RESUMO
The taro plant, Colocasia esculenta, contains bioactive proteins with potential as cancer therapeutics. Several groups have reported anti-cancer activity in vitro and in vivo of taro-derived extracts (TEs). We reported that TE inhibits metastasis in a syngeneic murine model of Triple-Negative Breast Cancer (TNBC). PURPOSE: We sought to confirm our earlier studies in additional models and to identify novel mechanisms by which efficacy is achieved. METHODS: We employed a panel of murine and human breast and ovarian cancer cell lines to determine the effect of TE on tumor cell viability, migration, and the ability to support cancer stem cells. Two syngeneic models of TNBC were employed to confirm our earlier report that TE potently inhibits metastasis. Cancer stem cell assays were employed to determine the ability of TE to inhibit tumorsphere-forming ability and to inhibit aldehyde dehydrogenase activity. To determine if host immunity contributes to the mechanism of metastasis inhibition, efficacy was assessed in immune-compromised mice. RESULTS: We demonstrate that viability of some, but not all cell lines is inhibited by TE. Likewise, tumor cell migration is inhibited by TE. Using 2 immune competent, syngeneic models of TNBC, we confirm our earlier findings that tumor metastasis is potently inhibited by TE. We also demonstrate, for the first time, that TE directly inhibits breast cancer stem cells. Administration of TE to mice elicits expansion of several spleen cell populations but it was not known if host immune cells contribute to the mechanism by which TE inhibits tumor cell dissemination. In novel findings, we now show that the ability of TE to inhibit metastasis relies on immune T-cell-dependent, but not B cell or Natural Killer (NK)-cell-dependent mechanisms. Thus, both tumor cell-autonomous and host immune factors contribute to the mechanisms underlying TE efficacy. Our long-term goal is to evaluate TE efficacy in clinical trials. Most of our past studies as well as many of the results reported in this report were carried out using an isolation protocol described earlier (TE). In preparation for a near future clinical trial, we have now developed a strategy to isolate an enriched taro fraction, TE-method 2, (TE-M2) as well as a more purified subfraction (TE-M2F1) which can be scaled up under Good Manufacturing Practice (GMP) conditions for evaluation in human subjects. We demonstrate that TE-M2 and TE-M2F1 retain the anti-metastatic properties of TE. CONCLUSIONS: These studies provide further support for the continued examination of biologically active components of Colocasia esculenta as potential new therapeutic entities and identify a method to isolate sufficient quantities under GMP conditions to conduct early phase clinical studies.
RESUMO
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.
RESUMO
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.
Assuntos
Movimento Celular , Dinoprostona/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Antineoplásicos/farmacologia , Transporte Biológico , Movimento Celular/efeitos dos fármacos , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Relação Dose-Resposta a Droga , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Hidroxiprostaglandina Desidrogenases/metabolismo , Células MCF-7 , Camundongos Endogâmicos BALB C , Camundongos SCID , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Metástase Neoplásica , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Propionatos/farmacologia , Quinazolinas/farmacologia , Quinolinas/farmacologia , Interferência de RNA , Receptores de Prostaglandina E Subtipo EP4/genética , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Transdução de Sinais , Fatores de Tempo , Transfecção , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Microambiente Tumoral , Tirfostinas/farmacologiaRESUMO
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.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Regulação Neoplásica da Expressão Gênica , Células-Tronco Neoplásicas/metabolismo , Receptores CXCR3/genética , Processamento Alternativo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Feminino , Inativação Gênica , Humanos , Metástase Neoplásica , Prognóstico , Isoformas de Proteínas , Receptores CXCR3/metabolismo , Esferoides Celulares , Células Tumorais CultivadasRESUMO
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.
Assuntos
Neoplasias da Mama/metabolismo , Células-Tronco Neoplásicas/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/metabolismo , Modelos Animais de Doenças , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Imuno-Histoquímica , Imunofenotipagem , Camundongos , Metástase Neoplásica , Células-Tronco Neoplásicas/efeitos dos fármacos , Receptores de Prostaglandina E Subtipo EP4/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP4/genética , Esferoides Celulares , Células Tumorais CultivadasRESUMO
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.
