Plexin-B1 Expression Research Articles

The metastasis-promoting P1597L mutation in PlexinB1 enhances Ras activity

BackgroundMetastatic prostate cancer is a leading cause of cancer-related morbidity and mortality in men, yet the underlying molecular mechanisms are poorly understood. Plexins are transmembrane receptors for semaphorins with divergent roles in many forms of cancer. We recently found that a single clinically relevant specific amino acid change (Proline1597Leucine, (P1597L)), found in metastatic deposits of prostate cancer patients, converts PlexinB1 from a metastasis suppressor to a gene that drives prostate cancer metastasis in vivo. However, the mechanism by which PlexinB1(P1597L) promotes metastasis is not known.MethodsPull down assays using GST-RalGDS or -GSTRaf1-RBD were used to reveal the effect of mutant or wild-type PlexinB1 expression on Rap and Ras activity respectively. Protein–protein interactions were assessed in GST pulldown assays, Akt/ERK phosphorylation by immunoblotting and protein stability by treatment with cycloheximide. Rho/ROCK activity was monitored by measuring MLC2 phosphorylation and actin stress fiber formation. PlexinB1 function was measured using cell-collapse assays.ResultsWe show here that the single clinically relevant P1597L amino acid change converts PlexinB1 from a repressor of Ras to a Ras activator. The PlexinB1(P1597L) mutation inhibits the RapGAP activity of PlexinB1, promoting a significant increase in Ras activity. The P1597L mutation also blocks PlexinB1-mediated reduction in Rho/ROCK activity, restraining the decrease in MLC2 phosphorylation and actin stress fiber formation induced by overexpression of wild-type PlexinB1. PlexinB1(P1597L) has little effect on the interaction of PlexinB1 with small GTPases or receptor tyrosine kinases and does not inhibit PlexinB1-stimulated Akt or ERK phosphorylation. These results indicate that the mutation affects Rho signalling via the Rap/Ras pathway. The PlexinB1(P1597L) mutation inhibits morphological cell collapse induced by wild-type PlexinB1 expression, suggesting that the mutation induces a loss of an inhibitory tumour suppressor function.ConclusionThese results suggest that the clinically relevant P1597L mutation in PlexinB1 may transform PlexinB1 from a suppressor to a driver of metastasis in mouse models of prostate cancer by reducing the RapGAP activity of PlexinB1, leading to Ras activation. These findings highlight the PlexinB1-Rap-Ras pathway for therapeutic intervention in prostate cancer.

Inhibition of the Semaphorin 4D-Plexin-B1 axis prevents calcification in vascular smooth muscle cells

Vascular calcification is common in cardiovascular diseases including atherosclerosis, and is associated with an increased risk of pathological events and mortality. Some semaphorin family members play an important role in atherosclerosis. In the present study, we show that Semaphorin 4D/Sema4D and its Plexin-B1 receptor were significantly upregulated in calcified aorta of a rat chronic kidney disease model. Significantly higher Sema4D and Plexin-B1 expression was also observed during inorganic phosphate-induced calcification of vascular smooth muscle cells. Knockdown of Sema4D or Plexin-B1 genes attenuated both the phosphate-induced osteogenic phenotype of vascular smooth muscle cells, through regulation of SMAD1/5 signaling, as well as apoptosis of vascular smooth muscle cells, through modulation of the Gas6/Axl/Akt survival pathway. Taken together, our results offer new insights on the role of Sema4D and Plexin-B1 as potential therapeutic targets against vascular calcification. [BMB Reports 2023; 56(3): 160-165].

Increased BMSC exosomal miR-140-3p alleviates bone degradation and promotes bone restoration by targeting Plxnb1 in diabetic rats

BackgroundDiabetes mellitus (DM) is considered to be an important factor for bone degeneration disorders such as bone defect nonunion, which is characterized by physical disability and tremendous economy cost to families and society. Exosomal miRNAs of BMSCs have been reported to participate in osteoblastogenesis and modulating bone formation. However, their impacts on the development of bone degeneration in DM are not yet known. The role of miRNAs in BMSCs exosomes on regulating hyperglycemia bone degeneration was investigated in the present study.ResultsThe osteogenic potential in bone defect repair of exosomes derived from diabetes mellitus BMSCs derived exosomes (DM-Exos) were revealed to be lower than that in normal BMSCs derived exosomes (N-Exos) in vitro and in vivo. Here, we demonstrate that miR-140-3p level was significantly altered in exosomes derived from BMSCs, ADSCs and serum from DM rats. In in vitro experiments, upregulated miR-140-3p exosomes promoted DM BMSCs differentiation into osteoblasts. The effects were exerted by miR-140-3p targeting plxnb1, plexin B1 is the receptor of semaphoring 4D(Sema4D) that inhibited osteocytes differentiation, thereby promoting bone formation. In DM rats with bone defect, miR-140-3p upregulated exosomes were transplanted into injured bone and accelerated bone regeneration. Besides, miR-140-3p in the exosomes was transferred into BMSCs and osteoblasts and promoted bone regeneration by targeting the plexin B1/RohA/ROCK signaling pathway.ConclusionsNormal-Exos and miR-140-3p overexpressed-Exos accelerated diabetic wound healing by promoting the osteoblastogenesis function of BMSCs through inhibition plexin B1 expression which is the receptor of Sema4D and the plexin B1/RhoA/ROCK pathway compared with diabetes mellitus-Exos. This offers a new insight and a new therapy for treating diabetic bone unhealing.Graphical

