David Ketelsen

Molecular Characterization of Human Breast Tumor Vascular Cells

A detailed understanding of the assortment of genes that are expressed in breast tumor vessels is needed to facilitate the development of novel, molecularly targeted anti-angiogenic agents for breast cancer therapies. Rapid immunohistochemistry using factor VIII-related antibodies was performed on sections of frozen human luminal-A breast tumors (n = 5) and normal breast (n = 5), followed by laser capture microdissection of vascular cells. RNA was extracted and amplified, and fluorescently labeled cDNA was synthesized and hybridized to 44,000-element long-oligonucleotide DNA microarrays. Statistical analysis of microarray was used to compare differences in gene expression between tumor and normal vascular cells, and Expression Analysis Systematic Explorer was used to determine enrichment of gene ontology categories. Protein expression of select genes was confirmed using immunohistochemistry. Of the 1176 genes that were differentially expressed between tumor and normal vascular cells, 55 had a greater than fourfold increase in expression level. The extracellular matrix gene ontology category was increased while the ribosome gene ontology category was decreased. Fibroblast activation protein, secreted frizzled-related protein 2, Janus kinase 3, and neutral sphingomyelinase 2 proteins localized to breast tumor endothelium as assessed by immunohistochemistry, showing significantly greater staining compared with normal tissue. These tumor endothelial marker proteins also exhibited increased expression in breast tumor vessels compared with that in normal tissues. Therefore, these genetic markers may serve as potential targets for the development of angiogenesis inhibitors.

Secreted Frizzle-Related Protein 2 Stimulates Angiogenesis via a Calcineurin/NFAT Signaling Pathway

Secreted frizzle-related protein 2 (SFRP2), a modulator of Wnt signaling, has recently been found to be overexpressed in the vasculature of 85% of human breast tumors; however, its role in angiogenesis is unknown. We found that SFRP2 induced angiogenesis in the mouse Matrigel plug assay and the chick chorioallantoic membrane assay. SFRP2 inhibited hypoxia induced endothelial cell apoptosis, increased endothelial cell migration, and induced endothelial tube formation. The canonical Wnt pathway was not affected by SFRP2 in endothelial cells; however, a component of the noncanonical Wnt/Ca2+ pathway was affected by SFRP2 as shown by an increase in NFATc3 in the nuclear fraction of SFRP2-treated endothelial cells. Tacrolimus, a calcineurin inhibitor that inhibits dephosphorylation of NFAT, inhibited SFRP2-induced endothelial tube formation. Tacrolimus 3 mg/kg/d inhibited the growth of SVR angiosarcoma xenografts in mice by 46% (P = 0.04). In conclusion, SFRP2 is a novel stimulator of angiogenesis that stimulates angiogenesis via a calcineurin/NFAT pathway and may be a favorable target for the inhibition of angiogenesis in solid tumors.

The Role of Calcineurin/NFAT in SFRP2 Induced Angiogenesis—A Rationale for Breast Cancer Treatment with the Calcineurin Inhibitor Tacrolimus

Tacrolimus (FK506) is an immunosuppressive drug that binds to the immunophilin FKBPB12. The FK506-FKBP12 complex associates with calcineurin and inhibits its phosphatase activity, resulting in inhibition of nuclear translocation of nuclear factor of activated T-cells (NFAT). There is increasing data supporting a critical role of NFAT in mediating angiogenic responses stimulated by both vascular endothelial growth factor (VEGF) and a novel angiogenesis factor, secreted frizzled-related protein 2 (SFRP2). Since both VEGF and SFRP2 are expressed in breast carcinomas, we hypothesized that tacrolimus would inhibit breast carcinoma growth. Using IHC (IHC) with antibodies to FKBP12 on breast carcinomas we found that FKBP12 localizes to breast tumor vasculature. Treatment of MMTV-neu transgenic mice with tacrolimus (3 mg/kg i.p. daily) (n = 19) resulted in a 73% reduction in the growth rate for tacrolimus treated mice compared to control (n = 15), p = 0.003; which was associated with an 82% reduction in tumor microvascular density (p<0.001) by IHC. Tacrolimus (1 µM) inhibited SFRP2 induced endothelial tube formation by 71% (p = 0.005) and inhibited VEGF induced endothelial tube formation by 67% (p = 0.004). To show that NFATc3 is required for SFRP2 stimulated angiogenesis, NFATc3 was silenced with shRNA in endothelial cells. Sham transfected cells responded to SFRP2 stimulation in a tube formation assay with an increase in the number of branch points (p<0.003), however, cells transfected with shRNA to NFATc3 showed no increase in tube formation in response to SFRP2. This demonstrates that NFATc3 is required for SFRP2 induced tube formation, and tacrolimus inhibits angiogenesis in vitro and breast carcinoma growth in vivo. This provides a rationale for examining the therapeutic potential of tacrolimus at inhibiting breast carcinoma growth in humans.

