset of CD14highCD16negCCR2low/neg monocytes functionally similar to myeloid-derived suppressor cells during SIV and HIV infection

Monocytes have been categorized in three main subpopulations based on CD14 and CD16 surface expression. Classical monocytes express the CD14(++)CD16(-)CCR2(+) phenotype and migrate to inflammatory sites by quickly responding to CCL2 signaling. Here, we identified and characterized the expansion of a novel monocyte subset during HIV and SIV infection, which were undistinguishable from classical monocytes, based on CD14 and CD16 expression, but expressed significantly lower surface CCR2. Transcriptome analysis of sorted cells demonstrated that the CCR2(low/neg) cells are a distinct subpopulation and express lower levels of inflammatory cytokines and activation markers than their CCR2(high) counterparts. They exhibited impaired phagocytosis and greatly diminished chemotaxis in response to CCL2 and CCL7. In addition, these monocytes are refractory to SIV infection and suppress CD8(+) T cell proliferation in vitro. These cells express higher levels of STAT3 and NOS2, suggesting a phenotype similar to monocytic myeloid-derived cells, which suppress expansion of CD8(+) T cells in vivo. They may reflect an antiproliferative response against the extreme immune activation observed during HIV and SIV infections. In addition, they may suppress antiviral responses and thus, have a role in AIDS pathogenesis. Antiretroviral therapy in infected macaque and human subjects caused this population to decline, suggesting that this atypical phenotype is linked to viral replication.

A truncated analogue of CCL2 mediates anti-fibrotic effects on murine fibroblasts independently of CCR2

The truncated [1 + 9–76] CCL2 analogue, also known as 7ND, has been described in numerous reports as an anti-inflammatory and anti-fibrotic agent in a wide spectrum of animal models, e.g. models of cardiovascular disease, graft versus host disease and bleomycin-induced pulmonary fibrosis. 7ND has been reported to function as a competitive inhibitor of CCL2 signaling via CCR2 in human in vitro systems. In contrast, the mechanistic basis of 7ND action in animal models has not been previously reported. Here we have studied how 7ND interacts with CCL2 and CCR2 of murine origin. Surprisingly, 7ND was shown to be a weak inhibitor of murine CCL2/CCR2 signaling and displaced murine CCL2 (JE) from the receptor with a K i > 1 μM. Using surface plasmon resonance, we found that 7ND binds murine CCL2 with a K d of 670 nM, which may indicate that 7ND inhibits murine CCL2/CCR2 signaling by a dominant negative mechanism rather than by competitive binding to the CCR2 receptor. In addition we observed that sub-nanomolar levels of 7ND mediate anti-fibrotic effects in CCR2 negative fibroblasts cultured from fibrotic lung of bleomycin-induced mice. Basal levels of extracellular matrix proteins were reduced (collagen type 1 and fibronectin) as well as expression levels of α-smooth muscle actin and CCL2. Our conclusion from these data is that the previously reported effects of 7ND in murine disease models most probably are mediated via mechanisms independent of CCR2.

Abstract A37: Development of biologics targeting soluble ligands: Novel biomarker and PK-PD analyzes examining free and total ligand profile following treatment with carlumab (anti-CCL2 mAb).

