IJC International Journal of Cancer
Transglutaminase 2 reprogramming of glucose metabolism in mammary epithelial cells via activation of inflammatory signaling pathways Santosh Kumar1, Taraka R. Donti2, Navneet Agnihotri1 and Kapil Mehta1 1
Cancer Cell Biology
2
Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX Department of Molecular and Human Genetics, One Baylor Plaza, Houston, TX
Aberrant glucose metabolism characterized by high levels of glycolysis, even in the presence of oxygen, is an important hallmark of cancer. This metabolic reprogramming referred to as the Warburg effect is essential to the survival of tumor cells and provides them with substrates required for biomass generation. Molecular mechanisms responsible for this shift in glucose metabolism remain elusive. As described herein, we found that aberrant expression of the proinflammatory protein transglutaminase 2 (TG2) is an important regulator of the Warburg effect in mammary epithelial cells. Mechanistically, TG2 regulated metabolic reprogramming by constitutively activating nuclear factor (NF)-jB, which binds to the hypoxia-inducible factor (HIF)21a promoter and induces its expression even under normoxic conditions. TG2/NF-jB-induced increase in HIF-1a expression was associated with increased glucose uptake, increased lactate production and decreased oxygen consumption by mitochondria. Experimental suppression of TG2 attenuated HIF-1a expression and reversed downstream events in mammary epithelial cells. Moreover, downregulation of p65/RelA or HIF-1a expression in these cells restored normal glucose uptake, lactate production, mitochondrial respiration and glycolytic protein expression. Our results suggest that aberrant expression of TG2 is a master regulator of metabolic reprogramming and facilitates metabolic alterations in epithelial cells even under normoxic conditions. A TG2-induced shift in glucose metabolism helps breast cancer cells to survive under stressful conditions and promotes their metastatic competence.
Increased conversion of glucose to lactic acid, even in the presence of oxygen, is a unique feature of tumors that was first described by Otto Warburg in 1956.1 This metabolic adaptation enables tumor cells to survive, proliferate and propagate in hostile environments.2 Rapidly growing tumors frequently become hypoxic and thus unable to meet their oxygen requirements. As a result, ATP production by mitochondria in the tumor cells is compromised. Such conditions would be lethal to normal cells. However, tumor cells, owing to genetic and epigenetic alterations in them, can successfully survive under these conditions via upregulation Key words: NF-jB, HIF-1a, metabolism, drug resistance, metastasis, EMT Additional Supporting Information may be found in the online version of this article. Grant sponsor: SK Agarwal Donation Funds to The University of Texas MD Anderson Cancer Center and the MD Anderson Cancer Center; Grant number: CA016672; Grant sponsor: Indian Council of Medical Research, India DOI: 10.1002/ijc.28623 History: Received 1 July 2013; Accepted 4 Nov 2013; Online 20 Nov 2013 Correspondence to: Dr. Kapil Mehta, Department of Experimental Therapeutics, Unit 1950, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA, Tel.: 17132540106, Fax: 1713-668-9032, E-mail: [email protected]
C 2013 UICC Int. J. Cancer: 134, 2798–2807 (2014) V
of glycolysis. This reprogramming of glucose metabolism provides a constant supply of energy as well as precursors for de novo biosynthesis of macromolecules, including DNA, RNA, fatty acids and amino acids that are essential for cell growth and proliferation. Such dependency of cancer cells on glucose metabolism for their growth and survival is being exploited in the development of novel anticancer drugs. The major mechanism responsible for metabolic reprogramming is upregulation of expression of hypoxia-inducible factor (HIF)21a, a transcription factor that shifts energy production from mitochondrial to glycolytic sources in hypoxic regions of tumors.3 HIF-1a expression can be upregulated under normoxic conditions in response to activation of certain oncogenic pathways, such as Ras, Src, Myc and phosphoinositide 3 kinase4,5 and loss of tumor suppressors, such as von Hippel-Lindau protein and phosphatase and tensin homolog.6 Free radicals and other metabolites, such as succinate and fumarate, can also stabilize HIF1a protein expression.7 Accumulation of HIF-1a in cells stimulates glucose uptake by transactivating glucose transporter genes, such as GLUT18 as well as glycolytic enzymes, such as hexokinase (HK) 1, HK2,9 phosphofructokinase 1, aldolase and lactate dehydrogenase A (LDHA).10 HK2 quickly phosphorylates glucose and traps it in cells whereas LDHA converts pyruvate into lactate to facilitate the use of lactate for lipid biosynthesis. Low energy production by this glycolytic pathway is
2799
Kumar et al.
compensated by increased glucose uptake by tumor cells. In addition, HIF-1a downregulates oxidative phosphorylation by inactivating the major enzymes that regulate Krebs cycle, such as pyruvate dehydrogenase. Moreover, HIF-1a activates pyruvate dehydrogenase kinase1, which phosphorylates pyruvate dehydrogenase leading to its inactivation.11 HIF-1a also reduces the demand for oxygen by regulating mitochondrial biogenesis.12 In view of these and our recent observations that overexpression of the proinflammatory protein transglutaminase 2 (TG2) is associated with constitutive activation of nuclear factor (NF)-jB and HIF-1a expression,13 we reasoned that TG2 expression may regulate glucose metabolism in cancer cells. Herein we present evidence that overexpression of TG2 results in increased expression and activation of HIF-1a in mammary epithelial cells under both normoxic and hypoxic conditions. TG2-induced HIF-1 expression prompts altered glucose metabolism by up regulating the expression of genes that encode for key glycolytic proteins and compromise mitochondrial respiration rates in transformed and nontransformed mammary epithelial cells. We observed that experimental suppression of TG2 or HIF-1a expression fully rescued cells from altered glucose metabolism and restored the mitochondrial respiration. Taken together, these results suggest that aberrant expression of TG2 represents a novel pathway that can facilitate altered glucose metabolism in cancer cells, even under normoxic conditions.
Material and Methods Cell lines and reagents
The non-transformed human mammary epithelial cell line MCF-10A, the breast cancer cell line MCF-7 and a drugresistant counterpart to MCF-7, MCF7-RT, were allowed to grow in RPMI-1640 medium and maintained as previously described.14 The full-length TG2 (TG2-WT) cDNA sequence was subcloned into a pCDH lentiviral vector (System Biosciences, Mountain View, CA) from a pcDNA3.1 vector as described previously.14 Lentiviral particles containing control or TG2-specific shRNA sequences were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). MCF-10A cells were stably transfected with an empty vector or a vector containing TG2 as described previously.14 Briefly, stable clones of TG2-overexpressing cells were selected via growth of transfected cells in a puromycin-containing medium (1 lg/mL). Multiple clones were used to rule out potential clonal effects. C 2013 UICC Int. J. Cancer: 134, 2798–2807 (2014) V
All experiments were performed from passage 3 to passage 10. Hypoxic exposure of cells was performed by placing tissue culture flasks in a modular incubator chamber (model MC-101; Billups-Rothenberg Inc., Del Mar, CA) flushed with premixed 94% N2, 5% CO2 and 1% O2. For transient transfection of cells with liposomal vector, SignalSilence p65/RelA, HIF-1a-specific small interfering RNA (siRNA) and control siRNA were purchased from Cell Signaling Technology (Danvers, MA). Anti-TG2, -GLUT-1, -LDHA and -b-actin antibodies were purchased from Abcam (Cambridge, MA); antiHK2 antibodies were purchased from Cell Signaling Technology; and Lipofectamine 2000, Oligofectamine and Stealth RNAi (negative control) were purchased from Invitrogen (Carlsbad, CA). Immunoblotting
For western blot analysis, cells were lysed on ice in NP-40 lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% NP-40 and pH 7.5). Forty micrograms of total protein from each sample were resolved on a 10–12% sodium dodecyl sulfatepolyacrylamide gel with running buffer and transferred onto nitrocellulose membranes. The membranes were probed with the appropriate primary antibody followed by horseradish peroxidase-conjugated secondary antibodies. Nuclear and cytosolic fractions of cells
Cytosolic fractions were prepared using the NE-PER nuclear and cytoplasmic extraction reagent kit (Pierce Biotechnology, Rockford, IL). Briefly, cells were harvested from tissue culture plates and suspended in CER1 buffer (including protease inhibitors) and kept on ice for 10 min. Next, 5.5% CER2 buffer was added and the mixture vortexed vigorously for 10 sec and centrifuged at 13,000 rpm for 5 min. The resulting supernatant was saved as the cytosolic fraction. The insoluble pellet was resuspended in NER buffer and continuously vortexed for 15 sec every 10 min for a total of 40 min. After centrifugation at 13,000 rpm for 10 min the resulting supernatant yielded the nuclear fraction. Glucose uptake
The glucose uptake by the study cells was measured using the glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]2-deoxyglucose (2-NBDG; Molecular Probes, Eugene, OR) as a fluorescent probe.15 To evaluate the kinetics of 2-NBDG uptake, equal numbers of cells were seeded in clear-bottomed
Cancer Cell Biology
What’s new? Increased conversion of glucose to lactic acid provides tumors with a constant supply of energy and biomass production, making altered glucose metabolism a target of particular interest in the development of novel anticancer drugs. Here, aberrant expression of the pro-inflammatory protein TG2 was found to reprogram glucose metabolism in mammary epithelial cells, providing new insight into the molecular mechanisms involved in the shift to altered metabolism. TG2 reprogramming induced constitutive activation of NF-jB and HIF-1a transcription factors, even under normoxic conditions. The findings help to explain how breast cancer cells survive stressful conditions and achieve metastatic competence.
2800
96-well microplates (BD Falcon, Franklin Lakes, NJ) at 20,000 cells/well in triplicate. Cells were allowed to adhere to the plates overnight at 37 C (for 12 hr) before assessing the glucose uptake. After overnight incubation, all of the wells were washed twice with phosphate-buffered saline (PBS) and incubated with 2-NBDG (100 lM) for 10 min at 37 C in a humidified atmosphere of 5% CO2. The reaction was stopped by adding ice-cold PBS to assay wells, followed by three washings with the same buffer. The fluorescent signal before (autofluorescence) and after adding 2-NBDG was measured in fluorometric mode (Perkin Elmer, Waltham, MA). The net increase in fluorescence was normalized according to the lowest signal (0 cells/ well) and was used as the ratiometric quantitation of 2-NBDG uptake by the cells.
