Matthew J. Craig

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.

Targeting CCL2 with systemic delivery of neutralizing antibodies induces prostate cancer tumor regression in vivo.

The identification of novel tumor-interactive chemokines and the associated insights into the molecular and cellular basis of tumor-microenvironment interactions have continued to stimulate the development of targeted cancer therapeutics. Recently, we have identified monocyte chemoattractant protein 1 (MCP-1; CCL2) as a prominent regulator of prostate cancer growth and metastasis. Using neutralizing antibodies to human CCL2 (CNTO888) and the mouse homologue CCL2/JE (C1142), we show that treatment with anti-CCL2/JE antibody (2 mg/kg, twice weekly i.p.) attenuated PC-3Luc-mediated overall tumor burden in our in vivo model of prostate cancer metastasis by 96% at 5 weeks postintracardiac injection. Anti-CCL2 inhibition was not as effective as docetaxel (40 mg/kg, every week for 3 weeks) as a single agent, but inhibition of CCL2 in combination with docetaxel significantly reduced overall tumor burden compared with docetaxel alone, and induced tumor regression relative to initial tumor burden. These data suggest an interaction between tumor-derived chemokines and host-derived chemokines acting in cooperation to promote tumor cell survival, proliferation, and metastasis.

CCL2 (Monocyte Chemoattractant Protein-1) in cancer bone metastases

The paradigm of cancer development and metastasis is a comprehensive, complex series of events that ultimately reflects a coordinated interaction between the tumor cell and the microenvironment within which the tumor cell resides. Despite the realization that this relationship has changed the current paradigm of cancer research, the struggle continues to more completely understand the pathogenesis of the disease and the ability to appropriately identify and design novel targets for therapy. A particular area of research that has added a significant understanding to cancer metastasis is the role of chemokines and chemokine receptors. Here we review the current concepts of CCL2 (monoctye chemoattractant protein 1) and its role in tumor metastasis with particular interest to its role in the development of bone metastases.

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.

PAR1-Mediated RhoA Activation Facilitates CCL2-Induced Chemotaxis in PC-3 Cells

Patients with advanced prostate cancer often exhibit increased activation of the coagulation system. The key activator of the coagulation cascade is the serine protease thrombin which is capable of eliciting numerous cellular responses. We previously reported that the thrombin receptor PAR1 is overexpressed in prostate cancer. To investigate further the role of PAR1 in prostate cancer metastasis, we examined the effects of thrombin activation on cell adhesion and motility in PC‐3 prostate cancer cells. Activation of PAR1‐induced dynamic cytoskeletal reorganization and reduced PC‐3 binding to collagen I, collagen IV, and laminin (P < 0.01) but not fibronectin. Expression of the cell surface integrin receptors did not change as assessed by flow cytometry. Immunofluorescence microscopy revealed that PAR1 stimulation caused reorganization of the focal adhesions, suggesting that PAR1 activation in PC‐3 cells may be modulating cell adhesion through integrin function but not expression. Furthermore, RhoA was activated upon stimulation with thrombin with subsequent cell contraction, decreased cell adhesion, and induced migration towards monocyte chemoattractant protein 1 (MCP‐1; CCL2). Thus, it appears that thrombin stimulation plays a role in prostate cancer metastasis by decreasing cell adhesion to the extracellular matrix and positioning the cell in a “ready state” for migration in response to a chemotactic signal. Further exploration is needed to determine whether PAR1 activation affects other signaling pathways involved in prostate cancer. J. Cell. Biochem. 101:1292–1300, 2007.

Co-inoculation of prostate cancer cells with U937 enhances tumor growth and angiogenesis in vivo.

Tumor‐associated macrophages (TAMs) have been implicated in promoting tumor growth and development. Here we present evidence that demonstrates that co‐inoculation of male athymic nude mice with PC‐3 prostate cancer cells and U937 promonocytic cells enhances tumor growth and increases tumor angiogenesis. Male athymic nude mice were co‐inoculated with PC‐3 and U937 cells (control or IL‐4 stimulated) and tumor growth was monitored over time. Immunohistochemical analysis of tumor specimens was performed for proliferation markers (e.g., Ki67) and the effects of IL‐4 stimulation on U937 cells were analyzed for chemokine expression. The presence of U937 cells increased the rate of tumor growth in vivo and stimulated increased microvascular density within the tumor bed. Stimulation of U937 cells with IL‐4 resulted in a significant increase in several pro‐angiogenic and pro‐tumor chemokines (e.g., CCL2). Co‐inoculation increases prostate cancer growth via upregulation of chemokines that induce angiogenesis within the tumor. J. Cell. Biochem. 103: 1–8, 2008.

