Ewing sarcoma-primitive neuroectodermal tumor (EWS) is associated with the most unfavorable prognosis of all primary musculoskeletal tumors. The objective of the present study was to investigate whether tumor-associated macrophages (TAMs) affect the development of EWS. TAMs were isolated from mouse xenografts using CD11b magnetic beads and examined for their cytokine expression and osteoclastic differentiation. To evaluate the role of TAMs in xenograft formation, liposome-encapsulated clodronate was used to deplete TAMs in mice. Macrophage infiltration and tumor microvascular density were histologically evaluated in 41 patients with EWS, and association with prognosis was examined using Kaplan-Meier survival analysis. In mouse EWS xenografts, TAMs expressed higher concentrations of cytokines including interleukin-6, keratinocyte-derived chemokine, and monocyte chemotactic protein-1. TAMs were more capable than normal monocytes of differentiating into tartrate-resistant acid phosphatase-positive giant cells. Depleting macrophages using liposome-encapsulated clodronate significantly inhibited development of EWS xenografts. In human EWS samples, higher levels of CD68-positive macrophages were associated with poorer overall survival. In addition, enhanced vascularity, increase in the amount of C-reactive protein, and higher white blood cell counts were also associated with poor prognosis and macrophage infiltration. TAMs seem to enhance the progression of EWS by stimulating both angiogenesis and osteoclastogenesis. Further investigation of the behavior of TAMs may lead to development of biologically targeted therapies for EWS.

EGF receptor (EGFR) kinase inhibitors, including gefitinib and erlotinib, exert potent therapeutic efficacy in non-small cell lung cancers harboring EGFR-activating mutations. However, most patients ultimately develop resistance to these drugs. Here, we report a novel mechanism of acquired resistance to EGFR tyrosine kinase inhibitors and the reversal of which could improve clinical outcomes. In erlotinib-resistant lung cancer cells harboring activating EGFR mutations that we established, there was increased expression of Src, integrin b1, a2, and a5 along with enhanced cell adhesion activity. Interestingly, RNAi-mediated silencing of integrin b1 restored erlotinib sensitivity and reduced activation of Src and Akt after erlotinib treatment. Furthermore, Src silencing inhibited Akt phosphorylation and cell growth, with this inhibitory effect further augmented by erlotinib treatment. Increased expression of integrin b1, a5, and/or a2 was also observed in refractory tumor samples from patients with lung cancer treated with erlotinib and/or gefitinib. Together, our findings identify the integrin b1/Src/Akt signaling pathway as a key mediator of acquired resistance to EGFR-targeted anticancer drugs. Cancer Res; 73(20); 6243-53. Ó2013 AACR.

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Most NSCLC patients with EGFR mutations benefit from treatment with EGFR-TKIs, but the clinical efficacy of EGFR-TKIs is limited by the appearance of drug resistance. Multiple kinase inhibitors of EGFR family proteins such as afatinib have been newly developed to overcome such drug resistance. We established afatinib-resistant cell lines after chronic exposure of activating EGFR mutation-positive PC9 cells to afatinib. Afatinib-resistant cells showed following specific characteristics as compared to PC9: [1] Expression of EGFR family proteins and their phosphorylated molecules was markedly downregulated by selection of afatinib resistance; [2] Expression of FGFR1 and its ligand FGF2 was alternatively upregulated; [3] Treatment with anti-FGF2 neutralizing antibody blocked enhanced phosphorylation of FGFR in resistant clone; [4] Both resistant clones showed collateral sensitivity to PD173074, a small-molecule FGFR-TKIs, and treatment with either PD173074 or FGFR siRNA exacerbated suppression of both afatinib-resistant Akt and Erk phosphorylation when combined with afatinib; [5] Expression of twist was markedly augmented in resistant sublines, and twist knockdown specifically suppressed FGFR expression and cell survival. Together, enhanced expression of FGFR1 and FGF2 thus plays as an escape mechanism for cell survival of afatinib-resistant cancer cells, that may compensate the loss of EGFR-driven signaling pathway.

