Ioannis Stasinopoulos

Silencing of Cyclooxygenase-2 Inhibits Metastasis and Delays Tumor Onset of Poorly Differentiated Metastatic Breast Cancer Cells

Cyclooxygenases (COX) are rate-limiting enzymes involved in the conversion of PLA2-mobilized arachidonic acid into prostaglandins and thromboxanes. COX-2 is a key mediator of inflammation during both physiologic and pathologic responses to endogenous stimuli and infectious agents. Its overexpression has been detected in different cancers, including that of the breast. Using RNA interference, we have reduced the expression of COX-2 in the highly malignant breast cancer cell line MDA-MB-231 below detectable levels in response to interleukin-1β or 12-O-tetradecanoylphorbol-13-acetate treatment. Microarray analysis showed that COX-2 silencing resulted in the loss of mRNA expression of several oncogenic markers, such as matrix metalloproteinase-1, chemokine (C-X-C motif) receptor 4, and interleukin-11, which have been correlated with poor disease outcome, and in the up-regulation of antimetastatic transcripts, such as thrombospondin-1 and Epstein-Barr-Induced 3. Cells lacking COX-2 were less able to invade reconstituted extracellular matrix than parental cells in vitro. Consistent with these changes, loss of COX-2 resulted in the abolition or the significant delay of tumor onset when the cells were injected in the mammary fat pad of severe combined immunodeficient mice. Finally, silencing of COX-2 resulted in the inhibition of metastasis to the lungs of severe combined immunodeficient mice after intravenous injection. These data show that silencing of COX-2 abolishes the metastatic potential of MDA-MB-231 cells in vivo. (Mol Cancer Res 2007;5(5):435–42)

Inflammation, but not hypoxia, mediated HIF-1α activation depends on COX-2

The COX pathway has been a target for pharmaceutical intervention in diseases with a high inflammatory component ranging from asthma and Alzheimers to arthritis and cancer. A major transcriptional promoter of the malignant phenotype, HIF-1α, has been observed to be regulated by the COX-2 product PGE2. Here we show that HIF-1α protein significantly accumulated in human breast cancer MDA-MB-231 cells in response to the pro-inflammatory cytokine IL-1β, but not in COX-2-silenced MDA-MB-231 cells. In contrast HIF-1α expression could be detected in COX-2-silenced cells in response to the hypoxia-mimetic agent CoCl2 and hypoxia. Gene expression profiling in COX-2-containing and COX-2-silenced cells showed that the hypoxia-induced transcriptional response is largely unaffected by COX-2 silencing. These data suggest that the profound effects of COX-2 silencing on inhibiting invasion, tumor growth, and metastasis from MDA-MB-231 cells are dependent on the induction of IL-1β-dependent COX-2 and HIF-1α but are independent of hypoxia.

COX-2 in cancer: Gordian knot or Achilles heel?

The networks of blood and lymphatic vessels and of the extracellular matrix and their cellular and structural components, that are collectively termed the tumor microenvironment, are frequently co-opted and shaped by cancer cells to survive, invade, and form distant metastasis. With an enviable capacity to adapt to continually changing environments, cancer represents the epitome of functional chaos, a stark contrast to the hierarchical and organized differentiation processes that dictate the development and life of biological organisms. The consequences of changing landscapes such as hypoxia and acidic extracellular pH in and around tumors create a cascade of changes in multiple pathways and networks that become apparent only several years later as recurrence and metastasis. These molecular and phenotypic changes, several of which are mediated by COX-2, approach the complexities of a “Gordian Knot.” We review evidence from our studies and from literature suggesting that cyclooxygenase-2 (COX-2) biology presents a nodal point in cancer biology and an “Achilles heel” of COX-2-dependent tumors.

