Vascular tumors have increased p70 S6-kinase activation and are inhibited by topical rapamycin

Abstract

Vascular tumors are endothelial cell neoplasms whose cellular and molecular mechanisms, leading to tumor formation, are poorly understood, and current therapies have limited efficacy with significant side effects. We have investigated mechanistic (mammalian) target of rapamycin (mTOR) signaling in benign and malignant vascular tumors, and the effects of mTOR kinase inhibitor as a potential therapy for these lesions. Human vascular tumors (infantile hemangioma and angiosarcoma) were analyzed by immunohistochemical stains and western blot for the phosphorylation of p70 S6-kinase (S6K) and S6 ribosomal protein (S6), which are activated downstream of mTOR complex-1 (mTORC1). To assess the function of S6K, tumor cells with genetic knockdown of S6K were analyzed for cell proliferation and migration. The effects of topical rapamycin, an mTOR inhibitor, on mTORC1 and mTOR complex-2 (mTORC2) activities, as well as on tumor growth and migration, were determined. Vascular tumors showed increased activation of S6K and S6. Genetic knockdown of S6K resulted in reduced tumor cell proliferation and migration. Rapamycin fully inhibited mTORC1 and partially inhibited mTORC2 activities, including the phosphorylation of Akt (serine 473) and PKCa, in vascular tumor cells. Rapamycin significantly reduced vascular tumor growth in vitro and in vivo. As a potential localized therapy for cutaneous vascular tumors, topically applied rapamycin effectively reduced tumor growth with limited systemic drug absorption. These findings reveal the importance of mTOR signaling pathways in benign and malignant vascular tumors. The mTOR pathway is an important therapeutic target in vascular tumors, and topical mTOR inhibitors may provide an alternative and well-tolerated therapy for the treatment of cutaneous vascular lesions.

Akt1 and Akt3 Exert Opposing Roles in the Regulation of Vascular Tumor Growth

“…21 and 23. ASM.5 cells were from Vera Krump-Konvalinkov (Ludwig Maximilian University Munchen, Germany) and EOMA cells were from ATCC as previously published (23)(24)(25). Cell line authentication and validation by short tandem repeats was performed.…”

“…Cell lysates were precleared with Protein A/G Agarose beads for 1 hour at 4 C and followed by addition of primary antibodies to 0.8 mg protein lysates and rotated overnight at 4 C. Protein A/G Agarose was then added to the lysates and rotated for 2 hours at 4 C. Immunoprecipitated proteins were denatured in Laemmli buffer and analyzed by Western blotting as described (23).…”

“…Quantitative PCR (qPCR) primers are provided in Supplementary Materials and Methods. Lentivirus packaging and qPCR were performed as described (23).…”

Inhibition of phosphoinositide 3-kinase is associated with reduced angiogenesis and an altered expression of angiogenic markers in endothelioma cells

“…12 In a recent study, Rapamycin, which regulates Akt phosphorylation, induced a decrease in vascular tumor growth and inhibited the phosphorylation of Akt in the same neoplastic cells. 1 In another study the inhibition of vascular tumor growth was associated with a decrease in Akt activity. 5 In the current study the suppression of PI3k, and ultimately Akt, lead to the inhibition of endothelioma cell growth and migration.…”

Educational paper: therapy of infantile haemangioma—history and current state (part II)

“…Many research efforts are directed at the pathogenesis of IH and hypoxia seems to be crucial [36,6,30]. Sirolimus, an mTOR inhibitor, probably has a good effect by blocking mammalian target of rapamycin (mTOR) signalling in the hypoxia pathway [16,44]. We have discussed the pathogenesis and sirolimus in more detail in part I in the previous issue [34].…”