Simon Lord-Dufour

Propranolol suppresses angiogenesis in vitro: inhibition of proliferation, migration, and differentiation of endothelial cells.

Propranolol, a non-selective β-adrenergic blocking drug, was recently reported to control the growth of hemangiomas, the most common vascular tumor of infancy. However, the mechanisms involved in this effect remain unknown. Here, we demonstrate that propranolol dose-dependently inhibited growth factor-induced proliferation of cultured human umbilical vein endothelial cells (HUVECs) through a G₀/G₁ phase cell cycle arrest. This was correlated to decreased cyclin D1, cyclin D3, and cyclin-dependent kinase CDK6 protein levels, while increases in the CDK inhibitors p15(INK4B), p21(WAF1/Cip1) and p27(Kip1) were observed. Chemotactic motility and differentiation of HUVECs into capillary-like tubular structures in Matrigel were also inhibited by propranolol. Furthermore, inhibition by propranolol of vascular endothelial growth factor (VEGF)-induced tyrosine phosphorylation of VEGF receptor-2 lead to inhibition of downstream signaling such as the activation of the extracellular signal-regulated kinase-1/2 and the secretion of the extracellular matrix degrading enzyme MMP-2. Taken together, these results demonstrate that propranolol interferes with several essential steps of neovascularization and opens up novel therapeutic opportunities for the use of β-blockers in the treatment of angiogenesis-dependent human diseases.

Improved Autograft Survival of Mesenchymal Stromal Cells by Plasminogen Activator Inhibitor 1 Inhibition

Mesenchymal stromal cells (MSCs) display robust reparative properties through their ability to limit apoptosis, enhance angiogenesis, and direct positive tissue remodeling. However, low in vivo survival of transplanted cells limits their overall effectiveness and significantly affects their clinical usage. Consequently, identifying strategies to improve cell survival in vivo are a priority. One explanation for their low survival is that MSCs are often transplanted into ischemic tissue, such as infarcted myocardium, where there is poor blood supply and low oxygen tension. Therefore, we examined how MSCs respond to a hypoxic, nutrient‐poor stress environment to identify trophic factors that could be manipulated in advance of MSC transplantation. Combining microarray and proteomic screens we identified plasminogen activator inhibitor 1 (PAI‐1) as one factor consistently upregulated in our in vitro ischemia‐mimicking conditions. Subsequent genetic and chemical manipulation studies define PAI‐1 as a negative regulator of MSC survival in vivo. Mechanistically, MSC‐derived PAI‐1 does not alter MSC survival through a plasmin‐dependent mechanism but rather directly impacts on the adhesiveness of MSCs to their surrounding matrices. Thus we can conclude that post‐transplantation, PAI‐1 negatively impacts MSC survival by promoting anoikis via matrix detachment. STEM CELLS 2008;27:467–477

Diet-derived polyphenols inhibit angiogenesis by modulating the interleukin-6/STAT3 pathway

Several epidemiological studies have indicated that abundant consumption of foods from plant origin is associated with a reduced risk of developing several types of cancers. This chemopreventive effect is related to the high content of these foods in phytochemicals, such as polyphenols, that interfere with several processes involved in cancer progression including tumor cell growth, survival and angiogenesis. In addition to the low intake of plant-based foods, increased body mass and physical inactivity have recently emerged as other important lifestyle factors influencing cancer risk, leading to the generation of low-grade chronic inflammatory conditions which are a key process involved in tumor progression. The objectives of the current study are to investigate the inhibitory effects of these polyphenols on angiogenesis triggered by an inflammatory cytokine (IL-6) and to determine the mechanisms underlying this action. We found that, among the tested polyphenols, apigenin and luteolin were the most potent angiogenesis inhibitors through their inhibitory effect on the inflammatory cytokine IL-6/STAT3 pathway. These effects resulted in modulation of the activation of extracellular signal-regulated kinase-1/2 signaling triggered by IL-6, as well as in a marked reduction in the proliferation, migration and morphogenic differentiation of endothelial cells. Interestingly, these polyphenols also modulated the expression of IL-6 signal transducing receptor (IL-6Rα) and the secretion of the extracellular matrix degrading enzyme MMP-2 as well as the expression of suppressor of cytokine signaling (SOCS3) protein. Overall, these results may provide important new information on the role of diet in cancer prevention.