Cancer Cell Biology
Lactate production
Cells were plated in six-well culture plates (3 3 105 cells/ well) and incubated for 2 days. The medium in the wells was replaced with fresh medium and incubation continued for an additional 2 days. At the end of the incubation, the medium was collected, and the lactate content was measured using a colorimetric enzymatic assay according to the manufacturer’s instructions (BioVision, Milpitas, CA). RNA isolation, reverse transcription-polymerase chain reaction and quantitative reverse transcriptase-ploymerase chain reaction
Total RNA was extracted from cells using a mini-RNA isolation kit (QIAGEN, Germantown, MD) according to manufacturer’s protocol. For reverse transcription-polymerase chain reaction (PCR), 2.5 lg of total RNA was reverse-transcribed to cDNA using an RT2 First Strand kit (SABiosciences, Valencia, CA). An equivalent volume (2 lL) of cDNA was used as the template for PCR with gene-specific primers (IDT, Coralville, IA). The relative change in signal was calculated after normalization according to glyceraldehyde-3-phosphate dehydrogenase. For whole genome microarray, total RNA from cells was isolated and quantified. The transcriptional microarray was performed using whole human genome oligo arrays with 44,000 60-mer probes (Agilent Technologies, Palo Alto, CA) as described earlier.16 Expression of selected genes from microarray data is shown in Supporting Information Table S1. Transfection with siRNA and shRNA
Cells (5 3 105 per well) were plated in six-well plates and allowed to adhere for 24 hr. The next day, cell monolayers were transfected with 30 nM p65/RelA or HIF-1a specific or control siRNA using Oligofectamine (Invitrogen) according to the manufacturer’s protocol. Briefly, 2 lL of a transfection reagent was added to 100 lL of culture medium, thoroughly mixed and incubated at room temperature for 15 min. Six microliters of siRNA was added to 150 lL of culture medium and then combined with diluted oligofectamine. The resulting solution was gently mixed, incubated at room temperature for 20 min, added to the cell culture and incubated for next
TG2-regulated metabolic reprogramming
48 hr. At the end of the incubation, cell lysates were prepared for further analysis. For shRNA transfection, cells (104 cells per well) were incubated with 5 lg/mL 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide (Polybrene) in a culture medium containing 5 3 104 lentiviral particles having TG2-shRNA or control shRNA sequences. Cells were then prepared for further analysis as described above. Cell viability
The viability of the study cells was determined using an MTT assay. Briefly, cells were seeded in quadruplicate wells in 96-well plates (3000 cells/well) and left to adhere to the plates overnight before placement of cells under hypoxic environment. After hypoxia exposure (94% N2, 5% CO2 and 1% O2) for 48 hr, the cell viability was assessed using MTT (0.5 mg/mL) as described previously.17 The cell survival under hypoxic conditions is expressed as the percent cell survival relative to survival of 100% of cells cultured in parallel under normal conditions (95% air and 5% CO2). Assessment of HIF-1a activity using a luciferase assay
To determine the effect of TG2 expression on HIF-1a transcriptional activation, cells at 40–50% confluence were transfected with a human VEGF 50 -flanking sequence (from 2985 to 2939 bp) inserted into the pGL2 basic luciferase vector as described previously.18 Transfected cells were cultured for 48 hr, washed once with PBS and lysed with a reporter lysis buffer. The luciferase activity in the cells was determined as described previously.18 Cell respiration assay
The XF analyzer (Seahorse Biosciences) was used to detect the oxygen consumption rate (OCR) to measure mitochondrial respiration, and the extracellular acidification rate (ECAR) to determine glycolysis in TG2 overexpressing and TG2-deficient cells. Briefly, cells were plated on XFe24 cell culture microplates at a density of 60,000 cells per well. The XFe24 cartridge was equilibrated with the calibration solution overnight at 37 C. For measuring OCR, XF medium (5 mM glucose and 2 mM Pyruvate in Dulbecco’s modified Eagle’s medium prepared and pH-adjusted to 7.0 on the day of the experiment) was used to prepare the cellular stress-inducing reagents, 500 nM oligomycin, 500 nM FCCP, 100 nM antimycin A and 100 nM rotenone (final concentration). For ECAR, the XF assay media (2 mM L-glutamine, 143 mM NaCl in DMEM, pH 7.4) was used to prepare glycolysis stress reagents, 10 mM Glucose, 500 nM Oligomycin and 100 mM 2-deoxyglucose (2-DG; final concentration). All the reagents were loaded in the ports, according to manufacturer’s instructions. The OCRs and ECARs were measured for 3 min with 3-min mixing and 2 min waiting periods. After the assay, cells in each well were counted using a ViCell counter and the cell counts were used to normalize the OCR (pmoles of oxygen per minute per 10,000 cells) and ECAR (mpH per minute per 10,000 cells). C 2013 UICC Int. J. Cancer: 134, 2798–2807 (2014) V
2801
Kumar et al.
Results TG2 upregulates HIF-1a expression
TG2 reprograms glucose uptake and metabolism
Because HIF-1a is known to shift energy production in cells by increasing glycolysis and because TG2 induces HIF-1a activation, we sought to determine the effect of TG2 expression on glucose uptake and lactate production. To that end, we compared glucose uptake by TG2-deficient and -overexpressing mammary epithelial cells. After normalization of the total cell count, we observed twofold greater glucose uptake by TG2-expressing 10A-TG2 and RT-Vec cells than by TG2deficient 10A-Vec and 7-WT cells (Fig. 2a). Conversely, we observed almost 60% less glucose uptake upon knockdown of TG2 expression in RT-TG2sh cells than in their parental
Cancer Cell Biology
In a recent study, we demonstrated that TG2 induces constitutive activation of NF-jB, which results in transcriptional regulation of the HIF-1a gene.13 To further investigate TG2mediated transcriptional regulation of HIF-1a we examined the nontransformed (MCF-10A) and transformed (MCF-7) mammary epithelial cells using loss- and gain-of-function techniques. We observed that TG2 expression in the cells was associated with a marked increase in HIF-1a protein expression and that experimental suppression of TG2 by short hairpin RNA (shRNA) attenuated this effect (Fig. 1a). A TG2induced increase in HIF-1a expression was evident even under normoxic conditions and was further increased under hypoxic conditions (Supporting Information Fig. S1). TG2regulated HIF-1a expression was transcriptionally active as suggested by its accumulation in the nucleus (Fig. 1b). The transcriptional activity of TG2-induced HIF-1a was further supported by the results of a reporter assay, which showed
that endogenous HIF-1a bound to the hypoxia-response elements in the vascular endothelial growth factor (VEGF) promoter, which was cloned upstream of the luciferase reporter gene. Indeed, the luciferase activity was strikingly higher in TG2-expressing cells than in those lacking TG2 expression (Fig. 1c).
Figure 1. TG2 expression in mammary epithelial cells induces increased accumulation of HIF-1a protein under normoxic conditions. (a) Immunoblot showing the basal HIF-1a and TG2 protein expression levels in nontransformed (MCF10A) and transformed (MCF-7/RT) mammary epithelial cells. MCF-10A cells were stably transfected with vector alone (10A-Vec) or a vector containing a TG2-coding sequence (10ATG2). Drug-resistant MCF-7/RT cells, which had high basal levels of TG2 expression, were transfected with control shRNA (RT-Vec) or TG2 shRNA (RT-TG2sh). (b) Cytosolic and nuclear fractions of the cells in (a) were isolated and subjected to western blotting for TG2 and HIF-1a protein expression. Membranes were stripped and reprobed with anti-b-actin (cytosolic fraction) or anti-histone-H1 (nuclear fraction) antibodies to ensure even protein loading in each lane. (c) TG2-expressing and -deficient cells in (a) and drug-sensitive counterparts of MCF-7/ RT cells (7-WT cells), which lack TG2 expression, were transfected with the VEGF 50 -flanking promoter sequence cloned upstream of a luciferase transcriptional reporter. Forty-eight hours after transfection, cells were assayed for luciferase activity. The results shown are from a representative experiments repeated at least twice with similar results.
C 2013 UICC Int. J. Cancer: 134, 2798–2807 (2014) V
Cancer Cell Biology
2802
Figure 2. Association of induced (10A-TG2 cells) and constitutive (RT-vec cells) expression of TG2 with (a) increased glucose uptake and (b) lactate production in mammary epithelial cells. The data are presented as the means 1 standard error of the mean for at least three independent experiments. ** p< 0.001.
TG2-expressing RT-Vec cells. These results suggested that TG2 expression facilitates glucose uptake, probably owing to increased HIF-1a expression. Lactate production, the end product of glycolytic energy metabolism, mirrored the glucose uptake in TG2-overexpressing and -deficient mammary epithelial cells. TG2-expressing cells had twofold to threefold greater lactate production than did their TG2-deficient counterparts (Fig. 2b). These results supported that TG2 expression can induce altered glucose metabolism in mammary epithelial cells. Effect of TG2 expression on mitochondrial respiration and Glycolysis
Under typical cell culture conditions, the rate of oxygen flux (OCR) is a direct measure of mitochondrial function. To determine the impact of TG2 expression on mitochondrial respiration, we measured the OCR in TG2-expressing and deficient mammary epithelial cells. For this purpose, we employed an XFe24 extracellular flux analyzer (Seahorse Biosciences, North Billerica, MA), which facilitates rapid realtime screening of the bioenergetic profiles of mitochondria in
TG2-regulated metabolic reprogramming
intact cells.19 When we compared the mitochondrial respiration rates of TG2-expressing cells and their TG2-deficient counterparts the latter cells exhibited the expected OCRs in response to successive treatment with oligomycin, carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), antimycin A and rotenone, which are well-defined small-molecule modulators of the electron transport chain (Fig. 3a).20 As expected, treatment with oligomycin decreased the OCR owing to blockage of ATP synthesis at mitochondrial complex V. Treatment with ionophore FCCP, which uncouples mitochondrial respiration from ATP synthesis, resulted in a rapid increase in OCR. Treatment with rotenone and antimycin A, which inhibit mitochondrial complexes I and III and stop the flow of oxygen in the electron transport chain, caused a drastic reduction in the OCR. TG2-overexpressing cells exhibited a low basal OCR and marked reduction in the FCCP-induced OCR (Fig. 3b). However, downregulation of TG2 expression completely rescued these cells from altered mitochondrial respiration. Next, we determined the effect of TG2 expression on glycolysis by measuring ECAR in response to three well-defined modulators of glycolysis. ECAR was significantly higher in TG2-expressing cells than their TG2-deficient counterparts (Figs. 3c and 3d). The inhibitor 2-DG, which inhibits HK— the first enzyme required in glycolysis, inhibited ECAR but it was stimulated by oligomycin and glucose. Moreover, the increased sensitivity of TG2 expressing cells to low glucose (Supporting Information Fig. 2) further supported TG2’s ability to enhance glycolytic flux. Taken, together, these results clearly support that TG2 expression promotes glycolytic phenotype in mammary epithelial cells as suggested by higher rates of proton production (ECAR). TG2 modulates glycolytic enzyme expression
The two proteins that are unequivocally associated with altered glucose metabolism are the glucose transporter GLUT-1 and HK, primarily its isoform HK2.21 In noncancerous cells, pyruvate (the end product of glycolysis) is metabolized in mitochondria. However, under hypoxic conditions, it is converted to lactate by the enzyme LDHA. HIF-1a can coordinate this conversion even under normoxic conditions by upregulating LDHA gene expression.22 Therefore, we next sought to determine the effect of TG2 on expression of GLUT-1, HK2 and LDHA. We observed that induced (10ATG2 cells) and constitutive (RT-vec cells) expression of TG2 in mammary epithelial cells was associated with increased expression of all three of these proteins (Fig. 4a). Their increased expression was accompanied by a corresponding increase in their transcript levels (Fig. 4b). As described previously, increased expression of TG2 was associated with increased expression of HIF-1a protein and transcription of this factor, whereas downregulation of TG2 expression by shRNA (RT-TG2sh cells) attenuated the expression of HIF1a, HK2, LDHA and GLUT1. These results suggested that a TG2-dependent increase in glucose uptake, lactate production C 2013 UICC Int. J. Cancer: 134, 2798–2807 (2014) V
Figure 3. (a,b) Real-time measurement of mitochondrial respiration rate (OCR; a, b) and glycolysis (ECAR; c and d) in mammary epithelial cells with induced (10A-TG2 cells; a and c) or constitutive (MCF-7/RT cells; b and d) overexpression of TG2. The small molecule-metabolic modulators oligomycin (500 nm), FCCP (100 nm) and antimycin A (100 nM) and rotenone (100 nM) were injected sequentially at the indicated time points after baseline OCR measurement. For ECAR determination, cells were glucose starved for 2 hr and subsequently treated with 10 mM D-glucose, 500 nM oligomycin and 100 mM 2-DG. ECAR in response to glucose treatment represents basal glycolytic activity, while in response to oligomycin the maximal glycolytic flux of the cells. ECAR prior to glucose treatment and following treatment with 2-DG represents ECAR due to non-glycolytic acidification. The data are presented as the mean 1 standard error for four independent experiments run in 8–10 replicate wells per experiment.
