Historically, drugs used in the treatment of cancers also tend to cause damage to healthy cells while affecting cancer cells. Therefore, the identification of novel agents that act specifically against cancer cells remains a high priority in the search for new therapies. In contrast with normal cells, most cancer cells contain multiple centrosomes which are associated with genome instability and tumorigenesis. Cancer cells can avoid multipolar mitosis, which can cause cell death, by clustering the extra centrosomes into two spindle poles, thereby enabling bipolar division. Kinesin-like protein KIFC1 plays a critical role in centrosome clustering in cancer cells, but is not essential for normal cells. Therefore, targeting KIFC1 may provide novel insight into selective killing of cancer cells. In the present study, we identified a small-molecule KIFC1 inhibitor, SR31527, which inhibited microtubule (MT)-stimulated KIFC1 ATPase activity with an IC50 value of 6.6 μM. By using bio layer interferometry technology, we further demonstrated that SR31527 bound directly to KIFC1 with high affinity (Kd=25.4 nM). Our results from computational modelling and saturation-transfer difference (STD)-NMR experiments suggest that SR31527 bound to a novel allosteric site of KIFC1 that appears suitable for developing selective inhibitors of KIFC1. Importantly, SR31527 prevented bipolar clustering of extra centrosomes in triple negative breast cancer (TNBC) cells and significantly reduced TNBC cell colony formation and viability, but was less toxic to normal fibroblasts. Therefore, SR31527 provides a valuable tool for studying the biological function of KIFC1 and serves as a potential lead for the development of novel therapeutic agents for breast cancer treatment.

Historically, drugs used in the treatment of certain cancers may cause damage to healthy cells while affecting cancer cells. Therefore, finding compounds that are specific toward cancer cells only is still a high priority in the search for new therapies. In contrast with normal cells, most cancer cells contain multiple centrosomes which are associated with genome instability and tumorigenesis. Cancer cells can avoid multipolar mitosis which can cause cell death by clustering the extra centrosomes into two spindle poles, thereby enabling bipolar division. Kinesin-like protein KIFC1 plays a critical role in centrosome clustering in cancer cells, but is not essential for normal cell survival. Therefore, targeting KIFC1 may give some insight into how a novel therapy can selectively target only cancer cells. Here, we report that KIFC1 up-regulation is a frequent event in human breast cancer and that KIFC1 is highly expressed in all 8 tested human breast cancer cell lines, but is absent in normal human mammary epithelial cells and weakly expressed in 2 human lung fibroblast lines. We also found that depletion of KIFC1 in breast cancer cells induced cell death. From a high throughput screen of 30,000 compounds, we identified a small molecule KIFC1 inhibitor, SRH06, which has an enzymatic IC50 value of 6.5 μM versus KIFC1 and binds directly to KIFC1 without interacting with the microtubule. Results from our computer modeling studies suggested that SRH06 binds to a novel allosteric site on KIFC1 that appears suitable for the development of selective KIFC1 inhibitors. Importantly, SRH06 prevented bipolar clustering of extra-centrosomes in breast cancer cells and significantly reduced colony formation and cell viability, but was less toxic to normal LL47 fibroblasts. Therefore, SRH06 provides a very valuable tool to study the biological function of KIFC1 and serves as a potential lead for the development of a novel therapeutic agent for the treatment of breast cancer. Citation Format: Wei Zhang, Ling Zhai, Wenyan Lu, Yimin Wang, Vandana V. Gupta, Indira Padmalayam, Robert J. Bostwick, Lucile White, Ross Larry, Joseph Maddry, Sam Ananthan, Mark Suto, Bo Xu, Rongbao Li, Yonghe Li. Discovery and evaluation of a small molecule KIFC1 inhibitor for breast cancer treatment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1746. doi:10.1158/1538-7445.AM2015-1746