ANG4043, a Novel Brain-Penetrant Peptide–mAb Conjugate, Is Efficacious against HER2-Positive Intracranial Tumors in Mice

Anti-HER2 monoclonal antibodies (mAb) have been shown to reduce tumor size and increase survival in patients with breast cancer, but they are ineffective against brain metastases due to poor brain penetration. In previous studies, we identified a peptide, known as Angiopep-2 (An2), which crosses the blood–brain barrier (BBB) efficiently via receptor-mediated transcytosis, and, when conjugated, endows small molecules and peptides with this property. Extending this strategy to higher molecular weight biologics, we now demonstrate that a conjugate between An2 and an anti-HER2 mAb results in a new chemical entity, ANG4043, which retains in vitro binding affinity for the HER2 receptor and antiproliferative potency against HER2-positive BT-474 breast ductal carcinoma cells. Unlike the native mAb, ANG4043 binds LRP1 clusters and is taken up by LRP1-expressing cells. Measuring brain exposure after intracarotid delivery, we demonstrate that the new An2–mAb conjugate penetrates the BBB with a rate of brain entry (Kin) of 1.6 × 10−3 mL/g/s. Finally, in mice with intracranially implanted BT-474 xenografts, systemically administered ANG4043 increases survival. Overall, this study demonstrates that the incorporation of An2 to the anti-HER2 mAb confers properties of increased uptake in brain endothelial cells as well as BBB permeability. These characteristics of ANG4043 result in higher exposure levels in BT-474 brain tumors and prolonged survival following systemic treatment. Moreover, the data further validate the An2–drug conjugation strategy as a way to create brain-penetrant biologics for neuro-oncology and other CNS indications. Mol Cancer Ther; 14(1); 129–40. ©2014 AACR.

Evidence for Transcriptional Regulation of the Glucose‐6‐Phosphate Transporter by HIF‐1α: Targeting G6PT with Mumbaistatin Analogs in Hypoxic Mesenchymal Stromal Cells

Mesenchymal stromal cell (MSC) markers are expressed on brain tumor‐initiating cells involved in the development of hypoxic glioblastoma. Given that MSCs can survive hypoxia and that the glucose‐6‐phosphate transporter (G6PT) provides metabolic control that contributes to MSC mobilization and survival, we investigated the effects of low oxygen (1.2% O2) exposure on G6PT gene expression. We found that MSCs significantly expressed G6PT and the glucose‐6‐phosphatase catalytic subunit β, whereas expression of the glucose‐6‐phosphatase catalytic subunit α and the islet‐specific glucose‐6‐phosphatase catalytic subunit‐related protein was low to undetectable. Analysis of the G6PT promoter sequence revealed potential binding sites for hypoxia inducible factor (HIF)‐1α and for the aryl hydrocarbon receptor (AhR) and its dimerization partner, the AhR nuclear translocator (ARNT), AhR:ARNT. In agreement with this, hypoxia and the hypoxia mimetic cobalt chloride induced the expression of G6PT, vascular endothelial growth factor (VEGF), and HIF‐1α. Gene silencing of HIF‐1α prevented G6PT and VEGF induction in hypoxic MSCs whereas generation of cells stably expressing HIF‐1α resulted in increased endogenous G6PT gene expression. A semisynthetic analog of the polyketide mumbaistatin, a potent G6PT inhibitor, specifically reduced MSC‐HIF‐1α cell survival. Collectively, our data suggest that G6PT may account for the metabolic flexibility that enables MSCs to survive under conditions characterized by hypoxia and could be specifically targeted within developing tumors. STEM CELLS 2009;27:489–497

Resveratrol Targeting of Carcinogen-Induced Brain Endothelial Cell Inflammation Biomarkers MMP-9 and COX-2 is Sirt1-Independent

