The Reverse Warburg Effect Aerobic Glycolysis in Cancer Associated Fibroblasts and the Tumor Stroma

Stephanos Pavlides,1,2,† Diana Whitaker-Menezes,1,2,† Remedios Castello-Cros,1,2 Neal Flomenberg,2,3 Agnieszka K. Witkiewicz,2,4 Philippe G. Frank,1,2 Mathew C. Casimiro,1,2 Chenguang Wang,1,2 Paolo Fortina,1,2 Sankar Addya,1,2 Richard G. Pestell,1,2 Ubaldo E. Martinez-Outschoorn,2,3 Federica Sotgia1,2,* and Michael P. Lisanti1-3,*

1Departments of Stem Cell Biology & Regenerative Medicine, and Cancer Biology; 2The Jefferson Stem Cell Biology and Regenerative Medicine Center; 3Department of Medical Oncology; and 4Department of Pathology, Anatomy & Cell Biology; Kimmel Cancer Center; Thomas Jefferson University; Philadelphia, PA USA

†These authors contributed equally to this work.

Keywords: caveolin-1, tumor stroma, myo-fibroblast, cancer-associated fibroblast, aerobic glycolysis, M2-isoform of pyruvate kinase, lactate dehydrogenase, Warburg effect

Here, we propose a new model for understanding the Warburg effect in tumor metabolism. Our hypothesis is that epithelial cancer cells induce the Warburg effect (aerobic glycolysis) in neighboring stromal fibroblasts. These cancer-associated fibroblasts, then undergo myo-fibroblastic differentiation, and secrete lactate and pyruvate (energy metabolites resulting from aerobic glycolysis). Epithelial cancer cells could then take up these energy-rich metabolites and use them in the mitochondrial TCA cycle, thereby promoting efficient energy production (ATP generation via oxidative phosphorylation), resulting in a higher proliferative capacity. In this alternative model of tumorigenesis, the epithelial cancer cells instruct the normal stroma to transform into a wound-healing stroma, providing the necessary energy-rich micro-environment for facilitating tumor growth and angiogenesis. In essence, the fibroblastic tumor stroma would directly feed the epithelial cancer cells, in a type of host-parasite relationship. We have termed this new idea the “Reverse Warburg Effect.” In this scenario, the epithelial tumor cells “corrupt” the normal stroma, turning it into a factory for the production of energy- rich metabolites. This alternative model is still consistent with Warburg’s original observation that tumors show a metabolic shift towards aerobic glycolysis. In support of this idea, unbiased proteomic analysis and transcriptional profiling of a new model of cancer-associated fibroblasts [caveolin-1 (Cav-1) deficient stromal cells], shows the upregulation of both (1) myo-fibroblast markers and (2) glycolytic enzymes, under normoxic conditions. We validated the expression of these proteins in the fibroblastic stroma of human breast cancer tissues that lack stromal Cav-1. Importantly, a loss of stromal Cav-1 in human breast cancers is associated with tumor recurrence, metastasis, and poor clinical outcome. Thus, an absence of stromal Cav-1 may be a biomarker for the “Reverse Warburg Effect,” explaining its powerful predictive value.

Activated myo-fibroblasts are critical for normal wound heal- Thus, one way to create a model of cancer-associated fibro- ing and are generated via the TGFb mediated differentiation blasts might be to cause constitutive activation of TGF signal- of normal fibroblasts.1 However, they have also been implicated ing. To test this hypothesis directly, we examined the phenotypic in a number of human diseases related to fibrosis, such as sys- behavior of fibroblasts derived from mice that specifically lack a temic sclerosis, interstitial pulmonary fibrosis, as well as renal known inhibitor of TGFb signaling, namely caveolin-1 (Cav-1). fibrosis following ischemia. Fibrosis and collagen deposition are Cav-1 is known to function as an inhibitor of the TGFb type I also known risk factors for malignancy, and it is, therefore, not receptor kinase.2 In accordance with this hypothesis, Cav-1 (-/-) surprising that cancer-associated fibroblasts share many charac- null mice are prone to the development of fibrotic disease, and teristics with activated myo-fibroblasts.1 In fact, cancer has been Cav-1 (-/-) null skin fibroblasts share characteristics with scle- referred to by many as a “wound that does not heal.”1 As such, the roderma fibroblasts,3,4 which also exhibit a constitutive myo- tumor micro-environment has been postulated to play a key role fibroblastic phenotype. Thus, a loss of Cav-1 expression may be in tumor initiation, progression, and metastasis. In this regard, sufficient to induce a constitutive myo-fibroblastic phenotype. cancer-associated fibroblasts may be thought of as activated myo- In accordance with this hypothesis, we previously demon- fibroblasts that cannot regress to the unactivated state. They are strated that Cav-1 expression is dramatically downregulated in stuck in the “on position.” human breast cancer-associated fibroblasts, relative to matched

*Correspondence to: Federica Sotgia and Michael P. Lisanti; Email: [email protected] and [email protected] Submitted: 10/02/09; Accepted: 10/05/09 Previously published online: www.landesbioscience.com/journals/cc/article/10238

