MYC regulates the unfolded protein response and glucose and glutamine uptake in endocrine resistant breast cancer

LCC1 (sensitive), LCC2 (TAM resistant; ICI sensitive), and LCC9 (ICI resistant and TAM cross-resistant) and LY2 (LY 117018 [Raloxifene analog] resistant and TAM and ICI cross-resistant) cells were established as previously described [17, 18]. Cells were grown in phenol red–free IMEM (Life Technologies, Grand Island, NY; A10488-01) with 5% charcoal-stripped calf serum (CCS); this media contains 2 mM L-glutamine and ~12 mM glucose. For glucose/glutamine-dependency assays, DMEM without glucose or glutamine (Life Technologies; A14430-01) was used supplemented with 5% CCS. LCC9Gln were derived from LCC9: cells were grown in DMEM without glucose but containing 2 mM L-glutamine (glutamine-only media) for 72 h; cells that survived (<5%) were continually grown in glutamine-only media for 12 weeks. All cells were authenticated by DNA fingerprinting and tested regularly for Mycoplasma infection. Faslodex and STF-31 were obtained from Tocris Bioscience (Ellisville, MO). Compound-968 was purchased from EMD Millipore (Billerica, MA). 10058-F4 was kindly provided by Dr. Steven Metallo (Georgetown University, Department of Chemistry). All other chemicals were purchased from Sigma-Aldrich.

Total protein (~20 μg) was isolated from cells following 48 h treatment or vehicle control (0.02% DMSO or ethanol) for protein analysis as previously described [19]. The following antibodies were used: MYC, MAX, NBR1, p62/SQSTM1, GRP78, IRE1α, phospho-JNK(Thr183/Tyr185), JNK, CHOP, cleaved Caspase-7 (CASP7), LC3B, (Cell Signaling, Danvers, MA); p62/SQSTM1 (BD Biosciences, San Jose, CA); GLS (Abnova, Taipei City, Taiwan; Abcam, Cambridge, England); GLUL (Origene, Rockville, MD); BCL2 (Enzo Life Sciences, Farmingdale, NY), XBP1s (BioLegend, San Diego, CA) β-actin and β- tubulin (Santa Cruz Biotechnology, Santa Cruz, CA).

For determination of cell number, cells were plated in 96-well plates at 5 × 10³ cells/well. At 24 h, cells were treated with specified drugs for 48 h (or otherwise indicated). After treatment, media were removed, and plates were stained with a solution containing 0.5% crystal violet and 25% methanol, rinsed, dried overnight, and resuspended in citrate buffer (0.1 M sodium citrate in 50% ethanol). Intensity of staining, assessed at 570 nm and quantified using a VMax kinetic microplate reader (Molecular Devices Corp., Menlo Park, CA), is directly proportional to cell number [20]. For apoptosis and necrosis, cells were treated for 48 h, and stained with an Annexin V-fluorescein isothiocyanate and propidium iodide, respectively (Trevigen, Gaithersburg, MD). Autophagy was detected by detecting SQSTM1/p62 and LC3II proteins by Western blotting. For the reactive species assay, cellular levels of total reactive species (RS; kit measures both oxygen and nitrogen species) were determined using the Total ROS detection kit (Enzo Lifesciences) and measured by Flow Cytometry and Cell Sorting Shared Resources.

Cells were cultured at 60-80% confluence in growth medium for 24 h. The following day, cells were treated with vehicle, ICI (100 nM), and/or 10058-F4 (25 μM) for an additional 72 h. Cells were then fixed in ethanol, and analyzed by the Flow Cytometry Shared Resource according to the method of Vindelov et al. [21].

Cells were plated at 60-80% confluence. 5 μM MYC siRNA (SMARTpool: ON-TARGETplus set of four MYC siRNA Dharmacon, Lafayette, CO), 10 GLS1, GRP78 (HSPA5), IRE1a or XBP1 (10 nM of 3 unique 27mer siRNA duplexes; Origene, Rockville, MD) or their respective control siRNA, were transfected using the TransIT-siQUEST (Mirus, Madison, WI) transfection reagent. At 48 h, 100 nM ICI or vehicle was added to the siRNA-transfected cells. For MYC overexpression, pcDNA3-MYC (plasmid 16011) was purchased from Addgene (Cambridge, MA) [22] and tranfected with TransIT-2020 (Mirus). Cells were lysed at 48 h post-transfection and subjected to Western blot analysis or cell number assay as described above.