and a shift from aerobic to anaerobic metabolism are controlled by the expression of some key genes. In this context, a genome-wide analysis of MCF-10A cells stably transfected with TG2 reveled altered expression of a large group of functional genes involved in glucose metabolism (Supporting Information Table S1). TG2-induced NF-jB activation and HIF-1a expression mediate shift in glucose metabolism
We recently reported that stable expression of TG2 results in constitutive activation of NF-jB, which in turn binds to the HIF-1a promoter and induces its expression.13 Therefore, we reasoned that TG2-induced activation of NF-jB and HIF-1a plays a pivotal role in reprogramming of glucose metabolism. To test this hypothesis, we first determined the effect of downregulation of HIF-1a expression on GLUT-1 and LDHA protein expression. As shown in Figure 5a, downregulation of HIF-1a expression by siRNA resulted in appreciably decreased expression of these proteins. Similarly, inhibition C 2013 UICC Int. J. Cancer: 134, 2798–2807 (2014) V
of NF-jB by transfection of the cells with p65/RelA siRNA attenuated GLUT-1 and LDHA expression (Fig. 5b). Importantly, inhibition of TG2-induced HIF-1a and p65/RelA expression resulted in greatly decreased glucose uptake by the cells (Fig. 5). Taken together, these results suggested that aberrant expression of TG2 plays an important role in reprogramming of glucose metabolism in mammary epithelial cells under normoxic conditions owing to its ability to activate NF-jB and to induce NF-jB mediated increases in HIF1a expression.
Discussion In the present study, we provide evidence for the first time that stable expression of the proinflammatory protein TG2 reprograms mammary epithelial cells to alter glucose metabolism. Assay of TG2-overexpressing MCF-10A cells (10ATG2) or MCF-7/RT cells with knockdown of TG2 expression (RT-TG2sh) along with control cells revealed that TG2 overexpression produced greater glucose uptake and lactate
Cancer Cell Biology
2803
Kumar et al.
Cancer Cell Biology
2804
TG2-regulated metabolic reprogramming
Figure 4. Increased expression of TG2 is associated with increased accumulation of HIF-1a and increased expression of its downstream target genes. (a) Total cell extracts from TG2-deficient (10A-Vec, MCF-7 and RT-TG2sh) and TG2-overexpressing (10A-TG2 and RT-Vec) cells were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with anti-HIF-1a, anti-LDHA, anti-GLUT-1, anti-HK2 and anti-TG2 antibodies. TG2-probed membrane was stripped and reprobed with anti-b-actin antibody to ensure even protein loading in each lane. (b) Reverse transcriptase-PCR analysis showing differential expression of LDHA, GLUT-1, HK2 and HIF-1a transcripts in TG2-expressing and -deficient mammary epithelial cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts in these cell extracts were amplified in parallel to ensure even RNA loading. The results shown are from a representative experiment repeated at least twice with similar results.
production in the MCF10A and MCF-7/RT cells than in control cells (Fig. 1). Western blot analysis indicated that TG2 expression increases accumulation of HIF-1a protein in mammary epithelial cells even under normoxic conditions; this is further pronounced under hypoxic conditions (Supporting Information Fig. S1). These observations suggest a possible role for HIF-1a in TG2-regulated shifts in glucose metabolism. Furthermore, expression of TG2 correlated with increased expression of glucose metabolic enzymes such as GLUT-1, HK2 and LDHA at both the transcript and protein level (Fig. 4). GLUT-1 is a glucose transporter whose expression is unequivocally associated with increased glucose uptake by cancer cells,23 whereas HK2 is a mitochondria-bound isoform of HK that plays a key role in glycolysis and cell survival in multiple type of cancer cells.24 Similarly, LDHA converts surplus pyruvate generated during glycolysis into lactate and fuels it into lipid biosynthesis.25 Taken together, these results suggest that TG2 regulates the expression of key genes that encode for functional glycolytic proteins and facilitates increased glucose uptake and consumption by mammary epithelial cells. TG2 is not a transcription factor; therefore, its ability to induce expression of HIF-1a and other glycolytic genes must depend on activation of another transcription factor or factors. Indeed, we recently showed that TG2 expression is associated with constitutive activation of NF-jB in epithelial cells.13 Thus, TG2 binds to the NF-jB inhibitory protein IjBa leading to the rapid degradation of IjBa via a nonproteasomal pathway. TG2-mediated depletion of IjBa in turn
results in the release of active NF-jB and its translocation to the nucleus, where it binds to specific sequence in the promoter and induces transcriptional regulation of target genes. Several lines of evidence suggest that TG2-mediated activation of NF-jB is a key event in promotion of cancer progression.26,27 Recently, a novel procancer feedback loop in which TG2 activates NF-jB, which in turn drives TG2 expression was proposed.28 In cancer cells, this TG2/NF-jB feedback loop may play a self-amplifying role, as high TG2 expression and constitutive NF-jB activation are frequently observed in advanced-stage cancer cells.29,30 For example, multiple tumors and tumor cell lines have exhibited elevated expression of TG2 when selected for resistance to chemotherapy or isolated from metastatic sites.31–33 Also, increased expression of TG2 in drug-resistant breast tumors and tumor cell lines is mediated by aberrant epigenetic regulation of the TGM2 gene.34 Moreover, stable expression of TG2 promotes the embryonic developmental program of epithelial-tomesenchymal transition (EMT)14 and acquisition of cancer stem cell traits in epithelial cancer cells.35,36 These are important observations, as increased TG2 expression in several types of tumors is associated with poor outcome, whereas experimental suppression of TG2 expression inhibits the invasion of cancer cells and sensitizes them to chemotherapy.37–39 Similar to TG2, NF-jB plays fundamental roles in cancer progression stemming from its ability to activate numerous pro-growth, anti-apoptotic and metastatic genes.30 Researchers have directed major efforts toward developing inhibitors that can block NF-jB activity. However, most of C 2013 UICC Int. J. Cancer: 134, 2798–2807 (2014) V
2805
Figure 5. TG2-induced increases in glucose uptake and GLUT-1 and LDHA expression are dependent on NF-jB activity and HIF-1a expression. (a, b) Increased glucose uptake (left panels) and increased GLUT-1 and LDHA expression (right panels) in TG2-overexpressing cells were attenuated in response to downregulation of (a) HIF-1a or (b) p65 expression by gene-specific siRNA. Membranes were stripped and reprobed with an anti-HIF-1a antibody to determine the extent of HIF-1a inhibition by gene-specific or control siRNA and with anti-b- actin antibodies to ensure even protein loading in each lane. Downregulation of p65/RelA or HIF1a expression did not cause any noticeable changes in TG2 expression (not shown).
these efforts have focused on the use of small molecules to block the IjB kinase kinase activity.40 Based on the findings that TG2-induced activation of NF-jB is mediated by IjB kinase-independent mechanism described above; these inhibitors are unlikely to disable the TG2/NF-jB feedback loop. An interesting feature of TG2-induced NF-jB activation is that TG2 translocates to the nucleus along with the p50/p65 NF-jB complex.13 We identified HIF-1a as one of the downstream target genes of TG2-regulated NF-jB activity. Specifically, TG2-p65/RelA complex co-occupied the NF-jB binding site in the HIF-1a promoter (2197 to 2188bp) and induced transcriptional regulation of HIF-1a.13 Like TG2 and NF-jB expression, HIF-1a expression is considered a negative prognostic factor for cancer because of its ability to promote chemoresistance, angiogenesis, invasiveness, metastasis, resistance to cell death and genomic stability.41 Initially, researchers considered HIF-1a to be a mediator of response only under hypoxic conditions because of its instability under normoxic conditions. However, more recent evidence suggests that HIF-1a expression can be upregulated even under normoxic conditions in the presence of certain hormones, growth factors and cytokines.41,42 These observations further C 2013 UICC Int. J. Cancer: 134, 2798–2807 (2014) V
support the contention that aberrant expression of TG2 in cancer cells induces oncogenic cues. HIF-1 is a heterodimeric transcription factor responsible for increasing glycolysis by regulating the expression of genes encoding for glucose transporters, glycolytic enzymes and LDHA while at the same time inhibiting oxidative phosphorylation and squelching pyruvate entry into Krebs cycle via PDK1 mediated inactivation of pyruvate dehydrogenase. Curiously, we found that TG2 expression, in addition to NFjB activation and enhanced HIF-1a expression is associated with increased GLUT-1, HK2 and LDHA expression at both the transcript and protein level (Fig. 4; Supporting Information Table 1). Importantly, downregulation of TG2, HIF-1a or p65 (NF-jB) expression downregulated GLUT-1, HK2 and LDHA expression and attenuated the glucose uptake in mammary epithelial cells (Fig. 5). In addition to regulating the expression of genes encoding for glycolytic proteins, HIF1a can orchestrate other events known to promote the adaptation of incipient tumors to metabolic stress. For examples HIF-1 is known to prompt E-cadherin loss, which, in concert with alterations of other genes, leads to transformation of epithelial cells into pseudomesenchymal cells (EMT).43 EMT
Cancer Cell Biology
Kumar et al.
