Selective targeting of metastatic ovarian cancer using an engineered anthrax prodrug activated by membrane-anchored serine proteases.

Treatments for advanced and recurrent ovarian cancer remain a challenge due to a lack of potent, selective, and effective therapeutics. Here, we developed the basis for a transformative anticancer strategy based on anthrax toxin that has been engineered to be selectively activated by the catalytic power of zymogen-activating proteases on the surface of malignant tumor cells to induce cell death. Exposure to the engineered toxin is cytotoxic to ovarian tumor cell lines and ovarian tumor spheroids derived from patient ascites. Preclinical studies demonstrate that toxin treatment induces tumor regression in several in vivo ovarian cancer models, including patient-derived xenografts, without adverse side effects, supportive of progression toward clinical evaluation. These data lay the groundwork for developing therapeutics for treating women with late-stage and recurrent ovarian cancers, utilizing a mechanism distinct from current anticancer therapies.

Combined inhibition of IL­6 and IL­8 pathways suppresses ovarian cancer cell viability and migration and tumor growth.

Ovarian cancer is the most lethal gynecological cancer type in the United States. The success of current chemotherapies is limited by chemoresistance and side effects. Targeted therapy is a promising future direction for cancer therapy. In the present study, the efficacy of co­targeting IL­6 and IL­8 in human ovarian cancer cells by bazedoxifene (Baze) + SCH527123 (SCH) treatment was examined. ELISA, cell viability, cell proliferation, cell migration, cell invasion, western blotting and peritoneal ovarian tumor mouse model analyses were performed to analyze the expression levels of IL­6 and IL­8, tumor growth, tumor migration and invasion, and the possible pathways of human ovarian cancer cell lines (SKOV3, CAOV3 and OVCAR3) and patient­derived OV75 ovarian cancer cells. Each cell line was treated by monotherapy or combination therapy. The results demonstrated that IL­6 and IL­8 were secreted by human ovarian cancer cell lines. Compared with the DMSO control, the combination of IL­6/glycoprotein 130 inhibitor Baze and IL­8 inhibitor SCH synergistically inhibited cell viability in ovarian cancer cells. Baze + SCH also inhibited cell migration and invasion, suppressed ovarian tumor growth and inhibited STAT3 and AKT phosphorylation, as well as survivin expression. Therefore, co­targeting the IL­6 and IL­8 signaling pathways may be an effective approach for ovarian cancer treatment.

Microtentacle Formation in Ovarian Carcinoma.

BACKGROUND: The development of chemoresistance to paclitaxel and carboplatin represents a major therapeutic challenge in ovarian cancer, a disease frequently characterized by malignant ascites and extrapelvic metastasis. Microtentacles (McTNs) are tubulin-based projections observed in detached breast cancer cells. In this study, we investigated whether ovarian cancers exhibit McTNs and characterized McTN biology. METHODS: We used an established lipid-tethering mechanism to suspend and image individual cancer cells. We queried a panel of immortalized serous (OSC) and clear cell (OCCC) cell lines as well as freshly procured ascites and human ovarian surface epithelium (HOSE). We assessed by Western blot ß-tubulin isotype, α-tubulin post-translational modifications and actin regulatory proteins in attached/detached states. We studied clustering in suspended conditions. Effects of treatment with microtubule depolymerizing and stabilizing drugs were described. RESULTS: Among cell lines, up to 30% of cells expressed McTNs. Four McTN morphologies (absent, symmetric-short, symmetric-long, tufted) were observed in immortalized cultures as well as ascites. McTN number/length varied with histology according to metastatic potential. Most OCCC overexpressed class III ß-tubulin. OCCC/OSC cell lines exhibited a trend towards more microtubule-stabilizing post-translational modifications of α-tubulin relative to HOSE. Microtubule depolymerizing drugs decreased the number/length of McTNs, confirming that McTNs are composed of tubulin. Cells that failed to form McTNs demonstrated differential expression of α-tubulin- and actin-regulating proteins relative to cells that form McTNs. Cluster formation is more susceptible to microtubule targeting agents in cells that form McTNs, suggesting a role for McTNs in aggregation. CONCLUSIONS: McTNs likely participate in key aspects of ovarian cancer metastasis. McTNs represent a new therapeutic target for this disease that could refine therapies, including intraperitoneal drug delivery.

