SHP2 mediates the activities of multiple receptor tyrosine kinase signaling and its function in endothelial processes has been explored extensively. However, genetic studies on the role of SHP2 in tumor angiogenesis have not been conducted. Here, we show that SHP2 is activated in tumor endothelia. Shp2 deletion and pharmacological inhibition reduce tumor growth and microvascular density in multiple mouse tumor models. Shp2 deletion also leads to tumor vascular normalization, indicated by increased pericyte coverage and vessel perfusion. SHP2 inefficiency impairs endothelial cell proliferation, migration, and tubulogenesis through downregulating the expression of proangiogenic SRY-Box transcription factor 7 (SOX7), whose re-expression restores endothelial function in SHP2-knockdown cells and tumor growth, angiogenesis, and vascular abnormalization in Shp2-deleted mice. SHP2 stabilizes apoptosis signal-regulating kinase 1 (ASK1), which regulates SOX7 expression mediated by c-Jun. Our studies suggest SHP2 in tumor associated endothelial cells is a promising anti-angiogenic target for cancer therapy.

Transforming growth factor β (TGFβ) signaling plays an important role in regulating tumor malignancy, including in non-small cell lung cancer (NSCLC). The major biological responses of TGFβ signaling are determined by the effector proteins SMAD2 and SMAD3. However, the regulators of TGFβ-SMADs signaling are not completely revealed yet. Here, we showed that the scaffolding protein PDZ and LIM domain protein 5 (PDLIM5, ENH) critically promotes TGFβ signaling by maintaining SMAD3 stability in NSCLC. Firstly, PDLIM5 was highly expressed in NSCLC compared with that in adjacent normal tissues, and high PDLIM5 expression was associated with poor outcome. Knockdown of PDLIM5 in NSCLC cells decreased migration and invasion in vitro, and lung metastasis in vivo. In addition, TGFβ signaling and TGFβ-induced epithelial-mesenchymal transition (EMT) was repressed by PDLIM5 knockdown. Mechanistically, PDLIM5 knockdown resulted in a reduction of SMAD3 protein levels. Overexpression of SMAD3 reversed the TGFβ-signaling-repressing and anti-migration effects induced by PDLIM5 knockdown. Notably, PDLIM5 interacted with SMAD3 but not SMAD2, and competitively suppressed the interaction between SMAD3 and its E3 ubiquitin ligase STUB1. Therefore, PDLIM5 protected SMAD3 from STUB1-mediated proteasome degradation. STUB1 knockdown restored SMAD3 protein levels, cell migration and invasion in PDLIM5-knockdown cells. Collectively, our findings indicate that PDLIM5 is a novel regulator of basal SMAD3 stability, with implications for controlling TGFβ signaling and NSCLC progression.

Objective: A decrease in nitric oxide, leading to vascular smooth muscle cell proliferation, is a common pathological feature of vascular proliferative diseases. Nitric oxide synthesis by eNOS (endothelial nitric oxide synthase) is precisely regulated by protein kinases including AKT1. ENH (enigma homolog protein) is a scaffolding protein for multiple protein kinases, but whether it regulates eNOS activation and vascular remodeling remains unknown. Approach and Results: ENH was upregulated in injured mouse arteries and human atherosclerotic plaques and was associated with coronary artery disease. Neointima formation in carotid arteries, induced by ligation or wire injury, was greatly decreased in endothelium-specific ENH-knockout mice. Vascular ligation reduced AKT and eNOS phosphorylation and nitric oxide production in the endothelium of control but not ENH-knockout mice. ENH was found to interact with AKT1 and its phosphatase PHLPP2 (pleckstrin homology domain and leucine-rich repeat protein phosphatase 2). AKT and eNOS activation were prolonged in VEGF (vascular endothelial growth factor)-induced ENH- or PHLPP2-deficient endothelial cells. Inhibitors of either AKT or eNOS effectively restored ligation-induced neointima formation in ENH-knockout mice. Moreover, endothelium-specific PHLPP2-knockout mice displayed reduced ligation-induced neointima formation. Finally, PHLPP2 was increased in the endothelia of human atherosclerotic plaques and blood cells from patients with coronary artery disease. Conclusions: ENH forms a complex with AKT1 and its phosphatase PHLPP2 to negatively regulate AKT1 activation in the artery endothelium. AKT1 deactivation, a decrease in nitric oxide generation, and subsequent neointima formation induced by vascular injury are mediated by ENH and PHLPP2. ENH and PHLPP2 are thus new proatherosclerotic factors that could be therapeutically targeted.

Vascular endothelial (VE)-cadherin junctional localization is known to play a central role in vascular development, endothelial barrier integrity, and homeostasis. The sarcoma (Src) homology (SH) domain containing protein tyrosine phosphatase (SHP)2 has been shown to be involved in regulating endothelial barrier function; however, the mechanisms remain largely unknown. In this work SHP2 knockdown in an HUVEC monolayer increased VE-cadherin internalization and endothelial barrier permeability. Loss of SHP2 specifically augmented the GTPase activity of ADP-ribosylation factor (ARF)-1. ARF1 knockdown or inhibition of its guanine nucleotide exchange factors (GEFs) markedly attenuated VE-cadherin internalization and barrier hyperpermeability induced by SHP2 deficiency. SHP2 knockdown increased the total and phosphorylated levels of MET, whose activity was necessary for ARF1 activation and VE-cadherin internalization. Furthermore, constitutive endothelium-specific deletion of Shp2 in mice led to disrupted endothelial cell junctions, massive hemorrhage, and lethality in embryos. Induced and endothelium-specific deletion of Shp2 in adult mice resulted in lung hyperpermeability. Inhibitors for ARF1-GEF or MET used in pregnant mice prevented the vascular leakage in endothelial Shp2-deleted embryos. Together, our findings define a novel role of SHP2 in stabilizing junctional VE-cadherin in the resting endothelial barrier through suppressing MET and ARF1 activation.-Zhang, J., Huang, J., Qi, T., Huang, Y., Lu, Y., Zhan, T., Gong, H., Zhu, Z., Shi, Y., Zhou, J., Yu, L., Zhang, X., Cheng, H., Ke, Y. SHP2 protects endothelial cell barrier through suppressing VE-cadherin internalization regulated by MET-ARF1.

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