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The role of polarity signaling in cancer metastasis is ill defined. Using two three-dimensional culture models of mammary epithelial cells and an orthotopic mouse model of breast cancer, we reveal that Par6 signaling, which is regulated directly by TGF[beta], plays a role in breast cancer metastasis. Interference with Par6 signaling blocked TGF[beta]-dependent loss of polarity in acini-like structures formed by non-transformed mammary cells grown in three-dimensional structures and suppressed the protrusive morphology of mesenchymal-like invasive mammary tumor cells without rescuing E-cadherin expression. Moreover, blockade of Par6 signaling in an in vivo orthotopic model of metastatic breast cancer induced the formation of ZO-1-positive epithelium-like structures in the primary tumor and suppressed metastasis to the lungs. Analysis of the pathway in tissue microarrays of human breast tumors further revealed that Par6 activation correlated with markers of the basal carcinoma subtype in BRCA1-associated tumors. These studies thus reveal a key role for polarity signaling and the control of morphologic transformation in breast cancer metastasis. epithelial-to-mesenchymal transition | cell polarity | metastasis | tumor invasion | epithelial plasticity

Activated EGF receptor (EGFR) plays an oncogenic role in several human malignancies. Although the intracellular effects of EGFR ate well studied, its ability to induce and modulate tumor angiogenesis is less understood. We found previously that oncogenic EGFR can be shed from cancer cells as cargo of membrane microvesicles (MVs), which can interact with surfaces of other cells. Here we report that MVs produced by human cancer cells harboring activated EGFR (A431, A549, DLD-1) can be taken up by cultured endothelial cells, in which they elicit EGFR-dependent responses, including activation of MAPK and Akt pathways. These responses can be blocked by annexin V and its homodimer, Diannexin, both of which cloak phosphatidylserine residues on the surfaces of MVs. Interestingly, the intercellular EGFR transfer is also accompanied by the onset of VEGF expression in endothelial cells and by autocrine activation of its key signaling receptor (VEGF receptor-2). In A431 human tumor xenografts in mice, angiogenic endothelial cells stain positively for human EGFR and phospho-EGFR, while treatment with Diannexin leads to a reduction of tumor growth rate and microvascular density. Thus, we propose that oncogene-containing tumor cell-derived MVs could act as a unique form of angiogenesis-modulating stimuli and are capable of switching endothelial cells to act in an autocrine mode. tumor angiogenesis | antiangiogenesis | exosomes | annexin V | oncogenes

Cancer patients treated with antiangiogenic multitargeted receptor tyrosine kinase (RTK) inhibitors show increased levels of plasma VEGF and placental growth factor and decreased levels of soluble VEGF receptor-2, thus implicating these overall changes as a possible class effect of such drugs and raising the possibility of their exploitation as surrogate biomarkers for pharmacodynamic drug activity/exposure and patient benefit. A postulated mechanism for these changes is that they are tumor-dependent, resulting from drug-induced decreases in vascular function, increases in tumor hypoxia, and changes in hypoxia-regulated genes. However, here we report that an identical pattern of change is observed in normal nontumor-bearing mice treated with SU11248/sunitinib, a small-molecule inhibitor of VEGF and PDGF RTKs. The changes were dose-dependent, plateaued after 4 days of consecutive treatment, reversed after discontinuation of therapy, and correlated with antitumor activity. Altered protein expression was found in a broad variety of tissues, and dose-dependent elevations were observed of several plasma proteins previously unassociated with this class of inhibitor, including G-CSF, SDF-1[alpha], SCF, and osteopontin. Our results suggest that observed sunitinib-induced molecular plasma changes, including those both directly and indirectly targeted by drug, represent a systemic tumor-independent response to therapy and may correlate with the most efficacious antitumor doses, potentially having utility for defining the optimal biologic dose range for this drug class but not as predictive markers of tumor response or clinical benefit. They may also be relevant to drug-associated toxicities, drug resistance, and observed rapid tumor (re)growth seen after cessation of therapy. angiogenesis | optimal biological dosing | SU11248 | surrogate biomarkers

Chemotherapeutic drugs chronically administered to tumor-bearing mice, using a frequent schedule at doses substantially lower than the maximum tolerated dose (MTD) (i.e, metronomic dosing), can cause sustained and potent antiangiogenic effects by targeting the endothelial cells of newly growing tumor blood vessels. These effects appear to occur in the absence of an increase in the severity of side effects caused by destruction of other cell types normally sensitive to MTD chemotherapy, suggesting a marked and selective sensitivity of activated endothelial cells, the basis of which is unknown. Here we report that protracted exposure of endothelial cells in vitro to low concentrations of several different anticancer agents, including microtubule inhibitors and an alkylating agent, caused marked induction of gene and protein expression of TSP-1, a potent and endothelial-specific inhibitor of angiogenesis. Increases in circulating TSP-1 were also detected in the plasma of human tumor-bearing severe combined immunodeficient mice treated with metronomic low-dose cyclophosphamide. Most importantly, the antiangiogenic and antitumor effects of low-dose continuous cyclophosphamide were lost in TSP-1-null C57BL/6 mice, whereas, in contrast, these effects were retained by using a MTD schedule of the same drug. Taken together, the results implicate TSP-1 as a secondary mediator of the antiangiogenic effects of at least some low-dose metronomic chemotherapy regimens.