Randomised phase II trial of weekly ixabepilone ± biweekly bevacizumab for platinum-resistant or refractory ovarian/fallopian tube/primary peritoneal cancer.

BACKGROUND: This multi-center RP2 study assessed activity/safety of ixabepilone + bevacizumab compared to ixabepilone in platinum-resistant/refractory ovarian/fallopian tube/primary peritoneal cancer. Additional objectives were to examine the role of prior bevacizumab and taxanes, and explore class III-ß-tubulin (TUBB3) as a predictive biomarker. METHODS: Participants were randomised to receive ixabepilone 20 mg/m2 days 1, 8, 15 with (IXA + BEV) or without (IXA) bevacizumab 10 mg/kg days 1, 15 every 28 days. Patients were stratified by prior BEV. The primary endpoint was PFS. OS, safety, and ORR served as secondary endpoints. RESULTS: Among 76 evaluable patients who received IXA + BEV (n = 39) compared to IXA (n = 37), the ORR was 33% (n = 13) versus 8% (n = 3)(P = 0.004), durable at 6 months in 37% (n = 14) and 3% (n = 1) (P < 0.001). BEV significantly improved PFS (median:5.5 vs 2.2 months, HR = 0.33, 95%CI 0.19-0.55, P < 0.001) and OS (median:10.0 vs 6.0 months, HR = 0.52, 95%CI 0.31-0.87, P = 0.006). Both regimens were well-tolerated. TUBB3 expression did not predict response. Subgroup analyses revealed minimal effect of prior BEV or taxane resistant/refractory status on response to IXA + BEV. CONCLUSIONS: IXA + BEV is a well-tolerated, effective combination for platinum/taxane-resistant ovarian cancer that extends PFS and likely OS relative to IXA monotherapy. Prior receipt of BEV should not preclude the use of IXA + BEV. TUBB3 is not a predictive biomarker. CLINICAL TRIAL REGISTRATION: NCT3093155.

Photodynamic Priming Improves the Anti-Migratory Activity of Prostaglandin E Receptor 4 Antagonist in Cancer Cells In Vitro.

The combination of photodynamic agents and biological inhibitors is rapidly gaining attention for its promise and approval in treating advanced cancer. The activity of photodynamic treatment is mainly governed by the formation of reactive oxygen species upon light activation of photosensitizers. Exposure to reactive oxygen species above a threshold dose can induce cellular damage and cancer cell death, while the surviving cancer cells are "photodynamically primed", or sensitized, to respond better to other drugs and biological treatments. Here, we report a new combination regimen of photodynamic priming (PDP) and prostaglandin E2 receptor 4 (EP4) inhibition that reduces the migration and invasion of two human ovarian cancer cell lines (OVCAR-5 and CAOV3) in vitro. PDP is achieved by red light activation of the FDA-approved photosensitizer, benzoporphyrin derivative (BPD), or a chemical conjugate composed of the BPD linked to cetuximab, an anti-epithelial growth factor receptor (EGFR) antibody. Immunoblotting data identify co-inhibition of EGFR, cAMP-response element binding protein (CREB), and extracellular signal-regulated kinase 1/2 (ERK1/2) as key in the signaling cascades modulated by the combination of EGFR-targeted PDP and EP4 inhibition. This study provides valuable insights into the development of a molecular-targeted photochemical strategy to improve the anti-metastatic effects of EP4 receptor antagonists.

Malignant Ascites in Ovarian Cancer: Cellular, Acellular, and Biophysical Determinants of Molecular Characteristics and Therapy Response.

