Transient mTOR Inhibition Facilitates Continuous Growth of Liver Tumors by Modulating the Maintenance of CD133+ Cell Populations

The mammalian target of the rapamycin (mTOR) pathway, which drives cell proliferation, is frequently hyperactivated in a variety of malignancies. Therefore, the inhibition of the mTOR pathway has been considered as an appropriate approach for cancer therapy. In this study, we examined the roles of mTOR in the maintenance and differentiation of cancer stem-like cells (CSCs), the conversion of conventional cancer cells to CSCs and continuous tumor growth in vivo. In H-Ras-transformed mouse liver tumor cells, we found that pharmacological inhibition of mTOR with rapamycin greatly increased not only the CD133+ populations both in vitro and in vivo but also the expression of stem cell-like genes. Enhancing mTOR activity by over-expressing Rheb significantly decreased CD133 expression, whereas knockdown of the mTOR yielded an opposite effect. In addition, mTOR inhibition severely blocked the differentiation of CD133+ to CD133- liver tumor cells. Strikingly, single-cell culture experiments revealed that CD133- liver tumor cells were capable of converting to CD133+ cells and the inhibition of mTOR signaling substantially promoted this conversion. In serial implantation of tumor xenografts in nude BALB/c mice, the residual tumor cells that were exposed to rapamycin in vivo displayed higher CD133 expression and had increased secondary tumorigenicity compared with the control group. Moreover, rapamycin treatment also enhanced the level of stem cell-associated genes and CD133 expression in certain human liver tumor cell lines, such as Huh7, PLC/PRC/7 and Hep3B. The mTOR pathway is significantly involved in the generation and the differentiation of tumorigenic liver CSCs. These results may be valuable for the design of more rational strategies to control clinical malignant HCC using mTOR inhibitors.


Introduction
Cancer stem-like cells (CSCs) are the most malignant subpopulations in tumors because of their resistance to drug therapy and association with poor outcomes. As in adult tissue-specific stem cells, which have extensive self-renewal abilities that ensure the continuous turnover of normal tissues, CSCs are required for continuous tumor growth. Great progress has been made in the identification and characterization of CSCs in several types of solid tumors, such as those of the brain, breast, colon and liver [1][2][3][4][5][6][7]. In addition, a few key signaling pathways, including Wnt/b-catenin, AKT and TGF-b [8][9][10][11][12][13], have been implicated in the maintenance of CSCs. However, the molecular events that preserve the pool size and stem cell properties of CSCs by maintaining the balance of proliferation, differentiation and self-renewal are still poorly understood. Elucidating the mechanistic differences between CSCs and conventional tumor cells may be valuable for developing strategies for cancer therapy.
The mammalian target of rapamycin (mTOR) is an essential serine/threonine kinase that regulates cell growth by controlling protein synthesis, autophagy, endocytosis, and metabolism in response to growth factors, nutrients, energy, and stress [14].
Increasing evidence indicates that the signaling pathways that activate mTOR are frequently improperly regulated in most human cancers [15][16][17][18][19][20][21][22][23]. Rapamycin, an inhibitor of mTOR, can block tumor growth and inhibit tumor cell motility [24][25][26]. These findings have promoted the clinical use of mTOR inhibitors for cancer therapy. However, the use of mTOR inhibitors alone has had limited clinical success [27,28]. The role of mTOR signaling in the maintenance of CSCs has been addressed recently, but the conclusions of these reports are controversial. In embryonic and adult stem cells, mTOR hyperactivation resulted in the differentiation and exhaustion of stem cells [29][30][31][32]. In the tumor development, mTOR signaling has been shown to enhance the survival of dormant tumor cells [33] and maintain the self-renewal and tumorigenicity of glioblastoma stem-like cells [34] and breast cancer stem-like cells [35]. In sharp contrast, mTOR inhibition by rapamycin has been shown to significantly increase CD133 expression in gastrointestinal cancer cells via down-regulation of HIF-1a [36]. However, there is a lack of in vivo data regarding the changes in CSC maintenance and tumorigenicity in tumor cells after transient exposure to rapamycin.
In this study, we examined the role of mTOR in the regulation of CD133+ CSCs differentiation, the conversion of CD133-conventional cancer cells to CD133+ CSCs, and the repopulation in vivo of mTOR-manipulated tumor cells. We found that mTOR inhibition increased the CD133+ subpopulation in liver tumor cells and potentiated the continuous growth of tumor cells in vivo via preventing differentiation, biased insensitivity of CD133+ subpopulation to rapamycin-induced apoptosis, and increasing the retrodifferentiation of CD133-to CD133+ cells.