RESUMO
Breast malignancies often have high levels of COX-2. The COX-2 product prostaglandin E2 (PGE2) contributes to the high metastatic capacity of breast tumors. Our published data indicate that inhibiting either PGE2 production or PGE2-mediated signaling through the PGE2 receptor EP4 (1 of 4 EP expressed on the malignant cell) reduces metastasis by a mechanism that requires natural killer (NK) cells. Tumor-derived PGE2 and exogenous PGE2 are known to have direct inhibitory effects on NK cell functions, but less is known regarding which EP receptors mediate these effects. We now show that several NK functions (lysis, migration, cytokine production) are compromised in tumor-bearing mice and that tumor-produced PGE2 interferes with NK cell functions. PGE2 inhibits the potential of NK cells to migrate, exert cytotoxic effects, and secrete interferon γ. The ability of PGE2 to inhibit NK cells from tumor-bearing mice is by acting on EP2 and EP4 receptors. NK cells from tumor-bearing mice were more sensitive to inhibition by EP4 and EP2 agonists compared with endogenous NK cells from healthy mice. PGE2 was inhibitory to most NK functions of either normal or tumor-bearing mice. In contrast, there was a trend for enhanced tumor necrosis factor α production in response to PGE2 by NK cells from tumor-bearing mice. We also report that a recently described EP4 antagonist, frondoside A, inhibits breast tumor metastasis in an NK-dependent manner and protects interferon γ production by NK cells from PGE2-mediated suppression. Taken together these data show that NK functions are depressed in tumor-bearing hosts relative to normal NK cells and that PGE2 suppresses NK functions by acting on EP2 and EP4 receptors.
Assuntos
Neoplasias da Mama/imunologia , Dinoprostona/imunologia , Células Matadoras Naturais/imunologia , Receptores de Prostaglandina E Subtipo EP2/imunologia , Receptores de Prostaglandina E Subtipo EP4/imunologia , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular/imunologia , Separação Celular , Dinoprostona/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Frondoside A, derived from the sea cucumber Cucumaria frondosa has demonstrable anticancer activity in several models, however, the ability of Frondoside A to affect tumor metastasis has not been reported. Using a syngeneic murine model of metastatic breast cancer, we now show that Frondoside A has potent antimetastatic activity. Frondoside A given i.p. to mice bearing mammary gland-implanted mammary tumors, inhibits spontaneous tumor metastasis to the lungs. The elevated Cyclooxygenase-2 activity in many malignancies promotes tumor growth and metastasis by producing high levels of PGE(2) which acts on the prostaglandin E receptors, chiefly EP4 and EP2. We examined the ability of Frondoside A to modulate the functions of these EP receptors. We now show that Frondoside A antagonizes the prostaglandin E receptors EP2 and EP4. (3)H-PGE(2) binding to recombinant EP2 or EP4-expressing cells was inhibited by Frondoside A at low µM concentrations. Likewise, EP4 or EP2-linked activation of intracellular cAMP as well as EP4-mediated ERK1/2 activation were also inhibited by Frondoside A. Consistent with the antimetastatic activity observed in vivo, migration of tumor cells in vitro in response to EP4 or EP2 agonists was also inhibited by Frondoside A. These studies identify a new function for an agent with known antitumor activity, and show that the antimetastatic activity may be due in part to a novel mechanism of action. These studies add to the growing body of evidence that Frondoside A may be a promising new agent with potential to treat cancer and may also represent a potential new modality to antagonize EP4.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Glicosídeos/farmacologia , Neoplasias Pulmonares/secundário , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP4/antagonistas & inibidores , Triterpenos/farmacologia , Animais , Antineoplásicos/uso terapêutico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , AMP Cíclico/metabolismo , Feminino , Glicosídeos/uso terapêutico , Humanos , Concentração Inibidora 50 , Neoplasias Pulmonares/prevenção & controle , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Triterpenos/uso terapêutico , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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, which 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 and spontaneous metastasis from mammary gland-implanted tumors, in a murine model of highly metastatic estrogen receptor, progesterone receptor and Her-2/neu-negative breast cancer. TE modestly inhibits the proliferation of some, but not all, breast and prostate cancer cell lines. Morphological changes including cell rounding were observed. Tumor cell migration was completely blocked by TE. TE treatment also inhibited prostaglandin E2 (PGE2) synthesis and downregulated cyclooxygenase 1 and 2 mRNA expression. We purified the active compound(s) to near homogeneity with antimetastatic activity comparable with stock TE. The active compound with a native size of approximately 25 kDa 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-kDa storage protein, tarin and taro lectin. All are similar in terms of amino acid sequence, posttranslational processing and all contain a carbohydrate-binding domain. This is the first report describing compound(s) derived from taro that potently and specifically inhibits tumor metastasis.