Stress and IGF-I Differentially Control Cell Fate through Mammalian Target of Rapamycin (mTOR) and Retinoblastoma Protein (pRB)

Abstract

Significant discoveries have recently contributed to our knowledge of intracellular growth factor and nutrient signaling via mTOR (mammalian target of rapamycin). This signaling pathway is essential in cellular metabolism and cell survival by enhancing protein translation through phosphorylation of 4EBP-1 and p70S6K. Growth factors like insulin-like growth factor-I induce mTOR to prevent cell death during cellular stress. Agents targeting mTOR are of major interest as anticancer agents. We show here, using human breast cancer cells, that certain types of stress activate mTOR leading to 4E-BP1 and p70S6K phosphorylation. UV treatment increased phosphorylation of the translation inhibitor eIF2␣, suggesting a potential mechanism for UV activation of Akt and mTOR. c-Myc, a survival protein regulated by cap-dependent protein translation, increased with IGF-I treatment, but this response was not inhibited by rapamycin. Additionally, UV treatment potently increased c-Myc degradation, which was reduced by co-treatment with the proteasomal inhibitor, MG-132. Together, these data suggest that protein translation does not strongly mediate cell survival in these models. In contrast, the phosphorylation status of retinoblastoma protein (pRB) was mediated by mTOR through its inhibitory effects on phosphatase activity. This effect was most notable during DNA damage and rapamycin treatment. Hypophosphorylated pRB was susceptible to inactivation by caspase-mediated cleavage, resulting in cell death. Reduction of pRB expression inhibited IGF-I survival effects. Our data support an important role of phosphatases and pRB in IGF-I/mTOR-mediated cell survival. These studies provide new directions in optimizing anticancer efficacy of mTOR inhibitors when used in combination with DNA-damaging agents.Overexpression of tyrosine kinase receptors (TKRs) has long been appreciated to contribute to tumorigenesis and resistance to treatment. Receptor activation of insulin-like growth factor-I (IGF-I) 2 , insulin, PDGF (platelet-derived growth factor), and some ErbB receptors induce Akt activity via PI3K (phosphatidylinositol 3-kinase). The p110 catalytic subunit of PI3K stimulates the phosphorylation of PI(4,5)P 2 to PI(3,4,5)P 3 activating PDK1 (3-phosphoinositol-dependent kinase-1). PDK1 then enhances the activity of several kinases including Akt, PKC isoforms, SGK (serum and glucocorticoid-induced protein kinase), mTOR (mammalian target of rapamycin), and p70S6K. Loss of PTEN function in cancer cells leads to similar signaling events as activation of TKRs. Moreover, many of IGF-I-mediated functions in breast cancer cells, such as proliferation and survival, are thought to be conveyed through PI3K and Akt.Given the Akt potency as a survival mediator, much attention has focused on how it conveys this response. Increased protein translation occurs via Akt and its downstream mTOR. mTOR contains an Akt phosphorylation site, but current evidence indicates that Akt induces mTOR activity indirectly by phosphorylating tuberous sclerosis 2 (TSC2) (1)...

A Role for p38 Stress-Activated Protein Kinase in Regulation of Cell Growth via TORC1

“…These stresses are all independent of p38. In contrast, UV radiation, which activates stress pathways such as p38 and JNK through the induction of DNA damage, activates TORC1 in a number of cell types (7,48,75).…”

Dephosphorylation of threonine-821 of the retinoblastoma tumor suppressor protein (Rb) is required for apoptosis induced by UV and Cdk inhibition

“…In several studies, phosphatase activity has been shown to be required to cause apoptosis by UV stress or chemotherapeutic agents, such as etoposide and Ara-C. 13,16,21 In these studies, phosphatase inhibitors blocked both Rb dephosphorylation and apoptosis. Protein phosphatase 1 (PP1) is the primary phosphatase of Rb, responsible for the dephosphorylation of Rb at mitotic exit.…”

“…[13][14][15][16] In addition, UV stress and cdk inhibition that leads to apoptosis results in dephosphorylation of Rb. 17,18,21 Here, we show in MCF7 breast cancer cells and HCT116 colon cancer cells that various apoptotic stimuli cause dephosphorylation of Rb (Fig. 1).…”

“…[13][14][15][16][17][18][19] Also, the specific activation of Rb-directed phosphatase activity has been shown to be required for apoptosis to occur. 13,14,16,20,21 These studies suggest that phosphorylated Rb protects against apoptosis, and dephosphorylation of Rb is involved in triggering cell death. In fact, Rb that lacks nine of the 16 phosphorylation sites, called PSM-RB (phosphorylation site-mutated Rb) does not protect cells from cell death initiated by apoptotic stimuli.…”

PNUTS knockdown potentiates the apoptotic effect of Roscovitine in breast and colon cancer cells

“…The anti-proliferative activity of Rb is mediated via interaction with the E2F family of transcription factors which regulate both the G1 to S phase transition and stimulation of apoptosis (9,11). However, when proliferating cells are treated with apoptotic stimuli, Rb becomes dephosphorylated due to the activation of PP1 which can lead to cell cycle arrest and/or apoptosis (12)(13)(14). Activity of PP1 is controlled by association with regulatory subunits responsible for the substrate specificity, localization and activity of the PP1 catalytic subunit (15).…”