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Inhibition of p70S6K does not mimic the enhancement of Akt phosphorylation by rapamycin

It has been suggested that the mTOR complex 1 (mTORC1)/p70S6K axis represses upstream PI3K/Akt signaling through phosphorylation of IRS-1 and its subsequent degradation. One potential and current model that explains Akt activation induced by the mTOR inhibitor rapamycin is the relief of mTORC1/p70S6K-mediated feedback inhibition of IRS-1/PI3K/Akt signaling, although this has not been experimentally proven. In this study, we found that chemical inhibition of p70S6K did not increase Akt phosphorylation. Surprisingly, knockdown of p70S6K even substantially inhibited Akt phosphorylation. Hence, p70S6K inhibition clearly does not mimic the activation of Akt by rapamycin. Inhibition or enforced activation of p70S6K did not affect the ability of rapamycin to increase Akt phosphorylation. Moreover, inhibition of mTORC1 with either rapamycin or raptor knockdown did not elevate IRS-1 levels, despite potently increasing Akt phosphorylation. Critically, knockdown or knockout of IRS-1 or IRS-2 failed to abolish the ability of rapamycin to increase Akt phosphorylation. Therefore, IRS-1 and IRS-2 are not essential for mediating rapamycin-induced Akt activation. Collectively, our findings suggest that Akt activation by rapamycin or mTORC1 inhibition is unlikely due to relief of p70S6K-mediated feedback inhibition of IRS-1/PI3K/Akt signaling.


Introduction
The ribosomal protein, 70 kDa ribosomal S6 kinase (p70S6K), is one of the best characterized downstream effectors of mTOR complex 1 (mTORC1). In response to mitogenic stimuli or nutrient availability, mTORC1 is activated, leading to phosphorylation of p70S6K. The first identified substrate of p70S6K is the ribosomal protein S6, a component of the 40S ribosome. It is generally recognized that the mTORC1-p70S6K axis controls fundamental cellular processes, including transcription, translation, protein and lipid synthesis, cell growth/size, and cell metabolism. Hence, p70S6K plays important roles in a number of pathologies, including obesity, diabetes, ageing, and cancer [1,2].
It is generally thought that mTORC1 functions downstream of the PI3K/Akt pathway and is phosphorylated (or activated) in response to stimuli that activate the PI3K/Akt pathway [3,4,5]. We and others have shown that the conventional mTOR inhibitor rapamycin and its analogues (rapalogs) activate Akt while suppressing mTORC1 signaling in different types of cancer cell lines and clinical human tumor samples [6,7,8]. Our previous study suggested that sustained Akt phosphorylation during rapamycin treatment seems associated with the development of rapamycin resistance [8]. Another study suggested that early increase in Akt phosphorylation predicts better response of cancer cells to rapalogs [9]. Moreover, prevention of Akt activation induced by mTOR inhibition exhibits enhanced cancer therapeutic efficacy in different preclinical studies [8,10,11,12].
Insulin receptor substrate-1 (IRS-1) is an important mediator of insulin receptordependent activation of PI3K. Chronic insulin stimulation causes the phosphorylation and degradation of IRS-1 protein in a rapamycin-sensitive manner [13]. Thus, studies on insulin signaling in mammalian skeletal muscle cells, adipocytes, and fibroblasts have suggested that mTORC1 activation by insulin initiates the feedback inhibition of PI3K and Akt, likely through p70S6K activation and its subsequent phosphorylation of IRS-1. The phosphorylation of IRS-1 promotes IRS-1 degradation and reduces its abundance, leading to decreased activity of PI3K and Akt. Thus, it has been proposed that rapalogs suppress p70S6K, thereby relieving this negative feedback inhibition of Akt and leading to activation of the PI3K/Akt survival pathway [7,13,14]. However, our recent work has suggested that protein phosphatase 2A and DNA-dependent protein kinase are involved in mediating rapamycin-induced Akt phosphorylation [12]. Moreover, Src-family kinase (SFK)-dependent Akt activation also occurs during rapamycin treatment [11].
The importance of p70S6K function in human diseases has led to the development of p70S6K-specific inhibitors by a number of companies as potential therapeutic agents [2,15]. Considering the central role of p70S6K in feedback inhibition of PI3K/Akt signaling activation as discussed above, we were interested in addressing the question of whether inhibition of p70S6K (e.g., by a chemical inhibitor) exerts similar effect on Akt activation as rapalogs do. Interestingly, we found that both chemical and genetic inhibition of p70S6K fails to increase Akt phosphorylation, suggesting that inhibition of p70S6K does not necessarily result in the activation of PI3K/Akt signaling.

