Karin Zins

Colony-Stimulating Factor-1 Blockade by Antisense Oligonucleotides and Small Interfering RNAs Suppresses Growth of Human Mammary Tumor Xenografts in Mice

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.

Colon Cancer Cell–Derived Tumor Necrosis Factor-α Mediates the Tumor Growth–Promoting Response in Macrophages by Up-regulating the Colony-Stimulating Factor-1 Pathway

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)-alpha mRNA expression with tumor growth. Incubation of macrophages with small interfering (si) RNAs directed against TNF-alpha or TNF-alpha-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-alpha gene silencing decreased mouse macrophage TNF-alpha, CSF-1, MMP-2, and VEGF-A mRNA expression in macrophages cocultured with human cancer cells. In addition, inhibition of human TNF-alpha 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-alpha stimulates TNF-alpha 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-alpha may provide an alternative therapeutic approach for the treatment of colon cancer.

A Rac1/Cdc42 GTPase-specific small molecule inhibitor suppresses growth of primary human prostate cancer xenografts and prolongs survival in mice.

Deregulated Rho GTPases Rac1 and Cdc42 have been discovered in various tumors, including prostate and Rac protein expression significantly increases in prostate cancer. The Rac and Cdc42 pathways promote the uncontrolled proliferation, invasion and metastatic properties of human cancer cells. We synthesized the novel compound AZA1 based on structural information of the known Rac1 inhibitor NSC23766. In the current study we investigated the effects of inhibition of these pathways by AZA1 on prostate tumorigenicity by performing preclinical studies using a xenograft mouse model of prostate cancer. In androgen-independent prostate cancer cells, AZA1 inhibited both Rac1 and Cdc42 but not RhoA GTPase activity in a dose-dependent manner and blocked cellular migration and proliferation. Cyclin D1 expression significantly decreased following Rac1/Cdc42 inhibition in prostate cancer cells. AZA1 treatment also down-regulated PAK and AKT activity in prostate cancer cells, associated with induction of the pro-apoptotic function of BAD by suppression of serine-112 phosphorylation. Daily systemic administration of AZA1 for 2 weeks reduced growth of human 22Rv1 prostate tumor xenografts in mice and improved the survival of tumor-bearing animals significantly. These data suggest a role of AZA1 in blocking Rac1/Cdc42-dependent cell cycle progression, cancer cell migration and increase of cancer cell apoptosis involving down-regulation of the AKT and PAK signaling pathway in prostate cancer cells. We therefore propose that a small-molecule inhibitor therapy targeting Rac1/Cdc42 Rho GTPase signaling pathways may be used as a novel treatment for patients with advanced prostate cancer.

Stromal cell-derived CSF-1 blockade prolongs xenograft survival of CSF-1-negative neuroblastoma

The molecular mechanisms of tumor–host interactions that render neuroblastoma (NB) cells highly invasive are unclear. Cancer cells upregulate host stromal cell colony‐stimulating factor‐1 (CSF‐1) production to recruit tumor‐associated macrophages (TAMs) and accelerate tumor growth by affecting extracellular matrix remodeling and angiogenesis. By coculturing NB with stromal cells in vitro, we showed the importance of host CSF‐1 expression for macrophage recruitment to NB cells. To examine this interaction in NB in vivo, mice bearing human CSF‐1‐expressing SK‐N‐AS and CSF‐1‐negative SK‐N‐DZ NB xenografts were treated with intratumoral injections of small interfering RNAs directed against mouse CSF‐1. Significant suppression of both SK‐N‐AS and SK‐N‐DZ NB growth by these treatments was associated with decreased TAM infiltration, matrix metalloprotease (MMP)‐12 levels and angiogenesis compared to controls, while expression of tissue inhibitors of MMPs increased following mouse CSF‐1 blockade. Furthermore, Tie‐2‐positive and ‐negative TAMs recruited by host CSF‐1 were identified in NB tumor tissue by confocal microscopy and flow cytometry. However, host‐CSF‐1 blockade prolonged survival only in CSF‐1‐negative SK‐N‐DZ NB. These studies demonstrated that increased CSF‐1 production by host cells enhances TAM recruitment and NB growth and that the CSF‐1 phenotype of NB tumor cells adversely affects survival.

