Tetrastatin, the NC1 Domain of the α4(IV) Collagen Chain: A Novel Potent Anti-Tumor Matrikine

BACKGROUND: NC1 domains from α1, α2, α3 and α6(IV) collagen chains were shown to exert anti-tumor or anti-angiogenic activities, whereas the NC1 domain of the α4(IV) chain did not show such activities so far. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate in the present paper that the NC1 α4(IV) domain exerts a potent anti-tumor activity both in vitro and in an experimental human melanoma model in vivo. The overexpression of NC1 α4(IV) in human UACC-903 melanoma cells strongly inhibited their in vitro proliferative (-38%) and invasive (-52%) properties. MT1-MMP activation was largely decreased and its cellular distribution was modified, resulting in a loss of expression at the migration front associated with a loss of migratory phenotype. In an in vivo xenograft model in athymic nude mice, the subcutaneous injection of NC1 α4(IV)-overexpressing melanoma cells induced significantly smaller tumors (-80% tumor volume) than the Mock cells, due to a strong inhibition of tumor growth. Exogenously added recombinant human NC1 α4(IV) reproduced the inhibitory effects of NC1 α4(IV) overexpression in UACC-903 cells but not in dermal fibroblasts. An anti-αvβ3 integrin blocking antibody inhibited cell adhesion on recombinant human NC1 α4(IV) substratum. The involvement of αvβ3 integrin in mediating NC1 α4(IV) effect was confirmed by surface plasmon resonance (SPR) binding assays showing that recombinant human NC1 α4(IV) binds to αvβ3 integrin (K(D) = 148 ± 9.54 nM). CONCLUSION/SIGNIFICANCE: Collectively, our results demonstrate that the NC1 α4(IV) domain, named tetrastatin, is a new endogenous anti-tumor matrikine

Matrikines : une nouvelle stratégie thérapeutique anti-cancéreuse

Le microenvironnement tumoral est un système complexe comportant une matrice extracellulaire largement modifiée et différents types cellulaires qui déterminent la réponse angiogénique et l’invasion locale. Sous l’influence de l’hypoxie, les cellules cancéreuses sécrètent des cytokines qui activent les cellules du stroma pour produire des protéases et des facteurs angiogéniques. Ces protéases dégradent la matrice extracellulaire stromale et participent à la libération de divers fragments de macromolécules matricielles, appelés matrikines ou matricryptines, capables de contrôler l’invasion tumorale et la dissémination métastatique. Nous focaliserons cet exposé sur les matrikines dérivées des domaines NC1 des différentes chaînes constitutives des collagènes associés aux membranes basales et notamment le collagène de type IV. Les cibles potentielles d’action des matrikines sont la prolifération et les propriétés invasives des cellules cancéreuses ou des cellules inflammatoires, ainsi que les réponses angiogéniques et lymphangiogéniques. Par exemple, la canstatine, la tumstatine et la tétrastatine, dérivant respectivement des domaines NC1 des chaînes α2, α3 and α4 du collagène IV, inhibent in vivo la croissance tumorale dans divers modèles expérimentaux de cancer. Leur activité anti-cancéreuse se manifeste par un effet anti-prolifératif sur les cellules tumorales et/ou les cellules endothéliales par induction de l’apoptose ou blocage du cycle cellulaire ainsi qu’en provoquant la perte de leur phénotype migratoire. Les matrikines constituent une nouvelle famille de puissants agents anticancéreux qui peuvent être utilisés dans différentes stratégies thérapeutiques : (i) induction de leur surexpression par les cellules cancéreuses ou par les cellules de l’hôte, (ii) utilisation de protéines recombinantes, de peptides synthétiques, d’analogues structuraux conçus à partir de la structure des séquences actives. Ces matrikines pourront être utilisées en combinaison avec des chimiothérapies conventionnelles ou la radiothérapie pour lutter contre la progression tumorale

NC1 α4(IV) overexpression inhibits <i>in vitro</i> melanoma cell proliferation.

<p>(A): Cell proliferation was measured using WST-1 reagent after 24, 48 and 72 h as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029587#s2" target="_blank">Materials and Methods</a> (mean of three clones±SD. ***: p<0.001) (A). (B): Cell proliferation of the three selected clones at T 72 h.</p

NC1 α4(IV) overexpression decreases MT1-MMP activation and modifies MT1-MMP distribution.