Ascites refers to the abnormal accumulation of fluid in the peritoneum resulting from an underlying pathology, such as metastatic cancer. Among all cancers, advanced-stage epithelial ovarian cancer is most frequently associated with the production of malignant ascites and is the leading cause of death from gynecologic malignancies. Despite decades of evidence showing that the accumulation of peritoneal fluid portends the poorest outcomes for cancer patients, the role of malignant ascites in promoting metastasis and therapy resistance remains poorly understood. This review summarizes the current understanding of malignant ascites, with a focus on ovarian cancer. The first section provides an overview of heterogeneity in ovarian cancer and the pathophysiology of malignant ascites. Next, analytical methods used to characterize the cellular and acellular components of malignant ascites, as well the role of these components in modulating cell biology, are discussed. The review then provides a perspective on the pressures and forces that tumors are subjected to in the presence of malignant ascites and the impact of physical stress on therapy resistance. Treatment options for malignant ascites, including surgical, pharmacological and photochemical interventions are then discussed to highlight challenges and opportunities at the interface of drug discovery, device development and physical sciences in oncology.

Evolutionary dynamics of cancer multidrug resistance in response to olaparib and photodynamic therapy.

P-glycoprotein (P-gp) is an adenosine triphosphate (ATP)-dependent drug efflux protein commonly associated with multidrug resistance in cancer chemotherapy. In this report, we used a dual-fluorescent co-culture model to study the population dynamics of the drug sensitive human ovarian cancer cell line (OVCAR-8-DsRed2) and its resistant subline that overexpresses P-gp (NCI/ADR-RES-EGFP) during the course of a photodynamic therapy (PDT)-olaparib combination regimen. Without treatment, OVCAR-8-DsRed2 cells grew more rapidly than the NCI/ADR-RES-EGFP cells. Olaparib treatment reduced the total number of cancer cells by 70±4% but selected for the resistant NCI/ADR-RES-EGFP population since olaparib is an efflux substrate for the P-gp pump. This study used the FDA-approved benzoporphyrin derivative (BPD) photosensitizer or its lipidated formulation ((16:0)LysoPC-BPD) to kill OVCAR-8 cells and reduce the likelihood that olaparib-resistant cells would have selective advantage. Three cycles of PDT effectively reduced the total cell number by 66±3%, while stabilizing the population ratio of sensitive and resistant cells at approximately 1:1. The combination of olaparib treatment and PDT enhanced PARP cleavage and deoxyribonucleic acid (DNA) damage, further decreasing the total cancer cell number down to 10±2%. We also showed that the combination of olaparib and (16:0)LysoPC-BPD-based PDT is up to 18-fold more effective in mitigating the selection of resistant NCI/ADR-RES-EGFP cells, compared to using olaparib and BPD-based PDT. These studies suggest that PDT may improve the effectiveness of olaparib, and the use of a lipidated photosensitizer formulation holds promise in overcoming cancer drug resistance.

The Role of Eicosanoids in Gynecological Malignancies.

