Interactions between FGFR2 and RSK2-implications for breast cancer prognosis.

We have previously demonstrated that fibroblast growth factor receptor 2 (FGFR2) activates ribosomal s6 kinase 2 (RSK2) in mammary epithelial cells and that this pathway promotes in vitro cell growth and migration. Potential clinical significance of FGFR2 and RSK2 association has never been investigated. Herein, we have undertaken an evaluation of a possible relationship between FGFR2/RSK2 interdependence and disease outcome in breast cancer (BCa) patients. The clinical analysis was complemented by an in vitro investigation of an involvement of RSK2 in the regulation of FGFR2 function. Primary tumour samples from 152 stage I-III BCa patients were examined for FGFR2 and RSK2 gene and protein expression. FGFR2 showed a positive correlation with RSK2 at both protein (p = 0.003) and messenger RNA (mRNA) (p = 0.001) levels. Lack of both FGFR2 and activated RSK (RSK-P) significantly correlated with better disease-free survival (DFS) (p = 0.01). Patients with tumours displaying immunoreactivity for either or both FGFR2 and RSK-P had 4.89-fold higher risk of recurrence when compared to the FGFR2/RSK-P-negative subgroup. FGFR2-RSK2 interactions were verified by co-immunoprecipitation and internalization assays in HB2 mammary epithelial cell line (characterized by high endogenous FGFR2 and RSK2 expression). In vitro analyses revealed that FGFR2 and RSK2 formed an indirect complex and that activated RSK exerted a significant impact on fibroblast growth factor 2 (FGF2)-triggered internalization of FGFR2. Our results suggest that the FGFR2-RSK2 signalling pathway is involved in pathophysiology of BCa and evaluation of FGFR2/RSK-P expression may be useful in disease prognostication.

Phosphorylation of RSK2 at Tyr529 by FGFR2-p38 enhances human mammary epithelial cells migration.

The members of p90 ribosomal S6 kinase (RSK) family of Ser/Thr kinases are downstream effectors of MAPK/ERK pathway that regulate diverse cellular processes including cell growth, proliferation and survival. In carcinogenesis, RSKs are thought to modulate cell motility, invasion and metastasis. Herein, we have studied an involvement of RSKs in FGF2/FGFR2-driven behaviours of mammary epithelial and breast cancer cells. We found that both silencing and inhibiting of FGFR2 attenuated phosphorylation of RSKs, whereas FGFR2 overexpression and/or its stimulation with FGF2 enhanced RSKs activity. Moreover, treatment with ERK, Src and p38 inhibitors revealed that p38 kinase acts as an upstream RSK2 regulator. We demonstrate for the first time that in FGF2/FGFR2 signalling, p38 but not MEK/ERK, indirectly activated RSK2 at Tyr529, which facilitated phosphorylation of its other residues (Thr359/Ser363, Thr573 and Ser380). In contrast to FGF2-triggered signalling, inhibition of p38 in the EGF pathway affected only RSK2-Tyr529, without any impact on the remaining RSK phosphorylation sites. p38-mediated phosphorylation of RSK2-Tyr529 was crucial for the transactivation of residues located at kinase C-terminal domain and linker-region, specifically, in the FGF2/FGFR2 signalling pathway. Furthermore, we show that FGF2 promoted anchorage-independent cell proliferation, formation of focal adhesions and cell migration, which was effectively abolished by treatment with RSKs inhibitor (FMK). These indicate that RSK2 activity is indispensable for FGF2/FGFR2-mediated cellular effects. Our findings identified a new FGF2/FGFR2-p38-RSK2 pathway, which may play a significant role in the pathogenesis and progression of breast cancer and, hence, may present a novel therapeutic target in the treatment of FGFR2-expressing tumours.

[Aspects of progesterone receptor (PR) activity regulation - impact on breast cancer progression]. / Aspekty regulacji aktywnosci receptora progesteronu (PR) - znaczenie w progresji raka gruczolu piersiowego.