Cells were transfected with 0.4 μg of MYC luciferase reporter plasmid (plasmid 16601) from Addgene and 0.1 μg pCMV-Renilla (Promega, Madison, WI) per well using the TransIT-2020 transfection reagent. Activation of the luciferase constructs was measured at 48 h post-transfection using the Dual Luciferase Assay Kit (Promega). Luciferase values were normalized to Renilla luminescence. Three independent experiments were performed in quadruplicate. Data are presented as the mean ± SE for all experiments.

Five week old ovariectomized athymic nude mice (Harlan, Fredrick, MD) were injected orthotopically with 1.0 × 10⁶ LCC1/LCC9 cells in 50% Matrigel into mammary fat pads. 17β-estradiol supplementation from a subcutaneous, 0.72 mg pellet (Innovative Research of America) with 60-day release was used. Mice were sacrificed after 9 weeks, tumors were fixed in formalin, and processed using routine histological methods as previously described [23]. Mice were housed and maintained under specific pathogen-free conditions and used in accordance with institutional guidelines approved by Georgetown University Animal Care and Use Committee (GUACUC).

Mammary tumors were induced in 50-day-old female Sprague–Dawley (Harlan) rats with 7,12-dimethylbenz[a]anthracene (10 mg; DMBA; Sigma-Aldrich) by oral gavage. Tumor (15 ± 3 mm, long axis) bearing rats were switched to AIN-93G diet containing 337 ppm tamoxifen citrate (Harlan; 15 mg/kg/day TAM). Tumors were classified by growth responsiveness to TAM treatment. Sensitive tumors completely regressed or stopped growing with TAM treatment; Acquired Resistant tumors stopped or regressed but then re-grew after ≥4 weeks; and de novo Resistant tumors continued to grow during treatment. Animals were euthanized at 38 weeks. Tumors used in this study were confirmed as adenocarcinomas by histopathological evaluation (ARUP Laboratories, Utah, IL) [23]. Rats were housed and maintained under specific pathogen-free conditions and used in accordance with institutional guidelines approved by Georgetown University Animal Care and Use Committee (GUACUC).

Tumors were fixed in formalin for 24 h prior to embedding in paraffin. Immunostaining was performed on 5 μm thick sections with an antibody to MYC (1:500) or a non-specific negative control antibody using the diaminobenzidine (DAB) method and photographed using an Olympus BX61 DSU microscope at the Histopathology and Tissue Shared Resource.

Extracts from six biological replicates from LCC1 and LCC9 cells were spiked with internal standards and extracted using the method described by Sheikh et al.[24]. Samples were reconstituted in MeOH:H₂O (1:1), and subsequently resolved on an Acquity ultra performance liquid chromatography (UPLC) column online with a triple quadrupole linear ion trap (QqQLIT) (Xevo-TQ-S, Waters Corporation, USA). The sample cone voltage and collision energies were optimized for each compound to obtain maximum ion intensity for parent and daughter ions using the “IntelliStart” feature of MassLynx software (Waters Corporation, USA). Data acquisition and analysis was done by the Proteomics and Metabolomics Shared Resource.

Glutamine and glucose uptake in LCC1 and LCC9 cells transfected with MYC siRNA was measured using a glutamine assay kit (BioAssay System, Hayward, CA); glucose uptake (2-NBDG, a fluorescently-labeled deoxy-glucose analog) was measured using a cell-based assay kit (#600470, Glucose uptake cell-based assay kit (Cayman Chemical, Ann Arbor, MI). In brief, differences in glucose or glutamine uptake, cells were transfected with MYC siRNA for 48 h. Glucose uptake was estimated by measuring the uptake of 2-NBDG by LCC1 and LCC9 cells in glucose-free media, as suggested by the protocol, for 30 min. Glutamine uptake was estimated by measuring the glutamine left in the media following the manufacturer’s protocol.

Statistical analyses were performed using the Sigmastat software package (Jandel Scientific, SPSS, Chicago, IL). Where appropriate, relative cellular metabolites, protein expression, cell growth, and apoptosis were compared using either a Student’s t test or ANOVA with a post hoc t-test for multiple comparisons. Differences were considered significant at p ≤ 0.05. RI values were obtained by calculating the expected cell survival (S_exp; the product of survival obtained with drug A alone and the survival obtained with drug B alone) and dividing S_exp by the observed cell survival in the presence of both drugs (S_obs). S_exp/S_obs > 1.0 indicates a synergistic interaction [25].