Eicosanoids, bio-active lipid molecules, evoke a multitude of biological effects that directly affect cancer cells and indirectly affect tumor microenvironment. An emerging role has been shown for eicosanoids in the pathogenesis of gynecological malignancies which include cancers of the vulva, vagina, cervix, uterine, and ovary. Eicosanoid biosynthesis pathways start at the metabolism of phospholipids by phospholipase A2 then proceeding to one of three pathways: the cyclooxygenase (COX), lipoxygenase (LOX), or P450 epoxygenase pathways. The most studied eicosanoid pathways include COX and LOX; however, more evidence is appearing to support further study of the P450 epoxygenase pathway in gynecologic cancers. In this review, we present the current knowledge of the role of COX, LOX and P450 pathways in the pathogenesis of gynecologic malignancies. Vulvar and vaginal cancer, the rarest subtypes, there is association of COX-2 expression with poor disease specific survival in vulvar cancer and, in vaginal cancer, COX-2 expression has been found to play a role in mucosal inflammation leading to disease susceptibility and transmission. Cervical cancer is associated with COX-2 levels 7.4 times higher than in healthy tissues. Additionally, HPV elevates COX-2 levels through the EGFR pathway and HIV promotes elevated COX-2 levels in cervical tissue as well as increases PGE2 levels eliciting inflammation and progression of cancer. Evidence supports significant roles for both the LOX and COX pathways in uterine cancer. In endometrial cancer, there is increased expression of 5-LOX which is associated with adverse outcomes. Prostanoids in the COX pathway PGE2 and PGF2α have been shown to play a significant role in uterine cancer including alteration of proliferation, adhesion, migration, invasion, angiogenesis, and the inflammatory microenvironment. The most studied gynecological malignancy in regard to the potential role of eicosanoids in tumorigenesis is ovarian cancer in which all three pathways have shown to be associated or play a role in ovarian tumorigenesis directly on the tumor cell or through modulation of the tumor microenvironment. By identifying the gaps in knowledge, additional pathways and targets could be identified in order to obtain a better understanding of eicosanoid signaling in gynecological malignancies and identify potential new therapeutic approaches.

Eicosanoids in Cancer: Prostaglandin E2 Receptor 4 in Cancer Therapeutics and Immunotherapy.

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 E2 (PGE2) 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-κB. 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. PGE2 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 PGE2 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 immune-based 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.

The Chemokine Receptor CXCR3 Isoform B Drives Breast Cancer Stem Cells.

We are seeking to identify molecular targets that are relevant to breast cancer cells with stem-like properties. There is growing evidence that cancer stem cells (CSCs) are supported by inflammatory mediators expressed in the tumor microenvironment. The chemokine receptor CXCR3 binds the interferon-γ-inducible, ELR-negative CXC chemokines CXCL9, CXCL10, and CXCL11 and malignant cells have co-opted this receptor to promote tumor cell migration and invasion. There are 2 major isoforms of CXCR3: CXCR3A and CXCR3B. The latter is generated from alternative splicing and results in a protein with a longer N-terminal domain. CXCR3 isoform A is generally considered to play a major role in tumor metastasis. When the entire tumor cell population is examined, CXCR3 isoform B is usually detected at much lower levels than CXCR3A and for this, and other reasons, was not considered to drive tumor progression. We have shown that CXCR3B is significantly upregulated in the subpopulation of breast CSCs in comparison with the bulk tumor cell population in 3 independent breast cancer cell lines (MDA-MB-231, SUM159, and T47D). Modulation of CXCR3B levels by knock in strategies increases CSC populations identified by aldehyde dehydrogenase activity or CD44+CD24- phenotype as well as tumorsphere-forming capacity. The reverse is seen when CXCR3B is gene-silenced. CXCL11 and CXCL10 directly induce CSC. We also report that novel CXCR3 allosteric modulators BD064 and BD103 prevent the induction of CSCs. BD103 inhibited experimental metastasis. This protective effect is associated with the reversal of CXCR3 ligand-mediated activation of STAT3, ERK1/2, CREB, and NOTCH1 pathways. We propose that CXCR3B, expressed on CSC, should be explored further as a novel therapeutic target.

EP4 and Class III ß-Tubulin Expression in Uterine Smooth Muscle Tumors: Implications for Prognosis and Treatment.

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 ß-tubulin has previously been shown to confer taxane resistance in a variety of human cancers. Prostaglandin E2 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 ß-tubulin have increased expression in LMS in comparison to normal myometrium or benign tumors and that expression of class III ß-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 ß-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 ß-tubulin was performed on patients with LMS, leiomyomatosis/STUMP, leiomyoma, and normal myometrium. Expression of EP4 and class III ß-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 ß-tubulin and EP4, treatment with EP4 inhibitor resulted in at least 2-fold sensitization to docetaxel. Expression of class III ß-tubulin and EP4 in LMS may identify patients at risk of resistance to standard chemotherapies and candidates for augmentation of therapy through EP4 inhibition.