2-methoxyestradiol inhibits the anaphase-promoting complex and protein translation in human breast cancer cells.

2-methoxyestradiol (2ME2), an estradiol metabolite with antiproliferative and antiangiogenic activities, is in phase I/II clinical trials for breast cancer. 2ME2 inhibits microtubule polymerization and causes cells to arrest in G2-M. The purpose of this study was to further elucidate the molecular mechanism of 2ME2. MDA-MB-435 breast cancer cells were treated with 2ME2 (2 micromol/L) or vehicle alone. RNA was extracted and genomic profiling was done using 22k Agilent microarrays. Expression Analysis Systematic Explorer was used to determine enrichment of Gene Ontology categories. Protein isolates were subjected to Western blot analysis. Protein synthesis was measured with a [35S]methionine pulse assay. An MDA-MB-435 cell line with two beta-tubulin mutations (2ME2R) was used to determine whether novel mechanisms were tubulin-dependent. Gene Ontology categories enriched include genes that regulate the mitotic spindle assembly checkpoint, apoptosis, and the cytosolic ribosome. The target of the mitotic spindle assembly checkpoint is the anaphase-promoting complex (APC). APC inhibition was confirmed by measuring protein levels of its targets securin and cyclin B1, which were increased in 2ME2-treated cells. Because gene expression in the cytosolic ribosome category was decreased, we evaluated whether 2ME2 decreases protein translation. This was confirmed with a pulse assay, which showed decreased isotope incorporation in 2ME2-treated cells, which was maintained in the tubulin-resistant 2ME2R cells. APC inhibition was not maintained in 2ME2R cells. 2ME2 induces tubulin-dependent cell cycle arrest through regulation of genes involved in the mitotic spindle assembly checkpoint, which results in inhibition of the APC and tubulin-independent inhibition of protein translation.

A Novel Monoclonal Antibody to Secreted Frizzled-Related Protein 2 Inhibits Tumor Growth

Secreted frizzled-related protein 2 (SFRP2) is overexpressed in human angiosarcoma and breast cancer and stimulates angiogenesis via activation of the calcineurin/NFATc3 pathway. There are conflicting reports in the literature as to whether SFRP2 is an antagonist or agonist of β-catenin. The aims of these studies were to assess the effects of SFRP2 antagonism on tumor growth and Wnt-signaling and to evaluate whether SFRP2 is a viable therapeutic target. The antiangiogenic and antitumor properties of SFRP2 monoclonal antibody (mAb) were assessed using in vitro proliferation, migration, tube formation assays, and in vivo angiosarcoma and triple-negative breast cancer models. Wnt-signaling was assessed in endothelial and tumor cells treated with SFRP2 mAb using Western blotting. Pharmacokinetic and biodistribution data were generated in tumor-bearing and nontumor-bearing mice. SFRP2 mAb was shown to induce antitumor and antiangiogenic effects in vitro and inhibit activation of β-catenin and nuclear factor of activated T-cells c3 (NFATc3) in endothelial and tumor cells. Treatment of SVR angiosarcoma allografts in nude mice with the SFRP2 mAb decreased tumor volume by 58% compared with control (P = 0.004). Treatment of MDA-MB-231 breast carcinoma xenografts with SFRP2 mAb decreased tumor volume by 52% (P = 0.03) compared with control, whereas bevacizumab did not significantly reduce tumor volume. Pharmacokinetic studies show the antibody is long circulating in the blood and preferentially accumulates in SFRP2-positive tumors. In conclusion, antagonizing SFRP2 inhibits activation of β-catenin and NFATc3 in endothelial and tumor cells and is a novel therapeutic approach for inhibiting angiosarcoma and triple-negative breast cancer. Mol Cancer Ther; 12(5); 685–95. ©2013 AACR.