MYC expression is increased in antiestrogen resistant breast tumors [10, 26]. To confirm activation of MYC gene in antiestrogen resistant cells, promoter luciferase activity was measured under basal conditions in ER + breast cancer cells that are either sensitive to antiestrogens (LCC1) or resistant to antiestrogens (LCC2, LY2 and LCC9). Relative to LCC1 cells, MYC promoter activation was 4-fold higher in LY2 and LCC2 cells and more than 6-fold higher in LCC9 cells (Figure 1A). Since the LCC9 cells showed the greatest upregulated MYC activation, LCC1 cells were compared with LCC9 cells for subsequent studies. Endogenous MYC protein was higher in LCC9 cells compared to LCC1 cells, while MAX levels remained unchanged (Figure 1B). In addition, untreated orthotopic xenografts showed upregulation of MYC protein in the antiestrogen resistant tumors (LCC9) (Figure 1C) when compared with sensitive tumors (LCC1). In the DMBA-induced rat mammary tumor model [23], MYC protein levels were higher in those tumors that acquired TAM resistance during treatment when compared with either TAM sensitive, de novo resistant, or untreated tumors (Figure 1D). These data strongly suggest that an increased MYC expression correlates with acquired antiestrogen resistance.

Knockdown of MYC with siRNA reduced MYC protein levels by 60% under basal conditions (results from LCC9 cells shown in Figure 2A and B) and significantly decreased cell number in both LCC1 and LCC9 cells compared with control siRNA (Figure 2C; p < 0.05). Treatment with ICI following MYC knockdown had an additive effect (RI = 1.11; see Experimental procedures) in LCC1 cells, while this combination did not further decrease cell number in LCC9 cells when compared with either treatment alone. LCC9 cells showed increased sensitivity to 10058-F4, a small molecule inhibitor of MYC-MAX heterodimer formation, compared with LCC1 cells at 48 h (Figure 2D). Cell number was significantly decreased for LCC9 cells treated with 20–60 μM of 10058-F4 compared with their LCC1 control cells (p < 0.05). In LCC1 cells, treatment with either 100 nM ICI or 25 μM 10058-F4 alone inhibited cell number; a combination of 10058-F4 and ICI significantly decreased cell number compared with the individual treatments (Figure 2E; p < 0.05). In LCC9 cells, while treatment with ICI had no effect, both 10058-F4 alone (p < 0.05) and a combination of ICI + 10058-F4 significantly (RI=1.51, a modest synergy) reduced the number of cells within 48 h (p < 0.05), suggesting a restoration of ICI sensitivity. Western blot analysis showed decreased levels of MYC, MAX, and BCL2 protein levels upon 10058-F4 treatments in both LCC1 and LCC9 cells (Figure 2F). LCC9 cells express lower levels of ERα under basal conditions compared with LCC1 cells [17, 27] and treatment with 10058-F4 alone did not change ERα levels. ICI, an antiestrogen that promotes degradation of ERα protein, and ICI + 10058 F4 decreased ERα levels (data not shown). Levels of cleaved Caspase-7 were highest in LCC9 cells treated with 10058-F4 and with the ICI + 10058-F4 combination, confirming induction of apoptosis under these conditions. 10058-F4 can decrease BCL2 protein levels [28]; BCL2 and other anti-apoptotic BCL2 proteins confer antiestrogen resistance in breast cancer cells [29]. Thus, the increased efficacy of 10058-F4, in comparison to MYC siRNA, in combination ICI may be due to a cumulative effect of its ability to downregulate MYC and other off-targets like BCL2.

To determine how 10058-F4 restored sensitivity of LCC9 cells to ICI, we studied changes in apoptosis. The proportion of cells undergoing apoptosis with combined ICI + 10058-F4 treatment was significantly higher in LCC9 compared with that in LCC1 cells (Figure 2G; p < 0.05). Dot plots for cells positive for apoptosis markers, Annexin-V-FITC and propidium iodide (PI), following different treatments are also shown in Figure 2H. Since MYC can regulate cell cycling [3], we analyzed the cell cycle profile of vehicle, 100 nM ICI, 25 μM 10058-F4, or the combination treatment at 48 h in LCC1 and LCC9 cells. ICI, 10058-F4, or the combination induced G1-phase cell cycle arrest in the antiestrogen sensitive LCC1 cells (Figure 3A). In the LCC9 cells (resistant), ICI or 10058-F4 treatment alone did not alter the cell cycle profile, whereas their combined treatment increased the percentage of cells in G1 arrest when compared with vehicle treated cells (Figure 3B; p < 0.001). These findings suggest that inhibition of MYC in LCC9 cells may restore sensitivity to ICI by both increasing apoptosis and inducing cell cycle arrest.

