The Molecular Mechanism Underlying In Inhibitory Effects Of Axon Guidance Molecule Sema3F On Proliferation And Metastasis Of Colorectal Carcinoma Through Its Receptor NRP2~1

IntroductionDistant dissemination is the most crucial cause of death in malignant cancer patients. Cancer cell departures from the primary site and further metastasizes through blood and lymphatic vasculatures. Regional lymphatic metastasis is frequently observed in early colorectal carcinoma. But the mechanism is not well known. Recently, it was found that tumor derived VEGF-C could induce tumor lymphangiogenesis via VEGFR signaling. But there are confusions remained. For instance, in some tumors, VEGF-C expression was detected without evident lymphangiogenesis, suggesting some alternative regulation mechanisms underlying in this process beside VEGF-C and VEGFR3.NRP1 and NRP2 were cell surface receptors involved in axon guidance repellent Semaphorin-3 (Sema3) signaling. However, NRP2 was also found to function critically on embryonic vasculogenesis expecially on developmental lymphangiogenesis. The NRP2 deficient mice exhibit a reduced capillary lymphatics but normal blood vessels and collective lymphatic vessels, suggesting a preferential participation of in embryonic lymphatic network development. There were also found that the primary saccus lymphaticus sprouting from vein does not need NRP2. But NRP2 is necessary for endothelial cells sprouting from primary saccus lymphaticus to form capillary networks, demonstrating that NRP2 is important for lymphatic vasculature maturation. In colorectal mucosa, NRP2 is abundantly expressed in lymphatic endothelial cells. So, we deduced that NRP2 expression might be crucial for colorectal cancer lymphangiogenesis. So far, the interactions between colorectal cancer cells and lymphatic endothelial cells highly expressing NRP2 is unknown. The roles of NRP2 on cancer lymphangiogenesis is still open to be explored.Recent studies have found two different functions of NRP. In nerve guidance, NRPs assists Sema3 binding with Plexins, forms a ligand-receptor complex and mediates Sema3 signal transductions. NRP2 binds with Sema3F alone and leads to chemorepulsion effect on Superior Cervical Ganglion. In vasculogenesis, NRP1 acts as a coreceptor of VEGF family to enhance the binding between VEGF165 and VEGFR2, and promotes the EC chemotaxis towards VEGF165 and cellular mitogen activity. Different ligands (Sema and VEGF) bind with identical receptor (NRP) that mediates totally different physiological processes of nerve guidance and vasculogenesis, revealing that these two processes share one same molecular basis.Sema also affects angiogenesis. Sema4D induces EC migration and tubulogenesis in vitro, and angiogenesis in vivo. But Sema3A plays a repressive role on angiogenesis due to the possible competitive antagonism against VEGF165 to bind with NRP1. Sema3F also suppress invasion, angiogenesis and dissemination in melanoma highly expressing functional NRP2. In spite of abundant expression of NRP2 in colorectal cancer LEC, the possibility and underlying mechanisms that Sema3F bonds with NRP2 might affect on cancer lymphangiogenesis is still unclear. Whether Sema3F could competitively antagonize VEGF to bind with NRP2 and impair the prolymphangiogenic effect of NRP2, or some other mechanisms deserve investigation.Therefore, we presumed that overexpression of Sema3F might be inhibitory for cancer cell proliferation, and through its receptor NRP2, cancer lymphangiogenesis might also be inhibited by Sema3F. In this study, based on the investigation results of Sema3F and NRP2 expression in colorectal adenocarcinoma, we established both full length form and shRNA form plasmid of Sema3F, transfected them into human colorectal cancer cell line SW480 and acquired stable transfectant. The effect of Sema3F on cancer cell proliferation, migration, adhesion capability was investigated in vitro. Tumor suppression and metastasis repression were also observed in orthotopic xenografts in vivo. Further, we isolated the characterized primary LEC from human skin, elevated the NRP2 level by retrovirus infection. The cell behaviors of LEC were investigated. We found that:Results1. Expression of Sema3F and RP2 in colorectal cancer is correlated with cancer progression and metastasis:①Sema3F was expressed in some cases. The mRNA level in tumor tissues were significantly lower than that of peritumoral tissues (P<0.05); compared with non-metastatic tumor, the metastasized cases exibited reduced Sema3F protein level (P<0.05);②The mRNA level of NRP2 in tumor tissue was significantly higher than peritumoral tissues, which is independent of tumor metastasis (P<0.05); but compared with non-metastatic tumor, the metastasized cases exibited reduced Sema3F protein level (P<0.05). The positive ratio of NRP2 expression was 54.2%. 