Cancer Cell Biology
2806
TG2-regulated metabolic reprogramming
is an embryonic developmental process that endows cancer cells with the ability to undergo anchorage-independent growth and enhances their motility and survival.44 Studies by our and other laboratories have demonstrated that TG2 expression in epithelial cancer cells is associated with EMT.14,35 More recently, we demonstrated that the EMTinducing ability of TG2 is dependent on NF-kB activation and is independent of the transamidation activity of TG2.13 Given the importance of glucose energy metabolism in cancer cells, that mitochondrial defects can be attributed to increased aerobic glycolysis is not surprising. Indeed, Warburg’s original hypothesis that increased aerobic glycolysis in cancer cells is related to defective mitochondrial functions supports this contention. In line with this hypothesis, our data demonstrated that overexpression of TG2 renders mammary epithelial cells unable to sense the presence of oxygen as revealed by decreased OCRs and stimulated ECARs under normoxic conditions (Fig. 3). Importantly, our study provided direct evidence that decreased mitochondrial respiration (OCR) and increased glycolysis (ECAR) is a dynamic process rather than related to mitochondrial damage. Downregulation of TG2 expression by shRNA fully restored the OCR and inhibited ECAR in drug-resistant MCF-7/RT cells (Fig. 3). Conversely, overexpression of TG2 in nontransformed MCF-10A cells greatly reduced the OCR and stimulated ECAR, suggesting that TG2 can induce alterations in mitochondria in an unknown way to influence their respiration rates. Indeed, authors have reported that TG2 is associated with the outer mitochondrial membrane and inner membrane space in various cell types.45 Moreover, researchers have implicated that TG2-catalyzed cross-linking of the mitochondrial matrix protein a-ketoglutarate dehydrogenase and aconitase plays a role in deterioration of energy metabolism.46,47 Additional studies are needed to establish the precise role of TG2 expression in mitochondrial energy metabolism. In summary, we found that TG2 expression reprograms energy metabolism in mammary epithelial cells by facilitating transcription of multiple genes involved in the glucose metabolic pathways. In addition, we observed a mechanistic relationship between TG2 and HIF-1a whereby TG2 facilitates HIF-1a synthesis via constitutive activation of the proinflammatory transcription factor NF-jB. As outlined in Figure 6, this interrelationship among TG2, NF-jb and HIF-1a facilitates oncogenic signaling networks that protect cells against stress-induced cell death under both normoxic and
Figure 6. Schematic representation of TG2-regulated metabolic pathways in mammary epithelial cells. Persistent inflammation or exposure to stressors causes epigenetic regulation of TG2. Aberrant expression of TG2 initiates a positive feedback loop, whereby TG2 activates NF-jB and NF-jB induces TG2 expression. After its activation, NF-jB binds to the HIF-1a promoter and induces its transcriptional regulation. HIF-1a upregulates expression of multiple genes, including those encoding for glycolytic proteins and EMT-related transcription repressors (Snail, Twist and Zeb). Together, these events compromise mitochondrial respiration rates (OCR), facilitate glucose uptake and metabolism via glycolysis, increase production of lactate, induce EMT and cancer stem cell phenotypes and render cancer cells highly invasive and resistant to exogenous (chemotherapy) and environmental (hypoxia) stresses.
hypoxic conditions (Supporting Information Fig. S3). In general, metabolic reprogramming is considered an essential mechanism for increased cell proliferation. Interestingly, TG2-mediated metabolic shift towards aerobic glycolysis did not have significant impact on cell proliferation rates. These findings suggest that the proposed association between altered metabolism and cell proliferation rate are either celltype dependent or metabolic shift signifies cellular response to increased proliferation rates. Taken together, these data suggest that aberrant expression of TG2 in cancer cells is a promising therapeutic target and can serve as a marker to evaluate the metabolic reprogramming of individual cancer cell types, their stage of transformation and metastatic potential.
Acknowledgement The authors thank Donald R. Norwood for critical reading of and editorial help with this manuscript.
References 1. 2.
3.
Warburg O. On respiratory impairment in cancer cells. Science 1956;124:269–70. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144: 646–74. Iyer NV, Kotch LE, Agani F, et al. Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1 alpha. Genes Dev 1998; 12:149–62.
4.
5.
6.
Denko NC. Hypoxia, HIF1 and glucose metabolism in the solid tumor. Nat Rev Cancer 2008;8: 705–13. Ferreira LM, Hebrant A, Dumont JE. Metabolic reprogramming of the tumor. Oncogene 2012;31: 3999–4011. Bardos JI, Ashcroft M. Hypoxia-inducible factor1 and oncogenic signaling. Bioessays 2004;26: 262–69.
7.
8.
9.
Gottlieb E, Tomlinson IP. Mitochondrial tumour suppressors: a genetic and biochemical update. Nat Rev Cancer 2005;5:857–66. Chen C, Pore N, Ismail-Behrooz F, et al. Regulation of glut1 mRNA by hypoxia-inducible factor1. Interaction between H-ras and hypoxia. J Biol Chem 2001;276:9519–25. Mathupala SP, Rempel A, Pedersen PL. Glucose catabolism in cancer cells: identification and
C 2013 UICC Int. J. Cancer: 134, 2798–2807 (2014) V
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
characterization of a marked activation response of the type II hexokinase gene to hypoxic conditions. J Biol Chem 2001;276:43407–12. Firth JD, Ebert BL, Ratcliffe PJ. Hypoxic regulation of lactate dehydrogenase A. Interaction between hypoxia-inducible factor 1 and cAMP response elements. J Biol Chem 1995;270:21021–27. Papandreou I, Cairns RA, Fontana L, et al. HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption. Cell Metab 2006;3:187–97. Zhang H, Gao P, Fukuda R, et al. HIF-1 inhibits mitochondrial biogenesis and cellular respiration in VHL-deficient renal cell carcinoma by repression of C-MYC activity. Cancer Cell 2007;11:407–20. Kumar S, Mehta K. Tissue transglutaminase constitutively activates HIF-1a promoter and nuclear factor-jB via a non-canonical pathway. PLoS One 2012;7:e49321. Kumar A, Xu J, Brady S, et al. Tissue transglutaminase promotes drug resistance and invasion by inducing mesenchymal transition in mammary epithelial cells. PLoS One 2010;15:e13390. Hassanein M, Weidow B, Koehler E, et al. Development of high-throughput quantitative assays for glucose uptake in cancer cell lines. Mol Imaging Biol 2011;13:840–52. Price ND, Trent J, El-Naggar AK, et al. Highly accurate two-gene classifier for differentiating gastrointestinal stromal tumors and leiomyosarcomas. Proc Natl Acad Sci USA 2007;104:3414–19. Wang P, Henning SM, Heber D. Limitations of MTT and MTS-based assays for measurement of antiproliferative activity of green tea polyphenols. PLoS One 2010;5:e10202. Forsythe JA, Jiang BH, Iyer NV, et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol 1996;16:4604–13. Malmgren S, Nicholls DG, Taneera J, et al. Tight coupling between glucose and mitochondrial metabolism in clonal beta-cells is required for robust insulin secretion. J Biol Chem 2009;284: 32395–404. Nicholls DG, Darley-Usmar VM, Wu M, et al. Bioenergetic profile experiment using C2C12 myoblast cells. J Vis Exp 2010;46:e2511. Gaillies RJ, Robey I, Gatenby RA. Causes and consequences of increased glucose metabolism of cancers. J Nucl Med 2008;49:24s–42s. Lum JJ, Bui T, Gruber M, et al. The transcription factor HIF-1alpha plays a critical role in the
23.
24.
25.
26.
27.
28.
29.
30. 31.
32.
33.
34.
35.
growth factor-dependent regulation of both aerobic and anaerobic glycolysis. Genes Dev 2007;21: 1037–49. Macheda ML, Rogers S, Best JD. Molecular and cellular regulation of glucose transporter (GLUT) proteins in cancer. J Cell Physiol 2005;202:654–62. Pedersen PL, Mathupala S, Rempel A, et al. Mitochondrial bound type II hexokinase: a key player in the growth and survival of many cancers and an ideal prospect for therapeutic intervention. Biochim Biophys Acta 2002;1555:14–20. Bauer DE, Hatzivassiliou G, Zhao F, et al. ATP citrate lyase is an important component of cell growth and transformation. Oncogene 2005;24: 6314–22. Kim DS, Park SS, Nam BH, et al. Reversal of drug resistance in breast cancer cells by transglutaminase 2 inhibition and nuclear factor-kappaB inactivation. Cancer Res 2006;66:10936–43. Mann AP, Verma A, Sethi G, et al. Overexpression of tissue transglutaminase leads to constitutive activation of nuclear factor-kappaB in cancer cells: delineation of a novel pathway. Cancer Res 2006;66:8788–95. Brown KD. Transglutaminase 2 and NF-^eB: an odd couple that shapes breast cancer phenotype. Breast Cancer Res Treat 2013;137:329–36. Mehta K, Kumar A, Kim HI. Transglutaminase 2: a multi-tasking protein in the complex circuitry of inflammation and cancer. Biochem Pharmacol 2010;80:1921–29. Karin M. Nuclear factor-kB in cancer development and progression. Nature 2006;441:431–36. Agnihotri N, Kumar S, Mehta K. Tissue transglutaminase as a central mediator in inflammationinduced progression of cancer. Breast Cancer Res 2013;15:202. Herman JF, Mangala LS, Mehta K. Implications of increased tissue transglutaminase (TG2) expression in drug-resistant breast cancer (MCF7) cells. Oncogene 2006;25:3049–58. Mangala LS, Fok JY, Zorrilla-Calancha IR, et al. Tissue transglutaminase expression promotes cell attachment, invasion and survival in breast cancer cells. Oncogene 2007;26:2459–70. Ai L, Kim WJ, Demircan B, et al. The transglutaminase 2 gene (TGM2), a potential molecular marker for chemotherapeutic drug sensitivity, is epigenetically silenced in breast cancer. Carcinogenesis 2008;29:510–8. Cao L, Shao M, Schilder J, et al. Tissue transglutaminase links TGF-b, epithelial to mesenchymal
C 2013 UICC Int. J. Cancer: 134, 2798–2807 (2014) V
36.
37.
38.
39.
40.
41. 42.
43.
44.
45.
46.
47.
transition and a stem cell phenotype in ovarian cancer. Oncogene 2012;31:2521–34. Kumar A, Gao H, Xu J, et al. Evidence that aberrant expression of tissue transglutaminase promotes stem cell characteristics in mammary epithelial cells. PLoS One 2011;6:e20701. Hwang JY, Mangala LS, Fok JY, et al. Clinical and biological significance of tissue transglutaminase in ovarian carcinoma. Cancer Res 2008;68: 5849–58. Verma A, Guha S, Diagaradjane P, et al. Therapeutic significance of elevated tissue transglutaminase expression in pancreatic cancer. Clin Cancer Res 2008;14:2476–83. Oh K, Ko E, Kim HS, et al. Transglutaminase 2 facilitates the distant hematogenous metastasis of breast cancer by modulating interleukin-6 in cancer cells. Breast Cancer Res 2011;13:R96. Gilmore TD, Herscovitch M. Inhibitors of NFkappaB signaling: 785 and counting. Oncogene 2006;25:6887–99. Wilson WR, Hay MP. Targeting hypoxia in cancer therapy. Nat Rev Cancer 2011;11:393–410. Bonello S, Z€ahringer C, BelAiba RS, et al. Reactive oxygen species activate the HIF-1alpha promoter via a functional NFkappaB site. Arterioscler Thromb Vasc Biol 2007;27:755–61. Thiery JP, Sleeman JP. Complex networks orchestrate epithelial–mesenchymal transitions. Nat Rev Mol Cell Biol 2006;7:131–42. Voulgari A, Pintzas A. Epithelial–mesenchymal transition in cancer metastasis: mechanisms, markers andstrategies to overcome drug resistance in the clinic. Biochim Biophys Acta 2009; 1796:75–90. Mastroberardino PG, Piacentini M. Type 2 transglutaminase in Huntington’s disease: a doubleedged sword with clinical potential. J Intern Med 2010;268:419–31. Cooper AJ, Sheu KR, Burke JR, et al. Transglutaminase-catalyzed inactivation of glyceraldehyde 3-phosphate dehydrogenase and alphaketoglutaratedehydrogenase complex by polyglutamine domains of pathological length. Proc Natl Acad Sci USA 1997;94:12604–109. Kim SY, Marekov L, Bubber P, et al. Mitochondrial aconitase is a transglutaminase 2 substrate: transglutamination is a probable mechanism contributing to high-molecular-weight aggregates of aconitase and loss of aconitase activity in Huntington disease brain. Neurochem Res 2005;30: 1245–55.
Cancer Cell Biology
2807
Kumar et al.