Progesterone receptor (PR) and its specific ligand play a key role in development and physiology of mammary gland. The role of PR in initiation and progression of breast carcinoma (BCa) is unquestionable, although molecular mechanism of PR action is complex and not fully understood. It is known that increased risk of breast cancer is associated with progestin-based (synthetic ligands of progesterone) hormonal contraception or hormone replacement therapies. It is estimated that ER/PR-positive tumours represent approximately 50-70% of all BCa cases, and the loss of PR is associated with resistance to hormonal therapy and increased tumour invasiveness. In classical, genomic signalling pathway cytoplasmic PR, following ligand binding, translocates to the nucleus and regulates expression of genes with the PRE sequence. PR is also involved in a large number of alternative, non-genomic signalling cascades, e.g. PR is able to activate MAPK and PI3K/AKT pathways, which leads to regulation of gene expression. The cross-talk between PR and Growth Factors Receptors (GFR) results in progesterone-independent activation of PR as well as PR-regulated GFR expression and activation. Growth factors signalling promotes formation of a pool of hypersensitive PR responsive to even very low ligand concentration. Transcriptional activity of PR as well as its dynamic impact on processes such as cell migration and adhesion are crucial for BCa progression. Further studies of multifaceted mechanisms of PR action may contribute to new PR-targeting therapeutic strategies for breast cancer patients.

Mechanism of cytotoxic action of perfluorinated acids. III. Disturbance in Ca²+ homeostasis.

The global distribution of perfluorinated acids (PFAs) in industry and in household is well known. Their increasing environmental occurrence and biomagnification in the living organisms have drawn growing interests in efforts to describe precisely the mechanisms of action in vitro and in vivo. Our previous investigations widely described lipophilicity-dependent cytotoxicity of PFAs as well as the effect of perfluorination of carbon chain on depolarization of plasma membrane potential, acidification or mitochondrial dysfunctions. In this study we presented in dose- and time-dependent manner the impact of PFAs on calcium homeostasis in HCT116 cells. Comparative analysis of cytosolic [Ca²+](c) and mitochondrial calcium [Ca²+](m) carried out by flow cytometry revealed distinct uptake of calcium into mitochondria in correlation to increasing lipophilicity of PFAs. Massive accumulation of [Ca²+](m) was not accompanied by equivalent loss of [Ca²+](c). Indeed, moderate changes of [Ca²+](c) were observed after incubation with 400 µM PFDoDA reaching 29.83% and 49.17% decrease at 4th and 72nd hour, respectively. At the same time, mitochondrial calcium uptake increased from 2- to more than 4-fold comparing with non-treated cells. Incubation with non-fluorinated decanoic acid (DA) did not cause any changes in calcium homeostasis. Presented data show that PFAs-induced perturbations in calcium distribution seem to be a missing link related to mitochondria dysfunction playing a crucial role in determination of apoptotic cell death. Complete scheme for the mechanism of cytotoxic action of PFAs has been included.

MET-Pyk2 Axis Mediates Acquired Resistance to FGFR Inhibition in Cancer Cells.

Deregulation of fibroblast growth factor receptors (FGFRs) signaling, as a result of FGFR amplification, chromosomal translocation, or mutations, is involved in both initiation and progression of a wide range of human cancers. Clinical data demonstrating the dependence of cancer cells on FGFRs signaling clearly indicate these receptors as the molecular targets of anti-cancer therapies. Despite the increasing number of tyrosine kinase inhibitors (TKIs) being investigated in clinical trials, acquired resistance to these drugs poses a serious therapeutic problem. In this study, we focused on a novel pan-FGFR inhibitor-CPL304110, currently being investigated in phase I clinical trials in adults with advanced solid malignancies. We analyzed the sensitivity of 17 cell lines derived from cancers with aberrant FGFR signaling, i.e. non-small cell lung cancer, gastric and bladder cancer to CPL304110. In order to explore the mechanism of acquired resistance to this FGFR inhibitor, we developed from sensitive cell lines their variants resistant to CPL304110. Herein, for the first time we revealed that the process of acquired resistance to the novel FGFR inhibitor was associated with increased expression of MET in lung, gastric, and bladder cancer cells. Overexpression of MET in NCI-H1703, SNU-16, RT-112 cells as well as treatment with HGF resulted in the impaired response to inhibition of FGFR activity. Moreover, we demonstrated that cells with acquired resistance to FGFR inhibitor as well as cells overexpressing MET displayed enhanced migratory abilities what was accompanied with increased levels of Pyk2 expression. Importantly, inhibition of both MET and Pyk2 activity restored sensitivity to FGFR inhibition in these cells. Our results demonstrate that the HGF/MET-Pyk2 signaling axis confers resistance to the novel FGFR inhibitor, and this mechanism is common for lung, gastric, and bladder cancer cells. Our study suggests that targeting of MET/Pyk2 could be an approach to overcome resistance to FGFR inhibition.

