Boaz Kigel

Successful Inhibition of Tumor Development by Specific Class-3 Semaphorins Is Associated with Expression of Appropriate Semaphorin Receptors by Tumor Cells

The class-3 semaphorins (sema3s) include seven family members. Six of them bind to neuropilin-1 (np1) or neuropilin-2 (np2) receptors or to both, while the seventh, sema3E, binds to the plexin-D1 receptor. Sema3B and sema3F were previously characterized as tumor suppressors and as inhibitors of tumor angiogenesis. To determine if additional class-3 semaphorins such as sema3A, sema3D, sema3E and sema3G possess anti-angiogenic and anti-tumorigenic properties, we expressed the recombinant full length semaphorins in four different tumorigenic cell lines expressing different combinations of class-3 semaphorin receptors. We show for the first time that sema3A, sema3D, sema3E and sema3G can function as potent anti-tumorigenic agents. All the semaphorins we examined were also able to reduce the concentration of tumor associated blood vessels although the potencies of the anti-angiogenic effects varied depending on the tumor cell type. Surprisingly, there was little correlation between the ability to inhibit tumor angiogenesis and their anti-tumorigenic activity. None of the semaphorins inhibited the adhesion of the tumor cells to plastic or fibronectin nor did they modulate the proliferation of tumor cells cultured in cell culture dishes. However, various semaphorins were able to inhibit the formation of soft agar colonies from tumor cells expressing appropriate semaphorin receptors, although in this case too the inhibitory effect was not always correlated with the anti-tumorigenic effect. In contrast, the anti-tumorigenic effect of each of the semaphorins correlated very well with tumor cell expression of specific signal transducing receptors for particular semaphorins. This correlation was not broken even in cases in which the tumor cells expressed significant concentrations of endogenous semaphorins. Our results suggest that combinations of different class-3 semaphorins may be more effective than single semaphorins in cases in which tumor cells express more than one type of semaphorin receptors.

Semaphorin-3B Is an Angiogenesis Inhibitor That Is Inactivated by Furin-Like Pro-Protein Convertases

Semaphorin-3B (sema3B) and semaphorin-3F (sema3F) are secreted tumor suppressors of lung cancer. Sema3F functions as an antiangiogenic factor that repels endothelial cells and compromises their proliferation/survival. However, tumor cells expressing either endogenous or recombinant sema3B fail to repel endothelial cells efficiently. Sema3B found in the conditioned medium of such cells is almost completely cleaved by furin-like pro-protein convertases, generating inactive 61- and 22-kDa fragments. We have generated a sema3B variant that was point mutated at the cleavage site (sema3B-m), thereby conferring partial resistance to cleavage. Conditioned medium from HEK293 cells expressing sema3b-m and conditioned medium of HEK293 cells expressing sema3B contained similar concentrations of semaphorin but sema3B-m was cleaved much less than sema3B. In contrast to HEK293 cells expressing native sema3B, cells expressing sema3b-m strongly repel endothelial cells. Conditioned medium from sema3B-m-expressing cells rapidly caused disassembly of focal adhesions and a collapse of the actin cytoskeleton of endothelial cells, inhibited vascular endothelial growth factor-induced phosphorylation of extracellular signal-regulated kinase 1/2, induced apoptosis of endothelial cells, and inhibited the formation of tubes from endothelial cells in an in vitro angiogenesis assay more potently than conditioned medium from cells expressing sema3B. Furthermore, HEK293 cells expressing sema3B-m inhibited basic fibroblast growth factor-induced angiogenesis in vivo much more potently than cells expressing sema3B. Repulsion of human umbilical vascular endothelial cells by sema3B-m was mediated primarily by the neuropilin-1 (np1) receptor but sema3B-m was also able to transduce signals via neuropilin-2 (np2). These results suggest that up-regulation of furin-like pro-protein convertases in malignant cells may enable tumors to evade the antiangiogenic effects of sema3B.

Plexin-A4 promotes tumor progression and tumor angiogenesis by enhancement of VEGF and bFGF signaling

Plexin-A4 is a receptor for sema6A and sema6B and associates with neuropilins to transduce signals of class-3 semaphorins. We observed that plexin-A1 and plexin-A4 are required simultaneously for transduction of inhibitory sema3A signals and that they form complexes. Unexpectedly, inhibition of plexin-A1 or plexin-A4 expression in endothelial cells using specific shRNAs resulted in prominent plexin type specific rearrangements of the actin cytoskeleton that were accompanied by inhibition of bFGF and VEGF-induced cell proliferation. The two responses were not interdependent since silencing plexin-A4 in U87MG glioblastoma cells inhibited cell proliferation and strongly inhibited the formation of tumors from these cells without affecting cytoskeletal organization. Plexin-A4 formed stable complexes with the FGFR1 and VEGFR-2 tyrosine-kinase receptors and enhanced VEGF-induced VEGFR-2 phosphorylation in endothelial cells as well as bFGF-induced cell proliferation. We also obtained evidence suggesting that some of the pro-proliferative effects of plexin-A4 are due to transduction of autocrine sema6B-induced pro-proliferative signals, since silencing sema6B expression in endothelial cells and in U87MG cells mimicked the effects of plexin-A4 silencing and also inhibited tumor formation from the U87MG cells. Our results suggest that plexin-A4 may represent a target for the development of novel anti-angiogenic and anti-tumorigenic drugs.