The occurrence of a functional relationship between the release of metalloproteinases (MMPs) and the expression of cyclooxygenase (COX)-2, two inducible pro-inflammatory biomarkers with important pro-angiogenic effects, has recently been inferred. While brain endothelial cells play an essential role as structural and functional components of the blood-brain barrier (BBB), increased BBB breakdown is thought to be linked to neuroinflammation. Chemopreventive mechanisms targeting both MMPs and COX-2 however remain poorly investigated. In this study, we evaluated the pharmacological targeting of Sirt1 by the diet-derived and antiinflammatory polyphenol resveratrol. Total RNA, cell lysates, and conditioned culture media from human brain microvascular endothelial cells (HBMEC) were analyzed using qRT-PCR, immunoblotting, and zymography respectively. Tissue scan microarray analysis of grade I–IV brain tumours cDNA revealed increased gene expression of Sirt-1 from grade I–III but surprisingly not in grade IV brain tumours. HBMEC were treated with a combination of resveratrol and phorbol 12-myristate 13-acetate (PMA), a carcinogen known to increase MMP-9 and COX-2 through NF-κB. We found that resveratrol efficiently reversed the PMA-induced MMP-9 secretion and COX-2 expression. Gene silencing of Sirt1, a critical modulator of angiogenesis and putative target of resveratrol, did not lead to significant reversal of MMP-9 and COX-2 inhibition. Decreased resveratrol inhibitory potential of carcinogen-induced IκB phosphorylation in siSirt1-transfected HBMEC was however observed. Our results suggest that resveratrol may prevent BBB disruption during neuroinflammation by inhibiting MMP-9 and COX-2 and act as a pharmacological NF-κB signal transduction inhibitor independent of Sirt1.

Cell-based evidence for aminopeptidase N/CD13 inhibitor actinonin targeting of MT1-MMP-mediated proMMP-2 activation

Recent profiling has identified the aminopeptidase N/CD13 inhibitor actinonin as a selective soluble secreted matrix metalloproteinase (MMP) inhibitor. Given that actinonins effects against membrane-bound MMPs remain unknown and that MT1-MMP has been linked to chemo- and radio-therapy resistance in brain tumor development, we therefore assessed MT1-MMP functional inhibition by actinonin in U87 glioblastoma cells. We show that actinonin inhibits concanavalin-A (ConA)-induced proMMP-2 activation, while it does not inhibit ConA-induced MT1-MMP gene expression suggesting post-transcriptional effects of the drug possibly mediated through the membrane-anchored protease regulator RECK. Specific gene silencing of MT1-MMP with siRNA abrogated the ability of ConA to activate proMMP-2. Functional recombinant MT1-MMP whose constitutive expression led to proMMP-2 activation was also efficiently antagonized by actinonin. We provide evidence for actinonins new therapeutic application in the direct targeting of MT1-MMP-mediated proMMP-2 activation, an essential step in both brain tumor infiltration and in brain tumor-associated angiogenesis.

Members of the low-density lipoprotein receptor-related proteins provide a differential molecular signature between parental and CD133(+) DAOY medulloblastoma cells

Members of the low‐density lipoprotein receptor‐related protein (LRP) family are involved in metabolic stress and resistance phenotypes of cancer cells. New breakthroughs in brain cancer therapy have exploited that molecular signature and proved that efficient delivery of therapeutic agents involve LRP‐mediated mechanisms. We performed gene expression profiling of CD133, a cell surface cancer stem cell marker, and of LRP in response to in vitro nutrient deprivation. We found that CD133 was selectively induced in serum‐starved DAOY medulloblastoma cells but not in U87MG glioblastoma cells. Such CD133 induction was correlated to increases in LRP‐1 and LRP‐1b gene and protein expression. When a specific CD133(+) DAOY cell population was sorted from parental DAOY, we found increases in LRP‐5 and LRP‐8. Uptake of α2‐macroglobulin, a specific LRP‐1/1b ligand, was increased in serum‐starved parental DAOY cells but not in CD133(+) DAOY cells, and receptor‐associated protein (RAP), which binds to all cell surface LRPs, was able to compete for that uptake. Conversely, RAP binding was increased in serum‐starved parental DAOY but α2‐macroglobulin was unable to compete for such uptake. Strategies aiming at targeting cancer stem cell metabolic adaptative responses, such as that through LRP differential expression within the brain tissue microenvironmental niche, can now be envisioned.

Inhibition of tubulogenesis and of carcinogen-mediated signaling in brain endothelial cells highlight the antiangiogenic properties of a mumbaistatin analog.