<p>(A): Mock or NC1 α4(IV)-overexpressing cells were incubated for 48 h without FBS. MT1-MMP expression and activation in whole cell extracts were analyzed by Western blot with an antibody directed against the hinge region of MT1-MMP. The membrane was dehybridized and reprobed with an anti-actin antibody. Quantifications were performed by densitometry using the Bio-1D software. Results were expressed as arbitrary units (AU). NS: Non significant. ***: p<0.001. (B): Cells were cultured on glass slides, fixed with paraformaldehyde and labelled with an anti-MT1-MMP directed against both the pro and the active form (green), anti-caveolin 1 (red). Yellow staining corresponds to areas where proMT1-MMP and caveolin-1 colocalized and white arrows in the insert underline the colocalization at the migration front at a higher magnification. Nuclei were conterstained with DAPI (blue). Scale bar: 5 µm.</p

Selection of NC1 α4(IV) overexpressing cell clones.

<p>UACC-903 cells were transfected with either p3xFLAG-CMV-9 (Mock 1, 2, 3) or p3xFLAG-NC1[α4(IV)] (NC1 α4(IV) 1, 2, 3). (A): RT-PCR: Total RNA was isolated from clones selected for G418 resistance and analyzed by RT-PCR using p3xFLAG-CMV-9 (1) and GAPDH specific primers (2). (B): Western-blot: Three clones were selected by RT-PCR for their high gene expression of FLAG epitope and FLAG-NC1[α4(IV)] fusion protein. Supernatant from Mock cells and from cells stably transfected with FLAG-NC1[α4(IV)] were tested for the 28 kDa fusion protein secretion by Western blot using an anti-FLAG monoclonal antibody or an anti-NC1 α4(IV) polyclonal antibody.</p

Recombinant human NC1 α4(IV) inhibits <i>in vitro</i> melanoma cell proliferation and invasion.

<p>(A): Recombinant human NC1 α4(IV) domain obtention: Recombinant human NC1 α4(IV) domain was expressed in <i>E. coli</i> JM109, DE3 strain (1) SDS-PAGE (lane M: low molecular weight markers; lane 1: T4h crude lysate, non-induced by IPTG; lane 2: T4h crude lysate, induced by IPTG, lane 3: recombinant human NC1 α4(IV) domain purified by chromatography on a Ni-NTA superflow resin. (2) Western blot using an anti-NC1 α4(IV) antibody. Lane 1, 2, 3: same as above. (3) Western blot using an anti-His tag antibody. Lane 1, 2, 3: same as above. (B): Cell proliferation: Melanoma cells and dermal fibroblasts were incubated without or with 10 or 20 µg/mL recombinant human NC1 α4(IV) for 48 h. Cell proliferation was measured as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029587#s2" target="_blank">Material and Methods</a> section. NS: Non significant. ***: p<0.001. (C): Cell invasion: UACC-903 melanoma cells were incubated without or with 20 µg/mL recombinant human NC1 α4(IV). Cell invasion was measured as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029587#s2" target="_blank">Material and Methods</a> section. ***: p<0.001.</p

Recombinant human NC1 α4(IV) binds to αvβ3 integrin.

<p>(A): Adhesion assays were performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029587#s2" target="_blank">Material and Methods</a> section. Cells were incubated or not with 5 mM EDTA. Cells were fixed after 30, 60 or 90 min with 1.1% glutaraldehyde and stained with crystal violet. After elution with 10% acetic acid, absorbance was read at 560 nm. *: p<0.05. Adhesion was restored by the addition of 1.3 mM Ca²⁺ or 0.5 mM Mg²⁺. (B): Adhesion assays were performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029587#s2" target="_blank">Material and Methods</a> section. Cells were preincubated for 30 min with an anti-αvβ3 blocking antibody or an irrelevant IgG (10 µg/mL) before seeding. Cells were fixed with 1.1% glutaraldehyde and stained with crystal violet. After elution with 10% acetic acid absorbance was read at 560 nm. *: p<0.05. (C): Surface plasmon resonance (SPR) binding assays were performed by injecting recombinant human NC1 α4(IV) (3.57 µM at 30 µL/min) over αvβ3 integrin immobilized on a CM5 sensor chip (2655 RU). Binding was expressed as resonance units.</p

NC1 α4(IV) overexpression by melanoma cells decreases tumor growth in a mouse xenograft model.

<p>Mock or NC1 α4(IV)-overexpressing UACC-903 cells (5×10⁶ cells) were subcutaneously injected into the left side of athymic mice. (A): Tumor size was measured at days 10, 14, 19 and 26. Tumor volumes were determined according to v = ½ A×B², where A denotes the largest dimension of the tumor and B represents the smallest dimension, and expressed as mean±SD (n = 10). ***: p<0.001. The insert shows example of tumors obtained in each mouse series after injection of Mock or NC1 α4(IV)-overexpressing cells. (B): Immunostaining of tumor section with an anti-Ki67 antibody. Quantification was performed with Image J. Scale bar: 50 µm.</p