Amy Fulton - Academia.edu (original) (raw)

Papers by Amy Fulton

Cancer Research, May 1, 2007

4664 Chemokine receptors are expressed by normal cells to enable migration in response to chemota... more 4664 Chemokine receptors are expressed by normal cells to enable migration in response to chemotactic gradients. Several chemokine receptors have now been identified on malignant cells and are implicated as determinants of tumor growth and metastasis. We have examined the expression and function of the chemokine receptor CXCR3 in metastatic breast cancer. We previously reported that malignant breast tumor cells express CXCR3 and that pharmacologic antagonism of CXCR3 inhibits experimental metastasis (Cancer Res. 66:7701, 2006). In the current study, we expressed an shRNA that targets CXCR3 in metastatic murine mammary tumor cells and show that, like pharmacologic antagonism, genetic inhibition of CXCR3 inhibits metastasis compared to vector-shRNA expressing cells. We have compared the expression and function of CXCR3 on normal versus malignant mammary epithelial cells. Each murine mammary cell type expresses surface CXCR3 although normal cells express significantly less CXCR3 than malignant cells. Likewise, normal human mammary epithelial cell line MCF10A expresses less CXCR3 than any malignant breast cell line examined. We also assessed the function of CXCR3 in migration assays in vitro . We observe that migration of normal epithelial cells in response to CXCR3 ligands requires much higher concentrations of ligand to induce significant migration. We are currently evaluating CXCR3 expression in a large series of breast tumors from women for whom longterm follow up information is available. Immunohistochemical examination of CXCR3 reveals a range of staining intensities with the majority of normal ducts expressing little or no CXCR3, whereas malignant epithelium is more highly positive. The relationship to clinical parameters is under examination. These studies confirm that inhibition of CXCR3 by either genetic or pharmacologic approaches reduces metastatic potential. These results identify CXCR3 as a determinant of tumor metastasis and as a potential therapeutic target in breast cancer.

2487 Several chemokine receptors have now been described in malignancy. The purpose of the curren... more 2487 Several chemokine receptors have now been described in malignancy. The purpose of the current study is to determine if the chemokine receptor CXCR3, which binds the ligands CXCL9, CXCL10 and CXCL11, is expressed in breast cancer and if this receptor contributes to more aggressive disease. By immunohistochemistry, early stage breast cancers express CXCR3 in malignant epithelium and expression levels are associated with significantly poorer survival. To examine the functional role of CXCR3 in breast tumor metastasis, we employed a syngeneic model of metastatic breast cancer. Like human breast cancer cells, metastatic murine mammary tumor cells express CXCR3 on the surface and, abundantly, in the cytoplasm. Using shRNA, we stably inhibited CXCR3 protein and mRNA in line 66.1 murine mammary tumor cells. When 66.1-shCXCR3 cells were implanted into the mammary fat pad of syngeneic Balb/cByJ mice, spontaneous metastasis to the lung was markedly reduced (by 72-94%) in comparison to vector control tumor cells. In contrast, growth of the mammary gland-injected tumors was not compromised by CXCR3 gene silencing. Lung colony formation by intravenously injected tumor cells was also significantly reduced by CXCR3 gene silencing. These data confirm our previous findings that a pharmacologic CXCR3 antagonist inhibits metastatic but not local mammary tumor growth. When CXCR3 expression and function on immortalized normal mammary epithelial (EpH4) and malignant breast cells are compared, levels of CXCR3 receptor protein are not different, however, quantitative and qualitative differences in receptor function are observed. CXCR3 ligands stimulate migration of tumor cells but EpH4 cells have a negligible chemotactic response to these ligands. Proliferation of EpH4 cells is inhibited by CXCR3 ligands in a dose-dependent manner, whereas growth of malignant cells is largely unaffected by the presence of CXCR3 ligands. Signal transduction pathway activation also differs between malignant and normal cells. Thus, CXCR3 activation alters the behavior of both malignant and normal cells, but in contrasting directions. Taken together, these studies indicate that CXCR3 contributes to metastatic potential in a model of metastatic breast cancer. The positive association of CXCR3 and poor longterm survival in women with breast cancer supports the further examination of this receptor as a therapeutic target.