3984 Cell Cycle Volume 8 Issue 23 Report Report

normal mammary fibroblasts isolated from the same patients.5 an absence of Cav-1, we subjected Cav-1 (-/-) stromal cells to Thus, we postulated that a loss of Cav-1 in fibroblasts is a new extensive unbiased proteomic analysis and genome-wide tran- marker of the cancer-associated fibroblast phenotype. To test scriptional profiling. Results from this screening approach were this hypothesis more directly and to establish a cause-effect then validated by the immuno-staining of human breast cancer relationship, we studied the phenotype of Cav-1 (-/-) null mam- samples that lack stromal Cav-1 expression. mar y stroma l fibroblasts in culture. 6 Specifica lly, we showed that Using a proteomic approach, we have now identified >25 can- genetic ablation of Cav-1 appeared to be sufficient to confer the didate stromal biomarkers that are upregulated by a loss of Cav-1 cancer-associated fibroblast phenotype. Cav-1 (-/-) mammary in stromal cells. Interestingly, these proteins include five myo- stromal fibroblasts behaved as myo-fibroblasts, exhibiting: fibroblast markers, three signaling molecules, one oncogene, eight (1) contraction/retraction; (2) the upregulation of muscle- metabolic and glycolytic enzymes, as well as three extra-cellular related genes; and (3) the upregulation of TGFb ligands, related matrix proteins—known to be associated with fibrosis and tum- factors, and responsive genes (TGFb-2/3, procollagen genes, origenesis. The eight glycolytic enzymes include the M2-isoform interleukin-11, and CTGF).6 Thus, a loss of Cav-1 in fibro- of pyruvate kinase and lactate dehydrogenase (Ldha), which are blasts appears to drive the onset of the myo-fibroblastic differ- key regulators known to mediate the Warburg effect.12,13 Two entiation program. Finally, unbiased genome-wide expression markers of oxidative stress were also upregulated, suggesting the profiling of Cav-1 (-/-) mammary stromal fibroblasts showed over-production of reactive oxygen species (ROS) in Cav-1 defi- significant transcriptional overlap with human breast cancer- cient stromal cells. associated fibroblasts.6 Thus, Cav-1 (-/-) mammary fibroblasts Based on these findings, we predict that a loss of stromal provide the first cell culture model for breast cancer-associated Cav-1 may be a novel biomarker for the Warburg effect (aerobic fibroblasts.6 glycolysis) in the tumor stromal compartment. We have termed Since a loss of Cav-1 is associated with and can confer the this new idea the “Reverse Warburg Effect” because it suggests cancer-associated fibroblast phenotype, we speculated that it that aerobic glycolysis may take place in the “fibroblastic” tumor may have clinical relevance as a biomarker.7 To test this hypoth- stromal compartment, rather than in the epithelial cancer cells esis directly, we immuno-stained a well-annotated breast cancer themselves. tumor micro-array containing 160 consecutive patients.8 Our Interestingly, previous metabolic studies with skin myo-fibro- results indicated that an absence of stromal Cav-1 was specifically blasts have clearly shown that they perform “aerobic glycolysis,” associated with a high rate of tumor recurrence, metastasis and with increased glucose utilization and lactate production/secre- tamoxifen-resistance, resulting in poor clinical outcome.8 An tion,14 suggesting that the “Reverse Warburg Effect” may be a absence of stromal Cav-1 had predictive value that was indepen- general feature of both myo-fibroblasts and cancer-associated dent of epithelial marker status, indicating that it was an effective fibroblasts (CAFs). biomarker for all the major subclasses of breast cancer, including ER+, PR +, HER2+, and triple-negative patients (ER-/PR-/HER2-).8 Results An absence of stromal Cav-1 expression was most predictive in lymph-node positive patients, where it showed an 11.5-fold strati- Proteomic analysis of Cav-1 (-/-) null stromal cell lysates: fication of recurrence-free survival at 5 years (Cav-1(+), 80% Identification of new potential tumor stromal biomarkers. survival versus Cav-1(-), 7% survival).8 Similar results were also Since a loss of Cav-1 protein expression in the breast tumor obtained with DCIS patients, indicating that a loss of stromal stroma, in both DCIS and breast cancer patients, is predictive Cav-1 may be linked both to tumor initiation and progression, as of poor clinical outcome,7-9 we hypothesized that molecules that well as metastasis.9 DCIS patients lacking stromal Cav-1 had an are upregulated in the absence of stromal Cav-1 may be novel overall recurrence rate of 100% and 80% of these patients under- biomarkers. went progression to invasive breast cancer.9 In prostate cancer To identify potential new biomarkers, we subjected Cav-1 (-/-) patients, an absence of stromal Cav-1 was specifically associated stromal cell lysates to extensive unbiased proteomic analysis. For with advanced prostate cancer and metastatic disease,10 indicat- this purpose, we used primary cultures of bone-marrow derived ing that a loss of stromal Cav-1 may have predictive value in mul- stromal cells (BMSCs) from WT and Cav-1 (-/-) null mice. We tiple forms of human cancer. The above results related to breast chose to use BMSCs, as CAFs are thought to originate from mes- cancer metastasis and survival have also now been independently enchymal stem cells of the bone marrow.15-17 Two-dimensional validated in a second unrelated patient cohort.11 separation of WT and Cav-1 (-/-) stromal cell lysates yielded at Based on the above mechanistic and clinical data, it is now least 60 protein spots which were differentially expressed. We used clear that Cav-1 (-/-) null mice are a new valuable resource for mass spec/protein micro-sequencing to determine the molecular studying how myo-fibroblasts/cancer-associated fibroblasts con- identity of 22 protein spots that were specifically upregulated. See tribute to tumor initiation, progression, and metastasis. For Supplemental Figure 1 for representative 2-D gel analysis. example, Cav-1 (-/-) stromal cells could be used to test new thera- Interestingly, five of the protein spots that were upregulated peutic strategies that target the tumor micro-environment. we identified as known markers of the myo-fibroblast and/or can- Here, we have used stromal cells derived from Cav-1 (-/-) null cer-associated fibroblast (CAF) phenotype (vimentin, calponin2, mice as an engine to drive new stromal biomarker discovery. In tropomyosin, gelsolin and prolyl 4-hydroxylase alpha) by mass spec- order to identify which proteins are selectively upregulated by trometry analysis (Table 1).

www.landesbioscience.com Cell Cycle 3985 Figure 1. Genetic ablation of Cav-1 in stromal cells upregulates eight metabolic enzymes associated with the glycolytic pathway. Enzymes and metabolites that are part of glycolysis, the pentose phosphate pathway, fatty acid synthesis, triglyceride synthesis, lactose synthesis and the TCA cycle are shown. Note that the protein spots identified by proteomics analysis/mass spec (listed in Table 1) are eight enzymes associated with the glycolytic pathway (highlighted in pink). This list includes pyruvate kinase, a key enzyme which is sufficient to mediate the Warburg effect, driving aerobic glycolysis in tumors. This diagram was modified (with permission) from Beddek et al. Proteomics 2008; 8:1502–15, an article on the proteomics of the lactating mammary gland, and is based on the KEGG pathway database (www.genome.jp/kegg/pathway.html).

3986 Cell Cycle Volume 8 Issue 23 Table 1. Proteomic analysis: identification of cellular proteins upregulated in Cav-1 (-/-) bone marrow stromal cells Fold change (KO/WT) Accession number Protein spot number Myo-fibroblast associated proteins gelsolin 2.21 gi|148676699 7 calponin 2 1.83 gi|6680952 44 tropomyosin 2; beta 1.86 gi|123227997 40 vimentin 1.82 gi|31982755 30 vimentin 1.75 gi|2078001 22 vimentin 1.7 gi|31982755 33 prolyl 4-hydroxylase alpha(I)-subunit (P4HA1) 1.7 gi|836898 11 Signaling molecules annexin A1 2.0 gi|124517663 38 annexin A2 1.86 gi|6996913 39 Rho, GDP dissociation inhibitor (GDI) beta 1.67 gi|33563236 50 Glycolytic and metabolic enzymes M2-type pyruvate kinase 2.78 gi|1405933 15 phosphoglycerate kinase 1 2.41 gi|70778976 31 lactate dehydrogenase A (Ldha) 2 .11 gi|13529599 43 fructose-bisphosphate aldolase A 1.87 gi|6671539 32 glycerol 3-phosphate dehydrogenase 2 1.83 gi|224922803 12 enolase 1 (Eno1) 1.77 gi|34784434 24 triosephosphate isomerase 1 1.7 gi|6678413 57 triosephosphate isomerase 1 1.65 gi|6678413 58 phosphoglycerate mutase 1 1.65 gi|10179944 54 Oncogenes elongation factor 1-delta; EF-1-delta 1.94 gi|13124192 41 Anti-oxidants associated with oxidative stress peroxiredoxin 1 1.88 gi|6754976 60 catalase 1.74 gi|442441 17 All peptide sequences used for protein identification correspond to the Mus musculus protein product, ruling out contamination by serum proteins.