Tenascin C interacts with ecto-5'-nucleotidase (eN) and regulates adenosine generation in cancer cells.

Tenascin C is expressed in invasive human solid tumors; however its specific role in cancer biology remains obscure. Previously, we have found that ecto-5'-nucleotidase (eN) is a marker of ER (-) breast carcinoma and elevated expression correlates with invasive mesenchymal cell phenotype. To investigate for the potential relationship between eN and protein components of the extracellular matrix (ECM) we measured adenosine generation from AMP in cells incubated with soluble ECM proteins. We found that tenascin C was the only ECM component that strongly inhibited ecto-5'-nucleotidase (eN) activity in situ and adenosine generation from AMP (75% inhibition, p < 0.01). The inhibition was comparable to that induced by concanavalin A, a well-defined and strong inhibitor of eN. Resin immobilized tenascin C, but not collagen, and only weakly fibronectin, specifically and quantitatively bound cell-extracted eN. We further developed breast cancer cell line with reduced eN expression and tested changes in cell adhesion on different ECM. Breast cancer cells expressing reduced eN attached 56% weaker (p < 0.05) to immobilized tenascin C. This difference was not detected with other ECM proteins. Finally, control breast cancer cells migrated slower on tenascin C when compared with clone with reduced eN expression. These data suggest that eN is a novel and specific receptor for tenascin C and that the interaction between these proteins may influence cell adhesion and migration and also lead to decreased generation of local adenosine.

Mechanism of cytotoxic action of perfluorinated acids. I. alteration in plasma membrane potential and intracellular pH level.

Perfluorinated (aliphatic) acids (PFAs) and congeners have many applications in various industrial fields and household for decades. Years later they have been detected in wildlife and this has spurred interest in environmental occurrence as well as influencing living organisms. PFAs were established as peroxisome proliferators and hepatocarcinogens. Amphipatic structure suggests that they may alter cell membrane potential (mbDeltaPsi) and/or induce changes in cytosolic pH (pHi). The aim of this study was to examine the correlation between changes of above parameters and PFAs structure (CF(6)-CF(12)) in human colon carcinoma HCT116 cells. mbDeltaPsi and pHi were measured by flow cytometry using fluorescence polarization of the plasma membrane probe 3,3'-dipentyloxacarbocyanine (DiOC(5)(3)) and fluorescein diacetate (FDA), respectively. Dose- and time-dependent manner analysis revealed relatively fast depolarization of plasma membrane and acidification of cytosol both positively correlated with fluorocarbon chain length. mbDeltaPsi depletion after 4 h of incubation reached 8.01% and 30.08% for 50 muM PFOA and 50 muM PFDoDA, respectively. Prolonged treatment (72 h) led to dramatic dissipation of membrane potential up to 21.65% and 51.29% and strong acidification to pHi level at 6.92 and 6.03 at the presence of above compounds, respectively. The data demonstrate that PFAs can alter plasma membrane protonotrophy with the mode dependent on the compound hydrophobicity.

Mechanism of cytotoxic action of perfluorinated acids II. Disruption of mitochondrial bioenergetics.

