Charlotta Dabrosin

Broad targeting of angiogenesis for cancer prevention and therapy

Deregulation of angiogenesis – the growth of new blood vessels from an existing vasculature – is a main driving force in many severe human diseases including cancer. As such, tumor angiogenesis is important for delivering oxygen and nutrients to growing tumors, and therefore considered an essential pathologic feature of cancer, while also playing a key role in enabling other aspects of tumor pathology such as metabolic deregulation and tumor dissemination/metastasis. Recently, inhibition of tumor angiogenesis has become a clinical anti-cancer strategy in line with chemotherapy, radiotherapy and surgery, which underscore the critical importance of the angiogenic switch during early tumor development. Unfortunately the clinically approved anti-angiogenic drugs in use today are only effective in a subset of the patients, and many who initially respond develop resistance over time. Also, some of the anti-angiogenic drugs are toxic and it would be of great importance to identify alternative compounds, which could overcome these drawbacks and limitations of the currently available therapy. Finding “the most important target” may, however, prove a very challenging approach as the tumor environment is highly diverse, consisting of many different cell types, all of which may contribute to tumor angiogenesis. Furthermore, the tumor cells themselves are genetically unstable, leading to a progressive increase in the number of different angiogenic factors produced as the cancer progresses to advanced stages. As an alternative approach to targeted therapy, options to broadly interfere with angiogenic signals by a mixture of non-toxic natural compound with pleiotropic actions were viewed by this team as an opportunity to develop a complementary anti-angiogenesis treatment option. As a part of the “Halifax Project” within the “Getting to know cancer” framework, we have here, based on a thorough review of the literature, identified 10 important aspects of tumor angiogenesis and the pathological tumor vasculature which would be well suited as targets for anti-angiogenic therapy: (1) endothelial cell migration/tip cell formation, (2) structural abnormalities of tumor vessels, (3) hypoxia, (4) lymphangiogenesis, (5) elevated interstitial fluid pressure, (6) poor perfusion, (7) disrupted circadian rhythms, (8) tumor promoting inflammation, (9) tumor promoting fibroblasts and (10) tumor cell metabolism/acidosis. Following this analysis, we scrutinized the available literature on broadly acting anti-angiogenic natural products, with a focus on finding qualitative information on phytochemicals which could inhibit these targets and came up with 10 prototypical phytochemical compounds: (1) oleanolic acid, (2) tripterine, (3) silibinin, (4) curcumin, (5) epigallocatechin-gallate, (6) kaempferol, (7) melatonin, (8) enterolactone, (9) withaferin A and (10) resveratrol. We suggest that these plant-derived compounds could be combined to constitute a broader acting and more effective inhibitory cocktail at doses that would not be likely to cause excessive toxicity. All the targets and phytochemical approaches were further cross-validated against their effects on other essential tumorigenic pathways (based on the “hallmarks” of cancer) in order to discover possible synergies or potentially harmful interactions, and were found to generally also have positive involvement in/effects on these other aspects of tumor biology. The aim is that this discussion could lead to the selection of combinations of such anti-angiogenic compounds which could be used in potent anti-tumor cocktails, for enhanced therapeutic efficacy, reduced toxicity and circumvention of single-agent anti-angiogenic resistance, as well as for possible use in primary or secondary cancer prevention strategies.

Flaxseed and Its Lignans Inhibit Estradiol-Induced Growth, Angiogenesis, and Secretion of Vascular Endothelial Growth Factor in Human Breast Cancer Xenografts In vivo