Also, several signaling molecules were overexpressed, such as for the first time, we have now identified that the Warburg Effect the annexins (A1 and A2), as well as RhoGDI. One of the proteins can originate in the tumor stroma. we identified is a known oncogene, Elongation factor 1-delta Two anti-oxidant markers of oxidative stress (peroxiredoxin 1 (EF-1-delta) (Table 1). Overexpression of EF-1-delta is sufficient and catalase) were also upregulated, suggesting the over-produc- to drive cell transformation and tumorigenesis.18,19 tion of reactive oxygen species (ROS) in Cav-1 deficient stromal Perhaps, most importantly, a loss of Cav-1 resulted in the cells, under normoxic conditions. upregulation of eight glycolytic/metabolic enzymes, including Proteomic analysis of Cav-1 (-/-) null stromal cell “condi- the M2-isoform of pyruvate kinase (Table 1). The M2-isoform of tioned media.” To identify secreted proteins that are upregulated pyruvate kinase is generated by gene splicing and is known to be in Cav-1 (-/-) stromal cells, we also subjected “conditioned media” sufficient to confer the “Warburg Effect”, i.e., aerobic glycolysis, from bone-marrow derived stromal cells (BMSCs) to proteomic which is thought to be a characteristic of tumor cells, stem cells, analysis. Two-dimensional separation of WT and Cav-1 (-/-) and cancer stem cells.12,13 The M1 isoform is the “adult” isoform, stromal cell “conditioned media” yielded greater than 60 protein while the M2 isoform is the corresponding “embryonic or devel- spots which were differentially expressed. We used mass spec/ opmental” isoform, both generated by alternate splicing from the protein micro-sequencing to determine the molecular identity of same gene. The M2-isoform of pyruvate kinase can also act a 8 protein spots that were specifically upregulated. Our results are nuclear co-factor to stimulate the transcriptional effects of Oct4, summarized in Table 2. See Supplemental Figure 2 for represen- an iPS transcription factor that confers pluripotency in ES cells.20 tative 2-D gel analysis. Figure 1 shows that all 8 of these enzymes sequentially map to Three of the protein spots that were upregulated we identified as the glycolytic pathway, which should result in the over-produc- extracellular matrix proteins [collagen I (Col1a1 and Col1a2)] and tion of the metabolites pyruvate and lactate, which could then be SPARC), that are known to be associated with fibrosis, the myo- secreted into the medium to “feed” adjacent tumor cells. Thus, fibroblast phenotype, and the tumor-associated stroma (Table 2).

www.landesbioscience.com Cell Cycle 3987 Table 2. Proteomic analysis: Identification of secreted proteins upregulated in Cav-1 (-/-) stromal cell “conditioned media” Fold change Accession Protein spot (KO/WT) number number Extracellular matrix proteins Col1a1 protein 3.92 gi|13096810 23 Col1a2 protein 2.88 gi|15214623 25 SPARC (secreted acidic cysteine rich glycoprotein) 2.59 gi|148701546 14 Albumin and alpha-fetoprotein: liver-specific secreted proteins albumin 5.77 gi|163310765 53 alpha-fetoprotein 4.39 gi|191765 43 alpha-fetoprotein 4.24 gi|191765 17 alpha-fetoprotein 2.42 gi|191765 4 Other unnamed protein product (Glutaredoxin (GRX) family, SH3BGR (SH3 domain binding 3.0 gi|74151816 58 glutamic acid-rich protein) subfamily)

All peptide sequences used for protein identification correspond to the Mus musculus protein product, ruling out contamination by serum proteins.

Interestingly, two liver-specific secreted proteins were also expressed in the tumor stromal compartment, using isoform-spe- upregulated, such as albumin and alpha-fetoprotein (Table 2). cific antibody probes that specifically recognize the M2-isoform. This is consistent with the idea that Cav-1 (-/-) null mesenchymal Furthermore, our proteomic studies using Cav-1 (-/-) null stromal stem cells may have an increased tendency towards liver-specific cells detected a peptide specific for the murine M2-isoform differentiation.21 Using in situ hybridization, alpha-fetoprotein (EAEAAIYHLQLFEELRR), but not the corresponding peptide expression has been previously localized selectively to myo-fibro- expected from the M1-isoform. blasts within the tumor stromal compartment in human breast Since annexin A2 and SPARC expression are associated with cancers, but not to the corresponding epithelial cancer cells.22 tenascin C overexpression in the extracellular matrix,23-25 we also Validation of stromal marker expression using human breast examined the expression of tenascin C in breast cancer tissues cancer tissues. We have now identified 19 candidate cell-based lacking Cav-1 expression. Interestingly, Figure 8 shows the robust biomarkers whose levels are upregulated in response to a loss of expression of tenascin C in the tumor stroma, as predicted. Cav-1 in stromal cells (Table 1). These include five myofibroblast Validation of stromal marker expression using transcriptional markers (such as vimentin), one oncogene (EF-1-delta), three gene profiling. In order to independently assess the expression signaling molecules (annexins A1, A2 and RhoGDI), eight gly- profiles of the gene products that are differentially expressed in colytic enzymes (including M2-pyruvate kinase) and two anti- WT and Cav-1 (-/-) stromal cells, we also performed gene expres- oxidant molecules related to oxidative stress (peroxiredoxin 1 and sion studies using transcriptome analysis. Briefly, mRNA species catalase). isolated from WT and Cav-1 (-/-) stromal cells were subjected to Similarly, we identified six secreted proteins that are upregu- transcriptional profiling using the Affymetrix GeneChip® Mouse lated in Cav-1 negative stromal cells (Table 2). For example, Cav-1 Exon 1.0 ST array. negative stromal cells showed the overexpression of both collagen Our results are summarized in Tables 3–5. Table 3 shows that I (Col1a1 and Col1a2) and SPARC. Liver-specific secretory pro- many of the cellular proteins that we identified as upregulated in teins were also overexpressed: albumin and alpha-fetoprotein. Cav-1 (-/-) stromal cells by proteomics were also transcription- To validate the potential clinical relevance of these candi- ally upregulated by gene profiling. These include myo-fibroblast date stromal biomarkers for human breast cancer, we immuno- markers (Gsn, Cnn2, Tpm2, Vim, P4ha1), signaling molecules stained sections from human breast cancer tumor tissues that (Anxa2, Arhgdib), glycolytic enzymes (Pkm2, Pgk1, Ldhal6b, were pre-selected based on a lack of stromal expression of Cav-1. Aldoa, Gpd2, Eno1, Tpi1, Pgam1), oncogenes (Eef1d) and anti- Figure 2 illustrates that stromal Cav-1 is absent or greatly oxidants (Cat). Also, additional glycolytic enzymes that were dimished in these samples (with the exception of the vascula- transcriptionally upregulated are included in this analysis. ture), and that vimentin is greatly increased. Similarly, Table 4 shows that the secreted proteins that we Using this immuno-histochemical approach, we also validated identified as upregulated in Cav-1 (-/-) stromal cells by proteom- the tumor stromal expression of other myo-fibroblast markers ics were also transcriptionally upregulated, such as extracellular (calponin and P4HA1), the annexins (A1 and A2), an oncogene matrix proteins (Col1a1, Col1a2, Spock1), and liver-specific pro- (EF-1-delta), two key glycolytic enzymes (M2-pyruvate kinase teins (Alb, Afp). and lactate dehyrogenase), and an extracellular matrix protein We also examined the expression of other functionally (SPARC) (Fig. 3–7). related gene products. Table 5 shows that TGFb receptor Importantly, M2-pyruvate kinase, the key glycolytic enzyme signaling molecules, glucose and lactate transporters, can- involved in conferring the “Warburg Effect” was abundantly cer-associated fibroblast markers, muscle-related genes, and

