Roles of prostaglandins in tumor-associated lymphangiogenesis with special reference to breast cancer.

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/ß1 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.

Tumor suppressor role of cytoplasmic polyadenylation element binding protein 2 (CPEB2) in human mammary epithelial cells.

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.

miR526b and miR655 Induce Oxidative Stress in Breast Cancer.

In eukaryotes, overproduction of reactive oxygen species (ROS) causes oxidative stress, which contributes to chronic inflammation and cancer. MicroRNAs (miRNAs) are small, endogenously produced RNAs that play a major role in cancer progression. We established that overexpression of miR526b/miR655 promotes aggressive breast cancer phenotypes. Here, we investigated the roles of miR526b/miR655 in oxidative stress in breast cancer using in vitro and in silico assays. miRNA-overexpression in MCF7 cells directly enhances ROS and superoxide (SO) production, detected with fluorescence assays. We found that cell-free conditioned media contain extracellular miR526b/miR655 and treatment with these miRNA-conditioned media causes overproduction of ROS/SO in MCF7 and primary cells (HUVECs). Thioredoxin Reductase 1 (TXNRD1) is an oxidoreductase that maintains ROS/SO concentration. Overexpression of TXNRD1 is associated with breast cancer progression. We observed that miR526b/miR655 overexpression upregulates TXNRD1 expression in MCF7 cells, and treatment with miRNA-conditioned media upregulates TXNRD1 in both MCF7 and HUVECs. Bioinformatic analysis identifies two negative regulators of TXNRD1, TCF21 and PBRM1, as direct targets of miR526b/miR655. We validated that TCF21 and PBRM1 were significantly downregulated with miRNA upregulation, establishing a link between miR526b/miR655 and TXNRD1. Finally, treatments with oxidative stress inducers such as H2O2 or miRNA-conditioned media showed an upregulation of miR526b/miR655 expression in MCF7 cells, indicating that oxidative stress also induces miRNA overexpression. This study establishes the dynamic functions of miR526b/miR655 in oxidative stress induction in breast cancer.

EP4 as a Therapeutic Target for Aggressive Human Breast Cancer.

G-protein-coupled receptors (GPCRs, also called seven-transmembrane or heptahelical receptors) are a superfamily of cell surface receptor proteins that bind to many extracellular ligands and transmit signals to an intracellular guanine nucleotide-binding protein (G-protein). When a ligand binds, the receptor activates the attached G-protein by causing the exchange of Guanosine-5'-triphosphate (GTP) for guanosine diphosphate (GDP). They play a major role in many physiological functions, as well as in the pathology of many diseases, including cancer progression and metastasis. Only a few GPCR members have been exploited as targets for developing drugs with therapeutic benefit in cancer. Present review briefly summarizes the signaling pathways utilized by the EP (prostaglandin E receptor) family of GPCR, their physiological and pathological roles in carcinogenesis, with special emphasis on the roles of EP4 in breast cancer progression. We make a case for EP4 as a promising newer therapeutic target for treating breast cancer. We show that an aberrant over-expression of cyclooxygenase (COX)-2, which is an inflammation-associated enzyme, occurring in 40-50% of breast cancer patients leads to tumor progression and metastasis due to multiple cellular events resulting from an increased prostaglandin (PG) E2 production in the tumor milieu. They include inactivation of host anti-tumor immune cells, such as Natural Killer (NK) and T cells, increased immuno-suppressor function of tumor-associated macrophages, promotion of tumor cell migration, invasiveness and tumor-associated angiogenesis, due to upregulation of multiple angiogenic factors including Vascular Endothelial Growth Factor (VEGF)-A, increased lymphangiogenesis (due to upregulation of VEGF-C/D), and a stimulation of stem-like cell (SLC) phenotype in cancer cells. All of these events were primarily mediated by activation of the Prostaglandin (PG) E receptor EP4 on tumor or host cells. We show that selective EP4 antagonists (EP4A) could mitigate all of these events tested with cells in vitro as well as in vivo in syngeneic COX-2 expressing mammary cancer bearing mice or immune-deficient mice bearing COX-2 over-expressing human breast cancer xenografts. We suggest that EP4A can avoid thrombo-embolic side effects of long term use of COX-2 inhibitors by sparing cardio-protective roles of PGI2 via IP receptor activation or PGE2 via EP3 receptor activation. Furthermore, we identified two COX-2/EP4 induced oncogenic and SLC-stimulating microRNAs-miR526b and miR655, one of which (miR655) appears to be a potential blood biomarker in breast cancer patients for monitoring SLC-ablative therapies, such as with EP4A. We suggest that EP4A will likely produce the highest benefit in aggressive breast cancers, such as COX-2 expressing triple-negative breast cancers, when combined with other newer agents, such as inhibitors of programmed cell death (PD)-1 or PD-L1.

