Colony-stimulating factor (CSF)-1 is the primary regulator of tissue macrophage production. CSF-1 expression is correlated with poor prognosis in breast cancer and is believed to enhance mammary tumor progression and metastasis through the recruitment and regulation of tumor-associated macrophages. Macrophages produce matrix metalloproteases (MMPs) and vascular endothelial growth factor, which are crucial for tumor invasion and angiogenesis. Given the important role of CSF-1, we hypothesized that blockade of CSF-1 or the CSF-1 receptor (the product of the c-fms proto-oncogene) would suppress macrophage infiltration and mammary tumor growth. Human MCF-7 mammary carcinoma cell xenografts in mice were treated with either mouse CSF-1 antisense oligonucleotide for 2 weeks or five intratumoral injections of either CSF-1 small interfering RNAs or c-fms small interfering RNAs. These treatments suppressed mammary tumor growth by 50%, 45%, and 40%, respectively, and selectively down-regulated target protein expression in tumor lysates. Host macrophage infiltration; host MMP-12, MMP-2, and vascular endothelial growth factor A expression; and endothelial cell proliferation within tumors of treated mice were decreased compared with tumors in control mice. In addition, mouse survival significantly increased after CSF-1 blockade. These studies demonstrate that CSF-1 and CSF-1 receptor are potential therapeutic targets for the treatment of mammary cancer.

Overexpression of colony-stimulating factor-1 (CSF-1) and its receptor in breast cancer is correlated with poor prognosis. Based on the hypothesis that blockade of CSF-1 would be beneficial in breast cancer treatment, we developed a murinized, polyethylene glycol–linked antigen-binding fragment (Fab) against mouse (host) CSF-1 (anti-CSF-1 Fab). Mice bearing human, chemoresistant MCF-7 breast cancer xenografts were treated with combination chemotherapy (CMF: cyclophosphamide, methotrexate, 5-fluorouracil; cycled twice i.p.), anti-CSF-1 Fab (i.p., cycled every 3 days for 14 days), combined CMF and anti-CSF-1 Fab, or with Ringer's solution as a control. Anti-CSF-1 Fab alone suppressed tissue CSF-1 and retarded tumor growth by 40%. Importantly, in combination with CMF, anti-CSF-1 Fab reversed chemoresistance of MCF-7 xenografts, suppressing tumor development by 56%, down-regulating expression of the chemoresistance genes breast cancer–related protein, multidrug resistance gene 1, and glucosylceramide synthase, and prolonging survival significantly. Combined treatment also reduced angiogenesis and macrophage recruitment and down-regulated tumor matrix metalloproteinase-2 (MMP-2) and MMP-12 expression. These studies support the paradigm of CSF-1 blockade in the treatment of solid tumors and show that anti-CSF-1 antibodies are potential therapeutic agents for the treatment of mammary cancer. (Cancer Res 2006; 66(8): 4349-56)

The Raf-1 kinase plays a key role in relaying proliferation signals elicited by mitogens or oncogenes. Raf-1 is regulated by complex and incompletely understood mechanisms including phosphorylation. A number of studies have indicated that phosphorylation of serines 259 and 621 can inhibit the Raf-1 kinase. We show that both serines are hypophosphorylated during early mitogenic stimulation and that hypophosphorylation correlates with peak Raf-1 activation. Concentrations of okadaic acid that selectively inhibit protein phosphatase 2A (PP2A) induce phosphorylation of these residues and prevent maximal activation of the Raf-1 kinase. This effect is mediated via phosphorylation of serine 259. The PP2A core heterodimer forms complexes with Raf-1 in vivo and in vitro. These data identify PP2A as a positive regulator of Raf-1 activation and are the first indication that PP2A may support the activation of an associated kinase.

