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Supplementary Figure Legends. Description of supplementary Figures 1-4

Supplementary Figure 4. Reduced COX-2 Expression after miR-526b knockdown. T

Supplementary Figure 3. Morphology of MCF7 cells before and after miRNA overexpression

Supplementary Table 1. List of miRNAs significantly down or up regulated in MCF7-COX-2 compared to MCF7-Mock cell line

Supplementary Figure 1. Validation of Transfection/COX-2 mRNA in breast cancer cell lines/ miR-526b expression levels in a panel of breast cancer cell lines

Supplementary Figure 2. Representative images for migrant MCF7 and SKBR3 miR526b over expressed cells

Placenta in certain species including the human has evolved as a highly invasive tumor-like organ invading the uterus aned its vasculature to derive oxygen and nutrients for the fetus and exchange waste products. While several excellent reviews have been written comparing hemochorial placentation with tumors, no comprehensive review is available dealing with mechanistic insights into what makes them different, and what tumor biologists can learn from placental biologists, and vice versa. In this review, we analyze the structure-function relationship of the human placenta, emphasizing the functional need of the spatio-temporally orchestrated trophoblast invasiveness for fetal development and growth, and pathological consequences of aberrant invasiveness for fetal and maternal health. We then analyze similarities and differences between the placenta and invasive tumors in terms of hallmarks of cancer, some key molecules regulating their invasive functions, and how placental cancers (choriocarcinomas) or other cancers become refractory or even addicted to these invasion-restraining molecules. We cite in vitro models of human trophoblast and choriocarcinoma cell lines utilized to study mechanisms in normal placental development as well as those responsible for tumor progression. We discuss the pathobiology of hyper-invasive placentas and show thattrophoblastic neoplasias are a unique and heterogeneous class of tumors. We delve into the questions as to why metastasis from other organs rarely occurs at the placental site and whether pregnancy makes the mother more or less vulnerable to cancer-related morbidity/mortality. We attempt to compare trophoblast stem cells and cancer stem cells. Finally, we leave the readers with some thoughts as foods of future investigations.

Two small leucine-rich proteoglycans (SLRP), decorin and biglycan, play important roles in structural–functional integrity of the placenta and fetal membranes, and their alterations can result in several pregnancy-associated diseases. In this review, we briefly discuss normal placental structure and functions, define and classify SLRPs, and then focus on two SLRPs, decorin (DCN) and biglycan (BGN). We discuss the consequences of deletions/mutations of DCN and BGN. We then summarize DCN and BGN expression in the pregnant uterus, myometrium, decidua, placenta, and fetal membranes. Actions of these SLRPs as ligands are then discussed in the context of multiple binding partners in the extracellular matrix and cell surface (receptors), as well as their alterations in pathological pregnancies, such as preeclampsia, fetal growth restriction, and preterm premature rupture of membranes. Lastly, we raise some unanswered questions as food for thought.

We reported that two microRNAs, miR526b and miR655, are oncogenic in breast cancer (BC). Overexpression of these two miRNAs in poorly metastatic BC cells promotes aggressive BC phenotypes in vitro and in vivo. High expression of each miRNA was associated with poor patient survival. In this pilot biomarker study, we report for the first time that miRNA precursor RNAs (pri-miRNAs) are robust and sensitive biomarkers for BC, detectable in both human blood plasma and biopsy tissues. Pri-miRNA detection and quantification do not require a special enrichment procedure, thus reducing specimen quantity. Blood plasma samples from 90 malignant tumor-bearing patients and 20 benign lesion-bearing participants (control) were analyzed for pri-miRNA expression with a quantitative real-time polymerase chain reaction. Results revealed that normalized expressions of plasma pri-miR526b and pri-miR655 are significantly upregulated in malignancy compared to benign plasmas (p = 0.002 and p = 0.03, respectively). Both pri-miRNAs showed more prominent results to distinguish stage I plasmas from benign plasmas (p = 0.001 for pri-miR526b and p = 0.0001 for pri-miR655). We have also validated pri-miRNA expression in independent tumor bank tissues, showing significant upregulation of both pri-miRNAs in BC, thus, pri-miRNAs are robust markers. The diagnostic relevance of pri-miRNAs was computed with the area under the curve (AUC). Pri-miR526b is a sensitive biomarker to distinguish cancer from control plasmas (sensitivity of 86%, AUC = 71.47%, p = 0.0027) with a positive predictive value of 88.89%, however, pri-miR655 did not show significant sensitivity. Furthermore, pri-miR526b could also significantly distinguish tumors as early as stage I from control (sensitivity of 75%, AUC = 72.71%, p = 0.0037). Therefore, pri-miR526b can be used as an early diagnostic biomarker. The expression of both pri-miRNAs was significantly high in ER-positive and HER2-negative subgroups of BC, hence, these biomarkers might play a role in the management of endocrine therapy designs. Additionally, with a case–control cohort study, we identified that high expression of pri-miR526b in the blood is also a risk factor associated with breast cancer (OR = 4.3, CI = 1.39–13.34, p = 0.01). Pri-miRNAs could be considered novel breast cancer blood biomarkers.

Simple Summary The formation of new blood (angiogenesis) and lymphatic (lymphangiogenesis) vessels are major events associated with most epithelial malignancies, including breast cancer. Inflammation is a key mediator of both processes, hijacked by many cancers by the aberrant expression of the inflammation-associated enzyme cyclo-oxygenase (COX)-2. In this review, we focus on breast cancer and show that COX-2 is a major promoter of both events, primarily resulting from the activation of prostaglandin (PG) E receptor EP4 on tumor cells, tumor-infiltrating immune cells, and endothelial cells; and induction of oncogenic microRNAs. The COX-2/EP4 pathway also promotes additional events in breast cancer progression, such as cancer cell migration, invasion, and the stimulation of stem-like cells. Based on a combination of studies using multiple breast cancer models, we show that EP4 antagonists hold a major promise in breast cancer therapy in combination with other modalities including immune check-point inhibitors. Abstract The formation of new blood (angiogenesis) and lymphatic (lymphangiogenesis) vessels are major events associated with most epithelial malignancies, including breast cancer. Angiogenesis is essential for cancer cell survival. Lymphangiogenesis is critical in maintaining tumoral interstitial fluid balance and importing tumor-facilitatory immune cells. Both vascular routes also serve as conduits for cancer metastasis. Intratumoral hypoxia promotes both events by stimulating multiple angiogenic/lymphangiogenic growth factors. Studies on tumor-associated lymphangiogenesis and its exploitation for therapy have received less attention from the research community than those on angiogenesis. Inflammation is a key mediator of both processes, hijacked by many cancers by the aberrant expression of the inflammation-associated enzyme cyclo-oxygenase (COX)-2. In this review, we focus on breast cancer and showed that COX-2 is a major promoter of both events, primarily resulting from the activation of prostaglandin (PG) E receptor EP4 on tumor cells, tumor-infiltrating immune cells, and endothelial cells; and the induction of oncogenic microRNAs. The COX-2/EP4 pathway also promotes additional events in breast cancer progression, such as cancer cell migration, invasion, and the stimulation of stem-like cells. Based on a combination of studies using multiple breast cancer models, we show that EP4 antagonists hold a major promise in breast cancer therapy in combination with other modalities including immune check-point inhibitors.