PLXNB1 mutations in the etiology of idiopathic hypogonadotropic hypogonadism.

Idiopathic hypogonadotropic hypogonadism (IHH) comprises a group of rare genetic disorders characterized by pubertal failure caused by gonadotropin-releasing hormone (GnRH) deficiency. Genetic factors involved in semaphorin/plexin signaling have been identified in patients with IHH. PlexinB1, a member of the plexin family receptors, serves as the receptor for semaphorin 4D (Sema4D). In mice, perturbations in Sema4D/PlexinB1 signaling leads to improper GnRH development, highlighting the importance of investigating PlexinB1 mutations in IHH families. In total, 336 IHH patients (normosmic IHH, n=293 and Kallmann syndrome, n=43) from 290 independent families were included in the present study. Six PLXNB1 rare sequence variants (p.N361S, p.V608A, p.R636C, p.V672A, p.R1031H, and p.C1318R) are described in eight normosmic IHH patients from seven independent families. These variants were examined using bioinformatic modeling and compared to mutants reported in PLXNA1. Based on these analyses, the variant p.R1031H was assayed for alterations in cell morphology, PlexinB1 expression, and migration using a GnRH cell line and Boyden chambers. Experiments showed reduced membrane expression and impaired migration in cells expressing this variant compared to the wild-type. Our results provide clinical, genetic, molecular/cellular, and modeling evidence to implicate variants in PLXNB1 in the etiology of IHH.

PlexinB1 Promotes Nuclear Translocation of the Glucocorticoid Receptor.

Androgen receptor (AR) and glucocorticoid receptor (GR) are nuclear receptors whose function depends on their entry into the nucleus where they activate transcription of an overlapping set of genes. Both AR and GR have a role in resistance to androgen deprivation therapy (ADT), the mainstay of treatment for late stage prostate cancer. PlexinB1, a receptor for semaphorins, has been implicated in various cancers including prostate cancer and has a role in resistance to ADT. We show here that activation of PlexinB1 by Sema4D and Sema3C results in translocation of endogenous GR to the nucleus in prostate cancer cells, and that this effect is dependent on PlexinB1 expression. Sema4D/Sema3C promotes the translocation of GR-GFP to the nucleus and mutation of the nuclear localization sequence (NLS1) of GR abrogates this response. These findings implicate the importin α/β system in the Sema4D/Sema3C-mediated nuclear import of GR. Knockdown of PlexinB1 in prostate cancer cells decreases the levels of glucocorticoid-responsive gene products and antagonizes the decrease in cell motility and cell area of prostate cancer cells upon dexamethasone treatment, demonstrating the functional significance of these findings. These results show that PlexinB1 activation has a role in the trafficking and activation of the nuclear receptor GR and thus may have a role in resistance to androgen deprivation therapy in late stage prostate cancer.

Plexin B1 expression on human Treg cells is required for regulation of their phenotypic stability by semaphorin 4A

