Placental alkaline phosphatase, insulin, and adenine nucleotides or adenosine synergistically promote long-term survival of serum-starved mouse embryo and human fetus fibroblasts

Earlier we showed that in serum-starved fibroblasts placental alkaline phosphatase (PALP) can exert growth factor-like effects. Here we report that in mouse embryo (NIH 3T3) and human fetus (HTB-157) fibroblasts, PALP (200 nM) alone provided full protection against serum starvation-induced cell death for 5 days. After 12 days, substantial effects of PALP on cell survival required the copresence of insulin (500 nM) and ATP or adenosine (100 microM). In serum-starved NIH 3T3 cells, PALP induced activating phosphorylation of p42/p44 mitogen-activated protein (MAP) kinases; insulin, but not ATP, had small additional effects. PALP also stimulated the expression of various cyclins; ATP both prolonged and enhanced PALP-induced expression of cyclins A and E. Finally, ATP/adenosine enhanced activation of Akt kinase by insulin. The results suggest that PALP may be a regulator of growth and remodeling of fetal tissues during the second and third trimester of pregnancy when it is expressed.     

DAP kinase activates a p19ARF/p53-mediated apoptotic checkpoint to suppress oncogenic transformation

DAP kinase is a pro-apoptotic calcium-regulated serine/threonine kinase, whose expression is frequently lost in human tumours. Here we show that DAP kinase counteracts oncogene-induced transformation by activating a p19ARF/p53-dependent apoptotic checkpoint. Ectopic expression of DAP kinase suppressed oncogenic transformation of primary embryonic fibroblasts by activating p53 in a p19ARF-dependent manner. Consequently, the fibroblasts underwent apoptosis, characterized by caspase activation and DNA fragmentation. In response to c-Myc or E2F-1, the endogenous DAP kinase protein was upregulated. Furthermore, functional or genetic inactivation of the endogenous DAP kinase reduced the extent of induction of p19ARF/p53 and weakened the subsequent apoptotic responses to c-Myc or E2F-1. These results establish a role for DAP kinase in an early apoptotic checkpoint designed to eliminate pre-malignant cells during cancer development.     

ATR-dependent activation of p38 MAP kinase is responsible for apoptotic cell death in cells depleted of Cdc7

Cdc7 is a serine/threonine kinase that plays essential roles in the initiation of eukaryotic DNA replication and checkpoint response. In previous studies, depletion of Cdc7 by small interfering RNA was shown to induce an abortive S phase that led to the cell cycle arrest in normal human fibroblasts and apoptotic cell death in various cancer cells. Here we report that stress-activated p38 MAP kinase was activated and responsible for apoptotic cell death in Cdc7-depleted HeLa cells. The activation of p38 MAP kinase in the Cdc7-depleted cells was shown to depend on ATR, a major sensor kinase for checkpoint or DNA damage responses. Only the p38 MAP kinase, and not the other stress-activated kinases such as JNK or ERK, was activated, and both caspase 8 and caspase 9 were activated for the induction of apoptosis. Activation of apoptosis in Cdc7-depleted cells was completely abolished in cells treated with small interfering RNA or an inhibitor of the p38 MAP kinase, suggesting that p38 MAP kinase activation was responsible for apoptotic cell death. Taken together, we suggest that the ATR-dependent activation of the p38 MAP kinase is a major signaling pathway that induces apoptotic cell death after depletion of Cdc7 in cancer cells.     

The first proline of PALP motif at the C terminus of presenilins is obligatory for stabilization, complex formation, and gamma-secretase activities of presenilins.

Mutations in presenilin (PS) genes cause early-onset familial Alzheimer's disease by increasing production of the amyloidogenic form of amyloid beta peptides ending at residue 42 (Abeta42). PS is an evolutionarily conserved multipass transmembrane protein, and all known PS proteins contain a proline-alanine-leucine-proline (PALP) motif starting at proline (P) 414 (amino acid numbering based on human PS2) at the C terminus. Furthermore, missense mutations that replace the first proline of PALP with leucine (P414L) lead to a loss-of-function of PS in Drosophila melanogaster and Caenorhabditis elegans. To elucidate the roles of the PALP motif in PS structure and function, we analyzed neuro2a as well as PS1/2 null fibroblast cell lines transfected with human PS harboring mutations at the PALP motif. P414L mutation in PS2 (and its equivalent in PS1) abrogated stabilization, high molecular weight complex formation, and entry to Golgi/trans-Golgi network of PS proteins, resulting in failure of Abeta42 overproduction on familial Alzheimer's disease mutant basis as well as of site-3 cleavage of Notch. These data suggest that the first proline of the PALP motif plays a crucial role in the stabilization and formation of the high molecular weight complex of PS, the latter being the active form with intramembrane proteolytic activities.     