Purpose: Vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis, which is crucial in cancer progression. We have previously shown that estradiol (E2) increases VEGF in breast cancer. Phytoestrogens are potential compounds in breast cancer prevention and treatment by poorly understood mechanisms. The main phytoestrogens in Western diet are lignans, and flaxseed is a rich source of the mammalian lignans enterodiol and enterolactone. Experimental Design: In the present study, ovariectomized mice were treated with continuous release of E2. MCF-7 tumors were established and mice were fed with basal diet or 10% flaxseed, and two groups that were fed basal diet received daily injections with enterodiol or enterolactone (15 mg/kg body weight). Results: We show that flaxseed, enterodiol, and enterolactone counteracted E2-induced growth and angiogenesis in solid tumors. Extracellular VEGF in vivo, sampled using microdialysis, in all intervention groups was significantly decreased compared with tumors in the basal diet group. Our in vivo findings were confirmed in vitro. By adding enterodiol or enterolactone, E2-induced VEGF secretion in MCF-7 cells decreased significantly without agonistic effects. The increased VEGF secretion by E2 in MCF-7 cells increased the expression of VEGF receptor-2 in umbilical vein endothelial cells, suggesting a proangiogenic effect by E2 by two different mechanisms, both of which were inhibited by the addition of lignans. Conclusions: Our results suggest that flaxseed and its lignans have potent antiestrogenic effects on estrogen receptor–positive breast cancer and may prove to be beneficial in breast cancer prevention strategies in the future.

Therapeutic effect of angiostatin gene transfer in a murine model of endometriosis.

Endometriosis, the growth of ectopic endometrial tissue, is a chronic recurrent disease affecting 10% of the female population causing dyspareunia, pelvic pain, dysmenorrhea, and infertility. Suppression of ovarian activity is the cornerstone of medical therapy with limited benefit and severe adverse effects. Angiogenesis plays a major role in the development of endometriosis suggesting that anti-angiogenic therapy would offer a new therapeutic approach. We report successful treatment of endometriosis in estrogen-supplemented ovariectomized mice by transient overexpression (6 to 10 days of duration) of the gene for a natural angiogenesis inhibitor angiostatin, delivered to the peritoneum by a replication-deficient adenovirus vector (AdAngiostatin). Established endometriosis was eradicated in 14 of 14 AdAngiostatin-treated animals, whereas 11 of 13 control animals showed full disease development. Administered to normal cycling mice for the same transient period, AdAngiostatin caused impaired ovarian function with suppressed corpus luteum development, decreased production of estradiol and progesterone, decreased ovarian and uterine weight, and increased body weight. AdAngiostatin treatment lowered the levels of sex steroids but did not induce total castration. Gene therapy with angiogenic inhibitors is a highly effective treatment for endometriosis, even in a host with preserved estrogen levels. However, local or targeted delivery of the gene must be considered to avoid prolonged systemic effects and impaired ovarian function.

Effects of oestradiol and tamoxifen on VEGF, soluble VEGFR-1, and VEGFR-2 in breast cancer and endothelial cells.

Angiogenesis is regulated by the balance between pro- and antiangiogenic factors. Vascular endothelial growth factor (VEGF), acting via the receptors VEGFR-1 and VEGFR-2, is a key mediator of tumour angiogenesis. The soluble form of the VEGF receptor-1 (sVEGFR-1) is an important negative regulator of VEGF-mediated angiogenesis. The majority of breast cancers are oestrogen dependent, but it is not fully understood how oestrogen and the antioestrogen, tamoxifen, affect the balance of angiogenic factors. Angiogenesis is a result of the interplay between cancer and endothelial cells, and sex steroids may exert effects on both cell types. In this study we show that oestradiol decreased secreted sVEGFR-1, increased secreted VEGF, and decreased the ratio of sVEGFR-1/VEGF in MCF-7 human breast cancer cells. The addition of tamoxifen opposed these effects. Moreover, human umbilical vein endothelial cells (HUVEC) incubated with supernatants from oestradiol-treated MCF-7 cells exhibited higher VEGFR-2 levels than controls. In vivo, MCF-7 tumours from oestradiol+tamoxifen-treated nude mice exhibited decreased tumour vasculature. Our results suggest that tamoxifen and oestradiol exert dual effects on the angiogenic environment in breast cancer by regulating cancer cell-secreted angiogenic ligands such as VEGF and sVEGFR-1 and by affecting VEGFR-2 expression of endothelial cells.

