EGCG Inhibits Adipose-Derived Mesenchymal Stem Cells Differentiation into Adipocytes and Prevents a STAT3-Mediated Paracrine Oncogenic Control of Triple-Negative Breast Cancer Cell Invasive Phenotype.

Obese subjects have an increased risk of developing triple-negative breast cancer (TNBC), in part associated with the chronic low-grade inflammation state. On the other hand, epidemiological data indicates that increased consumption of polyphenol-rich fruits and vegetables plays a key role in reducing incidence of some cancer types. Here, we tested whether green tea-derived epigallocatechin-3-gallate (EGCG) could alter adipose-derived mesenchymal stem cell differentiation into adipocytes, and how this impacts the secretome profile and paracrine regulation of the TNBC invasive phenotype. Here, cell differentiation was performed and conditioned media (CM) from preadipocytes and mature adipocytes harvested. Human TNBC-derived MDA-MB-231 real-time cell migration was performed using the exCELLigence system. Differential gene arrays and RT-qPCR were used to assess gene expression levels. Western blotting was used to assess protein expression and phosphorylation status levels. In vitro vasculogenic mimicry (VM) was assessed with Matrigel. EGCG was found to inhibit the induction of key adipogenic biomarkers, including lipoprotein lipase, adiponectin, leptin, fatty acid synthase, and fatty acid binding protein 4. Increased TNBC-derived MDA-MB-231 cell chemotaxis and vasculogenic mimicry were observed in response to mature adipocytes secretome, and this was correlated with increased STAT3 phosphorylation status. This invasive phenotype was prevented by EGCG, the JAK/STAT inhibitors Tofacitinib and AG490, as well as upon STAT3 gene silencing. In conclusion, dietary catechin-mediated interventions could, in part through the inhibition of adipogenesis and modulation of adipocytes secretome profile, prevent the onset of an obesogenic environment that favors TNBC development.

Anthocyanidins inhibit epithelial-mesenchymal transition through a TGFß/Smad2 signaling pathway in glioblastoma cells.

Epidemiological studies have convincingly demonstrated that diets rich in fruits and vegetables play an important role in preventing cancer due to their polyphenol content. Among polyphenols, the anthocyanidins are known to possess anti-inflammatory, cardioprotective, anti-angiogenic, and anti-carcinogenic properties. Despite the well-known role of transforming growth factor-ß (TGF-ß) in high grade gliomas, the impact of anthocyanidins on TGF-ß-induced epithelial-mesenchymal transition (EMT), a process that allows benign tumor cells to infiltrate surrounding tissues, remains poorly understood. The objective of this study is to investigate the impact of anthocyanidins such as cyanidin (Cy), delphinidin (Dp), malvidin (Mv), pelargonidin (Pg), and petunidin (Pt) on TGF-ß-induced EMT and to determine the mechanism(s) underlying such action. Human U-87 glioblastoma (U-87 MG) cells were treated with anthocyanidins prior to, along with or following the addition of TGF-ß. We found that anthocyanidins differently affected TGF-ß-induced EMT, depending on the treatment conditions. Dp was the most potent EMT inhibitor through its inhibitory effect on the TGF-ß Smad and non-Smad signaling pathways. These effects altered expression of the EMT mesenchymal markers fibronectin and Snail, as well as markedly reducing U-87 MG cell migration. Our study highlights a new action of anthocyanidins against EMT that supports their beneficial health and chemopreventive effects in dietary-based strategies against cancer. © 2016 Wiley Periodicals, Inc.

The enzyme L-isoaspartyl (D-aspartyl) methyltransferase is required for VEGF-dependent endothelial cell migration and tubulogenesis.

