Plexins as Regulators of Cancer Cell Proliferation, Migration, and Invasivity.

Plexins are a family of nine single-pass transmembrane receptors with a conserved GTPase activating protein (GAP) domain. The plexin family is divided into four subfamilies: Type-A, type-B, type-C, and type-D plexins. Plexins function as receptors for axon guidance factors of the semaphorin family. The semaphorin gene family contains 22 genes that are divided into eight subclasses of which subclasses three to seven represent vertebrate semaphorins. The plexins and their semaphorin ligands have important roles as regulators of angiogenesis, cancer proliferation, and metastasis. Class 3 semaphorins, with the exception of sema3E, are the only semaphorins that do not bind directly to plexins. In order to transduce their signals, they bind instead to complexes consisting of receptors of the neuropilin family and various plexins. Some plexins also form complexes with tyrosine-kinase receptors such as the epidermal growth factor receptor ErbB2, the mesenchymal epithelial transition factor receptor (MET), and the Vascular endothelial growth factor receptor 2 (VEGFR2) and, as a result, can modulate cell proliferation and tumor progression. This review focuses on the roles of the different plexins in the control of cancer cell proliferation and invasiveness. Plexins also affect tumor progression and tumor metastasis by indirect mechanisms, such as modulation of angiogenesis and immune responses. However, these topics are not covered in the present review.

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

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 ß-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.

Lysyl Oxidase Family Enzymes and Their Role in Tumor Progression.

The five genes of the lysyl oxidase family encode enzymes that covalently cross-link components of the extracellular matrix, such as various types of collagen and elastin, and, thus, promote the stabilization of extracellular matrixes. Several of these genes, in particular lysyl oxidase (LOX) and lysyl oxidase like-2 (LOXL2) were identified as genes that are upregulated by hypoxia, and promote tumor cells invasion and metastasis. Here, we focus on the description of the diverse molecular mechanisms by which the various lysyl oxidases affect tumor progression. We also describe attempts that have been made, and are still on-going, that focus on the development of efficient lysyl oxidase inhibitors for the treatment of various forms of cancer, and of diseases associated with abnormal fibrosis.

Semaphorin 3A Is Effective in Reducing Both Inflammation and Angiogenesis in a Mouse Model of Bronchial Asthma.

Semaphorin 3A (sema3A) belongs to the sub-family of the immune semaphorins that function as regulators of immune-mediated inflammation. Sema3A is a membrane associated molecule on T regulatory cells and on B regulatory cells. Being transiently ligated to the cell surface of these cells it is suggested to be a useful marker for evaluating their functional status. In earlier studies, we found that reduced sema3A concentration in the serum of asthma patients as well as reduced expression by Treg cells correlates with asthma disease severity. Stimulation of Treg cells with recombinant sema3A induced a significant increase in FoxP3 and IL-10 expression. To find out if sema3A can be of benefit to asthma patients, we evaluated the effect of sema3A injection in a mouse model of asthma. BALB\c-mice were sensitized using ovalbumin (OVA) + adjuvant for 15 days followed by OVA aerosol inhalation over five consecutive days. Four hours following air ways sensitization on each of the above days- 15 of these mice were injected intraperitoneally with 50 µg per mouse of recombinant human sema3A-FR and the remaining 15 mice were injected with a similarly purified vehicle. Five days later the mice were sacrificed, broncheo-alveolar lavage (BAL) was collected and formalin-fixed lung biopsies taken and analyzed. In sema3A treated mice, only 20% of the bronchioles and arterioles were infiltrated by inflammatory cells as compared to 90% in the control group (p = 0.0079). In addition, eosinophil infiltration was also significantly increased in the control group as compared with the sema3A treated mice. In sema3A treated mice we noticed only a small number of mononuclear and neutrophil cells in the BAL while in the control mice, the BAL was enriched with mononuclear and neutrophil cells. Finally, in the control mice, angiogenesis was significantly increased in comparison with sema3A treated mice as evidenced by the reduced concentration of microvessels in the lungs of sema3A treated mice. To conclude, we find that in this asthma model, sema3A functions as a potent suppressor of asthma related inflammation that has the potential to be further developed as a new therapeutic for the treatment of asthma.

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

Abstract: 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.

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.

The semaphorins and their receptors as modulators of tumor progression.