3988 Cell Cycle Volume 8 Issue 23 Figure 2. Vimentin is highly overexpressed in the stroma of human Figure 3. Validating the tumor stromal expression of myo-fibroblast breast cancers that lack stromal Cav-1 expression. We selected a num- markers, calponin and prolyl 4-hydroxylase, in breast cancers that lack ber of human breast cancer cases that lack stromal Cav-1 expression stromal Cav-1 expression. Paraffin-embedded tissue sections from for validating the stromal expression of new biomarkers. The upper human breast cancer samples were immuno-stained with antibod- panel shows a representative example of a loss of Cav-1 stromal stain- ies directed against calponin and prolyl 4-hydroxylase, alpha I subunit ing; however, note that the endothelial vasculature still remains Cav-1 (P4HA1). Slides were counterstained with hematoxylin. Note that positive (see arrows), as we have previously documented. Note that breast cancer tumor sections show the overexpression of calponin and sections from the same tumor show the overexpression of vimentin in P4HA1 in the tumor stromal compartment. Original magnification, 40X. the tumor stromal compartment. These results directly demonstrate the feasibility and success of our proteomics analysis. Original magnification, 40X. These studies provide critical independent validation for our results obtained by proteomic analysis of Cav-1 (-/-) stromal complement regulatory proteins, were all upregulated in Cav-1 cells. (-/-) stromal cells. The upregulation of TGFb receptor related signaling mol- Discussion ecules, cancer-associated fibroblast markers, and muscle-specific genes is consistent with the onset of a myo-fibroblastic pheno- The “Warburg Hypothesis” was first formulated by Otto H. type. Furthermore, the upregulation of glucose transporters, gly- Warburg in the early 1920s.26 He hypothesized that tumor colytic enzymes, and lactate transporters is consistent with a shift metabolism is different from normal metabolism, and relies on towards aerobic glycolysis, i.e., the “Warburg Effect.” glycolysis for the production of energy in the form of ATP, despite Finally, the overexpression of complement regulatory genes is in the presence of oxygen. Thus, aerobic glycolysis has come to be accordance with the idea that the Cav-1 negative tumor stroma would known as the “Warburg Effect,” and was originally attributed to provide a micro-environment like a “wound that does not heal.” mitochondrial mal-functioning.26 This is thought to result from

www.landesbioscience.com Cell Cycle 3989 Figure 4. Annexins (A1 and A2) are overexpressed in the stroma of human breast cancers that lack stromal Cav-1 expression. Paraffin-embedded tissue sections from human breast cancer samples were immuno-stained with antibodies directed against annexins (A1 and A2). Slides were counterstained with hematoxylin. Note that breast cancer tumor sections show the overexpression of annexins (A1 and A2) in the tumor stromal compartment. Also, note that tumor cell “nests” surrounded by annexin-positive stroma were observed (upper). For annexin A2, virtually identical results were obtained with antibodies directed against both the heavy and light chains (not shown). Upper panels, original magnification, 20X; Lower panels, original magnification, 40X. the adaptation of cancer cells to a hypoxic micro-environment. A a family of mono-carboxylate transporters (such as MCT1 and subset of proteins overexpressed in breast cancer (such as Cyclin MCT4). 29 This allows cancer cells undergoing aerobic glycolysis D1) have also been shown to inhibit mitochondrial metabolism, to “feed” adjacent cancer cells and other cells which are undergo- thereby driving epithelial cell aerobic glycolysis in vitro and in ing oxidative metabolism. vivo.27,28 Importantly, aerobic glycolysis results in the production Until recently, the “Warburg Effect” was thought to be con- of two metabolic end-products, pyruvate and lactate, which can fined to cancer cells. However, a recent paper by Vincent et al. then be secreted by cancer cells. Secreted lactate and pyruvate 2008 directly demonstrates that myo-fibroblasts from skin con- can be taken up by adjacent cancer cells and provides a feed- duct aerobic glycolysis much like cancer cells.14 As compared with forward mechanism for tumor growth, as these metabolites can normal fibroblasts, myo-fibroblasts consumed more glucose and then enter into the TCA cycle in cancer cells which are using oxi- produced more secreted lactate, behaving exactly like cancer cells.14 dative metabolism. Lactate dehyrogenase (LDH) is essential for Furthermore, pre-treatment with quercetin—a well-known lac- this process, as it is a bi-directional enzyme that coverts lactate tate transport inhibitor—is sufficient to prevent the conversion of to pyruvate and visa-versa. So LDH converts lactate to pyruvate, normal fibroblasts to myo-fibroblasts.30-34 Interestingly, the idea which enters the TCA cycle. Bi-directional transport of lactate that myo-fibroblasts exhibit the “Warburg Effect” has never been and pyruvate (into and out of cancer cells) is accomplished by considered in the context of tumorigenesis or Warburg’s original

3990 Cell Cycle Volume 8 Issue 23 Figure 5. The M2-isoform of pyruvate kinase (M2-PK) is overexpressed in the stroma of human breast cancers that lack stromal Cav-1 expression. Paraffin-embedded tissue sections from human breast cancer samples were immuno-stained with antibodies directed against the M2-isoform of pyruvate kinase (PK-M2). Slides were counterstained with hematoxylin. Note that breast cancer tumor sections show the overexpression of PK-M2 in the tumor stromal compartment (see arrow). Virtually identical results were obtained with two different monoclonal antibodies directed against PK-M2. Upper panel, original magnification, 40X; Middle and Lower panels, original magnification, 60X.

Moreover, these Cav-1 (-/-) deficient stromal cells also overexpress 8 glycolytic enzymes, including the M2-isoform of pyruvate kinase and lactate dehydrogenase. Transcriptome analysis provided independent validation for these proteomic studies, and showed the overexpression of cancer-associated fibroblast markers, muscle-related genes, glucose and lactate transporters, glycolytic enzymes, and TGFb related signaling molecules, in Cav-1 (-/-) deficient stromal cells. Thus, our current results with Cav-1 (-/-) deficient stromal cells provide genetic support for the hypothesis that aerobic glycolysis is a key feature of the myo- fibroblast phenotype. Cav-1 (-/-) deficient stromal cells also functionally behave like human cancer-associated fibroblasts.6 By unbiased genome-wide expression profiling, we have previously shown significant overlap between the transcriptomes of Cav-1 (-/-) mammary stromal fibroblasts and human breast cancer-associated fibroblasts.6 Furthermore, Cav-1 (-/-) mammary stromal fibroblasts overexpress a number of growth factors associated with cancer-associated fibroblasts (PDGF, VEGF, TGFb, IL-6, among others), and can stimulate, in a paracrine-fashion, the onset of an EMT in normal mammary epithelial cells.6 Finally, an absence of stromal Cav-1 expression in human breast cancers is associated with metastasis and recurrence, resulting in poor-clinical outcome.8,9 With this in mind, we used human breast cancer tissues which lack the stromal expression of Cav-1 to validate our results from the proteomic analysis of Cav-1 (-/-) deficient stromal cells. Here, we show that eight of these proteins are indeed expressed in the stromal or extracellular matrix compartment of human breast cancer tissues. Importantly, two of these proteins are key Warburg-related glycolytic enzymes (the M2-isoform of pyruvate kinase and lactate dehydrogenase) that are prominently expressed in cancer-associated fibroblasts seen within the tumor stroma, but not within the adjacent cancer cells. Although these observations are consistent with the idea that aerobic glycolysis is a key feature of tumor metabolism, they directly suggest that this effect can also be confined to the tumor stromal compartment. As an absence of stromal Cav-1 is powerful predictive biomarker, these results suggest that the presence hypothesis. Thus, aerobic glycolysis may be a key feature of the of aerobic glycolysis in the tumor stroma may have dire conse- myo-fibroblast phenotype. This has important implications for quences for patient survival. tumorigenesis, if we consider the striking similarities between Thus, based on these data, we would like to propose a new myo-fibroblasts and cancer-associated fibroblasts. model for understanding the Warburg effect in tumor metab- In direct support of this notion, we show here that Cav-1 (-/-) olism. Our hypothesis is that epithelial cancer cells induce the deficient stromal cells exhibit the characteristics of myo-fibro- Warburg effect (aerobic glycolysis) in neighboring stromal fibroblasts, as they overexpress five myo-fibroblast marker proteins. blasts. These cancer-associated fibroblasts, then undergo myo-