Although standard anticancer chemotherapeutic drugs have been designed to inhibit the survival or growth of rapidly dividing tumor cells, it is possible to enhance the efficacy of such drugs by targeting the proliferating host endothelial cells (ECs) of the tumor vasculature. A theoretical advantage of this strategy lies in the possibility of circumventing, or significantly delaying, acquired drug resistance driven by the genetic instability of tumor cells. Here, we show that both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor significantly reduce the pro-apoptotic potency of chemotherapy on both micro- and macrovascular ECs. This cytoprotection to drug toxicity was found to be phosphatidylinositol 3-kinase-dependent and could be recapitulated in the absence of VEGF by overexpressing the dominant-active form of the serine/threonine kinase protein kinase B/Akt. Downstream of phosphatidylinositol 3-kinase, we also show that survivin plays a pivotal role in VEGF-mediated EC protection by preserving the microtubule network. In this respect, its induction effectively protects ECs against chemotherapeutic damage, whereas overexpression of its dominant-interfering mutant (C84A) abrogates the protective effects of VEGF. Accordingly, the potency of VEGF as a chemoprotectant was more pronounced with drugs that interfere with microtubule dynamics than those that damage DNA. These studies implicate a role for survivin up-regulation as a novel mechanism of EC drug 'resistance' and support the notion that angiogenic factors that induce the expression of survivin may act to shield tumor ECs from the apoptotic effects of chemotherapy. Thus, exploiting chemotherapeutic drugs as antiangiogenics is likely to be compromised by the high concentrations of pro-angiogenic survival/growth factors present in the tumor micro-environment; targeting EC survival pathways should improve the antiangiogenic efficacy of antineoplastic agents, particularly microtubule-inhibitor drugs.

Targeted disruption of the single mutant K-ras allele in two human colorectal carcinoma cell lines (DLD-1 and HCT-116) leads to loss of tumorigenic competence in nude mice with retention of ability to grow indefinitely in monolayer culture. Because expression of the mutant K.ras oncogene in these cell lines is associated with marked up-regulation of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), we sought to determine whether this potent angiogenesis inducer plays a role in K.ras-dependent tumorigenic competence. Transfection of a [VEGF.sub.121] antisense expression vector into DLD-1 and HCT-116 cells resulted in suppression of VEGF/VPF production by a factor of 3- to 4-fold. The VEGF/VPF-deficient sublines, unlike the parental population or vector controls, were profoundly suppressed in their ability to form tumors in nude mice for as long as 6 months after cell injection. In contrast, in vitro growth of these sublines was unaffected, thus demonstrating the critical importance of VEGF/VPF as an angiogenic factor for HCT-116 and DLD-1 cells. Transfection of a full-length [VEGF.sub.121] cDNA into two nontumorigenic mutant K-ras knockout sublines resulted in a weak but detectable restoration of tumorigenic ability in vivo in a subset of the transfectants, with no consistent change in growth properties in vitro. The findings indicate that mutant ras-oncogene-dependent VEGF/VPF expression is necessary, but not sufficient, for progressive tumor growth in vivo and highlight the relative contribution of oncogenes, such as mutant K-ras, to the process of tumor angiogenesis.

The basis of the mechanism of acquired drug resistance observed in vivo in alkylating agent-resistant EMT-6 mouse mammary tumor sublines was investigated. It was demonstrated that the EMT-6 sublines manifest their resistance in tissue culture only when grown as three-dimensional multicellular spheroids. The results suggest a possible mechanism of acquired drug resistance in tumors that operate at the multicellular or tissue level.

Coculture of human melanoma cell lines in radial growth phase or early-stage vertical growth phase with irradiated human dermal fibroblasts results in tumor growth inhibition. An investigation was conducted to determine the factor present in fibroblasts that could regulate the growth of early-stage melanoma cells. The results showed that interleukin (IL)-6 could inhibit the growth of metastatically-incompetent melanoma cell lines. However, this growth inhibition effect was lost on metastatically-competent cells. These results suggest the potential of IL-6 as a growth regulator of certain types of early stage non-hematopoietic human solid tumors.

The role of polarity signaling in cancer metastasis is ill defined. Using two three-dimensional culture models of mammary epithehal cells and an orthotopic mouse model of breast cancer, we reveal that Par6 signaling, which is regulated directly by TGFβ, plays a role in breast cancer metastasis. Interference with Par6 signaling blocked TGFβ-dependent loss of polarity in acini-like structures formed by non-transformed mammary cells grown in three-dimensional structures and suppressed the protrusive morphology of mesenchymal-like invasive mammary tumor cells without rescuing E-cadherin expression. Moreover, blockade of Par6 signaling in an in vivo orthotopic model of metastatic breast cancer induced the formation of ZO-1-positive epithehium-like structures in the primary tumor and suppressed metastasis to the lungs. Analysis of the pathway in tissue microarrays of human breast tumors further revealed that Par6 activation correlated with markers of the basal carcinoma subtype in BRCA 1-associated tumors. These studies thus reveal a key role for polarity signaling and the control of morphologic transformation in breast cancer metastasis. [ABSTRACT FROM AUTHOR]

Presents a study which was conducted to determine whether endothelial growth factor/vascular permeability factor (VEGF/VPF) plays a role in K-ras-dependent tumorigenic competence. Materials and methods used; Importance of VEGF/VPF as an inducer of tumor angiogensis; Results and discussion.