Targeting Endothelial Connexin37 Reduces Angiogenesis and Decreases Tumor Growth.

Connexin37 (Cx37) and Cx40 form intercellular channels between endothelial cells (EC), which contribute to the regulation of the functions of vessels. We previously documented the participation of both Cx in developmental angiogenesis and have further shown that loss of Cx40 decreases the growth of different tumors. Here, we report that loss of Cx37 reduces (1) the in vitro proliferation of primary human EC; (2) the vascularization of subcutaneously implanted matrigel plugs in Cx37-/- mice or in WT using matrigel plugs supplemented with a peptide targeting Cx37 channels; (3) tumor angiogenesis; and (4) the growth of TC-1 and B16 tumors, resulting in a longer mice survival. We further document that Cx37 and Cx40 function in a collaborative manner to promote tumor growth, inasmuch as the injection of a peptide targeting Cx40 into Cx37-/- mice decreased the growth of TC-1 tumors to a larger extent than after loss of Cx37. This loss did not alter vessel perfusion, mural cells coverage and tumor hypoxia compared to tumors grown in WT mice. The data show that Cx37 is relevant for the control of EC proliferation and growth in different tumor models, suggesting that it may be a target, alone or in combination with Cx40, in the development of anti-tumoral treatments.

Targeting connexin37 alters angiogenesis and arteriovenous differentiation in the developing mouse retina.

Connexin37 (Cx37) forms intercellular channels between endothelial cells (EC), and contributes to coordinate the motor tone of vessels. We investigated the contribution of this protein during physiological angiogenesis. We show that, compared to WT littermates, mice lacking Cx37 (Cx37-/- ) featured (i) a decreased extension of the superficial vascular plexus during the first 4 days after birth; (ii) an increased vascular density at the angiogenic front at P6, due to an increase in the proliferative rate of EC and in the sprouting of the venous compartment, as well as to a somewhat displaced position of tip cells; (iii) a decreased coverage of newly formed arteries and veins by mural cells; (iv) altered ERK-dependent endothelial cells proliferation through the EphB4 signaling pathway, which is involved in the specification of veins and arteries. In vitro studies documented that, in the absence of Cx37, human venous EC (HUVEC) released less platelet-derived growth factor (PDGF) and more Angiopoietin-2, two molecules involved in the recruitment of mural cells. Treatment of mice with DAPT, an inhibitor of the Notch pathway, decreased the expression of Cx37, and partially mimicked in WT retinas, the alterations observed in Cx37-/- mice. Thus, Cx37 contributes to (i) the early angiogenesis of retina, by interacting with the Notch pathway; (ii) the growth and maturation of neo-vessels, by modulating tip, stalk, and mural cells; (iii) the regulation of arteriovenous specification, thus, representing a novel target for treatments of retina diseases.

Rebound pathway overactivation by cancer cells following discontinuation of PI3K or mTOR inhibition promotes cancer cell growth.

Whilst effects of anti-cancer drugs have been thoroughly explored, little is known about the repercussion of drug cessation. However, this has important clinical relevance since several clinical protocols such as intermittent drug scheduling lead to frequent drug discontinuation. In this study, we have thus investigated the consequences of withdrawal of agents that target the PI3K/AKT/mTOR signaling pathway in cancer cells. We report that washout of kinase inhibitors of mTOR or PI3K inhibitors led to a rapid and sustainable overactivation of AKT. Consequently, proliferation of tumor cells was significantly higher following drug washout in cancer cells that were pre-treated with mTOR or PI3K inhibitors compared to untreated cells. This effect was prevented by the addition of an AKT inhibitor following drug washout. Rebound AKT overactivation induced by mTOR or PI3K inhibitors discontinuation was mediated by IGF-1R, as demonstrated by its prevention in the presence of an IGF-1R inhibitor and by increased IGF-1R phosphorylation in treated cells versus control cells. Taken together, our results show that discontinuation of PI3K or mTOR inhibitors results in AKT overactivation that promotes tumor growth. They further highlight the benefit of adding an AKT inhibitor following cessation of PI3K or mTOR inhibitors.

mTOR and Tumor Cachexia.