Ovarian cancer has the highest mortality incidence of all gynecologic malignancies in the United ... more Ovarian cancer has the highest mortality incidence of all gynecologic malignancies in the United States. The majority of ovarian cancer cases lead to recurrent disease that is often incurable and fatal due to innate or acquired chemoresistance; therefore, novel therapeutic interventions are desperately needed. Cyclooxygenases–COX-1 and COX-2–are enzymes that catalyze the production of prostaglandin E2 (PGE2), an important inflammatory lipid mediator that is functionally linked to progression of many cancers, including breast and ovarian cancer. PGE2 is exported from the cell via multidrug resistance-associated protein 4 (MRP4) where it acts in a paracrine and autocrine manner by activating a family of four G-protein coupled receptors (EP1-4) that are linked to different intracellular signaling pathways. EP2 and EP4 can activate PKA/cAMP, PI3K and ERK pathways. We hypothesize that the EP4 receptor has increased expression in ovarian cancer and that binding of its cognate ligand, PGE2, will drive ovarian cancer progression. We also hypothesize that alternation of the tumor microenvironment via MRP4 will also lead to inhibition of EP4-mediated signaling and affect phenotypes associated with ovarian cancer progression. In order to test this hypothesis, we analyzed the expression of the EP4 and MRP4 in a human ovarian cancer tissue microarray (TMA) as well as human ovarian cancer cell lines. Immunohistochemical analysis of EP4 on the TMA composed of varying histologies, including serous, endometrioid, and clear-cell, as well as normal ovarian tissue, revealed that EP4 was expressed in 38.7% of ovarian cancer tissues, whereas EP4 had no or low expression in 10 normal ovarian tissue samples. Immunohistochemistry of MRP4 also revealed increased expression in ovarian cancer histologies compared to normal ovarian tissue. Serous, endometrioid, and clear-cell subtypes presented with a majority of 4+ and 3+ staining intensities compared to normal ovarian tissue, which presented with mostly 2+ and 1+ staining, and none of the normal ovarian tissue presented with 4+ intensity. EP4 and MRP4 also has increased expression in multiple ovarian cancer cells lines including those representing low-grade serous, clear-cell, and high-grade serous ovarian cancer. Treatment of these cell lines with an EP4 antagonist resulted in decreased proliferation and migration compared to vehicle control. Consistent with the pharmacologic data, treatment of ovarian cancer cell lines with siRNA directed against the EP4 receptor led to decreased proliferation and migration. Inhibition of PGE2 export via MRP4 inhibitor Ceefourin and probenecid results in increased sensitization of ovarian cancer cell lines to treatment with paclitaxel. Based on these data, targeting of the PGE2 EP4 receptor and PGE2 export via MRP4 should be investigated further for the treatment of ovarian cancer. Citation Format: Jocelyn Reader, McMillan Ching, Cong (Ava) Fan, Sulan Wu, Paul Staats, Teklu Legesse, Olga Goloubeva, Ningbo Jian, Mark Carey, Amy Fulton, Dana Roque, Gautam Rao. Analysis of function and inhibition of PGE2 pathway members MRP4 and EP4 in treatment of ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B67.

OBJECTIVES: Advanced ovarian clear cell carcinoma (OCCC) is associated with a survival disadvanta... more OBJECTIVES: Advanced ovarian clear cell carcinoma (OCCC) is associated with a survival disadvantage relative to ovarian serous carcinoma following platinum/taxane-based chemotherapy and optimal cytoreduction. Prostaglandin E2 (PGE2) contributes to disease progression through modulation of several G-protein coupled receptors (EP1-4) [Fig.1a]. The addition of upstream COX inhibition to platinum/taxane-based chemotherapy in the first-line phase II setting in ovarian cancer has been disappointing,a possibly due to compensatory upregulation of COX isoenzymes and negation of protective effects of EP1. Selective antagonism of EP4 may therefore pose a more rational strategy than global COX inhibition. Paclitaxel administration has been shown to upregulate components of the COX pathway.b Class III β-tubulin is a marker for paclitaxel resistance and is widely overexpressed in OCCC. The purpose of this study is to demonstrate that EP4 inhibition may overcome paclitaxel resistance in OCCC that overexpress class III β-tubulin. METHODS: Expression of EP4 receptor and class III β-tubulin was quantified using immunohistochemistry and Western blot in solid tissues and cell lines. Standard metabolic growth and migration assays were employed to test the effects of drug treatment (paclitaxel and EP4 inhibitors RQ-15986/AH-23848) with and without EP4 silencing using siRNA. RESULTS: OCCC overexpress class III β-tubulin/EP4 relative to normal ovary [Fig. 1b/c]. EP4 staining intensity was 2+ in 100% OCCC using an ovarian cancer tissue microarray (62 cores, 13% OCCC); this rate was only 26-56% among other histologies. EP4 inhibition reduces growth of paclitaxel-resistant cells [Fig. 1d]. Likewise, treatment with EP inhibitors [Fig.1 e-top] and silencing of EP4 resulted in reduced migration [Fig.1 e-bottom]. CONCLUSIONS: Selective antagonism of PGE2 through EP4 receptor inhibition may represent a powerful targeted therapy for paclitaxel-resistant OCCC. Further study including simultaneous treatment (EP4 inhibitor+paclitaxel) and larger samples sizes is required. REFERENCES: a Reyners et al. A randomized phase II study investigating the addition of the specific COX-2 inhibitor celecoxib to docetaxel plus carboplatin as first-line chemotherapy for stage IC to IV epithelial ovarian cancer, fallopian tube or primary peritoneal carcinomas: the DoCaCel study. Ann Oncol 2012; 23:2986-902. b Moos et al. Effects of taxol/taxotere on gene expression in macrophages: induction of the PGH synthetase-2 isoenzyme. J Immunol 1999;162:476-73. Citation Format: Dana M. Roque, Danielle Meir-Levi, Gautam G. Rao, Paul Staats, Amy Fulton, Jocelyn Reader. EP4 receptor antagonism in paclitaxel-resistant ovarian clear cell carcinomas that overexpress class III β-tubulin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1179. doi:10.1158/1538-7445.AM2017-1179