The protein L-isoaspartyl (D-aspartyl) methyltransferase (PIMT) methylates proteins carrying altered aspartyl residues in their structure. PIMT is postulated to limit the accumulation of these damaged proteins with abnormal aspartyl residues. However, little is known about the role of PIMT in tumor growth and almost nothing about its involvement in angiogenic processes. We previously reported that PIMT was up-regulated when endothelial cells were detached from extracellular matrix, leading us to postulate that PIMT could play a critical role during angiogenic steps, since the contacts between endothelial cells and the extracellular matrix are intensively regulated during this process. Here, we demonstrated that PIMT down-regulation by siRNA in human umbilical vein endothelial cells (HUVECs) inhibited both cell migration and tube formation in vitro when stimulated by vascular endothelial growth factor (VEGF). Conversely, overexpression of wild-type PIMT promoted HUVEC migration in the presence of VEGF, while this response was prevented in cells transfected with the inactive mutant PIMT(D83V). Similar results were obtained with the two forms of PIMT regarding their capacity to regulate the action of VEGF during the formation of capillary-like structures in vitro. Together, these data highlight the importance of the catalytic activity of PIMT to mediate VEGF effects during endothelial cell migration and tube formation in angiogenesis. Furthermore, these results identify a new function for PIMT as an enzyme involved in pro-angiogenic processes.

Olive oil compounds inhibit vascular endothelial growth factor receptor-2 phosphorylation.

Vascular endothelial growth factor (VEGF) triggers crucial signaling processes that regulate tumor angiogenesis and, therefore, represents an attractive target for the development of novel anticancer therapeutics. Several epidemiological studies have confirmed that abundant consumption of foods from plant origin is associated with reduced risk of developing cancers. In the Mediterranean basin, the consumption of extra virgin olive oil is an important constituent of the diet. Compared to other vegetable oils, the presence of several phenolic antioxidants in olive oil is believed to prevent the occurrence of a variety of pathological processes, such as cancer. While the strong antioxidant potential of these molecules is well characterized, their antiangiogenic activities remain unknown. The aim of this study is to investigate whether tyrosol (Tyr), hydroxytyrosol (HT), taxifolin (Tax), oleuropein (OL) and oleic acid (OA), five compounds contained in extra virgin olive oil, can affect in vitro angiogenesis. We found that HT, Tax and OA were the most potent angiogenesis inhibitors through their inhibitory effect on specific autophosphorylation sites of VEGFR-2 (Tyr951, Tyr1059, Tyr1175 and Tyr1214) leading to the inhibition of endothelial cell (EC) signaling. Inhibition of VEGFR-2 by these olive oil compounds significantly reduced VEGF-induced EC proliferation and migration as well as their morphogenic differentiation into capillary-like tubular structures in Matrigel. Our study demonstrates that HT, Tax and OA are novel and potent inhibitors of the VEGFR-2 signaling pathway. These findings emphasize the chemopreventive properties of olive oil and highlight the importance of nutrition in cancer prevention.

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.

The Peptide-Drug Conjugate TH1902: A New Sortilin Receptor-Mediated Cancer Therapeutic against Ovarian and Endometrial Cancers.

Sortilin (SORT1) receptor-mediated endocytosis functions were exploited for this new approach for effective and safe treatments of gynecological cancers. Here, high expression of SORT1 was found in >75% of the clinically annotated ovarian and endometrial tumors analyzed by immunohistochemistry. Therefore, the anticancer properties of the peptide-drug conjugate TH1902, a peptide that targets SORT1 and which is linked to docetaxel molecules, were investigated both in vitro using ovarian and endometrial cancer cell cultures and in vivo using xenograft models. In vitro, TH1902 inhibited cell proliferation and triggered higher SORT1-dependent cell apoptosis than unconjugated docetaxel did in ES-2 and SKOV3 ovarian cancer cell lines. The uptake of the Alexa488-TH19P01 peptide from TH1902 was reduced upon siRNA-mediated silencing of SORT1. In vivo, weekly administration of TH1902 showed better tolerability compared to equivalent docetaxel doses and inhibited tumor growth in ovarian and endometrial xenograft mice models. TH1902 as a single agent inhibited ovarian tumor growth more than either of the unconjugated taxanes or carboplatin. Furthermore, TH1902 combination with carboplatin also demonstrated better efficacy when compared to both taxanes-carboplatin combinations. Overall, TH1902 shows better in vivo efficacy, compared to that of docetaxel and even paclitaxel, against SORT1-positive ovarian and endometrial cancers and could be safely combined with carboplatin.

