Gannareddy V. Girish

Targeting COX-2 and EP4 to control tumor growth, angiogenesis, lymphangiogenesis and metastasis to the lungs and lymph nodes in a breast cancer model

We reported that cyclo-oxygenase (COX)-2 expression in human breast cancer stimulated cancer cell migration and invasiveness, production of vascular endothelial growth factor (VEGF)-C and lymphangiogenesis in situ, largely from endogenous PGE2-mediated stimulation of prostaglandin E (EP)1 and EP4 receptors, presenting them as candidate therapeutic targets against lymphatic metastasis. As human breast cancer xenografts in immuno-compromised mice have limitations for preclinical testing, we developed a syngeneic murine breast cancer model of spontaneous lymphatic metastasis mimicking human and applied it for mechanistic and therapeutic studies. We tested the roles of COX-2 and EP receptors in VEGF-C and -D production by a highly metastatic COX-2 expressing murine breast cancer cell line C3L5. These cells expressed all EP receptors and produced VEGF-C and -D, both inhibited with COX-2 inhibitors or EP4 (but not EP1, EP2 or EP3) antagonists. C3H/HeJ mice, when implanted SC in both inguinal regions with C3L5 cells suspended in growth factor-reduced Matrigel, exhibited rapid tumor growth, tumor-associated angiogenesis and lymphangiogenesis (respectively measured with CD31 and LYVE-1 immunostaining), metastasis to the inguinal and axillary lymph nodes and the lungs. Chronic oral administration of COX-1/COX-2 inhibitor indomethacin, COX-2 inhibitor celecoxib and an EP4 antagonist ONO-AE3-208, but not an EP1 antagonist ONO-8713 at nontoxic doses markedly reduced tumor growth, lymphangiogenesis, angiogenesis, and metastasis to lymph nodes and lungs. Residual tumors in responding mice revealed reduced VEGF-C and -D proteins, AkT phosphorylation and increased apoptotic/proliferative cell ratios consistent with blockade of EP4 signaling. We suggest that EP4 antagonists deserve clinical testing for chemo-intervention of lymphatic metastasis in human breast cancer.

Decorin Is a Novel VEGFR-2-Binding Antagonist for the Human Extravillous Trophoblast

Extravillous trophoblasts (EVT) of the human placenta invade the uterine decidua and its arteries to ensure successful placentation. We previously identified two decidua-derived molecules, TGF-β and a TGF-β-binding proteoglycan decorin (DCN), as negative regulators of EVT proliferation, migration, and invasiveness and reported that DCN acts via multiple tyrosine kinase receptors [epidermal growth factor-receptor (EGF-R), IGF receptor-1 (IGFR1), and vascular endothelial growth factor 2 receptor (VEGFR-2)]. Because binding of DCN to VEGFR-2 has never been reported earlier, present study explored this binding, the approximate location of VEGFR-2-binding site in DCN, and its functional role in our human first trimester EVT cell line HTR-8/SVneo. Based on far-Western blotting and coimmunoprecipitation studies, we report that DCN binds both native (EVT expressed) and recombinant VEGFR-2 and that this binding is abrogated with a VEGFR-2 blocking antibody, indicating an overlap between the ligand-binding and the DCN-binding domains of VEGFR-2. We determined that (125)I-labeled VEGF-E (a VEGFR-2 specific ligand) binds EVT with a dissociation constant (K(d)) of 566 pM, and DCN displaced this binding with an inhibition constant (K(i)) of 3.93-5.78 nM, indicating a 7- to 10-fold lower affinity of DCN for VEGFR-2. DCN peptide fragments derived from the leucine rich repeat 5 domain that blocked DCN-VEGFR-2 interactions or VEGF-E binding in EVT cells also blocked VEGF-A- and VEGF-E-induced EVT cell proliferation and migration, indicative of functional VEGFR-2-binding sites of DCN. Finally, DCN inhibited VEGF-E-induced EVT migration by interfering with ERK1/2 activation. Our findings reveal a novel role of DCN as an antagonistic ligand for VEGFR-2, having implications for pathophysiology of preeclampsia, a trophoblast hypoinvasive disorder in pregnancy, and explain its antiangiogenic function.