Neuro-oncology, Nov 1, 2015

Ion channels govern portals that facilitate ionic passage across membranes in all organisms. KCNK... more Ion channels govern portals that facilitate ionic passage across membranes in all organisms. KCNK9, a member of the family of two-pore domain potassium K+ channels, mediates "leak" currents critical to maintaining cell resting membrane potentials. Since its identification, KCNK9 has been implicated in various human disorders including cancer. KCNK9 overexpression has been observed in melanoma, colorectal, breast and lung cancers, and KCNK9 has been found to promote neoplastic cell survival and growth. However, KCNK9's underlying contributions to malignancy remain elusive due to the absence of specific modulators. Here, we describe the development of novel monoclonal antibodies directed against the KCNK9 extracellular domain and their functional effects. We show that one antibody (Y4) with the highest affinity binding, inhibits KCNK9 activity by inducing internalization of cell-surface channels. When tested on KCNK9-expressing cells, Y4 reduces cell viability; increases cell death and triggers complement-dependent cytotoxicity. Systemic administration of Y4 effectively inhibits growth of human lung cancer xenografts and breast cancer metastasis in a syngeneic murine model (P < 0.05). Since KCNK9 expression has been reported in human glioma and linked to regulation of glioblastoma cell survival, we extended our study to glioblastoma and examined KCNK9 expression in a panel of glioblastoma cell lines as well as primary cultures from patient-derived tumors. We found abundant KCNK9 expression in some aggressive glioblastoma models (e.g. Mayo39). We are currently investigating the functional effects of antibodies in these models. In conclusion, we have generated a high affinity KCNK9-specific monoclonal antibody that inhibits tumor cell survival, primary tumor formation and metastasis. Antibody-based KCNK9 targeting can shed light on KCNK9's significance in glioblastoma development and become promising therapeutic strategy in KCNK9-expressing brain malignancy.

Objectives: To examine the role of the cyclooxygenase (COX) pathway in ovarian cancer. Cyclooxyge... more Objectives: To examine the role of the cyclooxygenase (COX) pathway in ovarian cancer. Cyclooxygenases, specifically COX1 and COX2, are frequently overexpressed in malignancies and lead to the synthesis of prostaglandins. A particularly important prostaglandin, PGE2 is exported from the cell into the extracellular milieu by the multi-drug resistance protein 4 (MRP4) transporter where it acts in a paracrine and autocrine manner by activating a family of G-protein coupled receptors (EP1-4). PGE2 is transported into the cell by the prostaglandin transporter (PGT) and catabolized by 15-prostaglandin dehydrogenase (15-PGDH). It has been previously shown that COX1, rather than COX2, is overexpressed in ovarian cancer. Little is known about the role of other members of this pathway, including EP receptors, PGT, MRP4, and 15-PGDH, in this disease. Methods: HOSE (benign immortalized ovarian epithelial cells) and two ovarian cancer cell lines, SKOV3 and OVCAR3, were grown under standard conditions. Cells were harvested and RNA and protein were isolated. Quantitative RT-PCR was done using the SYBR Green protocol to examine the expression of members of the cyclooxygenase pathway and expressed relative to HOSE cells. Results: In both ovarian cancer cell lines relative to HOSE, expression of COX2, PGT, EP4, and EP1 was decreased. In SKOV3 cells, COX1 and 15-PGDH expression was decreased but MRP4 levels were comparable to levels detected in HOSE cells. In OVCAR3 cells, COX1 and 15-PGDH expression levels were increased and MRP4 was decreased relative to HOSE cells. PGE2 levels in media will be measured to determine the net effect of these changes. Protein levels of the members of the cyclooxygenase pathway will be determined by western blot. Conclusions: It is clear that several members of the cyclooxygenase pathway are expressed differently in malignant versus normal ovarian cells. Studies in progress will determine the functional significance of these differences. Citation Format: Gautam G. Rao, Tyler Kochel, Namita Kundu, Jocelyn Reader, Amy Fulton. Cyclooxygenase pathway in ovarian cancer. [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 524. doi:10.1158/1538-7445.AM2013-524