mTOR inhibition increases CD133+ subpopulations and retain stemness properties in liver tumor cells
To elucidate whether mTOR is involved in the maintenance of CSCs, H-Ras-transformed mouse liver tumor cells were employed in the study. We found that the percentage of CD133+ cells, which have been referred as a subpopulation containing CSCs [5,37,38], was approximately 0.2 to 1% in H-Ras-transformed mouse liver tumor cells (LPC-H) and 10 to 20% in the derived clone LPC-H12, both of which expressed markers of live progenitor cells, and the latter displayed much high potential of tumorigenicity ( Figure  S1). To examine whether there was a significant difference in mTOR activation between CD133-and CD133+ LPC-H12 cells, we purified these two population by FASC sorting and found that the CD133+ subsets exhibited lower activation of mTOR signaling compared with CD133-cells, as indicated by the levels of phosphorylated p70 S6 kinase (p-p70 S6) ( Figure 1A). We next examined whether mTOR inhibition could lead to a change in the proportion of CD133+ LPCs. LPC-H and LPC-H12 cells were treated with the mTOR inhibitor rapamycin and measured CD133 expression by FACS. Intriguingly, the CD133+ fraction of the cells treated with rapamycin was significantly greater than that of the cells without treatment ( Figure 1B and 1C). The influence of rapamycin on the maintenance of CD133+ subpopulations was dose-and time-dependent ( Figure S2A and S2B). Expression of p-p70 S6 was undetectable in cells after rapamycin treatment, demonstrating the efficiency of rapamycin in eliminating mTOR activity ( Figure S2C). Notably, we found that the expression of several stem cell-associated genes, such as Nanog, Klf4 and SOX2, were increased significantly after treatment with rapamycin ( Figure 1D). To further identify the role of mTOR in the CD133+ subpopulation, we knocked down mTOR expression in LPC-H cells using the retrovirus expressing short hairpin RNA (shRNA) ( Figure 1E). Downregulation of mTOR expression via mTOR shRNA-2 (mTOR-sh2) dramatically increased the CD133+ fraction in cells in a time-dependent manner ( Figure 1F and 1G). Similar results were obtained in LPC-H12 cells ( Figure  S2D).
In some human liver tumor cell lines, such as Hep3B, Huh7 and PLC/PRF/5, the enhanced lever of CD133 expression after treatment with rapamycin was also detected (Figure 2A-2D). Similarly, the expression of stem cell-associated genes, including Bmi1, Oct4 amd Nanog, were significantly increased in the human liver tumor cell lines treated with rapamycin, even in MHCC-97L cells that are no CD133 expression ( Figure 2E). Collectively, these data indicate that inhibition of mTOR could enrich CSCs in both mouse and human liver tumor cells.
To investigate whether the disparities exist in the sensitivity to rapamycin between CD133-and CD133+ cells, we directly examined the effect of rapamycin on cell growth of the two subsets among LPC-H12 cells. We found that rapamycinmediated inhibition in CD133-cells was greater than that in CD133+ cells ( Figure S3), indicating that a greater impairment of the CD133-subset upon rapamycin treatment might partially contribute to the enrichment of CD133+ cells.