Chemical inhibition of p70S6K does not increase Akt phosphorylation
FRI00705 is a specific inhibitor of p70S6K1 (IC 50 = 3 nM) and p70S6K2 (IC 50 = 35 nM) developed by Forest Research Institute (Jersey City, NJ). We first determined whether FRI00705 is able to increase Akt phosphorylation, as rapamycin does, by comparing the ability of FRI00705 and rapamycin to suppress p70S6K signaling and induce Akt phosphorylation. In A549 cells, rapamycin at both 1 nM and 10 nM clearly increased Akt phosphorylation while decreasing the levels of not only p-S6, but also p-rictor (T 1135 ), another novel substrate of p70S6K [16,17]. In contrast, FRI00705 at concentration ranges between 100 nM and 10 μM effectively reduced the levels of p-S6 and p-rictor, but did not increase Akt phosphorylation ( Fig. 1A and B). Similar results were also generated in other cancer cell lines including H157, Calu-1, H1299, Du145, and DLD1 (Fig. 1C).
These results clearly show that inhibition of p70S6K with FRI00705 does not necessarily lead to increased Akt phosphorylation.

Genetic inhibition of p70S6K1 does not increase Akt phosphorylation or impair the ability of rapamycin to increase Akt phosphorylation
We then determined whether genetic inhibition of p70S6K1 using a small interfering RNA (siRNA) approach increases Akt phosphorylation and impacts rapamycin-induced Akt phosphorylation. p70S6K1 siRNA substantially reduced the level of p70S6 K, indicating a successful gene silencing effect ( Fig. 2A). Moreover, the levels of p-S6 and p-Rictor were also reduced, indicating that p70S6K1 knockdown also inhibits downstream signaling. However, the level of basal p-Akt was decreased to a substantial degree in cells transfected with p70S6K1 siRNA ( Fig. 2A and B). In addition, IRS-1 levels were not altered ( Fig. 2B). Treatment with rapamycin increased the levels of p-Akt not only in control siRNA-transfected cells but also in p70S6K1 siRNA-transfected cells to comparable degrees. Rapamycin did not alter the level of IRS-1 (Fig. 2B). These results suggest that inhibition of p70S6K does not result in IRS-1 elevation or Akt Article No~e00378 activation and that rapamycin increases Akt phosphorylation under the condition that p70S6K is depleted.
Article No~e00378 however, rapamycin was equally potent in increasing the level of p-Akt in these cells transfected with empty vector, WT p70S6K1, or mutant p70S6K1, regardless  of whether p70S6K signaling was inhibited or not (Fig. 3). In this experiment, we also did not see any effect of rapamycin on the level of IRS-1. Thus, these data also do not support a role for p70S6K in rapamycin-induced Akt phosphorylation.

Rapalogs or raptor knockdown increases Akt phosphorylation without altering IRS-1 levels
Following the above experiments, we determined whether IRS-1 protein levels are elevated while Akt phosphorylation is increased in cells exposed to rapalogs. We compared the effects of rapamycin and RAD001 on the modulation of p-Akt and IRS-1 in a group of lung cancer cell lines after either a short (i.e., 1 h) or long (24 h) treatment time. Among the 8 tested cell lines, IRS-1 was not detected in two cell lines (i.e., H460 and H226 cells). In the remaining cell lines in which IRS-1 was present, rapamycin or RAD001 treatment did not alter IRS-1 levels regardless of whether the cells were treated for 1 h or 24 h. However, the level of p-Akt was increased in all cell lines treated with rapamycin or RAD001 regardless of IRS-1 presence or modulation and exposure time ( Fig. 4A and B). We also extended the study to other types of cancer cells and obtained similar results in HCT116 (colon cancer) and MCF-7 (breast cancer) cells. In Du145 (prostate cancer) cells, we  . 4C). Moreover, we knocked down raptor in A549 cells and found that this did not increase the level of IRS-1, but effectively elevated p-Akt and reduced p-p70S6K (Fig. 4D). Taken together, these data clearly indicate that mTORC1 inhibition induces Akt phosphorylation without the apparent elevation of IRS-1 levels.