Ovarian cancer is the leading cause of death from gynecologic malignancy in the United States. Mo... more Ovarian cancer is the leading cause of death from gynecologic malignancy in the United States. Most cases of ovarian cancer present in late stages, leading to recurrent disease that is incurable and often fatal due to innate or acquired chemoresistance; therefore, new therapies are desperately needed. Cyclo-oygenases, COX-1 and COX-2, are enzymes that catalyze the production of the prostaglandin E2 (PGE2), a lipid mediator that is functionally linked to progression of many cancers including breast and ovarian cancer. PGE2 is exported from the cell via multidrug resistance-associated protein 4 (MRP4) where it acts in a paracrine and autocrine manner by activating a family of four G-protein coupled receptors (EP1-4) that are linked to different intracellular signaling pathways. EP2 and EP4 can activate PKA/cAMP, PI3K, and ERK pathways. COX-1 and COX-2 have been shown to be overexpressed in primary ovarian cancer as well as in many ovarian cancer cell lines. We hypothesized that the EP4 receptor is overexpressed in ovarian cancer; that binding of its cognate ligand, PGE2, will drive ovarian cancer progression; and that inhibition of the EP4-mediated signaling will lead to inhibition of ovarian cancer growth and metastasis. In order to test this hypothesis, we analyzed the expression of the EP4 receptor in a human ovarian cancer tissue microarray (TMA) as well as human ovarian cancer cell lines. Immunohistochemical analysis of EP4 on the TMA composed of varying histologies, including serous, endometrioid, and clear cell, as well as normal ovarian tissue revealed that EP4 was expressed in 38.7% of ovarian cancer patients, whereas EP4 was not expressed in the 10 normal ovarian tissue samples. Additionally, in comparison to immortalized human ovarian surface epithelial (HOSE) cells, EP4 is overexpressed in many of the cell lines analyzed, including OVCAR-3, COAV-3, SKOV3, and Kuramochi cells. Blockade of the EP4 receptor via antagonist or siRNA results in a decrease in proliferation, migration, and invasion in ovarian cancer cell lines. In addition to overexpression of EP4, MRP4 was also found to be overexpressed in ovarian cancer cell lines when compared to HOSE cells. Given the role of MRP4 in exporting PGE2 and affecting drug resistance/ sensitivity in breast cancer cell lines, we hypothesize that MRP4 will similarly play a role in modulating the amount of PGE2 in the microenvironment in ovarian cancer, thereby affecting signaling via of EP4 receptor. Functional testing of dual EP4/ MRP4 inhibition may provide much-needed new therapies for the treatment of ovarian cancer. Citation Format: Mc Millan Ching, Cong Fan, Dana Roque, Gautam Rao, Paul Staats, Amy Fulton, Jocelyn Reader. Functional analysis of PGE2 pathway members EP4 and MRP4 in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A22.

Advanced ovarian clear cell carcinoma (OCCC) is associated with a survival disadvantage relative ... more Advanced ovarian clear cell carcinoma (OCCC) is associated with a survival disadvantage relative to ovarian serous carcinoma following platinum/taxane-based chemotherapy and optimal cytoreduction. Prostaglandin E2 (PGE2) contributes to disease progression through modulation of several G-protein coupled receptors (EP1-4). Selective EP4 antagonism is preferred over global COX inhibition due to concerns over cardiotoxicity and compensatory upregulation of COX isoenzymes. Class III β-tubulin is a marker for paclitaxel resistance and is widely overexpressed in OCCC. The purpose of this study is to demonstrate that EP4 inhibition may overcome paclitaxel resistance in OCCC that overexpress class III β-tubulin. Expression of EP4 receptor and class III β-tubulin was quantified using immunohistochemistry and Western blot in solid tissues and cell lines. OCCC overexpress class IIIβ-tubulin/EP4 relative to normal ovary. EP4 staining intensity was 2+ in 100% OCCC using an ovarian cancer tissue microarray (62 cores, 13% OCCC); this rate was only 26-56% among other histologies. Standard metabolic growth and migration assays were employed to test the effects of drug treatment and EP4 inhibition. EP4 inhibition reduces growth of paclitaxel resistant cells. Likewise, treatment with EP inhibitors and silencing of EP4 resulted in reduced migration. Selective antagonism of PGE2 through EP4 receptor inhibition may represent a powerful targeted therapy for paclitaxel-resistant OCCC. Further study including simultaneous treatment (EP4 inhibitor + paclitaxel) and larger samples sizes is required. Citation Format: Cong Fan, Jocelyn Reader, Gautam Rao, Paul Staats, Mc Millan Ching, Amy Fulton, Dana Roque. EP4 receptor antagonism in paclitaxel-resistant ovarian clear cell carcinomas. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A50.

Cancers, Oct 18, 2019

The microtubule-stabilizing agent docetaxel in combination with gemcitabine represents one of the... more The microtubule-stabilizing agent docetaxel in combination with gemcitabine represents one of the most effective regimens against the aggressive gynecologic tumor leiomyosarcoma (LMS). Upregulation of class III β-tubulin has previously been shown to confer taxane resistance in a variety of human cancers. Prostaglandin E 2 receptor EP4 is linked to progression of a variety of human cancers and may represent a novel target for tumor inhibition in LMS. We evaluated the hypotheses that EP4 and class III β-tubulin have increased expression in LMS in comparison to normal myometrium or benign tumors and that expression of class III β-tubulin correlates with resistance to taxanes and poor clinical outcome. Gene expression was examined using TCGA data and correlated with clinicopathologic outcome which demonstrated that class III β-tubulin is more highly expressed in more aggressive sarcomas with EP4 being widely expressed in all subtypes of sarcoma. Immunohistochemistry for EP4 and class III β-tubulin was performed on patients with LMS, leiomyomatosis/STUMP, leiomyoma, and normal myometrium. Expression of EP4 and class III β-tubulin were characterized for cell lines SK-UT-1, SK-UT-1B, and PHM-41 and these cell lines were treated with docetaxel alone and in combination with EP4 inhibitors. In taxane-resistant cell lines that overexpress class III β-tubulin and EP4, treatment with EP4 inhibitor resulted in at least 2-fold sensitization to docetaxel. Expression of class III β-tubulin and EP4 in LMS may identify patients at risk of resistance to standard chemotherapies and candidates for augmentation of therapy through EP4 inhibition.

