Rongbao Li

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.

Overexpression, purification and crystallographic analysis of a unique adenosine kinase from Mycobacterium tuberculosis

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.

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]

Discovery of a novel inhibitor of kinesin-like protein KIFC1

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.

Identification of the binding site of an allosteric ligand using STD-NMR, docking, and CORCEMA-ST calculations

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.

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 1746: Discovery and evaluation of a small molecule KIFC1 inhibitor 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

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