The interplay between malignant and stromal cells is essential in tumorigenesis. We have previously shown that colony-stimulating factor (CSF)-1, matrix metalloprotease (MMP)-2, and vascular endothelial growth factor (VEGF)-A production by stromal cells is enhanced by CSF-1–negative SW620 colon cancer cells. In the present study, the mechanisms by which colon cancer cells up-regulate host factors to promote tumorigenesis were investigated. Profiling of tumor cell cytokine expression in SW620 tumor xenografts in nude mice showed increased human tumor necrosis factor (TNF)-α mRNA expression with tumor growth. Incubation of macrophages with small interfering (si) RNAs directed against TNF-α or TNF-α–depleted SW620 cell conditioned medium versus SW620 cell conditioned medium failed to support mouse macrophage proliferation, migration, and expression of CSF-1, VEGF-A, and MMP-2 mRNAs. Consistent with these results, human TNF-α gene silencing decreased mouse macrophage TNF-α, CSF-1, MMP-2, and VEGF-A mRNA expression in macrophages cocultured with human cancer cells. In addition, inhibition of human TNF-α or mouse CSF-1 expression by siRNA reduced tumor growth in SW620 tumor xenografts in mice. These results suggest that colon cancer cell–derived TNF-α stimulates TNF-α and CSF-1 production by macrophages, and that CSF-1, in turn, induces macrophage VEGF-A and MMP-2 in an autocrine manner. Thus, interrupting tumor cell–macrophage communication by targeting TNF-α may provide an alternative therapeutic approach for the treatment of colon cancer. [Cancer Res 2007;67(3):1038–45]

—Cardiomyopathy (CM) comprises a heterogeneous group of diseases, including ischemic (ICM) and dilative (DCM) forms. The pathogenesis of primary DCM is not clearly understood. Recent studies in mice show that vascular endothelial growth factor (VEGF) is involved in ICM. Whether VEGF plays a role in human CM is unknown. We examined the mRNA and protein expression of VEGF and its receptors in hearts of patients with end-stage DCM and ICM and in healthy individuals using real-time polymerase chain reaction and Western blotting. Number of capillaries, area of myocytes, and collagen were calculated in cardiac biopsies using transmission electron microscopy. In DCM, except for VEGF-C, mRNA transcript levels of VEGF-A ₁₆₅ , VEGF-A ₁₈₉ , and VEGF-B and the protein level of VEGF-A and VEGF-R ₁ were downregulated compared with controls ( P <0.05). However, in ICM, mRNA transcript levels of VEGF isoforms and protein levels of VEGF-C were upregulated. The vascular density was decreased in DCM but increased in ICM compared with controls ( P <0.05). Muscular hypertrophy was not different for ICM and DCM, although DCM had more collagen ( P <0.05). Blunted VEGF-A and VEGF-R ₁ protein expression and downregulated mRNA of the predominant isoform of VEGF-A, VEGF-A ₁₆₅ , to our knowledge shown here for the first time, provide evidence that the VEGF-A defect in DCM is located upstream. Whether downregulation of certain VEGF isoforms in DCM is a cause or consequence of this disorder remains unclear, although upregulated VEGF levels in ICM are most likely the result of ischemia.

Mitogenic stimulation of Raf-1 is a complex yet incompletely understood process involving membrane relocalization and phosphorylation of activating residues. We recently reported that Raf-1-associated protein phosphatase 2A contributes to kinase activation, an effect mediated via Ser-259 of Raf-1. Here, we show that mitogens stimulate Ser-259 dephosphorylation and Raf-1/protein phosphatase 2A association concomitantly with membrane accumulation and activation of Raf-1. Blocking Ser-259 dephosphorylation inhibits the two latter events, but it does not prevent activation of a S259A Raf-1 mutant, which is preferentially localized at the membrane independently of mitogenic stimulation. Inhibition of Ser-259 dephosphorylation has no effect on the activation of membrane-tethered Raf-1 (Raf-1CAAX). These data show that Ser-259 dephosphorylation contributes to Raf-1 activation by supporting its membrane accumulation rather than by increasing the specific activity of the kinase and provide a mechanistic basis for the support of kinase activation by Raf-1-associated protein phosphatase 2A.