COX-2 Induces Breast Cancer Stem Cells via EP4/PI3K/AKT/NOTCH/WNT Axis.

Cancer stem-like cells (SLC) resist conventional therapies, necessitating searches for SLC-specific targets. We established that cyclo-oxygenase(COX)-2 expression promotes human breast cancer progression by activation of the prostaglandin(PG)E-2 receptor EP4. Present study revealed that COX-2 induces SLCs by EP4-mediated NOTCH/WNT signaling. Ectopic COX-2 over-expression in MCF-7 and SKBR-3 cell lines resulted in: increased migration/invasion/proliferation, epithelial-mesenchymal transition (EMT), elevated SLCs (spheroid formation), increased ALDH activity and colocalization of COX-2 and SLC markers (ALDH1A, CD44, ß-Catenin, NANOG, OCT3/4, SOX-2) in spheroids. These changes were reversed with COX-2-inhibitor or EP4-antagonist (EP4A), indicating dependence on COX-2/EP4 activities. COX-2 over-expression or EP4-agonist treatments of COX-2-low cells caused up-regulation of NOTCH/WNT genes, blocked with PI3K/AKT inhibitors. NOTCH/WNT inhibitors also blocked COX-2/EP4 induced SLC induction. Microarray analysis showed up-regulation of numerous SLC-regulatory and EMT-associated genes. MCF-7-COX-2 cells showed increased mammary tumorigenicity and spontaneous multiorgan metastases in NOD/SCID/IL-2Rγ-null mice for successive generations with limiting cell inocula. These tumors showed up-regulation of VEGF-A/C/D, Vimentin and phospho-AKT, down-regulation of E-Cadherin and enrichment of SLC marker positive and spheroid forming cells. MCF-7-COX-2 cells also showed increased lung colonization in NOD/SCID/GUSB-null mice, an effect reversed with EP4-knockdown or EP4A treatment of the MCF-7-COX-2 cells. COX-2/EP4/ALDH1A mRNA expression in human breast cancer tissues were highly correlated with one other, more marked in progressive stage of disease. In situ immunostaining of human breast tumor tissues revealed co-localization of SLC markers with COX-2, supporting COX-2 inducing SLCs. High COX-2/EP4 mRNA expression was linked with reduced survival. Thus, EP4 represents a novel SLC-ablative target in human breast cancer. Stem Cells 2016;34:2290-2305.

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

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.

The role of CCL21/CCR7 chemokine axis in breast cancer-induced lymphangiogenesis.

BACKGROUND: Tumor-induced lymphangiogenesis facilitates breast cancer progression by generating new lymphatic vessels that serve as conduits for tumor dissemination to lymph nodes and beyond. Given the recent evidence suggesting the implication of C-C chemokine ligand 21/chemokine receptor 7 (CCL21/CCR7) in lymph node metastasis, the aim of our study was to define the role of this chemokine pair in breast cancer-associated lymphangiogenesis. METHODS: The expression analysis of CCL21/CCR7 pair and lymphatic endothelial cell (LEC) markers in breast cancer specimens was performed by means of quantitative real-time PCR. By utilizing CCR7 and CCL21 gene manipulated breast cancer cell implants into orthotopic sites of nude mice, lymphatic vessel formation was assessed through quantitative real-time PCR, immunohistochemistry and immunofluorescence assays. Finally, the lymphangiogenic potential of CCL21/CCR7 was assessed in vitro with primary LECs through separate functional assays, each attempting to mimic different stages of the lymphangiogenic process. RESULTS: We found that CCR7 mRNA expression in human breast cancer tissues positively correlates with the expression of lymphatic endothelial markers LYVE-1, podoplanin, Prox-1, and vascular endothelial growth factor-C (VEGF-C). We demonstrated that the expression of CCL21/CCR7 by breast cancer cells has the ability to promote tumor-induced lymph-vascular recruitment in vivo. In vitro, CCL21/CCR7 chemokine axis regulates the expression and secretion of lymphangiogenic factor VEGF-C and thereby promotes proliferation, migration, as well as tube formation of the primary human LECs. Finally, we showed that protein kinase B (AKT) signaling pathway is the intracellular mechanism of CCR7-mediated VEGF-C secretion by human breast cancer cells. CONCLUSIONS: These results reveal that CCR7 and VEGF-C display a significant crosstalk and suggest a novel role of the CCL21/CCR7 chemokine axis in the promotion of breast cancer-induced lymphangiogenesis.

