Our earlier work [55] and the data presented in Physique 2 have shown that erlotinib treatment alone has no effects on DLD-1 cell growth and, thus, we omitted erlotinib treatment alone and saved a group of mice. was transient only in the first few hours of the PP242 treatment. Receptor tyrosine kinase arrays further revealed that PP242 treatment GSK1059615 increased the phosphorylated epidermal growth factor receptor (EGFR) at Tyr 1068 (EGFRT1068). The parallel increase of AKTS473 and EGFRT1068 in Hbb-bh1 the cells following PP242 treatment raised the possibility that EGFR phosphorylation might contribute to the PP242 incomplete inhibition of mTORC2. To test this notion, we showed that this combination of PP242 with erlotinib, an EGFR small molecule inhibitor, blocked both mTORC1 and mTORC2 kinase activity. In addition, we showed that this combination treatment inhibited colony formation, blocked cell growth and induced apoptotic cell death. A systemic administration of PP242 and erlotinib resulted in the progression suppression of colorectal carcinoma xenografts in mice. This study suggests that the combination of mTOR kinase and EGFR inhibitors may provide an effective treatment of colorectal carcinoma. Introduction Colorectal carcinoma is the third most common cancer in men and women but the second leading cause of cancer-related deaths GSK1059615 in the United States [1]. Recent advances in research suggest that targeting of mTOR pathway may provide novel therapies for clinical treatment of the carcinoma [2]. The mTOR is usually a conservative serine/threonine (S/T) protein kinase of the phosphatidylinositol 3-kinase (PI3K) family [3]. The mTOR kinase exists in two functional complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) [4]. Both the complexes contain the mTOR kinase but they are distinguished by unique regulatory proteins: the regulatory-associated protein of mTOR (RAPTOR) defines mTORC1 [5] whereas the rapamycin-insensitive companion of mTOR (RICTOR) is usually specific to mTORC2 [6]. The mTORC1 controls the rate of protein synthesis through phosphorylation and activation of its substrates, p70S6 ribosomal kinase 1 (p70S6K) and eukaryotic translation initiation factor 4E (eIF4E) binding protein-1 (4E-BP1) and once phosphorylated, p70S6K phosphorylates ribosomal protein S6 and 4E-BP1 becomes dissociated from eIF4 and promote mRNA translation and protein synthesis [7]. On the other hand, mTORC2 regulates cell survival and cell cycle progression through phosphorylation of AKT, serum- and glucocorticoid-regulated kinase (SGK) and protein kinase C (PKC) [8C11]. mTOR is usually a central integrator for upstream inputs from growth factors, nutrients and stress [12]. Insulin-like growth factor-1 (IGF1), for instance, can activate mTORC1 through its receptor tyrosine kinase (RTK)-mediated phosphorylation and activation of PI3K and AKT and AKT in turn mediates phosphorylation of tuberous sclerosis 2 (TSC2) and proline-rich AKT substrate 40 kDa (PRAS40), thus releasing their inhibition of mTORC1 [13,14]. RTKs also activate mTORC1 through Ras-extracellular signal-regulated kinase (ERK) pathway [15] and subsequent ERK phosphorylation of the mTORC1 inhibitor TSC2 [16] and RAPTOR [17]. This growth factor-mTORC1 pathway is usually regulated through two unfavorable feedback loops: mTORC1-p70S6K-mediated phosphorylation and degradation of insulin receptor substrate (IRS) [18,19] and mTORC1-mediated phosphorylation of growth factor receptor-bound protein 10 (GRB10) [20]. The mTOR pathway is usually overactive in cancers [21]; thus, mTOR inhibitors have been developed as cancer therapeutic brokers [22,23]. The first generation of mTOR inhibitors, rapamycin and its analogs (known as rapalogs) such as everolimus (RAD001), temsirolimus (CCI-779) and ridaforolimus (AP23573) have entered clinical trials but, unfortunately, shown limited clinic benefits against many types of cancers [24,25], even though temsirolimus has been approved for clinical treatment of renal cell carcinoma in United States [26]. Patients with advanced carcinoma, for instance, show a partial response to rapalog treatment in phase I trials [27,28]. The cancer resistance to the rapalog treatment is mainly due to the presence of unfavorable feedback loops. Rapamycin interacts with FK506 binding protein 12 (FKBP-12) and form a complex that binds and removes RAPTOR from mTORC1 [29]; thus, rapamycin inhibits mTORC1 but has little effect on mTORC2. By inhibiting mTORC1, rapalog prevents inhibitory IRS phosphorylation and degradation and activates PI3K/AKT [30,31] and ERK pathway through the feedback loops [32C34]. In addition, rapalogs incompletely inhibit the 4E-BP1 phosphorylation [35] and do not induce apoptosis in cancer cells [36] because the IGF1 pathway inhibits apoptosis [37]. The GSK1059615 second generation of mTOR inhibitors has been developed to target the adenosine triphosphate (ATP)-binding site of mTOR kinase [38]. These mTOR kinase inhibitors such as Torin1, PP242 and PP30 block p70S6K, 4E-BP1 and AKT phosphorylation and.