Knockdown or knockout of IRS-1, IRS-2, or both does not prevent rapamycin from increasing Akt phosphorylation
We examined the effects of rapamycin on Akt phosphorylation in cell lines in which IRS-1 was knocked down by IRS-1 siRNA or knocked out. Transfection of IRS-1 siRNA efficiently reduced the level of IRS-1 in three lung cancer cell lines (i.e., A549, H157 and Calu-1) as detected by Western blotting (Fig. 5A). Under such conditions, rapamycin treatment for 1 h did not alter the level of IRS-1, but increased p-Akt in both control siRNA-and IRS-1 siRNA-transfected cells to comparable degrees (Fig. 5A). We found that basal levels of p-Akt were lower in IRS-1/KO murine 3T3 cells than in IRS-1 WT cells. Treatment with rapamycin for 30 min or 3 h did not increase the levels of IRS-1 in IRS-1 WT cells, but increased p-Akt levels in both IRS-1 WT and knockout cell lines (Fig. 5B). We also examined the effects of rapamycin on Akt phosphorylation in WT, IRS-1/KO, and IRS-2/KO murine embryonic fibroblasts (MEFs) and found that rapamycin treatment increased the level of p-Akt in both IRS-1 and IRS-2 knockout cells (Fig. 5C). Furthermore, we knocked down IRS-2 or both IRS-1 and IRS-2 in human cancer cells and found that knockdown of IRS-2 or both IRS-1 and IRS-2 did not inhibit rapamycin-induced Akt phosphorylation (Fig. 5D). These results indicate that rapamycin is unlikely to increase Akt phosphorylation through IRS-1 and/or IRS-2.