PFAs and derivatives due to perfect technological properties are broadly applied in industry and consumer goods, and in consequence widely disseminated, environmentally bioaccumulative and found at ppb level in human serum. Earlier we revealed that in vitro cytotoxicity increases with chain length (CF(6)-CF(14)). The compounds dissipate plasma membrane potential and acidify of cytosol. Here we determine whether there is an association between the protonophoric uncoupling of respiration and disruption of bioenergetics caused by CF(6)-CF(12) on HCT116 cell apoptosis. Again the effects were stronger for longer molecules. Incubation of cells with CF(10) stimulated time-dependent generation of reactive oxygen species, opening of mitochondrial permeability transition (MPT) pore, release of cytochrome c, activation of caspases and depletion of intracellular level of ATP occurring in intrinsic pathway of apoptosis. Incubation with decanoic acid (DA) did not lead to mitochondrial dysfunctions neither to cell cycle disturbances. Synchronized removal of the phosphorylated state of Akt, ERK1/2 and PKCdelta/theta kinases by CF(10) suggests presence of concerted action to uninhibit Bad protein activation and a cascade of intrinsic pathway of apoptosis. Blocking MPT pore by cyclosporin A (CsA) led to a reduction of mitochondrial potential dissipation (mtDeltaPsi). Such cells neither showed cytochrome c release nor the downstream activation of caspase-9 and caspase-3. Our results confirm that mitochondria play a crucial role in perfluorochemicals induced apoptosis by releasing apoptotic signals through MPT pore.

Induction of the multixenobiotic/multidrug resistance system in various cell lines in response to perfluorinated carboxylic acids.

The multixenobiotic resistance (closely related to multidrug resistance) system controls transport across the plasma membrane as a defense against toxic molecules. Multixenobiotic resistance system consists of an efflux pump, ABCB1 (also named P-glycoprotein, P-gp), and/or a molecule of the ABCC family (also named multiple resistance associated protein, MRP). ABCB1 is able to increase efflux of many low-molecular foreign molecules. Measuring system induction may be used as a biomarker of cell/organism exposure to foreign substances. Various established cell lines were tested for constitutive and induced multixenobiotic resistance proteins by Western blotting immunodetection. The pumping function was indirectly assayed with Rhodamine B by visualization of cell fluorescence in the presence of verapamil. Changes in ABC proteins were measured by flow cytometry after exposition to various perfluorinated carboxylic acids. MCF7 and HeLa cells were found to contain the highest constitutive level of both ABCB1 and ABCC1. HEK293 exhibited much less ABCB1 and no activity of pumping out Rhodamine B. The pumping activity was found to be related to the amount of the cell-type specific 170 kDa ABCB1 protein. An 8-day exposure to 10(-4) M perfluorononanoic acid resulted in about 2-2.5-fold increase of ABCB1 level. That was confirmed also for short times by flow cytometry of cells exposed to perfluorinated acids and its natural congeners. Both ABCB1- and ABCC1-related fluorescence increased along with the carbon chain in acids from C(6) up to C(9) and decreased for C(10). Measuring of multixenobiotic resistance changes in vitro induced by chemicals may be a convenient test for screening for their potential toxicity.

Progesterone impairs Herceptin effect on breast cancer cells.

