Involvement of PTEN Promoter Methylation in Cerebral Cavernous Malformations

Zhu, Yuan; Wloch, Andreas; Wu, Qun; Peters, Christian; Pagenstecher, Axel; Bertalanffy, Helmut; Sure, Ulrich

doi:10.1161/strokeaha.108.526376

Cited by 30 publications

(13 citation statements)

References 35 publications

(36 reference statements)

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“…1,17 Recent findings that frequent epigenetic alteration in phosphatase and tensin homolog promoter led to deficiency of this protein expression in the ECs of familial CCMs may support this notion. 30,31 Furthermore, we (unpublished data, 2010) and others 2,29 have found that ECs derived from CCMs (CCM-ECs) exhibit unique angiogenesis properties, indicating a pathological background of CCM-ECs. Clinically, a different intracranial hemorrhage rate has been observed in CCMs carrying individual CCM gene mutations.…”

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confidence: 82%

Differential angiogenesis function of CCM2 and CCM3 in cerebral cavernous malformations

Zhu

et al. 2010

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Object Loss-of-function mutations in CCM genes are frequently detected in familial cerebral cavernous malformations (CCMs). However, the current functional studies of the CCM genes in vitro have been performed mostly in commercially purchased normal cell lines and the results appeared discrepant. The fact that the cerebral vascular defects are rarely observed in CCM gene-deficient animals suggests the requirement of additional pathological background for the formation of vascular lesions. Consistent with these data, the authors assumed that silencing CCM genes in the endothelium derived from CCMs (CCM-ECs) serves as a unique and valuable model for investigating the function of the CCM genes in the pathogenesis of CCMs. To this end, the authors investigated the role and signaling of CCM2 and CCM3 in the key steps of angiogenesis using CCM-ECs. Methods Endothelial cells (ECs) derived from CCMs were isolated, purified, and cultured from the fresh operative specimens of sporadic CCMs (31 cases). The CCM2 and CCM3 genes were silenced by the specific short interfering RNAs in CCM-ECs and in control cultures (human brain microvascular ECs and human umbilical vein ECs). The efficiency of gene silencing was proven by real-time reverse transcriptase polymerase chain reaction. Cell proliferation and apoptosis, migration, tube formation, and the expression of phosphor-p38, phosphor-Akt, and phosphor-extracellular signal-regulated kinase–1 and 2 (ERK1/2) were analyzed under CCM2 and CCM3 silenced conditions in CCM-ECs. Results The CCM3 silencing significantly promoted proliferation and reduced apoptosis in all 3 types of endothelium, but accelerated cell migration exclusively in CCM-ECs. Interestingly, CCM2 siRNA influenced neither cell proliferation nor migration. Silencing of CCM3, and to a lesser extent CCM2, stimulated the growth and extension of sprouts selectively in CCM-ECs. Loss of CCM2 or CCM3 did not significantly influence the formation of the tubelike structure. However, the maintenance of tube stability was largely impaired by CCM2, but not CCM3, silencing. Western blot analysis revealed that CCM2 and CCM3 silencing commonly activated p38, Akt, and ERK1/2 in CCM-ECs. Conclusions The unique response of CCM-ECs to CCM2 or CCM3 siRNA indicates that silencing CCM genes in CCM-ECs is valuable for further studies on the pathogenesis of CCMs. Using this model system, the authors demonstrate a distinct role of CCM2 and CCM3 in modulating the different processes of angiogenesis. The stimulation of endothelial proliferation, migration, and massively growing and branching angiogenic sprouts after CCM3 silencing may potentially contribute to the formation of enriched capillary-like immature vessels in CCM lesions. The severe impairment of the tube integrity by CCM2, but not CCM3, silencing is associated with the different intracranial hemorrhage rate observed from CCM2 and CCM3 mutation carriers. The activation of p38, ERK1/2, and Akt signal proteins in CCM2- or CCM3-silenced CCM-ECs suggests a possible involvement of these common pathways in the pathogenesis of CCMs. However, the specific signaling mediating the distinct function of CCM genes in the pathogenesis of CCMs needs to be further elucidated.

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confidence: 82%

Differential angiogenesis function of CCM2 and CCM3 in cerebral cavernous malformations

Zhu

et al. 2010

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“…Total protein extraction and the electrophoresis were performed as described previously [33]. The blots were incubated at 4°C overnight with the following first antibodies: DLL4, p-Erk1/2, p-Akt and GAPDH (each 1:1000 dilution, Cell Signaling, Frankfurt am Main, Germany); Notch4 [1:200, specifically detect the cleaved domain (active form) of the protein, Santa-Cruz Technology, Heidelberg, Germany], VEGF (1:1000; Abcam, Cambridge, UK), Hey1 (1:200, Abcam), CCM3 (1:400; Atlas Antibodies, Stockholm, Sweden) and actin (1:1000; Sigma-Aldrich, Seelze, Germany).…”

Section: Methodsmentioning

confidence: 99%

Loss of CCM3 impairs DLL4‐Notch signalling: implication in endothelial angiogenesis and in inherited cerebral cavernous malformations

