Targeting Angiogenesis and the Tumor Microenvironment

The role of the microenvironment during the initiation and progression of malignancy is appreciated to be of critical importance for improved molecular diagnostics and therapeutics. The tumor microenvironment is the product of a crosstalk between different cells types. Critical elements in the microenvironment include tumor associated fibroblasts, which provide an essential communication network via secretion of growth factors and chemokines, inducing an altered extracellular matrix (ECM), thereby providing additional oncogenic signals that enhance cancer-cell proliferation and invasion. Active contribution of tumor-associated stromal cells to cancer progression has been recognized. Stromal elements consist of the ECM, fibroblasts of various phenotypes, and a scaffold composed of immune and inflammatory cells, blood and lymph vessels, and nerves. This review will focus on therapeutic targets in the microenvironment related to tumor endothelium, tumor associated fibroblasts and the extracellular matrix

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

Genetic Variants of VEGFA and FLT4 Are Determinants of Survival in Renal Cell Carcinoma Patients Treated with Sorafenib

Molecular markers of sorafenib efficacy in patients with metastatic renal cell carcinoma (mRCC) are not available. The purpose of this study was to discover genetic markers of survival in patients with mRCC treated with sorafenib. Germline variants from 56 genes were genotyped in 295 patients with mRCC. Variant-overall survival (OS) associations were tested in multivariate regression models. Mechanistic studies were conducted to validate clinical associations. VEGFA rs1885657, ITGAV rs3816375, and WWOX rs8047917 (sorafenib arm), and FLT4 rs307826 and VEGFA rs3024987 (sorafenib and placebo arms combined) were associated with shorter OS. FLT4 rs307826 increased VEGFR-3 phosphorylation, membrane trafficking, and receptor activation. VEGFA rs1885657 and rs58159269 increased transcriptional activity of the constructs containing these variants in endothelial and RCC cell lines, and VEGFA rs58159269 increased endothelial cell proliferation and tube formation. FLT4 rs307826 and VEGFA rs58159269 led to reduced sorafenib cytotoxicity. Genetic variation in VEGFA and FLT4 could affect survival in sorafenib-treated patients with mRCC. These markers should be examined in additional malignancies treated with sorafenib and in other angiogenesis inhibitors used in mRCC. Significance: Clinical and mechanistic data identify germline genetic variants in VEGFA and FLT4 as markers of survival in patients with metastatic renal cell carcinoma.Peer reviewe

Young Women with Locally Advanced Breast Cancer Who Achieve Breast Conservation after Neoadjuvant Chemotherapy Have a Low Local Recurrence Rate

Women with locally advanced breast cancer (LABC) who are breast conservation (BCT) candidates after neoadjuvant chemotherapy have the best long-term outcome and low local–regional recurrence (LRR) rates. However, young women are thought to have a higher risk of LRR based on historical data. This study sought to evaluate LRR rates in young women who undergo BCT after neoadjuvant chemotherapy. We identified 122 women aged 45 years or younger with American Joint Committee on Cancer (AJCC) Stage II to III breast cancer, excluding T4d, treated with neoadjuvant chemotherapy from 1991 to 2007 from a prospective, Institutional Review Board-approved, single-institution database. Data were analyzed using Fisher eExact test, Wilcoxon tests, and the Kaplan-Meier method. Median follow-up was 6.4 years. Fifty-four (44%) patients had BCT and 68 (56%) mastectomy. Forty-six per cent were estrogen receptor-positivity and 28 per cent overexpressed Her2. Mean pretreatment T size was 5.6 cm in the BCT group and 6.7 cm in the mastectomy group (P = 0.04). LRR rates were no different after BCT compared with mastectomy (13 vs 18%, P = 0.6). Higher posttreatment N stage (P <0.001) and AJCC stage (P = 0.008) were associated with LRR but not pretreatment staging. Disease-free survival was better for patients achieving BCT, with 5-year disease-free survival rates of 82 per cent (95% CI, 69 to 90%) compared with 58 per cent (95% CI, 45 to 69%) for mastectomy (P = 0.03). Young women with LABC who undergo BCT after neoadjuvant chemotherapy appear to have similar LRR rates compared with those with mastectomy. This suggests that neoadjuvant chemotherapy may identify young women for whom BCT may have an acceptable risk of LRR

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

Cosmetic Outcomes for Accelerated Partial Breast Irradiation Prior to Surgical Excision of Early Stage Breast Cancer Using Single Dose Intraoperative Radiotherapy

Determine cosmetic outcome and toxicity profile of intraoperative radiation delivered prior-to tumor excision for patients with early stage breast cancer

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 non-canonical Wnt/Ca++ pathway was affected by SFRP2, as demonstrated by an increase in NFATc3 in the nuclear fraction of SFRP2-treated endothelial cells. Tacrolimus, a calcineurin inhibitor which inhibits dephosphorylation of NFAT, inhibited SFRP2-induced endothelial tube formation. Tacrolimus 3 mg/kg/daily 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

Quantifying the recruitment challenges with couple-based interventions for cancer: applications to early-stage breast cancer

Despite mounting evidence supporting the use of psychosocial interventions to promote adaptation to cancer, enrolling participants into these interventions is challenging. This is particularly salient for couple-based interventions, and newer, more targeted recruitment strategies to increase enrollment are needed. However, there have been few published empirical studies focused specifically on recruitment–related variables associated with enrollment into these types of interventions. To better understand how to encourage participation in couple-based psychosocial interventions for cancer, we examined facilitating and impeding factors to enrollment into a couple-based intervention for women with early stage breast cancer