Transglutaminase 2 reprogramming of glucose metabolism in mammary epithelial cells via activation of inflammatory signaling pathways Santosh Kumar1, Taraka R. Donti2, Navneet Agnihotri1 and Kapil Mehta1 1
Cancer Cell Biology
2
Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX Department of Molecular and Human Genetics, One Baylor Plaza, Houston, TX
Aberrant glucose metabolism characterized by high levels of glycolysis, even in the presence of oxygen, is an important hallmark of cancer. This metabolic reprogramming referred to as the Warburg effect is essential to the survival of tumor cells and provides them with substrates required for biomass generation. Molecular mechanisms responsible for this shift in glucose metabolism remain elusive. As described herein, we found that aberrant expression of the proinflammatory protein transglutaminase 2 (TG2) is an important regulator of the Warburg effect in mammary epithelial cells. Mechanistically, TG2 regulated metabolic reprogramming by constitutively activating nuclear factor (NF)-jB, which binds to the hypoxia-inducible factor (HIF)21a promoter and induces its expression even under normoxic conditions. TG2/NF-jB-induced increase in HIF-1a expression was associated with increased glucose uptake, increased lactate production and decreased oxygen consumption by mitochondria. Experimental suppression of TG2 attenuated HIF-1a expression and reversed downstream events in mammary epithelial cells. Moreover, downregulation of p65/RelA or HIF-1a expression in these cells restored normal glucose uptake, lactate production, mitochondrial respiration and glycolytic protein expression. Our results suggest that aberrant expression of TG2 is a master regulator of metabolic reprogramming and facilitates metabolic alterations in epithelial cells even under normoxic conditions. A TG2-induced shift in glucose metabolism helps breast cancer cells to survive under stressful conditions and promotes their metastatic competence.
Increased conversion of glucose to lactic acid, even in the presence of oxygen, is a unique feature of tumors that was first described by Otto Warburg in 1956.1 This metabolic adaptation enables tumor cells to survive, proliferate and propagate in hostile environments.2 Rapidly growing tumors frequently become hypoxic and thus unable to meet their oxygen requirements. As a result, ATP production by mitochondria in the tumor cells is compromised. Such conditions would be lethal to normal cells. However, tumor cells, owing to genetic and epigenetic alterations in them, can successfully survive under these conditions via upregulation Key words: NF-jB, HIF-1a, metabolism, drug resistance, metastasis, EMT Additional Supporting Information may be found in the online version of this article. Grant sponsor: SK Agarwal Donation Funds to The University of Texas MD Anderson Cancer Center and the MD Anderson Cancer Center; Grant number: CA016672; Grant sponsor: Indian Council of Medical Research, India DOI: 10.1002/ijc.28623 History: Received 1 July 2013; Accepted 4 Nov 2013; Online 20 Nov 2013 Correspondence to: Dr. Kapil Mehta, Department of Experimental Therapeutics, Unit 1950, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA, Tel.: 17132540106, Fax: 1713-668-9032, E-mail: [email protected]
C 2013 UICC Int. J. Cancer: 134, 2798–2807 (2014) V
of glycolysis. This reprogramming of glucose metabolism provides a constant supply of energy as well as precursors for de novo biosynthesis of macromolecules, including DNA, RNA, fatty acids and amino acids that are essential for cell growth and proliferation. Such dependency of cancer cells on glucose metabolism for their growth and survival is being exploited in the development of novel anticancer drugs. The major mechanism responsible for metabolic reprogramming is upregulation of expression of hypoxia-inducible factor (HIF)21a, a transcription factor that shifts energy production from mitochondrial to glycolytic sources in hypoxic regions of tumors.3 HIF-1a expression can be upregulated under normoxic conditions in response to activation of certain oncogenic pathways, such as Ras, Src, Myc and phosphoinositide 3 kinase4,5 and loss of tumor suppressors, such as von Hippel-Lindau protein and phosphatase and tensin homolog.6 Free radicals and other metabolites, such as succinate and fumarate, can also stabilize HIF1a protein expression.7 Accumulation of HIF-1a in cells stimulates glucose uptake by transactivating glucose transporter genes, such as GLUT18 as well as glycolytic enzymes, such as hexokinase (HK) 1, HK2,9 phosphofructokinase 1, aldolase and lactate dehydrogenase A (LDHA).10 HK2 quickly phosphorylates glucose and traps it in cells whereas LDHA converts pyruvate into lactate to facilitate the use of lactate for lipid biosynthesis. Low energy production by this glycolytic pathway is
2799
Kumar et al.
compensated by increased glucose uptake by tumor cells. In addition, HIF-1a downregulates oxidative phosphorylation by inactivating the major enzymes that regulate Krebs cycle, such as pyruvate dehydrogenase. Moreover, HIF-1a activates pyruvate dehydrogenase kinase1, which phosphorylates pyruvate dehydrogenase leading to its inactivation.11 HIF-1a also reduces the demand for oxygen by regulating mitochondrial biogenesis.12 In view of these and our recent observations that overexpression of the proinflammatory protein transglutaminase 2 (TG2) is associated with constitutive activation of nuclear factor (NF)-jB and HIF-1a expression,13 we reasoned that TG2 expression may regulate glucose metabolism in cancer cells. Herein we present evidence that overexpression of TG2 results in increased expression and activation of HIF-1a in mammary epithelial cells under both normoxic and hypoxic conditions. TG2-induced HIF-1 expression prompts altered glucose metabolism by up regulating the expression of genes that encode for key glycolytic proteins and compromise mitochondrial respiration rates in transformed and nontransformed mammary epithelial cells. We observed that experimental suppression of TG2 or HIF-1a expression fully rescued cells from altered glucose metabolism and restored the mitochondrial respiration. Taken together, these results suggest that aberrant expression of TG2 represents a novel pathway that can facilitate altered glucose metabolism in cancer cells, even under normoxic conditions.
Material and Methods Cell lines and reagents
The non-transformed human mammary epithelial cell line MCF-10A, the breast cancer cell line MCF-7 and a drugresistant counterpart to MCF-7, MCF7-RT, were allowed to grow in RPMI-1640 medium and maintained as previously described.14 The full-length TG2 (TG2-WT) cDNA sequence was subcloned into a pCDH lentiviral vector (System Biosciences, Mountain View, CA) from a pcDNA3.1 vector as described previously.14 Lentiviral particles containing control or TG2-specific shRNA sequences were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). MCF-10A cells were stably transfected with an empty vector or a vector containing TG2 as described previously.14 Briefly, stable clones of TG2-overexpressing cells were selected via growth of transfected cells in a puromycin-containing medium (1 lg/mL). Multiple clones were used to rule out potential clonal effects. C 2013 UICC Int. J. Cancer: 134, 2798–2807 (2014) V
All experiments were performed from passage 3 to passage 10. Hypoxic exposure of cells was performed by placing tissue culture flasks in a modular incubator chamber (model MC-101; Billups-Rothenberg Inc., Del Mar, CA) flushed with premixed 94% N2, 5% CO2 and 1% O2. For transient transfection of cells with liposomal vector, SignalSilence p65/RelA, HIF-1a-specific small interfering RNA (siRNA) and control siRNA were purchased from Cell Signaling Technology (Danvers, MA). Anti-TG2, -GLUT-1, -LDHA and -b-actin antibodies were purchased from Abcam (Cambridge, MA); antiHK2 antibodies were purchased from Cell Signaling Technology; and Lipofectamine 2000, Oligofectamine and Stealth RNAi (negative control) were purchased from Invitrogen (Carlsbad, CA). Immunoblotting
For western blot analysis, cells were lysed on ice in NP-40 lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% NP-40 and pH 7.5). Forty micrograms of total protein from each sample were resolved on a 10–12% sodium dodecyl sulfatepolyacrylamide gel with running buffer and transferred onto nitrocellulose membranes. The membranes were probed with the appropriate primary antibody followed by horseradish peroxidase-conjugated secondary antibodies. Nuclear and cytosolic fractions of cells
Cytosolic fractions were prepared using the NE-PER nuclear and cytoplasmic extraction reagent kit (Pierce Biotechnology, Rockford, IL). Briefly, cells were harvested from tissue culture plates and suspended in CER1 buffer (including protease inhibitors) and kept on ice for 10 min. Next, 5.5% CER2 buffer was added and the mixture vortexed vigorously for 10 sec and centrifuged at 13,000 rpm for 5 min. The resulting supernatant was saved as the cytosolic fraction. The insoluble pellet was resuspended in NER buffer and continuously vortexed for 15 sec every 10 min for a total of 40 min. After centrifugation at 13,000 rpm for 10 min the resulting supernatant yielded the nuclear fraction. Glucose uptake
The glucose uptake by the study cells was measured using the glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]2-deoxyglucose (2-NBDG; Molecular Probes, Eugene, OR) as a fluorescent probe.15 To evaluate the kinetics of 2-NBDG uptake, equal numbers of cells were seeded in clear-bottomed
Cancer Cell Biology
What’s new? Increased conversion of glucose to lactic acid provides tumors with a constant supply of energy and biomass production, making altered glucose metabolism a target of particular interest in the development of novel anticancer drugs. Here, aberrant expression of the pro-inflammatory protein TG2 was found to reprogram glucose metabolism in mammary epithelial cells, providing new insight into the molecular mechanisms involved in the shift to altered metabolism. TG2 reprogramming induced constitutive activation of NF-jB and HIF-1a transcription factors, even under normoxic conditions. The findings help to explain how breast cancer cells survive stressful conditions and achieve metastatic competence.
2800
96-well microplates (BD Falcon, Franklin Lakes, NJ) at 20,000 cells/well in triplicate. Cells were allowed to adhere to the plates overnight at 37 C (for 12 hr) before assessing the glucose uptake. After overnight incubation, all of the wells were washed twice with phosphate-buffered saline (PBS) and incubated with 2-NBDG (100 lM) for 10 min at 37 C in a humidified atmosphere of 5% CO2. The reaction was stopped by adding ice-cold PBS to assay wells, followed by three washings with the same buffer. The fluorescent signal before (autofluorescence) and after adding 2-NBDG was measured in fluorometric mode (Perkin Elmer, Waltham, MA). The net increase in fluorescence was normalized according to the lowest signal (0 cells/ well) and was used as the ratiometric quantitation of 2-NBDG uptake by the cells.