Cancer cachexia affects most patients with advanced forms of cancers. It is mainly characterized by weight loss, due to muscle and adipose mass depletion. As cachexia is associated with increased morbidity and mortality in cancer patients, identifying the underlying mechanisms leading to cachexia is essential in order to design novel therapeutic strategies. The mechanistic target of rapamycin (mTOR) is a major intracellular signalling intermediary that participates in cell growth by upregulating anabolic processes such as protein and lipid synthesis. Accordingly, emerging evidence suggests that mTOR and mTOR inhibitors influence cancer cachexia. Here, we review the role of mTOR in cellular processes involved in cancer cachexia and highlight the studies supporting the contribution of mTOR in cancer cachexia.

Acidic tumor microenvironment abrogates the efficacy of mTORC1 inhibitors.

BACKGROUND: Blocking the mechanistic target of rapamycin complex-1 (mTORC1) with chemical inhibitors such as rapamycin has shown limited clinical efficacy in cancer. The tumor microenvironment is characterized by an acidic pH which interferes with cancer therapies. The consequences of acidity on the anti-cancer efficacy of mTORC1 inhibitors have not been characterized and are thus the focus of our study. METHODS: Cancer cell lines were treated with rapamycin in acidic or physiological conditions and cell proliferation was investigated. The effect of acidity on mTORC1 activity was determined by Western blot. The anticancer efficacy of rapamycin in combination with sodium bicarbonate to increase the intratumoral pH was tested in two different mouse models and compared to rapamycin treatment alone. Histological analysis was performed on tumor samples to evaluate proliferation, apoptosis and necrosis. RESULTS: Exposing cancer cells to acidic pH in vitro significantly reduced the anti-proliferative effect of rapamycin. At the molecular level, acidity significantly decreased mTORC1 activity, suggesting that cancer cell proliferation is independent of mTORC1 in acidic conditions. In contrast, the activation of mitogen-activated protein kinase (MAPK) or AKT were not affected by acidity, and blocking MAPK or AKT with a chemical inhibitor maintained an anti-proliferative effect at low pH. In tumor mouse models, the use of sodium bicarbonate increased mTORC1 activity in cancer cells and potentiated the anti-cancer efficacy of rapamycin. Combining sodium bicarbonate with rapamycin resulted in increased tumor necrosis, increased cancer cell apoptosis and decreased cancer cell proliferation as compared to single treatment. CONCLUSIONS: Taken together, these results emphasize the inefficacy of mTORC1 inhibitors in acidic conditions. They further highlight the potential of combining sodium bicarbonate with mTORC1 inhibitors to improve their anti-tumoral efficacy.

Connexin37-Dependent Mechanisms Selectively Contribute to Modulate Angiotensin II -Mediated Hypertension.

Background Gap junction channels made of Connexin37 (Cx37) are expressed by aortic endothelial and smooth muscle cells of hypertensive mice, as well as by the renin-secreting cells of kidneys. Methods and Results To decipher whether Cx37 has any role in hypertension, angiotensin II (Ang II ) was infused in normotensive wild-type and Cx37-deficient mice (Cx37-/-). After 2 to 4 weeks, the resulting increase in blood pressure was lower in Cx37-/- than in wild-type mice, suggesting an alteration in the Ang II response. To investigate this possibility, mice were submitted to a 2-kidney, 1-clip procedure, a renin-dependent model of hypertension. Two weeks after this clipping, Cx37-/- mice were less hypertensive than wild-type mice and, 2 weeks later, their blood pressure had returned to control values, in spite of abnormally high plasma renin levels. In contrast, Cx37-/- and wild-type mice that received N-nitro-l-arginine-methyl-ester, a renin-independent model of hypertension, featured a similar and sustained increase in blood pressure. The data indicate that loss of Cx37 selectively altered the Ang II -dependent pathways. Consistent with this conclusion, aortas of Cx37-/- mice featured an increased basal expression of the Ang II type 2 receptors ( AT 2R), and increased transcripts levels of downstream signaling proteins, such as Cnksr1 and Ptpn6 ( SHP -1). Accordingly, the response of Cx37-/- mice aortas to an ex vivo Ang II exposure was altered, since phosphorylation levels of several proteins of the Ang II pathway ( MLC 2, ERK , and AKT ) remained unchanged. Conclusions These findings provide evidence that Cx37 selectively influences Ang II signaling, mostly via a modulation of the expression of the Ang II type 2 receptor.