Abstract Background: Despite the large numbers of antibodies targeting soluble ligands in development in oncology and other therapeutic areas, very few reports have described pharmacodynamic (PD)/ mechanism of action (MOA) biomarkers of free ligand or total (antibody-complexed) ligand and their PK-PD relationship. Carlumab is a human IgG1 monoclonal antibody that binds the chemokine CCL2, which is a potent chemoattractant for tumor associated macrophages and promotes tumor growth and metastasis. Therapeutic targeting of CCL2 was based on the hypothesis that carlumab binding would inhibit chemokine activity and block tumor inflammation and macrophage differentiation thereby impairing tumor growth and metastases. Because proof of concept (POC) is a major milestone in early drug development, the objective of this study was to develop key PD/MOA biomarkers to facilitate the clinical evaluation of POC for carlumab. This required the development of robust analytical assays to accurately measure free and total CCL2 concentrations in the serum, and mechanistic PK-PD modeling for data analysis. Methods: Carlumab was evaluated in three oncology clinical studies (as monotherapy in a Phase 1 first-in-human trial in patients with solid tumors, a Phase 1b chemotherapy combination study, and as a single agent Phase 2a study in patients with advanced prostate cancer). Biomarkers evaluated included free and total CCL2 concentrations in serum, a broad panel of soluble biomarkers, bone turnover markers, and circulating tumor cell (CTC) enumeration. Sequential tumor biopsies in ten patients from Phase 1 study yielded 4 paired specimens for immunohistochemical analysis of tumor associated macrophages (TAMs). A mechanistic PK-PD model that simultaneously characterized free and total CCL2, and carlumab concentrations in serum was used to estimate biologically effective doses that would inhibit CCL2 signaling. Results: Analysis of CCL2 levels following carlumab administration revealed a rapid fall in free CCL2 as expected; however, post-treatment free CCL2 levels rapidly rebounded and quickly exceeded pretreatment serum concentrations. Two different analytical methods showed that carlumab suppressed CCL2 levels for only ∼24 hrs followed by a sustained increase in CCL2 concentration with subsequent treatment. These surprising results suggest that carlumab was ineffective at neutralizing free CCL2. PK-PD modeling confirmed the transient suppression of free CCL2 consistent with a higher carlumab dissociation constant (Kd) for CCL2 binding in vivo. Finally, carlumab did not affect other biomarkers including bone turnover markers or CTC numbers, consistent with an inability to impact CCL2 signaling. No evidence of clinical efficacy was observed in any of carlumab clinical trials. Conclusion: Development of robust analytical assays to measure free and total CCL2 concentration and utilization of mechanistic PK-PD modeling facilitated the clinical evaluation of POC for carlumab. The unexpected increase in free CCL2 concentration and the lack of CCL2 target suppression likely explain the absence of carlumab clinical activity. Supportive biomarker analyzes and mechanistic PK-PD modeling are important components of the early development clinical plan for developing biologics against soluble ligands. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A37.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Identification of CCR2‐binding features in Cytl1 by a CCL2‐like chemokine model

Chemokines are small secreted proteins that play an important role in immune responses and have also been shown to be involved in cartilage development and contributing to pathogenesis of a variety of diseases. They present a conserved 3D structure, so-called IL8-like chemokine fold, which is supported by conserved cysteines forming intra-molecular disulfide bonds. These cysteine sequence motifs have often been used to find new chemokine family members by sequence-based database searches. However, it has been shown that different patterns can provide disulfide bonds fitting into an IL8-like architecture, which has been the key to identify new remote homologues of the IL8-like chemokine family. We report a structural-functional characterization of cytokine-like protein 1 (Cytl1) by a combination of different computational structure-based techniques. Previous studies based on sequence analysis and secondary structure predictions reported that Cytl1 might adopt a 4-helical cytokine fold. However, our detailed molecular modeling studies and structure-based functional analysis strongly suggest that Cytl1 is more likely to adopt an IL8-like chemokine fold, in particular similar to CCL2 (monocyte chemoattractant protein 1, MCP-1). Moreover, we identify in a CCL2-like 3D model of Cytl1 the necessary reported features to signal through the chemokine receptor CCR2. Those discovered structural features of Cytl1 as CCL2-like chemokine, together with the fact that both, CCL2 and Cytl1, are known to be involved in cartilage development and pathogenesis of osteoarthritis and rheumatoid arthritis, make us hypothesize that Cytl1 could be a structurally and functionally related analog of CCL2 signaling through the chemokine receptor CCR2.

CCL2/CCR2 and CX3CL1/CX3CR1 chemokine axes and their possible involvement in age-related macular degeneration

The causes of age-related macular degeneration (AMD) are not well understood. Due to demographic shifts in the industrialized world a growing number of people will develop AMD in the coming decades. To develop treatments it is essential to characterize the disease's pathogenic process. Over the past few years, numerous studies have focused on the role of chemotactic cytokines, also known as chemokines. Certain chemokines, such as CCL2 and CX3CL1, appear to be crucial in subretinal microglia and macrophage accumulation observed in AMD, and participate in the development of retinal degeneration as well as in choroidal neovascularization. This paper reviews the possible implications of CCL2 and CX3CL1 signaling in AMD. Expression patterns, single nucleotide polymorphisms (SNPs) association studies, chemokine and chemokine receptor knockout models are discussed. Future AMD treatments could target chemokines and/or their receptors.

Delineating the angiogenic gene expression profile before pulmonary vascular remodeling in a lamb model of congenital heart disease.