A better understanding of the metabolic adaptations of the vascular endothelial cells (EC) that mediate tumor vascularization would help the development of new drugs and therapies. Novel roles in cell survival and metabolic adaptation to hypoxia have been ascribed to the microsomal glucose‐6‐phosphate translocase (G6PT). While antitumorigenic properties of G6PT inhibitors such as chlorogenic acid (CHL) have been documented, those of the G6PT inhibitor and semi‐synthetic analog AD4‐015 of the polyketide mumbaistatin are not understood. In the present study, we evaluated the in vitro antiangiogenic impact of AD4‐015 on human brain microvascular endothelial cells (HBMEC), which play an essential role as structural and functional components in tumor angiogenesis. We found that in vitro HBMEC migration and tubulogenesis were reduced by AD4‐015 but not by CHL. The mumbaistatin analog significantly inhibited the phorbol 12‐myristate 13‐acetate (PMA)‐induced matrix‐metalloproteinase (MMP)‐9 secretion and gene expression as assessed by zymography and RT‐PCR. PMA‐mediated cell signaling leading to cyclooxygenase (COX)‐2 expression and IκB downregulation was also inhibited, further confirming AD4‐015 as a cell signaling inhibitor in tumor promoting conditions. G6PT functions may therefore account for the metabolic flexibility that enables EC‐mediated neovascularization. This process could be specifically targeted within the vasculature of developing brain tumors by G6PT inhibitors.

Abstract 2465: Targeting HER2-positive brain metastases by incorporating the brain-penetrant Angiopep-2 peptide to an anti-HER2 antibody and anti-HER2 antibody drug conjugate

Monoclonal antibodies directed against receptor tyrosine kinases such as HER2 have been demonstrated to reduce tumor size and increase survival. However, these agents achieve little to no brain penetration, making them ineffective against metastatic brain tumors. The blood-brain barrier (BBB), efficient at restricting entry of proteins such as mAbs and anticancer drugs into the brain, is comprised of capillary endothelial cells with tight junctions and efflux pumps. We have created a family of peptides (Angiopeps) which use receptor-mediated transcytosis to enter the brain. Conjugation of the Angiopep-2 (An2) to confer brain permeability has been validated for small molecules (ANG 1005, Phase II), peptides and proteins. The brain-penetrant An2 has also been incorporated to a humanized anti-HER2 mAb. This Angiopep-Antibody Conjugate, ANG4043, displays HER2 binding affinity and in vitro cytotoxic potency similar to that of native anti-HER2. ANG4043 demonstrates a high rate of entry into the brain. ANG4043 reduces the tumor size of BT-474 human breast cancer cells when implanted in the brain, consistent with achieving therapeutic concentrations. Here we describe chemical conjugation between three molecules: the An2, a cytotoxic drug (docetaxel or maytansine), and a mAb directed against HER2. These new An2-antibody-drug-conjugates (An2-ADCs) show a higher in vitro anti-proliferative potency than unconjugated mAb against HER2+ BT-474 and HC-19554 cells that are sensitive and resistant to Herceptin, respectively. Furthermore, they demonstrate a high rate of entry into the brain when compared to controls, leading to a reduction in brain tumor size and to an increase in the survival of mice bearing intracranial BT-474 tumors. Furthermore, An2-anti-HER2 derivatives are also efficacious in peripheral tissues since they inhibited the growth of subcutaneous BT-474 luciferase tumors. Overall, these data demonstrate that the conjugation of an Angiopep to therapeutic mAbs or ADCs can increase their efficacy in the CNS without affecting their anticancer properties outside of the brain. These results extend the validation of Angiopep conjugation beyond small anticancer drugs to include larger molecules such as therapeutic mAbs and ADCs for development of new brain-penetrant therapeutics for brain malignancies. Citation Format: Michel Demeule, Sanjoy Das, Christian Che, Gaoqiang Yang, Jean-Christophe Currie, Simon Lord-Dufour, Sasmita Tripathy, Anthony Regina, Jean-Paul Castaigne, Jean E. Lachowicz. Targeting HER2-positive brain metastases by incorporating the brain-penetrant Angiopep-2 peptide to an anti-HER2 antibody and anti-HER2 antibody drug conjugate. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2465. doi:10.1158/1538-7445.AM2015-2465