ras-induced Up-regulation of CTP:Phosphocholine Cytidylyltransferase α Contributes to Malignant Transformation of Intestinal Epithelial Cells*

Background: PC synthesis by the CDP-choline pathway is regulated by CCTα. Results: Increased expression of nuclear CCTα in ras-transformed intestinal epithelial contributes to PC synthesis that is required for anchorage-independent growth. Conclusion: An expanded pool of nuclear CCTα is involved in malignant transformation by ras. Significance: CCTα is a potential target to restore anchorage-dependent growth sensitivity of cancer cells. Cancer cells have enhanced lipogenic capacity characterized by increased synthesis of fatty acids and complex lipids, including phosphatidylcholine (PC). As the rate-limiting enzyme in the CDP-choline pathway for PC synthesis, CTP:phosphocholine cytidylyltransferase α (CCTα) is implicated in the provision of membranes and bioactive lipids necessary of cell proliferation. In this study, we assessed the role of CCTα in malignant intestinal epithelial cells transformed with activated H-ras (IEC-ras). Three IEC-ras clones had significant up-regulation CCTα expression, but PC synthesis and in vitro activity of CCTα were similar to control IEC. RNA interference of CCTα in adherent IEC-ras did not affect PC synthesis, confirming that the enzyme was relatively inactive. However, CCTα silencing in ras-transformed IEC reduced anchorage-independent growth, a criterion for malignant transformation, as well as tumorigenicity in mice. Relative to their adherent counterparts, detached IEC-ras had increased PC synthesis that was attenuated by inducible CCTα silencing. Detachment of IEC-ras was accompanied by increased CCTα phosphorylation and cytosolic enzyme activity. We conclude that the expanded pool of CCTα in IEC-ras is activated by detachment. This provides the increased PC biosynthetic capacity that contributes to malignant transformation of intestinal epithelial cells when detached from the extracellular matrix.

Tumor cells have profound alterations in metabolic pathways that facilitate survival in hypoxic/anoxic environments and provide cellular biomass for accelerated growth (1). Channeling of the glycolytic product pyruvate into acetyl-CoA and increased expression of biosynthetic genes for fatty acids, cholesterol, and other lipids provide cancer cells with enhanced lipogenic capacity (2). Increased activity of ATP-citrate lyase (3,4), acetyl-CoA carboxylase (5), fatty acid synthase (6,7), and stearoyl-CoA desaturase (8), as a consequence of Akt/phosphatidylinositol-3 kinase stimulation of sterol regulatory element-binding protein-1 activity (9), contributes to a dependence on de novo fatty acid synthesis for proliferation and evasion of apoptosis. Because this is a relatively early event in malignant transformation that affects multiple lipid metabolic pathways, pharmacological targeting of the lipogenic capacity of tumors could be a viable therapeutic approach.
The majority of de novo synthesized fatty acids in transformed cells are channeled into glycerophospholipids (10,11), the primary components of cellular membranes and precursors for signaling molecules that are essential for sustained cell proliferation. This raises the possibility that the sensitivity of tumor cells to inhibition of fatty acid synthesis could be due to reduced synthesis of complex lipids or products thereof. Phosphatidylcholine (PC), 2 the major glycerophospholipid in eukaryotic cells, comprises 40 -60% of membrane mass and is synthesized de novo by the CDP-choline (Kennedy) pathway (12). ras-transformed cells often display increased PC synthesis and catabolism (13,14), and tumor tissue has significantly elevated levels of choline metabolites, indicative of increased choline uptake and/or synthesis and catabolism of PC (15,16). Increased levels of phosphocholine (pCholine) in transformed cells and tumor tissue are linked to increased activity of the ␣-isoform of choline kinase (CK) (13,14,17,18). ras-dependent activation of choline kinase ␣ gene expression involves a phosphatidylinositol 3-kinase/RhoA pathway (19). In turn, elevated choline kinase activity is proposed to stimulate mitogen-activated protein kinase and Akt activity by production of a PC-derived lipid activator, possibly phosphatidic acid (20,21). Pharmacological or RNA interference (RNAi) inhibition of CK␣ suppresses cell proliferation and anchorage-independent growth, suggesting an important role in carcinogenesis (22,23).