Prostaglandin E2 receptor EP4 as the common target on cancer cells and macrophages to abolish angiogenesis, lymphangiogenesis, metastasis, and stem‐like cell functions

We previously established that COX‐2 overexpression promotes breast cancer progression and metastasis. As long‐term use of COX‐2 inhibitors (COX‐2i) can promote thrombo‐embolic events, we tested an alternative target, prostaglandin E2 receptor EP4 subtype (EP4), downstream of COX‐2. Here we used the highly metastatic syngeneic murine C3L5 breast cancer model to test the role of EP4‐expressing macrophages in vascular endothelial growth factor (VEGF)‐C/D production, angiogenesis, and lymphangiogenesis in situ, the role of EP4 in stem‐like cell (SLC) functions of tumor cells, and therapeutic effects of an EP4 antagonist RQ‐15986 (EP4A). C3L5 cells expressed all EP receptors, produced VEGF‐C/D, and showed high clonogenic tumorsphere forming ability in vitro, functions inhibited with COX‐2i or EP4A. Treating murine macrophage RAW 264.7 cell line with COX‐2i celecoxib and EP4A significantly reduced VEGF‐A/C/D production in vitro, measured with quantitative PCR and Western blots. Orthotopic implants of C3L5 cells in C3H/HeJ mice showed rapid tumor growth, angiogenesis, lymphangiogenesis (CD31/LYVE‐1 and CD31/PROX1 immunostaining), and metastasis to lymph nodes and lungs. Tumors revealed high incidence of EP4‐expressing, VEGF‐C/D producing macrophages identified with dual immunostaining of F4/80 and EP4 or VEGF‐C/D. Celecoxib or EP4A therapy at non‐toxic doses abrogated tumor growth, lymphangiogenesis, and metastasis to lymph nodes and lungs. Residual tumors in treated mice revealed markedly reduced VEGF‐A/C/D and phosphorylated Akt/ERK proteins, VEGF‐C/D positive macrophage infiltration, and proliferative/apoptotic cell ratios. Knocking down COX‐2 or EP4 in C3L5 cells or treating cells in vitro with celecoxib or EP4A and treating tumor‐bearing mice in vivo with the same drug reduced SLC properties of tumor cells including preferential co‐expression of COX‐2 and SLC markers ALDH1A, CD44, OCT‐3/4, β‐catenin, and SOX‐2. Thus, EP4 is an excellent therapeutic target to block stem‐like properties, angiogenesis, and lymphangiogenesis induced by VEGF‐A/C/D secreted by cancer cells and tumor infiltrating macrophages.

Mechanisms in Decorin Regulation of Vascular Endothelial Growth Factor-Induced Human Trophoblast Migration and Acquisition of Endothelial Phenotype

ABSTRACT Extravillous trophoblast (EVT) cells of the human placenta invade the uterine decidua and utero-placental arteries to establish an efficient exchange of key molecules between maternal and fetal blood. Trophoblast invasion is stringently regulated in situ both positively and negatively by a variety of factors at the fetal-maternal interface to maintain a healthy utero-placental homeostasis. One such factor, decorin, a transforming growth factor (TGF)-beta binding, leucine-rich proteoglycan produced by the decidua, negatively regulates EVT proliferation, migration, and invasiveness independent of TGF-beta. We reported that these decorin actions were mediated by its binding to multiple tyrosine kinase receptors, including vascular endothelial growth factor receptor (VEGFR)-2. The present study explores the mechanisms underlying decorin antagonism of VEGF (VEGF-A) stimulation of endovascular differentiation of EVT using our EVT cell line, HTR-8/SVneo. We observe that decorin inhibits VEGF-induced EVT cell migration and endothelial-like tube formation on matrigel. VEGF activates MAPKs (p38 MAPK, MEK3/6, and ERK1/2) in EVT cells, and the activation is blocked in both cases by decorin. Employing selective MAPK inhibitors, we show that both p38 and ERK pathways contribute independently to VEGF-induced EVT migration and capillary-like tube formation. VEGF upregulates the vascular endothelial (VE) markers VE-cadherin and beta-catenin in EVT and endothelial cells, and this upregulation is blocked by decorin and MAPK inhibitors. These results suggest that decorin inhibits VEGF-A stimulation of trophoblast migration and endovascular differentiation by interfering with p38 MAPK and ERK1/2 activation. Thus decorin-mediated dual impediment of endovascular differentiation of the EVT and angiogenesis may have implications for pathogenesis of preeclampsia, a hypoinvasive trophoblast disorder in pregnancy.