Our previous study showed that Akt phosphorylates TopBP1 at the Ser-1159 residue and induces its oligomerization. Oligomerization is required for TopBP1 to bind and repress E2F1 activity. However, the mechanism through which phosphorylation of TopBP1 by Akt leads to its oligomerization remains to be determined. Here, we demonstrate that binding between the phosphorylated Ser-1159 (pS1159) residue and the 7th and 8th BRCT domains of TopBP1 mediates TopBP1 oligomerization. Mutations within the 7th and 8th BRCT domains of TopBP1 that block binding to a pS1159-containing peptide block TopBP1 oligomerization and its ability to bind and repress E2F1 activities. The Akt-induced TopBP1 oligomerization is also directly demonstrated in vitro by size exclusion chromatography. Importantly, oligomerization perturbs the checkpoint-activating function of TopBP1 by preventing its recruitment to chromatin and ATR binding upon replicative stress. Hyperactivation of Akt inhibits Chk1 phosphorylation after hydroxyurea treatment, and this effect is dependent on TopBP1 phosphorylation at Ser-1159. Thus, Akt can switch the TopBP1 function from checkpoint activation to transcriptional regulation by regulating its quaternary structure. This pathway of regulation is clinically significant, since treatment of a specific Akt inhibitor in PTEN-mutated cancer cells inhibits TopBP1 oligomerization and causes its function to revert from promoting survival to checkpoint activation.

Singling out the truth: A combined application of STD-NMR, molecular docking, and CORCEMA-ST calculations is described as an attractive, easily applicable tool for the identification and validation of the binding site for allosteric ligands, with potential application as an aid in drug discovery research.

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 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 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

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 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 Proliferation and invasion of malignant tumor cells are mediated by multiple events, including activation of focal adhesion signalings and cytoskeleton remodeling. Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are cytoplasmic tyrosine kinases identified as key mediators in regulation of cytoskeleton proteins in focal adhesion. Pyk2 were found to be overexpressed in Glioblastoma and played a central role in brain tumor invasion. Studies also showed that FAK increased expression and activation in various human cancers. Recently, studies with patients’ tumor samples have demonstrated that Pyk2 and FAK are co-overexpressed in over 70% human breast cancers from the very early disease stage of in situ carcinoma to the advanced metastatic stage. Pyk2 was also involved in the cytoskeleton changes initiated by heregulin through the ErbB-2 receptor in breast cancer cell lines. To further investigate role of Pyk2 and FAK in breast cancer progression and metastasis, we studied protein expression and activation of Pyk2 and FAK, as well as their substrates and interacting proteins in human breast cancer and normal breast epithelial cell lines. Our results showed that activation of both Pyk2 and FAK are marked increased in breast cancer cell lines in comparison with normal breast epithelial cells. In addition, serum stimulation was able to further activate Pyk2 and FAK and to increase tyrosine phosphorylation of their substrates and interacting proteins, including paxillin, Src, and p130CAS. The activated Pyk2 and FAK formed protein complex with paxillin, Src, and p130CAS. Our results and other published studies support the cooperative function of Pyk2 and FAK in breast cancer progression and suggest that dual inhibition of Pyk2 and FAK is a potential therapeutic approach for invasive breast cancer. Lead compounds of Pyk2 and FAK inhibitors have been identified by Southern Research drug discovery team and will be further evaluated in vitro and in vivo as potential breast cancer treatment. 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 1562.

Abstract Human breast cancers contain high levels of fibroblastic stroma as a critical feature of desmoplasia. The complex interactions between tumor and stromal cells play important roles in tumor growth, metastasis and resistance to anticancer treatments. Our previous in vivo studies showed that increased levels of tumor-released VEGF were associated with increased desmoplastic responses and resulted in a hormonal resistance phenotype of ER+ breast tumor. Based on our findings, a clinical trial conducted at UAB with a new combinational treatment targeting both ER and VEGF signalings has achieved significant objective response in ER+ breast cancer patients. In our current studies, we further studied the role of VEGF in mediating tumor-stromal interactions and initiation of desmoplasia. Our results have shown that tumor released VEGF interacts with stromal fibroblasts which express VEGF receptors R1, R2 and Neuropilin-1 (NRP1), thereby stimulating their infiltration to promote desmoplastic response. Down regulation of NRP1 expression by siRNA significantly abolished the fibroblast chemo-attractive motility towards VEGF, while the VEGFR1 and VEGFR2 blocking antibodies had no effects on their migration mobility, suggesting that NRP1 expressed in fibroblast is involved in tumor-stroma cell interactions in breast cancer. Our studies using breast cancer xenograft mouse model with regulated tumor VEGF expression revealed that tumor VEGF expression was positively correlated with expression of NRP1 and alpha smooth muscle actin (α-SMA, a transformed/myo fibroblast marker) in the tumor stroma. In addition, levels of tumor VEGF, stromal NRP1 and α-SMA were all positively correlated with level of tumor stromal content and tumor growth. Blocking tumor VEGF with a neutralizing antibody (Avastin) as a treatment modality in xenograft mice reversed the VEGF increased desmoplasia. Our study results reveal a novel role of VEGF and NRP1 in tumor-stromal interactions and desmoplasia, indicating NRP1 can be a therapeutic target for breast cancer treatment. 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 563.

