Yulia Y. Maxuitenko

A Novel Sulindac Derivative That Does Not Inhibit Cyclooxygenases but Potently Inhibits Colon Tumor Cell Growth and Induces Apoptosis with Antitumor Activity

Nonsteroidal anti-inflammatory drugs such as sulindac have shown promising antineoplastic activity, although toxicity from cyclooxygenase (COX) inhibition and the suppression of prostaglandin synthesis limits their use for chemoprevention. Previous studies have concluded that the mechanism responsible for their antineoplastic activity may be COX independent. To selectively design out the COX inhibitory activity of sulindac sulfide (SS), in silico modeling studies were done that revealed the crucial role of the carboxylate moiety for COX-1 and COX-2 binding. These studies prompted the synthesis of a series of SS derivatives with carboxylate modifications that were screened for tumor cell growth and COX inhibitory activity. A SS amide (SSA) with a N,N-dimethylethyl amine substitution was found to lack COX-1 and COX-2 inhibitory activity, yet potently inhibit the growth of human colon tumor cell lines, HT-29, SW480, and HCT116 with IC50 values of 2 to 5 μmol/L compared with 73 to 85 μmol/L for SS. The mechanism of growth inhibition involved the suppression of DNA synthesis and apoptosis induction. Oral administration of SSA was well-tolerated in mice and generated plasma levels that exceeded its in vitro IC50 for tumor growth inhibition. In the human HT-29 colon tumor xenograft mouse model, SSA significantly inhibited tumor growth at a dosage of 250 mg/kg. Combined treatment of SSA with the chemotherapeutic drug, Camptosar, caused a more sustained suppression of tumor growth compared with Camptosar treatment alone. These results indicate that SSA has potential safety and efficacy advantages for colon cancer chemoprevention as well as utility for treating malignant disease if combined with chemotherapy.

Colon Tumor Cell Growth-Inhibitory Activity of Sulindac Sulfide and Other Nonsteroidal Anti-Inflammatory Drugs Is Associated with Phosphodiesterase 5 Inhibition

Nonsteroidal anti-inflammatory drugs (NSAID) display promising antineoplastic activity, but toxicity resulting from cyclooxygenase (COX) inhibition limits their clinical use for chemoprevention. Studies suggest that the mechanism may be COX independent, although alternative targets have not been well defined. Here, we show that the NSAID sulindac sulfide (SS) inhibits cyclic guanosine 3′,5′-monophosphate (cGMP) phosphodiesterase (PDE) activity in colon tumor cell lysates at concentrations that inhibit colon tumor cell growth in vitro and in vivo. A series of chemically diverse NSAIDs also inhibited cGMP hydrolysis at concentrations that correlate with their potency to inhibit colon tumor cell growth, whereas no correlation was observed with COX-2 inhibition. Consistent with its selectivity for inhibiting cGMP hydrolysis compared with cyclic AMP hydrolysis, SS inhibited the cGMP-specific PDE5 isozyme and increased cGMP levels in colon tumor cells. Of numerous PDE isozyme–specific inhibitors evaluated, only the PDE5-selective inhibitor MY5445 inhibited colon tumor cell growth. The effects of SS and MY5445 on cell growth were associated with inhibition of β-catenin–mediated transcriptional activity to suppress the synthesis of cyclin D and survivin, which regulate tumor cell proliferation and apoptosis, respectively. SS had minimal effects on cGMP PDE activity in normal colonocytes, which displayed reduced sensitivity to SS and did not express PDE5. PDE5 was found to be overexpressed in colon tumor cell lines as well as in colon adenomas and adenocarcinomas compared with normal colonic mucosa. These results suggest that PDE5 inhibition, cGMP elevation, and inhibition of β-catenin transcriptional activity may contribute to the chemopreventive properties of certain NSAIDs. Cancer Prev Res; 3(10); 1303–13. ©2010 AACR.

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.

Establishment of a Mammary Carcinoma Cell Line from Syrian Hamsters Treated with N-Methyl-N-Nitrosourea

Clearly new breast cancer models are necessary in developing novel therapies. To address this challenge, we examined mammary tumor formation in the Syrian hamster using the chemical carcinogen N-methyl-N-nitrosourea (MNU). A single 50mg/kg intraperitoneal dose of MNU resulted in a 60% incidence of premalignant mammary lesions, and a 20% incidence of mammary adenocarcinomas. Two cell lines, HMAM4A and HMAM4B, were derived from one of the primary mammary tumors induced by MNU. The morphology of the primary tumor was similar to a high-grade poorly differentiated adenocarcinoma in human breast cancer. The primary tumor stained positively for both HER-2/neu and pancytokeratin, and negatively for both cytokeratin 5/6 and p63. When the HMAM4B cell line was implanted subcutaneously into syngeneic female hamsters, tumors grew at a take rate of 50%. A tumor derived from HMAM4B cells implanted into a syngeneic hamster was further propagated in vitro as a stable cell line HMAM5. The HMAM5 cells grew in female syngeneic hamsters with a 70% take rate of tumor formation. These cells proliferate in vitro, form colonies in soft agar, and are aneuploid with a modal chromosomal number of 74 (the normal chromosome number for Syrian hamster is 44). To determine responsiveness to the estrogen receptor (ER), a cell proliferation assay was examined using increasing concentrations of tamoxifen. Both HMAM5 and human MCF-7 (ER positive) cells showed a similar decrease at 24h. However, MDA-MB-231 (ER negative) cells were relatively insensitive to any decrease in proliferation from tamoxifen treatment. These results suggest that the HMAM5 cell line was likely derived from a luminal B subtype of mammary tumor. These results also represent characterization of the first mammary tumor cell line available from the Syrian hamster. The HMAM5 cell line is likely to be useful as an immunocompetent model for human breast cancer in developing novel therapies.