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Colocasia/química , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Animais , Antineoplásicos Fitogênicos/isolamento & purificação , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cromatografia em Gel , Cromatografia de Fase Reversa , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Feminino , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos , Peso Molecular , Transplante de Neoplasias , Extratos Vegetais/isolamento & purificação , Raízes de Plantas/química , Prostaglandina-Endoperóxido Sintases/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The COX-2 product prostaglandin E(2) (PGE(2)) contributes to the high metastatic capacity of breast tumors. Our published data indicate that inhibiting either PGE(2) production or PGE(2)-mediated signaling through the PGE(2) receptor EP4 reduces metastasis by a mechanism that requires natural killer (NK) cells. It is known that NK cell function is compromised by PGE(2), but very little is known about the mechanism by which PGE(2) affects NK effector activity. We now report the direct effects of PGE(2) on the NK cell. Endogenous murine splenic NK cells express all four PGE(2) receptors (EP1-4). We examined the role of EP receptors in three NK cell functions: migration, cytotoxicity, and cytokine release. Like PGE(2), the EP4 agonist PGE(1)-OH blocked NK cell migration to FBS and to four chemokines (ITAC, MIP-1α, SDF-1α, and CCL21). The EP2 agonist, Butaprost, inhibited migration to specific chemokines but not in response to FBS. In contrast to the inhibitory actions of PGE(2), the EP1/EP3 agonist Sulprostone increased migration. Unlike the opposing effects of EP4 vs. EP1/EP3 on migration, agonists of each EP receptor were uniformly inhibiting to NK-mediated cytotoxicity. The EP4 agonist, PGE(1)-OH, inhibited IFNγ production from NK cells. Agonists for EP1, EP2, and EP3 were not as effective at inhibiting IFNγ. Agonists of EP1, EP2, and EP4 all inhibited TNFα; EP4 agonists were the most potent. Thus, the EP4 receptor consistently contributed to loss of function. These results, taken together, support a mechanism whereby inhibiting PGE(2) production or preventing signaling through the EP4 receptor may prevent suppression of NK functions that are critical to the control of breast cancer metastasis.
Assuntos
Dinoprostona/imunologia , Células Matadoras Naturais/imunologia , Receptores de Prostaglandina E Subtipo EP4/imunologia , Animais , Linhagem Celular Tumoral , Dinoprostona/farmacologia , Feminino , Linfoma de Células T/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
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.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/prevenção & controle , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Receptores de Prostaglandina E Subtipo EP1/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/biossíntese , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/biossíntese , Prognóstico , Receptores de Prostaglandina E Subtipo EP1/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP1/biossíntese , Taxa de Sobrevida , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/biossínteseRESUMO
Breast tumor cells express the chemokine receptor CXCR3, which binds the ligands CXCL9, CXCL10, and CXCL11. CXCR3 and other chemokine receptors may mediate tumor metastasis by supporting migration of tumor cells to sites of ligand expression including the lymph nodes, lungs, and bone marrow. We examined the relationship of CXCR3 expression to clinical outcome in 75 women diagnosed with early-stage breast cancer. We detected CXCR3 in malignant epithelium from all tumors. Twelve percent were weakly positive and 64% had moderate levels of CXCR3. Strong CXCR3-positive staining was observed in 24% of tumors. Kaplan-Meier survival curves showed that high CXCR3 expression was associated with poorer overall survival; the unadjusted hazard ratio was 1.56 and it was marginally significant (P=0.07). When interactions between lymph node status and CXCR3 were considered, the adjusted hazard ratio for CXCR3 was 2.62 (P=0.02) for women with node-negative disease at diagnosis, whereas the hazard ratio for CXCR3 was not significant for those with node-positive disease. CXCR3 gene silencing inhibited lung colonization and spontaneous lung metastasis from mammary gland-implanted tumors in a murine model. The size or growth rate of the locally growing tumors was not affected. The antimetastatic effect of CXCR3 gene silencing was compromised in mice depleted of Natural Killer cells or with mutations in IFN-gamma, suggesting that the role of CXCR3 is not simply to mediate tumor cell trafficking. These studies support the continued examination of CXCR3 as a potential therapeutic target in patients with breast cancer.
