The glycan-binding protein galectin-1 links hypoxia and angiogenesis in Kaposi Sarcoma.

DO. CROCI; M. SALATINO; N. RUBINSTEIN; JM. ILARREGUI; MA TOSCANO; GA. RABINOVICH

Viña del Mar Chile

Congreso; ALAI 2009- Latinoamerican Congress of Immunology,; 2009

Soc Latinoamericana de Inmunología

Angiogenesis is a critical process for tumor progression. We previously demonstrated that galectin-1 (Gal-1), a glycan-binding protein with immunosuppressive activity, controls tumor growth by favoring escape from T-cell-dependent immunity. The aim of this study was to investigate, using in vitro and in vivo strategies, the role of Gal-1 in the control of tumor angiogenesis and to analyze the mechanisms underlying this effect. To examine the contribution of Gal-1 to tumor angiogenesis in vivo we inhibited Gal-1 expression by silencing-gene strategies in human Kaposi sarcoma tumor cell lines. Targeted inhibition of gal-1 gene expression inhibited the formation new blood vessels (p<0,05 knockdown vs wild-type) and suppressed tumor growth in vivo (p<0,01 knockdown vs wild-type). Expression of Gal-1 in Kaposi cells was found to be up-regulated under hypoxic conditions, both in vitro (as evaluated by WB, Real Time RT-PCR and promoter activity) and in vivo (IHC).This over expression was dependent of NFkB but not  HIF-1a activity, since inhibitor of IkB phosphorylation BAY 11-7082 (1.5 µM) but not Hif-1a (2µM) prevented hypoxia-induced expression of Gal-1 (p<0.05). Moreover, conditioned media from KS cells (CM-KS) incubated 16 h under hypoxic conditions (O2 1 KPa) were more effective at inducing tubulogenesis (p<0.01) and invasion (p<0.05) than normoxic cells. Interestingly, this effect was abrogated when KS hypoxic cells were infected with retrovirus encoding shRNA to gal-1(p<0.01). This result was confirmed in vivo using a matrigel sponges model in which hypoxic MC-KS were more effective to induce hemoglobin accumulation in matrigel plugs (p<0.05 hypoxic MC-KS vs normoxic MC-KS). Remarkably, the presence of shRNA-gal-1 significantly reduced accumulation of hemoglobin in matrigel plugs either in normoxic or hypoxic conditions (p<0.05 vs empty vector). Recombinant Gal-1 bound human umbilical vein endothelial cells (HUVEC) in a dose and carbohydrate-dependent manner (p<0.01). Moreover, treatment of HUVEC with recombinant Gal-1 promoted tubulogenesis (p<0.01) proliferation (p<0.05) and invasion (p<0.05). A mechanistic analysis of the intracellular signals involved in this effects revealed a substantial increase in phosphorylated-Akt and Erk ½ following Gal-1 treatment (F.I>3). Pretreatment of HUVEC with either PI3K-inhibitor LY294.002 (2µM) or Erk1/2-inhibitor U0126 (5µM) but not NFkB, p38 or JNKs inhibitors, abolished the ability of Gal-1 to induce tubulogenesis (p<0.01) proliferation (p<0.05) and invasion (p<0.05) of HUVEC Our results suggest that hypoxia-regulated Gal-1 is a key modulator of tumor cell angiogenesis by promoting tubulogenesis, proliferation and invasion of endothelial cells. In addition, we propose a model in which Gal-1 regulated by hypoxia is secreted to the extracellular milieu to regulate angiogenesis and tumor-immune escape, thus influencing tumor progression.