Abstract The clinical successes of VEGF-targeted agents in recent years have demonstrated that antiangiogenic therapy can be developed as effective anti-cancer treatment. However, the field of oncology is facing a new challenge of overcoming the resistance to VEGF-targeted therapy; therefore, novel angiogenesis inhibitors are required for new therapeutic development. During the growth of new blood capillaries, following proliferation and migration, endothelial cells undergo morphological changes and adhere to each other forming tubular structures as a process of tubulogenesis, which presents a unique target for inhibitory action of antiangiogenic agents. Southern Research angiogenesis research team has identified a structurally related series of heterocyclic compounds SRI-24637 and its analogs as a new class of antiangiogenic agents. SRI-24637 demonstrated selective potent inhibition of endothelial tube-formation and significant activity in blocking tumor-induced blood vessel formation using an ex vivo human cancer xenograft-CAM assay model system. The mechanism of action of SRI-24637 is different from the clinical VEGF-targeted drugs; therefore, it may provide a complementary mechanism to overcome the resistance to VEGF-targeted therapies. Bioavailability studies showed that SRI-24637 dosed at 40 mg/kg in DMSO/PEG400 reached systemic concentration levels much higher than the IC50 concentration for in vitro endothelial tube-formation. Comparing with the clinical VEGF-targeted drug Sunitinib, the cytotoxic activities of SRI-24637 against normal human fibroblast and epithelial cells suggest that the toxic side effects of SRI-24637 may be within a tolerable range. Ongoing preclinical efficacy studies with animal tumor models will aid in the development of SRI-24637 as an anti-cancer drug. Based on the outcome of these studies, further lead optimization will be done if required, before entering it into the clinical Phase I and II trials using protocols similar to those employed for Sunitinib and Sorafenib, or other investigational small molecules targeting tumor angiogenesis. 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 5474.

1543 Imbalance in angiogenesis is involved in many pathological conditions, such as cancer, rheumatoid arthritis, and inflammation. Targeting angiogenesis has recently emerged as a proven therapeutic strategy for treatment of cancer and age-related macular degeneration. To understand the underlying biological signaling pathways of angiogenesis and to develop potential anti-angiogenic therapy, we have developed a system to discover small molecular probes that can selectively inhibit endothelial cell activation, and thereby block excessive angiogenesis. First, a high throughput screening (HTS) assay was developed for screening large libraries of chemical compounds against both primary human endothelia and fibroblasts to identify compounds with differential inhibitory activity against the endothelial cells versus fibroblasts. Second, HTS dose response-based EC50 values were determined to identify lead compounds with selectivity and potency against human endothelial activation. Third, a number of selected lead compounds were studied for their activities in endothelial cell tube-formation and migration, and to elucidate their mechanisms of action. Fourth, the selective compounds are being further studied in vivo by embryo CAM assay and mouse Matrigel plug assay. A number of compounds that exhibited specific inhibitory activities against human endothelial cells were identified from an 86,000 compound library screening. Analysis of structural clusters of the initial hits led to the identification of several chemical scaffolds as interesting leads. Structure-activity relationship (SAR) analyses and synthetic efforts on selected scaffolds for lead optimization are in progress. Our progress toward identification of specific anti-angiogenic agents will be presented.

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.

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. © 2007 Wiley‐Liss, Inc.