PGE2 promotes breast cancer-associated lymphangiogenesis by activation of EP4 receptor on lymphatic endothelial cells.

BackgroundLymphatic metastasis, facilitated by lymphangiogenesis is a common occurrence in breast cancer, the molecular mechanisms remaining incompletely understood. We had earlier shown that cyclooxygenase (COX)-2 expression by human or murine breast cancer cells promoted lymphangiogenesis and lymphatic metastasis by upregulating VEGF-C/D production by tumor cells or tumor-associated macrophages primarily due to activation of the prostaglandin receptor EP4 by endogenous PGE2. It is not clear whether tumor or host-derived PGE2 has any direct effect on lymphangiogenesis, and if so, whether EP4 receptors on lymphatic endothelial cells (LEC) play any role.MethodsHere, we address these questions employing in vitro studies with a COX-2-expressing and VEGF-C/D-producing murine breast cancer cell line C3L5 and a rat mesenteric (RM) LEC line and in vivo studies in nude mice.ResultsRMLEC responded to PGE2, an EP4 agonist PGE1OH, or C3L5 cell-conditioned media (C3L5-CM) by increased proliferation, migration and accelerated tube formation on growth factor reduced Matrigel. Native tube formation by RMLEC on Matrigel was abrogated in the presence of a selective COX-2 inhibitor or an EP4 antagonist. Addition of PGE2 or EP4 agonist, or C3L5-CM individually in the presence of COX-2 inhibitor, or EP4 antagonist, restored tube formation, reinforcing the role of EP4 on RMLEC in tubulogenesis. These results were partially duplicated with a human dermal LEC (HMVEC-dLyAd) and a COX-2 expressing human breast cancer cell line MDA-MB-231. Knocking down EP4 with shRNA in RMLEC abrogated their tube forming capacity on Matrigel in the absence or presence of PGE2, EP4 agonist, or C3L5-CM. RMLEC tubulogenesis following EP4 activation by agonist treatment was dependent on PI3K/Akt and Erk signaling pathways and VEGFR-3 stimulation. Finally in a directed in vivo lymphangiogenesis assay (DIVLA) we demonstrated the lymphangiogenic as well as angiogenic capacity of PGE2 and EP4 agonist in vivo.Discussion/conclusionsThese results demonstrate the roles of tumor as well as host-derived PGE2 in inducing lymphangiogenesis, at least in part, by activating EP4 and VEGFR-3 on LEC. EP4 being a common target on both tumor and host cells contributing to tumor-associated lymphangiogenesis reaffirms the therapeutic value of EP4 antagonists in the intervention of lymphatic metastasis in breast cancer.