Cancer Cell Biology
Lactate production
Cells were plated in six-well culture plates (3 3 105 cells/ well) and incubated for 2 days. The medium in the wells was replaced with fresh medium and incubation continued for an additional 2 days. At the end of the incubation, the medium was collected, and the lactate content was measured using a colorimetric enzymatic assay according to the manufacturer’s instructions (BioVision, Milpitas, CA). RNA isolation, reverse transcription-polymerase chain reaction and quantitative reverse transcriptase-ploymerase chain reaction
Total RNA was extracted from cells using a mini-RNA isolation kit (QIAGEN, Germantown, MD) according to manufacturer’s protocol. For reverse transcription-polymerase chain reaction (PCR), 2.5 lg of total RNA was reverse-transcribed to cDNA using an RT2 First Strand kit (SABiosciences, Valencia, CA). An equivalent volume (2 lL) of cDNA was used as the template for PCR with gene-specific primers (IDT, Coralville, IA). The relative change in signal was calculated after normalization according to glyceraldehyde-3-phosphate dehydrogenase. For whole genome microarray, total RNA from cells was isolated and quantified. The transcriptional microarray was performed using whole human genome oligo arrays with 44,000 60-mer probes (Agilent Technologies, Palo Alto, CA) as described earlier.16 Expression of selected genes from microarray data is shown in Supporting Information Table S1. Transfection with siRNA and shRNA
Cells (5 3 105 per well) were plated in six-well plates and allowed to adhere for 24 hr. The next day, cell monolayers were transfected with 30 nM p65/RelA or HIF-1a specific or control siRNA using Oligofectamine (Invitrogen) according to the manufacturer’s protocol. Briefly, 2 lL of a transfection reagent was added to 100 lL of culture medium, thoroughly mixed and incubated at room temperature for 15 min. Six microliters of siRNA was added to 150 lL of culture medium and then combined with diluted oligofectamine. The resulting solution was gently mixed, incubated at room temperature for 20 min, added to the cell culture and incubated for next
TG2-regulated metabolic reprogramming
48 hr. At the end of the incubation, cell lysates were prepared for further analysis. For shRNA transfection, cells (104 cells per well) were incubated with 5 lg/mL 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide (Polybrene) in a culture medium containing 5 3 104 lentiviral particles having TG2-shRNA or control shRNA sequences. Cells were then prepared for further analysis as described above. Cell viability
The viability of the study cells was determined using an MTT assay. Briefly, cells were seeded in quadruplicate wells in 96-well plates (3000 cells/well) and left to adhere to the plates overnight before placement of cells under hypoxic environment. After hypoxia exposure (94% N2, 5% CO2 and 1% O2) for 48 hr, the cell viability was assessed using MTT (0.5 mg/mL) as described previously.17 The cell survival under hypoxic conditions is expressed as the percent cell survival relative to survival of 100% of cells cultured in parallel under normal conditions (95% air and 5% CO2). Assessment of HIF-1a activity using a luciferase assay
To determine the effect of TG2 expression on HIF-1a transcriptional activation, cells at 40–50% confluence were transfected with a human VEGF 50 -flanking sequence (from 2985 to 2939 bp) inserted into the pGL2 basic luciferase vector as described previously.18 Transfected cells were cultured for 48 hr, washed once with PBS and lysed with a reporter lysis buffer. The luciferase activity in the cells was determined as described previously.18 Cell respiration assay
The XF analyzer (Seahorse Biosciences) was used to detect the oxygen consumption rate (OCR) to measure mitochondrial respiration, and the extracellular acidification rate (ECAR) to determine glycolysis in TG2 overexpressing and TG2-deficient cells. Briefly, cells were plated on XFe24 cell culture microplates at a density of 60,000 cells per well. The XFe24 cartridge was equilibrated with the calibration solution overnight at 37 C. For measuring OCR, XF medium (5 mM glucose and 2 mM Pyruvate in Dulbecco’s modified Eagle’s medium prepared and pH-adjusted to 7.0 on the day of the experiment) was used to prepare the cellular stress-inducing reagents, 500 nM oligomycin, 500 nM FCCP, 100 nM antimycin A and 100 nM rotenone (final concentration). For ECAR, the XF assay media (2 mM L-glutamine, 143 mM NaCl in DMEM, pH 7.4) was used to prepare glycolysis stress reagents, 10 mM Glucose, 500 nM Oligomycin and 100 mM 2-deoxyglucose (2-DG; final concentration). All the reagents were loaded in the ports, according to manufacturer’s instructions. The OCRs and ECARs were measured for 3 min with 3-min mixing and 2 min waiting periods. After the assay, cells in each well were counted using a ViCell counter and the cell counts were used to normalize the OCR (pmoles of oxygen per minute per 10,000 cells) and ECAR (mpH per minute per 10,000 cells). C 2013 UICC Int. J. Cancer: 134, 2798–2807 (2014) V
2801
Kumar et al.
Results TG2 upregulates HIF-1a expression
TG2 reprograms glucose uptake and metabolism
Because HIF-1a is known to shift energy production in cells by increasing glycolysis and because TG2 induces HIF-1a activation, we sought to determine the effect of TG2 expression on glucose uptake and lactate production. To that end, we compared glucose uptake by TG2-deficient and -overexpressing mammary epithelial cells. After normalization of the total cell count, we observed twofold greater glucose uptake by TG2-expressing 10A-TG2 and RT-Vec cells than by TG2deficient 10A-Vec and 7-WT cells (Fig. 2a). Conversely, we observed almost 60% less glucose uptake upon knockdown of TG2 expression in RT-TG2sh cells than in their parental
Cancer Cell Biology
In a recent study, we demonstrated that TG2 induces constitutive activation of NF-jB, which results in transcriptional regulation of the HIF-1a gene.13 To further investigate TG2mediated transcriptional regulation of HIF-1a we examined the nontransformed (MCF-10A) and transformed (MCF-7) mammary epithelial cells using loss- and gain-of-function techniques. We observed that TG2 expression in the cells was associated with a marked increase in HIF-1a protein expression and that experimental suppression of TG2 by short hairpin RNA (shRNA) attenuated this effect (Fig. 1a). A TG2induced increase in HIF-1a expression was evident even under normoxic conditions and was further increased under hypoxic conditions (Supporting Information Fig. S1). TG2regulated HIF-1a expression was transcriptionally active as suggested by its accumulation in the nucleus (Fig. 1b). The transcriptional activity of TG2-induced HIF-1a was further supported by the results of a reporter assay, which showed
that endogenous HIF-1a bound to the hypoxia-response elements in the vascular endothelial growth factor (VEGF) promoter, which was cloned upstream of the luciferase reporter gene. Indeed, the luciferase activity was strikingly higher in TG2-expressing cells than in those lacking TG2 expression (Fig. 1c).
Figure 1. TG2 expression in mammary epithelial cells induces increased accumulation of HIF-1a protein under normoxic conditions. (a) Immunoblot showing the basal HIF-1a and TG2 protein expression levels in nontransformed (MCF10A) and transformed (MCF-7/RT) mammary epithelial cells. MCF-10A cells were stably transfected with vector alone (10A-Vec) or a vector containing a TG2-coding sequence (10ATG2). Drug-resistant MCF-7/RT cells, which had high basal levels of TG2 expression, were transfected with control shRNA (RT-Vec) or TG2 shRNA (RT-TG2sh). (b) Cytosolic and nuclear fractions of the cells in (a) were isolated and subjected to western blotting for TG2 and HIF-1a protein expression. Membranes were stripped and reprobed with anti-b-actin (cytosolic fraction) or anti-histone-H1 (nuclear fraction) antibodies to ensure even protein loading in each lane. (c) TG2-expressing and -deficient cells in (a) and drug-sensitive counterparts of MCF-7/ RT cells (7-WT cells), which lack TG2 expression, were transfected with the VEGF 50 -flanking promoter sequence cloned upstream of a luciferase transcriptional reporter. Forty-eight hours after transfection, cells were assayed for luciferase activity. The results shown are from a representative experiments repeated at least twice with similar results.
C 2013 UICC Int. J. Cancer: 134, 2798–2807 (2014) V
Cancer Cell Biology
2802
Figure 2. Association of induced (10A-TG2 cells) and constitutive (RT-vec cells) expression of TG2 with (a) increased glucose uptake and (b) lactate production in mammary epithelial cells. The data are presented as the means 1 standard error of the mean for at least three independent experiments. ** p< 0.001.
TG2-expressing RT-Vec cells. These results suggested that TG2 expression facilitates glucose uptake, probably owing to increased HIF-1a expression. Lactate production, the end product of glycolytic energy metabolism, mirrored the glucose uptake in TG2-overexpressing and -deficient mammary epithelial cells. TG2-expressing cells had twofold to threefold greater lactate production than did their TG2-deficient counterparts (Fig. 2b). These results supported that TG2 expression can induce altered glucose metabolism in mammary epithelial cells. Effect of TG2 expression on mitochondrial respiration and Glycolysis
Under typical cell culture conditions, the rate of oxygen flux (OCR) is a direct measure of mitochondrial function. To determine the impact of TG2 expression on mitochondrial respiration, we measured the OCR in TG2-expressing and deficient mammary epithelial cells. For this purpose, we employed an XFe24 extracellular flux analyzer (Seahorse Biosciences, North Billerica, MA), which facilitates rapid realtime screening of the bioenergetic profiles of mitochondria in
TG2-regulated metabolic reprogramming
intact cells.19 When we compared the mitochondrial respiration rates of TG2-expressing cells and their TG2-deficient counterparts the latter cells exhibited the expected OCRs in response to successive treatment with oligomycin, carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), antimycin A and rotenone, which are well-defined small-molecule modulators of the electron transport chain (Fig. 3a).20 As expected, treatment with oligomycin decreased the OCR owing to blockage of ATP synthesis at mitochondrial complex V. Treatment with ionophore FCCP, which uncouples mitochondrial respiration from ATP synthesis, resulted in a rapid increase in OCR. Treatment with rotenone and antimycin A, which inhibit mitochondrial complexes I and III and stop the flow of oxygen in the electron transport chain, caused a drastic reduction in the OCR. TG2-overexpressing cells exhibited a low basal OCR and marked reduction in the FCCP-induced OCR (Fig. 3b). However, downregulation of TG2 expression completely rescued these cells from altered mitochondrial respiration. Next, we determined the effect of TG2 expression on glycolysis by measuring ECAR in response to three well-defined modulators of glycolysis. ECAR was significantly higher in TG2-expressing cells than their TG2-deficient counterparts (Figs. 3c and 3d). The inhibitor 2-DG, which inhibits HK— the first enzyme required in glycolysis, inhibited ECAR but it was stimulated by oligomycin and glucose. Moreover, the increased sensitivity of TG2 expressing cells to low glucose (Supporting Information Fig. 2) further supported TG2’s ability to enhance glycolytic flux. Taken, together, these results clearly support that TG2 expression promotes glycolytic phenotype in mammary epithelial cells as suggested by higher rates of proton production (ECAR). TG2 modulates glycolytic enzyme expression
The two proteins that are unequivocally associated with altered glucose metabolism are the glucose transporter GLUT-1 and HK, primarily its isoform HK2.21 In noncancerous cells, pyruvate (the end product of glycolysis) is metabolized in mitochondria. However, under hypoxic conditions, it is converted to lactate by the enzyme LDHA. HIF-1a can coordinate this conversion even under normoxic conditions by upregulating LDHA gene expression.22 Therefore, we next sought to determine the effect of TG2 on expression of GLUT-1, HK2 and LDHA. We observed that induced (10ATG2 cells) and constitutive (RT-vec cells) expression of TG2 in mammary epithelial cells was associated with increased expression of all three of these proteins (Fig. 4a). Their increased expression was accompanied by a corresponding increase in their transcript levels (Fig. 4b). As described previously, increased expression of TG2 was associated with increased expression of HIF-1a protein and transcription of this factor, whereas downregulation of TG2 expression by shRNA (RT-TG2sh cells) attenuated the expression of HIF1a, HK2, LDHA and GLUT1. These results suggested that a TG2-dependent increase in glucose uptake, lactate production C 2013 UICC Int. J. Cancer: 134, 2798–2807 (2014) V
Figure 3. (a,b) Real-time measurement of mitochondrial respiration rate (OCR; a, b) and glycolysis (ECAR; c and d) in mammary epithelial cells with induced (10A-TG2 cells; a and c) or constitutive (MCF-7/RT cells; b and d) overexpression of TG2. The small molecule-metabolic modulators oligomycin (500 nm), FCCP (100 nm) and antimycin A (100 nM) and rotenone (100 nM) were injected sequentially at the indicated time points after baseline OCR measurement. For ECAR determination, cells were glucose starved for 2 hr and subsequently treated with 10 mM D-glucose, 500 nM oligomycin and 100 mM 2-DG. ECAR in response to glucose treatment represents basal glycolytic activity, while in response to oligomycin the maximal glycolytic flux of the cells. ECAR prior to glucose treatment and following treatment with 2-DG represents ECAR due to non-glycolytic acidification. The data are presented as the mean 1 standard error for four independent experiments run in 8–10 replicate wells per experiment.
