SEMA3B is a candidate tumor suppressor because it frequently undergoes allele loss and reduced expression or function in lung and breast cancers (usually through epigenetic mechanisms), whereas reexpression decreases cell proliferation and induces apoptosis in lung, breast, and ovarian cancer cells (3
). Recently, our laboratory showed that SEMA3B exerts its tumor suppressor effects through competition with VEGF165
). This presumably occurs because SEMA3B and VEGF165
compete for neuropilin receptors on the tumor cell surface (1
). VEGF-A has been shown to promote tumor cell proliferation and survival in an autocrine manner through binding Np-1. Thus, a reasonable hypothesis is that SEMA3B mediates its tumor-suppressing effects, in part, by blocking VEGF-A autocrine activity in tumor cells. VEGF-A signaling includes activation of the PI3K/Akt pathway (32
). The link between the PI3K/Akt pathway and semaphorin-induced signaling in tumor cells has been previously suggested by the studies with SEMA3F, another member of the semaphorin family. SEMA3F, a protein with similar tumor suppressor properties as those of SEMA3B, is located in the 3p21.3 region that is ~75 kb away from SEMA3B (33
). SEMA3F has been shown to antagonize neurotrophin-induced PI3K and mitogen-activated protein kinase kinase signaling in sympathetic neurons leading to growth cone collapse (9
). Reintroduction of SEMA3F in H157 NSCLC cells suppressed tumor cell density and growth as well as ILK-extracellular signal-regulated kinase 1/2 and Akt/signal transducer and activator of transcription 3 signaling (35
). In the present report, we tested the hypothesis that SEMA3B, a member of the same semaphorin family, exerts similar tumor-inhibitory effects by binding Np-1 receptor that induces signals suppressing the PI3K/Akt pathway activation. The results of the studies reported here showed that exogenously added SEMA3B inhibits the PI3K/Akt pathway acting through Np-1 receptors in cancer cells, leading to decreased cell survival and increased apoptosis. Our studies showed that SEMA3B decreased phosphorylation of Akt and multiple other proteins related to the Akt pathway, including p85 (PI3K complex), PDK, PTEN, GSK-3β, FKHR, and MDM-2. These Akt pathway effects were observed in NSCLC and breast cancer cells that are sensitive to the antiproliferative effect of SEMA3B. By contrast, SEMA3B-driven changes in Akt phosphorylation or related pathway changes did not occur in SEMA3B-resistant tumor cells or in cells treated with a SEMA3B missense mutant protein, indicating the specificity of the effect of SEMA3B. The reduced level of Akt phosphorylation in sensitive cells after SEMA3B-CM treatment is associated with diminished Akt capacity to phosphorylate GSK-3α/β substrate. We confirmed these observations by the introduction of pAktDD, which overcame the SEMA3B effects on NSCLC cells. Furthermore, exogenous expression of Np-1 in ZR-75 cells (Np-1 negative) and knockdown of Np-1 in MDA-MB-231 cells (Np-1 positive) show that in these cells Np-1 mediates SEMA3B effects on the Akt pathway. However, the fact that SEMA3B-resistant lines H2009, H23, and H1993 express Np-1 suggests that Np-1 is necessary but not sufficient for SEMA3B to exert its effect on cell survival, inhibition of Akt kinase activity, and subsequently reduction of phosphorylation of survival-promoting proteins. The mechanism of SEMA3B resistance will be of great interest to unravel but, as described in Results, these “SEMA3B-resistant” tumor lines provide important specificity controls for all of our experiments. Thus, despite these exceptions between Np-1 expression and response to SEMA3B, in a major subset of NSCLC and breast cancer cells, the addition of SEMA3B, at picomolar concentrations, functions through Np-1 to inactivate Akt signaling activity, consequently decreasing proliferation and inducing apoptosis.
Np-1 is expressed at a high level in tumors of epithelial origin (carcinomas), whereas tumors from different origin such as melanomas strongly express Np-2 (36
). Increased expression of Np-1 and Np-2 correlates with tumor aggressiveness, advanced disease stage, and poor prognosis (39
). In contrast, several studies have reported that Np-1 overexpression in tumor cells reduces motility and tumorigenicity or prolonged patient survival (42
). Possible reasons for discrepancies between various studies may include the expression of functional receptor/ligand repertoire (e.g., plexins) and/or the ratio of VEGF-A to SEMA3 within the particular tumor microenvironment. Np-1 internalizes on ligand binding at different rates depending on a ligand type (46
). Preferential receptor binding and ligand-mediated internalization suggest a mechanism by which a common receptor, Np-1, can function in prioritizing conflicting signals (46
). These effects of neuropilins suggest their potential as targets for antitumor therapy.
The Akt pathway is activated in a variety of carcinomas of lung, breast, colon, and pancreatic and other tumor types through overexpression of Akt protein. Additionally, KRAS, EGFR
, and PI3K
mutations activate this pathway as well as loss of negative regulatory proteins such as PTEN that shuts down the PI3K pathway (47
). In the clinical setting, aberrations in the Akt pathway are associated with poor prognosis and a decreased patient survival rate after combined radiotherapy and chemotherapy (25
). Recent reports have shown that radiation activates the Akt pathway, which may promote endothelial cell survival (48
). Moreover, radiation-induced Akt activation also increased cell migration and subsequently metastasis in animal models of breast cancer and in cultured pancreatic carcinoma cells (49
). These studies collectively show the effect that the Akt pathway exerts on tumorigenesis, tumor angiogenesis, tumor progression, and metastasis as well as tumor responses to anticancer therapies. Drugs that negatively regulate the Akt pathway in either tumor cells or angiogenic endothelial cells could be of therapeutic value. In our experimental model, exogenously added SEMA3B induces apoptosis, in part, by inhibiting the Akt pathway in lung and breast cancer cells. SEMA3B effect on phosphorylation starts with the upstream protein, PI3K, and continues down the Akt pathway also affecting downstream proteins, such as GSK-3β, FKHR, and MDM-2. The inhibitory effect of SEMA3B on the Akt pathway in tumor cells with numerous genetic and epigenetic changes and the role of Np-1 in this effect suggest SEMA3B as a potential novel systemic anticancer therapeutic agent. Moreover, given the important role of Akt in cell survival, SEMA3B down-regulation of Akt activity might also sensitize tumor cells to chemotherapy, radiotherapy, or other types of therapy. Based on the data reported here, further assessment of antitumor and antimetastatic effects of soluble SEMA3B is warranted.