Formation of new blood (angiogenesis) and lymphatic (lymphangiogenesis) vessels are major events associated with most epithelial malignancies, including breast cancer. Angiogenesis is essential for cancer cell survival. Lymphangiogenesis is critical in maintaining tumoral interstitial fluid balance and importing tumor-facilitatory immune cells. Both vascular routes also serve as conduits for cancer metastasis. Intratumoral hypoxia promotes both events by stimulating multiple angiogenic/lymphangiogenic growth factors. Studies on tumor-associated lymphangiogenesis and its exploitation for therapy have received less attention from the research community than those on angiogenesis. Inflammation is a key mediator of both processes, hijacked by many cancers by aberrant expression of the inflammation-associated enzyme cyclo-oxygenase (COX)-2. In this review, we focus on breast cancer and show that COX-2 is a major promoter of both events, primarily resulting from the activation of Prostaglandin (PG) E receptor EP4 on tumor cells, tumor-infiltrating immune cells, and endothelial cells; and induction of oncogenic microRNAs. COX-2/EP4 pathway also promotes additional events in breast cancer progression, such as cancer cell migration, invasion, and stimulation of stem–like cells. Based on a combination of studies using multiple breast cancer models, we show that EP4 antagonists hold a major promise in breast cancer therapy in combination with other modalities including immune check-point inhibitors

Decidualization involves the proliferation and differentiation of fibroblast-like endometrial stromal cells into epithelioid-shaped and secretory ‘decidual’ cells in response to steroid hormones. Human decidual cells produce insulin-like growth factor-binding protein-1 and prolactin (PRL), two well-recognized markers of decidual cell maturation and a proteoglycan decorin (DCN). We reported that DCN restrains the human trophoblast renewal, migration, invasion and endovascular differentiation needed for uterine arterial remodeling during normal pregnancy. DCN overproduction by the decidua is associated with a hypo-invasive placenta and a serious pregnancy disorder, pre-eclampsia (PE). Furthermore, elevated maternal plasma DCN levels during the second trimester is a predictive biomarker of PE. While these paracrine roles of decidua-derived DCN on trophoblast physiology and pathology have been well-defined, it remains unknown whether DCN plays any autocrine role in decidual cell development. The objectives of this study were to examine: the kinetics of DCN production during decidualization of human endometrial stromal cells; gestational age-related changes in DCN production by the first trimester decidua; and a possible autocrine role of DCN on decidual cell maturation. We found that DCN production is enhanced during decidualization of both primary and immortalized human endometrial stromal cells in vitro and during early gestation in decidual samples tested ex vivo, and that it is important for endometrial stromal cell maturation into a decidual phenotype. Decorin-depleted human endometrial stromal cells exposed to decidualizing stimuli failed to mature fully, as evidenced by fibroblastoid morphology, reduced insulin-like growth factor-binding protein-1 and PRL expression, and reduction in cellular ploidy. We identified heart and neural crest derivatives-expressed protein 2, and progesterone receptor as potential downstream mediators of DCN effects.

Lymphangiogenesis (formation of new lymphatic vessels), unlike angiogenesis, has been a lesser-focused field in cancer biology, because of earlier controversy regarding whether lymphatic metastasis occurs via pre-existing or newly formed lymphatics. Recent evidence reveals that peri-tumoral or intra-tumoral lymphangiogenesis is a precursor for lymphatic metastasis in most carcinomas and melanomas. Two major lymphangiogenic factors, vascular endothelial growth factor (VEGF)-C and VEGF-D, are produced by cancer cells or immune cells such as macrophages in the tumor-stroma to promote sprouting of lymphatics from lymphatic endothelial cells (LEC) or LEC precursors (LECP) by binding to their primary (high affinity) receptor VEGF-R3 or secondary receptors VEGF-R2, neuropilin (NRP)2 and α9/β1 integrin. Many other growth factors/receptors such as VEGF-A/VEGF-R2, fibroblast growth factor (FGF)2/FGF-R, platelet-derived growth factor (PDGF)/PDGF-R, hepatocyte growth factor (HGF)/C-Met, angiopoietins (Ang)1, 2/Tie2, and chemokines/ chemokine receptors (CCL21/CCR7, CCL12/CCR4) can also stimulate LEC sprouting directly or indirectly. This review deals with the roles of prostaglandins (PG), in particular PGE2, in cancer-associated lymphangiogenesis, with special emphasis on breast cancer. We show that cyclooxygenase (COX)-2 expression by breast cancer cells or tumor stroma leading to high PGE2 levels in the tumor milieu promotes lymphangiogenesis and lymphatic metastases, resulting from binding of PGE2 to PGE receptors (EP, in particular EP4) on multiple cell types: tumor cells, tumor-infiltrating immune cells, and LEC. EP4 activation on cancer cells and macrophages upregulated VEGF-C/D production to stimulate LEC sprouting. Furthermore, ligation of EP4 with PGE2 on cancer or host cells can initiate a new cascade of molecular events leading to cross-talk between cancer cells and LEC, facilitating lymphangiogenesis and lympho-vascular transport of cancer cells. We make a case for EP4 as a potential therapeutic target for breast cancer.

Background Over-expression of cyclooxygenase (COX)-2 promotes breast cancer progression by multiple mechanisms, including induction of stem-like cells (SLC). Combined gene expression and microRNA microarray analyses of empty vector vs COX-2- transfected COX-2 low MCF7 breast cancer cell line identified two COX-2-upregulated microRNAs, miR-526b and miR-655, both found to be oncogenic and SLC-promoting. Cytoplasmic Polyadenylation Element-Binding Protein 2 (CPEB2) was the single common target of both microRNAs, the functions of which remain controversial. CPEB2 has multiple isoforms (A-F), and paradoxically, a high B/A ratio was reported to impart anoikis-resistance and metastatic phenotype in triple- negative breast cancer cells. We tested whether CPEB2 is a tumor suppressor in mammary epithelial cells. Methods We knocked-out CPEB2 in the non-tumorigenic mammary epithelial cell line MCF10A by CRISPR/Cas9-double nickase approach, and knocked-down CPEB2 with siRNAs in the poorly malignant MCF7 cell line, both lines being high CPEB2-expressing. The resultant phenotypes for oncogenity were tested in vitro for both lines and in vivo for CPEB2KO cells. Finally, CPEB2 expression was compared between human breast cancer and non-tumor breast tissues. Results CPEB2 (isoform A) expression was inversely correlated with COX-2 or the above microRNAs in COX-2-divergent breast cancer cell lines. CPEB2KO MCF10A cells exhibited oncogenic properties including increased proliferation, migration, invasion, EMT (decreased E-Cadherin, increased Vimentin, N-Cadherin, SNAI1, and ZEB1) and SLC phenotype (increased tumorsphere formation and SLC marker-expression). Tumor-suppressor p53 protein was shown to be a novel translationally-regulated target of CPEB2, validated with polysome profiling. CPEB2KO, but not wild-type cells produced lung colonies upon intravenous injection and subcutaneous tumors and spontaneous lung metastases upon implantation at mammary sites in NOD/SCID/IL2Rϒ-null mice, identified with HLA immunostaining. Similarly, siRNA-mediated CPEB2 knockdown in MCF7 cells promoted oncogenic properties in vitro. Human breast cancer tissues (n = 105) revealed a lower mRNA expression for CPEB2 isoform A and also a lower A/B isoform ratio than in non-tumour breast tissues (n = 20), suggesting that CPEB2A accounts for the tumor-suppressor functions of CPEB2. Conclusions CPEB2, presumably the isoform A, plays a key role in suppressing tumorigenesis in mammary epithelial cells by repressing EMT, migration, invasion, proliferation and SLC phenotype, via multiple targets, including a newly-identified translational target p53. Electronic supplementary material The online version of this article (10.1186/s12885-019-5771-5) contains supplementary material, which is available to authorized users.

