Localisation of PlexinB1 and Sema4D in dividing cells

HeLa cells expressing GFP-tubulin and mCherry-histone transfected with siRNA to PlexinB1, (time lapse video taken at same time as video 3). 5 min frames, total time of 150 min, x40 magnification.

Effect of Sema4D on mitosis in MCF7 cells

HeLa cells expressing GFP-tubulin and mCherry-histone transfected with control siRNA, (control for video 2). 10 min frames, total time of 90 min, x60 magnification.

Plexin depletion delays mitosis in HeLa cells

Control for video 4. HeLa cells expressing GFP-tubulin and mCherry-histone transfected with control siRNA, 5 min time frames, total time of 150 min, x40 magnification.

Depletion of PlexinB1 results in dividing cells with multipolar spindles HeLa cells expressing GFP-tubulin and mCherry-histone transfected with siRNA to PlexinB1, 10 min time frames, total time of 200 min, x60 magnification.

B-type Plexin depletion increases unattached kinetochores

Depletion of PlexinB1 in NP1542 cells increases the number of dividing cells with multipolar spindles.

Abstract Aberrant mitosis can result in aneuploidy and cancer. The small GTPase, Ras-related nuclear protein (Ran), is a key regulator of mitosis. B-type plexins regulate Ran activity by acting as RanGTPase-activating proteins and have been implicated in cancer progression. However, whether B-type plexins have a role in mitosis has not so far been investigated. We show here that Plexin B1 functions in the control of mitosis. Depletion of Plexin B1 affects mitotic spindle assembly, significantly delaying anaphase. This leads to mitotic catastrophe in some cells and prolonged application of the spindle assembly checkpoint. Plexin B1 depletion also promoted acentrosomal microtubule nucleation and defects in spindle pole refocusing and increased the number of cells with multipolar or aberrant mitotic spindles. An increase in lagging chromosomes or chromosomal bridges at anaphase was also found upon Plexin B1 depletion. Plexin B1 localizes to the mitotic spindle in dividing cells. The mitotic defects observed upon Plexin B1 depletion were rescued by an RCC1 inhibitor, indicating that Plexin B1 signals, via Ran, to affect mitosis. These errors in mitosis generated multinucleate cells and nuclei of altered morphology and abnormal karyotype. Furthermore, semaphorin 4D treatment increased the percentage of cells with micronuclei, precursors of chromothripsis. Implications: Defects in B-type plexins may contribute to the well-established role of plexins in cancer progression by inducing chromosomal instability.

Aberrant mitosis can result in aneuploidy and cancer. The small GTPase, Ras-related nuclear protein (Ran), is a key regulator of mitosis. B-type plexins regulate Ran activity by acting as RanGTPase-activating proteins and have been implicated in cancer progression. However, whether B-type plexins have a role in mitosis has not so far been investigated. We show here that Plexin B1 functions in the control of mitosis. Depletion of Plexin B1 affects mitotic spindle assembly, significantly delaying anaphase. This leads to mitotic catastrophe in some cells and prolonged application of the spindle assembly checkpoint. Plexin B1 depletion also promoted acentrosomal microtubule nucleation and defects in spindle pole refocusing and increased the number of cells with multipolar or aberrant mitotic spindles. An increase in lagging chromosomes or chromosomal bridges at anaphase was also found upon Plexin B1 depletion. Plexin B1 localizes to the mitotic spindle in dividing cells. The mitotic defects observed upon Plexin B1 depletion were rescued by an RCC1 inhibitor, indicating that Plexin B1 signals, via Ran, to affect mitosis. These errors in mitosis generated multinucleate cells and nuclei of altered morphology and abnormal karyotype. Furthermore, semaphorin 4D treatment increased the percentage of cells with micronuclei, precursors of chromothripsis. Implications: Defects in B-type plexins may contribute to the well-established role of plexins in cancer progression by inducing chromosomal instability.

Metastatic 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. Pull 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. We 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. These 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.

Metastases in Ptenfl/flKrasG12V mice

Metastatic prostate cancer is essentially incurable and 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 show here that prostate epithelial cell–specific expression of a mutant form of Plexin-B1 (P1597L) which was identified in metastatic deposits in patients with prostate cancer, significantly increases metastasis, in particular metastasis to distant sites, in two transgenic mouse models of prostate cancer (PbCre+Ptenfl/flKrasG12Vand PbCre+Ptenfl/flp53fl/fl). In contrast, prostate epithelial cell–specific expression of wild-type (WT) Plexin-B1 in PbCre+Ptenfl/flKrasG12V mice significantly decreases metastasis, showing that a single clinically relevant Pro1597Leu amino-acid change converts Plexin-B1 from a metastasis-suppressor to a metastasis-promoter. Furthermore, PLXNB1P1597L significantly increased invasion of tumor cells into the prostate stroma, while PLXNB1WTreduced invasion, suggesting that Plexin-B1 has a role in the initial stages of metastasis. Deletion of RhoA/C or PDZRhoGEF in Ptenfl/flKrasG12VPLXNB1P1597L mice suppressed metastasis, implicating the Rho/ROCK pathway in this phenotypic switch. Germline deletion of Plexin-B1, to model anti-Plexin-B1 therapy, significantly decreased invasion and metastasis in both models.Our results demonstrate that Plexin-B1 plays a complex yet significant role in metastasis in mouse models of prostate cancer and is a potential therapeutic target to block the lethal spread of the disease.Significance:Few therapeutic targets have been identified specifically for preventing locally invasive/oligometastatic prostate cancer from becoming more widely disseminated. Our findings suggest Plexin-B1 signaling, particularly from the clinically relevant P1597L mutant, is such a target.

Metastatic deposit in Ptenfl/flKrasG12VPLXNB1WT mouse

Proliferation and ROCK activation in Ptenfl/flKrasG12V PlxnB1-/- and Ptenfl/flp53fl/fl PlxnB1-/- mice

Metastatic deposits in Ptenfl/flKrasG12VPLXNB1P1597L mice

Generation of PLXNB1WT and PLXNB1P1597L mice

Summary of age of mice and weight of primary tumours

Metastatic deposits in Ptenfl/flp53fl/fl mice