Air pollution-associated fly ash particles induce fibrotic mechanisms in primary fibroblasts

Air pollution is associated with a variety of respiratory and cardiovascular disorders, including fibrosis. To understand the possible molecular mechanisms underlying this observation, we examined the effect of particulate matter on primary fibroblasts, the key regulators of the extracellular matrix. Fly ash collected in an experimental waste incinerator was used as model particles for fine and ultrafine pollution components. Brief treatment of fibroblasts isolated from adult male Wistar rat hearts with fly ash triggered the immediate formation of intracellular reactive oxygen species (ROS). Using phospho-specific antibodies we observed activation of p38 MAP kinase, p44/42 MAP kinase (ERK1/2) and p70(S6) kinase. Prolonged incubation with fly ash increased the expression of collagen 1 and TGF-beta1, but decreased mRNA levels of MMP9 and TNF-alpha. Cell proliferation was inhibited at high concentrations of fly ash. An increase in the level of advanced glycation endproduct (AGE) modification of various cellular proteins after long-term treatment of cultured fibroblasts with fly ash was observed. The results of our study demonstrate that direct activation of fibroblasts by combustion-derived particles is a mechanism that may contribute to the adverse health effects of particulate air pollution.     

Activation of p70 ribosomal protein S6 kinase is an essential step in the DNA damage-dependent signaling pathway responsible for the ultraviolet B-mediated increase in interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3) protein levels in human dermal fibroblasts

Ultraviolet B (UVB) irradiation has been shown to stimulate the expression of matrix-degrading metalloproteinases via generation of DNA damage and/or reactive oxygen species. Matrix-degrading metalloproteinases promote UVB-triggered detrimental long term effects like cancer formation and premature skin aging. Here, we were interested in identifying components of the signal transduction pathway that causally link UVB-mediated DNA damage and induction of matrix-degrading metalloproteinase (MMP)-1/interstitial collagenase and MMP-3/stromelysin-1 in human dermal fibroblasts in vitro. The activity of p70 ribosomal S6 kinase, a downstream target of the FK506-binding protein-12/rapamycin-associated protein kinase (FRAP) kinase (RAFT1, mTOR), was identified to be 4.8 +/- 0.8-fold, and MMP-1 and MMP-3 protein levels 2.4- and 11.5-fold increased upon UVB irradiation compared with mock-irradiated controls. The FRAP kinase inhibitor rapamycin and the DNA repair inhibitor aphidicolin significantly suppressed the UVB-mediated increase in p70 ribosomal S6 kinase activity by 50-65% and MMP-1 and MMP-3 protein levels by 34-68% and 42-88% compared with UVB-irradiated fibroblasts. By contrast, the interleukin-1beta-mediated increase in MMP-1 and MMP-3 protein levels could not be suppressed by rapamycin. Collectively, our data suggest that the FRAP-controlled p70 ribosomal S6 kinase is an essential component of a DNA damage-dependent, but not of the interleukin-1/cell membrane receptor-dependent signaling.     

gadd45 is not required for activation of c-Jun N-terminal kinase or p38 during acute stress.

Cells respond to environmental stress with activation of c-Jun N-terminal kinase (JNK) and p38. Recent studies have implicated Gadd45 and two related proteins, MyD118/Gadd45beta and CR6/Gadd45gamma, as initiators of JNK/p38 signaling via their interaction with an upstream kinase MTK1. It was proposed that stress-induced expression of the Gadd45-related proteins leads to MTK1 activation and subsequent JNK/p38 activation. Using embryo fibroblasts from gadd45-null mice, we have addressed the requirement for Gadd45 in mediating JNK/p38 activation during acute stress. Comparison of JNK/p38 activities in response to methyl methanesulfonate, hydrogen peroxide, UVC irradiation, sorbitol, and anisomycin treatment of gadd45(+/+) and gadd45(-/-) fibroblasts revealed no deficiency in JNK/p38 activation in gadd45(-/-) fibroblasts. In addition, in wild type cells, JNK and p38 activation significantly preceded gadd45 induction with all stresses. Examination of myd118/gadd45beta and cr6/gadd45gamma expression in gadd45(+/+) and gadd45(-/-) fibroblasts revealed similar induction patterns in the two cell types, which, like gadd45 expression, was delayed relative to JNK/p38 activation. We conclude that gadd45 expression is not required for activation of JNK/p38 by environmental stresses, nor are stress-induced increases in myd118/gadd45beta and cr6/gadd45gamma expression necessary for kinase activation in response to such insults.     