Estradiol increases extracellular levels of vascular endothelial growth factor in vivo in murine mammary cancer

Angiogenesis is essential for tumor growth and metastasis and an important prognostic factor in breast cancer. VEGF, a key factor for angiogenesis, has been correlated with tumor vessel density in breast cancer. Estrogen, another crucial factor in breast cancer, stimulates VEGF; and an ERE in the VEGF gene has been defined. VEGF is bioactive in the extracellular fluid, where it becomes available to endothelial cells. Whether E2 affects VEGF levels in the extracellular fluid is not known. We show, using intratumoral microdialysis in vivo, that E2 treatment increased tumor extracellular levels of VEGF in an estrogen‐dependent breast cancer model. Moreover, extracellular levels of VEGF in the tumor showed a strong correlation with total tumor VEGF, contrary to plasma levels of VEGF. Ninety‐three percent of measured VEGF in the extracellular fluid in the tumor was tumor‐derived, while only 45% of VEGF in circulating plasma originated from the tumor. We conclude that E2 increases extracellular VEGF and that microdialysis is a sensitive method for measurement of local VEGF production in vivo. Our results have potential application to the assessment of tumor characteristics in vivo in human tumors for individualized cancer therapy.

Gene transfer of matrix metalloproteinase-9 induces tumor regression of breast cancer in vivo

Matrix metalloproteinases (MMP) are important regulators of angiogenesis and tumor progression by degradation of extracellular matrix. Clinical trials using MMP inhibitors have failed and recent studies suggest that MMPs may in contrast suppress tumor growth. It is not known, however, if MMPs or their inhibitors, tissue inhibitor of metalloproteinases (TIMP), can be used as therapy of established cancer. Here, adenovirus vectors carrying the human genes for MMP-9, TIMP-1, or empty controls were injected intratumorally in breast cancers established in mice supplemented with estradiol and treated with tamoxifen. Microdialysis was used to quantify MMP activity and sampling of endostatin and vascular endothelial growth factor (VEGF) in situ. We show that AdMMP-9 increased MMP activity in vivo, decreased tumor growth rate, and decreased microvessel area significantly. AdMMP-9 therapy resulted in significantly increased levels of endostatin in vivo, whereas VEGF levels were unaffected. As previously shown, tamoxifen exposure by itself increased MMP activity in all treatment groups. Moreover, the combined therapy with AdMMP-9 and tamoxifen further reduced tumor growth and increased the endostatin levels compared with either treatment alone. Gene transfer of TIMP-1 had no effects on tumor progression and counteracted the therapeutic effect of tamoxifen in our breast cancer model. This is the first report showing that overexpression of MMP-9 results in increased generation of antiangiogenic fragments, decreased angiogenesis, and therapeutic effects of established breast cancer.

Positive Correlation between Estradiol and Vascular Endothelial Growth Factor but not Fibroblast Growth Factor-2 in Normal Human Breast Tissue In vivo

Purpose: Angiogenesis is crucial in tumor development and progression. Ovarian hormones regulate angiogenesis in the reproductive tract but very little is known about its regulation in the normal breast. Sex steroids play an important role in breast cancer development by poorly understood mechanisms. Vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) are potent stimulators of angiogenesis. Both VEGF and FGF-2 function in autocrine/paracrine pathways and there is a major contribution of bioactive proteins by a posttranslational activation of sequestered molecules in the extracellular space. A direct measurement of these molecules in the extracellular compartment is, therefore, needed. Experimental Design: In this study, microdialysis was used to measure extracellular VEGF and FGF-2 in normal human breast tissue in situ in 11 premenopausal and 5 postmenopausal women. Results: Significantly higher level of VEGF in breast tissue of premenopausal women was found. Plasma as well as local estradiol and breast tissue VEGF exhibited significant correlations, whereas progesterone had no correlation with breast VEGF. FGF-2 did not correlate with either estradiol or progesterone. Conclusion: The result suggests that estradiol is a more potent regulator of free VEGF levels than progesterone in the normal breast. The control of free FGF-2 seems to be independent of sex steroids in the breast. Estrogen induction of free extracellular VEGF may be one mechanism involved in sex steroid–dependent breast carcinogenesis.