The semaphorins were initially characterized as repulsive axon guidance factors. However, they are currently also recognized as important regulators of diverse biological processes which include regulation of immune responses, angiogenesis, organogenesis, and a variety of additional physiological and developmental functions. The semaphorin family consists of more than 20 genes divided into seven subfamilies, all of which contain the sema domain signature. They usually transduce signals by activation of receptors belonging to the plexin family, either directly, or indirectly following the binding of some semaphorins to receptors of the neuropilin family which subsequently associate with plexins. Additional receptors which form complexes with these primary semaphorin receptors are also frequently involved in semaphorin signalling, and can strongly influence the nature of the biological responses of cells to semaphorins. Recent evidence suggests that semaphorins play important roles in the etiology of multiple forms of cancer. Some semaphorins such as some semaphorins belonging to the class-3 semaphorin subfamily, have been found to function as bona fide tumor suppressors and to inhibit tumor progression by various mechanisms. Because these class-3 semaphorins are secreted proteins, these semaphorins may potentially be used as anti-tumorigenic drugs. Other semaphorins, such as semaphorin-4D, function as inducers of tumor progression and represent targets for the development of novel anti-tumorigenic drugs. The mechanisms by which semaphorins affect tumor progression are diverse, ranging from direct effects on tumor cells to modulation of accessory processes such as modulation of immune responses and inhibition or promotion of tumor angiogenesis and tumor lymphangiogenesis. This review focuses on the diverse mechanisms by which semaphorins affect tumor progression.

A SEMA3E mutant resistant to cleavage by furins (UNCL-SEMA3E) inhibits choroidal neovascularization.

Abnormal subretinal choroidal neovascularization (CNV) is a major cause of blindness in exudative age-related macular degeneration (AMD). Current anti-angiogenic treatments by VEGF sequestering agents have been successful, but a significant proportion of patients do not respond well to these treatments, and the response of others diminishes over time, suggesting that additional anti-angiogenic agents that function by separate mechanisms may be of use to such patients. We have previously found that a point mutated form of semaphorin-3E resistant to cleavage by furin like pro-protein convertases (UNCL-Sema3E) displays potent anti-angiogenic properties. We therefore determined if UNCL-Sema3E has potential as an inhibitor of CNV formation. We chose to study UNCL-Sema3E rather than wild type sema3E because unlike full length sema3E, the major p61-Sema3E peptide that is produced by cleavage of sema3E with furin like pro-protein convertases activates signal transduction mediated by the ErbB2 receptor and can promote tumor metastasis in addition to its anti-angiogenic activity. UNCL-Sema3E inhibited efficiently vascular endothelial growth factor-A (VEGF), platelet derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) signaling in human umbilical vein derived endothelial cells (HUVEC) and to a lesser extent hepatocyte growth factor (HGF) signal transduction. CNV that was induced in the eyes of C57 black mice by laser photocoagulation was inhibited by 65% (P < 0.01) following a single bolus intra-vitreal injection of 5 µg UNCL-Sema3E. This inhibitory effect was similar to the inhibition produced by a single bolus intra-vitreal injection of 5 µg aflibercept. A similar inhibition of CNV was observed following the injection of UNCL-Sema3E into the eyes of Long-Evans rats. However, a higher dose of UNCL-Sema3E (125 µg), partially due to the larger volume of the vitreous cavity of rats, was required to achieve maximal inhibition of CNV. Injection of UNCL-Sema3E into eyes of healthy mice did not have any adverse effect on retinal function as assessed by optic kinetic reflex (OKR) or by electroretinogram (ERG) assays nor did UNCL-Sema3E injection affect the structure of the retina as determined using histology. To conclude, our results suggest that UNCL-Sema3E may be useful for the treatment of exudative AMD, which does not respond well to conventional anti-VEGF therapy.

The role of the semaphorins in cancer.

The semaphorins were initially characterized as axon guidance factors, but have subsequently been implicated also in the regulation of immune responses, angiogenesis, organ formation, and a variety of additional physiological and developmental functions. The semaphorin family contains more then 20 genes divided into 7 subfamilies, all of which contain the signature sema domain. The semaphorins transduce signals by binding to receptors belonging to the neuropilin or plexin families. Additional receptors which form complexes with these primary semaphorin receptors are also frequently involved in semaphorin signaling. Recent evidence suggests that semaphorins also fulfill important roles in the etiology of multiple forms of cancer. Some semaphorins have been found to function as bona-fide tumor suppressors and to inhibit tumor progression by various mechanisms while other semaphorins function as inducers and promoters of tumor progression.