TARGETED INHIBITION OF mTOR SIGNALING INHIBITS TUMORIGENESIS OF COLORECTAL CANCER

Immunohistochemistry

Tissue microarrays containing normal and cancer tissues, A203 (VI), were purchased from ISUABXIS through Accurate Chemical & Scientific Corporation (Westbury, NY). Each array consisted of tissue derived from 45 patients: 90 tumor cores, 8 normal cores. Immunohistochemistry (IHC) was performed as described previously (10). For negative controls, primary antibody was omitted from the above protocol. Scoring was performed blindly by a pathologist according to a semi-quantitative seven-tier system developed by Allred et al. (20). This system assesses the percentage of positive cells (none = 0; <10% = 1; 10% to 50%, = 2; >50% = 3) and intensity of staining (none = 0; weak = 1; intermediate = 2; and strong = 3). The intensity and percentage scores are added to give a final immunoreactivity score ranging from 0 to 6. mTOR, Raptor and Rictor antibodies used for IHC in Fig. 1A were purchased from Bethyl Laboratories (Montgomery, TX), while pAkt^Ser473 antibody was purchased from Cell Signaling (Danvers, MA). mTOR, Raptor and Rictor antibodies used in Supplemental Fig. 1 were purchased from Abcam (Cambridge, MA).

Cell Lines, Plasmid Transfections and Lentiviral Transductions

The human colon cancer cell lines HCT116, KM20, SW480 and Caco-2 were used in these studies. HCT116, SW480 and Caco-2 cells were obtained from American Type Culture Collection (Manassas, VA); KM20 cells were kindly provided by Dr. Isaiah J. Fidler (MD Anderson Cancer Center, Houston, TX). HCT116 cells were cultured in McCoy’s 5A medium supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT). SW480 cells were cultured in DMEM supplemented with 10% FBS. Caco-2 cells were cultured in MEM medium supplemented with 10% FBS. KM20 cells were cultured in MEM medium supplemented with 10% FBS, 1% sodium pyruvate, 1% non-essential amino acids and 2% MEM essential vitamins. All tissue culture media were purchased from Invitrogen (Carlsbad, CA). Non-targeting control (NTC) and SMARTPool Raptor and Rictor siRNA were purchased from Dharmacon (Lafayette, CO).

For generation of stable knockdown HCT116 and SW480 cells, shRNAs for human mTOR, Raptor and Rictor genes were constructed in pLKO.1-puro vector and purchased from Addgene (Cambridge, MA). A plasmid carrying a non-targeting sequence was used to create the control cells. For virus packaging, the control or mTOR/Raptor/Rictor shRNA constructs were co-transfected with Mission lentiviral packing mix (Sigma-Aldrich) into HEK 293T cells using FuGene 6. The virus-containing medium was collected, filtered and overlaid onto the parental cells in the presence of polybrene (10µg/mL) for 24 h. The infected cells were then selected with puromycin (2.5µg/mL).

Cell proliferation and Apoptosis analyses

Equal numbers of cells were seeded onto 24-well plates at a density of 1 × 10⁴ cells per well in the appropriate culture medium with supplements. For proliferation assays, cells were treated with varying doses of rapamycin for 24–96 h. Cells were trypsinized and counted using a cell counter (Beckman-Coulter, Fullerton, CA) or Cell Proliferation ELISA (Roche, Indianapolis, IN). For apoptosis assays, cells were serum starved for 48 h and apoptosis was measured using the Cell Death Detection ELISA^plus (Roche, Indianapolis, IN) as detailed in the manufacturer’s instructions.

Cell Cycle analysis

Cells (1×10⁴) were trypsinized, washed with PBS and fixed in 70% methanol. Fixed cells were then washed with PBS, incubated with 100µg/ml RNAase for 30 min at 37°C, stained with propidium iodide (50µg/ml). Cells were subjected to flow cytometry analysis using Becton Dickinson FACScanto (Franklin Lakes, NJ). The percentages of cells in different cell cycle phases were analyzed using ModFit LT software (Verity Software House).

Western Blot analysis

Western blot analysis was performed as described previously (10). The following antibodies were from Cell Signaling (Danvers, MA): pAkt^Ser473, pAkt^Thr308, total Akt, β-Actin, p-mTOR^Ser2448, Tubulin, p-p70S6K^Thr389, pS6^Ser235/236. Antibodies for mTOR, Raptor and Rictor were obtained from Bethyl Labs (Montgomery, TX).

