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

Semaphorins in Angiogenesis and Tumor Progression

The semaphorins were initially described as axon guidance factors, but have recently been implicated in a variety of physiological and developmental functions, including regulation of immune response, angiogenesis, and migration of neural crest cells. The semaphorin family contains more than 30 genes divided into seven subfamilies, all of which are characterized by the presence of a sema domain. The semaphorins transduce their signals by binding to one of the nine receptors belonging to the plexin family, or, in the case of the class 3 semaphorins, by binding to one of the two neuropilin receptors. Additional receptors, which form complexes with these primary semaphorin receptors, are also frequently involved in semaphorin signaling. Recent evidence suggests that some semaphorins can act as antiangiogenic and/or antitumorigenic agents whereas other semaphorins promote tumor progression and/or angiogenesis. Furthermore, loss of endogenous inhibitory semaphorin expression or function on one hand, and overexpression of protumorigenic semaphorins on the other hand, is associated with the progression of some tumor types

Class-3 Semaphorins and Their Receptors: Potent Multifunctional Modulators of Tumor Progression

Semaphorins are the products of a large gene family containing 28 genes of which 21 are found in vertebrates. Class-3 semaphorins constitute a subfamily of seven vertebrate semaphorins which differ from the other vertebrate semaphorins in that they are the only secreted semaphorins and are distinguished from other semaphorins by the presence of a basic domain at their C termini. Class-3 semaphorins were initially characterized as axon guidance factors, but have subsequently been found to regulate immune responses, angiogenesis, lymphangiogenesis, and a variety of additional physiological and developmental functions. Most class-3 semaphorins transduce their signals by binding to receptors belonging to the neuropilin family which subsequently associate with receptors of the plexin family to form functional class-3 semaphorin receptors. Recent evidence suggests that class-3 semaphorins also fulfill important regulatory roles in multiple forms of cancer. Several class-3 semaphorins function as endogenous inhibitors of tumor angiogenesis. Others were found to inhibit tumor metastasis by inhibition of tumor lymphangiogenesis, by direct effects on the behavior of tumor cells, or by modulation of immune responses. Notably, some semaphorins such as sema3C and sema3E have also been found to potentiate tumor progression using various mechanisms. This review focuses on the roles of the different class-3 semaphorins in tumor progression

Knock-Out of the Five Lysyl-Oxidase Family Genes Enables Identification of Lysyl-Oxidase Pro-Enzyme Regulated Genes

The five lysyl-oxidase genes share similar enzymatic activities and contribute to tumor progression. We have knocked out the five lysyl-oxidase genes in MDA-MB-231 breast cancer cells using CRISPR/Cas9 in order to identify genes that are regulated by LOX but not by other lysyl-oxidases and in order to study such genes in more mechanistic detail in the future. Re-expression of the full-length cDNA encoding LOX identified four genes whose expression was downregulated in the knock-out cells and rescued following LOX re-expression but not re-expression of other lysyl-oxidases. These were the AGR2, STOX2, DNAJB11 and DNAJC3 genes. AGR2 and STOX2 were previously identified as promoters of tumor progression. In addition, we identified several genes that were not downregulated in the knock-out cells but were strongly upregulated following LOX or LOXL3 re-expression. Some of these, such as the DERL3 gene, also promote tumor progression. There was very little proteolytic processing of the re-expressed LOX pro-enzyme in the MDA-MB-231 cells, while in the HEK293 cells, the LOX pro-enzyme was efficiently cleaved. We introduced point mutations into the known BMP-1 and ADAMTS2/14 cleavage sites of LOX. The BMP-1 mutant was secreted but not cleaved, while the LOX double mutant dmutLOX was not cleaved or secreted. However, even in the presence of the irreversible LOX inhibitor β-aminoproprionitrile (BAPN), these point-mutated LOX variants induced the expression of these genes, suggesting that the LOX pro-enzyme has hitherto unrecognized biological functions

Plexin-A2 enables the proliferation and the development of tumors from glioblastoma derived cells

