Intratumoral COX‐2 inhibition enhances GM‐CSF immunotherapy against established mouse GL261 brain tumors

Immunotherapy has shown effectiveness against experimental malignant brain tumors, but the clinical results have been less convincing most likely due to immunosuppression. Prostaglandin E₂ (PGE₂) is the key immunosuppressive product of cyclooxygenase‐2 (COX‐2) and increased levels of PGE₂ and COX‐2 have been shown in several tumor types, including brain tumors. In the current study, we report enhanced cure rate of mice with established mouse GL261 brain tumors when immunized with granulocyte macrophage‐colony stimulating factor (GM‐CSF) secreting tumor cells and simultaneously treated with the selective COX‐2 inhibitors parecoxib systemically (5 mg/kg/day; 69% cure rate) or valdecoxib intratumorally (5.3 µg/kg/day; 63% cure rate). Both combined therapies induced a systemic antitumor response of proliferating CD4⁺ and CD8⁺ T cells, and further analysis revealed T helper 1 (T_h1) cell supremacy. The GL261 tumor cell line produced low levels of PGE₂ in vitro, and co‐staining at the tumor site demonstrated that a large fraction of the COX‐2⁺ cells were derived from CD45⁺ immune cells and more specifically macrophages (F4/80⁺), indicating that tumor‐infiltrating immune cells constitute the primary source of COX‐2 and PGE₂ in this model. We conclude that intratumoral COX‐2 inhibition potentiates GM‐CSF immunotherapy against established brain tumors at substantially lower doses than systemic administration. These findings underscore the central role of targeting COX‐2 during immunotherapy and implicate intratumoral COX‐2 as the primary target.     

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

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

Timosaponin AIII inhibits melanoma cell migration by suppressing COX‐2 and in vivo tumor metastasis

Melanoma is the leading cause of death from skin disease, due in large part to its propensity to metastasize. We examined the effects of timosaponin AIII, a compound isolated from Anemarrhena asphodeloides Bunge, on melanoma cancer cell migration and the molecular mechanisms underlying these effects using B16‐F10 and WM‐115 melanoma cells lines. Overexpression of COX‐2, its metabolite prostaglandin E₂ (PGE₂), and PGE₂ receptors (EP2 and EP4) promoted cell migration in vitro. Exposure to timosaponin AIII resulted in concentration‐dependent inhibition of cell migration, which was associated with reduced levels of COX‐2, PGE₂, and PGE₂ receptors. Transient transfection of COX‐2 siRNA also inhibited cell migration. Exposure to 12‐O‐tetradecanoylphorbal‐13‐acetate enhanced cell migration, whereas timosaponin AIII inhibited 12‐O‐tetradecanoylphorbal‐13‐acetate‐induced cell migration and reduced basal levels of EP2 and EP4. Moreover, timosaponin AIII inhibited activation of nuclear factor‐kappa B (NF‐κB), an upstream regulator of COX‐2 in B16‐F10 cells. Consistent with our in vitro findings, in vivo studies showed that timosaponin AIII treatment significantly reduced the total number of metastatic nodules in the mouse lung and improved histological alterations in B16‐F10‐injected C57BL/6 mice. In addition, C57BL/6 mice treated with timosaponin AIII showed reduced expression of COX‐2 and NF‐κB in the lung. Together, these results indicate that timosaponin AIII has the capacity to inhibit melanoma cell migration, an essential step in the process of metastasis, by inhibiting expression of COX‐2, NF‐κB, PGE_2, and PGE₂ receptors.     