In vivo studies

The stable knockdown HCT116 and SW480 CRC cells were collected in 50µL of sterile PBS and inoculated subcutaneously into 6 week old male athymic nude mice at 2 × 10⁶ cells per injection site (n=5 for each group). The tumor size was measured every 3–5 d with a vernier caliper, and the tumor volume was defined as (longest diameter) × (shortest diameter)² / 2. At the end of the experiment on day 25 post-injection, mice were sacrificed and tumors removed, weighed and then extracted for protein analysis. All animal procedures were performed in the nude mouse facility using protocols approved by the UTMB Animal Care and Use Committee.

Statistical Analysis

Association between IHC score and stage (Fig. 1B) was assessed using Fisher’s exact test. Effects of: (1) rapamycin dose on cell proliferation (Fig. 2); (2) siRNA treatment on cell proliferation (Fig. 3); (3) shRNA treatment on cell proliferation (Fig. 4B and Fig. 5B); (4) shRNA treatment on tumor volume (transformed to cubic root) and tumor weight (Fig. 6) were analyzed using one-way analysis of variance. Effects of combinations of shRNA treatment and serum (Fig. 4C and Fig. 5C) were analyzed using analysis of variance for a two-factor experiment. All tests were assessed at the 0.05 level of significance (experiment-wise). Multiple comparisons were conducted using Fisher’s least significant difference procedure with Bonferroni adjustment for the number of comparisons. Statistical computations were carried out using SAS 9.1^® (21).

mTORC1 and mTORC2 are overexpressed in CRC tissues and cells

To determine whether mTORC1 and mTORC2 proteins are overexpressed in CRCs, we examined CRCs and adjacent normal colonic tissue for expression of mTOR, Raptor and Rictor. Each sample was assigned an IHC immunoreactivity score ranging from 0–6. Representative patient samples for each protein are shown in Fig. 1A along with data analysis in Fig. 1B. The IHC score for tumor tissue was significantly higher than normal tissue for mTOR, Raptor and Rictor (p<0.0001). mTOR exhibits membranous and cytoplasmic staining; Raptor displays mixed cytoplasmic and nuclear staining of approximately equal intensity and Rictor exhibits granular cytoplasmic staining. These findings were confirmed using another set of antibodies (Supplemental Fig. 1); we found no differences in the localization and intensity of staining between the two sets of antibodies used for analysis. Furthermore, since mTORC2 has been implicated as the major kinase to phosphorylate the Ser⁴⁷³ residue of Akt, we also examined the expression of pAkt^Ser473 in the same set of CRC tissues. The IHC score of tumor tissue was found to be significantly higher than normal tissue for pAkt^Ser473 (p=0.002). Interestingly, expression of Rictor was found to correlate with elevated pAkt^Ser473 expression; all patient samples that stained positive for pAkt^Ser473 (regardless of staining intensity) exhibited elevated Rictor expression (IHC score=5 or 6). To further confirm our findings, we examined the expression levels of various mTOR complex components and their downstream effectors in a panel of CRC cell lines representing a spectrum of aberrations in this signaling pathway. Lysates were prepared from four human CRC cell lines (HCT116, KM20, SW480 and Caco-2) and probed with various antibodies using western blot analysis. As shown in Fig. 1C, we found that all cell lines expressed the mTORC1 components, mTOR and Raptor, with highest levels detected in HCT116 and KM20 cells. Moreover, all four cell lines expressed the mTORC2 protein, Rictor. Furthermore, all cell lines exhibit a decrease in levels of p-p70S6K^Thr389 upon rapamycin treatment (Fig. 1C and Supplemental Fig. 2). Taken together with the IHC results, these findings suggest that mTORC1 and mTORC2 proteins are overexpressed in CRCs. Furthermore, the mTORC2 kinase may contribute to the elevated pAkt^Ser473 levels commonly seen in CRCs.