Assuntos
Adenocarcinoma/diagnóstico , Adenocarcinoma/patologia , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Neoplasias Mamárias Experimentais/diagnóstico , Neoplasias Mamárias Experimentais/patologia , Receptores CXCR3/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Feminino , Inativação Gênica/fisiologia , Interferon gama/genética , Interferon gama/metabolismo , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/fisiologia , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/mortalidade , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Metástase Neoplásica , Prognóstico , Receptores CXCR3/antagonistas & inibidores , Receptores CXCR3/genética , Receptores CXCR3/fisiologia , Análise de Sobrevida , Células Tumorais CultivadasRESUMO
Cyclooxygenase-2 (COX-2) is associated with aggressive breast cancers. The COX-2 product prostaglandin E(2) (PGE(2)) acts through four G-protein-coupled receptors designated EP1-4. Malignant and immortalized normal mammary epithelial cell lines express all four EP. The EP4 antagonist AH23848 reduced the ability of tumor cells to colonize the lungs or to spontaneously metastasize from the mammary gland. EP4 gene silencing by shRNA also reduced the ability of mammary tumor cells to metastasize. Metastasis inhibition was lost in mice lacking either functional Natural Killer (NK) cells or interferon-gamma. EP4 antagonism inhibited MHC class I expression resulting in enhanced ability of NK cells to lyse mammary tumor target cells. These studies support the hypothesis that EP4 receptor antagonists reduce metastatic potential by facilitating NK-mediated tumor cell killing and that therapeutic targeting of EP4 may be an alternative approach to the use of COX inhibitors to limit metastatic disease.
Assuntos
Células Matadoras Naturais/imunologia , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/metabolismo , Invasividade Neoplásica/genética , Receptores de Prostaglandina E/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Neoplasias Mamárias Experimentais/genética , Camundongos , Invasividade Neoplásica/imunologia , RNA Interferente Pequeno , Receptores de Prostaglandina E/genética , Receptores de Prostaglandina E Subtipo EP4 , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Current immunotherapies are limited by several factors, including the failure to recruit sufficient numbers of immune effector cells to tumors. The chemokine monokine induced by gamma-interferon (Mig; CXCL9) attracts activated T cells and natural killer (NK) cells bearing the chemokine receptor CXCR3. We investigated Mig as an immunotherapeutic agent in a syngeneic murine model of metastatic breast cancer. We transfected the highly malignant murine mammary tumor cell line 66.1 to stably express murine Mig cDNA. Immune-competent mice injected with Mig-expressing tumor cells developed smaller local tumors and fewer lung metastases, and they survived longer than mice injected with vector-control tumor cells. Mig-mediated inhibition of local tumor growth was lost in the absence of host T cells. Mig-transduced tumors had increased numbers of CD4 T cells compared with vector-control tumors, consistent with the T-cell chemoattractant property of Mig, and many tumor-infiltrating host cells expressed CXCR3. NK cells had not been examined previously as a possible effector cell in Mig-based therapies. Our studies now show that NK cells are critical to the mechanism by which Mig limits metastasis. Inhibition of angiogenesis was not implicated as a mechanism of Mig-mediated therapy in this model. These studies support the hypothesis that by manipulating the Mig-CXCR3 gradient, it is possible to direct host immune effector cells to tumors, curtailing both local tumor growth and metastasis. These studies also implicate host NK cells as an additional effector cell critical for Mig-mediated control of metastasis.