Necrosis in DU145 prostate cancer spheroids induces COX‐2/mPGES‐1‐derived PGE2 to promote tumor growth and to inhibit T cell activation

Cyclooxygenase (COX)‐2‐derived prostaglandin E2 (PGE₂) supports the growth of a spectrum of cancers. The potential benefit of COX‐2‐inhibiting non‐steroidal anti‐inflammatory drugs (NSAIDs) for cancer treatment is however limited by their well‐known cardiovascular side‐effects. Therefore, targeting microsomal PGE synthase 1 (mPGES‐1), the downstream enzyme in the COX‐2‐dependent pathway of PGE₂ production might be attractive, although conflicting data regarding a potential tumor‐supporting function of mPGES‐1 were reported. We determined the impact of mPGES‐1 in human DU145 prostate cancer cell growth. Surprisingly, knockdown of mPGES‐1 did not alter growth of DU145 monolayer cells, but efficiently inhibited the growth of DU145 multicellular tumor spheroids (MCTS). Opposed to MCTS, monolayer cells did not secrete PGE₂ due to a lack of COX‐2 expression, which was induced during spheroid formation. Pharmacological inhibition of COX‐2 and mPGES‐1 supported the crucial role of PGE₂ for growth of MCTS. The functionality of spheroid‐derived PGE₂ was demonstrated by its ability to inhibit cytotoxic T cell activation. When investigating mechanisms of spheroid‐induced COX‐2 induction, we observed that among microenvironmental factors neither glucose deprivation, hypoxia nor tumor cell apoptosis enhanced COX‐2 expression. Interestingly, interfering with apoptosis in spheroids triggered a shift towards necrosis, thus augmenting COX‐2 expression. We went on to demonstrate that necrotic cells induced COX‐2 mRNA expression and PGE₂ secretion from live tumor cells. In conclusion, necrosis‐dependent COX‐2 upregulation in MCTS promoted PGE₂‐dependent tumor growth and inhibited activated cytotoxic T cells. Hence, blocking mPGES‐1 as a therapeutic option may be considered for COX‐2/mPGES‐1‐positive solid cancers.     

Antagonism of the prostaglandin E receptor EP4 inhibits metastasis and enhances NK function

Cyclooxygenase-2 (COX-2) is associated with aggressive breast cancers. The COX-2 product prostaglandin E₂ (PGE₂) acts through four G-protein-coupled receptors designated EP1–4. Malignant and immortalized normal mammary epithelial cell lines express all four EP. The EP4 antagonist AH23848 reduced the ability of tumor cells to colonize the lungs or to spontaneously metastasize from the mammary gland. EP4 gene silencing by shRNA also reduced the ability of mammary tumor cells to metastasize. Metastasis inhibition was lost in mice lacking either functional Natural Killer (NK) cells or interferon-γ. EP4 antagonism inhibited MHC class I expression resulting in enhanced ability of NK cells to lyse mammary tumor target cells. These studies support the hypothesis that EP4 receptor antagonists reduce metastatic potential by facilitating NK-mediated tumor cell killing and that therapeutic targeting of EP4 may be an alternative approach to the use of COX inhibitors to limit metastatic disease.     

Inflammation in gastric cancer: Interplay of the COX‐2/prostaglandin E2 and Toll‐like receptor/MyD88 pathways

Cyclooxygenase‐2 (COX‐2) and its downstream product prostaglandin E₂ (PGE₂) play a key role in generation of the inflammatory microenvironment in tumor tissues. Gastric cancer is closely associated with Helicobacter pylori infection, which stimulates innate immune responses through Toll‐like receptors (TLRs), inducing COX‐2/PGE₂ pathway through nuclear factor‐κB activation. A pathway analysis of human gastric cancer shows that both the COX‐2 pathway and Wnt/β‐catenin signaling are significantly activated in tubular‐type gastric cancer, and basal levels of these pathways are also increased in other types of gastric cancer. Expression of interleukin‐11, chemokine (C‐X‐C motif) ligand 1 (CXCL1), CXCL2, and CXCL5, which play tumor‐promoting roles through a variety of mechanisms, is induced in a COX‐2/PGE₂ pathway‐dependent manner in both human and mouse gastric tumors. Moreover, the COX‐2/PGE₂ pathway plays an important role in the maintenance of stemness with expression of stem cell markers, including CD44, Prom1, and Sox9, which are induced in both gastritis and gastric tumors through a COX‐2/PGE₂‐dependent mechanism. In contrast, disruption of Myd88 results in suppression of the inflammatory microenvironment in gastric tumors even when the COX‐2/PGE₂ pathway is activated, indicating that the interplay of the COX‐2/PGE₂ and TLR/MyD88 pathways is needed for inflammatory response in tumor tissues. Furthermore, TLR2/MyD88 signaling plays a role in maintenance of stemness in normal stem cells as well as gastric tumor cells. Accordingly, these results suggest that targeting the COX‐2/PGE₂ pathway together with TLR/MyD88 signaling, which would suppress the inflammatory microenvironment and maintenance of stemness, could be an effective preventive or therapeutic strategy for gastric cancer.     