Although elevated CK expression could increase flux through the CDP-choline pathway, it is generally not considered to be rate-limiting for PC synthesis because the pCholine pool in cells is large compared with other intermediates. In most cases, the rate-limiting step in the pathway is the conversion of pCholine to CDP-choline catalyzed by CTP:phosphocholine cytidylyltransferase (CCT), an amphitropic enzyme that is stimulated by reversible binding to membranes in response to increased fatty acids or type II lipids, such as diacylglycerol (24). The Pcyt1b gene encodes CCT␤2 and CCT␤3 isoforms in rodents as well as C-terminal truncated CCT␤1 in humans, all of which are cytoplasmic and have a restricted tissue distribution (25,26). Pcyt1a encodes the ubiquitously expressed CCT␣ isoform, a nuclear enzyme that is essential for cell viability and embryonic development (25,27,28). CCT␣ regulates PC synthesis during cell division (29,30) and programmed cell death (apoptosis) (27,(31)(32)(33), yet changes in enzyme activity and expression are not a consistent feature of oncogenic transformation. For instance, CCT activity in rastransformed keratinocytes and fibroblasts is increased (34) or decreased (13,14,35), respectively. Bakovic et al. (36) showed that H-ras-transformed fibroblasts have increased CCT␣ mRNA (2-4-fold) and protein (1.8-fold) expression, yet enzyme activity was decreased by 40%. Surprisingly, pharmacological inhibition of the Ras/MAPK signaling pathway reduced CCT␣ mRNA and protein expression but had no effect on enzyme activity, suggesting that ras transformation expanded an inactive pool of enzyme.
Cancer cells, particularly those transformed with oncogenic ras, have elevated pCholine as a result of increased choline kinase expression and increased PC synthesis and turnover. However, this seems at odds with reports of reduced CCT expression and/or activity in the same or related studies. Given these inconsistencies, we investigated the role of CCT␣ in ras transformation of intestinal epithelial cells (IEC)-18. Normal epithelial cells grow in contact with the extracellular matrix (ECM), and detachment of such cells from the ECM triggers a form of apoptosis called anoikis (37). By contrast, malignant cells are typically anoikis-resistant and grow as three-dimensional masses in the absence of adhesion to the ECM, one of the most stringent criteria for malignant transformation (38). Oncogenic ras is a well known inhibitor of anoikis, and numerous studies indicate that ras-induced anoikis-resistance is required for the malignant transformation of IEC into tumors in vivo (39,40). Here we provide evidence that oncogenic ras triggers CCT␣ up-regulation in non-malignant IEC-18 and that this is required for the ability of such cells to grow in the absence of adhesion to the ECM and form tumors in vivo. This dependence on CCT␣ expression was linked to increased PC synthesis in cells that were denied adhesion to the ECM. This represents a novel mechanism by which CCT␣ and the CDPcholine pathway contribute to the anoikis resistance of IEC-18 transformed with oncogenic ras.

EXPERIMENTAL PROCEDURES
Materials-AlexaFluor-conjugated donkey anti-goat antibodies, TRIzol, Lipofectamine 2000, and SYBR Green quantitative polymerase chain reaction (qPCR) master mix were pur-chased from Invitrogen. The CCT␣ antibody was generated in rabbits as described previously (41). The CCT␤2 antibody was generously provided by Dr. Suzanne Jackowski (St. Jude Children's Research Hospital, Memphis, TN). IRDye800-and IRDye680-conjugated secondary antibodies were purchased from LI-COR Biosciences (Lincoln, NE). A ␤-actin monoclonal antibody, and lentiviral pLKO.1 vectors encoding shRNAs directed against CCT␣ and CCT␤, as well as non-targeting (shNT) and shGFP controls, were purchased from Sigma-Aldrich. [ 3 H]Choline and phospho[ 14 C]choline was purchased from PerkinElmer Life Sciences. The lamin A/C antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A CK␣specific antibody was from Proteintech Group (Chicago IL). Eight-week-old female athymic mice (BALB/c) were from Charles River Laboratories (Wilmington, MA) and housed in the Carleton Animal Care Facility (Dalhousie University) for 1 week prior to experiments.