Discussion
It has been suggested that mTORC1/p70S6K represses upstream PI3K signaling through phosphorylation of IRS-1 and its subsequent degradation [14,18,19]. Therefore, one potential and widely accepted model [15] that may explain rapamycin-induced Akt activation is the relief of an mTORC1/p70S6K-mediated feedback inhibition of IRS-1/PI3K [7,19], although this has not been experimentally confirmed. If this model is correct, inhibition of p70S6K would mimic mTORC1 inhibition (e.g., by a rapalog) in activating Akt. However, we demonstrated that while both rapamycin treatment and chemical inhibition of p70S6K with FRI00705 effectively suppressed S6 and rictor phosphorylation, p-Akt levels were potently elevated by rapamycin but not by FRI00705 (Fig. 1).
Surprisingly, knockdown of p70S6K1 substantially suppressed Akt phosphorylation (Fig. 2). Hence it is clear that inhibition of p70S6 K does not mimic the activation of Akt by rapamycin or mTORC1 inhibition.
Our previous study clearly showed that rapamycin increases p-Akt rapidly, within 1 h of treatment [6]. Moreover, Akt seemed to be more sensitive than p70S6K to mTORC1 inhibition, since the increase in Akt phosphorylation in response to mTORC1 inhibition occurred under conditions in which p-p70S6K levels were not reduced: e.g., transient knockdown of raptor as we demonstrated previously [8]. This notion is further supported by our finding that knockdown of p70S6K itself actually decreased the basal level of p-Akt, which could be increased upon rapamycin treatment (Fig. 2). In agreement, overexpression of constitutively active and rapamycin-resistant p70S6K1 mutants attenuated the ability of rapamycin to inhibit S6 phosphorylation, but did not affect its ability to increase Akt phosphorylation (Fig. 3). Collectively, it is clear that inhibition of p70S6K is unlikely to be the mechanism leading to increased Akt phosphorylation. In agreement with our finding, a recent study reported that inhibition of mTORC1 by Article No~e00378 knocking down raptor in p70S6K1/p70S6K2 double knockout cells still increased Akt phosphorylation without affecting IRS-1 abundance [20].
Although O'Reilly et al. [7] reported that rapamycin increased IRS-1 abundance from 1 h to 24 h in the cell lines tested, we did not find that either rapamycin or RAD001 increased the level of IRS-1 in most of the tested cell lines, whereas p-Akt levels were increased in every tested cell line under our tested conditions (1 or 24 h treatment). Moreover, rapamycin still effectively increased p-Akt levels even in cell lines in which IRS-1 was not detected (e.g., H460 and H226 cells) or knocked down (e.g., A549, H157 and Calu-1). Importantly, rapamycin still increased Akt phosphorylation even in the IRS-1 knockout cell line; this finding is in agreement with a previous report by Wan et al. [21], showing that knockdown of IRS-1 failed to block the effects of mTOR inhibitors on increasing Akt phosphorylation. Collectively, we conclude that IRS-1 is not required for rapid Akt phosphorylation by rapalogs.
Since IRS-2 can also play a role in regulating PI3K and Akt signaling [22], we also examined whether IRS-2 is required for mTOR inhibitor-induced Akt phosphorylation. Both knockdown and knockout of IRS-2 failed to inhibit rapamycin-induced Akt phosphorylation. Even co-knockdown of both IRS-1 and IRS-2 did not suppress rapamycin-induced Akt phosphorylation. Thus, rapalogs also increase Akt phosphorylation independently of IRS-2.
We noted that rapamycin did increase the levels of p-Akt and IRS-1 in MDA-MB-435 cells after both 1 h and 24 h and in Du145 cells after 24 h of rapamycin treatment. Thus, we cannot rule out the possibility that sustained exposure to rapamycin may result in Akt phosphorylation through an IRS-1-dependent mechanism in certain cancer cell lines (e.g., MDA-MB-435).
Genetic inhibition of p70S6K through gene knockdown substantially decreased p-Akt levels in this study. The underlying mechanism is unknown. Rictor phosphorylation at T 1135 by p70S6K does not affect mTORC2 assembly and kinase activity [16,17,23]. However, it has been suggested that this phosphorylation inhibits mTORC2 and Akt S 473 phosphorylation albeit with no effects on phosphorylation of SGK1 or PKCα [16,23]. Our results clearly show that both chemical inhibition and genetic knockdown of p70S6K effectively inhibited rictor T 1135 phosphorylation, but generated different outcomes on Akt Akt phosphorylation. Further study of the mechanism accounting for the positive regulation of Akt activity by p70S6K is warranted.

Reagents
Rapamycin was purchased from LC Laboratories (Woburn, MA). RAD001 was provided by Novartis Pharmaceuticals Corporation (East Hanover, NJ).

Cell lines and cell culture
Human cancer cell lines used in this study were purchased from the American Type Culture Collection (ATCC; Manassas, VA) and were grown as described previously [6]. Murine WT and IRS-1 knockout (IRS-1-KO) 3T3 fibroblasts [26] were obtained from Dr.

Western blot analysis
The procedures for preparation of whole-cell protein lysates from cells and for Western blotting were described previously [8]. Since rapalog treatment does not alter the total levels of Akt, p70S6 and S6 based on our previous studies [6, 8, 12, 28], we did not detect these proteins in some experiments while their phosphorylation was detected. SMARTpool ® siRNAs were purchased from Upstate Biotechnology. p70S6K1 (Cat. #1027217) and control (non-silencing) (Cat. #1022076) siRNAs were ordered from Qiagen (Valencia, CA). Raptor siRNA that targets 5′-AAGGC-TAGTCTGTTTCGAAAT-3′ [29] was synthesized by Qiagen. The transfection of siRNA was conducted in a 12-well plate or 24-well plate using Lipofectamine 2000 (Invitrogen) or HiPerFect (Qiagen) following the manufacturer's instructions.
Forty-eight hours after transfection, cells were treated with DMSO or rapamycin for the given times and then subjected to detection of the given proteins by Western blot analysis.

Declarations
Author contribution statement