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 3730: A quick and cost effective 12-cell line panel assay to predict drug activity in human tumor xenograft models

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The procedure to identify and develop an anti-cancer drug first involves testing drug candidates in cell lines followed by human tumor xenograft models, usually selected based upon the histotype of the cell lines in which the drug showed optimal activity. Many drugs fail at this stage, as activity in cell lines does not often correlate with activity in xenograft models. This is not surprising, as we have previously shown that gene expression in xenograft models does not necessarily correlate with the cell line from which it was derived. In an attempt to improve the success rate of drugs tested in xenograft models, we have developed a fast and cost effective 12-panel human tumor cell line assay that represents the genetic diversity of all our xenograft models and several different cancer histotypes. Affymetrix genomic analysis was performed on 100 human tumor xenograft and cell line models. The genomic profiles obtained underwent Unsupervised Hierarchical Cluster Analysis to group models with similar genetic profiles. This analysis resulted in 12 distinct clusters; a representative cell line was chosen from each cluster. Stocks of each representative cell line were frozen and tested to ensure exponential growth immediately upon thawing, resulting in no waiting time for drug testing. It follows that if a candidate drug shows activity in one or more of these representative cell lines, other cell lines and/or xenograft models in the same cluster can also be tested. As the cell lines and xenograft models within the same cluster will have a similar genetic profile, the chances of success should thus be increased. To test the effectiveness of this approach, we used our database to further develop an internal compound. SRI-20900 had been tested previously in the CCRF-CEM and CAKI-1 xenograft models. The compound showed no activity in CCRF-CEM cells, but excellent activity in CAKI-1 cells. These models were in completely different clusters. So, based on these data, we tested the compound in the SKOV-3 and IGROV-1 xenograft models, as these clustered closely to the CAKI-1 model. The compound showed excellent activity in both SKOV-3 and IGROV-1 models. Although these data provide proof of principle, further work needs to be done by testing targeted compounds in the 12-cell line panel, followed by testing in xenograft models within the same cluster as the cell lines that show optimal activity. In addition, it would follow that a xenograft model within the same cluster as an inactive cell line should also be tested. We hope to start these studies early in 2014. Citation Format: Michael J. Roberts, Tommie A. Gamble, Richard D. May, Murray Stackhouse, Kristy L. Berry, Andrew D. Penman, Robert J. Rooney, Yulia Maxuitenko, Michael S. Koratich. A quick and cost effective 12-cell line panel assay to predict drug activity in human tumor xenograft models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3730. doi:10.1158/1538-7445.AM2014-3730

Abstract 2778: Tumor target vs. tissue of tumor origin: cluster analysis of genomic profile of 42 human tumor in vitro and in vivo models.

Traditionally, drug development has relied upon testing cancer drug candidates in cell lines. Active drugs are then tested in human tumor xenograft models, usually selected based upon the cell lines in which the drug showed activity. The majority of drugs fail at this stage as they do not show activity in the xenograft models chosen. We performed Affymetrix genomic analysis on 42 human tumor xenograft models and the original cell lines from which they were established. The genomic profiles obtained underwent Unsupervised Hierarchical Cluster Analysis to ascertain which cell lines and xenograft models had similar genomic profiles and which did not. The analysis showed that only 24 of 42 human tumor xenograft models clustered side-by-side with the cell line from which they were established. All 6 human leukemia/lymphoma xenograft models clustered very well with the cell lines from which they were established, and they clustered perfectly according to histological class. Five out of six human colon tumor xenograft models clustered well with the cell lines from which they were established and according to histotype. Of the 18 xenograft/cell line pairs that did not cluster side-by-side, 10 pairs remained in the same general cluster, whereas the partners of 8 other pairs were dispersed across different major clusters. Ovarian, breast, melanoma, and pancreatic human tumor xenograft models did not cluster according to histotype. Our data may explain why some drugs that show in vitro activity in some cell lines are not active in other cell lines of the same histological type, and also why some drugs that show activity in vitro then fail in xenograft models. In our laboratory, the PANC-1 cell line is very often chosen as a model of pancreatic cancer. A drug showing activity in the PANC-1 cell line would next be tested in other in vitro models of pancreatic cancer (e.g., MIA PaCa-2, CFPAC-1, and BxPC-3). However, none of these other pancreatic models have a similar genetic profile to PANC-1. Based upon our data, the cell line showing most similarity to the PANC-1 cell line is the breast cancer cell line MDA-MB-231. It is our suggestion that a drug showing activity in the PANC-1 cell line should be tested in other cell lines showing similar genetic profiles, not in cell lines based on histotype. Another example from our analysis is the LOX-IMV1 melanoma cell line. Not only does this cell line not cluster with its corresponding LOX-IMV1 xenograft model, it clusters most closely with the NCI/ADR-RES ovarian cell line. In summary, the genomic profiles of approximately 57% of the tumor xenograft models analyzed closely associate with the cell line from which they were established. Some of the tumor xenograft models show very little similarity to the cell lines from which they were established. Additionally, many of the models (both xenografts and cell lines), do not cluster according to their tissue of origin. Citation Format: Michael J. Roberts, Michael S. Koratich, Murray Stackhouse, Richard D. May, Andrew D. Penman, Tommie A. Gamble, Kristy L. Berry, Joseph F. Murphy, Robert J. Rooney, Yulia Y. Maxuitenko. Tumor target vs. tissue of tumor origin: cluster analysis of genomic profile of 42 human tumor in vitro and in vivo models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2778. doi:10.1158/1538-7445.AM2013-2778

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