CRCs show differential sensitivity to rapamycin treatment

Many cancers demonstrate variable sensitivity to rapamycin treatment (4). The effects of mTOR inhibition on CRC growth are not well defined. Therefore, we determined the sensitivity or resistance of the four human CRC cell lines to rapamycin treatment. Cells were treated with increasing doses of rapamycin over a 48 h time period and cell proliferation quantitated. We found differential sensitivity to the effects of rapamycin with significant inhibition of proliferation noted in HCT116 and KM20 cells (rapamycin sensitive) as shown in Fig. 2A and 2B respectively. Rapamycin treatment of both sensitive cell lines for 48 h resulted in a dose-dependent decrease in pAkt^Ser473 levels. In contrast, increasing doses of rapamycin had no significant effect on Caco-2 or SW480 cell proliferation (rapamycin resistant) as shown in Fig. 2C & 2D respectively. Interestingly, rapamycin treatment of both resistant cell lines for 48 h resulted in a dose-dependent increase in pAkt^Ser473 levels. In summary, HCT116 and KM20 cells were sensitive to the anti-proliferative effects of rapamycin and exhibited a decrease in pAkt^Ser473 levels with rapamycin treatment, while SW480 and Caco-2 cells were resistant to the anti-proliferative effects of rapamycin and exhibited an increase in pAkt^Ser473 levels with rapamycin treatment.

Transient Rictor knockdown inhibits proliferation of both rapamycin-sensitive and rapamycin-resistant CRCs

We were interested in determining whether selective inhibition of either mTOR complex could inhibit proliferation of CRC cells. We first examined the rapamycin-sensitive cell lines, HCT116 and KM20, to determine the effects of Raptor or Rictor knockdown using siRNA (Fig. 3A & 3B). Specific knockdown of Raptor and Rictor by their corresponding siRNA was confirmed by western blot analysis. Transfection with Raptor siRNA significantly decreased KM20 cell proliferation, but not HCT116, compared to NTC siRNA. In contrast, transfection with Rictor siRNA significantly inhibited cell proliferation in both cell lines. The levels of pAkt^Ser473 were dramatically reduced with Rictor knockdown, but not with Raptor knockdown. Next, we looked at rapamycin-resistant CRCs, Caco-2 and SW480, to determine the effects of Raptor or Rictor knockdown using specific siRNA (Fig. 3C & 3D). Transfection with Raptor siRNA had no effect on proliferation of either cell line. Surprisingly, transfection with Rictor siRNA significantly inhibited cell proliferation in both rapamycin-resistant cell lines. Levels of pAkt^Ser473 were dramatically reduced with Rictor knockdown, but not with Raptor knockdown. These findings suggest that knockdown of Rictor can significantly inhibit the proliferation of both rapamycin-sensitive and rapamycin-resistant CRC cells.

Functional effects of stable inhibition of mTORC1 and mTORC2 in rapamycin-sensitive CRCs

To further confirm whether targeted inhibition of mTOR signaling affects the oncogenic properties of rapamycin-sensitive CRCs, we generated HCT116 cells with stable shRNA-mediated knockdown of mTOR, Raptor or Rictor protein expression. Cells expressing shRNA targeting mTOR, Raptor or Rictor had significantly reduced levels (>90% reduction) of each of the targeted proteins (Fig. 4A). Cells expressing shRNA targeting mTOR or Rictor had significantly reduced levels of pAkt^Ser473, while levels of pAkt^Ser473 in cells expressing shRNA targeting Raptor remained unaffected compared to control cells. Next, we determined whether knockdown of mTOR, Raptor and Rictor affected proliferation of these cells. As shown in Fig. 4B, HCT116 cells with knockdown of mTOR, Raptor or Rictor proliferate at significantly slower rates as compared to control cells. The decreased cell proliferation produced by knockdown of these proteins may be due to decreased cell cycle progression and/or increased induction of apoptosis.

We then determined whether knockdown of mTOR, Raptor and Rictor increased apoptosis in these cell lines. As shown in Fig. 4C, HCT116 cells expressing shRNA targeting mTOR, Raptor or Rictor demonstrated significantly increased rates of apoptosis compared to control cells. Furthermore, knockdown cells were sensitized to serum starvation-induced apoptosis compared to control cells. We also assessed the effect of knockdown of these mTOR complex proteins on cell cycle progression. As shown in Fig. 4D, the percentage of cells in the S- and G2/M-phases decreased markedly and the percentage of cells in the G0/G1 phase increased markedly in the population of cells with reduced mTOR, Raptor or Rictor compared with control cells. Taken together, these results suggest that inhibition of mTORC1 and mTORC2 proteins inhibits cell proliferation by inducing apoptosis as well as G0/G1 growth arrest in rapamycin-sensitive HCT116 cells.