235 2-chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)adenine (clofarabine) is a new-generation nucleoside analog discovered by Southern Research Institute based on the experience with cladribine and fludarabrine. Clofarabine has demonstrated significant single-agent antitumor activity and has recently been approved by the FDA for the treatment of pediatric patients with relapsed or refractory acute lymphoblast leukemia. Clofarabine has also shown significant efficacy in xenograft models of solid tumors; its activity in solid tumors is presently being investigated clinically. It is known that clofarabine inhibits DNA synthesis and repair and induces apoptosis by disrupting the integrity of mitochondrial membranes. However, it has not been tested if clofarabine is active against tumor angiogenesis that is required for a solid tumor to grow beyond 2 mm in diameter. In this study, we have investigated the effect of clofarabine on endothelial cell proliferation, migration and differentiation into capillary vessels, the key steps in angiogenesis. The results show that clofarabine inhibits human endothelial cell proliferation, with an IC50 value in sub-micromolar range, and also their migration towards the angiogenic factors, an important functional property of endothelial cells to form the vascular network. Clofarabine, also, clearly interrupts the endothelial cell differentiation to form endothelial tubes. These results indicate that clofarabine is not only active against tumor cell growth, but also active against endothelial cells to inhibit its proliferation, migration and microvessel tube formation, suggesting an important role of clofarabine in antiangiogenesis. Further studies on the mechanism by which clofarabine inhibits neoangiogenesis are being undertaken. These findings highlight the potential of clofarabine as an effective agent against human solid tumors, which combines anti-proliferative and antiangiogenic activities in one molecule, and provide information to further optimize dosage and sequence of clofarabine treatment in future clinical trials.

4'-Thio-beta-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 IC(50)s. 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 anti proliferative 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.

1091 Clinical studies have shown that elevated levels of VEGF are associated with poor response to tamoxifen treatment for estrogen receptor (ER)-positive breast cancer. Our previous study provided the first direct in vivo evidence that tumor cytosolic VEGF expression at a clinically relevant level not only increases tumor angiogenesis, but also is sufficient to promote ER-positive, tamoxifen-responsive, and non-metastatic breast tumor to become tamoxifen resistant and metastatic. We have developed a xenograft mouse model of ER-positive human breast cancer, in which VEGF expression is tightly controlled by doxycycline in drinking water. The VEGF level detected in the tumor sample is within a range that is found in patient biopsy samples. This study suggested a new function of VEGF in tamoxifen resistance and metastasis and indicated that inhibition of both ER-dependent and VEGF-induced tumor cell growth would be a potential therapy for breast cancer. This study was designed to test this hypothesis using tamoxifen and bevacizumab (Avastin™), a recombinant humanized monoclonal antibody against VEGF. The efficacy of the tamoxifen-Avastin combined therapy was evaluated with two arms of mice designed for early and late stage settings of the disease using the newly developed animal model of ER-positive breast cancer. For the early stage setting, mice received the combined and mono treatments when their tumors reached about 100 mm3 in size. Significant inhibitory effect on primary tumor growth was observed for both the combined treatment and the Avastin mono treatment (P

Adenosine kinase from Mycobacterium tuberculosis is the only prokaryotic adenosine kinase that has been isolated and characterized. The enzyme catalyzes the phosphorylation of adenosine to adenosine monophosphate and is involved in the activation of 2-methyladenosine, a compound that has demonstrated selective activity against M. tuberculosis. The mechanism of action of 2-methyladenosine is likely to be different from those of current tuberculosis treatments and this compound (or other adenosine analogs) may prove to be a novel therapeutic intervention for this disease. The M. tuberculosis adenosine kinase was overexpressed in Escherichia coli and the enzyme was purified with activity comparable to that reported previously. The protein was crystallized in the presence of adenosine using the vapour-diffusion method. The crystals diffracted X-rays to high resolution and a complete data set was collected to 2.2 A using synchrotron radiation. The crystal belonged to space group P3(1)21, with unit-cell parameters a = 70.2, c = 111.6 A, and contained a single protein molecule in the asymmetric unit. An initial structural model of the protein was obtained by the molecular-replacement method, which revealed a dimeric structure. The monomers of the dimer were related by twofold crystallographic symmetry. An understanding of how the M. tuberculosis adenosine kinase differs from the human homolog should aid in the design of more potent and selective antimycobacterial agents that are selectively activated by this enzyme.