Assuntos
Adenocarcinoma/terapia , Quimiocinas CXC/uso terapêutico , Neoplasias Pulmonares/terapia , Neoplasias Mamárias Experimentais/terapia , Adenocarcinoma/imunologia , Adenocarcinoma/secundário , Animais , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular Tumoral , Quimiocina CXCL9 , Quimiocinas CXC/imunologia , Feminino , Imunoterapia , Células Matadoras Naturais/imunologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Neovascularização PatológicaRESUMO
It is well established that high cyclooxygenase-2 (COX-2) expression contributes to the aggressive behavior of breast and other malignancies. Due to concerns regarding the safety of long-term use of COX-2 inhibitors as well as a desire to seek more effective alternatives to prevent and treat metastatic disease, we tested the hypothesis that inhibition of downstream signaling by the COX-2 product prostaglandin E(2) (PGE(2)) would be as effective as inhibiting global prostaglandin synthesis. PGE(2) acts through four G-protein-coupled receptors designated EP1-4. Here, we summarize data from many laboratories regarding the role of individual E-series of prostaglandin (EP) receptors on cancer behavior and we discuss our own recent findings that antagonists of the PGE receptor subtype 4, EP4, inhibit experimental metastasis in a murine model of hormone-resistant, metastatic breast cancer. These initial results indicate that selective targeting of individual EP receptors should be investigated as an approach to exploit the high COX-2 activity in many epithelial malignancies.
Assuntos
Neoplasias/patologia , Receptores de Prostaglandina E/antagonistas & inibidores , Animais , Ciclo-Oxigenase 2/metabolismo , Humanos , Camundongos , Metástase Neoplásica , Neoplasias/metabolismo , Receptores de Prostaglandina E/metabolismo , Receptores de Prostaglandina E/fisiologia , Receptores de Prostaglandina E Subtipo EP4RESUMO
Tumor cells aberrantly express chemokines and/or chemokine receptors, and some may promote tumor growth and metastasis. We examined the expression and function of chemokine receptor CXCR3 in a syngeneic murine model of metastatic breast cancer. By flow cytometry, CXCR3 was detected in all murine mammary tumor cell lines examined. All human breast cancer cell lines examined also expressed CXCR3, as did the immortalized but nontumorigenic MCF-10A cell line. Interaction of CXCR3 ligands, CXCL9, CXCL10, and CXCL11, with CXCR3 on the highly malignant murine mammary tumor cell line 66.1 resulted in intracellular calcium mobilization and chemotaxis in vitro. To test the hypothesis that tumor metastasis is facilitated by CXCR3 expressed by tumor cells, we employed a small molecular weight antagonist of CXCR3, AMG487. 66.1 tumor cells were pretreated with AMG487 prior to i.v. injection into immune-competent female mice. Antagonism of CXCR3 on 66.1 tumor cells inhibited experimental lung metastasis, and this antimetastatic activity was compromised in mice depleted of natural killer cells. Systemic administration of AMG487 also inhibited experimental lung metastasis. In contrast to the antimetastatic effect of AMG487, local growth of 66.1 mammary tumors was not affected by receptor antagonism. These studies indicate that murine mammary tumor cells express CXCR3 which facilitates the development of lung metastases. These studies also indicate for the first time that a small molecular weight antagonist of CXCR3 has the potential to inhibit tumor metastasis.
