Maxime Lehmann

Antiangiogenic Concentrations of Paclitaxel Induce an Increase in Microtubule Dynamics in Endothelial Cells but Not in Cancer Cells

Microtubule-targeted drugs such as paclitaxel exhibit potent antiangiogenic activity at very low concentrations, but the mechanism underlying such an effect remains unknown. To understand the involvement of microtubules in angiogenesis, we analyzed the dynamic instability behavior of microtubules in living endothelial cells [human microvascular endothelial cells (HMEC-1) and human umbilical vascular endothelial cells (HUVEC)] following 4 hours of paclitaxel treatment. Unexpectedly, antiangiogenic concentrations of paclitaxel (0.1-5 nmol/L) strongly increased microtubule overall dynamicity in both HMEC-1 (86-193%) and HUVEC (54-83%). This increase was associated with increased microtubule growth and shortening rates and extents and decreased mean duration of pauses. The enhancement of microtubule dynamics by paclitaxel seemed to be specific to antiangiogenic concentrations and to endothelial cells. Indeed, cytotoxic concentration (100 nmol/L) of paclitaxel suppressed microtubule dynamics by 40% and 54% in HMEC-1 and HUVECs, respectively, as observed for all tested concentrations in A549 tumor cells. After 4 hours of drug incubation, antiangiogenic concentrations of paclitaxel that inhibited endothelial cell proliferation without apoptosis (1-5 nmol/L) induced a slight decrease in anaphase/metaphase ratio, which was more pronounced and associated with increased mitotic index after 24 hours of incubation. Interestingly, the in vitro antiangiogenic effect also occurred at 0.1 nmol/L paclitaxel, a concentration that did not alter mitotic progression and endothelial cell proliferation but was sufficient to increase interphase microtubule dynamics. Altogether, our results show that paclitaxel mediates antiangiogenesis by an increase in microtubule dynamics in living endothelial cells and suggest that the impairment of interphase microtubule functions is responsible for the inhibition of angiogenesis.

Regulation of urokinase plasminogen activator/plasmin- mediated invasion of melanoma cells by the integrin vitronectin receptor αVβ3

The integrin vitronectin receptor αvβ3 is a mediator of cellular migration and invasion and has been identified as a marker of progression in malignant melanoma. Using a human melanoma model, we have previously shown that this receptor was coordinately expressed with the receptor for the urokinase plasminogen activator (uPAR). In our present study, the link between these receptors was further investigated by assessing the effect of αvβ3 ligation on uPAR transcription and function. Using the reverse transcription‐polymerase chain reaction, we found that receptor ligation by immobilized monoclonal antibodies (MAbs) induced a rapid increase (up to 4.5 fold) in uPAR mRNA levels, which was maximal 4 hr after cell attachment. An increase was also noted in plasminogen activator inhibitor type‐1 (PAI‐1) mRNA levels (2.7‐fold), but none was noted in uPA levels. In addition, ligation of αvβ3 resulted in a significant increase in cell surface‐associated plasmin levels, which coincided with a 2‐ to 3‐fold increase in cell invasion as measured in the Matrigel invasion assay. This increase in invasion could in turn be abolished by antibodies directed to uPA and uPAR and by the plasmin inhibitors ϵ‐aminocaproic acid and aprotinin. Furthermore, ligation of the integrin αvβ3 triggered a rapid increase of up to 12‐fold in total cellular PKC activity, and this coincided with the redistribution of PKCβ, but not PKCα, from the cytosol to the membrane. Treatment of the cells with the PKCβ‐specific inhibitor LY379196 blocked uPAR and PAI‐1 mRNA induction and reduced the increase in cell invasion due to αvβ3 ligation, confirming the involvement of this isoform in the response. The results provide evidence that the vitronectin receptor can enhance invasion by regulating the uPAR/uPA/plasmin system of proteolysis and implicate PKCβ as an intermediate in the activation pathway.