The origin and regulation of stem cells sustaining trophoblast renewal in the human placenta remain unclear. Decorin, a leucine-rich proteoglycan restrains trophoblast proliferation, migration/invasiveness and endovascular differentiation, and local decorin overproduction is associated with preeclampsia (PE). Here, we tested the role of decorin in human trophoblast stem cell self-renewal and differentiation, using two models: an immortalized first trimester trophoblast cell line HTR-8/SVneo (HTR) and freshly isolated primary trophoblast (p-trophoblast) from early first trimester (6–9 weeks) placentas. Self-renewal capacity was measured by spheroid forming ability of single cells on ultra-low attachment plates for multiple generations. Markers of embryonic stem (ES) cells, trophoblast stem (TS) cells and trophoblast were used to identify stem cell hierarchy. Differentiation markers for syncytial and extravillous (EVT) pathways were employed to identify differentiated cells. Bewo cells were additionally used to explore DCN effects on syncytialization. Results reveal that the incidence of spheroid forming stem-like cells was 13–15% in HTR and 0.1–0.4%, in early first trimester p-trophoblast, including a stem cell hierarchy of two populations of ES and TS-like cells. DCN restrained ES cell self-renewal, promoted ES to TS transition and maintenance of TS cell stem-ness, but inhibited TS cell differentiation into both syncytial and EVT pathways.

The objective of the present review is to synthesize the information on the cellular and molecular players responsible for maintaining a homeostatic balance between a naturally invasive human placenta and the maternal uterus in pregnancy; to review the roles of decorin (DCN) as a molecular player in this homeostasis; to list the common maladies associated with a break-down in this homeostasis, resulting from a hypo-invasive or hyper-invasive placenta, and their underlying mechanisms. We show that both the fetal components of the placenta, represented primarily by the extravillous trophoblast, and the maternal component represented primarily by the decidual tissue and the endometrial arterioles, participate actively in this balance. We discuss the process of uterine angiogenesis in the context of uterine arterial changes during normal pregnancy and preeclampsia. We compare and contrast trophoblast growth and invasion with the processes involved in tumorigenesis with special emphasis on the roles of DCN and raise important questions that remain to be addressed. Decorin (DCN) is a small leucine-rich proteoglycan produced by stromal cells, including dermal fibroblasts, chondrocytes, chorionic villus mesenchymal cells and decidual cells of the pregnant endometrium. It contains a 40 kDa protein core having 10 leucine-rich repeats covalently linked with a glycosaminoglycan chain. Biological functions of DCN include: collagen assembly, myogenesis, tissue repair and regulation of cell adhesion and migration by binding to ECM molecules or antagonising multiple tyrosine kinase receptors (TKR) including EGFR, IGF-IR, HGFR and VEGFR-2. DCN restrains angiogenesis by binding to thrombospondin-1, TGFβ, VEGFR-2 and possibly IGF-IR. DCN can halt tumor growth by antagonising oncogenic TKRs and restraining angiogenesis. DCN actions at the fetal-maternal interface include restraint of trophoblast migration, invasion and uterine angiogenesis. We demonstrate that DCN overexpression in the decidua is associated with preeclampsia (PE); this may have a causal role in PE by compromising endovascular differentiation of the trophoblast and uterine angiogenesis, resulting in poor arterial remodeling. Elevated DCN level in the maternal blood is suggested as a potential biomarker in PE.

Background: We had shown that over-expression of the inflammation-associated enzyme cyclo-oxygenase (COX)-2 promotes breast cancer progression and metastasis and sustains stem-like cells (SLC), believed to evade traditional therapy and promote tumor reoccurrence. Stable transfection of the COX-2 gene into COX-2–, HER-2–, non-metastatic human MCF-7 breast cancer cell line (named MCF-7-COX-2) led to a highly aggressive phenotype in vitro, where we observed epithelial-mesenchymal transition, increased migration, invasion, clonogenic tumorsphere forming ability (surrogate of SLC) and expression of SLC marker ALDH. Furthermore, we observed an increase in the tumorigenic and metastatic capacity of the cells in immuno-compromised mice in vivo. Combined gene expression and microRNA (miRNA) micro-array analysis of MCF-7 and MCF-7-COX-2 cells revealed COX-2 induced up-regulation of two miRNAs, miR-526b and miR-655, and down-regulation of their common gene target, cytoplasmic polyadenylation element binding protein (CPEB)-2. While both miRNAs were found to be oncogenic in functional assays, the function of CPEB-2 in breast cancer remains untested. Hypothesis: miR-526b and miR-655 down-regulate CPEB-2 to promote breast cancer cell aggressiveness and the SLC phenotype. Approaches & Results: (1) To test if expression levels of COX-2 and CPEB-2 are inversely related, we measured both gene and protein expression in multiple COX-2 disparate breast cancer cell lines. (2) We also tested if there is an inverse relationship between miRNA expression and the expression levels of their target gene CPEB-2. We found that high COX-2/miRNA expressing cell lines such as MDA-MB-231 and MCF-7-COX-2 had significantly lower expression of CPEB-2 than MCF-7 cells (low COX-2/low miRNA). (3) To test the functional effects of CPEB-2 gene manipulation in vitro and in vivo, we knocked down CPEB-2 in CPEB-2-high MCF-7 cell line. MCF-7-CPEB-2-KD cells were found to be more migratory and invasive than control cells in transwell assays. Ongoing studies are to test (a) whether there is a cause and effect relationship between the expression of the miRNAs and CPEB-2 expression, that is, whether knocking down or knocking in the miRNAs respectively up-regulates and down-regulates CPEB-2 expression; and (b) whether CPEB-2 expression is lower in breast cancer tissues than in adjacent non-tumor tissues, and inversely correlated with the miRNAs in breast cancer tissues. Conclusion and Significance: If CPEB-2 is proven to be tumor suppressor, these oncogenic miRNAs and putatively anti-oncogenic CPEB-2 may serve as potential biomarkers in personalizing breast cancer therapy with COX-2 inhibitors as adjuvant. (Supported by funds of the OICR to PKL. AH and MM are CaRTT/TBCRU scholars). Citation Format: Asma Hasan, Mousumi Majumder, Peeyush K Lala. The role of cytoplasmic polyadenylation element binding protein-2 in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-29.

We show that Cyclooxygenase-2 over-expression induces an oncogenic microRNA miR655 in human breast cancer cells by activation of EP4. MiR655 expression positively correlated with COX-2 in genetically disparate breast cancer cell lines and increased in all cell lines when grown as spheroids, implicating its link with stem-like cells (SLCs). Ectopic miR655 over-expression in MCF7 and SKBR3 cells resulted in increased proliferation, migration, invasion, spheroid formation and Epithelial to Masenchymal transition (EMT). Conversely, knocking down miR655 in aggressive MCF7-COX2 and SKBR3-COX2 cells reverted these phenotypes. MCF7-miR655 cells displayed upregulated NOTCH/WNT genes; both pathway inhibitors abrogated miR655-induced spheroid formation, linking miR655 with SLC-related pathways. MiR655 expression was dependent on EP4 activity and EP4 downstream signaling pathways PI3K/AKT, ERK and NF-kB and led to TGFβ resistance for Smad3 phosphorylation. Tail vein injection of MCF7-miR655 and SKBR3-miR655 cells in NOD/SCID/GUSB-null mice revealed increased lung colony growth and micrometastases to liver and spleen. MiR655 expression was significantly high in human breast tumors (n = 105) compared to non-tumor tissues (n = 20) and associated with reduced patient survival. Thus miR655 could serve as a prognostic breast cancer biomarker.