Lentiviral shRNAs under the control of the doxycycline promoter (TRIPz-shCCT␣1, TGACTTTTCCTCCACATC; TripZ shCCT␣2, AAGTCAAATATTCCATCGG) were expressed in ras4 cells and selected with puromycin as described above. After culturing on agarose for 24 h, shRNAs were induced with or without 1 g/ml doxycycline for 48 h prior to the start of experiments (see Fig. 9, C and D) Immunofluorescence Microscopy-Cells cultured on glass coverslips were fixed with 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS) for 15 min and permeabilized with 0.5% (w/v) Triton X-100 for 10 min at 4°C. Coverslips were incubated sequentially with a primary antibody in PBS plus 1% (w/v) BSA at 20°C for 1 h followed by an AlexaFluor-conju-gated secondary antibody for 1 h at 20°C. Coverslips were mounted on glass slides with Mowiol 4-88, and images were captured using a Zeiss LSM510 META confocal microscope equipped with a ϫ100 oil immersion (1.4 numerical aperture) objective. Microscope settings for imaging of lamin A/C in IEC-18 and IEC-ras cells were identical, but the gain was adjusted when imaging CCT␣ in IEC-ras cells due to the level of overexpression.
Immunoblotting-Total cell lysates were prepared in SDS-PAGE buffer (12.5% SDS, 30 mM Tris-HCl (pH 6.8), 12.5% glycerol, and 0.01% bromphenol blue), sonicated for 20 s, and heated to 90°C for 5 min prior to SDS-8% PAGE and transfer to nitrocellulose. Proteins were visualized by immunoblotting with primary antibodies for 1-2 h at 20°C followed by secondary antibodies conjugated to horseradish peroxidase (enhanced chemiluminescence detection) or IRDye800 and IRDye680 (detection using an Odyssey infrared imaging system). Depending on the method, protein expression was quantified relative to load controls (actin or oxysterol-binding protein) using densitometry and ImageJ software version 1.4 or Odyssey Application software version 3.0.
Fluorescence-activated Cell Sorting (FACS)-Adherent or detached ras4 cells were harvested, washed in PBS, trypsinized to reduce aggregates, and fixed in 5 ml of ethanol. Prior to analysis, cells were resuspended in PBS containing 50 g/ml propidium iodide. Samples were analyzed using a FACSCalibur system (BD Biosciences), and cell cycle profiles were analyzed using ModiFit LT 3.0 software.
[ 3 H]Choline Incorporation into PC and Choline Metabolites-IEC-18 and IEC-ras cells growing in monolayers were incubated with choline-free IEC-MEM for 2 h prior to incubation with [ 3 H]choline (1.5 Ci/ml) for 3 h. For cells cultured in suspension on agarose monolayers, [ 3 H]choline (1.5 Ci/ml) was added directly to dishes without a medium change for the indicated times (see the legend to Fig. 9). Cells were harvested from dishes in 1 ml of methanol/water (5:4, v/v), an aliquot was removed for protein quantification, and 3 ml of CHCl 3 was added. After phase separation by centrifugation, the aqueous fraction containing CDP-choline, glycerophosphocholine (GPC), and pCholine was resolved by thin-layer chromatography in water/ethanol/ammonium hydroxide, (50:95:6, v/v/v) and visualized by spraying with 1% (w/v) phosphomolybdate in chloroform/methanol (1:1, v/v) and 1% (w/v) stannous chloride in 3 N HCl. Choline-labeled metabolites were identified using standards, and radioactivity was measured by liquid scintillation counting. [ 3 H]Choline incorporation into PC was determined by liquid scintillation counting of an aliquot of the CHCl 3 phase (Ͼ98% of radioactivity was in the [ 3 H]PC, as determined by thin layer chromatography).
Clonogenicity, Soft Agar, and Tumor Growth Assays-A clonogenicity assay was used to measure cell viability as a function of colony formation following anchorage-independent growth on SeaPlaque agarose-coated plates for up to 72 h (44). Briefly, IEC-18, ras3, ras4, and ras7 cells were trypsinized, and 500 cells were seeded onto 60-mm dishes coated with 2 ml of 1% (w/v) SeaPlaque agarose in MEM. To calculate survival, an equivalent number of cells were seeded onto dishes without agarose to determine maximum colony number under adherent growth conditions. Cells cultured on agarose for 24, 48, and 72 h were transferred to plastic dishes and cultured in IEC-MEM. After 3-4 days, colonies were visualized by staining with Crystal Violet, and survival was calculated relative to adherent controls.