AT‐101 (R‐(−)‐gossypol acetic acid) enhances the effectiveness of androgen deprivation therapy in the VCaP prostate cancer model

Prostate cancer remains a leading cause of cancer death in American men. Androgen deprivation therapy (ADT) is the most common treatment for advanced prostate cancer patients; however, ADT fails in nearly all cases resulting in castration resistant or androgen‐insensitive (AI) disease. In many cases, this progression results from dysregulation of the pro‐survival Bcl‐2 family proteins. Inhibition of pro‐survival Bcl‐2 family proteins, therefore, may be an effective strategy to delay the onset of AI disease. Gossypol, a small molecule inhibitor of pro‐survival Bcl‐2 family proteins, has been demonstrated to inhibit AI prostate cancer growth. The apoptotic effect of gossypol, however, has been demonstrated to be attenuated by the presence of androgen in a prostate cancer xenograft mouse model (Vertebral Cancer of Prostate [VCaP]) treated with AT‐101 (R‐(−)‐gossypol acetic acid). This study was undertaken to better understand the in vitro effects of androgen receptor (AR) on AT‐101‐induced apoptosis. VCaP cells treated with AT‐101 demonstrated an increase in apoptosis and downregulation of Bcl‐2 pro‐survival proteins. Upon AR activation in combination with AT‐101 treatment, apoptosis is reduced, cell survival increases, and caspase activation is attenuated. Akt and X inhibitor of apoptosis (XIAP) are downregulated in the presence of AT‐101, and AR stimulation rescues protein expression. Combination treatment of bicalutamide and AT‐101 increases apoptosis by reducing the expression of these pro‐survival proteins. These data suggest that combination therapy of AT‐101 and ADT may further delay the onset of AI disease, resulting in prolonged progression‐free survival of prostate cancer patients. J. Cell. Biochem. 110: 1187–1194, 2010. Published 2010 Wiley‐Liss, Inc.

Abstract 3369: Macrophages and their induced cytokines drive the epithelial to mesenchymal transition in prostate cancer cells

Macrophages derived from circulating monocytic myeloid precursors are a major component of leukocyte infiltrate found in tumors and their role in prostate cancer progression is now emerging. Here we investigate the potential of macrophages to induce the epithelial-mesenchymal transition (EMT) in prostate cancer cells. EMT is a critical process for metastasis, and the elucidation of factors that initiate EMT would be beneficial in the development of treatments to halt the dissemination of cancer cells throughout the body. CD14+-peripheral blood monocytes were isolated from healthy donors and stimulated with either interferon gamma (INFγ) or interleukin (IL)-4, factors known to promote the differentiation of monocytes into M1 or M2-type macrophages, respectively. After 48 hours, cells from prostate cancer cell lines PC3 Luc, DU145 Luc, and ARCAP Luc were co-cultured with the macrophages for four days. The cells were passaged three times by trypsination and protein lysates were analyzed by Western Blot for the expression of EMT markers: E-cadherin and vimentin. Our results revealed that cells co-cultured with IL-4-stimulated macrophages expressed lower levels of E-cadherin and higher levels of vimentin compared to control epithelial cells. In addition, three mesenchymal-type subpopulations that were isolated from PC3 cells interacting with IL-4-treated CD14+-cells exhibited a complete loss of E-cadherin. After more than 20 passages, these cells maintained the mesenchymal characteristics in culture and showed a striking up-regulation of the transcription factor ZEB 1, whose expression has been previously correlated with Gleason grade in human prostate tumors. Concurrently, we set out to study the secreted factors by which macrophages may trigger EMT. We treated DU145 Luc and PC3 Luc cells with several cytokines found to be differentially expressed in media containing cancer cells co-cultured with IL-4-stimulated macrophages and evaluated whether any of these factors induced EMT. It was discovered that cells grown in media containing the cytokine, IL-1B, in combination with IL-4 showed a greater display of mesenchymal markers than control cells. In conclusion, these results establish that macrophages can act as potent EMT-inducers in prostate cancer cells, although the exact mediators of this function remain unknown. The data suggests that CD14+ cells stimulated with IL-4 are more effective at triggering EMT than non-stimulated or INFγ-stimulated macrophages, which may pinpoint M2-type macrophages as a mediator of EMT in vivo. Furthermore, the cytokine study revealed that macrophage-induced IL-1B in combination with IL-4 may be an important factor in the mechanism of macrophage-induced EMT. As this transition may represent a major mechanism of prostate cancer metastasis, future research will be focused on elucidating the molecules involved in order to develop novel therapies. 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 3369. doi:10.1158/1538-7445.AM2011-3369

A scaleable multi-document centroid-based summarizer

We are presenting the most recent version of MEAD (v. 3.08), a large-scale public-domain summarizer that has been used in a number of applications, including the 2001 JHU summer workshop and the NewsInEssence project (www.newsinessence.com). A version of MEAD finished in first place on task 4 at DUC 2003 and finished in the top three on two other tasks.