and a shift from aerobic to anaerobic metabolism are controlled by the expression of some key genes. In this context, a genome-wide analysis of MCF-10A cells stably transfected with TG2 reveled altered expression of a large group of functional genes involved in glucose metabolism (Supporting Information Table S1). TG2-induced NF-jB activation and HIF-1a expression mediate shift in glucose metabolism
We recently reported that stable expression of TG2 results in constitutive activation of NF-jB, which in turn binds to the HIF-1a promoter and induces its expression.13 Therefore, we reasoned that TG2-induced activation of NF-jB and HIF-1a plays a pivotal role in reprogramming of glucose metabolism. To test this hypothesis, we first determined the effect of downregulation of HIF-1a expression on GLUT-1 and LDHA protein expression. As shown in Figure 5a, downregulation of HIF-1a expression by siRNA resulted in appreciably decreased expression of these proteins. Similarly, inhibition C 2013 UICC Int. J. Cancer: 134, 2798–2807 (2014) V
of NF-jB by transfection of the cells with p65/RelA siRNA attenuated GLUT-1 and LDHA expression (Fig. 5b). Importantly, inhibition of TG2-induced HIF-1a and p65/RelA expression resulted in greatly decreased glucose uptake by the cells (Fig. 5). Taken together, these results suggested that aberrant expression of TG2 plays an important role in reprogramming of glucose metabolism in mammary epithelial cells under normoxic conditions owing to its ability to activate NF-jB and to induce NF-jB mediated increases in HIF1a expression.
Discussion In the present study, we provide evidence for the first time that stable expression of the proinflammatory protein TG2 reprograms mammary epithelial cells to alter glucose metabolism. Assay of TG2-overexpressing MCF-10A cells (10ATG2) or MCF-7/RT cells with knockdown of TG2 expression (RT-TG2sh) along with control cells revealed that TG2 overexpression produced greater glucose uptake and lactate
Cancer Cell Biology
2803
Kumar et al.
Cancer Cell Biology
2804
TG2-regulated metabolic reprogramming
Figure 4. Increased expression of TG2 is associated with increased accumulation of HIF-1a and increased expression of its downstream target genes. (a) Total cell extracts from TG2-deficient (10A-Vec, MCF-7 and RT-TG2sh) and TG2-overexpressing (10A-TG2 and RT-Vec) cells were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with anti-HIF-1a, anti-LDHA, anti-GLUT-1, anti-HK2 and anti-TG2 antibodies. TG2-probed membrane was stripped and reprobed with anti-b-actin antibody to ensure even protein loading in each lane. (b) Reverse transcriptase-PCR analysis showing differential expression of LDHA, GLUT-1, HK2 and HIF-1a transcripts in TG2-expressing and -deficient mammary epithelial cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts in these cell extracts were amplified in parallel to ensure even RNA loading. The results shown are from a representative experiment repeated at least twice with similar results.
production in the MCF10A and MCF-7/RT cells than in control cells (Fig. 1). Western blot analysis indicated that TG2 expression increases accumulation of HIF-1a protein in mammary epithelial cells even under normoxic conditions; this is further pronounced under hypoxic conditions (Supporting Information Fig. S1). These observations suggest a possible role for HIF-1a in TG2-regulated shifts in glucose metabolism. Furthermore, expression of TG2 correlated with increased expression of glucose metabolic enzymes such as GLUT-1, HK2 and LDHA at both the transcript and protein level (Fig. 4). GLUT-1 is a glucose transporter whose expression is unequivocally associated with increased glucose uptake by cancer cells,23 whereas HK2 is a mitochondria-bound isoform of HK that plays a key role in glycolysis and cell survival in multiple type of cancer cells.24 Similarly, LDHA converts surplus pyruvate generated during glycolysis into lactate and fuels it into lipid biosynthesis.25 Taken together, these results suggest that TG2 regulates the expression of key genes that encode for functional glycolytic proteins and facilitates increased glucose uptake and consumption by mammary epithelial cells. TG2 is not a transcription factor; therefore, its ability to induce expression of HIF-1a and other glycolytic genes must depend on activation of another transcription factor or factors. Indeed, we recently showed that TG2 expression is associated with constitutive activation of NF-jB in epithelial cells.13 Thus, TG2 binds to the NF-jB inhibitory protein IjBa leading to the rapid degradation of IjBa via a nonproteasomal pathway. TG2-mediated depletion of IjBa in turn
results in the release of active NF-jB and its translocation to the nucleus, where it binds to specific sequence in the promoter and induces transcriptional regulation of target genes. Several lines of evidence suggest that TG2-mediated activation of NF-jB is a key event in promotion of cancer progression.26,27 Recently, a novel procancer feedback loop in which TG2 activates NF-jB, which in turn drives TG2 expression was proposed.28 In cancer cells, this TG2/NF-jB feedback loop may play a self-amplifying role, as high TG2 expression and constitutive NF-jB activation are frequently observed in advanced-stage cancer cells.29,30 For example, multiple tumors and tumor cell lines have exhibited elevated expression of TG2 when selected for resistance to chemotherapy or isolated from metastatic sites.31–33 Also, increased expression of TG2 in drug-resistant breast tumors and tumor cell lines is mediated by aberrant epigenetic regulation of the TGM2 gene.34 Moreover, stable expression of TG2 promotes the embryonic developmental program of epithelial-tomesenchymal transition (EMT)14 and acquisition of cancer stem cell traits in epithelial cancer cells.35,36 These are important observations, as increased TG2 expression in several types of tumors is associated with poor outcome, whereas experimental suppression of TG2 expression inhibits the invasion of cancer cells and sensitizes them to chemotherapy.37–39 Similar to TG2, NF-jB plays fundamental roles in cancer progression stemming from its ability to activate numerous pro-growth, anti-apoptotic and metastatic genes.30 Researchers have directed major efforts toward developing inhibitors that can block NF-jB activity. However, most of C 2013 UICC Int. J. Cancer: 134, 2798–2807 (2014) V
2805
Figure 5. TG2-induced increases in glucose uptake and GLUT-1 and LDHA expression are dependent on NF-jB activity and HIF-1a expression. (a, b) Increased glucose uptake (left panels) and increased GLUT-1 and LDHA expression (right panels) in TG2-overexpressing cells were attenuated in response to downregulation of (a) HIF-1a or (b) p65 expression by gene-specific siRNA. Membranes were stripped and reprobed with an anti-HIF-1a antibody to determine the extent of HIF-1a inhibition by gene-specific or control siRNA and with anti-b- actin antibodies to ensure even protein loading in each lane. Downregulation of p65/RelA or HIF1a expression did not cause any noticeable changes in TG2 expression (not shown).
these efforts have focused on the use of small molecules to block the IjB kinase kinase activity.40 Based on the findings that TG2-induced activation of NF-jB is mediated by IjB kinase-independent mechanism described above; these inhibitors are unlikely to disable the TG2/NF-jB feedback loop. An interesting feature of TG2-induced NF-jB activation is that TG2 translocates to the nucleus along with the p50/p65 NF-jB complex.13 We identified HIF-1a as one of the downstream target genes of TG2-regulated NF-jB activity. Specifically, TG2-p65/RelA complex co-occupied the NF-jB binding site in the HIF-1a promoter (2197 to 2188bp) and induced transcriptional regulation of HIF-1a.13 Like TG2 and NF-jB expression, HIF-1a expression is considered a negative prognostic factor for cancer because of its ability to promote chemoresistance, angiogenesis, invasiveness, metastasis, resistance to cell death and genomic stability.41 Initially, researchers considered HIF-1a to be a mediator of response only under hypoxic conditions because of its instability under normoxic conditions. However, more recent evidence suggests that HIF-1a expression can be upregulated even under normoxic conditions in the presence of certain hormones, growth factors and cytokines.41,42 These observations further C 2013 UICC Int. J. Cancer: 134, 2798–2807 (2014) V
support the contention that aberrant expression of TG2 in cancer cells induces oncogenic cues. HIF-1 is a heterodimeric transcription factor responsible for increasing glycolysis by regulating the expression of genes encoding for glucose transporters, glycolytic enzymes and LDHA while at the same time inhibiting oxidative phosphorylation and squelching pyruvate entry into Krebs cycle via PDK1 mediated inactivation of pyruvate dehydrogenase. Curiously, we found that TG2 expression, in addition to NFjB activation and enhanced HIF-1a expression is associated with increased GLUT-1, HK2 and LDHA expression at both the transcript and protein level (Fig. 4; Supporting Information Table 1). Importantly, downregulation of TG2, HIF-1a or p65 (NF-jB) expression downregulated GLUT-1, HK2 and LDHA expression and attenuated the glucose uptake in mammary epithelial cells (Fig. 5). In addition to regulating the expression of genes encoding for glycolytic proteins, HIF1a can orchestrate other events known to promote the adaptation of incipient tumors to metabolic stress. For examples HIF-1 is known to prompt E-cadherin loss, which, in concert with alterations of other genes, leads to transformation of epithelial cells into pseudomesenchymal cells (EMT).43 EMT
Cancer Cell Biology
Kumar et al.
