Zhican Qu

Vascular endothelial growth factor reduces tamoxifen efficacy and promotes metastatic colonization and desmoplasia in breast tumors.

Clinical studies have shown that decreased tamoxifen effectiveness correlates with elevated levels of vascular endothelial growth factor (VEGF)-A(165) in biopsy samples of breast cancers. To investigate the mechanisms underlying tamoxifen resistance and metastasis, we engineered the estrogen receptor (ER)-positive MCF-7 human breast cancer cell line to express VEGF to clinically relevant levels in a doxycycline-regulated manner. Induction of VEGF expression in orthotopically implanted xenografts that were initially tamoxifen responsive and noninvasive resulted in tamoxifen-resistant tumor growth and metastasis to the lungs. Lung metastases were also observed in a VEGF-dependent manner following tail vein injection of tumor cells. At both primary and metastatic sites, VEGF-overexpressing tumors exhibited extensive fibroblastic stromal content, a clinical feature called desmoplasia. VEGF-induced metastatic colonies were surrounded by densely packed stromal cells before detectable angiogenesis, suggesting that VEGF is involved in the initiation of desmoplasia. Because expression of VEGF receptors R1 and R2 was undetectable in these tumor cells, the observed VEGF effects on reduction of tamoxifen efficacy and metastatic colonization are most likely mediated by paracrine signaling that enhances tumor/stromal cell interactions and increases the level of desmoplasia. This study reveals new roles for VEGF in breast cancer progression and suggests that combination of antiestrogens and VEGF inhibitors may prolong tamoxifen sensitivity and prevent metastasis in patients with ER-positive tumors.

Prolonged Extracellular Signal-Regulated Kinase 1/2 Activation during Fibroblast Growth Factor 1- or Heregulin β1-Induced Antiestrogen-Resistant Growth of Breast Cancer Cells Is Resistant to Mitogen-Activated Protein/Extracellular Regulated Kinase Kinase Inhibitors

Increased growth factor receptor signaling is implicated in antiestrogen-resistant breast tumors suggesting that abrogation of such signaling could restore or prolong sensitivity to antihormonal agents. Activation of the mitogen-activated protein/extracellular regulated kinase kinase (MEK)-extracellular regulated kinase (ERK)1/2 cascade is a common component of such pathways. We investigated the ability of the MEK activation inhibitor U0126 to block the increased growth of estrogen receptor-positive MCF-7 breast cancer cells caused by fibroblast growth factor 1 (FGF-1), heregulin β1 (HRGβ1), and epidermal growth factor (EGF) in the presence of the pure antiestrogen ICI 182780 (Faslodex; fulvestrant). We found that either FGF-1 or HRGβ1 but not EGF substantially reduced the inhibitory effects of U0126 on growth and ERK1/2 activation, including the combined inhibitory effects of U0126 and ICI 182780. FGF-1 and HRGβ1 also reduced the inhibition of ERK1/2 phosphorylation by the MEK inhibitors PD98059 and PD184161. Interestingly, a transiently transfected dominant-negative MEK1 completely abrogated activation of a coexpressed green fluorescent protein-ERK2 reporter by all three of the factors. Despite a short-lived activation of Ras and Raf-1 by all three of the growth factors, both FGF-1 and HRGβ1, unlike EGF, induced a prolonged activation of MEK and ERK1/2 in these cells. Thus, activation of FGF-1- and HRGβ1-specific signaling causes MEK-dependent prolonged activation of ERK1/2, which is incompletely susceptible to known MEK inhibitors. We also demonstrate that the cytosolic phospholipase A2 inhibitor arachidonyl trifluoro methyl ketone and the pan PKC inhibitor bisindolymaleimide abrogated U0126-resistant phosphorylation of ERK1/2 induced by HRGβ1 but not by FGF-1. Phosphorylation of ERK5 by all three of the factors was also resistant to U0126 suggesting that its activation is not sufficient to overturn growth inhibition due to diminished ERK1/2 activation. Therefore, therapy combining antiestrogens and MEK inhibitors may be ineffective in some antiestrogen-resistant estrogen receptor-positive breast cancers.