Colony-stimulating factor-1 transfection of myoblasts improves the repair of failing myocardium following autologous myoblast transplantation

AIMS Skeletal myoblasts are used in repair of ischaemic myocardium. However, a large fraction of grafted myoblasts degenerate upon engraftment. Colony-stimulating factor-1 (CSF-1) accelerates myoblast proliferation and angiogenesis. We hypothesized that CSF-1 overexpression improves myoblast survival and cardiac function in ischaemia-induced heart failure. METHODS AND RESULTS Three weeks following myocardial infarction, rats developed heart failure and received intramyocardial injections of mouse CSF-1-transfected or untransfected primary autologous rat myoblasts, recombinant human CSF-1, mouse CSF-1 expressing plasmids, or culture medium. Tissue gene and protein expression was measured by quantitative RT-PCR (reverse transcription-polymerase chain reaction) and western blotting. Fluorescence imaging and immunocytochemistry were used to analyse myoblasts, endothelial cells, macrophages, and infarct wall thickening. Electrocardiograms were recorded online using a telemetry system. Left ventricular function was assessed by echocardiography over time, and improved significantly only in the CSF-1-overexpressing myoblast group. CSF-1-overexpression enhanced myoblast numbers and was associated with an increased infarct wall thickness, enhanced angiogenesis, increased macrophage recruitment and upregulated matrix metalloproteases (MMP)-2 and -12 in the zone bordering the infarction. Transplantation of CSF-1-overexpressing myoblasts did not result in major arrhythmias. CONCLUSION Autologous intramyocardial transplantation of CSF-1 overexpressing myoblasts might be a novel strategy in the treatment of ischaemia-induced heart failure.

Targeting Cdc42 with the small molecule drug AZA197 suppresses primary colon cancer growth and prolongs survival in a preclinical mouse xenograft model by downregulation of PAK1 activity

BackgroundRho GTPases play important roles in cytoskeleton organization, cell cycle progression and are key regulators of tumor progression. Strategies to modulate increased Rho GTPase activities during cancer progression could have therapeutic potential.MethodsWe report here the characterization of a Cdc42-selective small-molecule inhibitor AZA197 for the treatment of colon cancer that was developed based on structural information known from previously developed compounds affecting Rho GTPase activation. We investigated the effects of AZA197 treatment on RhoA, Rac1 and Cdc42 activities and associated molecular mechanisms in colon cancer cells in vitro. Therapeutic effects of AZA197 were examined in vivo using a xenograft mouse model of SW620 human colon cancer cells. After treatment, tumors were excised and processed for Ki-67 staining, TUNEL assays and Western blotting to evaluate proliferative and apoptotic effects induced by AZA197.ResultsIn SW620 and HT-29 human colon cancer cells, AZA197 demonstrated selectivity for Cdc42 without inhibition of Rac1 or RhoA GTPases from the same family. AZA197 suppressed colon cancer cell proliferation, cell migration and invasion and increased apoptosis associated with down-regulation of the PAK1 and ERK signaling pathways in vitro. Furthermore, systemic AZA197 treatment reduced tumor growth in vivo and significantly increased mouse survival in SW620 tumor xenografts. Ki-67 staining and tissue TUNEL assays showed that both inhibition of cell proliferation and induction of apoptosis associated with reduced PAK/ERK activation contributed to the AZA197-induced therapeutic effects in vivo.ConclusionsThese data indicate the therapeutic potential of the small-molecule inhibitor AZA197 based on targeting Cdc42 GTPase activity to modulate colorectal cancer growth.

Analysis of the rs10046 Polymorphism of Aromatase (CYP19) in Premenopausal Onset of Human Breast Cancer

The CYP19 gene encodes aromatase, an enzyme catalyzing the conversion of androgens to estrogens. Studies analyzing associations between single nucleotide polymorphisms in CYP19 and breast cancer risk have shown inconsistent results. The rs10046 polymorphism is located in the 3′ untranslated region of the CYP19 gene, but the influence of this polymorphism on breast cancer risk is unclear. In this study, we investigated the impact of rs10046 SNP on breast cancer risk, age at onset and association with clinical characteristics in an Austrian population of 274 breast cancer patients and 253 controls. The results show that a significantly increased fraction of patients with the TT genotype of rs10046 develop breast cancer under the age of 50 (41.8% of TT patients, compared to 26.6% of C carriers; p = 0.018, Chi-square test). No rs10046 genotypes were significantly associated with increased breast cancer risk or patient characteristics other than age at onset. These results suggest that the rs10046 polymorphism in the CYP19 gene may have an effect on breast cancer susceptibility at an age under 50 in the investigated population.