mTOR activation by Rheb overexpression decreases the CD133+ subpopulations
To test whether activation of mTOR signaling could decrease the CD133+ subpopulations, Rheb, an activator of mTOR signaling [39], was overexpressed in LPC-H and LPC-H12 cells by retroviral infection. We found that Rheb expression significantly enhanced mTOR activity, as determined by increased phosphorylation of p70 S6 ( Figure 3A). As expected, mTOR activation resulted in significant exhaustion of the CD133+ subsets ( Figure 3B). A time-course analysis of CD133 expression by FACS showed that Rheb overexpression gradually decreased the percentage of CD133+ cells in LPC-H12 cells (from 19.2262.09% to 3.9460.86%), whereas cells infected with empty retrovirus showed only a slight decrease in CD133 expression (from 22.8562.17% to 16.9961.28%) ( Figure 3C). Furthermore, the expression of all detected stem cell-associated genes were significantly reduced in Rheb-overexpressed cells ( Figure 3D). These results suggested that mTOR activation might enhance the differentiation of CD133+ to CD133-liver tumor cells.
Single-cell culture experiments define a critical role for mTOR in the conversion of CD133-to CD133+ liver tumor cells Previous studies have shown that conventional cancer cells can be induced by certain events to express stem cell markers and acquire properties of CSCs [40,41]. To address whether CD133cells can be converted to CD133+ tumor cells without adding exogenous TGF-b, and if so, whether mTOR signaling is involved in the process, we employed a single-cell long-term culture system ( Figure 4A). CD133-cells of LPC-H12 were FACS-sorted, and each single cell was robotically plated into individual wells of 96well plates. After 10 days in 96-well plates, single-cell derived clones were individually trypsinized and seeded into 24-well plates for expansion. After 11 days, the cells were harvested for FACS analysis of CD133 expression. Of significant interest, 46.7% of colonies derived from single CD133-LPC-H12 cells expressed CD133, and the percentage of CD133+ cells in the positive colonies was approximately 1 to 7%. Notably, we found that rapamycin treatment significantly increased both the formation of CD133+ cell colonies and the percentage of CD133+ cells in positive colonies ( Figure 4B and 4C). Accordingly, using the same protocol, downregulation of mTOR expression by shRNA in the LPC-H12 cells dramatically increased the number of colonies that contained CD133+ cells and the proportion of CD133+ cells in the positive colonies ( Figure 4D and 4E). Similar results were obtained using LPC-H cells (data not shown). In agreement with the FACS results, the level of CD133 mRNA in the single-cell-derived colonies expressing mTOR-sh2 was significantly higher than that in the colonies containing control shRNA ( Figure 4F and 4G).

Repression of mTOR inhibits differentiation of CD133+ liver tumor cells
On the basis of recent findings that mTOR activity mediates the exhaustion and differentiation of stem cells [29][30][31], we hypothesized that the increase in the proportion of CD133+ cells in the presence of rapamycin might be partially due to blockage of differentiation of CD133+ cells. To test this hypothesis, we isolated CD133+ cells from LPC-H12 by FACS sorting, and further treated cells with rapamycin for 10 days. Strikingly, we found that CD133+ expression declined substantially in the control group, whereas the proportion of CD133+ cells in the rapamycin-treated group was maintained ( Figure 5A and 5B). Moreover, we sorted CD133+ cells and seeded them for single-cell culture. At day 10 of culture, 32.8% (63 of 192 wells) and 54.6% (105 of 192 wells) single-cell-derived colonies were formed in the culture medium with or without rapamycin, respectively. These colonies were then transferred into 24-well plates. After one week of culture, we found high levels of CD133+ cells in the colonies that had been treated with rapamycin ( Figure 5C). In agreement with these results, high CD133 expression was associated with colonies derived from single CD133+ cells with expression of shRNAs against mTOR ( Figure 5D). Collectively, these results illustrate that inhibition of mTOR can maintain CD133+ subpopulations by suppressing the differentiation of CD133+ cells.

mTOR inhibition maintains CD133+ subpopulations in vivo and enhances re-propagation of Ras-dependent liver tumors
To assess the maintenance of CD133+ subpopulations in tumor cells in vivo by inhibition of mTOR, we injected LPC-H cells expressing mTOR-sh2 or control shRNA into nude mice. After two weeks, both types of cells resulted in tumor formation. However, using FACS analysis to detect GFP+ tumor cells in the xenotransplants on day 24, a high percentage of CD133+ cells were preserved in tumors derived from LPC-H cells expressing shRNA against mTOR ( Figure 6A). No significant difference was found in tumor size between the two types of cells ( Figure 6B). Increased proportion of CD133+ tumor cells was also found in the tumor tissues after treatment of the tumor-bearing mice with rapamycin for 10 days ( Figure 6C).
The increase in CD133+ subsets induced by mTOR inhibition led us to speculate that transient treatment with rapamycin might result in the enhanced continuous growth of Ras-dependent mouse liver tumors in vivo. We tested this hypothesis by taking advantage of the secondary tumor xenograft model ( Figure 7A). GFP+ LPC-H cells were subcutaneously injected into nude mice. The nude mice bearing xenografts were then treated with rapamycin or control diluents beginning 3 weeks after tumor implantation. After 10 days of rapamycin treatment, tumor growth was significantly reduced ( Figure 7B and 7C). The tumor tissues were enzymatically dissociated into single-cell suspensions, and CD133 expression was analyzed in the GFP+ LPC-H cells. The significant increase in CD133 expression in tumor cells from the first implantation after rapamycin treatment was similar to the effect of downregulation of mTOR in vitro. The tumor cells, harvested by FACS sorting of GFP+ population, were then transplanted into the secondary recipients. We found that as few as 2.5610 3 tumor cells with rapamycin treatment were sufficient to initiate tumor development, whereas up to 1610 5 tumor cells without rapamycin treatment failed to initiate subcutaneous tumors in secondary recipient mice ( Figure 7D and 7E). Thus, mTOR inhibition by rapamycin could block tumor growth from the first implantation but enhance continuous growth of the secondary tumors derived from H-Ras-transformed mouse liver cells.