Transgenic insulin-like growth factor-1 stimulates activation of COX-2 signaling in mammary glands

Studies show that elevated insulin‐like growth factor‐1 (IGF‐1) levels are associated with an increased risk of breast cancer; however, mechanisms through which IGF‐1 promotes mammary tumorigenesis in vivo have not been fully elucidated. To assess the possible involvement of COX‐2 signaling in the pro‐tumorigenic effects of IGF‐1 in mammary glands, we used the unique BK5.IGF‐1 mouse model in which transgenic (Tg) mice have significantly increased incidence of spontaneous and DMBA‐induced mammary cancer compared to wild type (WT) littermates. Studies revealed that COX‐2 expression was significantly increased in Tg mammary glands and tumors, compared to age‐matched WTs. Consistent with this, PGE₂ levels were also increased in Tg mammary glands. Analysis of expression of the EP receptors that mediate the effects of PGE₂ showed that among the four G‐protein‐coupled receptors, EP3 expression was elevated in Tg glands. Up‐regulation of the COX‐2/PGE₂/EP3 pathway was accompanied by increased expression of VEGF and a striking enhancement of angiogenesis in IGF‐1 Tg mammary glands. Treatment with celecoxib, a selective COX‐2 inhibitor, caused a 45% reduction in mammary PGE₂ levels, attenuated the influx of mast cells and reduced vascularization in Tg glands. These findings indicate that the COX‐2/PGE₂/EP3 signaling pathway is involved in IGF‐1‐stimulated mammary tumorigenesis and that COX‐2‐selective inhibitors may be useful in the prevention or treatment of breast cancer associated with elevated IGF‐1 levels in humans. © 2011 Wiley Periodicals, Inc.     

Epidermal growth factor‐induced COX‐2 regulates metastasis of head and neck squamous cell carcinoma through upregulation of angiopoietin‐like 4

Epidermal growth factor receptor (EGFR) expression and activation are the major causes of metastasis in cancers such as head and neck squamous cell carcinoma (HNSCC). However, the reciprocal effect of EGF‐induced COX‐2 and angiopoietin‐like 4 (ANGPTL4) on HNSCC metastasis remains unclear. In this study, we revealed that the expression of ANGPTL4 is essential for COX‐2‐derived prostaglandin E₂ (PGE₂)‐induced tumor cell metastasis. We showed that EGF‐induced ANGPTL4 expression was dramatically inhibited with the depletion and inactivation of COX‐2 by knockdown of COX‐2 and celecoxib treatment, respectively. Prostaglandin E₂ induced ANGPTL4 expression in a time‐ and dose‐dependent manners in various HNSCC cell lines through the ERK pathway. In addition, the depletion of ANGPTL4 and MMP1 significantly impeded the PGE₂‐induced transendothelial invasion ability of HNSCC cells and the binding of tumor cells to endothelial cells. The induction of molecules involved in the regulation of epithelial‐mesenchymal transition was also dependent on ANGPTL4 expression in PGE₂‐treated cells. The depletion of ANGPTL4 further blocked PGE₂‐primed tumor cell metastatic seeding of lungs. These results indicate that the EGF‐activated PGE₂/ANGPTL4 axis enhanced HNSCC metastasis. The concurrent expression of COX‐2 and ANGPTL4 in HNSCC tumor specimens provides insight into potential therapeutic targets for the treatment of EGFR‐associated HNSCC metastasis.     