Estradiol Promotes Growth and Angiogenesis in Polyoma Middle T Transgenic Mouse Mammary Tumor Explants

Estrogen is important for breast carcinogenesis and the majority of breast cancers maintain hormone dependency. Estrogen has the ability to stimulate both breast epithelial cell growth and angiogenesis, and a well-characterized in vivo cancer model where these functional interactions can be studied is lacking. We demonstrate estrogen dependent angiogenesis, growth in vivo, and proliferation in vitro, in explants from polyoma middle T transgenic mouse mammary tumors. Thus, in addition to genetic similarities, this model also exerts a sex hormone, and angiogenic phenotype similar to human breast cancer. This immune-competent animal model offers the opportunity to study molecular events in estrogen dependent breast cancer.

Estradiol and Tamoxifen Regulate Endostatin Generation via Matrix Metalloproteinase Activity in Breast Cancer In vivo

Matrix metalloproteinases (MMP) are important regulators of tumor progression and angiogenesis. MMPs generate both proangiogenic and antiangiogenic fragments, such as vascular endothelial growth factor and endostatin. The in vivo activation of MMPs and endostatin generation occur mainly in the extracellular environment by interactions of different cell types. Therefore, these processes are necessary to study in the extracellular space in vivo. Sex steroids play a dominant role in breast carcinogenesis, by largely unknown mechanisms. In the present study, we used in vivo microdialysis to directly quantify MMP-2 and MMP-9 activity and sample endostatin from both stroma (murine) and tumor (human) cells in vivo in solid MCF-7 tumors in nude mice. We found that tamoxifen in combination with estradiol increased tumor MMP-2/MMP-9 in vivo activity, endostatin levels, and decreased tumor vascularization compared with estradiol treatment only. The stroma-derived endostatin was three to five times higher than cancer cell-generated endostatin. After inhibition of MMP-2/MMP-9, endostatin levels decreased, providing evidence that these proteases are highly involved in the generation of endostatin. Our results support the previously reported concept that MMPs may serve as negative regulators of angiogenesis. The regulation of endostatin generation by modulation of MMP-2/MMP-9 activities suggests a previously unrecognized mechanism of estradiol and tamoxifen, which may have implications for the pathogenesis of breast cancer.

Tamoxifen, flaxseed and the lignan enterolactone increase stroma and cancer cell derived IL-1Ra and decrease tumor angiogenesis in estrogen dependent breast cancer

The proinflammatory cytokines IL-1α and IL-1β promote tumor angiogenesis that might be counteracted by the IL-1 receptor antagonist (IL-1Ra), anakinra, a clinically approved agent. A diet with high amounts of phytoestrogens, such as flaxseed (Flax), genistein (GEN), and the mammalian lignan enterolactone (ENL), may affect breast cancer progression in a similar fashion as the antiestrogen tamoxifen. Both cancer cells and tumor stroma may be targets for cancer therapy. By using microdialysis in a model of human breast cancers in nude mice, we could perform species-specific analyses of released proteins in the microenvironment. We show that tumors treated with tamoxifen and fed Flax or ENL exhibited decreased in vivo release of IL-1β derived from the murine stroma and decreased microvessel density whereas dietary GEN had no effects. Cancer cell-released IL-1Ra were approximately 5 times higher than stroma-derived IL-1Ra. Tamoxifen, Flax, and ENL increased IL-1Ra levels significantly whereas GEN did not. The tumor stroma contained macrophages, which expressed the estrogen receptor. In vitro, estradiol decreased IL-1Ra released from breast cancer cells and from cultured macrophages. IL-1Ra decreased endothelial cell proliferation significantly in vitro whereas breast cancer cell proliferation was unaffected in presence of estradiol. Finally, IL-1Ra therapy of tumor-bearing mice opposed estrogen-dependent breast cancer growth and decreased angiogenesis. We conclude that the release of IL-1s both by cancer cells and the stroma, where macrophages are a key component, may offer feasible targets for antiestrogen therapy and dietary interventions against breast cancer.