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. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Background: Lymphatic 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. Methods: Here, 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. Results: RMLEC 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/conclusions: These 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.

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. © 2012 USCAP, Inc All rights reserved.

Workshops are an important part of the IFPA annual meeting. At IFPA Meeting 2010 diverse topics were discussed in twelve themed workshops, six of which are summarized in this report. 1. The placental pathology workshop focused on clinical correlates of placenta accreta/percreta. 2. Mechanisms of regulation of trophoblast invasion and spiral artery remodeling were discussed in the trophoblast invasion workshop. 3. The fetal sex and intrauterine stress workshop explored recent work on placental sex differences and discussed them in the context of whether boys live dangerously in the womb. 4. The workshop on parasites addressed inflammatory responses as a sign of interaction between placental tissue and parasites. 5. The decidua and embryonic/fetal loss workshop focused on key regulatory mediators in the decidua, embryo and fetus and how alterations in expression may contribute to different diseases and adverse conditions of pregnancy. 6. The trophoblast differentiation and syncytialisation workshop addressed the regulation of villous cytotrophoblast differentiation and how variations may lead to placental dysfunction and pregnancy complications.

The invasion of maternal decidua and uterine spiral arteries by a trophoblast subpopulation called extravillous trophoblast (EVT) is essential for the establishment of a normal placenta and an adequate blood flow toward the fetus. Derangements in these processes underlie pregnancy-related diseases like preeclampsia and intrauterine growth restriction. Many growth factors, growth factor binding proteins, and extracellular matrix components can positively or negatively regulate the proliferation, migration, and/or invasiveness of these EVT cells. RHO GTPases, including RHOA, RAC1, and CDC42, are ubiquitous proteins that control cytoskeletal changes by forming stress fibers and projecting lamellipodia and filopodia during cellular migration. We had previously shown that prostaglandin (PG) E(2) produced in abundance by the decidua promotes the migration of first-trimester human EVTs by increasing the intracellular concentration of calcium and activating calpain. Using our well-characterized immortalized EVT cell line, HTR-8/SVneo, as well as villus explants from first-trimester placentae, this study examined the role of RHO GTPases RAC1 and CDC42 in PGE(2)-mediated migratory responses of these cells. Though a RAC1 inhibitor, NSC23766 as well as RAC1 knockdown by siRNA decreased the migration of HTR-8/SVneo cells in a Transwell migration assay, this inhibition could not be restored by PGE(2) or 17-phenyl trinor PGE(2) (PGE receptor PTGER1 agonist) or PGE(1) Alcohol (PGE receptor PTGER4 agonist). Similar results were noted for EVT cell spreading in villus explants. Furthermore, CDC42 silencing using siRNA inhibited PGE(2)-induced migration of HTR-8/SVneo cells. Finally, the treatment of EVT cells with PGE(2), PTGER1 agonist, or PTGER4 agonist activated RAC1 and CDC42 at 10 min, suggesting that RAC1 and CDC42 play an essential role in PGE(2)-mediated migration of human EVTs.

Prostaglandin (PG) E(2) may regulate invasiveness of human placenta because we previously reported stimulation of migration of placental trophoblasts by PGE(2) acting through PGE receptor (EP)-1 and activating calpain. RhoA GTPase and its important effector Rho kinase (ROCK) have also been previously shown to regulate trophoblast migration. Using immortalized HTR-8/SVneo trophoblast cells and first-trimester human chorionic villus explant cultures on matrigel, we further examined the role of RhoA/ROCK and MAPK (ERK1/2) pathways on PGE(2)-mediated stimulation of trophoblast migration. Migration of cytotrophoblasts was shown to be inhibited by treatment of the trophoblast cell line and chorionic villus explants with either cell-permeable C3 transferase or selective RhoA small interfering RNA. These inhibitions were significantly mitigated by the addition of PGE(2), an EP1/EP3 agonist or an EP3/EP4 agonist, suggesting that RhoA plays an important role in trophoblast migration but may not be obligatory for PGE(2) action. Treatment of HTR-8/SVneo cells with nonselective ROCK inhibitor Y27632 or ROCK small interfering RNAs inhibited migration of these cells, which could not be rescued with PGE(2) or the other two EP agonists, suggesting the obligatory role of ROCK in PGE(2)-induced migratory response. Furthermore, U0126, an inhibitor of MAPK kinases MEK1 and MEK2, abrogated PGE(2)-induced migration of trophoblasts, and PGE(2) or the other two EP agonists stimulated ERK1/2 activation in trophoblasts, which was not abrogated by pretreatment with C3 transferase, indicating that ERK signaling pathway is an efficient alternate pathway for RhoA in PGE(2)-mediated migration of trophoblasts. These results suggest that ROCK and ERK1/2 play more important roles than RhoA in PGE(2)-mediated migration stimulation of first-trimester trophoblasts.

Vascular endothelial growth factor C (VEGF-C) is a lymphangiogenic factor over-expressed in highly metastatic, cyclooxygenase (COX)-2 expressing breast cancer cells. We tested the hypothesis that tumour-derived VEGF-C may play an autocrine role in metastasis by promoting cellular motility through one or more VEGF-C-binding receptors VEGFR-2, VEGFR-3, neuropilin (NRP)-1, NRP-2, and integrin alpha9beta1. We investigated the expression of these receptors in several breast cancer cell lines (MDA-MB-231, Hs578T, SK-BR-3, T-47D, and MCF7) and their possible requirement in migration of two VEGF-C-secreting, highly metastatic lines MDA-MB-231 and Hs578T. While cell lines varied significantly in their expression of above VEGF-C receptors, migratory activity of MDA-MB-231 and Hs578T cells was linked to one or more of these receptors. Depletion of endogenous VEGF-C by treatments with a neutralising antibody, VEGF-C siRNA or inhibitors of Src, EGFR/Her2/neu and p38 MAP kinases which inhibited VEGF-C production, inhibited cellular migration, indicating the requirement of VEGF-C for migratory function. Migration was differentially attenuated by blocking or downregulation of different VEGF-C receptors, for example treatment with a VEGFR-2 tyrosine kinase inhibitor, NRP-1 and NRP-2 siRNA or alpha9beta1 integrin antibody, indicating the participation of one or more of the receptors in cell motility. This novel role of tumour-derived VEGF-C indicates that breast cancer metastasis can be promoted by coordinated stimulation of lymphangiogenesis and enhanced migratory activity of breast cancer cells.