Abstract We previously reported that neuroimmune semaphorin (Sema) 4A regulates the severity of experimental allergic asthma and increases regulatory T-cell (Treg) numbers in vivo, however the mechanisms of Sema4A action remain unknown. It was also reported that Sema4A controls murine Treg cell function and survival acting through Neuropilin 1 (NRP-1) receptor. To clarify Sema4A action on human T cells, we employed T-cell lines, HuT78 and HuT102, as well as human PBMCs and CD4+ T cells in phenotypic and functional assays. We found that HuT78 demonstrated a T-effector-like phenotype (CD4+CD25lowFoxp3-), while HuT102 expressed a Treg-like phenotype (CD4+CD25hi Foxp3+). Neither cell line expressed NRP-1. However, HuT102 cells expressed high levels of the Sema4A-counter receptor, Plexin B1 whereas HuT78 cells demonstrated high cell-surface expression of Sema4A. All human peripheral blood CD4+ T cells, including Treg cells, expressed PlexinB1 and lacked both, NRP-1 and −2. Culture of HuT cells with soluble Sema4A led to an upregulation of CD25 and Foxp3 markers on HuT102 cells. Addition of soluble Sema4A increased the relative numbers of CD4+CD25+Foxp3+ cells in PBMC and CD4+ T cells which did not express NRP-1 but were PlexinB1+, suggesting the role of this receptor in Treg cell stability. The inclusion of anti-PlexinB1 blocking Ab in cultures before rSema4A addition significantly decreased Treg cell numbers as compared to cultures with rSema4A alone. Sema4A was as effective as TGF-b in iTreg induction from CD4+CD25depleted cells but did not affect Treg cell suppressive activity in vitro. These results suggest strategies for the development of new Sema4A-based therapeutic measures to combat allergic inflammatory diseases.

Role of Plexin B1 in a Breast Cancer Cohort of Pakistani Patients and its Contribution Towards Cancer Metastasis as Indicated by an In Vitro Model.

In the current study, the role of plexin B1 in breast cancer metastasis was explored. Freshly-excised tumours along with background tissues of affected patients (n=121) were collected from Pakistani hospitals and processed for RNA isolation and cDNA synthesis. Using quantitative polymerase chain reaction, expression of plexin B1 was evaluated and correlated with clinicopathological parameters. Furthermore, involvement of plexin B1 in metastasis was explored by generating gene knockdown in MDA-MB-231 and MCF-7 breast cancer cells. Poorly-differentiated tumours showed low plexin B1 expression in comparison to well-differentiated ones. Similarly, reduced plexin B1 expression correlated positively with advanced tumour stage and metastasis. Loss of plexin B1 significantly reduced cell adhesion in comparison with respective control cell lines (p<0.05). Knockdown of plexin B1 in MDA-MB-231 cells led to a remarkable increase in cell motility in contrast to the respective control. Loss of plexin B1 expression might play a pivotal role in enhancing the metastatic potential of breast cancer cells.

Endometrial expression of β3 integrin, calcitonin and plexin-B1 in the window of implantation in women with unexplained infertility

Endometrial receptivity plays a key role in the establishment of successful implantation and its impairment may contribute to subfertility and limit the assisted reproduction techniques (ART) success. The aim of present study was to investigate endometrial receptivity in terms of β3 integrin, calcitonin and plexin-B1 expression in women with unexplained infertility. We evaluated expression of β3 integrin, calcitonin and plexin-B1 through mRNA level measurement with real-time RT-PCR, in the endometrium of 16 infertile women with unexplained infertility and 10 fertile women. Endometrial biopsies were collected during a single menstrual cycle on postovulatory day LH+7 in each subject. Significant differences regarding β3 integrin and calcitonin expression levels found between patients with unexplained infertility and the fertile women. Endometrial plexin-B1 expression levels showed no significant difference between fertile and infertile women. There were significant correlations between expression of β3 integrin with calcitonin and plexin-B1 in fertile and infertile women. Reduced in endometrial expression of β3 integrin and calcitonin alone or together may contribute to unexplained infertility and these genes could account as the potential molecular markers of infertility.

Identification of TMPRSS2-ERG mechanisms in prostate cancer invasiveness: Involvement of MMP-9 and plexin B1.

The relationship of TMPRSS2-ERG fusion gene with matrix metalloproteinase-9 (MMP-9) and PLXNB1 (plexin B1) in regulation of prostate cancer (PCa) aggressiveness was investigated. Fluorescence in situ hybridization (FISH) assays, qRT-PCR and western blot analysis were employed to detect the expression of TMPRSS2-ERG fusion gene, ERG, MMP-9 and PLXNB1 of 135 human tissues, which included 55 metastatic PCa cases, 50 localized PCa cases and 30 BPH cases. Then using siRNA (anti-ERG, MMP-9 and PLXNB1, respectively) downregulation of the target gene of VCaP and PC-3 cells, MTT and Transwell were performed. The results showed that the positive rate of TMPRSS2-ERG fusion was 38.1% (40/105) in total PCa samples, 47.3% (26/55) of metastatic PCa, 28.0%(14/50) of localized PCa, while 0.0% (0/30) in BPH samples. The mRNA and protein expression of ERG, MMP-9 and PLXNB1 were higher in metastatic PCa (P<0.0001), and the mRNA expression of the three genes were positively correlated with TMPRSS2-ERG fusionin PCa group (P<0.0001). siRNA transfected PCa cells can effectively downregulate the target gene expression, and we identified that MMP-9 and PLXNB1 expression were all regulated by TMPRSS2-ERG fusion gene. While only PLXNB1 contributed to TMPRSS2-ERG mediated enhancements of VCaP cell migration and invasion. The results demonstrated that PLXNB1, but not MMP-9, was the target gene directly related to TMPRSS2-ERG in PCa cell migration and invasion.