Review of Immune Therapies Targeting Ovarian Cancer.

OPINION STATEMENT: The rise of immunotherapy is the greatest advance in oncology to occur over the last several years, but applications in gynecologic malignancies lag behind other tumors. The term "immunotherapy" envelops monoclonal antibodies as receptor mediators, including immune checkpoint inhibitors (ICPI), cancer vaccines, and adoptive immunotherapies alone or in combination with other therapeutic approaches. The purpose of this review is to summarize the status of immunotherapy trials in ovarian cancer and to specifically highlight data published in the last 1-2 years.

Multiple drug resistance-associated protein (MRP4) exports prostaglandin E2 (PGE2) and contributes to metastasis in basal/triple negative breast cancer.

Cyclooxygenase-2 (COX-2) and its primary enzymatic product, prostaglandin E2 (PGE2), are associated with a poor prognosis in breast cancer. In order to elucidate the factors contributing to intratumoral PGE2 levels, we evaluated the expression of COX-2/PGE2 pathway members MRP4, the prostaglandin transporter PGT, 15-PGDH (PGE2 metabolism), the prostaglandin E receptor EP4, COX-1, and COX-2 in normal, luminal, and basal breast cancer cell lines. The pattern of protein expression varied by cell line reflecting breast cancer heterogeneity. Overall, basal cell lines expressed higher COX-2, higher MRP4, lower PGT, and lower 15-PGDH than luminal cell lines resulting in higher PGE2 in the extracellular environment. Genetic or pharmacologic suppression of MRP4 expression or activity in basal cell lines led to less extracellular PGE2. The key finding is that xenografts derived from a basal breast cancer cell line with stably suppressed MRP4 expression showed a marked decrease in spontaneous metastasis compared to cells with unaltered MRP4 expression. Growth properties of primary tumors were not altered by MRP4 manipulation. In addition to the well-established role of high COX-2 in promoting metastasis, these data identify an additional mechanism to achieve high PGE2 in the tumor microenvironment; high MRP4, low PGT, and low 15-PGDH. MRP4 should be examined further as a potential therapeutic target in basal breast cancer.

Divergent roles of CXCR3 isoforms in promoting cancer stem-like cell survival and metastasis.

There is growing evidence that several chemokine receptors including CXCR3 contribute to metastasis of breast and other cancers, however, in order to target CXCR3 effectively, it is critical to understand the relative contribution of each CXCR3 isoform. Furthermore, the possible contribution of either major CXCR3 isoform (CXCR3-A, CXCR3-B) to cancer stem cell behavior has not been reported. We employed primary invasive ductal carcinomas, a panel of breast cell lines, and a xenograft model of metastatic breast cancer to examine the role of CXCR3 isoforms in the behavior of breast cancer stem-like cells and the contribution of each isoform to metastasis. In primary human breast cancer specimens as well as established breast cancer cell lines, CXCR3-A is more highly expressed than CXCR3-B. Conversely, immortalized normal MCF10A cells express more CXCR3-B relative to CXCR3-A. Overexpression of CXCR3-B in MDA-MB-231 basal-like cells inhibits CXCR3 ligand-stimulated proliferation, which is accompanied by reduced ligand-mediated activation of ERK1/2 and p38 kinases. Likewise, metastatic capacity is reduced in vivo by higher levels of CXCR3-B, and migratory and invasive properties are inhibited in vitro; conversely, silencing of CXCR3-B enhances lung colonization. In contrast to the anti-metastatic and anti-proliferative roles of CXCR3-B in the non-stem cell population, this isoform supports a cancer stem-like cell phenotype. CXCR3-B is markedly elevated in mammosphere-forming parental cells and overexpressing CXCR3-B further enhances mammosphere-forming potential as well as growth in soft agar; stem-like behavior is inhibited in MDA-MB-231shCXCR3-B cells. Targeting of both CXCR3 isoforms may be important to block the stem cell-promoting actions of CXCR3-B, while inhibiting the pro-proliferative and metastasis-promoting functions of CXCR3-A.