Activation loop phosphorylation-independent kinase activity of human protein kinase C ζ

Atypical protein kinase C ζ (PKCζ) plays an important role in cell proliferation and survival. PKCζ and its truncated form containing only the kinase domain, CATζ, have been reported to be activated by the phosphorylation of threonine 410 in the activation loop. We expressed both the full length PKCζ and CATζ in a baculovirus/insect cell over‐expression system and purified the proteins for biochemical characterization. Ion exchange chromatography of CATζ revealed three species with different levels of phosphorylation at Thr‐410 and allowed the isolation of the CATζ protein devoid of phosphorylation at Thr‐410. All three species of CATζ were active and their activity was not correlated with phosphorylation at Thr‐410, indicating that the kinase activity of CATζ did not depend solely on activation loop phosphorylation. Tyrosine phosphorylation was detected in all three species of CATζ and the full length PKCζ. Homology structural modeling of PKCζ revealed a conserved, predicted‐to‐be phosphorylated tyrosine residue, Tyr‐428, in the close proximity of the RD motif of the catalytic loop and of Thr‐410 in the activation loop. The structural analysis indicated that phospho‐Tyr‐428 would interact with two key, positively‐charged residues to form a triad conformation similar to that formed by phospho‐Thr‐410. Based on these observations, it is possible that the Thr‐410 phosphorylation‐independent kinase activity of CATζ is regulated by the phosphorylation of Tyr‐428. This alternative mode of PKCζ activation is supported by the observed stimulation of PKCζ kinase activity upon phosphorylation at the equivalent site by Abl, and may be involved in resistance to drug‐induced apoptosis. Proteins 2007.

Antiangiogenic activity of 4′-thio-β-d-arabinofuranosylcytosine

4′-Thio-β-d-arabinofuranosylcytosine (T-araC), a new-generation deoxycytidine nucleoside analogue, showed significant efficacy against numerous solid tumors in preclinical studies and entered clinical development for cancer therapy. It is a structural analogue of cytarabine (araC), a clinically used drug in the treatment of acute myelogenous leukemia, which has no or very limited efficacy against solid tumors. In comparison with araC, the excellent in vivo activity of T-araC against solid tumors suggests that, in addition to inhibition of DNA synthesis, T-araC may target cellular signaling pathways, such as angiogenesis, in solid tumors. We studied T-araC and araC for their antiangiogenic activities in vitro and in vivo. Both compounds inhibited human endothelial cell proliferation with similar IC50s. However, only T-araC inhibited endothelial cell migration and differentiation into capillary tubules. T-araC also abrogated endothelial cell extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, a key signaling molecule involved in cellular processes of angiogenesis. Results from chick chorioallantoic membrane angiogenesis assays revealed that T-araC significantly inhibited the development of new blood vessels in vivo, whereas araC showed much less effect. The findings of this study show a role of T-araC in antiangiogenesis and suggest that T-araC combines antiproliferative and antiangiogenic activity in one molecule for a dual mechanism of drug action to achieve the excellent in vivo efficacy against several solid tumors. This study also provides important information for optimizing dosage and sequence of T-araC administration in clinical investigations by considering T-araC as both an antiproliferative and an antiangiogenic agent. [Mol Cancer Ther 2006;5(9):2218–24]

Abstract 5154: Serotonin signaling as a novel target of tumor angiogenesis

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Angiogenesis is a well-controlled process that is regulated by multiple factors that are secreted by the cancer cells as well as other cells within the tumor microenvironment. Angiogenesis inhibitors are showing therapeutic efficacy in an increasing number of human cancers. However, in both preclinical and clinical settings, the benefits are transitory and are followed by resistance and a restoration of tumor growth and progression. Therefore, novel anti-angiogenic strategies with complementary mechanisms are needed to maximize efficacy and minimize resistance to current angiogenesis inhibitors. Activation of platelets and blood coagulation frequently occurs in cancer patients. Apart from VEGF, platelets contain several other angiogenic growth factors and inhibitors that are released upon activation and promote tumor neoangiogenesis. Although considerable attention has been focused on platelet peptide growth factors, little is known about the mitogenic effects of nonpeptide platelet products such s serotonin (5-HT), considering that 99% of 5-HT in blood is found in platelets and is released at blood clotting sites. In previous studies, we have shown that endothelial cells express 5-HT receptors and 5-HT has growth stimulatory effcts on multiple types of endothelial cells. We have also demonstrated that 5-HT binds to inhibitory type of G-protein coupled receptors and stimulates the phosphorylation of PYK2/PI3K/AKT/mTOR signaling pathway, the same signaling pathway, which has been activated by most angiogenic factors, including VEGF. In our recent studies, we explored angiogenic promoting activity of 5-HT in the mouse matrigel plug assay (in vivo angiogenesis model system) and antiangiogenic potential of antagonists against 5-HT receptors in xenograft-CAM assay (ex vivo tumor angiogenesis model system). The most importantly, an antagonist against a specific 5-HT receptor demonstrated the synergistic effect in blocking tumor-induced new blood vessel formation when it was applied in combination with Sutent, a FDA approved antiangiogenic drug against VEGF receptor. The results of this study suggest that 5-HT signaling pathway constitute a novel target of tumor angiogenesis in anticancer therapeutic development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5154. doi:10.1158/1538-7445.AM2011-5154

Abstract 3727: Targeting autotaxin to reduce chemotherapy resistance in ovarian cancer