Cancer cells with an aberrantly high expression of MYC often have deregulated cellular metabolism, particularly increased glycolysis and glutaminolysis [13]. To compare status of glutamine metabolism in LCC9 versus LCC1 cells, the relative concentration of glutamine metabolites were measured: glutamine, glutamate (immediate metabolite catalyzed by glutaminase, GLS and releasing ammonia; Figure 4A), and proline (downstream metabolite) using ultra performance liquid chromatography/mass spectrometry (UPLC/MS). While glutamine levels were not significantly different (p = 0.206), glutamate (p = 0.002), and proline levels (p = 0.032) were significantly higher in LCC9 compared with LCC1 cells (Figure 4B-D). In addition, uptake of glucose was significantly higher in LCC9 cells compared to LCC1 cells (Figure 4E; p = 0.005). Knockdown of MYC with siRNA inhibited cellular uptake of both glutamine (Figure 4F; p = 0.05) and glucose (Figure 4G; p = 0.011) more significantly in LCC9 cells than in LCC1 cells. Moreover, MYC knockdown reduced expression of glutamine transporter ASCT2 (SLC1A5), glutamate transporter EAAT2 (SLC1A2), and the glucose transporter GLUT1 (SLC2A1) in LCC9 cells (Figure 4H). Thus, MYC controls uptake of glutamine and glucose seen in antiestrogen resistant cells.

Since LCC9 cells showed increased glutamine metabolism and glucose uptake, we determined whether inhibitors of these pathways differentially affected cell survival in LCC1 versus LCC9 cells. Cell number was significantly decreased in LCC9 compared with LCC1 cells in response to the GLS/GAC inhibitor compound-968 (Figure 5A; p < 0.05). Moreover, increasing doses of the GLUT1 inhibitor STF-31, an inhibitor of glycolysis, produced a significant decrease in cell number in LCC9 cells relative to LCC1 cells (Figure 5B; p < 0.05). While LCC9 cells showed significantly increased sensitivity to both STF-31 (p < 0.05) and compound-968 compared with LCC1 cells at 48 h (p < 0.05), adding ICI to either drug did not resensitize LCC9 cells to the antiestrogen (Figure 5C). Thus, specific inhibitors of glutamine and glucose metabolism are potent inhibitors of cell proliferation in both ER + sensitive and antiestrogen resistant breast cancer cells. Knockdown of GLS in LCC9 cells significantly decreased cell numbers within 24 h post transfection with GLS siRNA compared with that in LCC1 cells (Figure 5D). Western blot analysis of total GLS protein following siRNA mediated knockdown within 24 h is shown in Figure 5E.

GLS has two splice variants resulting from alternate splicing: KGA (~66 kDa; full-length) and GAC (~53 kDa; truncated form). GLS/GAC is the predominant form found in tumors [30] and is the variant present in the models used in this study. To show whether MYC regulates GLS/GAC levels in antiestrogen resistant cells, we inhibited MYC with siRNA or 10058-F4 in LCC9 (Figure 5E); and with MYC siRNA in LY2 and LCC2 cells (Figure 5F). In all three antiestrogen resistant cells, MYC inhibition increased GLS/GAC but inhibited glutamine synthase (GLUL), an enzyme that converts glutamate to glutamine. Thus, MYC can regulate GLS/GAC-GLUL enzyme levels to control glutamine metabolism in antiestrogen resistant cells.