Gα(q/11)-coupled P2Y2 nucleotide receptor inhibits human keratinocyte spreading and migration

Reepithelialization is a critical step in wound healing. It is initiated by keratinocyte migration at the wound edges. After wounding, extracellular nucle‐otides are released by keratinocytes and other skin cells. Here, we report that activation of P2Y2 nucleotide receptor by ATP/UTP inhibits keratinocyte cell spreading and induces lamellipodium withdrawal. Kymogra‐phy analysis demonstrates that these effects correlate with a durable decrease of lamellipodium dynamics. P2Y2 receptor activation also induces a dramatic dismantling of the actin network, the loss of a3 integrin expression at the cell periphery, and the dissolution of focal contacts as indicated by the alteration of av integrins and focal contact protein distribution. In addition, activation of P2Y2R prevents growth factor‐induced phosphorylation of Erk1,2 and Akt/PkB. The use of a specific pharmacological inhibitor (YM‐254890), the depletion of Ga(q/11) by siRNA, or the expression of a constitutively active Ga(q/11) mutant (Q209L) show that activation of Ga(q/11) is responsible for these ATP/ UTP‐induced effects. Finally, we report that ATP delays growth factor‐induced wound healing of keratino‐cyte monolayers. Collectively, these findings provide evidence for a unique and important role for extracellular nucleotides as efficient autocrine/paracrine regulators of keratinocyte shape and migration during wound healing.— Taboubi, S., Milanini, J., Delamarre, E., Parat, F., Garrouste, F., Pommier, G., Takasaki, J., Hubaud, J‐C., Kovacic, H., Lehmann, M. Ga(q/11)‐coupled P2Y2 Nucleotide Receptor Inhibits Human Keratinocyte Spreading and Migration. FASEB J. 21, 4047–4058 (2007)

Deficient Processing and Activity of Type I Insulin-Like Growth Factor Receptor in the Furin-Deficient LoVo-C5 Cells1

To investigate endoproteolytic processing of the type I insulin-like growth factor receptor (IGF-IR), we have examined its structure and activity in the furin-deficient LoVo-C5 cell line. Immunoprecipitation experiments using the monoclonal anti-IGF-IR antibody (α-IR3) showed that LoVo-C5 cells expressed a major high molecular mass receptor (200 kDa) corresponding to the unprocessed α/β pro-receptor. A small amount of successfully cleaved α/β heterodimers was also produced, indicating a residual endoproteolytic cleavage activity in these cells. In vitro, a soluble form of recombinant furin was able to cleave the pro-IGF-IR (200 kDa) into α-subunit (130 kDa) andβ -subunit (97 kDa). Measurement of IGF binding parameters in LoVo-C5 cells indicated a low number of typical type I IGF-binding sites (binding capacity, 5 × 103 sites/cell; Kd, 1.9 nm for IGF-I and 7.0 nm for IGF-II). These findings in LoVo-C5 contrast with those in HT29-D4 cells, which have active furin, and where IGF-IR (2.8 × 104 sites/cell) was...

Up-regulation of insulin/insulin-like growth factor-I hybrid receptors during differentiation of HT29-D4 human colonic carcinoma cells.

To assess the autocrine function of insulin-like growth factor II (IGF-II) in the balance of proliferation and differentiation in HT29-D4 human colonic cancer cells, we studied the expression of IGF-I receptors (IGF-IR) and insulin receptors (IR) in relation to the state of cell differentiation. IGF-IR and IR were expressed in both undifferentiated and enterocyte-like differentiated HT29-D4 cells. IGF-IR had two isoforms with a 97-kDa and a 102-kDa β-subunit. In addition, HT29-D4 cells expressed hybrid receptors (HR) formed by the association of two αβ heterodimers from both IR and IGF-IR. HR were evidenced through 1) inhibition of IGF-I binding by the B6 anti-IR antibody and 2) immunoprecipitation with the α-IR3 anti-IGF-IR antibody, which revealed an additional 95-kDa IR β-subunit that disappeared when the heterotetrameric receptor was dissociated by disulfide reduction into αβ heterodimers before immunoprecipitation. Like IGF-IR, HR had a high affinity for IGF-I (Kd, ∼1.5 nm), but did not bind insulin ...

β1 Integrins as Therapeutic Targets to Disrupt Hallmarks of Cancer.