Breast cancer (BCa) is the most common cancer affecting women worldwide. Overexpression of human epidermal growth factor receptor 2 (HER2) occurs in ~20-25% of invasive ductal breast carcinomas and is associated with the more aggressive phenotype. Herceptin, a humanized antibody against HER2, is a standard therapy in HER2-overexpressing cases. Approximately one-third of patients relapse despite treatment. Therefore numerous studies have investigated the molecular mechanisms associated with Herceptin resistance. An interaction between HER2 signalling and steroid hormone receptor signalling pathways has been previously investigated, but the effect of this relationship on Herceptin resistance has never been studied. The present study analysed an impact of the steroid hormone, progesterone (PG), on Herceptin-dependent cell growth inhibition. Results indicated that Herceptin-inhibited proliferation of breast cancer cell lines overexpressing HER2 (BT474 and MCF/HER2) in 3D culture is abolished by PG. Furthermore, results demonstrated that PG led to the activation of HER2/HER3-mediated signalling. Moreover, PG treatment induced a shift of Herceptin-dependent cell cycle arrest in G1 phase towards S and G2 phases with concomitant upregulation of cyclin-dependent kinase 2 (CDK2) and downregulation of CDK inhibitor p27Kip1. These results demonstrate for the first time PG involvement in the failure of Herceptin treatment in vitro. The present observations suggest that cross-talk between PG- and HRG/HER2-initiated signalling pathways may lead to the acquisition of resistance to Herceptin in patients with BCa.

Analysis of structure-cytotoxicity in vitro relationship (SAR) for perfluorinated carboxylic acids.

Perfluorinated carboxylic acids (PFAs) represent derivatives of naturally occurring compounds and have been widely used in various industrial fields for decades. They are known to be environmentally persistent. Thus far numerous reports have been focused on reproductive toxicity of PFAs in animals but few studies have been carried out on toxicity towards human cells. Viability tests were performed here at varying time-exposures on C6-C18 PFAs with human colon carcinoma (HCT116) cells. These cells were found earlier as the most useful line for in vitro assays. A chain length-EC50 dependence has been clearly observed. Estimated values of EC50 decreased with elongation of fluorocarbon chain (PFHxA > PFHpA > PFOA > PFNA > PFDA > PFDoA > PFTeDA). Further elongation (C16 and C18) did not deepen the effect but even partially reversed it. The effect was intensified after longer exposure (72 h); at relatively low 40 microM PFTeDA, the viability decreased to approximately 50%. It seems that PFAs are not acutely toxic at the cellular level. Even so, however, they can trigger cell apoptosis, which is prominent in the case of myristic acid perfluorinated analogue.

Tetraspanin CD151 mediates communication between PC3 prostate cancer cells and osteoblasts.

Invasion and migration of cancer cells are crucial for the formation of secondary lesions. These require activation of signalling cascades modulated by the number of regulatory molecules. One such molecule is CD151, a member of evolutionary conserved tetraspanin family. CD151 is involved in cell adhesion, motility and cancer progression due to formation of complexes with laminin-binding integrins and regulation of growth factor receptors function (e.g. HGFR, TGFßR, EGFR). Recent studies point to correlation between CD151 expression and high tumour grade in prostate cancer (PCa). Herein, we investigated a possible role of CD151 in communication between PC3 cancer cells and either cancer-associated fibroblasts (CAFs) or osteoblasts, an interplay which is significant for metastasis. The analysis showed that although CAFs strongly enhanced both migration and invasion of PC3 prostate cancer cells, the effect was not dependent on CD151. On the other hand, CD151 was found to promote 3D migration as well as invasive growth in response to osteoblasts-secreted growth factors. Obtained data revealed that knockdown of CD151 abolished activation of pro-migratory/pro-survival kinases (i.e FAK, Src, HSP27) triggered by osteoblasts, along with expression of matrix metalloproteinase-13. This suggests that CD151 participates in communication between PC3 cells and bone microenvironment and the process can be considered as a significant step of PCa progression and metastasis.

FGFR2-Driven Signaling Counteracts Tamoxifen Effect on ERα-Positive Breast Cancer Cells.

Signaling mediated by growth factors receptors has long been suggested as one of the key factors responsible for failure of endocrine treatment in breast cancer (BCa). Herein we present that in the presence of tamoxifen, FGFs (Fibroblast Growth Factors) promote BCa cell growth with the strongest effect being produced by FGF7. FGFR2 was identified as a mediator of FGF7 action and the FGFR2-induced signaling was found to underlie cancer-associated fibroblasts-dependent resistance to tamoxifen. FGF7/FGFR2-triggered pathway was shown to induce ER phosphorylation, ubiquitination and subsequent ER proteasomal degradation which counteracted tamoxifen-promoted ER stabilization. We also identified activation of PI3K/AKT signaling targeting ER-Ser167 and regulation of Bcl-2 expression as a mediator of FGFR2-promoted resistance to tamoxifen. Analysis of tissue samples from patients with invasive ductal carcinoma revealed an inversed correlation between expression of FGFR2 and ER, thus supporting our in vitro data. These results unveil the complexity of ER regulation by FGFR2-mediated signaling likely to be associated with BCa resistance to endocrine therapy.