Assuntos
Neoplasias da Mama/patologia , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Receptores de Quimiocinas/antagonistas & inibidores , Adenocarcinoma/imunologia , Adenocarcinoma/prevenção & controle , Adenocarcinoma/secundário , Animais , Neoplasias da Mama/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos SCID , Receptores CXCR3 , Receptores de Quimiocinas/biossíntese , Receptores de Quimiocinas/imunologiaRESUMO
Cyclooxygenase-2 (COX-2) expression in epithelial tumors is frequently associated with a poor prognosis. In a murine model of metastatic breast cancer, we showed that COX-2 inhibition is associated with decreased metastatic capacity. The COX-2 product, prostaglandin E(2) (PGE(2)), acts through a family of G protein-coupled receptors designated EP1-4 that mediate intracellular signaling by multiple pathways. We characterized EP receptor expression on three murine mammary tumor cell lines and show that all four EP isoforms were detected in each cell. Stimulation of cells with either PGE(2) or the selective EP4/EP2 agonist PGE(1)-OH resulted in increased intracellular cyclic AMP and this response was inhibited with either EP2 or EP4 antagonists. Nothing is known about the function of EP receptors in tumor metastasis. We tested the hypothesis that the prevention of EP receptor signaling would, like inhibition of PGE(2) synthesis, inhibit tumor metastasis. Our results show for the first time that antagonism of the EP4 receptor with either AH23848 or ONO-AE3-208 reduced metastasis as compared with vehicle-treated controls. The therapeutic effect was comparable to that observed with the dual COX-1/COX-2 inhibitor indomethacin. EP3 antagonism had no effect on tumor metastasis. Mammary tumor cells migrated in vitro in response to PGE(2) and this chemotactic response was blocked by EP receptor antagonists. Likewise, the proliferation of tumor cells was also directly inhibited by antagonists of either EP4 or EP1/EP2. These studies support the hypothesis that EP receptor antagonists may be an alternative approach to the use of COX inhibitors to prevent tumor metastasis.
Assuntos
Neoplasias Mamárias Experimentais/tratamento farmacológico , Receptores de Prostaglandina E/antagonistas & inibidores , Animais , Compostos de Bifenilo/farmacologia , Inibidores de Ciclo-Oxigenase 2/farmacologia , Feminino , Indometacina/farmacologia , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Naftalenos/farmacologia , Metástase Neoplásica , Fenilbutiratos/farmacologia , Isoformas de Proteínas , Receptores de Prostaglandina E/biossíntese , Receptores de Prostaglandina E/classificação , Receptores de Prostaglandina E Subtipo EP4RESUMO
Cyclooxygenase (COX) inhibitors have demonstrated efficacy in models of human cancer but the relevant mechanisms have not all been elucidated. Both Cox-dependent as well as Cox-independent mechanisms have been implicated. Using a syngeneic model of metastatic breast cancer, we have investigated the effect of Cox inhibitors on NK functions that are critical to the control of metastatic disease. NK recognition of target cells is governed by a balance of activating and inhibiting receptors that bind ligands including MHC class I. We now show that treatment of tumor cells with the nonselective COX-1/COX-2 inhibitor indomethacin or the selective COX-2 inhibitor celecoxib leads to decreased expression of the MHC class I molecules Ld and Kd . Downregulated class I expression is associated with concomitant increased sensitivity to NK cell-mediated lysis. Both COX inhibitors limit tumor metastasis and this therapeutic effect is dependent on NK but not T cell function. Antimetastatic activity is also lost in the absence of interferon- gamma (IFN-gamma). Both COX inhibitors also suppress local tumor growth of subcutaneously implanted mammary tumor cells in immune competent Balb/cByJ mice. This therapeutic activity is lost in the absence of either CD4+ or CD8+ T cells, but is not compromised by the loss of NK activity. Thus, the mechanism of tumor inhibition differs in the context of local versus metastatic disease. Taken together, these findings are consistent with a mechanism not previously described, whereby COX inhibitors may relieve MHC-mediated inhibition of NK cytotoxicity leading to recognition and lysis of metastatic tumor cells.
