Abstract
Phosphatidylinositol 3'-kinase (PI3K) activity is required for Ras- mediated transformation of intestinal epithelial cells (IECs). The mammalian target of rapamycin (mTOR) and its downstream pathways control the translation of specific mRNAs that are required for cell proliferation and transformation. Here, we elucidated the roles of PI3K and mTOR in K-Ras-mediated transformation of IECs (IEC-6). Induction of K-Ras activated PI3K and mTOR in IECs. p70 ribosomal protein S6 kinase activity was induced by K-Ras in a PI3K- and mTOR-dependent manner. K-Ras did not significantly alter the phosphorylation of eukaryotic initiation factor 4E-binding protein 1. Treatment with either LY-294002 or rapamycin inhibited IEC proliferation and resulted in G(1) growth arrest. However, it was noted that inhibition of mTOR enhanced K-Ras-mediated morphological transformation and increased invasiveness of IECs in a mitogen-activated protein/extracellular signal-regulated kinase-dependent manner. Furthermore, inhibition of PI3K or mTOR impaired the growth of an array of colon cancer cells. Spindle transformation, reduced E-cadherin, and increased invasiveness were observed in LY-294002-treated Moser cells. Thus, our results suggest that K-Ras-mediated transformation of IECs involves activation of the PI3K/mTOR pathway. Inhibition of PI3K/mTOR activity leads to G(1) growth arrest of transformed IECs. On the other hand, inhibition of PI3K or mTOR may induce the epithelial to mesenchymal transdifferentiation of IECs under certain circumstances.
Highlights
About 50% of colorectal carcinomas contain K-Ras mutations [1], and the K-Ras oncogene plays a key role during the adenoma to carcinoma sequence of events involved in the neoplastic transformation of colonic epithelial cells [2]
We show the critical roles of phosphatidylinositol 3Ј-kinase (PI3K)/mammalian target of rapamycin (mTOR) in intestinal epithelial growth and transformation; this pathway can be targeted for cancer treatment
We have shown that induction of Ha-Ras activates PI3K/Akt, which is critical for Ras-mediated growth and transformation of rat intestinal epithelial (RIE) cells [16]
Summary
About 50% of colorectal carcinomas contain K-Ras mutations [1], and the K-Ras oncogene plays a key role during the adenoma to carcinoma sequence of events involved in the neoplastic transformation of colonic epithelial cells [2]. The Raf/mitogen-activated protein/ extracellular signal-regulated kinase (MEK)/extracellular signalregulated kinase (ERK) pathway has been implicated as a critical effector of Ras function [3]. The phosphatidylinositol 3Ј-kinase (PI3K)/protein kinase B (Akt) pathway lies downstream of receptor tyrosine kinases and can be directly activated by Ras [7]. The IEC-i-K-Ras cell line with an inducible K-RasVal cDNA was generated by using LacSwitch eukaryotic expression system (Stratagene, La Jolla, CA) and grown in DMEM containing 10% fetal bovine serum [27]. Isopropyl-1-thio--D-galactopyranoside (IPTG; Life Technologies, Inc., Gaithersburg, MD) at a concentration of 5 mM was used to induce the expression of mutated K-Ras. The RIE-i-myr-p110␣ cell line has been described previously [28]. The anti-pan-Ras antibody was purchased from Calbiochem (La Jolla, CA). The DNA was separated on 1.6% agarose gels
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