Colony formation in soft agar was determined by spreading 500 IEC-18 or IEC-ras cells suspended in 0.3% (w/v) bactoagar/IEC-MEM onto 60-mm dishes coated with 0.5% (w/v) agar/IEC-MEM. Colonies were counted after 3-4 days and expressed as a percentage relative to the number of colonies formed by an equivalent number of cells cultured on plastic dishes.
To assess whether CCT␣ expression was required for solid tumor growth, ras4 cells (2 ϫ 10 5 cells) transduced with lentiviral shGFP, shCCT␣1, or shCCT␣2 were injected into the right flank of athymic mice. Tumor volume was monitored daily using a skin fold caliper and calculated using an ellipsoidal volume approximation (0.5 ϫ (length ϫ width 2 )) (45). All procedures were certified by the Dalhousie University Committee on Laboratory Animals (protocol number 11-022).

Phosphatidylcholine Metabolism in H-ras-transformed IEC-
Epithelial cell-derived carcinomas account for Ͼ90% of all cancers, of which 25-50% are characterized by expression of oncogenic forms of ras (46). To assess whether PC metabolism is altered in ras-transformed epithelial cells, we utilized non-malignant spontaneously immortalized rat IEC-18 and three independently derived clones of this cell line that are transformed with an oncogenic form of H-ras (ras3, ras4, and ras7) (39). IEC-18 and its ras-transformed clones were cultured in monolayer and pulse-labeled with [ 3 H]choline to measure incorporation into PC and other choline metabolites (Fig. 1) Although choline metabolism was not robustly affected in ras-transformed IEC-18, this may not reflect changes in CCT␣ expression because most cells contain a substantial pool of soluble inactive enzyme (47), and the membrane remodeling activity of CCT␣ does not require catalytic activity (48,49). Indeed, the expression of CCT␣ mRNA in ras3 and ras7 cells was increased 8-fold compared with expression in IEC-18, whereas expression in ras4 cells was unaffected ( Fig. 2A). CCT␣ protein expression was also increased in ras3 (20-fold), ras4 (5-fold), and ras7 cells (8-fold) relative to IEC-18 (Fig. 2B). qPCR analysis using a primer set that detects all CCT␤ transcripts revealed a 5-7-fold increase in ras3 and ras7 cells but not in ras4 cells ( Fig. 2A). CCT␤ protein was detected by immunoblotting using an antibody specific for CCT␤2 that should also detect the CCT␤3 protein (Fig. 2B). IEC and ras cells expressed a single peptide at ϳ40 kDa that was tentatively identified as CCT␤2 because there was no evidence of an additional lower molecular mass protein corresponding to N-terminal truncated CCT␤3 (50). CCT␤2 protein expression is significantly increased in ras7 cells (2.4-fold) but not in ras3 and ras4 cells, indicating a lack of correlation with mRNA levels.
CK␣ expression and activity is increased in ras-transformed cells (16,19), suggesting that discrepant choline metabolite profiles in ras3, -4, and -7 cells could be due to altered expression of this enzyme. However, immunoblotting for CK␣ revealed a 30 -50% increase in protein expression for the ␣1 and ␣2 isoforms in all three ras cells compared with IEC-18 (Fig. 2C).
CCT␣ is localized in the nucleoplasm of most cultured cells but translocates to the nuclear envelope and nucleoplasmic reticulum upon fatty acid activation (41,48,51). Similarly, CCT␣ was detected by indirect immunofluorescence throughout the interior of uniform, elliptically shaped IEC-18 nuclei with no evidence of co-localization at the nuclear envelope with lamin A/C (Fig. 3). In contrast, the nuclei of IEC-ras cells were irregularly shaped with numerous folds, indentations, and protrusions that stained for lamin A/C (Fig. 3). Localization of CCT␣ in IEC-ras cells was heterogeneous both within individual cells and between cell lines; there was evidence of nucleoplasmic CCT␣ as well as co-localization with lamin A/C at the nuclear envelope and in nuclear folds and protrusions, particularly in ras4 and ras7 cells.