www.landesbioscience.com Cell Cycle 3991 fibroblastic differentiation, and secrete lactate and pyruvate (energy metabolites resulting from aerobic glycolysis). Epithelial cancer cells could then take up these energy-rich metabolites and use them in the mitochondrial TCA cycle, thereby promoting efficient energy production (ATP generation via oxidative phosphorylation), resulting in a higher proliferative capacity. This new model is illustrated schematically in Figure 9. In this alterative model of tumorigenesis, the epithelial cancer cells instruct the normal stroma to transform into a wound-healing stroma, providing the necessary energy-rich micro-environment for facilitating tumor growth and angiogenesis. In essence, the tumor stroma would directly feed the epithelial cancer cells, in a type of host-parasite relationship. We have termed this new idea the “Reverse Warburg Effect.” In this scenario, the tumor cells “corrupt” the normal stroma, turning it into a factory for the production of energy-rich metabolites. This alternative hypothesis is still consistent with Warburg’s original observation that tumors show a metabolic shift towards aerobic glycolysis. Thus, we should consider that the Warburg effect may be a stromal phenomenon. Consistent with this new stromal hypothesis, lactate by itself is sufficient to promote aerobic glycolysis, fibrosis and angiogenesis.35-37 So, secreted lactate and pyruvate derived from cancer-associated fibroblasts may also be used by endothelial progenitor cells, pericytes, and endothelial cells to drive angiogenesis. Similarly, a high tumor content of lactate is a powerful predictive biomarker for recurrence, metastasis and poor clinical outcome.38-41 Finally, it has been recognized as early as 1985 that TGFb treatment is sufficient to induce aerobic glycolysis and lactate production/secretion in fibroblasts.42 TGFb is also sufficient to induce the fibroblast-to-myofibroblast transition. This hormonal connection via TGFb also provides key evidence linking myo- Figure 6. Lactate dehydrogenase, a key glycolytic enzyme, is highly expressed in the breast cancer tumor stroma. Paraffin-embedded tissue fibroblasts with aerobic glycolysis and lactate production. sections from human breast cancer samples were immuno-stained with A simple prediction of the “Reverse Warburg Effect” is that antibodies directed against lactate dehydrogenase (LDH). Slides were tumors with an increased percentage of stroma would have a worse counterstained with hematoxylin. Note that breast cancer tumor sec- prognosis, because they would be expected to have increased lactate tions show the overexpression of LDH in the tumor stromal compart- production/secretion. In fact, when colon cancer patients are strati- ment (see boxed area). Upper panel, original magnification, 40X; Lower 43,44 panel, boxed area shown at higher magnification. Arrow points at LDH- fied based on tumor stromal content, patients with high tumor positive stromal fibroblasts. stromal content show dramatically increased tumor progression and poor survival, without the need for any biomarker analysis. The “Reverse Warburg Effect” may have important new impli- five myo-fibroblast markers and eight glycolytic enzymes. cations for novel therapies targeting the tumor stromal micro- Interestingly, an informatics analysis of our proteomics results environment. If the “Reverse Warburg Effect” is correct, then reveals that many of these protein products (including myo- lactate transport inhibitors would be a promising therapeutic fibroblast markers and glycolytic enzymes) are normally upregu- strategy, as they would metabolically uncouple the stromal fibro- lated by hypoxia and/or are targets of the HIF genes.45 Thus, blasts from the epithelial cancer cells. Lactate transport inhibitors stromal induction of HIF-responsive genes could possibly explain would be predicted to kill cancer-associated fibroblasts, as they our current findings. Notably, the stromal expression of HIF2- would prevent the secretion of lactate, leading towards intracel- alpha is associated with progression and poor clinical outcome in lular acidification. Similarly, lactate transport inhibitors would human colon cancer patients.46,47 starve epithelial tumor cells by eliminating their extracellular Interestingly, the over-production of reactive oxygen species source of lactate and pyruvate. Non-metabolizable derivatives of (ROS) is sufficient to induce HIF through its stabilization under glucose, such as 2-deoxy-glucose, could also be used to therapeu- normoxic conditions.48,49 Thus, a loss of stromal Cav-1 may some- tically target the fibroblastic tumor stroma. These ideas undoubt- how induce oxidative stress. In accordance with this hypothesis, edly deserve further study. Cav-1 (-/-) deficient stromal cells also show the upregulation of Further mechanistic experiments will be necessary to deter- two anti-oxidant proteins (peroxiredoxin 1 and catalase) nor- mine exactly how a loss of stromal Cav-1 coordinately induces mally associated with ROS-production and oxidative stress.

3992 Cell Cycle Volume 8 Issue 23 Figure 8. Expression of tenascin C in the extracellular matrix in breast cancer tumor tissue lacking stromal Cav-1. Paraffin-embedded tissue sections from human breast cancer samples were immuno-stained with antibodies directed against tenascin C. Slides were counterstained with hematoxylin. Note that breast cancer tumor sections show the overexpression of tenascin C in the extracellular matrix. Note the presence of tumor cell nests outlined by tenascin C. Original magnification, 20X.

are present in the tumor stromal compartment of human breast cancer tissue sections. This is consistent with previous studies showing that annexins A1 and A2 are localized to myo-fibroblasts within human breast cancers.53 In accordance with our overall hypothesis, both annexins A1 and A2 have been shown to be upregulated in response to hypoxia and/or are HIF target genes.54,55 Importantly, annexin A1 may also be a therapeutic target as radio-labeled antibodies directed against annexin A1 are sufficient to block tumor growth and prolong survival in an animal model, using a rat mammary adenocarcinoma cell Figure 7. Validating the tumor stromal expression of an oncogene (EF- 56 1-delta) and an extracellular matrix protein (SPARC). Paraffin-embed- line. ded tissue sections from human breast cancer samples were immuno- Interestingly, RhoGDI was also increased nearly two- stained with antibodies directed against EEF1D (eukaryotic translation fold in Cav-1 (-/-) stromal cells. Originally, RhoGDI was elongation factor 1 delta) and SPARC (Secreted Protein Acidic and Rich thought to function as a negative regulator of the Rho family in Cysteine). Slides were counterstained with hematoxylin. Note that of small GTPases (Rho/Rac/Cdc42). However, it appears that breast cancer tumor sections show the overexpression of EEF1D in the 57 tumor stromal compartment and SPARC in the extracellular matrix. RhoGDI is required for proper Rac-activation and targeting. Original magnification, 40X. Thus, RhoGDI is now considered to be a positive regulator of down-stream targets, such as NADPH oxidase.57,58 As such, an increase in RhoGDI expression would be expected to stimulate Previous unrelated studies have shown the Cav-1 expression is NADPH oxidase, leading to the increased production of reac- essential for liver regeneration. Thus, Cav-1 (-/-) null mice sub- tive oxygen species (ROS) and oxidative stress. This may also jected to partial hepatectomy have extremely low survival rates.50- explain why we see the concomitant upregulation of two anti- 52 This defect can be rescued by dietary supplementation of Cav-1 oxidant proteins (peroxiredoxin 1 and catalase) in Cav-1 (-/-) (-/-) deficient mice with glucose, but not fatty acids, as an energy stromal cells. source.50,51 As such, these functional/physiological observations are also consistent with the idea that Cav-1 (-/-) mice are more Materials and Methods metabolically-dependent on glucose and aerobic glycolysis for routine energy production. Materials. Antibodies for immuno-staining were obtained from The annexins are a family of membrane-targeted calcium- commercial sources: anti-annexin A1 (#610066, BD Biosciences); binding proteins. Interestingly, we show here that annexins A1 anti-annexin A2 [#610070 (LC, light chain) and #610068 and A2 are both upregulated in Cav-1 (-/-) null stromal cells and (HC, heavy chain), BD Biosciences]; anti-calponin 1/2/3