Plexin-B1 indirectly affects glioma invasiveness and angiogenesis by regulating the RhoA/αvβ3 signaling pathway and SRPK1.

Gliomas are one of the most common primary brain tumors in adults. They display aggressive invasiveness, are highly vascular, and have a poor prognosis. Plexin-B1 is involved in numerous cellular processes, especially cellular migration and angiogenesis. However, the role and regulatory mechanisms of Plexin-B1 in gliomas are not understood and were thus investigated in this study. By using multiple and diverse experimental techniques, we investigated cell apoptosis, mitochondrial membrane potential, cell migration and invasion, angiogenesis, PI3K and Akt phosphorylation, and also the levels of SRPK1 and αvβ3 in glioma cells and animal glioma tissues. The results indicated that Plexin-B1 expression in glioma cell lines is increased compared to normal human astrocytes. Plexin-B1 mediates RhoA/integrin αvβ3 involved in the PI3K/Akt pathway and SRPK1 to influence the growth of glioma cell, angiogenesis, and motility in vitro and in vivo. Thus, Plexin-B1 signaling regulates the Rho/αvβ3/PI3K/Akt pathway and SRPK1, which are involved in glioma invasiveness and angiogenesis. Therefore, the new drug research should focus on Plexin-B1 as a target for the treatment of glioma invasion and angiogenesis.

Plexin-B1 and semaphorin 4D cooperate to promote cutaneous squamous cell carcinoma cell proliferation, migration and invasion

Background objectivesSemaphorin 4D (Sema4D) and its receptor, Plexin-B1, are involved in the pathogenesis of squamous cell carcinoma (SCC) by mediating angiogenesis or perineural invasion through the interaction between Sema4D expression on SCC cells and Plexin-B1 expression on endothelial cells or nerves. Plexin-B1 was also recently found to be expressed on SCC cells. Plexin-B1 expression on several types of tumor cells could mediate various, and occasionally opposing, effects, including tumor cell survival, proliferation, angiogenesis, invasion, and metastasis. However, whether Sema4D exerts paracrine or autocrine effects on SCC via Plexin-B1 remains unclear. ObjectivesThe aim of this study is to explore the effects of Sema4D/Plexin-B1 interaction on SCC via Plexin-B1 expressed on the tumor cells. MethodsIn the present study, we detected the expression of Plexin-B1 and Sema4D in cutaneous SCC (cSCC) tissues and in the cSCC cell line A431 and analyzed the effects of the Sema4D/Plexin-B1 interaction on cSCC cell proliferation, migration, and invasion, as well as on the signaling pathway downstream of Plexin-B1. ResultsWe observed significantly increased Plexin-B1 and Sema4D expression in keratinocytes in cSCC lesions and in A431 cells compared with that in normal skin tissue and in non-malignant keratinocytes. Plexin-B1 silencing reduced the growth, proliferation, migration, and invasion of A431 cells and inhibited the phosphorylation of Akt and extracellular signal-regulated protein kinase (Erk). Soluble recombinant Sema4D promoted the growth, proliferation, migration, and invasion of A431 cells; Akt and Erk phosphorylation is also involved in these processes with a Plexin-B1 dependent manner. ConclusionPlexin-B1 induces cSCC cell proliferation, migration, and invasion by interacting with Sema4D. Plexin-B1 might serve as a useful biomarker and/or as a novel therapeutic target for cSCC.

Semaphorin 4D induces vaginal epithelial cell apoptosis to control mouse postnatal vaginal tissue remodeling.