Increased CCT␣ protein expression and partial localization on the nuclear envelope in IEC-ras cells suggests that enzyme activity is elevated. On the other hand, PC synthesis and choline metabolites in IEC-ras cells were not significantly affected (Fig.  1). To resolve this, we assayed CCT activity in the soluble (cytosolic) and particulate (membrane) fractions of IEC-18, ras3, ras4, and ras7 cells using the digitonin permeabilization method (33) (Fig. 4A). Despite increased CCT␣ expression in both the soluble and membrane fractions of IEC-ras cells (Fig.  4B), CCT activity in the absence of oleate was no different from that in IEC-18 controls. With the exception of ras4 cytosolic activity, enzyme activity in the soluble and membrane fractions from all four cell lines was not activated by PC/oleate vesicles. Control experiments confirmed that the activity of recombinant CCT␣ was increased 4 -5-fold by PC/oleate vesicle under the same conditions (results not shown). Thus, the increased nuclear pool of CCT␣ in IEC-ras cells appears to be catalytically inactive.
CCT␣ Is Necessary for the Anchorage-independent Growth of IEC-ras Cells-The increased pool of CCT␣ in monolayer cultures of IEC-ras cells did not contribute to PC synthesis but  could be involved in anchorage-independent growth and anoikis. IEC-ras cells are resistant to anoikis and grow as anchorage-independent, three-dimensional multicellular spheroids due to activation and suppression of anti-and pro-apoptotic pathways, respectively (40,52,53). As a first step in assessing whether increased expression of CCT␣ contributes to anchorage-independent growth of IEC-ras cells, we identified two lentiviral shRNAs that provided optimal silencing of gene expression. As an example, shCCT␣1 transduction into IEC-18 and ras cells reduced CCT␣ expression by 80 -90%, which in the case of ras4 and ras7 cells was similar to the level of expression in IEC-18 transduced with non-targeting (shNT) lentivirus (Fig. 5A). Silencing in ras4 cells using shCCT␣1 or shCCT␣2 (two separate shRNAs complementary to different regions of the CCT␣ mRNA) reduced expression of CCT␣ to the level of shNT-transduced IEC-18 but did not affect CCT␤2 expression (Fig. 5B) (data not shown). Based on these experiments, ras4 cells were used in subsequent experiments. As well, CCT␤ mRNA and protein expression in ras4 cells were similar to IEC-18, thus precluding potential involvement of this CCT isoform (Fig. 2, A and B). FIGURE 3. CCT␣ is localized to the nucleoplasm and nuclear envelope in IECras cells. IEC-18, ras3, ras4, and ras7 cells cultured on glass coverslips were immunostained with the following primary and secondary antibodies: rabbit anti-CCT␣ and AlexaFluor488-conjugated goat anti-rabbit; goat anti-lamin A/C and AlexaFluor594-conjugated donkey anti-goat. Images are 1-m optical sections through the mid-nuclei captured using a Zeiss LSM520 Meta confocal microscope.  We further tested whether CCT␣ knockdown affected [ 3 H]choline incorporation into PC and metabolites of the CDP-choline pathway. Following lentiviral transduction of shCCT␣1, adherent IEC-18 and ras4 cells were pulse-labeled with [ 3 H]choline, and incorporation into PC and water-soluble metabolites was measured (Fig. 5C). Consistent with its ratelimiting role in the pathway, CCT␣ silencing in IEC-18 resulted in a significant 40% reduction in PC synthesis and accumulation of pCholine compared with shNT controls. Reduced PC synthesis was also partially balanced by reduced degradation to [ 3 H]GPC. In contrast, [ 3 H]choline incorporation into PC and pCholine were unaffected in shCCT␣1-transduced ras4 cells. There was a slight reduction in [ 3 H]GPC, but this accounted for only 4% of [ 3 H]choline incorporation into PC. This further suggests that the large reservoir of nuclear enzyme in adherent IEC-ras cells has minimal impact on PC synthesis.
To determine if CCT␣ overexpression was involved in anchorage-independent growth, ras4 cells transduced with control and shCCT lentivirus were subjected to clonogenicity and soft agar growth assays (Fig. 6, A and B). When suspended in agar for 5 days, IEC-18 produced Ͻ1% of the colonies formed by adherent controls (results not shown). In contrast, ras4 cells expressing control shNT formed colonies in soft agar (deprived of adhesion to the ECM) to a similar extent as adherent counterparts. However, this growth was reduced by 50% when CCT␣ expression was targeted with either shCCT␣1 or shCCT␣2 (Fig. 6A). Using a clonogenicity assay that measures that ability of detached cells to form colonies when provided with a suitable substrate on which to grow, IEC-18 had a progressive loss of viability when cultured in suspension for up to 72 h that was not affected by reducing CCT␣ expression (Fig.  6B). Similar to the soft agar growth assay, the Ͼ90% viability of detached ras4 cells was suppressed to 20 -40% when CCT␣ expression was reduced with shCCT␣1.