Generation of Monoclonal Antibody MS17-57 Targeting Secreted Alkaline Phosphatase Ectopically Expressed on the Surface of Gastrointestinal Cancer Cells

Background

Therapeutic antibody development is one of the fastest growing areas of the pharmaceutical industry. Generating high-quality monoclonal antibodies against a given therapeutic target is crucial for successful drug development. However, due to immune tolerance, making it difficult to generate antibodies using conventional approaches.

Methodology/Findings

Mixed four human gastric cancer (GC) cell lines were used as the immunogen in A/J mice; sixteen highly positive hybridoma colonies were selected via fluorescence-activated cell sorting-high throughput screening (FACS-HTS) using a total of 20,000 colonies in sixty-seven 96-well plates against live cells (mixed human GC cells versus human PBMC controls). MS17-57 and control commercial Alkaline Phosphatase (ALP) mAbs were used to confirm the target antigens (Ags), which were identified as ALPs expressed on the GC cell surface through a combination of western blot, immunoprecipitation and mass spectrometry (MS).MS identified the Ags recognized by MS17-57 to be two variants of a secreted ALP, PALP and IALP (Placental and intestinal ALP). These proteins belong to a hydrolase enzyme family responsible for removing phosphate groups from many types of molecules. Immunofluorescence staining using MS17-57 demonstrated higher staining of gastrointestinal (GI) cancer tissues compared to normal GI tissues (P<0.03), and confirmed binding of MS17-57 to be restricted to a functional epitope expressed on the cancer cell surface. Proliferation assays using the PALP/IALP-expressing GC cell lines demonstrated that MS17-57 inhibited cell growth by 32±8%. Transwell cell migration assays documented that MS17-57 can inhibit PALP/IALP-expressing GI cancer cell migration by 25±5%. MS17-57 mAb inhibited tumor growth in nude mice.

Conclusions

Our findings indicate that PALP and IALP can be ectopically expressed on extracellular matrix of GI cancers, and that MS17-57 directed against PALP/IALP can inhibit GI cancer cells growth and migration in vitro and in vivo. This investigation provides an example of identification of cancer biomarkers representing promising therapeutic targets using mAb generated through a novel HTS technology.     

Demonstration of gene dosage effects for AK3 and GALT in fibroblasts from a fetus with 9p trisomy

After prenatal detection of a de novo unbalanced translocation 46,XX,18p+, fetal fibroblasts were obtained for further studies. Chromosome banding suggested that the fetus might have trisomy of the short arm of chromosome 9. This tentative diagnosis could be confirmed by demonstration of gene dosage effects for galactose-1-phosphate uridyl transferase (GALT, EC 2.7.7.12) and nucleoside triphosphate adenylate kinase (AK3, EC 2.7.4.10) both known to be controlled by genes assigned to 9p. These findings demonstrate for the first time a gene dosage effect for AK3 in a case of 9p trisomy and show again that gene mapping information can be used to define partial aneuploidies in fetal fibroblasts.     

Association of type II cAMP-dependent protein kinase with p34cdc2 protein kinase in human fibroblasts

Previous independent studies suggested that type II cAMP-dependent protein kinase and the p34cdc2 protein kinase cell cycle regulator co-localize at centrosomes. In order to investigate whether there is an association of type II cAMP-dependent protein kinase with p34cdc2 in human fibroblasts, we used three different approaches. First, the regulatory subunits RI and RII were photoaffinity-labeled with 8-N3-[32P]cAMP, and anti-p34cdc2 immunoprecipitates were screened for the presence of either RI or RII regulatory subunits by one- or two-dimensional gel electrophoresis. Second, anti-RII alpha immunoprecipitates were screened for the presence of p34cdc2 by Western blot using three different affinity-purified antibodies recognizing different domains of human p34cdc2. Conversely, anti-p34cdc2 immunoprecipitates (three different antibodies), as well as the material retained on p13suc1-Sepharose Bio-Beads, which binds specifically p34cdc2, were screened for the presence of RII alpha. Finally, we have looked for cAMP-dependent protein kinase activity specifically inhibited by PKI in immunoprecipitates obtained from extracts treated with different anti-p34cdc2 antibodies. All these experiments gave concordant results and demonstrate that at least at G0/G1, human fibroblasts contain a complex of active type II cAMP-dependent protein kinase associated through its RII alpha subunit with p34cdc2.     