You

Sandalcioglu

Dammann

et al. 2013

J Cellular Molecular Medi

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CCM3, a product of the cerebral cavernous malformation 3 or programmed cell death 10 gene (CCM3/PDCD10), is broadly expressed throughout development in both vertebrates and invertebrates. Increasing evidence indicates a crucial role of CCM3 in vascular development and in regulation of angiogenesis and apoptosis. Furthermore, loss of CCM3 causes inherited (familial) cerebral cavernous malformation (CCM), a common brain vascular anomaly involving aberrant angiogenesis. This study focused on signalling pathways underlying the angiogenic functions of CCM3. Silencing CCM3 by siRNA stimulated endothelial proliferation, migration and sprouting accompanied by significant downregulation of the core components of Notch signalling including DLL4, Notch4, HEY2 and HES1 and by activation of VEGF and Erk pathways. Treatment with recombinant DLL4 (rhDLL4) restored DLL4 expression and reversed CCM3-silence-mediated impairment of Notch signalling and reduced the ratio of VEGF-R2 to VEGF-R1 expression. Importantly, restoration of DLL4-Notch signalling entirely rescued the hyper-angiogenic phenotype induced by CCM3 silence. A concomitant loss of CCM3 and the core components of DLL4-Notch signalling were also demonstrated in CCM3-deficient endothelial cells derived from human CCM lesions (CCMEC) and in a CCM3 germline mutation carrier. This study defined DLL4 as a key downstream target of CCM3 in endothelial cells. CCM3/DLL4-Notch pathway serves as an important signalling for endothelial angiogenesis and is potentially implicated in the pathomechanism of human CCMs.

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“…Methylation specific PCR (MSP) was performed at four independent CpG sites within the PTEN promoter previously shown to be prone to methylation in CRC (ST-3) [33, 39–41], using ZymoTaq Premix (Zymo Research) [42]. Reactions were carried out on a Peltier thermocycler in a 25 µL volume consisting of 12.5 µL of ZymoTaq premix solution, 2 µL each of 10 µM forward and reverse primers, 2 µL of bisulfite treated DNA template with approximately 25ng of DNA and 6.5 µL of DNAse, RNAse free water.…”

Section: Methodsmentioning

confidence: 99%

PTEN Gene Expression and Mutations in the PIK3CA Gene as Predictors of Clinical Benefit to Anti-Epidermal Growth Factor Receptor Antibody Therapy in Patients With KRAS Wild-Type Metastatic Colorectal Cancer

Sood

McClain

Maitra

et al. 2012

Clinical Colorectal Cancer

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PURPOSE To identify novel genetic markers predictive of clinical benefit from epidermal growth factor receptor-directed antibody therapy in patients with metastatic colorectal cancer (mCRC). PATIENTS AND METHODS Seventy-six consecutive patients who received cetuximab or panitumumab, either alone or in combination with chemotherapy, with available tumor tissue were included. Tumor tissue was tested for mutations at known hotspots in the KRAS, BRAF, PIK3CA, PIK3R1, AKT1, and PTEN genes by pyrosequencing. PTEN promoter methylation status was analyzed by methylation-specific PCR, and expression determined by immunohistochemistry (IHC). Forty-four patients had ≥ 4 weeks of therapy and were considered for clinical correlates. RESULTS Consistent with previous studies, KRAS gene mutations were associated with a shorter progression free (PFS) and overall survival (OS). Among the KRAS wild type patients, preservation of PTEN expression and PIK3CA WT status was associated with improved OS (median OS, 80.4 vs 32.5 weeks, HR: 0.33, p=0.0008) and a trend towards improved PFS (median PFS, 24.8 vs 15.2 weeks, HR: 0.51, p=0.06), compared to PTEN negative or PIK3CA mutant tumors. PTEN methylation was more common in the metastases than the primary (p=0.02). Simultaneous presence of methylation and mutation in the PTEN gene was associated with IHC negativity (p=0.026). CONCLUSION In addition to KRAS mutation, loss of PTEN expression (by IHC) and PIK3CA mutation is likely to be predictive of lack of benefit to anti-EGFR therapy in mCRC. PTEN promoter methylation and mutation status was predictive of PTEN expression, and may be utilized as an alternative means of predicting response to EGFR-targeted therapy.

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Involvement of PTEN Promoter Methylation in Cerebral Cavernous Malformations

Cited by 30 publications

References 35 publications

Differential angiogenesis function of CCM2 and CCM3 in cerebral cavernous malformations

Differential angiogenesis function of CCM2 and CCM3 in cerebral cavernous malformations

Loss of CCM3 impairs DLL4‐Notch signalling: implication in endothelial angiogenesis and in inherited cerebral cavernous malformations

PTEN Gene Expression and Mutations in the PIK3CA Gene as Predictors of Clinical Benefit to Anti-Epidermal Growth Factor Receptor Antibody Therapy in Patients With KRAS Wild-Type Metastatic Colorectal Cancer

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