Background Decorin, a leucine-rich proteoglycan that is produced by decidual cells, limits invasion and endovascular differentiation of extravillous trophoblast cells during early placentation by binding to multiple tyrosine kinase receptors, in particular, vascular endothelial growth factor receptor-2. Objective Because many studies have reported an association between poor trophoblast invasion and endovascular differentiation with preeclampsia, the studies reported here tested (1) whether decorin over-expression in the chorionic villi and/or basal decidua is associated with preeclampsia and, if so, (2) whether this association results in a hypoinvasive placenta, and (3) whether elevated plasma decorin concentration in the second trimester is a predictive biomarker for preeclampsia. Study Design Decorin messenger RNA expression was measured with quantitative polymerase chain reaction at the tissue level and with in situ hybridization at the cellular level using 35 S-labeled antisense complimentary RNA probe in placentas from healthy control subjects and subjects with preeclampsia (14 each, 23-40 weeks of gestation). Tissue sections of the same placentas were also immunostained for decorin protein. A decorin over-expressing human endometrial stromal cell line was tested for invasion-regulatory effects on an invasive first-trimester extravillous trophoblast cell line HTR-8/SVneo plated in cocultures that were separated by a semipermeable membrane. Furthermore, we conducted retrospective measurements of plasma decorin levels during the second trimester (15-18 weeks of gestation) in a cohort of 28 body mass index–matched pairs of control subjects and subjects with preeclampsia before the onset of clinical disease. Results First, decorin messenger RNA expression at the cellular level measured with in situ hybridization exhibited profoundly higher expression levels in basal plate decidual cells within the placentas from preeclamptic subjects than those from control subjects at all gestational ages, whereas no difference between the 2 subject groups was noted in villus mesenchymal cells. Similarly decorin messenger RNA expression at the tissue level in chorionic villi (primarily resulting from fetally derived mesenchymal cells) did not differ significantly between control and preeclampsia placentas. These findings were validated with immunostaining for decorin protein. Second, knocking down decorin gene in a decorin over-expressing endometrial cell line (used as an in vitro surrogate of decorin over-expressing decidual cells) in cocultures with extravillous trophoblast cells abrogated its invasion-restraining actions on trophoblast cells, which indicated paracrine contribution of decorin over-expressing decidua to the poor trophoblast invasiveness in situ. Finally, retrospective measurement of plasma decorin levels during the second trimester in 28 body mass index–matched pairs of control subjects and subjects with preeclampsia revealed elevated plasma decorin levels in all subjects with preeclampsia in all body mass index groups. A receiver operating characteristic curve analysis revealed strong diagnostic performance of plasma decorin in the prediction of preeclampsia status. Although there was no significant gestational age-related change in decorin levels during the second trimester in control or subjects with preeclampsia, we found that plasma decorin had a significant inverse relationship with body mass index or bodyweight. Conclusion We conclude that decorin over-expression by basal decidual cells is associated with hypoinvasive phenotype and poor endovascular differentiation of trophoblast cells in preeclampsia and that elevated plasma decorin concentration is a potential predictive biomarker for preeclampsia before the onset of clinical signs.

Decorin (DCN) is a leucine-rich, TGF-β binding proteoglycan produced by mesenchymal cells including chondrocytes, dermal fibroblasts, and uterine decidual cells. It exerts multiple physiological functions including collagen fibrillogenesis, myogenesis, angiostasis, and restraining placental invasiveness. We discovered that decidua-derived DCN restrains proliferation, migration, and invasion of extravillous trophoblast (EVT) cells of the human placenta in a TGF-β-independent manner. These functions were differentially mediated by binding of DCN to multiple tyrosine kinase receptors (TKR) including EGFR, IGFR1, and VEGFR2. DCN blocked VEGFR-2 dependent EVT cell migration and endovascular differentiation by inhibiting P38MAPK and ERK1/2 pathways.We identified the avid VEGFR2 binding site in DCN protein as a 12 amino acids (LGTNPLKSSGIE) span in the Leucine-rich-repeat (LRR) 5 region of domain III. A single amino acid mutation (substitution of K to A) of DCN at this site abrogated VEGFR-2- dependent DCN actions. Also, DCN mRNA expression, measured with in situ hybridization, was selectively upregulated in decidual cells in placentas from mothers suffering from pre-eclampsia (PE), whereas the expression levels remained unchanged in chorionic villus mesenchymal cells. This difference between PE and control placentas was present at all gestational ages, indicating the pathogenic role of DCN in PE. We hypothesize that increased blood DCN levels could be a candidate biomarker for PE.

Increased expression of COX-2 or VEGF-C has been correlated with progressive disease in certain cancers. Present study utilized several human breast cancer cell lines (MCF-7, T-47D, Hs578T and MDA-MB-231, varying in COX-2 expression) as well as 10 human breast cancer specimens to examine the roles of COX-2 and prostaglandin E (EP) receptors in VEGF-C expression or secretion, and the relationship of COX-2 or VEGF-C expression to lymphangiogenesis. We found a strong correlation between COX-2 mRNA expression and VEGF-C expression or secretion levels in breast cancer cell lines and VEGF-C expression in breast cancer tissues. Expression of LYVE-1, a selective marker for lymphatic endothelium, was also positively correlated with COX-2 or VEGF-C expression in breast cancer tissues. Inhibition of VEGF-C expression and secretion in the presence of COX-1/2 or COX-2 inhibitors or following downregulation of COX-2 with COX-2 siRNA established a stimulatory role COX-2 in VEGF-C synthesis by breast cancer cells. EP1 as well as EP4 receptor antagonists inhibited VEGF-C production indicating the roles of EP1 and EP4 in VEGF-C upregulation by endogenous PGE2. Finally, VEGF-C secretion by MDA-MB-231 cells was inhibited in the presence of kinase inhibitors for Her-2/neu, Src and p38 MAPK, indicating a requirement of these kinases for VEGF-C synthesis. These results, for the first time, demonstrate a regulatory role of COX-2 in VEGF-C synthesis (and thereby lymphangiogenesis) in human breast cancer, which is mediated at least in part by EP1/EP4 receptors.

The root cause of preeclampsia in the human lies in the placenta, where a subpopulation of cytotrophoblast cells called extravillous trophoblasts (EVT), known to be involved in the invasion of the uterine endometrium and utero-placental arteries, become less invasive, resulting in poor perfusion of maternal blood into placenta.Because EVT migrate into the prostaglandin (PG) E2-rich decidua, we tested the roles of PGE2 and PGE2-mediated signaling in EVT migration, using our well-characterized EVT line HTR-8/Svneo as well as first trimester villus explants in culture.mRNA expression of different PGE2 receptors (EPs) in HTR-8/Svneo cells was studied using RT-PCR. To characterize the functional significance of EP receptors in EVT, different EP receptor agonists and antagonists were used in our migration assay systems and in the measurements of intracellular concentration of Ca2+ ([Ca2+]i) and calpain activity.Exogenous PGE2 stimulated EVT migration both in vitro and in the villus explant cultures. Although EVT expressed mRNA for all EP receptors (EP 1-4), a functional predominance of EP1 and EP4 was demonstrated in migration assays using specific EP agonists and antagonists. EP1-receptor-mediated signaling events such as activation of phospholipase C and elevation of cytosolic free [Ca2+]i were confirmed by the following findings: 1) exogenous PGE2 or an EP1 agonist, but not an EP4 agonist, increased [Ca2+]i, which could be blocked with an EP1 antagonist as well as BAPTA and thapsigargin; 2) phospholipase C inhibitor U73122, BAPTA, and thapsigargin inhibited PGE2-mediated migratory response of EVT; and 3) PGE2-mediated EVT migration was shown to be dependent on a class of Ca2+-dependent proteases called calpains, known to be involved in cell detachment from substratum during migratory responses. The presence of PGE2 stimulated calpain activity, whereas two calpain inhibitors, calpastatin and N-Ac-Leu-Leu-methioninal (ALLM), blocked EVT migration.PGE2 stimulates EVT migration by signaling through EP1 receptors, increasing [Ca2+]i, and activating calpain.