Cancer Pharmacology, 2019

Systemic treatments for breast carcinoma have improved substantially over the past quarter centur... more Systemic treatments for breast carcinoma have improved substantially over the past quarter century. New insights into cancer biology, refinements in biotechnology, and bioengineering of macromolecules hold the promise of even greater reductions in breast and other cancer mortality as a result of biologicals. As exemplified by the clinical results with the monoclonal antibody to HER-2 for antigen-specific passive immunotherapy, biological therapies for breast carcinoma hold substantial promise. The objective of this report is to highlight aspects of preclinical and clinical research on other biologicals for breast carcinoma that also hold potential for improving patient survival. As examples of the potential of cytokines to modulate breast carcinoma cell proliferation and tumor growth, data on cytokines (interferons) with pleiotropic effects and a lymphokine (interleukin-10) acting on T cells and macrophages will be reviewed. HER-2 has promise as a vaccine for active specific immunotherapy; these data will be summarized. Progress on these and other biologicals promises that this will be another modality of therapy resulting in improved survival for patients with both early and metastatic breast carcinoma in the next millennium.

Cancer Research, 2009

Background: The cyclooxygenase-2 (COX-2) enzyme is highly expressed in breast cancer and is assoc... more Background: The cyclooxygenase-2 (COX-2) enzyme is highly expressed in breast cancer and is associated with a poorer prognosis. Past efforts have focused on targeting the COX-2 enzyme with pharmacologic COX-2 inhibitors including celecoxib and rofecoxib. Recent safety concerns have led us to test the hypothesis that targeting downstream members of the COX-2 pathway will be more efficacious and safer. The principle COX-2 product in tumors is prostaglandin E2 (PGE2) which mediates cellular effects by binding to four distinct G-protein-coupled receptors designated EP1, EP2, EP3 and EP4.Materials and Methods: Using pharmacologic and genetic approaches and a preclinical model of metastatic, triple-negative (ER, PR, Her-2 negative) breast cancer, we have examined the role of EP4 and EP1 in malignant behavior. We determined the effect of antagonizing either EP1 or EP4 with the pharmacologic antagonists AH23848, ONO-AE3-208 or SC19220 on metastatic ability of line 66.1 or line 410.4 mammary...

Frontiers in Pharmacology, May 29, 2020

The cyclooxygenase-2 (COX-2) enzyme is frequently overexpressed in epithelial malignancies includ... more The cyclooxygenase-2 (COX-2) enzyme is frequently overexpressed in epithelial malignancies including those of the breast, prostate, lung, kidney, ovary, and liver and elevated expression is associated with worse outcomes. COX-2 catalyzes the metabolism of arachidonic acid to prostaglandins. The COX-2 product prostaglandin E 2 (PGE 2) binds to four G-protein-coupled EP receptors designated EP1-EP4. EP4 is commonly upregulated in cancer and supports cell proliferation, migration, invasion, and metastasis through activation of multiple signaling pathways including ERK, cAMP/PKA, PI3K/AKT, and NF-kB. EP4 antagonists inhibit metastasis in preclinical models. Cancer stem cells, that underlie therapy resistance and disease relapse, are driven by the expression of EP4. Resistance to several chemotherapies is reversed in the presence of EP4 antagonists. In addition to tumor cell-autonomous roles of EP4, many EP4-positive host cells play a role in tumor behavior. Endothelial cell-EP4 supports tumor angiogenesis and lymphangiogenesis. Natural Killer (NK) cells are critical to the mechanism by which systemically administered EP4 antagonists inhibit metastasis. PGE 2 acts on EP4 expressed on the NK cell to inhibit tumor target cell killing, cytokine production, and chemotactic activity. Myeloid-derived suppressor cells (MDSCs), that inhibit the development of cytotoxic T cells, are induced by PGE 2 acting on myeloid-expressed EP2 and EP4 receptors. Inhibition of MDSC-EP4 leads to maturation of effector T cells and suppresses the induction of T regulatory cells. A number of EP4 antagonists have proven useful in dissecting these mechanisms. There is growing evidence that EP4 antagonism, particularly in combination with either chemotherapy, endocrine therapy, or immunebased therapies, should be investigated further as a promising novel approach to cancer therapy. Several EP4 antagonists have now progressed to early phase clinical trials and we eagerly await the results of those studies.