Exploiting the tumor microenvironment for theranostic imaging

The integration of chemistry and molecular biology with imaging is providing some of the most exciting opportunities in the treatment of cancer. The field of theranostic imaging, where diagnosis is combined with therapy, is particularly suitable for a disease as complex as cancer, especially now that genomic and proteomic profiling can provide an extensive ‘fingerprint’ of each tumor. Using this information, theranostic agents can be shaped for personalized treatment to target specific compartments, such as the tumor microenvironment (TME), whilst minimizing damage to normal tissue. These theranostic agents can also be used to target multiple pathways or networks by incorporating multiple small interfering RNAs (siRNAs) within a single agent. A decade ago genetic alterations were the primary focus in cancer research. Now it is apparent that the tumor physiological microenvironment, interactions between cancer cells and stromal cells, such as endothelial cells, fibroblasts and macrophages, the extracellular matrix (ECM), and a host of secreted factors and cytokines, influence progression to metastatic disease, aggressiveness and the response of the disease to treatment. In this review, we outline some of the characteristics of the TME, describe the theranostic agents currently available to target the TME and discuss the unique opportunities the TME provides for the design of novel theranostic agents for cancer therapy. Copyright

Noninvasive imaging identifies new roles for cyclooxygenase-2 in choline and lipid metabolism of human breast cancer cells.

The expression of cyclooxygenase‐2 (COX‐2) is observed in approximately 40% of breast cancers. A major product of the COX‐2‐catalyzed reaction, prostaglandin E2, is an inflammatory mediator that participates in several biological processes, and influences invasion, vascularization and metastasis. Using noninvasive MRI and MRS, we determined the effect of COX‐2 downregulation on the metabolism and invasion of intact poorly differentiated MDA‐MB‐231 human breast cancer cells stably expressing COX‐2 short hairpin RNA. Dynamic tracking of invasion, extracellular matrix degradation and metabolism was performed with an MRI‐ and MRS‐compatible cell perfusion assay under controlled conditions of pH, temperature and oxygenation over the course of 48 h. COX‐2‐silenced cells exhibited a significant decrease in invasion relative to parental cells that was consistent with the reduced expression of invasion‐associated matrix metalloproteinase genes and an increased level of the tissue inhibitor of metalloproteinase‐1. We identified, for the first time, a role for COX‐2 in mediating changes in choline phospholipid metabolism, and established that choline kinase expression is partly dependent on COX‐2 function. COX‐2 silencing resulted in a significant decrease in phosphocholine and total choline that was detected by MRS. In addition, a significant increase in lipids, as well as lipid droplet formation, was observed. COX‐2 silencing transformed parental cell metabolite patterns to those characteristic of less aggressive cancer cells. These new functional roles of COX‐2 may identify new biomarkers and new targets for use in combination with COX‐2 targeting to prevent invasion and metastasis. Copyright

ADAM17: the new face of breast cancer-promoting metalloprotease activity.

Commentary to: ADAM17 promotes breast cancer cell malignant phenotype through EGFR-PI3K-AKT activation Xuguang Zheng, Feng Jiang, Mark Katakowski, Zheng Gang Zhang, Qing-e Lu, Michael Chopp

COX-2 dependent regulation of mechanotransduction in human breast cancer cells

The ability of living cells to exert physical forces upon their surrounding is a necessary prerequisite for diverse biological processes, such as local cellular migrations in wound healing to metastatic-invasion of cancer. How forces are coopted in metastasis has remained unclear, however, because the mechanical interplay between cancer cells and the various stromal components has not been experimentally accessible. Current dogma implicates inflammation in these mechanical processes. Using Fourier transform traction microscopy, we measured the force-generating capacity of human breast cancer cells occupying a spectrum of invasiveness as well as basal and inducible COX-2 expression (MCF-7<SUM-149<MDA-MB-231). Compared with non-invasive MCF-7 and moderately-invasive SUM-149, poorly-differentiated MDA-MB-231 cells showed increased cellular dispersion on collagen matrix that was accompanied by emergent distribution of contractile stresses at the interface between the adherent cell and its substrate, defined herein as the traction field. In metastatic MDA-MB-231 cells, the local tractions were precisely tuned to the surrounding matrix rigidity in a physiologic range with the concomitant expression of mechanosensitive integrin β1. These discrete responses at the single-cell resolution were correlated with PGE2 secretion and were ablated by shRNA-mediated knockdown of COX-2. Both COX-2-silenced and COX-2-expressing cells expressed EP2 and EP4 receptors, but not EP1 and EP3. Exogenous addition of PGE2 increased cell tractions and stiffened the underlying cytoskeletal network. To our knowledge this is the first report linking the expression of COX-2 with mechanotransduction of human breast cancer cells, and the regulation of COX-2-PGE2-EP signaling with physical properties of the tumor microenvironment. Drug treatments aimed at reducing this mechanical interplay may have therapeutic potential in the treatment of breast cancer.