Aberrant upregulation of COX-2 enzyme resulting in accumulation of PGE2 in a cancer cell environment is a marker for progression of many cancers, including breast cancer. Four subtypes of cell surface receptors (EP1, EP2, EP3, and EP4), which are coupled with different G-proteins, mediate PGE2 actions. Since migration is an essential step in invasion and metastasis, in the present study we defined the expression of EP receptors and their roles in migratory function of breast cancer cells of murine (C3L5) and human (MDA-MB-231 and MCF-7) origin. Highly metastatic C3L5 and MDA-MB-231 cells, found to be highly migratory in a Transwell migration assay, were shown to accumulate much higher levels of PGE2 in culture media in comparison with nonmetastatic and poorly migrating MCF-7 cells; the levels of PGF2alpha and 6-keto-PGF1alpha were low in all cases. The elevated PGE2 production by metastatic cancer cells was due to COX-2 activity since dual COX-1/2 inhibitor indomethacin and selective COX-2 inhibitor NS-398 equally suppressed both basal and inducible (by IFN-gamma/LPS or Ca2+-ionophores) PGE2 accumulation. RT-PCR analysis revealed that murine C3L5 cells expressed mRNA of EP1, EP3, and EP4 but not EP2 receptors. On the other hand, human MDA-MB-231 and MCF-7 cells expressed all the above receptors. High levels of expression of functional EP4 receptors coupled with Gs-protein was confirmed in C3L5 cells by biochemical assay showing a dose-dependent increase of intracellular cAMP synthesis in response to PGE2. EP receptor antagonists SC-19220, AH-6809, and AH-23848B, having highest affinity for EP1, EP1/EP2/DP, and EP4 receptors, respectively, variably inhibited migration of metastatic breast cancer cells. An autocrine PGE2-mediated migratory activity of these cells appeared to be associated predominantly with EP4 receptor-mediated signaling pathway, which uses cAMP as a second messenger. This conclusion is based on several observations: (1) selective EP4 antagonist AH-23848B effectively inhibited migration of both C3L5 and MDA-MB-231 cells in a dose-dependent manner; (2) exogenous PGE2 and EP4 agonist PGE1 alcohol increased migration of C3L5 cells; (3) forskolin, a potent activator of adenylate cyclase, as well as membrane-permeable analogues of cAMP (8-bromo-cAMP, dibutyryl-cAMP) stimulated migration of C3L5 cells; and (4) Rp-cAMPS, a selective protein kinase A inhibitor, reduced migration of C3L5 cells. Migration of poorly migratory MCF-7 cells remained unaffected with either PGE2 or EP4 antagonist. These findings are relevant for designing therapeutic strategies against breast cancer metastasis.

Impaired trophoblast invasiveness and spiral arterial remodelling, which results in poor placental perfusion during early pregnancy, is believed to cause fetal injury and growth retardation, and also endothelial cell activation/dysfunction in a susceptible mother, leading to clinical manifestations of pre-eclampsia. This article briefly reviews the regulatory roles of certain locally active factors in trophoblast migration and invasiveness. This background is then used to discuss and debate whether derangements or dysfunction of some of these factors can manifest as early serum markers predictive of the disease, as opposed to the intermediate and late stage markers which may reflect manifestations and consequences of the disease. Of particular significance are the observed derangements in uPA/uPAR/PAI system, IGFBP-1, HGF, HB-EGF and TGFbeta, factors which are known to regulate trophoblast migration and invasiveness in situ. An emphasis is placed on the need for longitudinal studies in order to identify predictive serum markers which may help strategies for prevention or amelioration of fetal injury and pre-eclampsia.

The serine protease urokinase-type plasminogen activator (uPA) promotes matrix degradation by many cell types, including the invasive extravillous trophoblast (EVT) of the human placenta. The noncatalytic amino-terminal end of uPA binds to uPA receptors (uPARs) expressed by these cells. A highly polarized expression of uPAR-bound uPA at the migration front of EVT cells in situ suggests a functional role of uPA:uPAR interaction in EVT cell migration. The present study examined whether uPA stimulates EVT cell migration, independent of proteolytic function, and investigated some of the signaling pathways involved. Using in vitro-propagated EVT cells in Transwell migration assays, both uPA and its noncatalytic amino-terminal fragment (ATF) were shown to stimulate migration through multiporous polycarbonate (pore size 8 μm) membranes. A uPAR-blocking antibody inhibited basal and ATF-stimulated migration. Migration was found to be stimulated by hypoxic conditions, which upregulates uPAR expression; this stimulation was abrogated with the uPAR-blocking antibody, indicating the role of endogenous uPA in EVT cell migration. Spectrofluorometric measurement of cytosolic calcium in cells treated with uPA and ATF demonstrated a rapid rise in [Ca2+]i, which was prevented by pretreatment of cells with thapsigargin, indicating a release from intracellular stores. Both basal and ATF-mediated migratory responses were suppressed in the presence of selective pharmacological inhibitors LY294002, U73122, and U0126, implicating the respective roles of phosphatidinylinositol 3-kinase (PI 3-K), phospholipase C (PLC), and MEK1/2 in basal and ATF-stimulated migratory capacity. Taken together, these results demonstrate that uPA:uPAR interaction stimulates EVT cell migration, independent of uPA enzymatic activity, using the mitogen-activated protein kinase pathway and calcium signaling events including the participation of PI 3-K and PLC. These findings are relevant to clinical conditions of aberrant trophoblast migration, including spontaneous abortion, preeclampsia, and choriocarcinoma.

A highly proliferative, migratory and invasive subpopulation of human placental trophoblasts, known as extravillous trophoblasts (EVT), invades the uterus and its vasculature, to establish an adequate exchange of key molecules between the maternal and fetal circulation. Our earlier studies provided evidence for a positive regulation of migration/invasion of EVT by an autocrine factor IGFII and a paracrine, decidua-derived factor IGFBP1. The present study examined the role played by endothelin (ET)-1, also produced at the fetal-maternal interface, and its receptor subtypes ET(A) and ET(B) in the regulation of human EVT cell functions. We utilized an in vitro propagated EVT cell line (HTR-8/SVneo) which exhibits the phenotypic and functional characteristics of EVT in situ. Reverse transcription-PCR with primers specific for prepro-ET-1, ET(A) and ET(B) cDNAs demonstrated the expression of all these genes in HTR-8/SVneo cells. While proliferation was not influenced, migration of these cells through porous Transwell membranes was stimulated by exogenous ET-1. ET-1 also induced biphasic elevation of cytosolic free Ca(2+) concentrations ([Ca(2+)](i)) consisting of an initial transient followed by a sustained plateau, as measured by spectrofluorimetry. The dependence of the Ca(2+) response on phospholipase C (PLC) was demonstrated by its abrogation in the presence of PLC inhibitor U73122. Furthermore, ET-1 treatment of EVT cells rapidly stimulated phosphorylation of MAP kinase (ERK1/2). By using ET receptor antagonists and agonists, it was shown that both ET(A) and ET(B) receptors were responsible for the effects of ET-1 on migration, [Ca(2+)](i) and MAPK phosphorylation. Thus, ET-1 may represent an autocrine/paracrine mediator of invasive trophoblast function.