100% expression of NRP2 was found in cases with lymphatic involvement and regional lymph node metastasis.③Sema3F expression is correlated with malignant features including invasion, differentiation, lymphatic involvement, regional metastasis and Dukes Stage. Besides correlation with above features, NRP2 expression was also correlated with blood vessel involvement.④Cancer lymphangiogenesis was heterogeneous in distribution, morphology and quantity (microlymphatic density) of lymphovasculature. There was negative correlation between tumor MLD and Sema3F expression. Sema3F decreased but MLD elevated as cancer Dukes stage progressed.2. RP2 promotes cell biological functions of LECs:①Using MACS, we successfully isolated and cultured a sort of LECs which has typical morphology, D2-40, LYVE-1, podoplanin and VEGFR3 positive immunophenotype, and biological features of response to VEGF-C;②Without 10 ng/ml VEGF-C stimulation, the proliferation of pLEC strain in which NRP2 was upregulated was enhanced. When 10ng/ml VEGF were added, pLEC showed the most proliferative acitivity in all groups. When NRP2 expression was downregulated by shRNA, the proliferation of iLEC strain was not affected. VEGF-C works on iLEC proliferation, but there was no significance between iLEC treatment group and cLEC treatment group.③When NRP2 was elevated, tubulogenesis capability was also enhanced in pLEC in early 48h, but iLEC can not form any typical tubular-like structures even though the investigation was terminated.④pLEC was most migratory in all groups. pLEC migrated cell number was 149% to cLEC. When NRP2 expression reduced, iLEC migration was also reduced; the migrated cell number was 84% to control cells.⑤Using 200 ng/ml VEGF-C as chemotaxis source, pLEC presents higher response to chemotaxis compared with control and siRNA groups.⑥The integrinα9 mRNA was not significantly changed in pLEC strain, butβ1 subunit mRNA was increased (P<0.05); in siRNA group, all integrinα9β1 mRNA was downregulated (P<0.05). Further investigation on translational level got similar results.3. Sema3F was demonstrated as a tumor suppressor in vitro and in vivo:①Sema3F expression significantly lowered the proliferative activity of SW480 cancer cell line (P<0.05), but shRNA targeting on Sema3F could attenuate this effects (P<0.05). When NRP2 was simultaneously transfected into SW480 and siRNA SW480, there was no change of proliferation affected by NRP2 expression;②The cell ratio at S-phase of cell cycle was significantly reduced in Sema3F overexpressing cells, siRNA could also reverse this effects. But the total apoptosis ratio was not changed in both cell transfectants.③Xenografts derived from Sema3F overexpressing cells had a lower growth rate and smaller tumor volumn; further studies found that these tumors had less activated cell proliferation using PCNA staining.④60 min after seeding, the cancer cells overexpressing Sema3F showed reduced adhesion capability compared with control cells and siRNA cells. But there was no significant change of adhesion when NRP2 level was modulated.⑤siRNA targeting on Sema3F leads to reduced cell migration compared with control and Sema3F overexpression groups;⑥Sema3F could induce the expression of integrinαvβ3, and siRNA could attenuate this effect.4. Sema3F expression has an inhibitory effect on xenograft lymphangiogenesis and metastasis:①There was no significance of xenograft MLD between Sema3F highly expressing cell formed xenografts and control xenografts. But when NRP2 was simultaneously elevated, Sema3F expression exhibited evident suppression of cancer lymphangiogenesis (P<0.05); on the contrary, siRNA targeting of Sema3F alone, resulted in much higher MLD in xenograft than control xenografts, while simultaneously NRP2 elevation resulted the highest MLD in resultant xenografts (P<0.05);②Overexpression of Sema3F also inhibited tumor metastasis, but this effect is NRP2-dependent. On the other side, when Sema3F expression was interfered, tumor metastasis raised evidently.Conclusion1. The loss or downregulation of Sema3F in colorectal cancer is tightly related to tumor progression. Overexpression of Sema3F in malignant cells leads to a reduced proliferation, migration, adhesion capabilities, resulted in tumor suppression and metastasis prevention.2. NRP2 expression enhances the proliferation, migration, chemotaxis and tubulogenesis capabilities of LECs and is considered as a prolymphangiogenic factors in NRP2 positive tumors.These results demonstrates that Sema3F as a candidate tumor suppressor has an inhibitory effect on proliferation and metastasis of colorectal carcinoma through its receptor NRP2.