Adenosine as a metabolic regulator of tissue function: production of adenosine by cytoplasmic 5'-nucleotidases.

Adenosine is a product of complete dephosphorylation of adenine nucleotides which takes place in various compartments of the cell. This nucleoside is a significant signal molecule engaged in regulation of physiology and modulation of the function of numerous cell types (i.e. neurons, platelets, neutrophils, mast cells and smooth muscle cells in bronchi and vasculature, myocytes etc.). As part a of purinergic signaling system, adenosine mediates neurotransmission, conduction, secretion, vasodilation, proliferation and cell death. Most of the effects of adenosine help to protect cells and tissues during stress conditions such as ischemia or anoxia. Adenosine receptors and nucleoside transporters are targets for potential drugs in many pathophysiological situations. The adenosine-producing system in vertebrates involves a cascade dephosphorylating ATP and ending with 5'-nucleotidase (EC 3.1.3.5) localized either on the membrane or inside the cell. In this paper the cytoplasmic variants of 5'-nucleotidase are broadly characterized as well as their clinical relevance. The role of AMP-selective 5'-nucleotidase (cN-I) in the heart, skeletal muscle and brain is highlighted. cN-I action is crucial during ischemia and important for the efficacy of some nucleoside-based drugs and in the regulation of the substrate pool for nucleic acids synthesis. Inhibitors used in studying the roles of cytoplasmic and membrane-bound 5'-nucleotidases are also described.

Expression of ecto-5'-nucleotidase (eN, CD73) in cell lines from various stages of human melanoma.

Ecto-5'-nucleotidase is a GPI-anchored enzyme localized in cell membrane lipid rafts. Although it is highly expressed in many tumour cells, its specific function during tumorigenesis is unclear. We have found that, among different melanoma cells, upregulated expression of ecto-5'-nucleotidase is associated with a highly invasive phenotype. Analysis of other cell membrane proteins involved in melanoma adhesion and metastasis demonstrated that expression of alpha5, beta1, beta3-integrin subunits and CD44 was elevated gradually in accordance with increasing metastatic potential. Expression of alphav-integrin and caveolin-1 was seen mostly in cells derived from metastatic melanomas. Furthermore, in contrast to N-cadherin, which was unaltered in all lines, we could not detect E-cadherin in any cell type. Functional assays demonstrated that highly expressed ecto-5'-nucleotidase is a catalytically competent protein that is very sensitive to inhibition by concanavalin A. The interaction with concanavalin A also caused increased association of ecto-5'-nucleotidase-rich lipid rafts with much heavier cytoskeletal complexes as determined by density gradient centrifugation. A similar shift towards heavier cytoskeletal fractions also took place with other proteins coexpressed with ecto-5'-nucleotidase, such as alphav, alpha5, beta1 and beta3-integrins, caveolin-1 and CD44. As ConA-induced clustering may reflect the interactions of membrane proteins with extracellular matrix, we also analysed the effect of several extracellular matrix proteins on the in-situ activity of ecto-5'-nucleotidase in WM9 cells and found that tenascin C strongly inhibited ecto-5'-nucleotidase activity and adenosine generation from AMP. We also developed WM9 cells with reduced ecto-5'-nucleotidase expression and tested differences in cell adhesion on various extracellular matrix proteins. WM9 cells attached significantly weaker to tenascin C layer. These observations indicate that expression of ecto-5'-nucleotidase correlates with a number of metastasis-related markers and thus may have a function in this process. Furthermore, our data suggest that, in addition to generating adenosine, ecto-5'-nucleotidase may have independent roles in adhesion and interaction with extracellular matrix components in melanoma.