New Peptide-Drug Conjugates for Precise Targeting of SORT1-Mediated Vasculogenic Mimicry in the Tumor Microenvironment of TNBC-Derived MDA-MB-231 Breast and Ovarian ES-2 Clear Cell Carcinoma Cells.

Vasculogenic mimicry (VM) is defined as the formation of microvascular channels by genetically deregulated cancer cells and is often associated with high tumor grade and cancer therapy resistance. This microcirculation system, independent of endothelial cells, provides oxygen and nutrients to tumors, and contributes also in part to metastasis. VM has been observed in ovarian cancer and in triple negative breast cancer (TNBC) and shown to correlate with decreased overall cancer patient survival. Thus, strategies designed to inhibit VM may improve cancer patient treatments. In this study, sortilin (SORT1) receptor was detected in in vitro 3D capillary-like structures formed by ES-2 ovarian cancer and MDA-MB-231 TNBC-derived cells when grown on Matrigel. SORT1 gene silencing or antibodies directed against its extracellular domain inhibited capillary-like structure formation. In vitro, VM also correlated with increased gene expression of matrix metalloproteinase-9 (MMP-9) and of the cancer stem cell marker CD133. In vivo ES-2 xenograft model showed PAS+/CD31- VM structures (staining positive for both SORT1 and CD133). TH1904, a Doxorubicin-peptide conjugate that is internalized by SORT1, significantly decreased in vitro VM at low nM concentrations. In contrast, VM was unaffected by unconjugated Doxorubicin or Doxil (liposomal Doxorubicin) up to µM concentrations. TH1902, a Docetaxel-peptide conjugate, altered even more efficiently in vitro VM at pM concentrations. Overall, current data evidence for the first time that 1) SORT1 itself exerts a crucial role in both ES-2 and MDA-MB-231 VM, and that 2) VM in these cancer cell models can be efficiently inhibited by the peptide-drug conjugates TH1902/TH1904. These new findings also indicate that both peptide-drug conjugates, in addition to their reported cytotoxicity, could possibly inhibit VM in SORT1-positive TNBC and ovarian cancer patients.

Periostin, a signal transduction intermediate in TGF-ß-induced EMT in U-87MG human glioblastoma cells, and its inhibition by anthocyanidins.

Periostin is a secreted protein that is highly expressed in glioblastoma cells as compared to normal brain tissue, and is therefore considered as a potential biomarker in therapeutic modalities. Its contribution in the cancer cells invasive phenotype is, however, poorly understood. This work investigates the role of periostin in U-87 MG glioblastoma cell invasion, cell migration and in Transforming Growth Factor ß (TGF-ß)-induced epithelial-mesenchymal transition (EMT). Periostin gene silencing, using small interfering RNA, decreased TGF-ß-induced mesenchymal marker expression of fibronectin and vimentin, partly through reduced Smad2, Akt and Fak phosphorylation as well as U-87 MG cell invasion and migration. The effects of anthocyanidins, the most abundant diet-derived flavonoids, were examined on periostin-mediated downstream signaling pathways. Anthocyanidins were found to decrease periostin expression whether added under pre-, co- or post-treatment conditions along with TGF-ß, and altered the Akt and Fak signaling pathways. These effects were similar to Galunisertib (LY2157299), a small molecule inhibitor of the TGF-ß receptor I kinase. Taken together, our data demonstrate that periostin acts as a central element in TGF-ß-induced EMT, which can be prevented by diet-derived anthocyanidins.