Ultrasound molecular imaging of secreted frizzled related protein-2 expression in murine angiosarcoma

Angiosarcoma is a biologically aggressive vascular malignancy with a high metastatic potential. In the era of targeted medicine, knowledge of specific molecular tumor characteristics has become more important. Molecular imaging using targeted ultrasound contrast agents can monitor tumor progression non-invasively. Secreted frizzled related protein 2 (SFRP2) is a tumor endothelial marker expressed in angiosarcoma. We hypothesize that SFRP2-directed imaging could be a novel approach to imaging the tumor vasculature. To develop an SFRP2 contrast agent, SFRP2 polyclonal antibody was biotinylated and incubated with streptavidin-coated microbubbles. SVR angiosarcoma cells were injected into nude mice, and when tumors were established the mice were injected intravenously with the SFRP2 -targeted contrast agent, or a control streptavidin-coated contrast agent. SFRP2 -targeted contrast agent detected tumor vasculature with significantly more signal intensity than control contrast agent: the normalized fold-change was 1.6±0.27 (n = 13, p = 0.0032). The kidney was largely devoid of echogenicity with no significant difference between the control contrast agent and the SFRP2-targeted contrast agent demonstrating that the SFRP2-targeted contrast agent was specific to tumor vessels. Plotting average pixel intensity obtained from SFRP2-targeted contrast agent against tumor volume showed that the average pixel intensity increased as tumor volume increased. In conclusion, molecularly-targeted imaging of SFRP2 visualizes angiosarcoma vessels, but not normal vessels, and intensity increases with tumor size. Molecular imaging of SFRP2 expression may provide a rapid, non-invasive method to monitor tumor regression during therapy for angiosarcoma and other SFRP2 expressing cancers, and contribute to our understanding of the biology of SFRP2 during tumor development and progression.

The Calcineurin Inhibitor Tacrolimus Inhibits Breast Tumor Growth In Vivo.

Background: We recently reported a novel angiogenesis factor, secreted frizzle-related protein 2 (SFRP2), that stimulates angiogenesis via a calcineurin/ NFAT pathway and is highly expressed in human breast tumor endothelium (Courtwight et. al., Cancer Research 2009). NFAT is a transcription factor that mediates vascular development and VEGF and bFGF-induced angiogenesis. Tacrolimus (FK506) is a calcineurin inhibitor that inhibits NFAT activation and is FDA approved to prevent organ transplant rejection. Based on its mechanism of action, we hypothesized that tacrolimus would inhibit angiogenesis and breast tumor growth.Materials and Methods:Tumor growth in vivo: MMTV-neu transgenic mice were treated with tacrolimus 3 mg/kg/day i.p. for 21 days or control when tumors became palpable. Tumor volumes were measured with 3D ultrasound. Endothelial tube formation in vitro: ECMatrix was solidified into wells of a 96 well plate and endothelial cells were seeded onto the matrix at a concentration of 1x104 cells/well with SFRP2 7nM or VEGFA 60ng/ml. Tacrolimus 1µM or 1.5% DMSO control was added to the wells. The number of branch points were counted after 8 hours. MMTV-neu migration in vitro: Breast tumor cells were plated at 10,000 cells/well into a 96 well plate and allowed to become confluent. The cells were quiesced for 18 hours and tacrolimus (1µM) or 0.5% DMSO control was added to the wells and the wound was formed using a 1 ml pipette tip. Migration distance was measured with an ocular micrometer after 16 hours. Western blot analyses: Endothelial cells were treated for 1 hour with negative control, SFRP2 7nM, or SFRP2 7nM plus tacrolimus 10 µM. Nuclear protein was extracted and Western blot for NFATc3 was performed. Statistics: Means were compared with a two-tailed t-test.Results:Tumor growth in vivo: Tacrolimus inhibited the growth rate of MMTV-neu tumors by 49% (n=12 treated, n=9 controls, p=0.04), without signs of toxicity. Endothelial tube formation: Tacrolimus (1µM) inhibited SFRP2 induced endothelial tube formation by 64% (0.002, n=4), and VEGF induced tube formation by 69% (p=0.004, n=4). MMTV-neu tumor cell migration: Tacrolimus (1µM) inhibited MMTV-neu cell migration by 36% (p Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3170.

P3-16-04: A Novel Monoclonal Antibody to Secreted Frizzled Related Protein 2 Inhibits Triple Negative Breast Carcinoma Growth Rate In Vivo.