To determine how CCT␣ silencing prevented detachmentindependent growth of ras4 cells, we initially tested whether apoptosis was increased. However, CCT␣ silencing did not increase apoptosis in ras4 cells as measured by several different methods (results not shown). In contrast, FACS analysis of detached ras4 cells transduced with two different CCT␣ shRNAs showed a significant 10 -15% increase in G 0 /G 1 and a reduction in the G 2 /M component compared with control (Fig.  6C). This partial G 0 /G 1 block due to CCT␣ silencing was not observed in adherent ras4 cells (Fig. 6C).
Finally, we tested whether reduction of CCT␤ expression in IEC-18 and ras4 cells with two different shRNAs affected cell viability as measured by a clonogenicity assay (Fig. 6D). Both CCT␤ shRNAs reduced mRNA expression by Ͼ85% in IEC-18 and ras4 cells as quantified by qPCR (results not shown). Relative to the control shRNA, reduction of CCT␤ expression did not affect the viability and anchorage-independent growth of 48-h detached IEC-18 and ras4 cells. CCT␤ silencing in IEC-18 or ras4 cells also did not affect [ 3 H]choline incorporation into PC (Fig. 6E) or other choline metabolites (results not shown).
Because increased CCT␣ expression was correlated with the ability of ras4 cells to grow anchorage-independently, we tested whether CCT␣ overexpression and/or activation with oleate had a similar effect in IEC-18 (Fig. 7). Overexpression of GFP-tagged CCT␣ (8 -10-fold) and/or activation with oleate did not improve the detachment-independent growth of IEC-18 as measured by the clonogenicity assay, indicating that CCT␣ is not sufficient to promote anoikis-resistance in the absence of ras transformation.
Capacity for anchorage-independent growth is critical for the ability of malignant cells to form primary tumors. We thus examined the effect of reducing CCT␣ expression on the tumorigenicity of ras4 cells in mice. To this end, ras4 cells expressing control shGFP, shCCT␣1, or shCCT␣2 were injected subcutaneously into the flanks of immunocompromised BALB/c mice, and tumor growth was measured (Fig. 8). Although ras4 cells transduced with shCCT␣1 and shCCT␣2 produced tumors similar in size to controls at day 1-7, expression of shCCT␣1 caused a significant ϳ50% reduction in tumor volume at days 8 -12. ras4 cells transduced with shCCT␣2 produced tumors with a significant 20% in reduction volume at day 12. The results of this xenograft experiment indicate that CCT␣ silencing reduces the tumorigenic potential of ras4 cells.

CCT␣-dependent PC Synthesis Facilitates Anchorage-independent Growth of ras Cells-We next used [ 3 H]choline label-
ing to compare PC metabolism between adherent and anchorage-deprived IEC-18 and ras cells, as well as the influence of CCT␣ silencing on PC synthesis in detached ras cells (Fig. 9). IEC cells showed a progressive loss of viability after growth in suspension culture using a clonogenicity assay, whereas ras3, ras4, and ras7 cells displayed robust anchorage-independent growth after 72 h (Fig. 9A). These cells cultured in suspension on agarose-coated dishes were labeled with [ 3 H]choline for 24, 48, or 72 h, and incorporation into PC and choline metabolites was compared with that of similarly treated adherent cells (Fig.  9B). Consistent with reduced IEC-18 viability, [ 3 H]choline incorporation into PC and choline metabolites was almost completely inhibited by 72 h (Fig. 9B). IEC-ras cells had an initial decline in [ 3 H]PC synthesis at 24 h that increased to Ͻ1.5-fold above adherent controls by 72 h. [ 3 H]pCholine levels also declined at 24 h and remained lower than levels of their adherent counterparts, consistent with increased conversion to PC. With the exception of ras3, isotope incorporation into CDP-choline and GPC was similar in adherent and detached ras cells.