Cancer Cell Biology
2806
TG2-regulated metabolic reprogramming
is an embryonic developmental process that endows cancer cells with the ability to undergo anchorage-independent growth and enhances their motility and survival.44 Studies by our and other laboratories have demonstrated that TG2 expression in epithelial cancer cells is associated with EMT.14,35 More recently, we demonstrated that the EMTinducing ability of TG2 is dependent on NF-kB activation and is independent of the transamidation activity of TG2.13 Given the importance of glucose energy metabolism in cancer cells, that mitochondrial defects can be attributed to increased aerobic glycolysis is not surprising. Indeed, Warburg’s original hypothesis that increased aerobic glycolysis in cancer cells is related to defective mitochondrial functions supports this contention. In line with this hypothesis, our data demonstrated that overexpression of TG2 renders mammary epithelial cells unable to sense the presence of oxygen as revealed by decreased OCRs and stimulated ECARs under normoxic conditions (Fig. 3). Importantly, our study provided direct evidence that decreased mitochondrial respiration (OCR) and increased glycolysis (ECAR) is a dynamic process rather than related to mitochondrial damage. Downregulation of TG2 expression by shRNA fully restored the OCR and inhibited ECAR in drug-resistant MCF-7/RT cells (Fig. 3). Conversely, overexpression of TG2 in nontransformed MCF-10A cells greatly reduced the OCR and stimulated ECAR, suggesting that TG2 can induce alterations in mitochondria in an unknown way to influence their respiration rates. Indeed, authors have reported that TG2 is associated with the outer mitochondrial membrane and inner membrane space in various cell types.45 Moreover, researchers have implicated that TG2-catalyzed cross-linking of the mitochondrial matrix protein a-ketoglutarate dehydrogenase and aconitase plays a role in deterioration of energy metabolism.46,47 Additional studies are needed to establish the precise role of TG2 expression in mitochondrial energy metabolism. In summary, we found that TG2 expression reprograms energy metabolism in mammary epithelial cells by facilitating transcription of multiple genes involved in the glucose metabolic pathways. In addition, we observed a mechanistic relationship between TG2 and HIF-1a whereby TG2 facilitates HIF-1a synthesis via constitutive activation of the proinflammatory transcription factor NF-jB. As outlined in Figure 6, this interrelationship among TG2, NF-jb and HIF-1a facilitates oncogenic signaling networks that protect cells against stress-induced cell death under both normoxic and
Figure 6. Schematic representation of TG2-regulated metabolic pathways in mammary epithelial cells. Persistent inflammation or exposure to stressors causes epigenetic regulation of TG2. Aberrant expression of TG2 initiates a positive feedback loop, whereby TG2 activates NF-jB and NF-jB induces TG2 expression. After its activation, NF-jB binds to the HIF-1a promoter and induces its transcriptional regulation. HIF-1a upregulates expression of multiple genes, including those encoding for glycolytic proteins and EMT-related transcription repressors (Snail, Twist and Zeb). Together, these events compromise mitochondrial respiration rates (OCR), facilitate glucose uptake and metabolism via glycolysis, increase production of lactate, induce EMT and cancer stem cell phenotypes and render cancer cells highly invasive and resistant to exogenous (chemotherapy) and environmental (hypoxia) stresses.
hypoxic conditions (Supporting Information Fig. S3). In general, metabolic reprogramming is considered an essential mechanism for increased cell proliferation. Interestingly, TG2-mediated metabolic shift towards aerobic glycolysis did not have significant impact on cell proliferation rates. These findings suggest that the proposed association between altered metabolism and cell proliferation rate are either celltype dependent or metabolic shift signifies cellular response to increased proliferation rates. Taken together, these data suggest that aberrant expression of TG2 in cancer cells is a promising therapeutic target and can serve as a marker to evaluate the metabolic reprogramming of individual cancer cell types, their stage of transformation and metastatic potential.
Acknowledgement The authors thank Donald R. Norwood for critical reading of and editorial help with this manuscript.
References 1. 2.
3.
Warburg O. On respiratory impairment in cancer cells. Science 1956;124:269–70. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144: 646–74. Iyer NV, Kotch LE, Agani F, et al. Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1 alpha. Genes Dev 1998; 12:149–62.
4.
5.
6.
Denko NC. Hypoxia, HIF1 and glucose metabolism in the solid tumor. Nat Rev Cancer 2008;8: 705–13. Ferreira LM, Hebrant A, Dumont JE. Metabolic reprogramming of the tumor. Oncogene 2012;31: 3999–4011. Bardos JI, Ashcroft M. Hypoxia-inducible factor1 and oncogenic signaling. Bioessays 2004;26: 262–69.
7.
8.
9.
Gottlieb E, Tomlinson IP. Mitochondrial tumour suppressors: a genetic and biochemical update. Nat Rev Cancer 2005;5:857–66. Chen C, Pore N, Ismail-Behrooz F, et al. Regulation of glut1 mRNA by hypoxia-inducible factor1. Interaction between H-ras and hypoxia. J Biol Chem 2001;276:9519–25. Mathupala SP, Rempel A, Pedersen PL. Glucose catabolism in cancer cells: identification and
C 2013 UICC Int. J. Cancer: 134, 2798–2807 (2014) V
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
characterization of a marked activation response of the type II hexokinase gene to hypoxic conditions. J Biol Chem 2001;276:43407–12. Firth JD, Ebert BL, Ratcliffe PJ. Hypoxic regulation of lactate dehydrogenase A. Interaction between hypoxia-inducible factor 1 and cAMP response elements. J Biol Chem 1995;270:21021–27. Papandreou I, Cairns RA, Fontana L, et al. HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption. Cell Metab 2006;3:187–97. Zhang H, Gao P, Fukuda R, et al. HIF-1 inhibits mitochondrial biogenesis and cellular respiration in VHL-deficient renal cell carcinoma by repression of C-MYC activity. Cancer Cell 2007;11:407–20. Kumar S, Mehta K. Tissue transglutaminase constitutively activates HIF-1a promoter and nuclear factor-jB via a non-canonical pathway. PLoS One 2012;7:e49321. Kumar A, Xu J, Brady S, et al. Tissue transglutaminase promotes drug resistance and invasion by inducing mesenchymal transition in mammary epithelial cells. PLoS One 2010;15:e13390. Hassanein M, Weidow B, Koehler E, et al. Development of high-throughput quantitative assays for glucose uptake in cancer cell lines. Mol Imaging Biol 2011;13:840–52. Price ND, Trent J, El-Naggar AK, et al. Highly accurate two-gene classifier for differentiating gastrointestinal stromal tumors and leiomyosarcomas. Proc Natl Acad Sci USA 2007;104:3414–19. Wang P, Henning SM, Heber D. Limitations of MTT and MTS-based assays for measurement of antiproliferative activity of green tea polyphenols. PLoS One 2010;5:e10202. Forsythe JA, Jiang BH, Iyer NV, et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol 1996;16:4604–13. Malmgren S, Nicholls DG, Taneera J, et al. Tight coupling between glucose and mitochondrial metabolism in clonal beta-cells is required for robust insulin secretion. J Biol Chem 2009;284: 32395–404. Nicholls DG, Darley-Usmar VM, Wu M, et al. Bioenergetic profile experiment using C2C12 myoblast cells. J Vis Exp 2010;46:e2511. Gaillies RJ, Robey I, Gatenby RA. Causes and consequences of increased glucose metabolism of cancers. J Nucl Med 2008;49:24s–42s. Lum JJ, Bui T, Gruber M, et al. The transcription factor HIF-1alpha plays a critical role in the
23.
24.
25.
26.
27.
28.
29.
30. 31.
32.
33.
34.
35.
growth factor-dependent regulation of both aerobic and anaerobic glycolysis. Genes Dev 2007;21: 1037–49. Macheda ML, Rogers S, Best JD. Molecular and cellular regulation of glucose transporter (GLUT) proteins in cancer. J Cell Physiol 2005;202:654–62. Pedersen PL, Mathupala S, Rempel A, et al. Mitochondrial bound type II hexokinase: a key player in the growth and survival of many cancers and an ideal prospect for therapeutic intervention. Biochim Biophys Acta 2002;1555:14–20. Bauer DE, Hatzivassiliou G, Zhao F, et al. ATP citrate lyase is an important component of cell growth and transformation. Oncogene 2005;24: 6314–22. Kim DS, Park SS, Nam BH, et al. Reversal of drug resistance in breast cancer cells by transglutaminase 2 inhibition and nuclear factor-kappaB inactivation. Cancer Res 2006;66:10936–43. Mann AP, Verma A, Sethi G, et al. Overexpression of tissue transglutaminase leads to constitutive activation of nuclear factor-kappaB in cancer cells: delineation of a novel pathway. Cancer Res 2006;66:8788–95. Brown KD. Transglutaminase 2 and NF-^eB: an odd couple that shapes breast cancer phenotype. Breast Cancer Res Treat 2013;137:329–36. Mehta K, Kumar A, Kim HI. Transglutaminase 2: a multi-tasking protein in the complex circuitry of inflammation and cancer. Biochem Pharmacol 2010;80:1921–29. Karin M. Nuclear factor-kB in cancer development and progression. Nature 2006;441:431–36. Agnihotri N, Kumar S, Mehta K. Tissue transglutaminase as a central mediator in inflammationinduced progression of cancer. Breast Cancer Res 2013;15:202. Herman JF, Mangala LS, Mehta K. Implications of increased tissue transglutaminase (TG2) expression in drug-resistant breast cancer (MCF7) cells. Oncogene 2006;25:3049–58. Mangala LS, Fok JY, Zorrilla-Calancha IR, et al. Tissue transglutaminase expression promotes cell attachment, invasion and survival in breast cancer cells. Oncogene 2007;26:2459–70. Ai L, Kim WJ, Demircan B, et al. The transglutaminase 2 gene (TGM2), a potential molecular marker for chemotherapeutic drug sensitivity, is epigenetically silenced in breast cancer. Carcinogenesis 2008;29:510–8. Cao L, Shao M, Schilder J, et al. Tissue transglutaminase links TGF-b, epithelial to mesenchymal
C 2013 UICC Int. J. Cancer: 134, 2798–2807 (2014) V
36.
37.
38.
39.
40.
41. 42.
43.
44.
45.
46.
47.
transition and a stem cell phenotype in ovarian cancer. Oncogene 2012;31:2521–34. Kumar A, Gao H, Xu J, et al. Evidence that aberrant expression of tissue transglutaminase promotes stem cell characteristics in mammary epithelial cells. PLoS One 2011;6:e20701. Hwang JY, Mangala LS, Fok JY, et al. Clinical and biological significance of tissue transglutaminase in ovarian carcinoma. Cancer Res 2008;68: 5849–58. Verma A, Guha S, Diagaradjane P, et al. Therapeutic significance of elevated tissue transglutaminase expression in pancreatic cancer. Clin Cancer Res 2008;14:2476–83. Oh K, Ko E, Kim HS, et al. Transglutaminase 2 facilitates the distant hematogenous metastasis of breast cancer by modulating interleukin-6 in cancer cells. Breast Cancer Res 2011;13:R96. Gilmore TD, Herscovitch M. Inhibitors of NFkappaB signaling: 785 and counting. Oncogene 2006;25:6887–99. Wilson WR, Hay MP. Targeting hypoxia in cancer therapy. Nat Rev Cancer 2011;11:393–410. Bonello S, Z€ahringer C, BelAiba RS, et al. Reactive oxygen species activate the HIF-1alpha promoter via a functional NFkappaB site. Arterioscler Thromb Vasc Biol 2007;27:755–61. Thiery JP, Sleeman JP. Complex networks orchestrate epithelial–mesenchymal transitions. Nat Rev Mol Cell Biol 2006;7:131–42. Voulgari A, Pintzas A. Epithelial–mesenchymal transition in cancer metastasis: mechanisms, markers andstrategies to overcome drug resistance in the clinic. Biochim Biophys Acta 2009; 1796:75–90. Mastroberardino PG, Piacentini M. Type 2 transglutaminase in Huntington’s disease: a doubleedged sword with clinical potential. J Intern Med 2010;268:419–31. Cooper AJ, Sheu KR, Burke JR, et al. Transglutaminase-catalyzed inactivation of glyceraldehyde 3-phosphate dehydrogenase and alphaketoglutaratedehydrogenase complex by polyglutamine domains of pathological length. Proc Natl Acad Sci USA 1997;94:12604–109. Kim SY, Marekov L, Bubber P, et al. Mitochondrial aconitase is a transglutaminase 2 substrate: transglutamination is a probable mechanism contributing to high-molecular-weight aggregates of aconitase and loss of aconitase activity in Huntington disease brain. Neurochem Res 2005;30: 1245–55.
Cancer Cell Biology
2807
Kumar et al.