Adenoviral-Mediated Endothelial Precursor Cell Delivery of Soluble CD115 Suppresses Human Prostate Cancer Xenograft Growth in Mice

Prostate cancer tumor growth and neovascularization is promoted by an interplay between migratory tumor stromal cells such as specialized tumor‐associated macrophages (TAMs) and circulating endothelial precursor cells (CEPs). As vehicles for tumor therapy, human CEPs are relatively easy to isolate from peripheral blood, are able to proliferate long‐term in vitro, are amenable to viral manipulation, and preferentially home to regions of ischemia found in growing tumors. We show here that human peripheral blood CEPs expanded ex vivo migrate to prostate cancer cells in vitro and efficiently home to human prostate tumor xenografts in vivo. Infection of precursors ex vivo with an adenovirus constructed to secrete a soluble form of the colony‐stimulating factor‐1 receptor CD115 that inhibits macrophage viability and migration in vitro significantly decreases the number of TAMs in xenografts (p < .05), reduces proliferation (p < .01) and vascular density (p < .03), and suppresses the growth of xenografts (p < .03). These data show for the first time that targeting stromal cell processes with cellular therapy has the potential to retard prostate tumor growth. STEM CELLS 2009;27:2342–2352

Host CD147 blockade by small interfering RNAs suppresses growth of human colon cancer xenografts

Tumor cells can stimulate matrix metalloproteinase (MMP) production by stromal cells through cell-cell interactions mediated by cell adhesion molecules such as extracellular matrix metalloproteinase inducer (human CD147/EMMPRIN, mouse CD147/Basigin). This study sought to characterize whether specific tumor-stromal cell interactions mediated by CD147 promote colon cancer growth by utilizing small interfering (si)RNAs directed against human CD147/EMMPRIN or mouse CD147/Basigin in co-cultures of cancer cells with macrophages and fibroblasts and established human SW620 colon cancer xenograft models in immune deficient mice. We show that blockade of host (mouse) CD147/Basigin expression, but not cancer cell-derived CD147/EMMPRIN, suppresses tumor growth in human colon cancer xenografts. Experiments in vitro indicated that colon cancer cell-stromal cell interactions mediated by CD147 lead to increased MMP-2 expression in fibroblasts but not macrophages. Furthermore, expression of host VEGF-A in both fibroblasts and macrophages is independent of CD147 in vitro and in vivo. Interestingly, inhibition of cancer cell-derived EMMPRIN leads to increased MMP-9 levels in vivo. Our findings provide new insights into CD147-mediated tumor-host interactions mediating colon cancer growth.

Toll-like receptor 3 signalling mediates angiogenic response upon shock wave treatment of ischaemic muscle

AIMS Shock wave therapy (SWT) represents a clinically widely used angiogenic and thus regenerative approach for the treatment of ischaemic heart or limb disease. Despite promising results in preclinical and clinical trials, the exact mechanism of action remains unknown. Toll-like receptor 3, which is part of the innate immunity, is activated by binding double-stranded (ds) RNA. It plays a key role in inflammation, a process that is needed also for angiogenesis. We hypothesize that SWT causes cellular cavitation without damaging the target cells, thus liberating cytoplasmic RNA that in turn activates TLR3. METHODS AND RESULTS SWT induces TLR3 and IFN-β1 gene expression as well as RNA liberation from endothelial cells in a time-dependant manner. Conditioned medium from SWT-treated HUVECs induced TLR3 signalling in reporter cells. The response was lost when the medium was treated with RNase III to abolish dsRNAs or when TLR3 was silenced using siRNAs. In a mouse hind limb ischaemia model using wt and TLR3(-/-) mice (n = 6), SWT induced angiogenesis and arteriogenesis only in wt animals. These effects were accompanied by improved blood perfusion of treated limbs. Analysis of main molecules of the TLR3 pathways confirmed TLR3 signalling in vivo following SWT. CONCLUSION Our data reveal a central role of the innate immune system, namely Toll-like receptor 3, to mediate angiogenesis upon release of cytoplasmic RNAs by mechanotransduction of SWT.