D-loop somatic mutations and ∼5 kb "common" deletion in mitochondrial DNA: important molecular markers to distinguish oral precancer and cancer.

Apart from genomic DNA, mutations at mitochondrial DNA (mtDNA) have been hypothesized to play vital roles in cancer development. In this study, ∼5 kb deletion and D-loop mutations in mtDNA and alteration in mtDNA content were investigated in buccal smears from 104 healthy controls and 74 leukoplakia and 117 cancer tissue samples using Taqman-based quantitative assay and re-sequencing. The ∼5 kb deletion in mtDNA was significantly less (9.8 and 10.5 folds, P < 0.0001) in cancer tissues compared to control and leukoplakia tissues, respectively. On the other hand, somatic mutations in D-loop, investigated in 54 controls, 50 leukoplakias and 56 cancer patients, were found to be significantly more in cancer tissues, but not in leukoplakia tissues, compared to control (Z-score = 5.4). MtDNA contents were observed to be significantly more in leukoplakia (2.1 folds, P = 0.004) and cancer (1.6 folds, P = 0.03) tissues compared to control tissues. So, D-loop somatic mutations and ∼5 kb deletion patterns could be used as distinguishing markers between precancer and cancer tissues. This observation further suggests that somatic mutations in D-loop may facilitate carcinogenesis and cancer cells with less ∼5 kb deletion, i.e., intact mtDNA, may become resistant to apoptosis.

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, ß-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.

A practical and sensitive method of quantitating lymphangiogenesis in vivo.

To address the inadequacy of current assays, we developed a directed in vivo lymphangiogenesis assay (DIVLA) by modifying an established directed in vivo angiogenesis assay. Silicon tubes (angioreactors) were implanted in the dorsal flanks of nude mice. Tubes contained either growth factor-reduced basement membrane extract (BME)-alone (negative control) or BME-containing vascular endothelial growth factor (VEGF)-D (positive control for lymphangiogenesis) or FGF-2/VEGF-A (positive control for angiogenesis) or a high VEGF-D-expressing breast cancer cell line MDA-MD-468LN (468-LN), or VEGF-D-silenced 468LN. Lymphangiogenesis was detected superficially with Evans Blue dye tracing and measured in the cellular contents of angioreactors by multiple approaches: lymphatic vessel endothelial hyaluronan receptor-1 (Lyve1) protein (immunofluorescence) and mRNA (qPCR) expression and a visual scoring of lymphatic vs blood capillaries with dual Lyve1 (or PROX-11 or Podoplanin)/Cd31 immunostaining in cryosections. Lymphangiogenesis was absent with BME, high with VEGF-D or VEGF-D-producing 468LN cells and low with VEGF-D-silenced 468LN. Angiogenesis was absent with BME, high with FGF-2/VEGF-A, moderate with 468LN or VEGF-D and low with VEGF-D-silenced 468LN. The method was reproduced in a syngeneic murine C3L5 tumor model in C3H/HeJ mice with dual Lyve1/Cd31 immunostaining. Thus, DIVLA presents a practical and sensitive assay of lymphangiogenesis, validated with multiple approaches and markers. It is highly suited to identifying pro- and anti-lymphangiogenic agents, as well as shared or distinct mechanisms regulating lymphangiogenesis vs angiogenesis, and is widely applicable to research in vascular/tumor biology.

Breast cancer cell secretome analysis to decipher miRNA regulating the tumor microenvironment and discover potential biomarkers.