www.landesbioscience.com Cell Cycle 3993 Table 3. Transcriptome analysis: validation of cellular proteins upregulated in Cav-1 (-/-) bone marrow stromal cells, continued Fold change (KO/WT) Accession number p-value Myo-fibroblast associated proteins Gsn gelsolin 2.05 NM_146120 0.02 Cnn2 calponin 2 1.95 NM_007725 0.006 Cnn1 calponin 1 1.37 NM_009922 0.09 Tpm3 tropomyosin 3 2.30 NM_022314 0.02 Tpm2 tropomyosin 2 1.71 BC014809 0.04 Tpm4 tropomyosin 4 1.60 NM_001001491 0.05 Vim vimentin 1.58 ENSMUST00000028062 0.09 P4htm prolyl 4-hydroxylase, transmembrane (ER) 1.70 NM_028944 0.03 P4ha1 prolyl 4-hydroxylase, alpha polypeptide I 1.35 NM_011030 0.1 P4hb prolyl 4-hydroxylase, beta polypeptide 1.42 NM_011032 0.02 Signaling molecules Anxa6 annexin A6 1.70 NM_013472 0.02 Anxa3 annexin A3 1.65 NM_013470 0.03 Anxa2 annexin A2 1.62 NM_007585 0.1 Anxa8 annexin A8 1.60 NM_013473 0.04 Anxa7 annexin A7 1.50 NM_009674 0.03 Anxa11 annexin A11 1.38 NM_013469 0.01 Arhgdib Rho GDP dissociation inhibitor (GDI) beta 1.49 NM_007486 0.03 Arhgdig Rho GDP dissociation inhibitor (GDI) gamma 1.38 NM_008113 0.01 Glycolytic and related metabolic enzymes Pkm2 pyruvate kinase, muscle 1.50 NM_011099 0.01 Pgk1 phosphoglycerate kinase 1 2.05 NM_008828 0.01 Pgk2 phosphoglycerate kinase 2 1.56 NM _031190 0.1 Ldhal6b lactate dehydrogenase A-like 6B 2.04 NM_175349 0.007 Ldhb lactate dehydrogenase B 2.44 NM_008492 0.02 Ldhc lactate dehydrogenase C 1.48 NM_013580 0.04 Ldhd lactate dehydrogenase D 1.53 NM_027570 0.04 Aldoa aldolase A, fructose-bisphosphate 1.84 NM_007438 0.05 Aldob aldolase B, fructose-bisphosphate 1.43 NM_144903 0.03 Aldoc aldolase C, fructose-bisphosphate 2.82 NM_009657 0.01 Gpd2 glycerol-3-phosphate dehydrogenase 2 2.50 NM_001145820 0.005 Gpd1l glycerol-3-phosphate dehydrogenase 1-like 1.94 NM_175380 0.02 Eno1 enolase 1, (alpha) 2 .11 NM _023119 0.0002 Eno2 enolase 2 (gamma, neuronal) 1.31 NM_013509 0.07 Tpi1 triosephosphate isomerase 1 2.02 NM_009415 0.006 Pgam1 phosphoglycerate mutase 1 (brain) 1.93 NM_023418 0.02 Pgam2 phosphoglycerate mutase 2 (muscle) 1.67 NM_018870 0.1 Pgam5 phosphoglycerate mutase family member 5 1.63 NM_028273 0.05 Hk1 hexokinase 1 1.87 NM_001146100 0.004 Hk2 hexokinase 2 2.40 NM_013820 0.04 Gck glucokinase (hexokinase 4) 1.48 NM_010292 0.002 Gpi1 glucose phosphate isomerase 1 1.81 NM_008155 0.01 Pgm1 phosphoglucomutase 1 1.25 NM_025700 0.04 Pgm2 phosphoglucomutase 2 1.76 NM_028132 0.02 Pgm3 phosphoglucomutase 3 2.06 NM_028352 0.03 Pfkl phosphofructokinase, liver 1.85 NM_008826 0.04 Gene products that were also identified by proteomics analysis are shown in bold; Other related genes and family members are also shown.

3994 Cell Cycle Volume 8 Issue 23 Table 3. Transcriptome analysis: validation of cellular proteins upregulated in Cav-1 (-/-) bone marrow stromal cells, continued Pfkm phosphofructokinase, muscle 1.94 NM_021514 0.03 Pfkfb3 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase3 1.36 NM_133232 0.02 Gapdh glyceraldehyde-3-phosphate dehydrogenase 1.58 NM_008084 0.04 Oncogenes Eef1d eukaryotic translation elongation factor 1 delta 1.61 NM_029663 0.03 Anti-oxidants associated with oxidative stress Prdx4 peroxiredoxin 4 1.74 NM_016764 0.095 Prdx5 peroxiredoxin 5 1.64 NM_012021 0.1 Prdx2 peroxiredoxin 2 1.44 NM _011563 0.08 Cat catalase 1.34 NM_009804 0.1 Gene products that were also identified by proteomics analysis are shown in bold; Other related genes and family members are also shown.

Table 4. Transcriptome analysis: validation of secreted proteins upregulated in Cav-1 (-/-) stromal cell “conditioned media” Fold change (KO/WT) Accession number p-value Extracellular matrix proteins Col1a1 collagen, type I, alpha1 1.59 NM_007742 0.05 Col1a2 collagen, type I, alpha2 1.69 NM_007743 0.03 Spock1 sparc/osteonectin, proteoglycan (testican)1 1.93 NM_009262 0.0005 Spock3 sparc/osteonectin, proteoglycan (testican)3 1.43 NM_023689 0.02 Sparcl1 SPARC-like 1 (hevin) 1.61 NM_010097 0.007 Smoc1 SPARC related modular calcium binding 1 2.24 NM _0 01146217 0.02 Albumin and alpha-fetoprotein: liver-specific secreted proteins Alb albumin 1.93 NM_009654 0.09 Afp alpha-fetoprotein 1.49 NM_007423 0.06 Other Sh3bgrl3 SH3 domain binding glutamic acid-rich protein like 3 1.78 NM_080559 0.003 Gene products that were also identified by proteomics analysis are shown in bold; Other related genes and family members are also shown.