CD44+ slow‐cycling tumor cell expansion is triggered by cooperative actions of Wnt and prostaglandin E2 in gastric tumorigenesis

(Cancer Sci 2010; 101: 673–678) Similar to normal tissue stem cells, cancer stem cells (CSCs) are thought to be quiescent or slow‐cycling and, thereby, insensitive to chemo‐ and radiotherapies. CD44, a cell surface component that interacts with the extracellular matrix, has been found to be highly expressed in CSCs of several solid tumors. However, the relevancy between CD44⁺ cells and slow‐cycling cells and the underlying mechanisms for the emergence of CD44⁺ CSCs during tumorigenesis have not been elucidated. Here we show that a gastric gland residing at the squamo‐columnar junction (SCJ) in normal mouse stomach contains CD44⁺ stem cell‐like slow‐cycling cells and that this characteristic CD44⁺ gland was expanded by prostaglandin E2 (PGE₂) and Wnt signaling in K19‐Wnt1/C2mE mouse, a genetic mouse model for gastric tumorigenesis. The analysis of three transgenic mouse lines, K19‐Wnt1, K19‐C2mE and K19‐Wnt1/C2mE, revealed that the expansion of CD44⁺ SCJ cells is triggered by PGE₂‐mediated signaling and is prominently enhanced by the addition of Wnt activation. Furthermore, each expanded CD44⁺ gland in gastric tumor of K19‐Wnt1/C2mE mouse contains a few BrdU label‐retaining quiescent or slow‐cycling cells, suggesting that the CD44⁺ SCJ cells in normal mouse are candidates for the cell‐of‐origin of gastric CSCs. These observations suggest that PGE₂‐mediated inflammatory signaling and Wnt signaling cooperatively trigger the expansion of CD44⁺ slow‐cycling stem‐like cells in SCJ, leading to development of lethal gastric tumors in mice.     

Inhibition of prostaglandin synthesis and actions by genistein in human prostate cancer cells and by soy isoflavones in prostate cancer patients

Soy and its constituent isoflavone genistein inhibit the development and progression of prostate cancer (PCa). Our study in both cultured cells and PCa patients reveals a novel pathway for the actions of genistein, namely the inhibition of the synthesis and biological actions of prostaglandins (PGs), known stimulators of PCa growth. In the cell culture experiments, genistein decreased cyclooxygenase‐2 (COX‐2) mRNA and protein expression in both human PCa cell lines (LNCaP and PC‐3) and primary prostate epithelial cells and increased 15‐hydroxyprostaglandin dehydrogenase (15‐PGDH) mRNA levels in primary prostate cells. As a result genistein significantly reduced the secretion of PGE₂ by these cells. EP4 and FP PG receptor mRNA were also reduced by genistein, providing an additional mechanism for the suppression of PG biological effects. Further, the growth stimulatory effects of both exogenous PGs and endogenous PGs derived from precursor arachidonic acid were attenuated by genistein. We also performed a pilot randomised double blind clinical study in which placebo or soy isoflavone supplements were given to PCa patients in the neo‐adjuvant setting for 2 weeks before prostatectomy. Gene expression changes were measured in the prostatectomy specimens. In PCa patients ingesting isoflavones, we observed significant decreases in prostate COX‐2 mRNA and increases in p21 mRNA. There were significant correlations between COX‐2 mRNA suppression, p21 mRNA stimulation and serum isoflavone levels. We propose that the inhibition of the PG pathway contributes to the beneficial effect of soy isoflavones in PCa chemoprevention and/or treatment. © 2008 Wiley‐Liss, Inc.     

Effect of Eicosapentaenoic Acid on E-type Prostaglandin Synthesis and EP4 Receptor Signaling in Human Colorectal Cancer Cells