Disordered angiogenesis is implicated in pulmonary vascular remodeling secondary to congenital heart diseases (CHD). However, the underlying genes are not well delineated. We showed previously that an ovine model of CHD with increased pulmonary blood flow (PBF, Shunt) has an "angiogenesis burst" between 1 and 4 wk of age. Thus we hypothesized that the increased PBF elicited a proangiogenic gene expression profile before onset of vessel growth. To test this we utilized microarray analysis to identify genes that could be responsible for the angiogenic response. Total RNA was isolated from lungs of Shunt and control lambs at 3 days of age and hybridized to Affymetrix gene chips for microarray analyses (n = 8/group). Eighty-nine angiogenesis-related genes were found to be upregulated and 26 angiogenesis-related genes downregulated in Shunt compared with control lungs (cutting at 1.2-fold difference, P < 0.05). We then confirmed upregulation of proangiogenic genes FGF2, Angiopoietin2 (Angpt2), and Birc5 at mRNA and protein levels and upregulation of ccl2 at mRNA level in 3-day Shunt lungs. Furthermore, we found that pulmonary arterial endothelial cells (PAEC) isolated from fetal lambs exhibited increased expression of FGF2, Angpt2, Birc5, and ccl2 and enhanced angiogenesis when exposed to elevated shear stress (35 dyn/cm²) compared with cells exposed to more physiological shear stress (20 dyn/cm²). Finally, we demonstrated that blocking FGF2, Angpt2, Birc5, or ccl2 signaling with neutralizing antibodies or small interfering RNA (siRNA) significantly decreased the angiogenic response induced by shear stress. In conclusion, we have identified a "proangiogenic" gene expression profile in a lamb model of CHD with increased PBF that precedes onset of pulmonary vascular remodeling. Our data indicate that FGF2, Angpt2, Birc5, and ccl2 may play important roles in the angiogenic response.

Utilizing mechanistic PK/PD modeling to simultaneously examine free CCL2, total CCL2, and CNTO 888 serum concentration time data.

3029 Background: The chemokine (C-C motif) CCL2 promotes angiogenesis, tumor proliferation, migration, and metastasis. CNTO 888 is a human IgG1κ monoclonal antibody with high CCL2 binding affinity. A mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model was developed to simultaneously characterize the time course of CNTO 888, free CCL2, and the CNTO 888-CCL2 complex in the serum. Comprehensive PK/PD modeling of oncology biologics targeting soluble ligands have been rare to date. The goal of the analysis was to predict a dose that will maximally affect CCL2 signaling in the serum and utilize that dose for the clinical development of CNTO 888. Methods: PK and PD data from 21 cancer patients with refractory tumors were examined. CNTO 888 dose levels examined were 0.3, 1, 3, 10 and 15 mg/kg. 3 subjects were treated at each dose level for dose levels < 3 mg/kg and 6 subjects each for the 10 and 15 mg/kg dose levels. All dose levels tested were well tolerated with no dose limiting toxicities. CNTO 888, free CCL2, and CNTO 888-CCL2 complex concentrations in serum were determined following the first and fourth dose. PK/PD modeling was conducted using population-based methods. Results: A mechanistic PK/PD model simultaneously described the temporal relationships among serum concentrations of CNTO 888, free CCL2, and the CNTO 888-CCL2 complex. Dose-dependent increases in total CCL2 concentrations were observed and are consistent with shifting free CCL2, which is rapidly cleared in the absence of CNTO 888 to the form bound to CNTO 888 which is cleared much slower. Free CCL2 declined rapidly after the end of the infusion and returned to baseline concentration within 7 days. Mean predicted half-lives of CNTO 888 and the CCL2-CNTO 888 complex were both 2.4 days and only 1 hour for free CCL2. The mean (%SEM) dissociation constant (KD) was 2.4 nM (72%). Conclusions: A mechanistic PK/PD model was developed that simultaneously describes CNTO 888, free CCL2 and the CNTO 888-CCL2 complex concentration-time data in serum. Model parameter estimates are physiologically plausible. This PK/PD model has the potential to predict clinically relevant changes in free CCL2 and the CNTO 888-CCL2 complex. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Centocor Ortho Biotech Centocor Ortho Biotech Centocor Ortho Biotech

Upregulation of P2X4R expression on surface of fibronectin-stimulated microglia is mediated by CCL2 signaling

Upregulation of P2X4R expression on surface of fibronectin-stimulated microglia is mediated by CCL2 signaling

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 microm) or the Akt-specific inhibitor-X (Akti-X; 2.5 microm). 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.