Modification of collagen by 3-deoxyglucosone alters wound healing through differential regulation of p38 MAP kinase

Background

Wound healing is a highly dynamic process that requires signaling from the extracellular matrix to the fibroblasts for migration and proliferation, and closure of the wound. This rate of wound closure is impaired in diabetes, which may be due to the increased levels of the precursor for advanced glycation end products, 3-deoxyglucosone (3DG). Previous studies suggest a differential role for p38 mitogen-activated kinase (MAPK) during wound healing; whereby, p38 MAPK acts as a growth kinase during normal wound healing, but acts as a stress kinase during diabetic wound repair. Therefore, we investigated the signaling cross-talk by which p38 MAPK mediates wound healing in fibroblasts cultured on native collagen and 3DG-collagen.

Methodology/Principal Findings

Using human dermal fibroblasts cultured on 3DG-collagen as a model of diabetic wounds, we demonstrated that p38 MAPK can promote either cell growth or cell death, and this was dependent on the activation of AKT and ERK1/2. Wound closure on native collagen was dependent on p38 MAPK phosphorylation of AKT and ERK1/2. Furthermore, proliferation and collagen production in fibroblasts cultured on native collagen was dependent on p38 MAPK regulation of AKT and ERK1/2. In contrast, 3DG-collagen decreased fibroblast migration, proliferation, and collagen expression through ERK1/2 and AKT downregulation via p38 MAPK.

Conclusions/Significance

Taken together, the present study shows that p38 MAPK is a key signaling molecule that plays a significantly opposite role during times of cellular growth and cellular stress, which may account for the differing rates of wound closure seen in diabetic populations.     

Phosphorylation of p16INK4A correlates with Cdk4 association

Progression through the eukaryotic cell cycle is driven by the activity of cyclin-dependent kinases. The cyclin D-dependent kinase Cdk4 promotes progression through the G(1) phase of the cell cycle and is deregulated in many human tumors. The tumor suppressor protein p16(INK4A) (p16) forms a complex with Cdk4 and inhibits kinase activity. Here we report that p16 is phosphorylated, and the phosphorylated form of p16 is preferentially associated with Cdk4 in normal human fibroblasts. We mapped phosphorylation sites on exogenously overexpressed p16 to serines 7, 8, 140, and 152 and found that endogenous p16 associated with Cdk4 is phosphorylated at serine 152. All mapped phosphorylation sites lie outside of the conserved kinase-binding domain of p16 but in regions of the protein affected by mutations in familial and sporadic cancer. Our results suggest a novel regulation of p16 activity.     

cAMP antagonizes p21ras-directed activation of extracellular signal-regulated kinase 2 and phosphorylation of mSos nucleotide exchange factor.

In fibroblasts, stimulation of receptor tyrosine kinases results in the activation of the extracellular signal-regulated kinase 2 (ERK2). The major signalling pathway employed by these receptors involves the activation of p21ras and raf-1 kinase. Here we show that in NIH3T3 and rat-1 fibroblasts, elevation of the intracellular cAMP level results in the inhibition of ERK2 activation induced by PDGF, EGF and insulin treatment. Analysis of various signalling intermediates shows that cAMP interferes at a site downstream of p21ras, but upstream of raf-1 kinase. Inhibition by cAMP depends on both the cAMP concentration and the absolute amount of p21ras molecules bound to GTP, suggesting a mechanism of competitive inhibition. Also TPA-induced, p21ras-independent, activation of raf-1 kinase and ERK2 is inhibited by cAMP. We have used the inhibitory effect of cAMP to investigate whether phosphorylation of mSos, a p21ras nucleotide exchange factor, is dependent on the activity of the raf-1 kinase/ERK2 pathway. We found that phosphorylation of mSos, as monitored by a mobility shift, is delayed with respect to p21ras and ERK2 activation and is inhibited by cAMP in a similar cell type- and concentration-dependent manner as the inactivation of ERK2. These results provide evidence for a model of p21ras-directed signalling towards ERK2 that feeds back on mSos by regulating its phosphorylation status and that can be negatively modulated by protein kinase A and positively modulated by protein kinase C action.     