To confirm whether MYC is responsible for the increased dependency on glutamine and glucose, MYC was either overexpressed in LCC1 cells (lower endogenous MYC expression/activation) or knocked down in LCC9 cells (higher endogenous MYC expression/activation) (see Figure 1). Figure 6A shows a significant decrease in cell number in LCC1 cells overexpressing MYC (p < 0.01), while Figure 6B shows a significant increase in cell survival is seen in LCC9 cells when MYC expression is reduced by RNAi (p ≤ 0.001) in the absence of both glucose and glutamine. Next, we determined number of LCC1 versus LCC9 cells in the presence or absence of glucose and glutamine at 24, 48, and 72 h. Cell growth was significantly greater in LCC9 compared with that in LCC1 cells at 48 and 72 h in complete media (Figure 6C; ANOVA p ≤ 0.001; p < 0.05). In incomplete media, LCC9 cells showed a significant increase in cell growth at 48 h compared with control (0 h; p < 0.05) or to LCC1 cells at 48 h (p < 0.05). However, at 72 h, cell growth in LCC9 was significantly decreased compared with control (p < 0.05) or LCC1 cells (Figure 6D; p < 0.05). In glucose-only conditions, LCC9 cells again showed an increase in cell growth at 48 h compared with either control (0 h) or LCC1 cells at 48 h. At 72 h, however, cell growth in LCC9 showed a significant decrease compared to either control (p < 0.05) or LCC1 cells at 72 h (Figure 6E; p < 0.05). Interestingly, in glutamine-only conditions, growth in LCC9 cells was significantly decreased compared with control or LCC1 cells at both 48 (p < 0.05) and 72 h (p < 0.05). LCC1 cells exhibited a similar but relatively slower response at 72 h when compared with the respective control (Figure 6F; p < 0.05). To delineate whether MYC directly regulated cell fate in the presence of glutamine-alone in glucose-deprived conditions, we investigated cell number following MYC inhibition in these conditions. Knockdown of MYC increased cell number in the absence of both glucose and glutamine in LCC9 cells as shown before in Figure 6B, and also when glutamine alone was present in glucose-deprived conditions, confirming the critical role of MYC (Figure 6G) in regulating cell fate in this condition.

We next examined how the presence of glutamine in glucose-deprived conditions triggered a rapid decrease in cell number in antiestrogen resistant cells. To determine whether the decrease in cell survival in the presence of glutamine in glucose-deprived conditions was caused by induction of apoptosis, we measured apoptosis following 48 h of glutamine-only treatment in LCC1 and LCC9 cells. Apoptosis was significantly increased in LCC9 compared with LCC1 cells in the absence of both glutamine and glucose (Figure 7A). Moreover, in the presence of glutamine-only conditions, cells underwent significantly higher levels of apoptosis in LCC9 cells than in LCC1 cells. To determine autophagic flux, total protein from both LCC1 and LCC9 cells in the differ conditions (glucose + glutamine, glucose-only, glutamine-only, no glucose + no glutamine) were analyzed at 0, 24 and 48 h for p62/SQSTM1, LC3II and actin (Figure 7B). p62/SQSTM1 are adapter proteins that are autophagosome cargo markers used to determine activity within autolysosomes [31, 32]; however, each protein is selectively degraded by autophagy depending on the signaling cues and nature of stress [31]. An increase in LC3II expression is a marker of increased autophagosome formation and enlargement [33]. Increase in number of autophagosomes in the absence cargo degradation indicates interrupted autophagy that can promote apoptosis [34]. Moreover, Western blot analysis of total proteins from LCC9 cells treated with increasing concentrations of glutamine had higher levels of MYC, MAX and LC3II expression when compared with LCC1 cells; p62/SQSTM1 levels did not change (Figure 7C). Thus, while formation of autophagosomes may be triggered by the glutamine-only condition, autophagy-mediated degradation of cellular substrates is halted. Moreover, the induction of MYC suggests a possible role for this protein in regulating autophagy (see next section and Figure 8B). Disruption in cellular metabolic processes can lead to accumulation of reactive oxygen species (ROS) [35] and reactive nitrogen species (RNS) [36]. Figure 7D shows that deprivation of both glucose and glutamine significantly increased total reactive species (RS) levels in LCC9 cells. However, in both LCC1 and LCC9 cells, the presence of either glucose alone or glutamine alone did not change cellular RS levels compared with conditions where both metabolites are present. Thus, the decrease in cell number in glutamine-only conditions is independent of RS.