Acetyl salicyclic acid (aspirin) improves synthesis of maspin and lowers incidence of metastasis in breast cancer patients

Maspin, a 42 kDa protein produced in normal breast cells, has been shown to inhibit the invasion and metastasis of breast cancer in an animal model. Ingestion of acetylsalicylic acid (aspirin) by breast cancer patients has been reported to restore the systemic synthesis of maspin through the stimulation of systemic nitric oxide production. Studies were carried out to determine the effect of aspirin on the incidence of breast cancer metastasis, which is reported to occur in 50% of patients who have previously received chemotherapy, radiation, and/or surgery. Thirty‐five female patients (aged 41–65 years) with breast cancer who had previously received these therapies took one 75 mg/70 kg body weight enteric‐coated aspirin tablet every 24 h, after an adequate meal, for 3 years. Their plasma nitric oxide and maspin levels were measured. The occurrence of metastasis was ascertained monthly by a qualified oncologist, and confirmed, if necessary, by biopsy. Daily ingestion of aspirin by participants resulted in an increase in maspin levels from 0.95 ± 0.04 to 4.63 ± 0.05 nM after 24 h. These levels were maintained for 3 years. These studies suggest that daily ingestion of aspirin might significantly reduce the incidence of breast cancer metastasis in patients who have previously received anticancer therapies. (Cancer Sci 2010)

The role of insulin dependent NO synthesis in the impaired production of maspin in human breast cancer

Purpose: The synthesis of maspin, a protein with various anti-breast cancer properties has been reported to be produced in normal breast tissue but not in breast cancer cells. We investigated the role of insulin receptors and their upregulation by prostaglandin E1 in vitro for the stimulation of NO synthesis by the hormone, and its consequence on maspin production in normal neutrophils and malignant cells in breast cancer. Methods: Maspin and NO were determined by ELISA and methemoglobin method, respectively. Results: The treatment of neutrophils and malignant breast cancer cells with prostaglandin E1 resulted in partial restoration of the impaired receptor numbers, and resulted in partial normalization of NO and maspin production in these cells compared to normal counterparts. It was not feasible to stimulate NO synthesis to normal ranges by the upregulation of receptor number due to intrinsic decrease of insulin receptors in breast cancer cells. However, aspirin, which was found to stimulate NO synthesis to normal ranges, normalized maspin production in these cells in breast cancer. Conclusion: The impaired maspin production was found to be the consequence of impaired insulin induced NO production in breast cancer due to the decrease of insulin receptor binding. Restoration of NO production by aspirin can be useful for the restoration of maspin production in breast cancer.

Abstract LB-67: Breast cancer-associated lymphangiogenesis: roles of PGE2 and EP4 receptor on lymphatic endothelial cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Lymphatic metastasis is a common event in breast cancer, facilitated by tumor-associated lymphangiogenesis, molecular mechanisms of which remain poorly defined. We had earlier shown that endogenous PGE2 resulting from elevated COX-2 expression by breast cancer cells promotes tumor progression and metastasis by multiple mechanisms: inactivation of host immune cells, stimulation of tumor cell migration, tumor-associated angiogenesis and lymphangiogenesis. The latter was due to upregulation of VEGF-C or -D secretion due to activation of EP4 receptors on breast cancer cells and cancer-infiltrating macrophages. Objectives: To examine whether PGE2 in the tumor micro-environment directly promotes lymphangiogenesis by stimulating EP4 receptors on lymphatic endothelial cells (LEC).Approaches: In vitro studies utilized a rat mesenteric lymphatic endothelial cell line (RMLEC) and a COX-2 expressing, VEGF-C/-D producing murine breast cancer cell line C3L5; in vivo studies measured lymphangiogenesis in nude mice. Results RMLEC expressed all EP receptors and responded to PGE2, an EP4 agonist (EP4a) PGE1-OH, or C3L5 cell conditioned media (C3L5-CM) by increased proliferation, migration and accelerated tube formation on growth factor-reduced matrigel. Tube formation on matrigel alone was completely abrogated in the presence of COX 1/2 inhibitor indomethacin (10uM), COX-2 inhibitor (COX-2i) NS-398 (15uM), and a selective EP4 antagonist (EP4A) RQ15986 (2.5 nM), indicating the roles of COX-2 and EP4. Addition of PGE2, EP4a, or C3L5-CM individually in presence of the COX-2i, or EP4A, partially restored the tube formation, reinforcing the role of EP4 on RMLEC. RMLEC grown in the presence of PGE2 showed upregulation of COX2, EP4, VEGF-C and VEGF-D mRNAs. Knocking down EP4 with shRNA in RMLEC abrogated tube formation on matrigel in the absence or presence of PGE-2, EP4a, or C3L5-CM. EP4 silenced RMLEC also became unresponsive to these agents in stimulating PGE2, VEGF-C, and -D production. Finally in a directed in vivo lymphangiogenesis assay (DIVLA) using implants of angioreactors including PGE-2, EP4a or VEGF-C in the dorsal flanks of nude mice, we simultaneously measured lymphangiogenesis and angiogenesis using dual immunostaining of the tissue contents for Lyve-1 and cd31, or prox-1 and cd31. Results demonstrated the lymphangiogenic as well as angiogenic capacity of PGE2 and EP4a in vivo by recruitment of new vessels. Conclusions: Tumor or host-derived PGE2 stimulates lymphangiogenesis, at least in part, by activating EP4 on the LEC. Combined with earlier studies we show that EP4 is a common therapeutic target on tumor and host cells (macrophages and LEC) in abrogating multiple events in breast cancer progression. This may spare cardio-protective prostanoids like PGI2, inhibited by COX-2 inhibitors Citation Format: Peeyush K. Lala, Gannareddy V. Girish, Xiping Xin, Mousumi Majumder, Elena Tutunea-Fatan, Pinki Nandi. Breast cancer-associated lymphangiogenesis: roles of PGE2 and EP4 receptor on lymphatic endothelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-67. doi:10.1158/1538-7445.AM2014-LB-67