Assuntos
Inibidores de Ciclo-Oxigenase/farmacologia , Células Matadoras Naturais/imunologia , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/secundário , Animais , Celecoxib , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Citotoxicidade Imunológica/efeitos dos fármacos , Regulação para Baixo , Feminino , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/metabolismo , Indometacina/farmacologia , Interferon gama/genética , Interferon gama/fisiologia , Neoplasias Mamárias Experimentais/imunologia , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Prostaglandina-Endoperóxido Sintases/química , Pirazóis/farmacologia , Sulfonamidas/farmacologiaRESUMO
INTRODUCTION: Overexpression of cyclooxygenase (COX-2) is commonly observed in human cancers. In a murine model of metastatic breast cancer, we observed that COX-2 expression and enzyme activity were associated with enhanced tumorigenic and metastatic potential. In contrast to the high COX-2 expression in metastatic tumors, transplantation of poorly tumorigenic tumor cell lines to syngeneic mice results in less COX-2 expression and less COX-2 activity in vivo. Aberrant CpG island methylation, and subsequent silencing of the COX-2 promoter, has been observed in human cancer cell lines and in some human tumors of the gastrointestinal tract. METHODS: Using bisulfite modification and a methylation-specific PCR, we examined the methylation status of the COX-2 promoter in a series of four closely-related murine mammary tumors differing in COX-2 expression and metastatic potential. RESULTS: We showed that line 410, which does not express COX-2 in vivo, exhibited evidence of promoter methylation. Interestingly, the metastatic counterpart of this cell (line 410.4) displayed only the unmethylated COX-2 promoter, as did two additional cell lines (lines 66.1 and 67). The methylation patterns observed in vitro were maintained when these murine mammary tumor lines were transplanted to syngeneic mice. Treatment with the DNA demethylating agent 5-aza-deoxycytidine increased COX-2 mRNA, increased protein and increased enzyme activity (prostaglandin synthesis). CONCLUSIONS: These results indicate that COX-2 promoter methylation may be one mechanism by which tumor cells regulate COX-2 expression. Upregulation of COX-2 expression in closely related metastatic lesions versus nonmetastatic lesions may represent a shift towards the unmethylated phenotype.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Metilação de DNA , Regulação Enzimológica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Isoenzimas/genética , Regiões Promotoras Genéticas/genética , Prostaglandina-Endoperóxido Sintases/genética , Animais , Neoplasias da Mama/secundário , Linhagem Celular Tumoral , Ciclo-Oxigenase 2 , DNA de Neoplasias/metabolismo , Modelos Animais de Doenças , Humanos , Isoenzimas/fisiologia , Neoplasias Mamárias Animais/enzimologia , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/secundário , Proteínas de Membrana , Camundongos , Prostaglandina-Endoperóxido Sintases/fisiologiaRESUMO
We have shown previously in a model of metastatic breast cancer that murine mammary tumor cells express both cyclooxygenase-1 (Cox-1) and Cox-2 isoforms. Growth and metastasis of these tumors in syngeneic hosts are inhibited by either selective Cox-1 (SC560) or selective Cox-2 (celecoxib) inhibitors. To gain insight into the relevant mechanisms involved in the therapeutic response, we determined the effect of Cox inhibitors on tumor cell behavior in vitro. We now report that either selective Cox-1 or Cox-2 drugs inhibited cell replication, but only at concentrations that are no longer selective for either isoform. Growth delay by either nonselective or selective inhibitors was associated with changes in cell morphology including cell rounding; these changes were reversed upon removal of drug. Unlike many other cell types examined, treatment of these mammary tumor cells with Cox inhibitors was not associated with detectable apoptosis. Growth inhibition, induced by either selective or nonselective Cox inhibitors, was accompanied by increased intracellular levels of the sphingolipid ceramide by 1.7-2.6-fold in comparison to vehicle-treated cells. Ceramide changes are associated with cell cycle arrest and we observed that all the Cox inhibitors examined increased significantly the number of cells in G0/G1 and reduced the S phase fraction. Likewise, addition of a cell-permeable form of ceramide (C6-ceramide) could mimic the effect of Cox inhibitors on both cell cycle and cell growth inhibition. Thus, mammary tumor cells are growth restricted by Cox inhibitors. These effects are associated with changes in ceramide levels and a block in cell cycle progression.