The opening of the mouse vaginal cavity to the skin is a postnatal tissue remodeling process that occurs at approximately five weeks of age for the completion of female genital tract maturation at puberty. The tissue remodeling process is primarily composed of a hormonally triggered apoptotic process predominantly occurring in the epithelium of the distal section of the vaginal cavity. However, the detailed mechanism underlying the apoptotic induction remains to be elucidated. In the present study, it was observed that the majority of BALB/c mice lacking the class 4 semaphorin, semaphorin 4D (Sema4D), developed imperforate vagina and hydrometrocolpos resulting in a perpetually unopened vaginal cavity regardless of a normal estrogen level comparable with that in wild-type (WT) mice. Administration of β-estradiol to infant Sema4D-deficient (Sema4D−/−) mice did not induce precocious vaginal opening, which was observed in WT mice subjected to the same β-estradiol administration, excluding the possibility that the closed vaginal phenotype was due to insufficient estrogen secretion at the time of vaginal opening. In order to assess the role of Sema4D in the postnatal vaginal tissue remodeling process, the expression of Sema4D and its receptor, plexin-B1, was examined as well as the level of apoptosis in the vaginal epithelia of five-week-old WT and Sema4D−/− mice. Immunohistochemical analyses confirmed the localization of Sema4D and plexin-B1 in the mouse vaginal epithelia. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunohistochemistry detecting activated caspase-3 revealed significantly fewer apoptotic cells in situ in the vaginal mucosa of five-week-old Sema4D−/− mice compared with WT mice. The addition of recombinant Sema4D to Sema4D−/− vaginal epithelial cells in culture significantly enhanced apoptosis of the vaginal epithelial cells, demonstrating the apoptosis-inducing activity of Sema4D. The experimental reduction of plexin-B1 expression in vaginal epithelial cells demonstrated the integral role of plexin-B1 in Sema4D-induced apoptotic cell death. These results suggest a non-redundant role of Sema4D in the postnatal tissue remodeling process in five-week-old BALB/c mice, which involves the induction of vaginal epithelial cell apoptosis through Sema4D binding to plexin-B1.

Changes of Temporomandibular Joint and Semaphorin 4D/Plexin-B1 Expression in a Mouse Model of Incisor Malocclusion

To investigate the changes in condylar cartilage and subchondral bone of the temporomandibular joint (TMJ) in a mouse model of incisor malocclusion. By bonding a single (single group) or a pair (pair group) of metal tube(s) to the left incisor(s), a crossbite-like relationship was created between left-side incisors in mice. The morphological changes in the TMJ condyles were examined by hematoxylin and eosin and toluidine blue staining. Indices of osteoclastic activity, including tartrate-resistant acid phosphatase (TRAP) staining and macrophage colony stimulating factor (M-CSF) were investigated by histochemistry or real-time polymerase chain reaction (PCR). The osteoblastic activity was indexed by osteocalcin expression. Expressions of semaphorin 4D and its receptor, Plexin-B1, were detected by real-time PCR. Two-way analysis of variance was used to assess the differences between groups. One week and 3 weeks after bonding the metal tube(s), cartilage degradation and subchondral bone loss were evident histologically. Both indices of osteoclastic activity (TRAP and M-CSF) were significantly increased in cartilage and subchondral bone after bonding the metal tube(s). Osteocalcin expression in cartilage was significantly increased at week 3, while its expression in subchondral bone was significantly increased at week 1 but decreased at week 3. The semaphorin 4D expression in cartilage and subchondral bone was significantly decreased at week 1 but significantly increased at week 3. For Plexin-B1 expression, a significant increase was detected in subchondral bone at week 3. Bonding a single or a pair of metal tube(s) to left incisor(s) is capable of inducing remodeling in the TMJ, which involved cartilage degradation and alteration of osteoclastic and osteoblastic activity.

Abstract 1245: Reduction of tumor growth and metastasis by a humanized IgG4 monoclonal antibody to SEMA4D (VX15/2503).