Integrins belong to a large family of αβ heterodimeric transmembrane proteins first recognized as adhesion molecules that bind to dedicated elements of the extracellular matrix and also to other surrounding cells. As important sensors of the cell microenvironment, they regulate numerous signaling pathways in response to structural variations of the extracellular matrix. Biochemical and biomechanical cues provided by this matrix and transmitted to cells via integrins are critically modified in tumoral settings. Integrins repertoire are subjected to expression level modifications, in tumor cells, and in surrounding cancer-associated cells, implicated in tumor initiation and progression as well. As critical players in numerous cancer hallmarks, defined by Hanahan and Weinberg (2011), integrins represent pertinent therapeutic targets. We will briefly summarize here our current knowledge about integrin implications in those different hallmarks focusing primarily on β1 integrins.

Evidences that β1 integrin and Rac1 are involved in the overriding effect of laminin on myelin-associated glycoprotein inhibitory activity on neuronal cells

During neurite elongation, migrating growth cones encounter both permissive and inhibitory substrates, such as laminin and MAG (myelin-associated glycoprotein), respectively. Here, we demonstrated on two neuronal cell lines (PC12 and N1E-115), that laminin and collagen hampered, in a dose-dependent manner, MAG inhibitory activity on several integrin functions, i.e., neurite growth, cell adhesion and cell spreading. Using a function blocking antibody, in PC12 cells, we showed that alpha1beta1 integrin is required in these phenomena. In parallel, we observed that MAG perturbs actin dynamics and lamellipodia formation during early steps of cell spreading. This seemed to be independent of RhoA activation, but dependent of Rac-1 inhibition by MAG. Laminin overrode MAG activity on actin and prevented MAG inhibition NGF-induced Rac1 activation. In conclusion, we evidenced antagonistic signaling between MAG receptors and beta1 integrins, in which Rac-1 may have a central function.

Role of αvβ5 and αvβ6 integrin glycosylation in the adhesion of a colonic adenocarcinoma cell line (HT29‐D4)

We have previously characterized the expression of the αvβ5 and αvβ6 integrins as major receptors for the human colonic adenocarcinoma cell line (HT29‐D4), on vitronectin and fibronectin, respectively [Lehmann et al. (1994): Cancer Res 54:2102–2107]. In the present work we investigated the glycosylation role of these integrins in their adhesive functions. To this end, we used glycohydrolases to show that cell surface integrins were N‐glycosylated and sialylated, and that only the αv subunit carried some immature oligosaccharide side chains. To alter the glycosylation state of the cell surface αvβ5 and αvβ6 integrins, we used two oligosaccharide‐processing inhibitors: 1‐deoxymannojirimycin (dMNJ) and tunicamycin (TM). Following treatment of HT29‐D4 cells with dMNJ, cell surface αvβ5 and αvβ6 carried only high‐mannose‐type sugar chains, while TM‐treated cells expressed de‐N‐glycosylated integrins. Neither α/β heterodimers assembly nor cell surface expression were impaired in the presence of the drugs. Finally, we established that adhesion of dMNJ‐ or TM‐treated cells was altered on both vitronectin and fibronectin substrata, whereas the adhesion of these cells on laminin or collagen type I was virtually unchanged.

Combination of culture on collagen gels and glucose starvation for cloning human colon cancer cells

Glucose starvation has been widely used to select differentiated subpopulations from the heterogenous human colon cancer cell line HT29. We observed that the important cell loss elicited by culturing these cells in glucose-free medium could be limited when type I collagen gel was used as substratum instead of conventional plastic support. We took advantage of this property to develop a new protocol, which combined glucose starvation and culture on collagen gels, for cloning HT29 cells. Using this procedure we have isolated four clones that were characterized on the basis of morphological (optical and transmission electron microscopy), electrophysiological (determination of transepithelial electrical parameters) and biochemical (detection of villin, sucrase-isomaltase and carcinoembryonic antigen) criteria. These four clones expressed different patterns of enterocytic differentiation regarding to these criteria. These results confirmed the heterogeneity of the HT29 cell line. One of these clones, HT29-A7, which displayed numerous intercellular cysts that disappeared at confluency, appears as a complementary model in the study of epithelial biogenesis.