Unlike p53, p27 failed to exhibit an anti-tumor genetic interaction with Ku80

Ku80 is often referred to as a tumor suppressor since it maintains the genome by repairing DNA double-strand breaks (DSBs) via the nonhomologous end joining (NHEJ) pathway. Even though Ku80 deletion causes hypersensitivity to γ-radiation, DNA damage and chromosomal rearrangements, Ku80-mutant mice exhibit very low cancer levels. We previously hypothesized these low cancer levels were caused by enhanced cell cycle checkpoints that responded to inefficiently repaired DNA damage because Ku80-mutant fibroblasts exhibit premature cellular senescence that was dependent on a p53-mediated DNA damage response. In addition, Ku80 and p53 show a genetic interaction to suppress pro-B cell lymphoma and medulloblastoma. Here we tested for a similar anti-tumor genetic interaction between Ku80 and the cyclin kinase inhibitor, p27^Kip1 (p27) since p27 mutant mice showed elevated levels of pituitary adenoma that were exacerbated by γ-radiation-induced DNA damage (damage repaired by Ku80). We found that deleting both Ku80 and p27 did not exacerbate cancer as compared to either single mutant. In addition, fibroblasts deleted for both exhibited premature cellular senescence similar to Ku80-mutant fibroblasts. Thus, p27 did not exhibit an obvious genetic interaction with Ku80 to suppress tumors. This observation suggests that DNA damage (or DNA damage responses) induced by either γ-radiation or Ku80 deletion are not equivalent since γ-radiation exacerbates oncogenesis in mice deleted for either p53 or p27 while Ku80 deletion exacerbates oncogenesis for only the former genotype.     

Constitutively active mitogen-activated protein kinase kinase 6 (MKK6) or salicylate induces spontaneous 3T3-L1 adipogenesis

Although much has been learned regarding the importance of p38 mitogen-activated protein kinase in inflammatory and stress responses, relatively little is known concerning its role in differentiation processes. Recently, we demonstrated that p38 mitogen-activated protein kinase activity is necessary for the differentiation of 3T3-L1 fibroblasts into adipocytes (Engelman, J. A., Lisanti, M. P., and Scherer, P. E. (1998) J. Biol. Chem. 273, 32111-32120). p38 activity is high during the initial stages of differentiation but decreases drastically as the fibroblasts undergo terminal differentiation into adipocytes. However, it remains unknown whether activation of p38 is sufficient to stimulate adipogenesis and whether the down-regulation of p38 activity in mature adipocytes is critical for maintaining adipocyte homeostasis. In this report, we have directly addressed these questions by analyzing 3T3-L1 cell lines harboring a specific upstream activator of p38 (a constitutively active mitogen-activated protein kinase kinase 6 (MKK6) mutant, MKK6(Glu)) under the control of an inducible promoter. Induction of MKK6(Glu) in 3T3-L1 fibroblasts spurs adipocyte conversion in the absence of the hormonal mixture normally required for efficient differentiation of wild-type cells. However, activation of p38 in adipocytes leads to cell death. Furthermore, treatment of 3T3-L1 fibroblasts with salicylate, a potent stimulator of p38, produces adipocyte-specific changes consistent with those observed with induction of MKK6(Glu). Expression of MKK6(Glu) in NIH-3T3 fibroblasts (cells that do not differentiate into adipocytes under normal conditions) is capable of converting these fibroblasts into lipid-laden fat cells following hormonal stimulation. Thus, p38 activation has pro-adipogenic effects in multiple fibroblast cell lines.     

The Ataxia telangiectasia gene product is required for oxidative stress-induced G1 and G2 checkpoint function in human fibroblasts