Abstract The semaphorin guidance factors receptor plexin-A2 transduces sema6A and sema6B signals and may mediate, along with plexin-A4, the anti-angiogenic effects of sema6A. When associated with neuropilins plexin-A2 also transduces the anti-angiogenic signals of sema3B. Here we show that inhibition of plexin-A2 expression in glioblastoma derived cells that express wild type p53 such as U87MG and A172 cells, or in primary human endothelial cells, strongly inhibits cell proliferation. Inhibition of plexin-A2 expression in U87MG cells also results in strong inhibition of their tumor forming ability. Knock-out of the plexin-A2 gene in U87MG cells using CRISPR/Cas9 inhibits cell proliferation which is rescued following plexin-A2 re-expression, or expression of a truncated plexin-A2 lacking its extracellular domain. Inhibition of plexin-A2 expression results in cell cycle arrest at the G2/M stage, and is accompanied by changes in cytoskeletal organization, cell flattening, and enhanced expression of senescence associated β-galactosidase. It is also associated with reduced AKT phosphorylation and enhanced phosphorylation of p38MAPK. We find that the pro-proliferative effects of plexin-A2 are mediated by FARP2 and FYN and by the GTPase activating (GAP) domain located in the intracellular domain of plexin-A2. Point mutations in these locations inhibit the rescue of cell proliferation upon re-expression of the mutated intracellular domain in the knock-out cells. In contrast re-expression of a plexin-A2 cDNA containing a point mutation in the semaphorin binding domain failed to inhibit the rescue. Our results suggest that plexin-A2 may represent a novel target for the development of anti-tumorigenic therapeutics

A Sema3C Mutant Resistant to Cleavage by Furin (FR-Sema3C) Inhibits Choroidal Neovascularization.

In age-related macular degeneration (AMD), abnormal sub retinal choroidal neovascularization (CNV) is a major cause of blindness. FR-sema3C is a point mutated form of semaphorin-3C that is resistant to cleavage by furin like pro-protein convertases (FPPC). We have found in previous work that FR-sema3C functions as an anti-angiogenic factor. In this study we investigated the possible use of FR-sema3C as an inhibitor of CNV. FR-sema3C inhibits VEGF as well as PDGF-BB signal transduction in endothelial cells and to less extent bFGF induced signal transduction using a mechanism that does not depend upon the binding of VEGF like the drugs that are currently the mainstay treatment for AMD. CNV was induced in eyes of C57 black mice by laser photocoagulation. Intravitreal injection of FR-Sema3C or aflibercept (VEGF-trap) was then used to inhibit CNV formation. Invading choroidal vessels were visualized a week later by injection of FITC-dextran into the circulation, followed by the measurement of the area of the invading blood vessels. Injection of 0.1 μg FR-Sema3C inhibited CNV by 55% (P<0.01) and was as effective as 5 μg aflibercept. FR-sema3C did not display any adverse effects on retinal function following its injection into eyes of healthy mice as assessed by optokinetic reflex (OKR) and Electro-retinogram (ERG) criteria. Furthermore, FR-sema3C did not induce apoptosis in the retina as determined by TUNEL nor was there any discernable structural damage to the retina as assessed by several immuno-histochemical criteria. Our results suggest that FR-sema3C could perhaps be used for the treatment of AMD, and that it may perhaps be of benefit to patients that do not respond well to current treatments relying on VEGF sequestering agents

Semaphorin3A: A Potential Therapeutic Tool for Lupus Nephritis

BackgroundThe immune regulatory properties of semaphorin3A (sema3A) (both innate and adaptive) are well established in many in vitro studies. The injection of sema3A into a mice model of rheumatoid arthritis was proven to be highly beneficial, both in attenuating clinical symptoms and in decreasing inflammatory mechanisms.ObjectivesThis study was designed in order to assess the possible therapeutic benefits of sema3A following its injection into female NZB/W mice.MethodsForty-eight NZB/W mice were recruited for this study. Thirty mice were treated as a “prevention group” and 18 were used as a “treatment group.” Eight-week-old mice were acclimated and then divided into the two abovementioned groups.ResultsThe injection of sema3A into young mice (at week 12) before the onset of disease (the prevention group) delayed the appearance of proteinuria. Here, the median time to severe proteinuria was 110 days, 95% CI: 88–131. However, in mice in which the empty vector was injected, the median time to severe proteinuria was 63 days, 95% CI: 0–139. sema3A treatment, significantly reduced renal damage, namely, it prevented the deposition of immune complexes in the glomeruli. When sema3A was injected at the onset of proteinuria (the treatment group), aiming to treat rather than to prevent disease in these mice, survival was increased and the deterioration of proteinuria was delayed.ConclusionSemaphorin3A is highly beneficial in reducing lupus nephritis in NZB/W mice. It delays the appearance and deterioration of proteinuria, and increases the survival rates in these mice. The regulatory mechanisms of sema3A involve both innate and adaptive immune responses. Further studies will establish the idea of applying sema3A in the treatment of lupus nephritis