Tumour growth inhibition and anti‐metastatic activity of a mutated furin‐resistant Semaphorin 3E isoform

Secreted Semaphorin 3E (Sema3E) promotes cancer cell invasiveness and metastatic spreading. The pro‐metastatic activity of Sema3E is due to its proteolytic fragment p61, capable of transactivating the oncogenic tyrosine kinase ErbB2 that associates with the Sema3E receptor PlexinD1 in cancer cells. Here, we show that a mutated, uncleavable variant of Sema3E (Uncl‐Sema3E) binds to PlexinD1 like p61‐Sema3E, but does not promote the association of PlexinD1 with ErbB2 nor activates the ensuing signalling cascade leading to metastatic spreading. Furthermore, Uncl‐Sema3E competes with endogenous p61‐Sema3E produced by tumour cells, thereby hampering their metastatic ability. Uncl‐Sema3E also acts independently as a potent anti‐angiogenic factor. It activates a PlexinD1‐mediated signalling cascade in endothelial cells that leads to the inhibition of adhesion to extracellular matrix, directional migration and cell survival. The putative therapeutic potential of Uncl‐Sema3E was validated in multiple orthotopic or spontaneous tumour models in vivo, where either local or systemic delivery of Uncl‐Sema3E‐reduced angiogenesis, growth and metastasis, even in the case of tumours refractory to treatment with a soluble vascular endothelial growth factor trap. In summary, we conclude that Uncl‐Sema3E is a novel inhibitor of tumour angiogenesis and growth that concomitantly hampers metastatic spreading.

Semaphorin-3D and semaphorin-3E inhibit the development of tumors from glioblastoma cells implanted in the cortex of the brain.

Class-3 semaphorins are secreted axon guidance factors. Some of these semaphorins have recently been characterized as suppressors of tumor progression. To determine if class-3 semaphorins can be used to inhibit the development of glioblastoma-multiforme tumors, we expressed recombinant sema-3A, 3B, 3D, 3E, 3F or 3G in U87MG glioblastoma cells. Sema3A and sema3B expressing cells contracted and changed shape persistently while cells expressing other semaphorins did not. Sema3A and sema3F differed from other semaphorins including sema3B as they also inhibited the proliferation of the cells and the formation of soft agar colonies. With the exception of sema3G and sema3B, expression of these semaphorins in U87MG cells inhibited significantly tumor development from subcutaneously implanted cells. Strong inhibition of tumor development was also observed following implantation of U87MG cells expressing each of the class-3 semaphorins in the cortex of mouse brains. Sema3D and sema3E displayed the strongest inhibitory effects and their expression in U373MG or in U87MG glioblastoma cells implanted in the brains of mice prolonged the survival of the mice by more then two folds. Furthermore, most of the mice that died prior to the end of the experiment did not develop detectable tumors and many of the mice survived to the end of the experiment. Most of the semaphorins that we have used here with the exception of sema3D were characterized previously as inhibitors of angiogenesis. Our results indicate that sema3D also functions as an inhibitor of angiogenesis and suggest that the anti-tumorigenic effects are due primarily to inhibition of tumor angiogenesis. These results indicate that class-3 semaphorins such as sema3D and sema3E could perhaps be used to treat glioblastoma patients.

Abstract 3268: Targeting Semaphorin signaling to inhibit tumor growth and metastatic spreading

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The Semaphorins and their receptors, Neuropilins and Plexins, were originally implicated as guidance signals in the development of the central nervous system. However, a growing body of evidence indicates that semaphorins may regulate tumor progression, by promoting or inhibiting tumor angiogenesis, tumor cell survival and metastatic spreading, in fact mediating a crosstalk between tumor cells and multiple stromal cell types in the surrounding microenvironment. Here, we provide two examples of the multifaceted activity of semaphorins during tumor progression. We found that Semaphorin 3A (SEMA3A) inhibits primary tumor growth and its metastatic dissemination by employing three different experimental approaches in mouse tumor models: (i) over-expression of SEMA3A in tumor cells; (ii) systemic expression of SEMA3A following gene transfer in mice; (iii) tumor-targeted release of SEMA3A using gene-modified Tie2-expressing monocytes (TEMs) as delivery vehicles. In each of these experimental settings, SEMA3A efficiently inhibited tumor growth by disrupting vessel function and inducing tumor hypoxia and necrosis, without promoting metastatic dissemination. Moreover, we have recently shown that another secreted semaphorin known to regulate angiogenesis, Semaphorin 3E (SEMA3E), is abundantly expressed in metastatic human tumors, where it promotes cancer cell invasiveness and metastatic spreading. Notably, this biological activity of SEMA3E depends on a proteolytic conversion by furin-like convertases, generating the active fragment “p61”-SEMA3E. In cancer cells, p61-SEMA3E induces the association of the receptor PlexinD1 with the oncogenic tyrosine-kinase receptor ErbB2, leading to ErbB2 activation and enhanced tumor invasion and metastasis. Here we show that a mutated uncleavable variant of SEMA3E (Uncl-SEMA3E) binds the receptor PlexinD1 but it is unable to trigger ErbB2 signaling in cancer cells and lacks any pro-metastatic activity. On the other hand, Uncl-SEMA3E functions as a potent anti-angiogenic factor and displays striking tumor-suppressor properties in vivo. In summary, SEMA3A and Uncl-SEMA3E are potent inhibitors of tumor angiogenesis and tumor growth which concomitantly block the metastatic spreading of tumor cells. These data provide evidence that support interfering with semaphorin-mediated signals as a promising anti-cancer strategy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3268. doi:10.1158/1538-7445.AM2011-3268