Development of resistance to chemotherapy presents the biggest challenge in the treatment of ovarian cancer. Autotaxin (ATX) is a secreted enzyme that catalyzes lysophosphatidic acid (LPA) production and is responsible for the up-regulation of LPA in ovarian cancer. The ATX-LPA axis has been identified to be one of the mechanisms of chemotherapy resistance in ovarian cancer. Thus, inhibition of autotaxin may be a potential strategy to increase the chemotherapy efficacy in this disease context. At Southern Research, we previously identified a known anti-parasitic small molecule, Bithionol as a potent antiangiogenic agent, which inhibits endothelial cell proliferation, migration and tubular morphogenesis in vitro and directly inhibits autotaxin enzyme activity. Our recent results have shown that Bithionol not only directly inhibits the enzyme activity; it also reduces autotaxin secretion from human endothelial and ovarian cancer cells. Recently, using a human ovarian cancer xenograft mouse model, Biothionol was shown to have in vivo anti-tumor activity as a single drug treatment. In addition, in combination therapy studies in mice, Bithionol significantly increased the efficacy of Paclitaxel and Cisplatin against ovarian tumor growth. These results suggest that Bithionol may provide a promising approach for reducing chemotherapy associated resistance in ovarian cancer. Additional preclinical studies are in progress to assess the potential clinical utility of Bithionol in combination with current ovarian cancer therapy. (This work is supported by a pilot grant from Norma Livingston Foundation and SRI SIP fund). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3727. doi:1538-7445.AM2012-3727

Abstract 2333: Serotonin: A known neurotransmitter functions as an angiokine to support cancer progression

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Platelet aggregation leads to significant serotonin release from its major storage and results in increasing of serotonin levels at injury site and thrombotic tumor environment. Many studies have shown that platelet activation plays a crucial role in tumor progression; however, the role of serotonin in angiogenesis and tumor progression has not been well studied. In this study, we have tested the hypothesis that 5-HT promotes angiogenesis, and antagonizing 5-HT activity has anti-angiogenic benefit in controlling tumor growth. Real time PCR and western blot studies showed that endothelial cell expressed 5-HT receptor 1B (HTR1B) in higher level than other 5-HT receptors. Endothelial cell proliferation and tube formation were significantly affected by blocking HTR1B and the cAMP and IP1 assays have revealed the HTR1B as the inhibitory type of GPCR. Stimulation of endothelial cell with 5-HT or HTR1B agonist has led to activation of two individual signaling pathways: ERK and Akt / mTOR. In further studies, p70S6K was recognized as the merging point of these signaling. These kinases have been also activated by known angiogenic factors (VEGF and FGF) but the mechanism of activation was different from serotonin and it was through their tyrosine kinase receptors. In contrast, pretreatment of endothelial cell with a selective HTR1B antagonist have led to blockade of the 5-HT induced kinases’ activation. We also demonstrated angiogenic promoting activity of 5-HT with a mouse Matrigel plug (in vivo angiogenesis model system) and antiangiogenic potential of antagonists against HTR1B with an ex vivo tumor angiogenesis model (xenograft CAM assay) and xenograft mice model of human ovary cancer (SKOV-3). Selective HTR1B antagonist displayed the synergistic effect (combination index analysis) in blocking tumor-induced new blood vessel formation when it was applied in combination with Sutent (VEGFR2 antagonist). HTR1B antagonist also showed preclinical efficacy in the xenograft model of human ovary cancer by reducing over 50% in tumor size and blood vessel density marker.The results of this study enhanced our understanding of the serotonin-signaling pathway in human endothelial cells during angiogenesis. This research also revealed the potential of 5-HT signaling as the new target for antiangiogenic development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2333. doi:1538-7445.AM2012-2333

Abstract 3114: Effects of tumor-stromal interactions on gene expression in panel of mouse tumor models