Induction of UPR has been reported in various cell models following a decrease in energy sources [37‐39]. Western blot analyses of proteins associated with UPR showed increased GRP78, IRE1α, phospho-JNK(Thr183/Tyr185) and CHOP in glucose-deprived/glutamine-only conditions in LCC9 cells relative to LCC1 cells. Interestingly, while levels of MYC were highest when both glucose and glutamine are present, MYC is undetectable when these metabolites are absent. MYC expression in the presence of glutamine-only, but not in presence of glucose-only, conditions correlated with an increase in the UPR-related proteins. BCL2, an anti-apoptotic protein, was decreased in glucose-deprived glutamine only conditions (Figure 8A). No change in protein expression levels was detected for PERK or ATF6 (data not shown). GRP78, XBP1(s), and phospho-JNK were robustly induced in glutamine-only and no glucose + no glutamine conditions. Knockdown of MYC with siRNA (Figure 8B) increased: (i) GRP78 in all conditions (expect in glutamine-only conditions where high GRP78 expression likely prevented any effect of MYC siRNA on total GRP78 protein levels), (ii) IRE1α in all conditions, (iii) phospho-JNK (Thr183/Tyr185) in glutamine-only conditions without altering total JNK levels, and (iv) LC3II and p62/SQSTM1 levels in glutamine-only conditions. Thus, MYC directly controls the UPR and autophagy to control cell fate in ER + breast cancer cells under specific cellular signals that may be initiated by changes in intracellular glucose or glutamine.

Since the GRP78-IRE1α arm of the UPR is activated in glutamine-only conditions, we further investigated the role of these molecules in cell fate, especially since this particular pathway can drive both cell death via JNK activation, or cell survival via XBP1(s) splicing [37, 40, 41]. Knockdown of GRP78, IRE1α, XBP1, or MYC followed by growth in either glucose + glutamine or glutamine-alone media was compared (Figure 9A-F; J-O). SP600125, a small molecule inhibitor of JNK activation [42, 43] was used (Figure 9G-I) since we observed an increase in phospho-JNK (activation) in glutamine-only conditions (Figure 8A). Inhibition of GRP78 did not significantly affect the inhibition of cell number in glutamine-only conditions in both LCC1 and LCC9 cell lines (Figure 9A). Western blot analyses of total GRP78 protein are shown in both cell lines in different conditions in Figure 9B and C. Knockdown of IRE1α (Figure 9D; Westerns, E-F) and XBP1 (Figure 9J; Westerns, K-L) significantly increased inhibition of cell growth in glutamine-only conditions in LCC9 cells. XBP1 splicing to XBP1(s) by IRE1α promotes cell survival in breast cancer cells [23, 37, 41, 44‐46], and thus, protein levels of XBP1(s) was determined. Inhibition of JNK activation with SP600125, however, significantly decreased the inhibition of cell growth in glutamine-only conditions (Figure 9J, Westerns, K-L). Finally, knockdown of MYC (Figure 9M, Westerns, N-O) significantly decreased inhibition of cell growth in glutamine-only conditions (as shown in Figure 6G). Thus, MYC may control an IRE1α-XBP1(s) pathway to promote survival during glutamine-only conditions, and also an IRE1α-phospho-JNK pathway to promote cell death under this condition; the balance between these two actions may determine individual cell fate.

UPR is a complex adaptive mechanism that can have both pro-death and pro-survival outcomes in breast cancer cells [23, 37]. Since we detected both pro-survival XBP1(s) and pro-death (JNK) pathways in LCC9 cell in glutamine-only condition, we examined cell survival in these cells beyond 72 h. We followed cell growth in LCC9 cells beyond 72 h for all four conditions: (i) glutamine + glucose, (ii) no glucose + no glutamine, (iii) glucose + no glutamine, and (iv) no glucose + glutamine. While 100% of the cells survived in glutamine + glucose conditions, no cells survived in no glucose + no glutamine or glucose + no glutamine conditions. Most LCC9 cells underwent apoptosis in no glucose + glutamine conditions within 72 h, however, a small number (<5%) of cells survived. We followed the growth of these cells (LCC9Gln) for 12 weeks. Cell number in LCC9Gln cells was significantly slower than in LCC9 (control) cells grown in complete media (Figure 10A; p ≤ 0.001). Moreover, LCC9Gln cells showed an increased in GLS/GAC expression but a decrease in GLUL, MYC, and MAX expression (Figure 10B). Table 1 summarizes the levels of MYC protein and cell fate at 72 h (short-term) and >72 h (long-term) in LCC9 cells in the presence of glutamine and/or glucose. In summary, when glutamine and glucose are abundant, MYC promotes their uptake and uniquely controls GLS and GLUL expression in antiestrogen resistant breast cancer cells (Figure 10). In glucose-deprived conditions when glutamine is present, the UPR is triggered and apoptosis is induced through GRP78-IRE1α-JNK-CHOP within 72 h. However, a small number of cells use the UPR to maintain survival beyond 72 h through GRP78-IRE1α-XBP1(s), albeit at a lower growth rate, by adjusting MYC to promote glutamine metabolism.