Journal of the National Cancer Institute, Apr 17, 1996

Background: Interleukin 10 (IL-10) is a potent immunoregulatory cytokine. It inhibits some cell f... more Background: Interleukin 10 (IL-10) is a potent immunoregulatory cytokine. It inhibits some cell functions, including T-helper (Thl) cell activity (i.e., interleukin 2 and interferon gamma production), and stimulates other functions such as natural killer (NK) activity. In mice, IL-10 suppresses tumorigenicity in a xenograft system using a nonmetastasizing hamster cell line. Purpose: We evaluated the antitumor and antimetastatic properties of IL-10 in syngeneic immunocompetent and immunocompromised murine hosts. Methods: Using the plasmids pBMGneo and pBMGneo.IL-10, we transfected the highly malignant murine mammary tumor cell lines 410.4 and 66.1 (transfectants designated as 410.4-IL10 and 66.1-IL10, respectively) to stably express IL-10 (2-100 U IL-10/2.5 x 10 5 cells per 48 hours). Tumorigenic and metastatic activities of the parent and transfected cells were measured in immunocompetent, syngeneic BALB/cByJ mice as well as in immunocompromised C.B-17/IcrCrl-SCID/BR and C.B-17/IcrCrl-SCID/Beige mice. Results: Tumor growth was completely inhibited following inoculation of 5 x 10 6 410.4-IL10 cells in immunocompetent, syngeneic BALB/cByJ mice. This inoculum contained 100 times the minimum cell number required for 100% tumor incidence. In contrast, tumor growth following the inoculation of parental 410.4 or 410.4-neo cells was progressive, resulting in death of animals from pulmonary metastases at days 40-50 after transplantation. The tumorigenicity of 66.1-IL10, compared with that of its parent cell line, was also significantly abrogated by IL-10 expression. Furthermore, in immunocompetent mice, the metastatic potential of both 410.4-IL10 and 66.1-IL10 was also completely inhibited. In immunocompromised C.B-17/IcrCrl-SCID/BR or C.B-17/IcrCrl-SCID/Beige mice, subcutaneous implants of 410.4-IL10 grew progressively, but growth was inhibited significantly in comparison to that produced by the parental 410.4 or 410.4-neo cells. In spite of the more limited efficacy of IL-10 against tumor growth in immunocompromised mice, spontaneous metastasis of 410.4-IL10 cells in C.B-17/IcrCrl-SCID/BR mice was inhibited by 90%. When NK activity was suppressed by asialoGMl ganglioside antibody in BALB/cByJ mice or in C.B-17/IcrCrl-SCID/Beige mice, the antimetastatic effect of IL-10 was lost. Conclusions: These data show for the first time that IL-10 is a potent antimetastatic agent that is effective in immunocompromised hosts. This effect thus appears to be relatively independent of T-cell function but is dependent on NK activity. In contrast, the inhibitory effect of IL-10 on tumorigenicity relies on T-cell function. Implications: Based on the recent observation of others that IL-10 has little toxicity when administered systemically to human volunteers and also on the findings of this study that it has antitumor and antimetastatic properties in mice, possible use of IL-10 in the treatment of human metastatic cancers deserves consideration.

There is an urgent need to identify molecular targets that are relevant to metastatic and/or stem... more There is an urgent need to identify molecular targets that are relevant to metastatic and/or stem-like cancer cells (CSC). Theoretically, surface-expressed G-protein coupled receptors on CSC are attractive therapeutic targets. There is considerable evidence that the inflammatory milieu of the tumor microenvironment drives CSC and we now show that two isoforms of the chemokine receptor CXCR3 play important but distinct roles in cancer behavior. In the bulk tumor cell population, CXCR3, isoform A is highly expressed relative to CXCR3B. CXCR3A is critical to metastatic success. CXCR3, isoform B, is highly upregulated in breast cancer CSC. When CXCR3B is gene-silenced in a model of basal-type, metastatic breast cancer, tumorsphere-forming capacity is reduced and the aldehyde dehydrogenase-positive (ALDH1+) population is correspondingly decreased. Conversely, overexpression of CXCR3B enhances these CSC properties. Thus, CXCR3B is critical to maintenance of the phenotype and function of breast CSC. CXCR3 ligands CXCL9, CXCL10 and CXCL11 all bind with high affinity to CXCR3, however, each ligand is coupled to different intracellular signaling pathways. For example, others have reported that, in HEK cells, CXCL10 strongly induces ERK activation, but CXCL11 does not. In our hands, CXCL11 more potently induces an ALDH1+ population in MDA-MB-231 cells compared to CXCL10. We have developed novel allosteric modulators of CXCR3 that demonstrate probe (ligand)-dependence. BD64 preferentially inhibits CXCL11-mediated β-arrestin 2 recruitment relative to inhibition of GPCR signaling; BD103 is significantly more effective at blocking CXCL11 versus CXCL10-mediated GPCR signaling. Neither BD64 nor BD103 affects the proliferation of non-CSC, however both compounds inhibit the ability of CSC to form tumorspheres. Furthermore, both CXCR3 antagonists reverse ligand-mediated induction of the ALDH1+ fraction. Most interesting is that both BD64 and BD103 effectively inhibit tumor cell colonization of the lung. These data suggest that optimal tumor control will be achieved when both metastatic, CXCR3Ahigh as well as CXCR3Bhigh CSC populations are inhibited by highly specific CXCR3 modulators. Note: This abstract was not presented at the meeting. Citation Format: Namita Kundu, Regine Brox, Xinrong Ma, Jocelyn Reader, Nuska Tschammer, Amy M. Fulton. Novel CXCR3 allosteric modulators inhibit breast cancer stem cells and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4770. doi:10.1158/1538-7445.AM2017-4770