MicroRNA (miRNA/miR) 526 b- and miR655-overexpressed tumor cell-free secretions regulate the breast cancer tumor microenvironment (TME) by promoting tumor-associated angiogenesis, oxidative stress, and hypoxic responses. Additionally, premature miRNA (pri-miR526b and pri-miR655) are established breast cancer blood biomarkers. However, the mechanisms of how these miRNAs regulate the TME has yet to be investigated. Mass spectrometry analysis of miRNA-overexpressed cell lines MCF7-miR526b, MCF7-miR655, and miRNA-low MCF7-Mock cell-free secretomes identified 34 differentially expressed proteins coded by eight genes. In both miRNA-high cell secretomes, four markers are upregulated: YWHAB, SFN, TXNDC12, and MYL6B, and four are downregulated: PEA15, PRDX4, PSMB6, and FN1. All upregulated marker transcripts are significantly high in both total cellular RNA pool and cell-free secretions of miRNA-high cell lines, validated with quantitative RT-PCR. Bioinformatics tools were used to investigate these markers' roles in breast cancer. These markers' top gene ontology functions are related to apoptosis, oxidative stress, membrane transport, and motility supporting oncogenic miR526b- and miR655-induced functions. Gene transcription factor analysis tools were used to show how these miRNAs regulate the expression of each secretory marker. Data extracted from the Human Protein Atlas showed that YWHAB, SFN, and TXNDC12 expression could distinguish early and late-stage breast cancer in various breast cancer subtypes and are associated with poor patient survival. Additionally, immunohistochemistry analysis showed the expression of each marker in breast tumors. A stronger correlation between miRNA clusters and upregulated secretory markers gene expression was found in the luminal A tumor subtype. YWHAB, SFN, and MYL6B are upregulated in breast cancer patient's blood, showing biomarker potential. Of these identified novel miRNA secretory markers, SFN and YWHAB successfully passed all validations and are the best candidates to further investigate their roles in miRNA associated TME regulation. Also, these markers show the potential to serve as blood-based breast cancer biomarkers, especially for luminal-A subtypes.

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.

Differential haplotype amplification leads to misgenotyping of heterozygote as homozygote when using single nucleotide mismatch primer.

Mismatches at the 3'end of /or within a primer are reported to affect the efficiency of PCR and cause allele drop. Here, we report preferential amplification of one haplotype and misgenotyping, when double heterozygotes at NAT1 (rs1057126 and rs15561) were genotyped by sequencing and PCR-RFLP methods using mismatch reverse primers located next to the target SNP. Detailed study revealed highest (100%) and lowest (0%) misgenotyping when the mismatch was at the 3rd and 15th nucleotide positions from 3' end of the primer, respectively. But, the same primers, without any mismatch genotyped heterozygotes correctly. Homozygotes can always be detected correctly irrespective of mismatch position in the primer. Similar results were observed for two SNPs (rs12947788 and rs 12951053) at TP53. Using mismatch NAT1 reverse primers, located three nucleotides away from the target SNP, both TaqMan and sequencing methods showed preferential synthesis of one haplotype strand and misgenotyping in heterozygotes, respectively. So, mismatch primer, located next to target SNP, should be avoided to genotype heterozygotes, since, PCR and sequencing based genotyping methods may lead the investigators to report faulty allelic and genotypic frequencies. This study mimics a situation when an unknown variation is present in the primer-binding sites of both chromosomes.

Association between polymorphisms at N-acetyltransferase 1 (NAT1) & risk of oral leukoplakia & cancer.

BACKGROUND & OBJECTIVES: N-acetyltransferases 1 and 2 (NAT1 and NAT2) are important enzymes for metabolism of tobacco carcinogens. Due to polymorphisms, improper activities of these enzymes might lead to the formation of DNA adducts that may modulate risk of tobacco related oral precancer and cancer. Previously, it was shown that NAT2 polymorphisms did not modulate the risk of oral precancer and cancer. We undertook this study to check whether polymorphisms at NAT1 can modulate the risk of oral leukoplakia and cancer either alone or in combination with NAT2. METHODS: Genotypes at four SNPs on NAT1 were determined by TaqMan method in 389 controls, 224 leukoplakia and 310 cancer patients. Genotype data were analyzed to know haplotypes and acetylation status of individuals and, then to estimate the risk of diseases. Using our previously published NAT2 data, combination of NAT1 and NAT2 acetylation genotypes of patients and controls were also analyzed to estimate the risk of diseases. RESULTS: Analysis of NAT1 genotype data revealed that 1088T and 1095C alleles exist in strong linkage disequilibrium (r 2 =0.97, P<0.0001) and SNPs are in Hardy-Weinberg Equilibrium (P=0.1). Wild type or normal acetylating and variant or rapid acetylating alleles were two major alleles (frequencies 0.62 and 0.36, respectively) present in the control population. NAT1 rapid acetylation could not modulate the risk of leukoplakia and cancer (OR=0.9, 95% CI: 0.6-1.3; OR=1.0, 95% CI: 0.7-1.4, respectively). Analysis of combined NAT1 and NAT2 acetylating data also showed no significant enhancement of the risk of diseases. INTERPRETATION & CONCLUSIONS: NAT1 rapid acetylation alone as well as combination of NAT1 rapid-NAT2 slow acetylation did not modulate the risk of oral precancer and cancer in our patient population. So, NAT1/NAT2 metabolized carcinogen products may not be involved in tobacco related oral precancer and cancer. It may be interpreted that large sample size as well as combination of polymorphisms at other candidate loci may be important to estimate the risk of a complex disease like oral cancer.