(#sc-28545, FL-297, Santa Cruz Biotech); anti-caveolin-1 (#sc- Isolation and culture of bone-marrow derived stromal cells 894, N-20, Santa Cruz Biotech); anti-EF-1-delta (#ab13962, (BMSCs). Bone marrow cells were collected from 10-week-old EEF1D, Abcam); anti-lactate dehydrogenase (#ab47010, Abcam), wild-type and Cav-1 (-/-) mice by flushing the hind leg femurs anti-prolyl 4-hydroxylase, alpha subunit (#12658-1-AP, P4HA1, and tibias. Bone marrow cells were washed twice, and plated Proteintech Group); anti-pyruvate kinase M2 (#S-1, clone DF-4, in 10 cm tissue culture dishes with Minimum Essential Media ScheBo Biotech; and ab55602, Abcam); anti-SPARC (ab14174, alpha (alpha-MEM; A10490-01, Gibco-Invitrogen), containing Abcam); tenascin C (NCL-TENAS-C, Novocastra/Leica 10% FBS. Once the culture reached 80–90% confluency, cells Microsystems); and anti-vimentin (#M0725, clone V9, Dako; were trypsinized and replated. Bone-Marrow Derived Stromal and ab8545, Abcam). Cells (BMSCs) were used for experiments at passage 3. BMSCs Animal studies. All animals were housed and maintained in a prepared in this fashion are also referred to in the literature as pathogen-free environment/barrier facility at the Kimmel Cancer mesenchymal stem cells (MSCs). Center at Thomas Jefferson University under National Institutes Preparation of “conditioned media” from bone-marrow of Health (NIH) guidelines. Mice were kept on a 12-hour light/ derived stromal cells. WT and Cav-1 (-/-) stromal cells were cul- dark cycle with ad libitum access to chow and water. Cav-1 (-/-) tured in normal medium until they reached confluence. Then, the deficient mice were generated, as we previously described.6,59 All cells were washed 3 times with PBS and cultured in low-serum wild-type and Cav-1 knockout (KO) mice used in this study α-MEM (supplemented with 0.1% FBS). After 48 hours, tissue were in the FVB/N genetic background. For most of the studies, culture supernatants were collected, filtered through a 0.45 μm 2.5-month-old virgin mice were used, unless stated otherwise. pore filter, and concentrated by Centriprep YM-10 (Millipore), Animal protocols used for this study were pre-approved by the according to the manufacturer’s instructions. Concentrated sam- institutional animal care and use committee. ples were stored at -80°C until proteomic analysis.

www.landesbioscience.com Cell Cycle 3995 Table 5. Transcriptome analysis of Cav-1 (-/-) stromal cells: TGFb signaling, glucose and lactate transporters, cancer-associated fibroblast markers and complement regulatory proteins, continued. Fold change (KO/WT) Accession number p-value TGFbeta receptor signaling, ligands and target genes Ctgf connective tissue growth factor 2.16 NM_010217 0.02 Tgfbi transforming growth factor, beta-induced, 68 kDa 2.83 NM_009369 0.002 Tgfbr2 transforming growth factor, beta receptor II (70/80 kDa) 1.82 NM_009371 0.01 Tgfbr3 transforming growth factor, beta receptor III 1.70 NM _011578 0.03 Tgfb1 transforming growth factor, beta1 1.67 NM _011577 0.01 Tgfb1i1 transforming growth factor beta1 induced transcript 1 1.43 NM_009365 0.04 Smad3 SMAD family member 3 1.75 NM_016769 0.03 Smad6 SMAD family member 6 1.61 NM_008542 0.03 Smad9 SMAD family member 9 1.62 NM_019483 0.02 Smad5 SMAD family member 5 1.48 NM_008541 0.04 Twist2 twist homolog 2 (Drosophila) 1.39 NM_007855 0.002 Snai2 snail homolog 2 (Drosophila); Slug 1.30 NM _011415 0.03 Loxl1 lysyl oxidase-like 1 1.80 NM_010729 0.05 Loxl3 lysyl oxidase-like 3 1.87 NM_013586 0.05 Loxl4 lysyl oxidase-like 4 1.75 NM_053083 0.007 Family of facilitated glucose transporters (GLUTs) Slc2a6 solute carrier family 2, GLUT6 2.03 NM_172659 0.01 Slc2a5 solute carrier family 2, GLUT5 1.87 NM_019741 0.008 Slc2a3 solute carrier family 2, GLUT3 1.77 NM_011401 0.02 Slc2a8 solute carrier family 2, GLUT8 1.30 NM_019488 0.01 Family of mono-carboxylate transporters (MCT1/4) Slc16a1 solute carrier family 16, member 1 (MCT1) 1.62 NM_009196 0.04 Slc16a3 solute carrier family 16, member 3 (MCT4) 1.53 NM_030696 0.03 Cancer-associated fibroblast markers Cxcl9 chemokine (C-X-C motif) ligand 9 7.99 NM_008599 0.04 Mme CD10 membrane metallo-endopeptidase 6.48 NM_008604 0.01 Cxcl12 chemokine (C-X-C motif) ligand 12 (SDF1) 5.14 NM_021704 0.002 Cxcl11 chemokine (C-X-C motif) ligand 11 3.97 NM_019494 0.01 Ly6e lymphocyte antigen 6 complex, locus E; stem cell antigen-2 3.78 NM_008529 0.009 Ccl5 chemokine (C-C motif) ligand 5 (RANTES) 3.37 NM_013653 0.03 Hgf hepatocyte growth factor (hepapoietin A; scatter factor) 3.09 NM_010427 0.04 Met met proto-oncogene (Hgf receptor) 1.36 NM_008591 0.04 Vegfa vascular endothelial growth factor A 2.56 NM_001025250 0.04 Cdh11 cadherin 11, type 2, OB-cadherin (osteoblast) 2.37 NM_009866 0.004 Pdpn podoplanin 2.10 NM_010329 0.01 Pdgfrl platelet-derived growth factor receptor-like 2.02 NM_026840 0.05 Pdgfrb platelet-derived growth factor receptor, beta polypeptide 1.78 NM _0 01146268 0.03 Pdgfra platelet-derived growth factor receptor, alpha polypeptide 1.57 NM_011058 0.05 Pdgfa platelet derived growth factor A 1.32 NM_008808 0.008 Cd34 Cd34 molecule 1.92 NM _0 01111059 0.003 Smtn smoothelin 1.78 NM _0 01159284 0.02 Igf2 insulin-like growth factor 2 (somatomedin A) 1.76 NM_010514 0.0004 Cd248 Cd248 molecule, endosialin, Tem1 1.53 NM_054042 0.04 Pecam1 platelet/endothelial cell adhesion molecule; Cd31 1.47 NM_008816 0.04 Retnla resistin like alpha; Fizz1 1.34 NM_020509 0.03