Abstract Semaphorin 4D (SEMA4D; CD100) has been implicated in several key mechanisms of tumor progression, including transactivation of several oncogenes, metastasis, tumor invasion, and neovascularization. Expression of SEMA4D and its receptor plexin-B1 (PLXNB1) correlates with invasive disease in humans. SEMA4D is over-expressed in a wide array of tumor types and is also produced by inflammatory cells recruited to the tumor microenvironment. SEMA4D binding to PLXNB1 on endothelial cells activates RhoA and AKT signaling pathways, which promotes formation of new blood vessels and tumor growth in vivo. In addition to its effects on endothelial cells, the interaction of PLXNB1 with MET and ERBB2 can lead to SEMA4D-mediated transactivation of these membrane receptor kinases with a direct effect on tumor cell migration and invasive growth. It is well known that tumor growth and metastasis involve a complex process of cross talk amongst the tumor cells, stroma and immune infiltrate, as well as the endothelial cells and vasculature. Our understanding of the role of SEMA4D in this process is evolving. We selected a humanized IgG4 antibody, VX15/2503, that blocks SEMA4D interaction with the high affinity receptor PLXNB1 and a lower affinity receptor, CD72, expressed on immune cells. The antibody binds with between 1-5 nM affinity to rat, mouse, primate, and human recombinant SEMA4D. Affinity to native cell-associated SEMA4D on primary human T cells was, however, determined to be 0.5 nM. We demonstrate that antibody-mediated SEMA4D neutralization delays tumor growth in several primary and metastatic in vivo models. Inhibition of SEMA4D regulates angiogenesis and vascular permeability in these models. Additionally, tumor growth delay results from modulation of the tumor microenvironment, such as infiltrating immune cells. We also demonstrate direct effects of SEMA4D/PLXNB1 interaction acting as a guidance signal for tumors, as previously described for axons, whereby tumor migration is affected ∼80%. Antibody blockade restores these functional activities. Moreover, using a genetic fingerprinting approach, we identified a unique gene signature that is related to changes in PLXNB1 expression and sensitivity to existing targeted therapies. This signature sheds light on combination therapies with anti-SEMA4D antibodies that may produce additive anti-tumor effects. In summary, blockade of SEMA4D reduces tumor growth through effects on tumor microenvironment, angiogenesis and vascular permeability, as well as direct effects on tumor. Therefore, antibody neutralization of SEMA4D represents a new therapeutic strategy for cancer treatment. The humanized antibody, VX15/2503, has successfully completed IND-enabling toxicology testing and a Phase I trial is currently being conducted in adult patients with advanced solid tumors. Citation Format: Elizabeth E. Evans, Alan S. Jonason, Mark Paris, Terrence L. Fisher, Sebold Torno, Holm Bussler, Jessica Decker, Maria Scrivens, He Huang, Laurie A. Winter, Tracy Pandina, Leslie Balch, Michael A. Doherty, Renee Kirk, Alan Howell, Jennifer Seils, Christine Reilly, Maurice Zauderer, John E. Leonard, Ernest S. Smith. Reduction of tumor growth and metastasis by a humanized IgG4 monoclonal antibody to SEMA4D (VX15/2503). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1245. doi:10.1158/1538-7445.AM2013-1245 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

ErbB-2 signals through Plexin-B1 to promote breast cancer metastasis

Diagnosis of metastatic breast cancer is associated with a very poor prognosis. New therapeutic targets are urgently needed, but their development is hampered by a lack of understanding of the mechanisms leading to tumor metastasis. Exemplifying this is the fact that the approximately 30% of all breast cancers overexpressing the receptor tyrosine kinase ErbB-2 are characterized by high metastatic potential and poor prognosis, but the signaling events downstream of ErbB-2 that drive cancer cell invasion and metastasis remain incompletely understood. Here we show that overexpression of ErbB-2 in human breast cancer cell lines leads to phosphorylation and activation of the semaphorin receptor Plexin-B1. This was required for ErbB-2-dependent activation of the pro-metastatic small GTPases RhoA and RhoC and promoted invasive behavior of human breast cancer cells. In a mouse model of ErbB-2-overexpressing breast cancer, ablation of the gene encoding Plexin-B1 strongly reduced the occurrence of metastases. Moreover, in human patients with ErbB-2-overexpressing breast cancer, low levels of Plexin-B1 expression correlated with good prognosis. Our data suggest that Plexin-B1 represents a new candidate therapeutic target for treating patients with ErbB-2-positive breast cancer.

Expression of neuroimmune semaphorins 4A and 4D and their receptors in the lung is enhanced by allergen and vascular endothelial growth factor

BackgroundSemaphorins were originally identified as molecules regulating a functional activity of axons in the nervous system. Sema4A and Sema4D were the first semaphorins found to be expressed on immune cells and were termed "immune semaphorins". It is known that Sema4A and Sema4D bind Tim-2 and CD72 expressed on leukocytes and PlexinD1 and B1 present on non-immune cells. These neuroimmune semaphorins and their receptors have been shown to play critical roles in many physiological and pathological processes including neuronal development, immune response regulation, cancer, autoimmune, cardiovascular, renal, and infectious diseases. However, the expression and regulation of Sema4A, Sema4D, and their receptors in normal and allergic lungs is undefined.ResultsAllergen treatment and lung-specific vascular endothelial growth factor (VEGF) expression induced asthma-like pathologies in the murine lungs. These experimental models of allergic airway inflammation were used for the expression analysis of immune semaphorins and their receptors employing immunohistochemistry and flow cytometry techniques. We found that besides accessory-like cells, Sema4A was also detected on bronchial epithelial and smooth muscle cells, whereas Sema4D expression was high on immune cells such as T and B lymphocytes. Surprisingly, under inflammation various cell types including macrophages, lymphocytes, and granulocytes in the lung expressed Tim-2, a previously defined marker for Th2 cells. CD72 was found on lung immune, inflammatory, and epithelial cells. Bronchial epithelial cells were positive for both plexins, whereas some endothelial cells selectively expressed Plexin D1. Plexin B1 expression was also detected on lung DC. Both allergen and VEGF upregulated the expression of neuroimmune semaphorins and their receptors in the lung tissue. However, the lung tissue Sema4A-Tim2 expression was rather weak, whereas Sema4D-CD72 ligand-receptor pair was vastly upregulated by allergen. Soluble Sema4D protein was present in the lung lysates and a whole Sema4A protein plus its dimer were readily detected in the bronchoalveolar (BAL) fluids under inflammation.ConclusionsThis study clearly shows that neuroimmune semaphorins Sema4A and Sema4D and their receptors might serve as potential markers for the allergic airway inflammatory diseases. Our current findings pave the way for further investigations of the role of immune semaphorins in inflammation and their use as potential therapeutic targets for the inflammatory lung conditions.