Prostaglandin E receptor EP4 is a therapeutic target in breast cancer cells with stem-like properties.

The cyclooxygenase pathway is strongly implicated in breast cancer progression but the role of this pathway in the biology of breast cancer stem/progenitor cells has not been defined. Recent attention has focused on targeting the cyclooxygenase 2 (COX-2) pathway downstream of the COX-2 enzyme by blocking the activities of individual prostaglandin E (EP) receptors. Prostaglandin E receptor 4 (EP4) is widely expressed in primary invasive ductal carcinomas of the breast and antagonizing this receptor with small molecule inhibitors or shRNA directed to EP4 inhibits metastatic potential in both syngeneic and xenograft models. Breast cancer stem/progenitor cells are defined as a subpopulation of cells that drive tumor growth, metastasis, treatment resistance, and relapse. Mammosphere-forming breast cancer cells of human (MDA-MB-231, SKBR3) or murine (66.1, 410.4) origin of basal-type, Her-2 phenotype and/or with heightened metastatic capacity upregulate expression of both EP4 and COX-2 and are more tumorigenic compared to the bulk population. In contrast, luminal-type or non-metastatic counterparts (MCF7, 410, 67) do not increase COX-2 and EP4 expression in mammosphere culture. Treatment of mammosphere-forming cells with EP4 inhibitors (RQ-15986, AH23848, Frondoside A) or EP4 gene silencing, but not with a COX inhibitor (Indomethacin) reduces both mammosphere-forming capacity and the expression of phenotypic markers (CD44(hi)/CD24(low), aldehyde dehydrogenase) of breast cancer stem cells. Finally, an orally delivered EP4 antagonist (RQ-08) reduces the tumor-initiating capacity and markedly inhibits both the size of tumors arising from transplantation of mammosphere-forming cells and phenotypic markers of stem cells in vivo. These studies support the continued investigation of EP4 as a potential therapeutic target and provide new insight regarding the role of EP4 in supporting a breast cancer stem cell/tumor-initiating phenotype.

A prostaglandin E (PGE) receptor EP4 antagonist protects natural killer cells from PGE2-mediated immunosuppression and inhibits breast cancer metastasis.

Cyclooxygenase-2 is frequently upregulated in epithelial tumors and contributes to poor outcomes in multiple malignancies. The COX-2 product prostaglandin E2 (PGE2) promotes tumor growth and metastasis by acting on a family of four G protein-coupled receptors (EP1-4). Using a novel small molecule EP4 antagonist (RQ-15986) and a syngeneic murine model of metastatic breast cancer, we determined the effect of EP4 blockade on innate immunity and tumor biology. Natural killer (NK)-cell functions are markedly depressed in mice bearing murine mammary tumor 66.1 or 410.4 cells owing to the actions of PGE2 on NK cell EP4 receptors. The EP4 agonist PGE1-OH inhibits NK functions in vitro, and this negative regulation is blocked by RQ-15986. Likewise, the treatment of tumor-bearing mice with RQ-15986 completely protected NK cells from the immunosuppressive effects of the tumor microenvironment in vivo. RQ-15986 also has direct effects on EP4 expressed by tumor cells, inhibiting the PGE2-mediated activation of adenylate cyclase and blocking PGE2-induced tumor cell migration. The pretreatment of tumor cells with a non-cytotoxic concentration of RQ-15986 inhibited lung colonization, a beneficial effect that was lost in mice depleted of NK cells. The oral administration of RQ-15986 inhibited the growth of tumor cells implanted into mammary glands and their spontaneous metastatic colonization to the lungs, resulting in improved survival. Our findings reveal that EP4 antagonism prevents tumor-mediated NK-cell immunosuppression and demonstrates the anti-metastatic activity of a novel EP4 antagonist. These observations support the investigation of EP4 antagonists in clinical trials.