To test whether the detachment-dependent increase in PC synthesis in ras cells was dependent on CCT␣, the enzyme was silenced using two inducible lentiviral shRNAs. For these experiments, ras4 cells transduced with doxycycline-inducible lentiviral shRNAs were cultured on agarose for 24 h, incubated with or without doxycycline for 48 h, and pulse-labeled with [ 3 H]choline for 3 h. Relative to non-induced controls, [ 3 H]choline incorporation into PC was significantly reduced by both CCT␣-specific shRNAs (Fig. 9C). [ 3 H]GPC was reduced with one shRNA, but all other metabolites were unaffected. Immu-noblotting revealed that CCT␣ expression was suppressed by Ͼ70% in doxycycline-treated cells compared with uninduced controls (Fig. 9D). These results suggest that ras-induced proliferation of detached IEC is linked to increased CCT␣ expression and PC synthesis.
To determine how detachment from the ECM affects CCT expression, total cell extracts of IEC-18 and ras cells cultured under detached conditions for up to 72 h were immunoblotted for CCT␣ and ␤2 (Fig. 10A). CCT␣ and CCT␤2 in adherent IEC-18 and ras cells resolved as a single major species, suggesting that the proteins are hypophosphorylated (Fig. 2A). When IEC were denied anchorage by culturing on agarose, there was an immediate shift to a higher mass CCT␣ and CCT␤2 species. As cell viability and protein expression declined, the relative expression of these two species oscillated (Fig. 10A). In anoikisresistant ras cells, a single species of CCT␣ and CCT␤2 was evident immediately after detachment (0 h), but at 6 -24 h, a higher mass species appeared, and it become prominent by 72 h. Alkaline phosphatase treatment of ras cell lysates confirmed that the increase in molecular mass is due to hyperphosphorylation (results not shown). This shift in apparent molecular mass of CCT␣ on SDS-PAGE is due to phosphorylation of the C-terminal domain, which negatively regulates membrane association and activity (54). Thus, expression of CCT␣ and CCT␤2 is sustained in detached ras-transformed cells but eventually shifts to a hyperphosphorylated species.  CCT␣ activity was assayed in the soluble and particulate fractions (membrane) of 48-h detached and adherent ras4 cells (Fig. 10B). There was a significant 80% increase in soluble CCT activity from detached ras4 cells compared with adherent cells, but membrane-associated activities were comparable. In these experiments, enzyme activity in soluble and membrane fractions was not affected by PC/oleate vesicles (results not shown).

DISCUSSION
Increased fatty acid synthase expression and channeling of glycolytic products into fatty acid synthesis are a hallmark of transformed cells and are frequently associated with more aggressive tumors (55). Increased lipogenesis could provide an energy supply and storage format that enhances cell proliferation. Alternatively, fatty acids could be utilized for membrane biogenesis in rapidly dividing cells. For example, the CDP-cho-line pathway is exquisitely sensitive to fatty acid activation by virtue of the membrane-sensing properties of CCT␣ (47), and PC synthesis and choline metabolites are elevated in various cancer cells (15). A comparison between IEC-18 and three rastransformed clones identified an unusual dichotomy within the CDP-choline pathway, a dramatic elevation of nuclear CCT␣ expression that that was inactive in adherent cells but contributed to anchorage-independent growth of IEC-ras cells by a mechanism that was dependent on PC biosynthesis.
In adherent IEC-18 and IEC-ras cells, [ 3 H]choline incorporation into PC was similar, as was the total amount of radiolabeled choline incorporated into all metabolites. Radiolabeled water-soluble metabolites were variably altered between the three ras-transformed clones. Despite having apparently normal rates of PC biosynthesis, CCT␣ mRNA and protein were increased by 5-20-fold in all IEC-ras clones. However, increased CCT␣ expression in IEC-ras cells was not correlated with the levels of [ 3 H]choline metabolites. For instance, CCT␣ expression was increased 8-fold in ras7 cells, but choline metabolites and PC were unchanged compared with IEC-18, whereas ras4 cells had less CCT␣ overexpression but significant changes in pCholine and GPC. The reason for this apparent discrepancy between CCT expression versus CDP-choline pathway activity was revealed when CCT was assayed in vitro; enzyme activity in the cytosolic and membrane fractions of IEC-ras cells was similar to control IEC-18, indicating that the large pool of enzyme was inactive and insensitive to activation by oleate. This was unexpected because CCT␣ in IEC-ras was partially localized with lamin A/C at the nuclear envelope and fractionated with membranes after digitonin permeabilization, a situation that should be correlated with increased activity (47). Our results agree with a previous study that showed modest increases in CCT␣ mRNA and protein but inhibition of CCT activity in H-ras-transformed murine fibroblasts (36). The conclusion that IEC-ras cells overexpress a biosynthetically inactive pool of CCT␣ is supported by the lack of effect of lentiviral shRNA silencing of CCT␣ on PC synthesis or the level of [ 3 H]choline metabolites in ras4 cells cultured in monolayer.