The ω-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA), in the free fatty acid (FFA) form, has been demonstrated to reduce adenoma number and size in patients with familial adenomatous polyposis. However, the mechanistic basis of the antineoplastic activity of EPA in the colorectum remains unclear. We tested the hypothesis that EPA-FFA negatively modulates synthesis of and signaling by prostaglandin (PG) E₂ in human colorectal cancer (CRC) cells. EPA-FFA induced apoptosis of cyclooxygenase (COX)-2-positive human HCA-7 CRC cells in vitro. EPA-FFA in cell culture medium was incorporated rapidly into phospholipid membranes of HCA-7 human CRC cells and acted as a substrate for COX-2, leading to reduced synthesis of PGE₂ and generation of PGE₃. Alone, PGE₃ bound and activated the PGE₂ EP4 receptor but with reduced affinity and efficacy compared with its “natural” ligand PGE₂. However, in the presence of PGE₂, PGE₃ acted as an antagonist of EP4 receptor-dependent 3′,5′ cyclic adenosine monophosphate induction in naturally EP4 receptor-positive LoVo human CRC cells and of resistance to apoptosis in HT-29-EP4 human CRC cells overexpressing the EP4 receptor. We conclude that EPA-FFA drives a COX-2-dependent “PGE₂-to-PGE₃ switch” in human CRC cells and that PGE₃ acts as a partial agonist at the PGE₂ EP4 receptor.     

MPGES-1-derived PGE2 suppresses CD80 expression on tumor-associated phagocytes to inhibit anti-tumor immune responses in breast cancer

Prostaglandin E₂ (PGE₂) favors multiple aspects of tumor development and immune evasion. Therefore, microsomal prostaglandin E synthase (mPGES-1/-2), is a potential target for cancer therapy. We explored whether inhibiting mPGES-1 in human and mouse models of breast cancer affects tumor-associated immunity. A new model of breast tumor spheroid killing by human PBMCs was developed. In this model, tumor killing required CD80 expression by tumor-associated phagocytes to trigger cytotoxic T cell activation. Pharmacological mPGES-1 inhibition increased CD80 expression, whereas addition of PGE₂, a prostaglandin E2 receptor 2 (EP2) agonist, or activation of signaling downstream of EP2 reduced CD80 expression. Genetic ablation of mPGES-1 resulted in markedly reduced tumor growth in PyMT mice. Macrophages of mPGES-1^−/− PyMT mice indeed expressed elevated levels of CD80 compared to their wildtype counterparts. CD80 expression in tumor-spheroid infiltrating mPGES-1^−/− macrophages translated into antigen-specific cytotoxic T cell activation. In conclusion, mPGES-1 inhibition elevates CD80 expression by tumor-associated phagocytes to restrict tumor growth. We propose that mPGES-1 inhibition in combination with immune cell activation might be part of a therapeutic strategy to overcome the immunosuppressive tumor microenvironment.     

Synergistic activation by p38MAPK and glucocorticoid signaling mediates induction of M2‐like tumor‐associated macrophages expressing the novel CD20 homolog MS4A8A

Tumor‐associated macrophages (TAMs) represent alternatively activated (M2) macrophages that support tumor growth. Previously, we have described a special LYVE‐1⁺ M2 TAM subset in vitro and in vivo; gene profiling of this TAM subset identified MS4A8A as a novel TAM molecule expressed in vivo by TAM in mammary carcinoma and malignant melanoma. In vitro, Ms4a8a mRNA and MS4A8A protein expression was strongly induced in bone marrow‐derived macrophages (BMDMs) by combining M2 mediators (IL‐4, glucocorticoids) and tumor‐conditioned media (TCM). Admixture of MS4A8A⁺ TCM/IL‐4/GC‐treated BMDM significantly enhanced the tumor growth rate of subcutaneously transplanted TS/A mammary carcinomas. Upon forced overexpression of MS4A8A, Raw 264.7 macrophage‐like cells displayed a special gene signature. Admixture of these MS4A8A⁺ Raw 264.7 cells also significantly enhanced the tumor growth rate of subcutaneously transplanted mammary carcinomas. To identify the signaling pathways involved in synergistic induction of MS4A8A, the major signaling cascades with known functions in TAM were analyzed. Although inhibitors of NF‐κB activation and of the MAPK JNK and ERK did not show relevant effects, the p38α/β MAPK inhibitor SB203580 strongly and highly significantly (p > 0.001) inhibited MS4A8A expression on mRNA and protein level. In addition, MS4A8A expression was restricted in M2 BMDM from mice with defective GC receptor (GR) dimerization indicating that classical GR gene regulation is mandatory for MS4A8A induction. In conclusion, expression of MS4A8A within the complex signal integration during macrophage immune responses may act to fine tune gene regulation. Furthermore, MS4A8A⁺ TAM may serve as a novel cellular target for selective cancer therapy.     