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The focus of the present study was whether and how infiltrating macrophages play a role in angiogenesis and the growth of cancer cells in response to the inflammatory cytokine interleukin (IL)-1β β β β. Lewis lung carcinoma cells overexpressing IL-1β β β β grew faster and induced greater neovascularization than a low IL-1β β β β-expressing counterpart in vivo. When macrophages were depleted using clodronate liposomes, both neovascularization and tumor growth were reduced in the IL-1β β β β-expressing tumors. Co-cultivation of IL-1β β β β-expressing cancer cells with macrophages synergistically augmented neovascularization and the migration of vascular endothelial cells. In these co-cultures, production of the angiogenic factors vascular endothelial growth factor-A and IL-8, monocyte chemoattractant protein-1, and matrix metalloproteinase-9 were increased markedly. The production of these factors, induced by IL-1β β β β-stimulated lung cancer cells, was blocked by a nuclear factor (NF)-κ κ κ κB Inflammatory cells, cytokines, and chemokines in malignant tumors affect the stromal microenvironment, suggesting that inflammation and cancer may be interrelated through the angiogenic process.(4,5) During inflammation, angiogenesis often coincides with the infiltration of inflammatory cells such as neutrophils, monocytes, and macrophages, which secrete key cytokines and growth factors. (3,5) Tumor-associated macrophages produce various pro-angiogenic cytokines and matrix-degrading proteinases, providing cancer cells and vascular endothelial cells with favorable conditions for proliferation, migration, angiogenesis, and tissue remodeling. We have previously reported that Sp1 and AP-1 motifs and NF-κB are prerequisites for the transcription of VEGF-A in response to inflammatory cytokines in cancer cells and vascular endothelial cells. (11,12) Expression of IL-8 is also highly inducible by inflammatory cytokines and oxygen stress, and NF-κB, AP-1, and NF-IL-6 have been implicated in this. (12,(15)(16)(17) We previously demonstrated that IL-1α and IL-1β enhance the production of VEGF and IL-8 in monocytes and macrophages, which results in the promotion of vascular endothelial cell migration.(15) Moreover, IL-1β can induce angiogenesis in vitro and in vivo by activating the COX2-prostanoid pathway following the expression of prostaglandin E2 and thromboxane A2, and this angiogenesis is blocked specifically by COX2-selective inhibitors.(18) We further demonstrated that the infiltration of macrophages is a prerequisite for IL-1β-induced angiogenesis. (18) Although this evidence has established a role for the inflammatory responses induced by TAM in the acquisition of malignant characteristics during cancer progression, exactly how TAM affect tumor angiogenesis and growth by interacting with cancer cells is still unclear. In the present study, we further investigated the mechanisms by which TAM affect tumor growth and angiogenesis. Materials and MethodsCell culture and reagents. The macrophage cell line U937 and human lung canc...

Y-box binding protein-1 (YB-1) plays pivotal roles in acquisition of global drug resistance and cell growth promotion through transcriptional activation of genes for both drug resistance and growth factor receptors. In this study, we investigated whether YB-1 is involved in regulation of the cell cycle and cell proliferation of human cancer cells. Treatment with YB-1 siRNA caused a marked suppression of cell proliferation and expression of a cell cycle related gene, CDC6 by cancer cells. Of cell cycle of cancer cells, S phase content was specifically reduced by knockdown of YB-1. The overexpression of CDC6 abrogated this inhibition of both cell proliferation and S phase entry. ChIP assay demonstrated that YB-1 binds to a Y-box located in the promoter region of the CDC6 gene. Expression of cyclin D1, CDK1 and CDK2 was also reduced with increased expression of p21(Cip1) and p16(INK4A) when treated with YB-1 siRNA. Furthermore, the nuclear YB-1 expression was significantly associated with the level of CDC6 nuclear expression in patients with breast cancer. In conclusion, YB-1 plays an important role in cell cycle progression at G1/S of human cancer cells. YB-1 thus could be a potent biomarker for tumour growth and cell cycle in its close association with CDC6.

Y‐box binding protein‐1 ( YBX 1), a multifunctional oncoprotein containing an evolutionarily conserved cold shock domain, dysregulates a wide range of genes involved in cell proliferation and survival, drug resistance, and chromatin destabilization by cancer. Expression of a multidrug resistance‐associated ATP binding cassette transporter gene, ABCB 1 , as well as growth factor receptor genes, EGFR and HER 2/ErbB2 , was initially discovered to be transcriptionally activated by YBX 1 in cancer cells. Expression of other drug resistance‐related genes, MVP / LRP , TOP 2A , CD 44 , CD 49f , BCL 2 , MYC , and androgen receptor ( AR ), is also transcriptionally activated by YBX 1, consistently indicating that YBX 1 is involved in tumor drug resistance. Furthermore, there is strong evidence to support that nuclear localization and/or overexpression of YBX 1 can predict poor outcomes in patients with more than 20 different tumor types. YBX 1 is phosphorylated by kinases, including AKT , p70S6K, and p90 RSK , and translocated into the nucleus to promote the transcription of resistance‐ and malignancy‐related genes. Phosphorylated YBX 1, therefore, plays a crucial role as a potent transcription factor in cancer. Herein, a novel anticancer therapeutic strategy is presented by targeting activated YBX 1 to overcome drug resistance and malignant progression.