Abstract 2310: Targeting EP4 receptor on cancer cells and macrophages to abrogate stem-like functions, lymphangiogenesis and lymphatic metastasis in a breast cancer model.

Background: We established that high COX-2 expression resulting in elevated prostaglandin (PG) E2 level in the tumor milieu promotes breast cancer progression and metastasis via multiple events: inactivation of immune cells, stimulation of tumor cell migration/invasiveness, tumor-associated angiogenesis and lymphangiogenesis. The lymphangiogenic action of COX-2 resulted from high VEGF-C/D production by tumor cells. These events were primarily due to activation of the PGE receptor EP4 on cancer cells, making it an excellent therapeutic target. Objectives: Present study tested the additional role of tumor-infiltrating macrophages in VEGF-C/D production and lymphangiogenesis in situ, the role of EP4 in stem-like tumor cell functions, and therapeutic effects of an EP4 antagonist. Since human cancer xenografts in immuno-compromised mice have limitations for preclinical testing, we developed a COX-2 expressing syngeneic murine breast cancer model of spontaneous lymphatic metastasis mimicking human and applied it for mechanistic and therapeutic studies. Methods and results: We tested the functional roles of COX-2 and EP4 in a highly metastatic COX-2 expressing murine breast cancer cell line C3L5. These cells expressed all EP receptors, produced VEGF-C and -D and showed high tumorsphere forming ability in ultra-low attachment plates by single cells for successive generations (surrogate of stem-like cell function) in vitro. Migration, tumorsphere formation and VEGF-C/D production by these cells were inhibited with Celecoxib or NS398 (COX-2 inhibitors) and a selective EP4 antagonist RQ-15986 (RaQualia Pharma). C3H/HeJ mice, when implanted SC in both inguinal regions with C3L5 cells suspended in growth factor-reduced Matrigel, exhibited rapid tumor growth, tumor-associated angiogenesis and lymphangiogenesis (respectively measured with CD31 and LYVE-1 immunostaining), metastasis to inguinal and axillary lymph nodes and the lungs. Tumors revealed high incidence of F4/ 80 +ve macrophages which also expressed EP4 and produced VEGF-C and -D in situ as evidenced by dual immunostaining of F4/80 and EP4, VEGF-C and -D. Chronic oral administration of Celecoxib or RQ-15986 at nontoxic doses abrogated tumor growth, lymphangiogenesis, angiogenesis, and metastasis to lymph nodes and lungs. Residual tumors in treated mice revealed markedly reduced VEGF-C/D proteins and incidence of VEGF-C/D +ve macrophages in situ. Conclusions: EP4 is an excellent therapeutic target to block stem-like properties, lymphangiogenesis induced by VEGF-C/D production by cancer cells and tumor infiltrating macrophages, deserving clinical testing for chemo-intervention in human breast cancer. (MM and XX contributed equally. Supported by grants of the CBCF, Ontario Chapter and the OICR to PKL, a TBCRU fellowship to MM and a gift of RQ-15986 by RaQualia Pharma). Citation Format: Mousumi Majumder, Xiping Xin, GannaReddy V. Girish, Peeyush K. Lala. Targeting EP4 receptor on cancer cells and macrophages to abrogate stem-like functions, lymphangiogenesis and lymphatic metastasis in a breast cancer model. [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 2310. doi:10.1158/1538-7445.AM2013-2310