Background: Secreted frizzled related protein 2 (SFRP2) is a novel angiogenesis factor expressed in the endothelium of a wide variety of human tumors including triple negative breast cancer and angiosarcoma. We previously reported generating a monoclonal antibody against SFRP2 that inhibits endothelial cell and angiosarcoma tube formation in vitro , and decreased tumor volume of the SVR angiosarcoma in vivo. The objectives of these studies were to determine pharmacokinetic (PK) and pharmacodynamic (PD) parameters of the SFRP2 MAb, and evaluate its efficacy in a triple negative breast cancer xenograft. Methods: 125 I-SFRP2 MAb was administered to nude mice i.v. via tail vein injections at 0.4 mg/kg, 4 mg/kg, or 10 mg/kg in mice with or without tumor. Blood organ, and tumor samples were collected at various time points from 5 min to 21 days. Radiolabeled SFRP2 MAb in serum and tissues was determined using a gamma counter. PK parameters were determined based on mean concentration values for 3–5 animals per time point. In vivo efficacy study: MDA-MB-231 human breast cancer xenografts were established in 6-week-old female nude mice. Mice were inoculated with 1 × 10 6 cells s.c.. Treatment began on day 16 after tumor inoculation when average tumor size was 200 mm 3 . Animals were randomly assigned (n = 12 per group) to buffer control, SFRP2 MAb 4 mg/kg iv twice weekly; Avastin (Roch) 5 mg/kg iv twice weekly, or IGg control 4 mg/kg iv twice weekly. Tumors were harvested when the tumor diameter reached 2 cm or at 28 days. Tumor volumes were measured with a caliper. Growth rates (percent change per day) were compared with the formula ((Final volume- initial volume)/ initial volume) x 100 / number of days. Differences in growth rate between treated and control were analyzed with a two-tailed t-test. Results: PK and PD: SFRP2 MAb was long circulating in the blood with an average t 1/2 in the range of 53–89 hr. In addition, the SFPR2 MAb was found to preferentially target the tumors versus all other organs except for the liver. For example, in tumor bearing mice, the blood/tissue ratio on day 14 was smallest in the liver (15:1) and tumor (16:1) as compared to all other organs (range: 39:1 to 255:1) proving that the tumor was a prime organ for accumulation of the SFRP2 MAb. The SFPRP2 MAb in tumor bearing and non-tumor bearing mice exhibited dose-independent kinetics as a one-way ANOVA analysis comparing t 1/2 at different dose levels was not statistically significant (p=0.2847 and 0.1204, respectively). However, there was statistically significant difference in t 1/2 of the SFPR2 MAb in tumor-bearing and non-tumor bearing mice (p=0.0386). Efficacy in triple negative breast cancer: There was a 40% decrease in growth rate between SFRP2 MAb and control (p=0.03) and a 20% inhibition of growth rate between Avastin and control (p=0.40). The IgG negative control had no effect on tumor growth. Conclusion: The SFRP2 MAb was long circulating and the tumor was a prime organ for accumulation of the SFRP2 MAb. SFRP2 MAb slowed the growth of a human triple negative breast cancer xenograft in a tumor model that was not sensitive to Avastin. We conclude that SFRP2 is a novel therapeutic target for breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-16-04.

Abstract 4410: Apoptosis in Ovarian Cancer Cells Induced by Polyclonal Antibodies to Secreted Frizzle-related Protein 2

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Objectives: We have previously discovered a novel angiogenesis factor, secreted frizzle-related protein 2 (SFRP2), that is strongly expressed in many human tumors including ovarian carcinoma. SFRP2 protects against endothelial cell apoptosis, and induces endothelial tube formation and migration. We hypothesize that in addition to its paracrine role in stimulating angiogenesis, SFRP2 also has a direct effect in tumor cells, and therefore blocking SFRP2 will induce ovarian cancer apoptosis. Methods: Protein expression of SFRP2 in ovarian cancer cells: SKOV-3 and OVCA −3 ovarian cancer cells were cultured in McCoys medium with 10% bovine serum albumin. Whole cell protein lysates were obtained and Western blot analysis was performed probing for SFRP2. Apoptosis assay: SKOV3 cells were plated in 96 well plates and allowed to attach overnight. Rabbit polyclonal antibody to SFRP2 (SFRP2 Ab) was run through a column with a saline buffer to remove the sodium azide preservative. Cells were treated with antibody in 1:10, 1:100, and 1:1000 concentrations. McCoys culture medium with 1% saline was used as a negative control. Cells were incubated at 37°C for 48 hours. Apoptosis was measured by the activity of cleaved caspase-3 by using a caspase-specific fluorogenic substrate. Statistical analysis was performed with a two-tailed students t-test. Results: SKOV-3 and OVCA-3 ovarian cancer cells strongly express SFRP2 on Western Blot analysis. When SKOV-3 cells were treated with polyclonal antibody to SFRP2 (1:10) there was a 47% increase in apoptosis as compared with negative controls (control = 1509 RFU ± 50, SFRP2 Ab =2221 RFU ± 39, n=4 for both groups, p<0.0001). Conclusions: SFRP2 is a protein endogenously expressed by ovarian carcinoma. Blocking SFRP2 with a polyclonal antibody induces apoptosis in SKOV-3 ovarian cancer cell line, suggesting that this protein may be a potential target in the treatment of ovarian cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4410.