Modulation of CXCR3 ligand secretion by prostaglandin E2 and cyclooxygenase inhibitors in human breast cancer

Introduction

In murine breast cancer models, the two interferon-gamma (IFN-γ) inducible chemokines and CXC-chemokine receptor 3 (CXCR3) receptor ligands, monokine induced by γ-interferon (CXCL9) and interferon-γ-inducible protein-10 (CXCL10) impair tumor growth and metastasis formation through recruitment of natural killer (NK) cells and tumor-suppressive T lymphocytes. In human breast cancer, CXCL9 mRNA overexpression correlates with the number of tumor infiltrating lymphocytes and predicts response to different chemotherapeutic regimens. Raising the intratumoral CXCR3 ligand concentration is therefore a possible way to enhance immune intervention in breast cancer. Little is known, however, about expression levels and regulation of these chemokines in human breast cancer. Since the inhibition of cyclooxygenases (COX) has been shown to reduce tumor growth and incidence of metastases in a lymphocytic and IFN-γ dependent manner, we argued that COX isoenzymes are a pharmacologic target to increase intratumoral CXCR3 ligand concentration in human breast cancer.

Methods

CXCL9 was visualized in breast cancer specimens by immunohistochemistry, expression levels of CXCL9 and cyclooxygenases were determined by ELISA and western blotting, respectively. For regulation studies, Michigan Cancer Foundation-7 (MCF-7) and M.D. Anderson - Metastatic Breast 231 (MDA-MB 231) breast cancer cells were stimulated with IFN-γ with or without prostaglandin E₂ (PGE₂) or COX inhibitors (indomethacin, acetylsalicylic acid (ASA), celecoxib). CXCR3 ligand release from cells was measured by ELISA.

Results

Within the tumor microenvironment, cancer cells are the major source of CXCL9. PGE₂ impairs IFN-γ mediated CXCL9 and CXCL10 release from MCF-7 and MDA-MB 231 cells, and inhibition of endogenous cyclooxygenases by indomethacin or ASA correspondingly increases this secretion. Otherwise, high concentrations of the Cyclooxygenase-2 (COX-2) specific antagonist celecoxib have opposite effects and impair CXCL9 and CXCL10 release. In human breast cancer tissue specimens there is an inverse correlation between COX-2 overexpression and CXCL9 concentration, suggesting that the observed in vitro effects are of importance in vivo as well.

Conclusions

Suppressing endogenous PGE₂ synthesis by cyclooxygenase inhibition increases CXCL9 and CXCL10 release from breast cancer cells and is therefore a pharmacologic candidate to enhance intratumoral immune infiltration. Yet, to this end the unselective COX inhibitors ASA and indomethacin seem preferable to celecoxib that at higher concentrations reduces CXCR3 ligand release most probably due to COX independent mechanisms.     

Cell transforming genes and tumor progression: In vivo unified secondary phenotypic cell changes

We have earlier shown that Syrian hamster cells spontaneously transformed in vitro during in vivo progression, acquire in 1 step, along with highly increased tumorigenicity, 2 new properties characterizing the [H₂O₂^CA + tPGE^S] phenotype, i.e., a high H₂O₂ catabolizing (antioxidant) activity and the ability to release PGE₂ upon contact with NK cells. In contrast, RSV-SR-(v-src)-transformed cells acquire the [H₂O₂^CA + PGE^S] phenotype and high tumorigenicity during in vitro transformation, i.e., without preliminary in vivo selection. In the present study, the possible influence of different transforming genes on the rates of subsequent in vivo tumor progression was studied using cells in vitro transformed by SV40, BAV-3, or transduced by activated genes Ha-ras, p53, myc and bcl-2. The expression of the [H₂O₂^CA + PGE^S] phenotype, the extent of tumorigenic and spontaneous metastasizing activities were examined before and during in vivo cells selection in s.c. growing tumors. Our results demonstrate that: (1) after in vitro transformation all cell lines (except v-src) were negative for the expression of [H₂O₂^CA + PGE^S] phenotype and remained equally low-tumorigenic; (2) independently of the types of genes initially transforming the cells, in vivo tumor progression was consistently leading to the replacement of parental cells by cells expressing the [H₂O₂^CA + PGE^S] phenotype, to 30–200 times increased tumorigenicity and less frequently to metastasizing; (3) the time necessary for selection of cells expressing this phenotype was the same (about 180 days in vivo) for all transformants, except bcl-2; the latter reaching similar values after a significant delay. Thus, common secondary src-like phenotypic cell changes, regardless of initially cell transforming genes are necessarily selected during tumor progression in vivo. Int. J. Cancer 75:277–283, 1998. © 1998 Wiley-Liss, Inc.     