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by neuronal degeneration accompanied by ataxia, telangiectasias, acute cancer predisposition, and sensitivity to ionizing radiation (IR). Cells from individuals with AT show unusual sensitivity to IR, severely attenuated cell cycle checkpoint functions, and poor p53 induction in response to IR compared with normal human fibroblasts (NHFs). The gene mutated in AT (ATM) has been cloned, and its product, pATM, has IR-inducible kinase activity. The AT phenotype has been suggested to be a consequence, at least in part, of an inability to respond appropriately to oxidative damage. To test this hypothesis, we examined the ability of NHFs and AT dermal fibroblasts to respond to t-butyl hydroperoxide and IR treatment. AT fibroblasts exhibit, in comparison to NHFs, increased sensitivity to the toxicity of t-butyl hydroperoxide, as measured by colony-forming efficiency assays. Unlike NHFs, AT fibroblasts fail to show G(1) and G(2) phase checkpoint functions or to induce p53 in response to t-butyl hydroperoxide. Treatment of NHFs with t-butyl hydroperoxide activates pATM-associated kinase activity. Our results indicate that pATM is involved in responding to certain aspects of oxidative damage and in signaling this information to downstream effectors of the cell cycle checkpoint functions. Our data further suggest that some of the pathologies seen in AT could arise as a consequence of an inability to respond normally to oxidative damage.     

Loss of p53 promotes RhoA-ROCK-dependent cell migration and invasion in 3D matrices

In addition to its role in controlling cell cycle progression, the tumor suppressor protein p53 can also affect other cellular functions such as cell migration. In this study, we show that p53 deficiency in mouse embryonic fibroblasts cultured in three-dimensional matrices induces a switch from an elongated spindle morphology to a markedly spherical and flexible one associated with highly dynamic membrane blebs. These rounded, motile cells exhibit amoeboid-like movement and have considerably increased invasive properties. The morphological transition requires the RhoA-ROCK (Rho-associated coil-containing protein kinase) pathway and is prevented by RhoE. A similar p53-mediated transition is observed in melanoma A375P cancer cells. Our data suggest that genetic alterations of p53 in tumors are sufficient to promote motility and invasion, thereby contributing to metastasis.     

Matrix survival signaling: from fibronectin via focal adhesion kinase to c-Jun NH(2)-terminal kinase

Most transformed cells have lost anchorage and serum dependence for growth and survival. Previously, we established that when serum is absent, fibronectin survival signals transduced by focal adhesion kinase (FAK), suppress p53-regulated apoptosis in primary fibroblasts and endothelial cells (Ilić et al. 1998. J. Cell Biol. 143:547–560). The present goals are to identify survival sequences in FAK and signaling molecules downstream of FAK required for anchorage-dependent survival of primary fibroblasts. We report that binding of the SH3 domain of p130Cas to proline-rich region 1 of FAK is required to support survival of fibroblasts on fibronectin when serum is withdrawn. The FAK–p130Cas complex activates c-Jun NH2-terminal kinase (JNK) via a Ras/Rac1/Pak1/MAPK kinase 4 (MKK4) pathway. Activated (phospho-) JNK colocalizes with FAK in focal adhesions of fibroblasts cultured on fibronectin, which supports their survival, but not in fibroblasts cultured on collagen, which does not. Cells often survive in the absence of extracellular matrix if serum factors are provided. In that case, we confirm work of others that survival signals are transduced by FAK, phosphatidylinositol 3′-kinase (PI3-kinase), and Akt/protein kinase B (PKB). However, when serum is absent, PI3-kinase and Akt/PKB are not involved in the fibronectin-FAK-JNK survival pathway documented herein. Thus, survival signals from extracellular matrix and serum are transduced by FAK via two distinct pathways.     

Stromal COX-2 signaling activated by deoxycholic acid mediates proliferation and invasiveness of colorectal epithelial cancer cells

COX-2 is a major regulator implicated in colonic cancer. However, how COX-2 signaling affects colonic carcinogenesis at cellular level is not clear. In this article, we investigated whether activation of COX-2 signaling by deoxycholic acid (DCA) in primary human normal and cancer associated fibroblasts play a significant role in regulation of proliferation and invasiveness of colonic epithelial cancer cells. Our results demonstrated while COX-2 signaling can be activated by DCA in both normal and cancer associated fibroblasts, the level of activation of COX-2 signaling is significantly greater in cancer associated fibroblasts than that in normal fibroblasts. In addition, we discovered that the proliferative and invasive potential of colonic epithelial cancer cells were much greater when the cells were co-cultured with cancer associated fibroblasts pre-treated with DCA than with normal fibroblasts pre-treated with DCA. Moreover, COX-2 siRNA attenuated the proliferative and invasive effect of both normal and cancer associate fibroblasts pre-treated with DCA on the colonic cancer cells. Further studies indicated that the activation of COX-2 signaling by DCA is through protein kinase C signaling. We speculate that activation of COX-2 signaling especially in cancer associated fibroblasts promotes progression of colonic cancer.