Unlike CCT␣, increased CCT␤ expression was not a consistent feature of ras-transformed IEC-18. In addition, there was discordance between total mRNA and protein expression of the full-length CCT␤2 isoform, suggesting additional levels of post-transcriptional regulation or expression of an additional isoform that is not detected by the CCT␤2 antibody. This last explanation seems unlikely because the antibody should detect both rodent CCT␤2 and CCT␤3 species (26). CCT␤ also did not appear to have a major role in PC synthesis relative to CCT␣; CCT␤ silencing in IEC-18 cells had no effect on PC synthesis (Fig. 6E), whereas reducing CCT␣ expression caused a 40% reduction (Fig. 5C). Although the ras4 cells chosen for anchorage-independent growth experiments did not have significantly elevated CCT␤ expression, further reduction by RNAi did not affect ras4 cell viability or PC synthesis (Fig. 6, D  and E). Collectively, this suggests that CCT␤ expression is not increased by H-ras transformation and does not contribute to anoikis resistance, and it is subordinate to CCT␣ in terms of providing PC biosynthetic capacity.
A major finding of this study is that ras-induced CCT␣ upregulation in IEC-18 is required to promote anchorage-independent growth and malignant transformation. Choline-labeling experiments showed that detached ras cells have increased PC synthesis and reduced [ 3 H]pCholine levels indicative of CCT␣ activation (Fig. 9B). Unlike adherent ras4 cells, inducible RNAi silencing of CCT␣ after detachment from the ECM significantly attenuated PC synthesis, suggesting that ras-induced CCT␣ is activated by these growth conditions. The partial G 1 arrest caused by CCT␣ silencing in detached ras4 cells is also consistent with inhibition of PC synthesis. CCT␣ is activated in early G 1 by phospholipid degradation products and contributes to net PC synthesis during S phase (25,29,56). Cessation of PC synthesis in CHO cells expressing temperature-sensitive CCT␣ resulted in G 1 arrest prior to induction of apoptosis (27). In the case of ras4 cells cultured on agarose, reducing CCT␣ expression by 70% caused partial inhibition of PC synthesis (Fig. 9C) and cell cycle arrest without induction of apoptosis (Fig. 6C) (results not shown).
Despite the apparent increase in PC synthesis in ras cells deprived of attachment to the ECM, CCT␣ and CCT␤ underwent a time-dependent increase in phosphorylation, which is usually associated with the cytosolic inactive form of the enzymes. It is feasible that detachment triggers an essential phosphorylation event required for enzyme activation. However, phosphorylation of CCT␣ occurred 6 -24 h after cells were cultured on agarose, suggesting that it is secondary to other factors that affect enzyme activity (e.g. release of lipid activators upon disengagement from the ECM). Despite the apparent increase in CCT␣-dependent PC synthesis, there was only a modest increase in cytosolic CCT activity in detached ras4 cells compared with adherent cells. CCT activity in ras cells did not correlate well with protein expression and metabolic labeling; nor was it activated by exogenous oleate, suggesting a novel mode of enzyme regulation. The factors regulating CCT␣ in ras-transformed cells will require an in depth analysis of covalent modifications, nuclear localization, and lipid activation in both adherent and detached cell populations.
CCT␣ has been implicated in rat hepatoma proliferation (57) and several human cancers (58). Our results indicate that ras induction of CCT␣ promotes carcinogenesis by increasing the PC biosynthetic capacity of cells after detachment from the ECM. In this context, CCT␣ could promote metastasis by increasing the survival and anoikis resistance of cancer cells detached from the ECM.
Acknowledgment-Robert Zwicker provided excellent technical assistance with cell culture.