Discussion
Aberrant activation of the mTOR pathway has been implicated in the growth of various human cancers [42,43]. mTOR signaling promotes cell proliferation, and, in the context of cancer development, it is highly correlated with tumor expansion and rate of tumor growth. Remarkably, several reports have highlighted the critical importance of mTOR signaling in driving differentiation of adult progenitor cells accompanied by rapid cell expansion [29][30][31][32]. Recently, it has been established that the preservation of small subpopulations of CSCs within tumors is critical for tumors to assume malignant behavior and resistance to therapy. To date, whether mTOR signaling is critically involved in maintaining the balance between proliferation and differentiation is still poorly defined. Because mTOR inhibitors have been utilized for cancer therapy, and only modest or unremarkable success has been achieved in some clinical trials [27,28], it is urgent to investigate the long-term effects of transient mTOR treatment on tumor re-emergence and malignant outcome. We demonstrated here that targeted inhibition of mTOR significantly enhanced the generation and maintenance of CD133+ subpopulations and promoted secondary tumor re-propagation of H-Rastransformed mouse liver tumor cells in vivo.
CD133 is considered a universal marker of stem cells and CSCs [44][45][46]. CD133+ tumor cells exhibit properties of CSCs in human hepatocellular carcinomas and mouse liver cancer [47][48][49][50][51][52]. In this study, we found that H-Ras-transformed mouse liver tumor cells expressed the surface marker CD133. To our surprise, by exploiting the difference in the status of mTOR activation between CD133+ and CD133-populations, we found elevated mTOR signaling in the CD133-subset. Although the mTOR pathway has been reported to be important for the viability and maintenance of breast and glioblastoma CSCs [34,35], inhibition of mTOR with rapamycin in H-Ras-transformed mouse liver cancer cells significantly increased the proportion of CD133+ cells and retained the stemness properties. Using RNAi-mediated knockdown of mTOR and overexpression of Rheb, an activator of the mTOR kinase [53], we confirmed a critical role for mTOR in exhaustion and differentiation of the CD133+ subsets. Interestingly, these data are consistent with the notion that elevated p70 S6 kinase activation induces differentiation of human embryonic stem cells (hESCs) [30]. Furthermore, a previous study has shown that persistent activation of mTOR in normal epithelial stem cells results in exhaustion of these stem cells [29]. Notably, mTOR pathway is also significantly important in the process of cellular senescence in multiple human and rodent cells [54][55][56]. Rapamycin and MEK inhibitor that can target mTOR/S6 pathway, decelerate cellular senescence. Moreover, mTOR inhibition attenuates cellular senescence but favors quiescence in p53-arrested cells. These important findings are principally supportive to our observation since the impaired capacity in proliferation and the great potential to regain propagation  C, D qPCR analysis shows enrichment of CD133 expression in human liver tumor cell lines after exposed to rapamycin (100 nM). Relative expression of CD133 in Hep3B, Huh7, and PLC/PRF/5 with or without treatment of rapamycin for 48 h is shown in C. Sustained increase of CD133 expression in Huh7 after treatment of rapamycin with 10 nM or 100 nM is illustrated in D. The sustained GAPDH was used as an internal control. E qPCR ananlysis was performed for stem cell-associated genes in multiple human liver tumor cell lines. Cells without treatment of rapamycin (100 nM) were defined as 1, and the relative expression of each gene in cells with treatment of rapamycin for 48 h was expressed as the fold difference over this baseline. GAPDH was used as an internal control. doi:10.1371/journal.pone.0028405.g002 (rejuvenation) are basically the features of CSCs. We must note that the similar effect of rapamycin was also found in multiple human liver tumor cell lines, such as Huh7, PLC/PRF/5, and Hep3B, suggesting that, even it was able to inhibit growth of tumor, rapamycin might enrich liver cancer stem-like cells in a clinical setting.
Several studies have revealed that cellular stress (such as hypoxia), cytokines (such as TGF-b), or activation of the mTOR pathway are able to increase the expression of CSC surface markers and phenotypes in certain bulk tumor cells [36,48,57,58], indicating that cell-extrinsic environmental factors may reprogram conventional tumor cells to cells with stem cell-like properties in the course of cancer development. In the present study, using single-cell culture experiments that can bring out conclusive results, we unexpectedly revealed that CD133-liver tumor cells were capable of converting to CD133+ cells, and blocking intracellular mTOR activity with rapamycin or RNAi against mTOR indeed promoted the conversion of CD133-to CD133+ cells. Notably, certain stem cell-associated genes are upregulated by rapamycin prior to the increasing of CD133 expression in LPCs, suggesting that these genes might function in the rapamycin-mediated enrichment of CD133+ subpopulations. Previous studies have shown that HIF-1a is a positive downstream target of the mTOR signaling pathway [59]. In human glioma cells, the activation of HIF-1a enhances CD133+ glioma-derived cancer stem cell expansion by increasing self-renewal activity and   [60]. Thus, the relationship between HIF-1a and CD133 expression in the process of conversion requires further investigation.
In human liver tumor cells, the induced CD133+ cells display significant potential for tumorigenesis [48]. Indeed, we also found that CD133 expression and certain stemness genes were upregulated with the treatment of rapamycin. The relevant significance of the regulation mediated by rapamycin on CSCs maintenance was illustrated by the in vivo study, in which we demonstrated that the tumor cells harvested from the first implantation with treatment of rapamycin were significantly higher in secondary tumorigenicity than those from the control, as manifested by the increased CD133 expression. Notably, these results are in agreement with the evidence derived from RNAi-C. D-G Knockdown of mTOR expression enhanced the rate of conversion of CD133-to CD133+ cells. The percentages of CD133+ colonies derived from single CD133-LPC-H12 cells infected with retrovirus containing mTOR-sh2 and control, respectively, are shown in D (n = 12 per group). The proportions of CD133+ cells in each colony are shown in E. The expression of CD133 mRNA in each clone examined by Real-time PCR experiments in triplicate, and all samples were normalized to GAPDH expression (F). The result of RT-PCR is shown in G. doi:10.1371/journal.pone.0028405.g004 The single CD133+ cell-derived colonies cultured in the media with rapamycin had more CD133+ cells than those in rapamycin-free media. Single CD133+ LPC-H12 cells were sorted into 96-well plates and cultured with or without rapamycin (10 nM). Each symbol represents an individual single-cell-derived colony; small horizontal lines indicate the means (n = 8). D The percentage of CD133+ cells in each colony derived from single CD133+ LPC-H12 stably expressing mTOR-sh2 was higher than that from single CD133+ LPC-H12 infected with control retrovirus. Data are shown as the means6SD (n = 8). doi:10.1371/journal.pone.0028405.g005 mediated downregulation of mTOR expression. These data suggest that mTOR signaling is involved in regulation of the balance of proliferation and differentiation of CSCs in Rasdependent tumorigenesis and that transient inhibition of mTOR promotes tumor re-emergence via an increased CD133+ subpopulation.
Taken together, our observations show that mTOR inhibition enriches CD133+ subpopulations. This enrichment is most likely achieved through blocking differentiation of the CD133+ subpopulations, enhancing apoptosis in the CD133-subsets, and triggering the conversion of CD133-to CD133+ cells. The maintenance of CD133+ cells in vivo by rapamycin leads to high continuous tumorigenic potential. Therefore, therapeutic design strategies should consider the possibility that complex regulatory events in CSC generation may be triggered by mTOR inhibition.