Pri-miR526b and Pri-miR655 Are Potential Blood Biomarkers for Breast Cancer.

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.

Prostaglandin E2 Receptor 4 (EP4) as a Therapeutic Target to Impede Breast Cancer-Associated Angiogenesis and Lymphangiogenesis.

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.

Chemically Induced Hypoxia Enhances miRNA Functions in Breast Cancer.

In aggressively growing tumors, hypoxia induces HIF-1α expression promoting angiogenesis. Previously, we have shown that overexpression of oncogenic microRNAs (miRNAs, miRs) miR526b/miR655 in poorly metastatic breast cancer cell lines promotes aggressive cancer phenotypes in vitro and in vivo. Additionally, miR526b/miR655 expression is significantly higher in human breast tumors, and high miR526b/miR655 expression is associated with poor prognosis. However, the roles of miR526b/miR655 in hypoxia are unknown. To test the relationship between miR526b/miR655 and hypoxia, we used various in vitro, in silico, and in situ assays. In normoxia, miRNA-high aggressive breast cancer cell lines show higher HIF-1α expression than miRNA-low poorly metastatic breast cancer cell lines. To test direct involvement of miR526b/miR655 in hypoxia, we analyzed miRNA-high cell lines (MCF7-miR526b, MCF7-miR655, MCF7-COX2, and SKBR3-miR526b) compared to controls (MCF7 and SKBR3). CoCl2-induced hypoxia in breast cancer further promotes HIF-1α mRNA and protein expression while reducing VHL expression (a negative HIF-1α regulator), especially in miRNA-high cell lines. Hypoxia enhances oxidative stress, epithelial to mesenchymal transition, cell migration, and vascular mimicry more prominently in MCF7-miR526b/MCF7-miR655 cell lines compared to MCF7 cells. Hypoxia promotes inflammatory and angiogenesis marker (COX-2, EP4, NFκB1, VEGFA) expression in all miRNA-high cells. Hypoxia upregulates miR526b/miR655 expression in MCF7 cells, thus observed enhancement of hypoxia-induced functions in MCF7 could be attributed to miR526b/miR655 upregulation. In silico bioinformatics analysis shows miR526b/miR655 regulate PTEN (a negative regulator of HIF-1α) and NFκB1 (positive regulator of COX-2 and EP4) expression by downregulation of transcription factors NR2C2, SALL4, and ZNF207. Hypoxia-enhanced functions in miRNA-high cells are inhibited by COX-2 inhibitor (Celecoxib), EP4 antagonist (ONO-AE3-208), and irreversible PI3K/Akt inhibitor (Wortmannin). This establishes that hypoxia enhances miRNA functions following the COX-2/EP4/PI3K/Akt pathways and this pathway can serve as a therapeutic target to abrogate hypoxia and miRNA induced functions in breast cancer. In situ, HIF-1α expression is significantly higher in human breast tumors (n = 96) compared to non-cancerous control tissues (n = 20) and is positively correlated with miR526b/miR655 expression. In stratified tumor samples, HIF-1α expression was significantly higher in ER-positive, PR-positive, and HER2-negative breast tumors. Data extracted from the TCGA database also show a strong correlation between HIF-1α and miRNA-cluster expression in breast tumors. This study, for the first time, establishes the dynamic roles of miR526b/miR655 in hypoxia.

Polymorphisms at p53, p73, and MDM2 loci modulate the risk of tobacco associated leukoplakia and oral cancer.