Growing evidence has demonstrated that the two major isoforms of chemokine receptor CXCR3 (CXCR3-... more Growing evidence has demonstrated that the two major isoforms of chemokine receptor CXCR3 (CXCR3-A, CXCR3-B) both contribute to breast cancer pathogenesis and metastasis, however, in different ways. Understanding the relative contribution of each isoform is critical to optimize therapeutic strategies targeting CXCR3. Furthermore, the possible involvement of either CXCR3 isoform in cancer stem-like properties has not been reported. We examined the contribution of each isoform to metastasis using a panel of breast cell lines and a xenograft model of breast cancer; we also explored the role of major CXCR3 isoforms in the behavior of stem-like cells. CXCR3-A is more abundantly expressed than CXCR3-B in both primary human breast cancer tissue and in breast cancer cell lines. On the contrary, immortalized normal MCF-10A cells are CXCR3-B dominant. In basal-like MDA-MB-231 cells, CXCR3 ligand-stimulated proliferation is inhibited by CXCR3-B overexpression, with concurrent reduced activation of ERK1/2 and p38 kinases. Similarly, higher levels of CXCR3-B inhibit migration and invasion in vitro and metastasis in vivo. Accordingly, reduced CXCR3-B expression by gene-silencing enhances lung colonization in the xenograft model. Although exhibiting anti-proliferative and anti-metastatic roles in the non-stem cell population, CXCR3-B supports a cancer stem-like cell phenotype. In mammosphere-forming MDA-MB-231 cells, CXCR3-B is markedly up-regulated and mammosphere-forming capacity is further increased when overexpressing CXCR3-B. Likewise, soft agar growth is promoted by CXCR3-B overexpression. Accordingly, silencing CXCR3-B by shRNA inhibits stem-like properties. In conclusion, both isoforms need to be targeted to inhibit the pro-proliferative and pro-metastasis functions of CXCR3-A and the stem cell-promoting actions of CXCR3-B. Citation Format: Yanchun Li, Jocelyn C. Reader, Xinrong Ma, Namita Kundu, Tyler Kochel, Amy M. Fulton. Distinct roles of CXCR3 isoforms in promoting breast cancer stem-like cell properties and metastasis. [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 1531. doi:10.1158/1538-7445.AM2015-1531

Cyclooxygenases (COX-1 and COX-2) catalyze the formation of prostaglandins and play a role in the... more Cyclooxygenases (COX-1 and COX-2) catalyze the formation of prostaglandins and play a role in the pathogenesis of breast cancer. Prostaglandin E2 (PGE2), the chief COX product in tumors, is the predominant protumorigenic prostanoid and mediates biological effects by binding to each of four EP receptors (EP1-4). Each receptor is coupled to different intracellular signaling pathways; and EP1 is coupled to calcium mobilization and PKC activation. Our published studies indicate that EP1 was detected in the cytoplasm and nucleus of benign ducts and malignant cells in invasive ductal carcinomas, and overall survival for women with tumors negative for nuclear EP1 was significantly worse than for women with any nuclear EP1 expression. Pharmacologic antagonism or reduction of EP1 expression increased metastatic capacity in a murine model of metastatic breast cancer. These data support our hypothesis that EP1 functions as a metastasis suppressor. We now report that murine metastatic mammary tumor cell lines 410.4 and 66.1 have decreased EP1 mRNA expression compared to the non-metastatic cell line 410. Western blot analysis of total and subcellular fractions of EpH4 (normal, immortalized mammary epithelial) and malignant 410, 410.4 and 66.1 cell lines demonstrates that EP1 protein is present in the total and cytoplasmic fractions for all cell lines examined. Nuclear EP1 is detected in EpH4 and non-metastatic 410 cells; however, very little to no nuclear protein is detected in metastatic cell lines 410.4 and 66.1. The absence of nuclear EP1 in metastatic cell lines is consistent with the prognostic data that overall survival for women with tumors negative for nuclear EP1 was significantly worse. Previously, we determined that reduction of EP1 expression leads to increased metastatic capacity; therefore, we investigated the effect of EP1 overexpression on lung colonization in 410.4 and 66.1 cells via tail vein injection in a syngeneic murine model of breast cancer. In 410.4 cells, EP1 overexpression leads to a 57%-97% decrease in metastasis, and in 66.1 cells EP1 overexpression resulted in a 10% - 38% decrease in lung tumor burden compared to vector control mice. The inverse correlation between EP1 expression and metastatic capacity supports our hypothesis that EP1 functions as a metastasis suppressor. We explored potential mechanisms leading to the alteration in the metastatic behavior in response to manipulation of the expression of EP1. We previously published that an EP1 antagonist altered adhesion to laminin in 410.4 cells. Overexpression of EP1 altered the expression of several integrin receptors in 410.4 and 66.1 cell lines including integrins alpha-V, alpha-6 and alpha-3. Also, overexpression of EP1 led to altered adhesion of these cells to several extracellular matrices including fibronectin, collagen, laminin and fibrinogen. Alteration of the adhesive properties of these cells by EP1 could contribute to the metastasis suppressor function of the EP1 receptor. Citation Format: Jocelyn C. Reader, Xinrong Ma, Namita Kundu, Olga Goloubeva, Amy Fulton. Prostaglandin E2 receptor EP1 suppresses breast cancer metastasis. [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 3250. doi:10.1158/1538-7445.AM2015-3250