Evolving Significance and Future Relevance of Anti-Angiogenic Activity of mTOR Inhibitors in Cancer Therapy.

mTOR inhibitors have demonstrated remarkable anti-tumor activity in experimental models, mainly by reducing cancer cell growth and tumor angiogenesis. Their use in cancer patients as monotherapy has, however, generated only limited benefits, increasing median overall survival by only a few months. Likewise, in other targeted therapies, cancer cells develop resistance mechanisms to overcome mTOR inhibition. Hence, novel therapeutic strategies have to be designed to increase the efficacy of mTOR inhibitors in cancer. In this review, we discuss the present and future relevance of mTOR inhibitors in cancer therapy by focusing on their effects on tumor angiogenesis.

Acidic pH reduces VEGF-mediated endothelial cell responses by downregulation of VEGFR-2; relevance for anti-angiogenic therapies.

Anti-angiogenic treatments targeting the vascular endothelial growth factor or its receptors have shown clinical benefits. However, impact on long-term survival remains limited. Solid tumors display an acidic microenvironment that profoundly influences their biology. Consequences of acidity on endothelial cells and anti-angiogenic therapies remain poorly characterized and hence are the focus of this study. We found that exposing endothelial cells to acidic extracellular pH resulted in reduced cell proliferation and migration. Also, whereas VEGF increased endothelial cell proliferation and survival at pH 7.4, it had no effect at pH 6.4. Furthermore, in acidic conditions, stimulation of endothelial cells with VEGF did not result in activation of downstream signaling pathways such as AKT. At a molecular level, acidity significantly decreased the expression of VEGFR-2 by endothelial cells. Consequently, anti-angiogenic therapies that target VEGFR-2 such as sunitinib and sorafenib failed to block endothelial cell proliferation in acidic conditions. In vivo, neutralizing tumor acidity with sodium bicarbonate increased the percentage of endothelial cells expressing VEGFR-2 in tumor xenografts. Furthermore, combining sodium bicarbonate with sunitinib provided stronger anti-cancer activity than either treatment alone. Histological analysis showed that sunitinib had a stronger anti-angiogenic effect when combined with sodium bicarbonate. Overall, our results show that endothelial cells prosper independently of VEGF in acidic conditions partly as a consequence of decreased VEGFR-2 expression. They further suggest that strategies aiming to raise intratumoral pH can improve the efficacy of anti-VEGF treatments.

Targeting carbonic anhydrase IX improves the anti-cancer efficacy of mTOR inhibitors.

The inhibition of the mechanistic target of rapamycin complex 1 (mTORC1) by chemical inhibitors, such as rapamycin, has demonstrated anti-cancer activity in preclinical and clinical trials. Their efficacy is, however, limited and tumors eventually relapse through resistance formation. In this study, using two different cancer mouse models, we identify tumor hypoxia as a novel mechanism of resistance of cancer cells against mTORC1 inhibitors. Indeed, we show that the activity of mTORC1 is mainly restricted to the non-hypoxic tumor compartment, as evidenced by a mutually exclusive staining pattern of the mTORC1 activity marker pS6 and the hypoxia marker pimonidazole. Consequently, whereas rapamycin reduces cancer cell proliferation in non-hypoxic regions, it has no effect in hypoxic areas, suggesting that cancer cells proliferate independently of mTORC1 under hypoxia. Targeting the hypoxic tumor compartment by knockdown of carbonic anhydrase IX (CAIX) using short hairpin RNA or by chemical inhibition of CAIX with acetazolamide potentiates the anti-cancer activity of rapamycin. Taken together, these data emphasize that hypoxia impairs the anti-cancer efficacy of rapalogs. Therapeutic strategies targeting the hypoxic tumor compartment, such as the inhibition of CAIX, potentiate the efficacy of rapamycin and warrant further clinical evaluation.