Ethics statement
All animal experiments were conducted in accordance with protocols approved by the Shanghai Medical Experimental Animal Care Commission (approval ID. ShCI-11-020).

Cell culture
Isolation, culturing and retroviral infection of purified p53-/-mouse fetal liver progenitor cells were performed as described by Zender et al. [61,62]. Retroviruses were based on MSCV-IRES-GFP vectors containing cDNAs encoding H-Ras and used for infection of the purified cells. H-Ras-transformed mouse liver tumor cells were cultured in a maintenance medium containing 1:1 DMEM:F12 (GIBCO, Grand Island, NY) with 10% fetal bovine serum (GIBCO)

Retroviral transfection
A Rheb cDNA was Flag-tagged and cloned into the retroviral expression vector MSCV (MSCV-Rheb). Recombinant retroviral particles were generated from the Phoenix packaging cell line and harvested 2 consecutive times for infection. Transfected cells were pooled for further analysis after selection with 10 mg/ml of puromycin (Sigma-Aldrich) for 1 week and then with 5 mg/ml for additional 1-2 weeks. Short-hairpin RNAs targeting mTOR (mTOR-sh1 and mTOR-sh2), based on mTOR coding sequences 5'-AAGAATGTTGACCAATGCT-3' and 5'-TGATGGACA-CAAATACCAA-3' respectively, were expressed from the LTR promoter of MSCV retroviruses.