Tumor growth is not determined solely by the tumor cells but is governed by interactions between tumor cells and host stromal cells, including endothelial cell activation and fibroblastic stroma response. Tumor stroma profoundly influences many steps of tumor progression. In many human cancers, such as breast, prostate, and colon, the stroma comprises the majority of the tumor mass, as a hallmark of the clinical feature called desmoplasia. Numerous studies have showed that tumor-stromal cell interactions play crucial roles in supporting cancer progression and in promoting anticancer drug resistance by alternating gene expression profiles in both tumor and stromal cells through network tumor-stromal interactions in the tumor microenvironments. It has been challenging to obtain separate gene profiles for tumor and stroma with human tumor samples as both tumor and stromal cells share the same genome. In xenograft mouse tumor models, human tumor cells are supported by mouse host stromal microenvironment. Therefore, effects of tumor stromal integrations on gene expression can be profiled separately by taking advantage of this heterogeneous genetic makeup. To understand the underlying biological process of stroma in cancer and select relevant in vitro and in vivo model systems for various targeted anticancer drug discovery and development projects, we selected a panel of 30 commonly used xenograft tumor mouse models that are derived from human tumor cell lines of various cancer histotypes and conducted analyses of differential gene expression in both human cancer cells and mouse host stromal cells before and after their interactions in vivo by using quantitative PCR with mouse and human specific primers. Growing the human tumors as a continuous in vivo passage subcutaneously in immunodeficient mice permits stroma infiltration over a long time. We have examined a number of anticancer target genes involved in different signaling pathways, such as tumor angiogenesis, apoptosis and survival (Akt/mTOR signaling pathway). Our study results have demonstrated that tumor stromal interactions significantly regulate expression levels of various genes important in tumor progression and development of resistance to treatment in both tumor and stromal cells. Tumor animal models play a critical role in translating the bench science to the bedside medical care of cancer patients. Decisions for moving new anticancer agents into costly clinical investigations are mostly based on the preclinical results using xenograft mouse models. The results of this gene profiling approach could provide tools for studying tumor microenvironment and tumor stromal interactions in vivo to advance anticancer drug development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3114. doi:10.1158/1538-7445.AM2011-3114

Abstract 4265: Bithionol as an inhibitor of tumor angiogenesis

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Tumor progression depends on angiogenesis. The clinical successes in recent years have demonstrated that antiangiogenic agents can be developed as effective treatment for cancer patients. However, the resistance to the current FDA approved antiangiogenic therapies is emerging, which presents challenges in cancer research and urgent needs for novel angiogenesis inhibitors with different mechanisms of drug actions to overcome the resistance. In our previous studies, we have identified a small molecule bithionol as a potential antiangiogenic agent through a chemical diversity library screening with a cell-based angiogenesis assay. Bithionol is a FDA approved and current clinically used anthelmintics, which has been found to directly inhibit activity of autotaxin, a secreted enzyme that catalyzes lysophosphatidic acid (LPA) production. LPA, as a lipid signaling molecule, is a potent angiogenic and cancer cell motility stimulating factor. Therefore, autotaxin plays important role in tumor angiogenesis and metastasis and has been recently identified as an attractive angiogenesis and cancer target. Our recent studies have shown that autotaxin is expressed at high levels in human endothelial and number of human cancer cell lines. Bithionol has demonstrated inhibitory activities against human endothelial cell proliferation, migration, and tube-formation as the three key cellular steps in angiogenesis process. The effect of bithionol on inhibition of tumor angiogenesis was also evaluated with an ex vivo xenograft chicken embryo chorioallantoic membrane (xenograft-CAM) model system. Bithionol is able to inhibit tumor-induced new blood vessel formation in a dose dependent manner and has demonstrated an additive inhibitory activity when it is applied in combination with Sutent, a current clinically used antiangiogenic drug. The results of this research suggest that bithionol provide a unique promising opportunity in developing new combinational treatment to increase the anticancer efficacy and minimize the resistance in cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4265. doi:10.1158/1538-7445.AM2011-4265

Abstract 2390: Serotonin signal transduction in human endothelial cells

Angiogenesis is a highly regulated biological complex process, which consist of endothelial cells proliferation, migration, and tubulogenesis as key cellular steps to form new blood capillaries from preexisting vessels. Imbalance in angiogenesis contributes to a variety of human diseases. Tumor growth depends on angiogenesis and antiangiogenic agents have been developed as effective anti-cancer medicine. Recently, we have demonstrated that serotonin (5-HT), a well-recognized neurotransmitter, has a promoting effect on endothelial cells proliferation, migration and tubulogenesis, suggesting serotonin plays a role in regulation of angiogenesis. To study the role of serotonin in angiogenesis, we examined the expression of serotonin receptor isoforms in human endothelial cells. Real time PCR studies have showed that, serotonin receptor 1B (HTR1B), 1D (HTR1D), 1A (HTR1A) and 2B (HTR2B) are expressed in primary human endothelial cell lines. Blocking serotonin 1B receptor had significant inhibitory effect on endothelial cells proliferation and tubulogenesis. We have further explored the 1B-mediated signal transduction in endothelial cells. The results have revealed that the serotonin 1B receptor as a G protein-coupled receptor (GPCR) is coupled to Gi protein and decreased intracellular cAMP level upon its receptor binding. Both serotonin and 1B receptor agonist were able to activate ERK, p38, Akt, RhoA, Rac1 and PYK2/FAK signaling in endothelial cells. In contrast, a selective antagonist for serotonin 1B receptor blocks the serotonin-stimulated Akt and PYK2/FAK activation. Experiments with pertussis toxin (general Gi blocker) further demonstrated that heterotrimeric Gi-protein is required for the activation of Akt and PYK2/FAK by serotonin. These findings suggest that Akt and PYK2/FAK are involved in signaling transduction in serotonin-activated endothelial cells. 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 2390.