Association of EP2 receptor and SLC19A3 in regulating breast cancer metastasis

Breast cancer is the most common cancer in women worldwide. Triple-negative breast cancer patients have higher metastatic rate than patients with other breast cancer subtypes. Distant metastasis is one of the causes leading to the high mortality rates. Cyclooxygenase-2 (COX2) is associated with breast cancer metastasis and the downstream prostaglandin E₂ (PGE₂) exerted its effect through EP receptors (EP1-EP4). However, the exact molecular events of EP receptors in breast cancer metastasis remain undefined. Expressions of EP receptors were determined during cancer development in NOD-SCID mice inoculated with MB-231 and MB-231-EP2 clone. EP2 overexpressing stable clone was constructed to investigate the proliferation and invasion potentials in vivo and in vitro. Drug transporter array was used to identify EP2 receptor-associated drug transported genes in breast cancer metastasis. Localization of EP2 receptor in primary tissues and xenografts were examined by immunostaining. Stable EP2-expression cells formed larger tumors than parental cells in mice model and was highly expressed in both primary and metastatic tissues. Silencing of EP2 receptor by siRNA and antagonist (AH 6809) significantly decreased cell proliferation and invasion, concomitant with reduced MMP-2 and MMP-9 expressions. Results from array data showed that expression of SLC19A3 was markedly increased in EP2 siRNA transfected cells. Ectopic expression of SLC19A3 retarded cell proliferation, invasion and MMPs expressions. Notably, SLC19A3 had a lower expression in primary tissues and was negatively correlated with EP2 receptor expression. Our novel finding revealed that EP2 receptor regulated metastasis through downregulation of SLC19A3. Thus, targeting EP2-SLC19A3 signaling is a potential therapeutic therapy for treating metastatic breast cancer.     

IgG1 anti‐epidermal growth factor receptor antibodies induce CD8‐dependent antitumor activity

Anti‐EGFR monoclonal antibodies (mAb) like Cetuximab are commonly used for treatment of EGFR⁺ solid tumors mainly by exerting their therapeutic effect through inhibition of signal transduction. Additionally, IgG1 is a potent mediator of antibody‐dependent cytotoxicity (ADCC). In case of the IgG1, Cetuximab induction of ADCC in vivo is controversially discussed. In our study, we investigated the efficiency of Cetuximab‐mediated ADCC in a humanized mouse tumor model in vivo and analyzed the contribution of immunologic processes toward antitumor activity. Therefore, we used immunodeficient NOD/Scid mice transgenic for human MHC class I molecule HLA‐A2 and adoptively transferred human HLA‐A2⁺ PBMC after engraftment of human epidermoid cell carcinoma A431. Here, we show that high doses of anti‐EGFR mAb induced strong tumor regression independent of the immune system. However, tumor regression by low doses of anti‐EGFR mAb treatment was ADCC dependent and mediated by tumor infiltrating CD8⁺ T effector cells. This novel mechanism of ADCC conducted by CD8⁺ T effector cells was restricted to IgG1 anti‐EGFR mAb, dependent of binding to CD16 on T cells and could be inhibited after EGFR blockade on tumor cells. Furthermore, CD8⁺ T effector cell‐mediated ADCC was enhanced in the presence of IL‐15 and strongly improved after glycosylation of anti‐EGFR mAb indicating the potential of glycoengineered therapeutic mAb as efficient biologicals in cancer therapy.