Differential regulation of vascular endothelial growth factor by Akt and mammalian target of rapamycin inhibitors in cell lines derived from childhood solid tumors

Kurmasheva, Raushan T.; Harwood, Franklin C.; Houghton, Peter J.

doi:10.1158/1535-7163.mct-06-0646

Cited by 44 publications

(41 citation statements)

References 30 publications

(31 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As anticipated, rapamycin completely suppressed mTORC1 signaling, as shown by the absence of detectable phospho-S6K1(T 389 ) [pS6K1(T 389 )] and pS6(S 235/236 ). Of note, however, was that in C2C12 myoblasts rapamycin induced a decrease in pAKT(S 473 ) as reported previously (26), in contrast to its effects on malignant muscle cells or other malignant cells, where rapamycin treatment enhances phosphorylation at this residue (18,21,28).…”

Section: Resultssupporting

confidence: 78%

“…However, the downregulation of ROCK1 activity in cells expressing AKT(S473D) was quite refractory to treatment with rapamycin. mTOR catalytic activity has been shown to be essential for fusion, which results in mature myotubes (18,23). Taken together, our results strongly imply a role for mTOR in the mTORC2 complex, rather than in the rapamycin-sensitive mTORC1 complex, in the terminal differentiation of C2C12 myoblasts to form myotubes.…”

Section: Discussionsupporting

confidence: 59%

See 1 more Smart Citation

The mTORC2 Complex Regulates Terminal Differentiation of C2C12 Myoblasts

Shu

Houghton

2009

Molecular and Cellular Biology

Self Cite

View full text Add to dashboard Cite

Rapamycin, a selective inhibitor of mTORC1 signaling, blocks terminal myoblast differentiation. We found that downregulation of rictor, a component of the mTORC2 complex, but not downregulation of raptor, a component of the mTORC1 complex, prevented terminal differentiation (fusion) of C2C12 myoblasts. Both rapamycin and rictor downregulation suppressed the phosphorylation of AKT(S 473 ), and rapamycin treatment of C2C12 myoblasts disrupted the mTORC2 complex. Importantly, downregulation of rictor inhibited TORC2 signaling without inhibiting mTORC1 signaling, suggesting that inhibition of mTORC1 by rapamycin may not be the cause of arrested differentiation. In support of this, expression of a phosphomimetic mutant AKT(S473D) in rictor-deficient cells rescued myoblast fusion even in the presence of rapamycin. mTORC2 signaling to AKT appears necessary for downregulation of the Rho-associated kinase (ROCK1) that occurs during myogenic differentiation. Rapamycin treatment prevented ROCK1 inactivation during differentiation, while suppression of ROCK1 activity during differentiation and myoblast fusion was restored through expression of AKT(S473D), even in the presence of rapamycin. Further, the ROCK inhibitor Y-27632 restored terminal differentiation in rapamycin-treated myoblasts. These results provide the first evidence of a specific role for mTORC2 signaling in terminal myogenic differentiation.Differentiation of skeletal muscle cells involves highly coordinated processes involving myogenic determination of pluripotent mesodermal precursors, withdrawal from the cell cycle, subsequent expression of myotube-specific genes, and cell fusion to form multinucleated myotubes (5,19,23,33). AKT represents a nodal point, signaling to several pathways to positively or negatively regulate growth, proliferation, survival, and myogenic differentiation (34,35). AKT phosphorylates the FoxO1a transcription factor required for myotube fusion of primary myoblasts (3), causing cytoplasmic localization. However, in primary myoblasts and C2C12 myoblasts, phosphorylation of FoxO1a appears partially independent of the PI3K/ AKT pathway, possibly regulated through the Rho-associated kinase ROCK1. Inactivation of ROCK1 has been shown to be necessary for FoxO1a nuclear translocation and C2C12 cell fusion (20). AKT also activates mTOR (mammalian target of rapamycin) through phosphorylation and inactivation of the tuberous sclerosis complex (36) and phosphorylation of PRAS40, an endogenous inhibitor of mTOR (31, 32). Recent studies have shown that the TOR kinase(s) exists in two complexes that are conserved (25,36). In mammalian cells, TORC1 comprises mTOR, raptor, and mLST8 and is part of a pathway that senses nutrient (amino acids, O 2 , AMP) and growth factor status. mTORC1 activates S6K1 and phosphorylates and inactivates 4E-BPs, promoting association of eIF4E, the RNA cap-binding protein, with the eIF4G scaffolding protein and assembly of the eIF4F preinitiation translation complex (36). Importantly, rapamycin in complex with the immuno...

show abstract

Section: Resultssupporting

confidence: 78%

Section: Discussionsupporting

confidence: 59%

The mTORC2 Complex Regulates Terminal Differentiation of C2C12 Myoblasts

Shu

Houghton

2009

Molecular and Cellular Biology

Self Cite

View full text Add to dashboard Cite

show abstract

“…The unrestricted proliferation of rhabdomyosarcoma cells is supported by a redundancy of autocrine circuits, including basic fibroblast growth factor, insulin-like growth factors, epidermal growth factor, hepatocyte growth factor, connective tissue growth factor, and vascular endothelial growth factor (7)(8)(9)(10)(11)(12). This led us and others to explore the induction of rhabdomyosarcoma differentiation through a blockade of autocrine circuits by means of monoclonal antibodies, antisense oligonucleotides, and small interfering RNAs (8,9,11,13).…”

Section: Introductionmentioning

confidence: 99%

Opposing control of rhabdomyosarcoma growth and differentiation by myogenin and interleukin 4

Nanni

Nicoletti

Palladini

et al. 2009

Molecular Cancer Therapeutics

View full text Add to dashboard Cite

Rhabdomyosarcoma is a tumor of striated muscle origin that displays defective myogenic differentiation. Terminal myogenesis switches off cell proliferation and migration, hence, the promotion of rhabdomyosarcoma differentiation should antagonize tumor growth and metastasis. Terminal myogenesis is controlled by cell-intrinsic myogenic transcription factors like myogenin and environmental mediators like interleukin 4 (IL-4). We studied whether the expression of myogenin or exposure to IL-4 could promote the myogenesis of poorly differentiating human rhabdomyosarcoma cells RD/12. Forced expression of myogenin amplified myosin expression and the formation of myotube-like elements, inhibited cell migration, and reduced the growth of local tumors and liver metastases in immunodepressed mice. In contrast, exposure to IL-4 promoted cell proliferation and survival, especially at high cell density, inhibited myogenin expression, and myogenesis. Moreover, IL-4 stimulated the directed migration of cells with low myogenin levels, but not of cells with higher (spontaneous or forced) levels. Thus, IL-4, which was known to promote late stages of normal myogenesis, favors growth and migration, and inhibits further differentiation of the myogenic stages attained by rhabdomyosarcoma cells. Strategies to increase myogenin expression and block IL-4 could simultaneously reduce growth and migration, and enhance terminal differentiation of rhabdomyosarcoma, thus contributing to the control of tumor growth and metastatic spread. [Mol Cancer Ther 2009;8(4):754-61]

show abstract

“…However, we noted that in all of our cell lines, rapamycin-induced inhibition of VEGF was significant only in the presence of IGF-1. Recently, Kurmasheva et al (2007) postulated that VEGF expression in unstimulated NB cells depends on PI3K/Akt-driven signaling that most likely bypasses mTOR. They observed that HIF-1a and VEGF expression was strongly decreased with an Akt inhibitor, but was inadequately affected by rapamycin treatment in standard (10%) serum conditions.…”

Section: Discussionmentioning

confidence: 99%