Plexin-B1 silencing inhibits ovarian cancer cell migration and invasion

BackgroundElevated Plexin-B1 expression has been found in diverse human cancers and in non-neoplastic tissues, and it mediates diverse biological and pathological activities. However, whether or not Plexin-B1 expression is involved in human ovarian tumors remains unclear. In the present study, Plexin-B1 expression was explored in benign and malignant human ovarian tumor tissues. In addition, the impact of Plexin-B1 expression on ovarian cancer cell proliferation, migration and invasion were investigated in vitro.MethodsPlexin-B1 expression was analyzed in normal and benign ovarian tissues and serous ovarian tumors (both borderline and malignant) by immunohistochemical staining, as well as in four human ovarian cancer cell lines (A2780, C13*, SKOV3, and OV2008) by RT-PCR and western blot analyses. Furthermore, endogenous Plexin-B1 expression was suppressed by Plexin-B1 siRNA in SKOV3 cells, which overexpress Plexin-B1. Protein levels of Plexin-B1, AKT and AKTSer473 were examined by western blot analysis. Cell proliferation, migration and invasion were measured with MTT, wound healing and boyden chamber assays, respectively, and the cytoskeleton was monitored via F-actin staining.ResultsExpression levels of Plexin-B1 protein were significantly higher in serous ovarian carcinomas than in normal ovaries or benign ovarian neoplasms, and in the former, Plexin-B1 expression was positively correlated with lymphatic metastasis, and the membrane and cytoplasm of cancer cells stained positively. SKOV3 cells displayed the highest Plexin-B1 expression at both the mRNA and protein levels among the four tested human ovarian cancer cell lines and was selected as a cell model for further in vitro experiments. Plexin-B1 siRNA significantly suppressed phosphorylation of AKT at Ser473 in SKOV3 cells, but it did not alter total AKT expression. In addition, silencing of Plexin-B1 in SKOV3 cells inhibited cell migration and invasion and reorganized the cytoskeleton, whereas cell proliferation was not affected.ConclusionPlexin-B1 expression correlates with malignant phenotypes of serous ovarian tumors, probably via phosphorylation of AKT at Ser473, suggesting that Plexin-B1 might be a useful biomarker and/or a novel therapeutic target.

Abstract 3183: Dysregulated expression and function of Plexin-B1 in ovarian cancers

Abstract Background: Ovarian cancer has a tendency to spread within the peritoneal cavity early in the disease course. Understanding the metastatic pathways of ovarian cancer is thus of high importance for devising ovarian cancer treatment. Plexin-B1 is a cognate receptor of Semaphorin 4D (Sema4D). This study aims at investigating the role of Plexin-B1 in ovarian cancer. Materials and Methods: Expression of Plexin-B1 and its ligand in ovarian epithelial cell lines was examined by western blot and qPCR. Their expression in benign, borderline, and malignant tumors was revealed by immunohistochemistrty of paraffin embedded tissues. High-resolution melting (HRM) curve analysis was used to screen for mutation in Rac binding region of PLXNB1. Recombinant Sema4D was purified and used to treat OVCA420 cells which express high level of Plexin-B1. siRNA was used to knock-down Plexin-B1 in OVCA420. Cell migration and invasion ability was assayed by transwell migration and invasion assay. Results: Plexin-B1 was most highly overexpressed in serous ovarian cancer cell lines OVCA420, OVCA429, and SW626, whereas Sema4D was ovexpressed in OVCAR3, TOV21G, and PAI. In contrast, Plexin-B1 and Sema4D were not expressed in immortalized normal ovarian epithelial cell lines. Immunohistochemical studies also revealed high Plexin B1 expression in ovarian borderline tumors and invasive cancers while no Plexin B1 expression was detectable in benign tumor. On the other hand, downregulation of Plexin-B1 was observed in the metastatic foci compared with their corresponding primary tumors. There is also a tendency of higher Plexin-B1 expression in serous histotype than in other histotypes. A preliminary HRM screening of Plexin-B1 cDNA from 28 ovarian cancers detected no mutation in the Rac binding region. Knock-down of Plexin-B1 by siRNA caused increased migration and invasion of ovarian cancer cells. In contrast to Sema4D, treatment of ovarian cancer cells OVCA420 by follicle-stimulating hormone which enhances ovarian cancer growth, led to Plexin-B1 downregulation. Conclusion: Our study suggested that Plexin-B1 may contribute to early ovarian carcinogenesis. Its dyregulation in ovarian cancers is paradoxically associated with altered cancer cell invasion and migration. The role of Plexin B1 in ovarian cancer progression is complex. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3183.