Specific Activation of A3, A2A and A1 Adenosine Receptors in CD73-Knockout Mice Affects B16F10 Melanoma Growth, Neovascularization, Angiogenesis and Macrophage Infiltration.

CD73 (ecto-5'-nucleotidase), a cell surface enzyme hydrolyzing AMP to adenosine, was lately demonstrated to play a direct role in tumor progression including regulation of tumor vascularization. It was also shown to stimulate tumor macrophage infiltration. Interstitial adenosine, accumulating in solid tumors due to CD73 enzymatic activity, is recognized as a main mediator regulating the production of pro- and anti-angiogenic factors, but the engagement of specific adenosine receptors in tumor progression in vivo is still poorly researched. We have analyzed the role of high affinity adenosine receptors A1, A2A, and A3 in B16F10 melanoma progression using specific agonists (CCPA, CGS-21680 and IB-MECA, respectively). We limited endogenous extracellular adenosine background using CD73 knockout mice treated with CD73 chemical inhibitor, AOPCP (adenosine α,ß-methylene 5'-diphosphate). Activation of any adenosine receptor significantly inhibited B16F10 melanoma growth but only at its early stage. At 14th day of growth, the decrease in tumor neovascularization and MAPK pathway activation induced by CD73 depletion was reversed by all agonists. Activation of A1AR primarily increased angiogenic activation measured by expression of VEGF-R2 on tumor blood vessels. However, mainly A3AR activation increased both the microvessel density and expression of pro-angiogenic factors. All agonists induced significant increase in macrophage tumor infiltration, with IB-MECA being most effective. This effect was accompanied by substantial changes in cytokines regulating macrophage polarization between pro-inflammatory and pro-angiogenic phenotype. Our results demonstrate an evidence that each of the analyzed receptors has a specific role in the stimulation of tumor angiogenesis and confirm significantly more multifaceted role of adenosine in its regulation than was already observed. They also reveal previously unexplored consequences to extracellular adenosine signaling depletion in recently proposed anti-CD73 cancer therapy.

Fibroblast growth factor signalling induces loss of progesterone receptor in breast cancer cells.

We have recently demonstrated that, fibroblast growth factor 2 (FGFR2), signalling via ribosomal S6 kinase 2 (RSK2), promotes progression of breast cancer (BCa). Loss of progesterone receptor (PR), whose activity in BCa cells can be stimulated by growth factor receptors (GFRs), is associated with poor patient outcome. Here we showed that FGF7/FGFR2 triggered phosphorylation of PR at Ser294, PR ubiquitination and subsequent receptor`s degradation via the 26S proteasome pathway in BCa cells. We further demonstrated that RSK2 mediated FGF7/FGFR2-induced PR downregulation. In addition, a strong synergistic effect of FGF7 and progesterone (Pg), reflected in the enhanced anchorage-independent growth and cell migration, was observed. Analysis of clinical material demonstrated that expression of PR inversely correlated with activated RSK (RSK-P) (p = 0.016). Patients with RSK-P(+)/PR(-) tumours had 3.629-fold higher risk of recurrence (p = 0.002), when compared with the rest of the cohort. Moreover, RSK-P(+)/PR(-) phenotype was shown as an independent prognostic factor (p = 0.006). These results indicate that the FGF7/FGFR2-RSK2 axis promotes PR turnover and activity, which may sensitize BCa cells to stromal stimuli and contribute to the progression toward steroid hormone negative BCa.

Isolation and characterization of pigeon breast muscle cytosolic 5'-nucleotidase-I (cN-I).