Functional effects of stable inhibition of mTORC1 and mTORC2 in rapamycin-resistant CRCs

In order to determine the effects of inhibiting mTOR signaling on oncogenic properties of rapamycin-resistant CRCs, we generated SW480 cells with stable shRNA-mediated knockdown of mTOR, Raptor or Rictor protein expression. Cells expressing shRNA targeting mTOR, Raptor or Rictor had significantly reduced levels (>90% reduction) of each of the targeted proteins (Fig. 5A). Cells expressing shRNA targeting Rictor had significantly reduced levels of pAkt^Ser473, while levels of pAkt^Ser473 in cells expressing shRNA targeting mTOR and Raptor were increased in comparison to control cells. Next, we determined whether knockdown of mTOR, Raptor and Rictor affected the proliferation of these cells. As shown in Fig. 5B, SW480 cells with knockdown of mTOR, Raptor or Rictor proliferate at significantly slower rates as compared to control cells.

We then determined whether knockdown of mTOR, Raptor and Rictor increased apoptosis in these cells. Surprisingly, SW480 cells expressing shRNA targeting mTOR or Raptor had significantly reduced rates of apoptosis, while cells with stable Rictor knockdown demonstrated increased apoptosis compared to control cells (Fig. 5C). Furthermore, Rictor knockdown sensitized cells to serum starvation-induced apoptosis compared to control cells. We also assessed the effect of knockdown of these mTOR complex proteins on cell cycle progression. As shown in Fig. 5D, the percentage of cells in the S-phase decreased markedly and the percentage of cells in the G0/G1 phase increased markedly in the population of cells with reduced mTOR and Raptor compared with control cells. However, cells with reduced levels of Rictor did not undergo G0/G1 arrest; instead, the percentage of cells in S-phase was slightly decreased while the percentage of cells in the G2/M-phases was slightly increased. Taken together, these results suggest that inhibition of mTOR and Raptor components inhibits cell proliferation mainly by G0/G1 growth arrest, while inhibition of Rictor inhibits cell proliferation mainly by inducing apoptosis in rapamycin-resistant SW480 cells.

Stable knockdown of mTORC1 and mTORC2 inhibits xenograft tumor growth

To examine whether reduced mTOR, Raptor and Rictor expression in rapamycin-sensitive and rapamycin-resistant CRC cells affects growth in vivo, we injected highly tumorigenic, rapamycin-sensitive HCT116 cells and moderately tumorigenic, rapamycin-resistant SW480 cells with stable knockdown of each of the mTOR complex proteins subcutaneously into athymic nude mice and monitored tumor growth over a period of 25 days. Tumors derived from control HCT116 cells formed tumors with sizes of approximately 9500 mm³ within 25 days (Fig. 6A). In contrast, knockdown of mTOR, Raptor or Rictor significantly reduced tumor growth over the same time period; smaller tumors with sizes ranging from 750–1250 mm³ were detected at sacrifice (day 25). Lysates from HCT116-derived tumors expressing shRNA targeting mTOR or Rictor had significantly reduced levels of pAkt^Ser473, while levels of pAkt^Ser473 in lysates expressing shRNA targeting Raptor remained unaffected compared to the control (Supplemental Fig. 3A). Moreover, tumors derived from control SW480 cells formed tumors with sizes of approximately 690 mm³ within 25 days (Fig. 6B). In contrast, knockdown of mTOR, Raptor or Rictor significantly reduced tumor growth over the same period; smaller tumors with sizes ranging from 10–135 mm³ were detected at sacrifice (day 25). Lysates from SW480-derived tumors expressing shRNA targeting Rictor had significantly reduced levels of pAkt^Ser473, while levels of pAkt^Ser473 in lysates expressing shRNA targeting mTOR and Raptor were increased in comparison to the control (Supplemental Fig. 3B). Taken together, our results demonstrate that knockdown of mTOR, Raptor and Rictor inhibits growth of rapamycin-sensitive and -resistant CRC xenografts in nude mice.