Molecular and functional imaging of invasion and metastasis: Windows into the metastatic cascade

The ability of cancer cells to invade, metastasize, and form distant colonies, is one of the key characteristics that confers lethality to cancer. Metastatic cancer cells typically become refractory to treatment. The metastatic cascade is a multi-step process that is governed by events within the cancer cell, the tumor microenvironment, and the distant environments that are invaded and colonized by the cancer cells. Noninvasive imaging techniques are facilitating a close examination of the stepwise journey of the cancer cell from the primary tumor to the distant metastatic site. Here we have discussed the metastatic process, and how molecular and functional imaging of cancer are providing new insights into the metastatic cascade that can be exploited for treatment of metastatic disease.

Prostaglandin E2 promotes the nuclear accumulation of lymphoid enhancer factor-1 in poorly differentiated breast cancer cells ☆

Products of the COX reaction are frequently elevated in solid tumors and their roles in the malignant phenotype have been extensively investigated. We have shown that COX-2 is essential for the growth of MDA-MB-231 cells in the fat pad of SCID mice and for their extrapulmonary colonization following injection in the tail vein of SCID mice. The molecular changes that follow shRNA-mediated silencing of COX-2 include a significant downregulation of LEF-1, a transcription factor normally activated during development following the Wnt-induced nuclear translocation of β-catenin. We also report that COX-2-silenced cells have reduced nuclear accumulation of LEF-1 protein and that the COX-2 product PGE(2) partially restored nuclear LEF-1 expression in COX-2-silenced cells. Further, we demonstrate that, like parental COX-2 containing MDA-MB-231 cells, COX-2-silenced cells maintain nuclear localization of β-catenin.

Abstract 5162: COX-2 downregulation protects SUM-149 cells following targeting of choline phospholipid metabolism

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Enzymes in the choline phospholipid metabolism pathway such as choline kinase (Chk) and phopholipase D1 (PLD1) are being evaluated as novel targets in cancer therapy since these enzymes are overexpressed in aggressive cancers including breast cancer. We have previously shown that single and double downregulation of Chk and PLD1 significantly reduced cell proliferation in breast cancer cells. We also observed a close association between Chk and cyclooxygenase 2 (COX-2), an inducible enzyme that mediates the inflammatory response and plays a role in cancer invasion, angiogenesis and metastasis. COX-2 inhibition is also being considered as a strategy for cancer prevention or treatment. Here we investigated the effect of the COX-2 downregulation combined with PLD1 and Chk downregulation using siRNA, on cell proliferation to achieve improved control and minimize compensatory responses that allow the cell to survive or adapt. The triple negative inflammatory breast cancer cell line, SUM149, was used in this study. Cells were transfected with 25 nM siRNA against COX-2 (siRNA-cox2), PLD1 (siRNA-pld1) or Chk (siRNA-chk) either singly or combined for 48 h. Untreated cells and the transfection reagent (DharmaFECT) treatment were used as negative controls. Immunoblot analysis was done after 48 h transfection using antibodies specific to COX-2, PLD1, Chk-α, and GAPDH (as a loading control). Proliferation/viability of cells was determined using the CCK-8 assay (cell counting kit-8, Dojindo) at day 2 and day 5 after transfection. After 48 h transfection with siRNA-pld1 or siRNA-chk, PLD1 or Chk-α protein were downregulated significantly and COX-2 protein was upregulated. When we combined downregulation of Chk or PLD1 with COX-2 dowregulation, we established significantly lower COX-2 protein levels compared to single knockdown of Chk and PLD1. COX-2 downregulation alone did not significantly reduce proliferation. Downregulation of PLD1 or Chk, however, significantly reduced proliferation compared to untreated cells (PLD1: –75%, Chk: –68%). When we combined COX-2 downregulation with PLD1 or Chk downregulation, cell survival was significantly higher than with just PLD1 or Chk alone (PLD1: –46%. Chk: –28%). These results suggest that COX-2 downregulation rescues cells following downregulation of PLD1 or Chk in SUM149 cells, identifying a different facet of COX-2 in the choline phospholipid pathway. This work supported by R01 CA82337 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5162. doi:1538-7445.AM2012-5162