5'-Nucleotidase specific towards dCMP and AMP was isolated from avian breast muscle and characterized. It was found to be similar to a type-I form (cN-I) identified earlier as the AMP-selective 5'-nucleotidase responsible for adenosine formation during ATP breakdown in transfected COS-7 cells. Expression pattern of the cN-I gene in pigeon tissues indicated breast muscle as a rich source of the transcript. We purified the enzyme from this source using two-step chromatography and obtained an active homogenous preparation, free of ecto-5'-nucleotidase activity. The tissue content of the activity was calculated at 0.09 U/g wet weight. The specific activity of the enzyme preparation was 4.33 U/mg protein and it preferred dCMP and AMP to dAMP and IMP as a substrate. Its kinetic properties were very similar to those of the enzyme purified earlier from heart tissue. It was strongly activated by ADP. Inhibition by inorganic phosphate was more pronounced than in heart-isolated cN-I. Despite this difference, a similar physiological function is suggested for cN-I in both types of muscle.

CD73 on B16F10 melanoma cells in CD73-deficient mice promotes tumor growth, angiogenesis, neovascularization, macrophage infiltration and metastasis.

Ecto-5'-nucleotidase (CD73), an enzyme providing interstitial adenosine, mediates diverse physiological and pathological responses. In tumor progression, it has primarily an immunosuppressive role but is also thought to regulate neovascularization. However, the latter role is still in debate. When B16F10 melanoma was subcutaneously injected into CD73 knockout mice, changes in the tumor vasculature were not always observed. However, we demonstrated earlier that the growth and vascularization of B16F10 melanoma in CD73 knockout mice depend on the low presence of CD73 on tumor cells. To further analyze the role of CD73 on tumor growth and vascularization, we compared the changes in B16F10 melanoma subcutaneously injected into right flank of wild-type mice, CD73 knockout mice lacking host CD73 only, and CD73 knockout mice with tumor cell CD73 either inhibited with AOPCP (adenosine α,ß-methylene 5'-diphosphate) or permanently knocked down through genetic modification. We report here that both inhibition and knockdown of tumor CD73 further inhibited tumor growth compared to host CD73 knockout alone. MAP-kinase signaling pathway activation also decreased more strongly in the stable knockdown. There was a significant reduction in the angiogenic activation of blood microvessels as observed by decreased anti-VEGFR2 staining. Stable CD73 knockdown also reduced endothelial cell proliferation as measured by anti-CD105 staining. However, only chemical inhibition with AOPCP significantly augmented the reduction in intratumoral microvessel density induced by host CD73 knockout. Such reduction was not observed when tumor CD73 was knocked down due to the much slower tumor growth and decreased oxygen demand as indicated by the low expression of Bad, a hypoxia marker. Decreased CD73 activity also led to the decreased expression of angiogenic factors, including VEGF and bFGF that was only partially reversed by hypoxia in tumors treated with AOPCP. Both inhibition and knockdown of tumor CD73 significantly decreased tumor macrophage infiltration and induced microenvironment changes, thereby influencing MI or MII macrophage polarization. Additionally, tumor cell CD73 is important in metastasis formation through adenosine-independent attachment to endothelium. We conclude that even low tumor cell CD73 expression has an undeniable role in melanoma progression, including the regulation of many aspects of angiogenesis. CD73 is thus a viable target in anti-angiogenic melanoma therapy.

Inhibition of CD73 stimulates the migration and invasion of B16F10 melanoma cells in vitro, but results in impaired angiogenesis and reduced melanoma growth in vivo.

The role of ecto-5'-nucleotidase (CD73), an enzyme providing interstitial adenosine, was investigated in B16F10 melanoma progression. Chemical inhibition of CD73 decreased adherence of cells to extracellular matrix proteins in vitro and led to enhanced migration and invasion. Both processes were reversed by adenosine receptor agonists. In CD73­deficient mice, tumor growth was decreased in comparison with that of wild-type animals. Additionally, the vasculature of CD73-inhibited tumors was impaired and neoangiogenesis in Matrigel plugs was reduced. It is, therefore, proposed that although CD73 shows anti-invasive and antimigratory function in B16F10 melanoma cells, its proangiogenic action is prevalent in vivo and may contribute to increased tumor growth.