Extravillous trophoblast (EVT) cells of the human placenta progressively lose their proliferative activity in situ as EVT cell columns migrate into and invade the decidua. It remains unclear whether this is due to a terminal differentiation of EVT cells along the invasive pathway with concomitant loss of proliferative ability, or a negative regulation by decidua-derived factors, or both mechanisms. Our earlier studies provided evidence for a negative regulation by a decidua-derived factor, transforming growth factor (TGF)-β, which inhibited proliferation, migration, and invasiveness of first-trimester EVT cells in vitro. We further discovered that decidua also produces decorin, a proteoglycan that binds TGF-β (and in some cases, inactivates TGF-β), which is colocalized with TGF-β in the decidual extracellular matrix. The present study used in vitro-propagated EVT cell lines to examine whether EVT cells retain their capacity for proliferation after the process of invasion; and whether decorin exer...

MicroRNAs (miRs) are small regulatory molecules emerging as potential biomarkers in cancer. Previously, it was shown that COX-2 expression promotes breast cancer progression via multiple mechanisms, including induction of stem-like cells (SLC), owing to activation of the prostaglandin E2 receptor EP4 (PTGER4). COX-2 overexpression also upregulated microRNA-526b (miR-526b), in association with aggressive phenotype. Here, the functional roles of miR-526b in breast cancer and the mechanistic role of EP4 signaling in miR-526b upregulation were examined. A positive correlation was noted between miR-526b and COX-2 mRNA expression in COX-2 disparate breast cancer cell lines. Stable overexpression of miR-526b in poorly metastatic MCF7 and SKBR3 cell lines resulted in increased cellular migration, invasion, EMT phenotype and enhanced tumorsphere formation in vitro, and lung colony formation in vivo in immunodeficient mice. Conversely, knockdown of miR-526b in aggressive MCF7-COX-2 and SKBR3-COX-2 cells reduced oncogenic functions and reversed the EMT phenotype, in vitro. Furthermore, it was determined that miR-526b expression is dependent on EP4 receptor activity and downstream PI3K–AKT and cyclic AMP (cAMP) signaling pathways. PI3K–AKT inhibitors blocked EP4 agonist–mediated miR-526b upregulation and tumorsphere formation in MCF7 and SKBR3 cells. NF-κB inhibitor abrogates EP agonist–stimulated miRNA expression in MCF7 and T47D cells, indicating that the NF-κB pathway is also involved in miR-526b regulation. In addition, inhibition of COX-2, EP4, PI3K, and PKA in COX-2–overexpressing cells downregulated miR-526b and its functions in vitro. Finally, miR-526b expression was significantly higher in cancerous than in noncancerous breast tissues and associated with reduced patient survival. In conclusion, miR-526b promotes breast cancer progression, SLC-phenotype through EP4-mediated signaling, and correlates with breast cancer patient survival. Implications: This study presents novel findings that miRNA 526b is a COX-2 upregulated, oncogenic miRNA promoting SLCs, the expression of which follows EP4 receptor-mediated signaling, and is a promising biomarker for monitoring and personalizing breast cancer therapy. Mol Cancer Res; 13(6); 1022–33. ©2015 AACR.

Clinical application of interleukin (IL)-2-based immunotherapy of cancer has been limited by a major side-effect known as 'capillary leak syndrome', resulting from nitric oxide (NO) overproduction. A galactoside-specific lectin from Viscum album L. (VAA) has been reported to induce certain lymphokines and upregulate IL-2 receptors on lymphocytes. Present study was, therefore, designed to compare the effects of combination therapy with IL-2 (10(4) Cetus units/mouse, intraperitoneal (i.p). every 8 h, given as 5 day rounds per week, for one or two rounds) and VAA (1 ng/kg subcutaneous (s.c.), biweekly) with those of IL-2 or VAA therapy alone in C3H/HeJ female mice bearing s.c. transplants of a highly metastatic C3L5 mammary adenocarcinoma. IL-2 therapy alone reduced tumour growth and metastasis, but caused significant water retention indicative of capillary leakage in the kidneys after both rounds of therapy, whereas pleural effusion was only evident after the first round and not the second round. A sharp rise in the systemic NO levels after the first round, followed by a decline after the second round of IL-2 therapy suggested a causal relationship of increased NO levels to pleural effusion. A strong immunostaining for nitrotyrosine (a marker for the production of peroxynitrite) was noted in the renal tubules at the end of both rounds of therapy suggestive of a causal association of this toxic NO-metabolite with capillary leakage in the kidneys. Addition of VAA to IL-2 therapy had no effect on any of the above parameters. Unexpectedly, however, VAA therapy alone stimulated tumour growth as well as lung metastases. NO induction in the C3L5 cells by VAA was excluded as a possible reason for this stimulation. Present results suggest the need for exercising caution in the use of VAA as an immunoadjuvant in human cancer therapy.

We had earlier shown that TGF-beta controls proliferation, migration, and invasiveness of normal human trophoblast cells, whereas premalignant and malignant trophoblast cells are resistant to TGF-beta. To identify signaling defects responsible for TGF-beta resistance in premalignant and malignant trophoblasts, we have compared the expression of TGF-beta signaling molecules in a normal trophoblast cell line (HTR-8), its premalignant derivative (RSVT2/C), and two choriocarcinoma cell lines (JAR and JEG-3). RT-PCR analysis revealed that all these cell lines expressed the mRNA of TGF-beta1, -beta2, and -beta3, TGF-beta receptors type I, II, and III, and post-receptor signaling genes smad2, smad3, smad4, smad6, and smad7 with the exception that TGF-beta2 and smad3 were undetectable in JAR and JEG-3 cells. Immunoblot analysis confirmed the absence of smad3 protein in choriocarcinoma cells. Treatment with TGF-beta1 induced smad3 phosphorylation and smad3 translocation to the nucleus in the normal and premalignant trophoblast cells. These results suggest that loss of smad3 may account for a functional disruption in the TGF-beta signaling pathway in choriocarcinomas, but not in the premalignant trophoblast.

A highly migratory subpopulation of the human placental trophoblast, known as the extravillous trophoblast (EVT), invades the uterus and its vasculature, to establish adequate exchange of key molecules between the maternal and fetal circulations. During their formation, EVT cells selectively acquire alpha 5 beta 1 integrin. We had shown that alpha 5 beta 1 is required for their migratory function, and that EVT cell migration is stimulated by insulin-like growth factor-binding protein (IGFBP)-1 produced by the uterine decidua. The present study examined whether this stimulation is dependent on binding of the Arg-Gly-Asp (RGD) domain of IGFBP-1 to an RGD binding site on the alpha 5 beta 1 integrin, followed by activation of focal adhesion kinase (FAK) and stimulation of the mitogen-activated protein kinase (MAPK) pathway. IGFBP-1 treatment increased migration of EVT cells, whereas an anti-alpha 5 beta 1 integrin antibody blocked migration regardless of IGFBP-1 treatment. Migration stimulation by IGFBP-1 was abrogated by pretreatment with a Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), but not a Gly-Arg-Gly-Glu-Ser-Pro (GRGESP) hexapeptide, and by mutation of the RGD domain of IGFBP-1 to Trp-Gly-Asp (WGD). IGFBP-1 treatment caused a rapid localization of immunoreactive FAK to cellular lamellipodia, a rapid increase in phosphorylation of FAK and extracellular-signal regulated kinases 1 and 2. Preincubation of EVT cells with Herbimycin A, a tyrosine kinase inhibitor, abrogated IGFBP-1 effects; whereas an MAPK kinase inhibitor, PD 98059, reduced migration regardless of IGFBP-1 treatment. These results indicate that IGFBP-1 stimulation of EVT cell migration occurs by binding of its RGD domain to the alpha 5 beta 1 integrin, leading to activation of FAK and stimulation of MAPK pathway.