Cancer is becoming the most prevalent disease in developed countries due to aging populations. Br... more Cancer is becoming the most prevalent disease in developed countries due to aging populations. Breast cancer is the most frequently diagnosed cancer among women accounting for 23% of total cancer diagnoses and 14% of cancer-related deaths. As with many solid tumors, cyclooxygenase 2 (COX-2) and its enzymatic product, prostaglandin E2 (PGE2), are elevated in breast cancer, and are associated with a poor prognosis. PGE2 initiates various signaling pathways when it binds to each of its four cognate EP receptors. We have previously shown that PGE2 signaling through the EP4 receptor increases the metastatic potential of breast cancer cells. Multiple drug resistance-associated protein 4 (MRP4) is responsible for the active export of PGE2 from the tumor cells, while the prostaglandin transporter (PGT) imports PGE2 for 15-hydroxyprostaglandin dehydrogenase (15-PGDH)-mediated degradation. The role of neither MRP4 nor PGT has been investigated in breast cancer progression. We hypothesize that increased expression of MRP4 would cause increased PGE2 signaling and, therefore, increased metastatic potential in breast cancer. Using a panel of breast cancer cell lines, we demonstrate that highly metastatic tumor cells can express low levels of COX-2, but still achieve high extracellular levels of PGE2. We used MCF10A (immortalized normal breast epithelium), MCF7 (luminal), T47D (luminal), MDA-MB-231 (basal B), 4175 (basal B), MDA-MD-468 (basal A), MDA-MB-436 (Basal B), and SKBR3 (HER2-enriched) cell lines. These cell lines span not only a range of molecular subtypes, but also a range of metastatic potential. MRP4 mRNA and protein expression is increased in tumor cell lines with high metastatic potential while expression of PGT mRNA and protein is decreased in these cells when compared to cells with lower metastatic potential. This inverse relationship between MRP4 and PGT should lead to higher concentrations of extracellular PGE2 in the tumor microenvironment, and this hypothesis is being tested through pharmacologic and genetic approaches. Pharmacologic inhibition of MRP4 with MK571 in vitro results in decreased export of two substrates of MRP4, PGE2 and cyclic-AMP (cAMP). Likewise, genetic suppression by MRP4 shRNA shows that decreased MRP4 expression also results in reduced PGE2 export. This data supports the hypothesis that MRP4 is a critcal step in the PGE2 signaling pathway that leads to high extracellular PGE2, and implicates MRP4 as a possible therapeutic target. Citation Format: Tyler J. Kochel, Jocelyn Reader, Namita Kundu, Yanchun Li, Xinrong Ma, Dawn Holt, Amy Fulton. Multiple drug resistance-associated protein 4 (MRP4) may contribute to breast cancer progression by exporting the COX-2 product PGE2. [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 5119. doi:10.1158/1538-7445.AM2013-5119

Breast cancer, 2019

Materials and Methods Cell lines Human breast cancer cell lines MCF7, MDA-MB-231, and T47D are gr... more Materials and Methods Cell lines Human breast cancer cell lines MCF7, MDA-MB-231, and T47D are grown in Dulbecco's Modified Eagle Medium; SKBR3 cells are grown in McCoy's 5A media; all media are supplemented with 10% fetal bovine serum (FBS; Gemini

Anti-Cancer Drugs, Feb 1, 2012

Breast cancer mortality is primarily due to the occurrence of metastatic disease. We have identif... more Breast cancer mortality is primarily due to the occurrence of metastatic disease. We have identified a novel potential therapeutic agent derived from an edible root of the plant Colocasia esculenta, commonly known as taro, that has demonstrable activity in a preclinical model of metastatic breast cancer and that should have minimal toxicity. We have shown for the first time that a water-soluble extract of taro (TE) potently inhibits lung colonizing ability as well as spontaneous metastasis from mammary gland-implanted tumors, in a murine model of highly metastatic ER, PR and Her-2/neu negative breast cancer. TE modestly inhibits proliferation of some, but not all, breast and prostate cancer cell lines. Morphologic changes including cell rounding were observed. Tumor cell migration was completely blocked by TE. TE treatment also inhibited prostaglandin E 2 (PGE 2) synthesis and downregulated cyclooxygenase (COX) 1 and 2 mRNA expression. We purified the active compound(s) to near homogeneity with antimetastatic activity comparable to stock TE. The active compound with a native size of approximately 25 kD contains two fragments of nearly equal size. The N-terminal amino acid sequencing of both fragments reveals that the active compound is highly related to three taro proteins; 12 kD storage protein, tarin and lectin. All are similar in terms of amino acid sequence, post-translational processing and all contain a carbohydrate-binding domain. This is the first report describing a compound(s) derived from taro, that potently and specifically inhibits tumor metastasis.