RNA extraction, reverse-transcription, and RT-qPCR
Total RNA was extracted from cultured cells with RNAiso Reagent (TaKaRa, Dalian, China) and converted to cDNA using the PrimeScript TM RT reagent kit (TaKaRa) according to the manufacturer's protocol. Gene expression analysis was determined using primers for CD133, AFP, albumin (Alb), cytokeratin 19 (CK19), and GAPDH, as listed in Table S1. For detection of CD133 expression, relative quantitative PCR (qPCR) was performed on a 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA) using Premix Ex TaqTM (TaKaRa). Quantitative real-time PCR was performed using SYBR green mix from TaKaRa for Bmi1, Klf4, Nanog, Oct4, and Sox2. All samples were normalized to the level of GAPDH. The primers are listed in Table S1.

Fluorescence-activated cell sorting and single-cell analysis
Cells were dissociated and resuspended in PBS containing 0.5% BSA. Flow cytometry was performed with PE-conjugated anti-mouse CD133 antibody (eBioscience, San Diego, CA) or PEconjugated anti-human CD133 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) incubated at 4uC for 30 min. Rat IgG1/k antibody conjugated to phycoerythrin was used as an isotype control. Preincubation with 7-AAD (Sigma-Aldrich) was used to exclude dead cells.
For cell sorting, samples were stained with PE-conjugated anti-mouse CD133 antibody and analyzed using a MoFlo XDP (Beckman Coulter, Fullerton, CA) with rat IgG1/k antibody conjugated to phycoerythrin as isotype controls. Isolated CD133-or CD133+ cells were plated in 6-well plates. For single-cell experiments, single CD133-or CD133+ cells were stringently gated, isolated using a FACSVantage set for Single Cell Purity, and robotically plated into a 96-well plate with 150 ml medium/well. After 24 h, an additional 50 ml medium with or without rapamycin was added to each well. After 1-2 weeks in culture, robust single-cell-derived colonies that filled more than 50% of the well area were transferred into 24-well plates for expansion.

Tumor cell preparation
Tumor tissue was mechanically cut into small pieces with sterile scalpel blades, then enzymatically dissociated for 1 h with a mixture of collagenase type I (0.05 mg/ml; Sigma-Aldrich), collagenase type IV (0.05 mg/ml; Sigma-Aldrich), hyaluronidase (0.025 mg/ml; Sigma-Aldrich), and DNase I (0.01 mg/ml; Roche) at 37uC and shaken repeatedly. The dissociated sample was then filtered (70-mm pore size) and washed with PBS. Cells were then injected into nude BALB/c mice for secondary tumor propagation or stained with mouse CD133-PE antibody for assessing CD133 expression using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA).

Subcutaneous implantation into nude BALB/c mice and rapamycin treatment
H-Ras-transformed mouse liver tumor cells (1610 6 ) were suspended in 100 ml PBS and injected subcutaneously into male nude BALB/c mice (5 to 6 weeks of age). Tumor size was serially measured using calipers. Tumor volume was estimated using the following formula: (w 1 6w 2 6w 2 )/2, where w 1 represents the length and w 2 represents the width of the tumor. In some experiments, rapamycin (LC Laboratories, Woburn, MA) was reconstituted in absolute ethanol at 10 mg/ml and diluted in 5% PEG-400 (Sigma-Aldrich) and 5% Tween-80 (Sigma-Aldrich) for treatment of mice. Each treatment group consisted of 4 or 5 animals. Nude mice bearing xenografts received 1.5 mg/kg rapamycin by i.p. injection daily starting 3 weeks after tumor transplantation. Control animals bearing tumors were treated with diluent at equivalent doses and schedules to the experimental animals. Tumors were removed and injected into secondary recipients by limiting dilution experiments.

Apoptosis assay
For quantification of apoptosis, Pharmingen TM PE Aneexin V Apoptosis Detection Kit (BD Biosciences) was performed according to the manufacturer's instructions.

Statistical analysis
Statistical analyses were performed using SPSS 11.5.0. A paired, two-tailed Student's t test was used to determine the significance between two groups. A p value less than 0.05 was considered statistically significant.