Abstract 1601: COX-2 and EP4 receptor as targets to ameliorate tumor growth, lymphangiogenesis, and metastasis in a murine breast cancer model

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC We had earlier shown that elevated cyclo-oxygenase (COX)-2 expression by breast cancer cells promotes tumor progression and metastasis by multiple mechanisms: inactivation of host immune cells, stimulation of tumor cell migration and tumor-associated angiogenesis. Furthermore, COX-2 was causally associated with increased VEGF-C expression/secretion in human breast cancer cell lines, corroborated with in situ studies on breast cancer tissue specimens, showing a strong correlation of COX-2 with VEGF-C and lymphangiogenic markers or lymphovascular density, implicated in lymphatic metastasis. VEGF-C upregulation resulted from PGE2-mediated stimulation of EP4, and to a smaller extent, EP1 receptors on breast cancer cells. Since human tumor xeno-transplants in immno-compromised hosts are less than ideal for a therapeutic testing of these findings, in the present study, we developed a syngeneic murine breast cancer model exhibiting lymphangiogenesis and lymphatic metastasis in C3H/HeJ mice, using a highly metastatic, COX-2 expressing C3L5 cell line. This cell line was found to express VEGF-C and VEGF-D mRNA and proteins, and all four EP receptors. Expression of VEGF-C and VEGF-D were suppressed by treatment of cells with COX-2 inhibitors or EP4 antagonist in vitro. C3L5 cells suspended in growth factor-reduced Matrigel were implanted in both inguinal regions in mice that received oral treatments with COX-1/2 inhibitor indomethacin, COX-2 inhibitor celecoxib, EP1 antagonist ONO-8713 or EP4 antagonist ONO-AE3-208, to evaluate tumor growth, metastasis to lymph nodes and the lungs and tumor-associated angiogenesis (CD31 immuno-staining) and lymphangiogenesis (LYVE1 immunostaining). Mice were killed on days 8, 12 and 16. Tumor growth in control vehicle-treated mice was associated with high levels of angiogenesis and lymphangiogenesis at all intervals. VEGF-C protein expression was noted in 20-30% of tumor cells as well as macrophages in situ. Micro-metastasis to the lungs and local lymph nodes was noted as early as day 8 and increased thereafter. Metastasis to distant axillary nodes was seen at day16. Significant reduction in tumor growth, tumor-associated angiogenesis and lymphangiogenesis, and metastasis to the lungs and lymph nodes was achieved by therapies with indomethacin, celecoxib and ONO-AE3-208 but not ONO-8713 as compared to respective vehicle treatments. In conclusion, we have devised a syngeneic mouse breast cancer model mimicking human, showing successful chemo-intervention of tumor growth, tumor-associated angiogenesis/lymphangiogenesis and metastasis to the lungs and lymph nodes with COX inhibitors and EP 4 antagonist. (Supported by grants from the Canadian Breast Cancer Foundation, Ontario chapter and the Ontario Institute of Cancer Research to PKL. EP antagonists were kindly provided by ONO pharmaceuticals, Japan and Celecoxib by Pfizer.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1601.