Polymorphisms at loci controlling cellular processes such as cell cycle, DNA repair, and apoptosis may modulate the risk of cancer. We examined the association of two linked polymorphisms (G4C14-A4T14) at p73 and one polymorphism (309G > T) at MDM2 promoter with the risk of leukoplakia and oral cancer. The p73 and MDM2 genotypes were determined in 197 leukoplakia patients, 310 oral cancer patients and in 348 healthy control subjects. The p73 GC/AT genotype increased the risk of leukoplakia (OR = 1.6, 95% CI = 1.1-2.3) and oral cancer (OR = 2.4, 95% CI = 1.7-3.3) but the 309G > T MDM2 polymorphism independently could not modify the risk of any of the diseases. Stratification of the study population into subgroups with different tobacco habits showed that the risk of the oral cancer is not modified further for the individuals carrying p73 risk genotype. However, leukoplakia patients with smokeless tobacco habit showed increased risk with combined GC/AT and AT/AT (OR = 3.0, 95% CI = 1.3-7.0) genotypes. A combined analysis was done with our previous published data on p53 codon 72 pro/arg polymorphism. Analysis of pair wise genotype combinations revealed increase in risk for specific p73-MDM2 and p73-p53 genotype combinations. Finally, the combined three loci analyses revealed that the presence of at least one risk allele at all three loci increases the risk of both leukoplakia and oral cancer.

Variant haplotypes at XRCC1 and risk of oral leukoplakia in HPV non-infected samples.

BACKGROUND: One of the mechanisms in human papillomavirus (HPV)-related carcinogenesis is inhibition of DNA repair by HPV oncoprotein. In this study, we investigated whether polymorphisms at XRCC1, one of the DNA repair loci, could modulate the risk of tobacco-related leukoplakia and cancer in HPV-infected individuals. METHODS: Tissue DNA from 83 oral cancer, 91 leukoplakia and 100 healthy controls were screened for HPV 16/18 infection and polymorphisms at XRCC1 by PCR-RFLP to estimate the risk of diseases independently and jointly. RESULTS: Human papillomavirus infection was significantly associated with increased risk of leukoplakia and cancer (OR = 2.8, 95% CI = 1.2-6.5 and OR = 5.5, 95% CI = 1.6-19, respectively). Independently, genotypes at three polymorphic sites on XRCC1 did not modulate the risk of diseases but pooled variant haplotypes increased the risk of leukoplakia in overall and HPV non-infected (OR = 1.8, 95% CI = 1.2-2.8; OR = 2.2, 95% CI = 1.2-4.0, respectively) samples but not that of cancer. CONCLUSION: The association between variant haplotypes at XRCC1 and risk of leukoplakia is pronounced in non-infected individuals since HPV oncoprotein could inhibit directly the DNA repair activity of XRCC1. But more samples of leukoplakia and cancer are essential to validate these results.

Mir526b and Mir655 Promote Tumour Associated Angiogenesis and Lymphangiogenesis in Breast Cancer.

MicroRNAs (miRNAs) are small endogenously produced RNAs, which regulate growth and development, and oncogenic miRNA regulate tumor growth and metastasis. Tumour-associated angiogenesis and lymphangiogenesis are processes involving the release of growth factors from tumour cells into the microenvioronemnt to communicate with endothelial cells to induce vascular propagation. Here, we examined the roles of cyclo-oxygenase (COX)-2 induced miR526b and miR655 in tumour-associated angiogenesis and lymphangiogenesis. Ectopic overexpression of miR526b and miR655 in poorly metastatic estrogen receptor (ER) positive MCF7 breast cancer cells resulted in upregulation of angiogenesis and lymphangiogenesis markers vascular endothelial growth factor A (VEGFA); VEGFC; VEGFD; COX-2; lymphatic vessel endothelial hyaluronan receptor-1 (LYVE1); and receptors VEGFR1, VEGFR2, and EP4. Further, miRNA-high cell free conditioned media promoted migration and tube formation by human umbilical vein endothelial cells (HUVECs), and upregulated VEGFR1, VEGFR2, and EP4 expression, showing paracrine stimulation of miRNA in the tumor microenvironment. The miRNA-induced migration and tube formation phenotypes were abrogated with EP4 antagonist or PI3K/Akt inhibitor treatments, confirming the involvement of the EP4 and PI3K/Akt pathway. Tumour supressor gene PTEN was found to be downregulated in miRNA high cells, confirming that it is a target of both miRNAs. PTEN inhibits hypoxia-inducible factor-1 (HIF1α) and the PI3K/Akt pathway, and loss of regulation of these pathways through PTEN results in upregulation of VEGF expression. Moreover, in breast tumors, angiogenesis marker VEGFA and lymphangiogenesis marker VEGFD expression was found to be significantly higher compared with non-adjacent control, and expression of miR526b and miR655 was positively correlated with VEGFA, VEGFC, VEGFD, CD31, and LYVE1 expression in breast tumour samples. These findings further strengthen the role of miRNAs as breast cancer biomarkers and EP4 as a potential therapeutic target to abrogate miRNA-induced angiogenesis and lymphangiogenesis in breast cancer.