Abstract 1014: Semaphorin 4D involves in the regulation of cell motility through its receptor Plexin B1 in pancreatic cancer

Abstract Introduction: Semaphorins are a large family of either membrane-bound or secreted proteins that provide axonal guidance in neuronal tissues and regulate cell motility in many types of cells. Recent articles reported that Semaphorin 4D (Sema 4D) enhanced in vitro motility and invasiveness through its receptor Plexin B1 and Met, and increased in vivo metastasis potential in breast cancer. However, these expression and function in pancreatic cancer is not known. Purpose: In the present study, we investigated the clinicopathological significance of Sema 4D and Plexin B1 expression in surgically resected pancreatic ductal adenocarcinoma sections and the regulation of cell motility through the Sema 4D - Plexin B1 signaling pathway. Material &amp; Methods: The mRNA expression level of Sema 4D and Plexin B1 were evaluated by Quantitative Real Time PCR from 12 surgical specimens and 10 normal pancreatic tissues. Expression of Sema 4D and Plexin B1 were also evaluated immunohistochemically from 90 patients. Using pancreatic cancer cell lines, the expression of endogenous Plexin B1 and Met protein was investigated by western blotting. And the phosphorylation of Met protein after stimulating by Sema4D was evaluated. As functional analysis, scratch assay and MTT assay were carried out. Results: The mRNA expression level of Sema 4D and Plexin B1 were significantly high in cancer tissues compared to normal pancreas tissues. Immunostaining showed that Sema 4D strongly expressed in lymphocytes and macrophages, as previously reported. Ninety two percent (83 out of 90 cases) pancreatic cancer samples overexpressed Plexin B1 compared to normal pancreas tissues. Endogenous Plexin B1 and Met protein were detected in eight pancreatic cancer cell lines, with a variety of expression levels. Sema 4D stimulation activated Met protein, and significantly induced cell migration. Discussion: Here we show that Sema 4D involves in the regulation of cell motility through its receptor Plexin B1. And the results of immunostaining suggest that Sema 4D could play a role in the interaction between tumor cells and tumor microenvironment in pancreatic cancer tissues. As many attempts are now ongoing to target the tumor stroma, these findings provide a new insight into molecular targeted therapies for pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1014.

Plexin-B1, glycodelin and MMP7 expression in the human fallopian tube and in the endometrium

BackgroundTo study the expression of Plexin-B1, Glycodelin, and MMP7 during the menstrual cycle in the endometrium and in the fallopian tube.MethodsThe research included women undergoing hysterectomy, tubal sterilization or salpingo-oophoerectomy. Total RNA from endometrial and fallopian tube tissues was extracted using a total RNA isolation kit. Semi-quantitative RT-PCR was performed to examine mRNA relative expression.ResultsPlexin-B1 expression in the endometrium was significantly higher on days 19 - 23 compared to days 12 - 14 (1.166 +/- 0.42 versus 0.523 +/- 0.299), P < 0.005. In the fallopian tube the level of plexin-B1 did not change significantly throughout the menstrual cycle. Glycodelin expression was significantly higher on days 19 - 23 compared with days 12-14, both in the endometrium (0.819 +/- 0.564 versus 0.072 +/- 0.343, P < 0.05) and the fallopian tube (0.796 +/- 0.196 versus 0.329 +/- 0.398, P < 0.05). Although the level of MMP7 secretion was the highest in the secretory phase the difference from the proliferative phase did not reach statistical significance, neither in the endometrium nor in the fallopian tube. This could result from a lack of power.ConclusionsIn the endometrium, both Glycodelin and Plexin-B1 are exhibiting a cyclic pattern suggesting a possible steroid regulation and a role in endometrial receptivity.