Prostaglandin E2 EP receptors as therapeutic targets in breast cancer.

Prostaglandins are lipid compounds that mediate many physiological effects. Prostaglandin E2 (PGE(2)) is the most abundant prostanoid in the human body, and synthesis of PGE(2) is driven by cyclooxygenase enzymes including COX-2. Both elevated expression of COX-2 and increased PGE(2) levels have been associated with many cancers including breast cancer. PGE(2) exerts its effect by binding to the E series of prostaglandin receptors (EP) which are G protein-coupled receptors. Four EP receptor subtypes exist, EP1-4, and each is coupled to different intracellular signaling pathways. As downstream effectors of the COX-2 pathway, EP receptors have been shown to play a role in breast and other malignancies and in cancer metastasis. The role of each EP receptor in malignant behavior is complex and involves the interplay of EP receptor signaling on the tumor cell, on stromal cells, and on host immune effector cells. While preclinical and epidemiological data support the use of nonsteroidal anti-inflammatory drugs and selective COX-2 inhibitors (COXibs) for the prevention and treatment of malignancy, toxicities due to COXibs as well as less than promising results from clinical trials have laboratories seeking alternative targets. As knowledge concerning the role of EP receptors in cancer grows, so does the potential for exploiting EP receptors as therapeutic targets for the treatment or prevention of cancer and cancer metastasis.

Prostaglandin E receptor EP1 suppresses breast cancer metastasis and is linked to survival differences and cancer disparities.

Cyclooxygenase-2 is frequently overexpressed and associated with poor prognosis in breast cancer. The cyclooxygenase-2 product prostaglandin E(2) elicits cellular responses through four G-protein-coupled receptors, designated EP1 to EP4, coupled to distinct intracellular signaling pathways. EP4, expressed on malignant breast cells, promotes metastasis; however, a role for EP1 in metastasis has not been investigated. Using a murine model of metastatic breast cancer, we now show that pharmacologic antagonism of EP1 with SC19220 or AH6809 promoted lung colonization of mammary tumor cells by 3.7- to 5.4-fold. Likewise, reducing EP1 gene expression by shRNA also increased metastatic capacity relative to cells transfected with nonsilencing vector but did not affect the size of transplanted tumors. Examination of invasive ductal carcinomas by immunohistochemistry shows that EP1 was detected in both the cytoplasm and nucleus of benign ducts as well as malignant cells in some samples, but was absent or limited to either the nucleus or cytoplasm in other malignant samples. Overall survival for women with tumors that were negative for nuclear EP1 was significantly worse than for women with EP1 expression (P = 0.008). There was no difference in survival for women with differences in cytoplasmic EP1 expression (P = 0.46). Comparing EP1 mRNA in breast tumors from African American and European American women revealed that many more African American breast tumors lacked detectable EP1 mRNA (P = 0.04). These studies support the hypothesis that EP1 functions as a metastasis suppressor and that loss of nuclear EP1 is associated with poorer overall survival and may contribute to disparities in outcome in different populations.

Regulation of differentiation by a PHD domain in the NUP98-PHF23 fusion protein.

Acute myeloid leukemia (AML) is frequently associated with chromosomal translocations. These translocations produce specific fusion genes that play crucial roles in leukemogenesis. We recently identified a novel NUP98-PHF23 fusion in AML. In this study, we attempt to determine the role of NUP98-PHF23 protein and its plant homeodomain (PHD) and coiled-coil domain in regulation of cellular differentiation and protein distribution. We provide evidence that NUP98-PHF23, through its PHD domain, impairs TPA-induced differentiation of K562 cells. While the fusion protein localizes to the nucleus, its deletion mutant without the PHD domain resides exclusively in the nucleolus, suggesting a potential link between chromatin-binding PHD domain and nuclear architecture.