Summary Nitric oxide(NO) is a short-lived molecule required for many physiological functions, produced from Larginine by NO synthases (NOS). It is a free radical, producing many reactive intermediates that account for its bioactivity. Sustained induction of the inducible form of NOS (iNOS) in chronic inflammation may be mutagenic, through NO-mediated DNA damage or hindrance to DNA repair, and thus potentially carcinogenic. Expression of iNOS is positively associated with P53 mutation in tumours of the colon, lung, and oropharynx. Progression of a large majority of human and experimental tumours seems to be stimulated by NO resulting from activation of iNOS or constitutive NOS, whereas inhibition is documented in others. This discrepancy is largely explained by differential sensitivity of tumour cells to NO-mediated cytostasis or apoptosis and clonal evolution of NO resistant and NO-dependent cells. P53 mutation or loss is one of many events linked with NO resistance and dependence. NO can stimulate tumour growth and metastasis by promoting migratory, invasive, and angiogenic abilities of tumour cells, which may also be triggered by activation of cyclo-oxygenase (COX)-2. Thus, selective inhibitors of NOS, COX, or both may have a therapeutic role in certain cancers.

Tumorigenesis results from genetic alterations that occur in a stepwise manner giving rise to cells with increasingly cancer-like characteristics. We used in vitro propagated first trimester human extravillous trophoblast (EVT) cells to identify genetic changes responsible for the transition of the EVT from a normal to premalignant stage. The model used consisted of a normal invasive EVT (HTR8) cell line and its premalignant derivative (RSVT2/C) generated by transfection with the SV40 Tag and selected using a forced crisis regimen. RSVT2/C display increased proliferative, migratory and invasive behavior, unresponsiveness to anti-proliferative and anti-invasive signals of TGFbeta and a deficiency in gap junctional intercellular communication. These cells, however, were unable to form colonies on soft agar or tumors in nude mice and are thus defined as premalignant. Differential display revealed 18 gene sequences, 7 with unknown and 11 with known identity, showing altered expression between the normal HTR8 and premalignant RSVT2/C cell lines. The known sequences include the potential tumor suppressors insulin-like growth factor binding protein (IGFBP)-5 and fibronectin (FN) and potential protooncogenes such as chromokinesin (KIF4), alternative splicing factor (SF2), dynein, DNA polymerase epsilon (DNApol epsilon) and NF-kappaB activating kinase (NAK). The role of the remaining 4 genes upregulated in the premalignant EVT is presently unknown and these are FK506 binding protein (FKBP) 25, histone protein (HP1Hs)-gamma, nucleoporin (Nup) 155 and an 82 kDa acidic human protein. The functional role of IGFBP-5 was examined in the control of proliferation, migration and invasiveness of RSVT2/C cells measured in vitro. IGFBP-5 alone had no effect on these properties of RSVT2/C cells. Furthermore, unlike normal EVT cells, RSVT2/C cells exhibited refractoriness to the migration stimulating signals of IGF-II, which was explained by the loss or downregulation of the IGF type 2 receptor (IGF-R2). RSVT2/C cells, however, expressed the IGF type 1 receptor (IGF-R1) and responded to IGF-I by increased proliferation. This response was blocked with increasing concentrations of IGFBP-5. These results suggest that the loss of IGFBP-5 and possibly IGF-R2, both of which can sequester IGF-I from IGF-R1, permits unhindered proliferation of the premalignant EVT in an IGF-I rich environment of the fetal-maternal interface. The functions of the other differentially expressed genes, some of which are essential for cell cycle progression or cell survival require further investigation.

The contributory role of nitric oxide (NO) on tumour growth and metastasis was evaluated in a murine mammary tumour model. NO synthase (NOS) protein expression levels were examined in spontaneously arising C3H/HeJ mammary adenocarcinomas and respective lung metastases. In addition, 2 clonal derivatives of a single spontaneous tumour differing in metastatic phenotype (C3L5 and C10; highly and weakly metastatic, respectively) were utilised to investigate (i) the relationship between NOS expression levels and the biological behaviour of tumour cells (e.g., in vitro migratory and invasive capacities, in vivo tumour growth rate and metastatic and angiogenic capacities) and (ii) whether tumour-derived NO stimulated the invasive, migratory and angiogenic capacities of tumour cells. A heterogeneous pattern of endothelial NOS (eNOS) expression was observed in tumour cells in spontaneous primary tumours, and eNOS expression was higher in undifferentiated relative to differentiated tumour zones. However, tumour cells in lung metastatic sites were always strongly eNOS-positive, suggesting that eNOS expression facilitated metastasis. Findings using clonal derivatives supported this notion; s.c. primary tumour growth rate, efficiency of spontaneous metastasis and eNOS expression were higher for C3L5 relative to C10 cell lines. Nevertheless, lung metastases derived from both tumour cell lines were always strongly and homogeneously eNOS-positive. C3L5 cells were more invasive than C10 cells in vitro, but the migratory capacities of the cell lines did not differ. However, migration and invasiveness of both cell lines were inhibited with L-NAME and restored with excess L-arginine. Tumour-associated angiogenesis, measured in Matrigel implants inclusive of tumour cells, was higher for C3L5 relative to C10 cells, and C3L5-induced angiogenesis was reduced with chronic L-NAME treatment of host animals. These findings suggest that tumour-derived eNOS promoted tumour growth and metastasis by multiple mechanisms: stimulation of tumour cell migration, invasiveness and angiogenesis.

Purified human first trimester extravillous trophoblast (EVT) cell lines HTR-8 and HT-116 were examined for susceptibility to natural killer (NK) cell-mediated lysis. Based upon nucleic acid sequencing of an amplified fragment of cDNA, Western blot analysis and immunostaining of fixed and live cells, it was shown that both EVT cell lines expressed HLA-G mRNA and protein within the cytoplasm when cultured on laminin-coated plates. Very weak HLA-G expression was detectable on the cell surface under these conditions. However, strong cell surface expression of a classical MHC class I molecule (most likely HLA-C) was exhibited by these EVT cell lines when grown on laminin, as indicated by W6/32 FACS analysis (Ab recognizing pan MHC class I), and Western immunoblotting with HC10 (Ab recognizing HLA-B/C). When these EVT cells, cultured on laminin, were used as targets for peripheral blood natural killer (NK) cells in a standard chromium release assay, both HTR-8 and HT-116 cells were lysed by NK cells in a dose-dependent manner. The respective percentage specific lysis at an effector to target (E/T) ratio of 100 was 28+/-7, and 48+/-14. The choriocarcinoma cell lines JAR and JEG-3 which were respectively MHC class I negative and HLA-G positive were resistant to NK cell lysis. Thus, there was no clear relationship between the MHC class I expression and NK cell resistance or susceptibility among the EVT cell lines and choriocarcinoma cells. These findings raise the possibility that NK cells may take part in the surveillance of the invasive EVT cells during normal placentation.