3996 Cell Cycle Volume 8 Issue 23 Table 5. Transcriptome analysis of Cav-1 (-/-) stromal cells: TGFb signaling, glucose and lactate transporters, cancer-associated fibroblast markers and complement regulatory proteins, continued. Cav1 caveolin-1; caveolae-associated protein 0.048 NM_007616 0.0001 Muscle-related genes Acta1 actin, alpha1, skeletal muscle 1.65 NM_009606 0.006 Actc1 actin, alpha, cardiac muscle 1 1.68 NM_009608 0.006 Actg1 actin, gamma1 2.68 NM_009609 0.004 Actl6a actin-like 6A 1.80 NM_019673 0.002 Actl6b actin-like 6B 1.63 NM_031404 0.01 Actn2 actinin, alpha2 2.50 NM_033268 0.03 Actn3 actinin, alpha3 1.45 NM_013456 0.04 Actn4 actinin, alpha4 1.41 NM_021895 0.03 Actr1a ARP1 actin-related protein 1 homolog A, centractin (yeast) 1.70 NM_016860 0.03 Actr6 ARP6 actin-related protein 6 homolog (yeast) 1.90 NM_025914 0.02 Myo10 myosin X 2.15 NM_019472 0.02 Myo15 myosin XV 2.08 NM_010862 0.02 Myo18a myosin XVIIIA 2.44 NM _011586 0.01 Myo1b myosin IB 1.74 NM_010863 0.03 Myo1c myosin IC 1.66 NM_001080775 0.02 Myo1e myosin IE 1.47 NM_181072 0.03 Myo1g myosin IG 1.49 NM_178440 0.0004 Myo1h myosin IH 1.71 ENSMUST00000031566 0.04 Myo5b myosin VB 1.45 NM_201600 0.02 Myo5c myosin VC 1.29 NM_001081322 0.03 Myo6 myosin VI 1.99 NM_001039546 0.04 Myo7a myosin VIIA 1.94 NM_008663 0.006 Myo7b myosin VIIB 1.64 NM_032394 0.03 Myo9b myosin IXB 1.93 NM _0 01142322 0.01 Myod1 myogenic differentiation 1 1.52 NM_010866 0.01 Myom1 myomesin 1, 185 kDa 1.75 NM_010867 0.02 Myom2 myomesin (M-protein) 2, 165 kDa 1.59 NM_008664 0.02 Myh10 myosin, heavy chain 10, non-muscle 1.41 NM_175260 0.004 Myh11 myosin, heavy chain 11, smooth muscle 3.12 NM_013607 0.04 Myh14 myosin, heavy chain 14 1.88 AY363100 0.04 Myh6 myosin, heavy chain 6, cardiac muscle, alpha 2.34 NM_010856 0.04 Myh7 myosin, heavy chain 7, cardiac muscle, beta 1.96 NM_080728 0.05 Myh9 myosin, heavy chain 9, non-muscle 3.14 NM_022410 0.008 Myl10 myosin, light chain 10, regulatory 1.75 NM_021611 0.03 Myl2 myosin, light chain 2, regulatory, cardiac, slow 1.89 NM_010861 0.03 Mylk myosin light chain kinase 1.34 NM_139300 0.001 Mylk3 myosin light chain kinase 3 1.48 NM_175441 0.02 Mylpf myosin light chain, phosphorylatable, fast skeletal muscle 1.27 NM_016754 0.04 Complement regulatory proteins Cfb complement factor B 7.80 NM_008198 0.01 C3 complement component 3 4.35 NM_009778 0.004 C1r complement component 1, r subcomponent 2.04 NM_023143 0.02 C1ql1 complement component 1, q subcomponent-like 1 1.87 NM _011795 0.01 C1ql2 complement component 1, q subcomponent-like 2 1.40 NM_207233 0.03 C1ql3 complement component 1, q subcomponent-like 3 1.85 AB044560 0.004

www.landesbioscience.com Cell Cycle 3997 Table 5. Transcriptome analysis of Cav-1 (-/-) stromal cells: TGFb signaling, glucose and lactate transporters, cancer-associated fibroblast markers and complement regulatory proteins, continued. C1qtnf2 C1q and tumor necrosis factor related protein 2 1.66 NM_026979 0.02 C1qtnf5 C1q and tumor necrosis factor related protein 5 1.99 NM_145613 0.005 C2 complement component 2 1.65 NM_013484 0.02 C4b complement component 4B 1.49 NM_009780 0.01 C8a complement component 8, alpha polypeptide 1.56 NM_146148 0.03 C8b complement component 8, beta polypeptide 1.27 NM_133882 0.04 C8g complement component 8, gamma polypeptide 1.35 NM_027062 0.03 C9 complement component 9 1.42 NM_013485 0.003

Proteomic analysis. 2-D DIGE (two-dimensional differ- RNA was ethanol precipitated and quantified on a NanoDrop ence gel electrophoresis)60 and mass spectrometry protein iden- ND-1000 spectrophotometer, followed by RNA quality assess- tification were run by Applied Biomics (Hayward, CA). Image ment by analysis on an Agilent 2100 Bioanalyzer (Agilent Inc., scans were carried out immediately following SDS-PAGE using Palo Alto, CA). RNA amplification and labeling was performed Typhoon TRIO (Amersham BioSciences) following the pro- by the WT-Ovation Pico RNA amplification system (NuGen tocols provided. The scanned images were then analyzed by Technologies, Inc.,). Briefly, 500 pg to 50 ng of total RNA was Image QuantTL software (GE-Healthcare), and then subjected reverse transcribed using a chimeric cDNA/mRNA primer, and to in-gel analysis and cross-gel analysis using DeCyder soft- a second complementary cDNA strand was synthesized. Purified ware version 6.5 (GE-Healthcare). The ratio of protein differ- cDNA was then amplified with ribo-SPIA enzyme and SPIA ential expression was obtained from in-gel DeCyder software DNA/RNA primers (NuGEN Technologies, Inc.). Amplified analysis. The selected spots were picked by an Ettan Spot Picker DNA was purified with the QIAquick PCR purification kit (GE-Healthcare) following the DeCyder software analysis and (Qiagen). Sense transcript cDNA (ST-cDNA) was generated from spot picking design. The selected protein spots were subjected to 3 μg amplified cDNA using WT-Ovation Exon module (NuGen in-gel trypsin digestion, peptides extraction, desalting and fol- Technologies, Inc.). Purified ST-cDNA was assed for yield using lowed by MALDI-TOF/TOF (Applied Biosystems) analysis to the Nanodrop Spectrophotometer (NanoDrop Technologies, determine the protein identity. Inc.). 2.5 μg ST-cDNAs were fragmented and chemically labeled Immuno-histochemical analysis. Immunohistochemical with biotin to generate biotinylated ST-cDNA using FL-Ovation staining was performed essentially as we previously described.8,9 cDNA biotin module V2 (NuGen Technologies, Inc.,). Each Briefly, 5-μm sections from paraffin-embedded breast cancer Affymetrix GeneChip® Mouse Exon 1.0 ST array (Affymetrix, tissue were de-paraffinized and rehydrated by passage through Santa Clara, CA) was hybridized with fragmented and biotin- a graded series of ethanol. Antigen retrieval was performed by labeled target (2.5 μg) in 110 μl of hybridization cocktail. Target heating the slides in 10 mM sodium citrate buffer, pH 6.0, for 10 denaturation was performed a 99°C for 2 min. and then 45°C minutes using a pressure cooker. Endogenous peroxidase activ- for 5 min., followed by hybridization for 18 hrs. Arrays then ity was quenched with 3% H2O2 for 10 minutes. Then, slides were washed and stained using Genechip Fluidic Station 450, were washed with phosphate-buffered saline (PBS) and blocked and hybridization signals were amplified using antibody ampli- with 10% goat serum in PBS for 1 hour at room temperature. fication with goat IgG (Sigma-Aldrich) and anti-streptavidin Samples were incubated with the primary antibodies diluted in biotinylated antibody (Vector Laboratories, Burlingame, CA). 1% BSA in PBS overnight at 4°C. After washing in PBS (three Chips were scanned on an Affymetrix Gene Chip Scanner 3000, times, 5 minutes each), slides were stained with the LSAB2 sys- using Command Console Software. Background correction and tem kit (Dako Cytomation, Glostrup, Denmark), according to normalization were done using the Robust Multichip Average the manufacturer’s recommendations. Briefly, samples were incu- (RMA) with Genespring V 10.0 software (Agilent, Palo Alto, bated with biotinylated linker antibodies for 30 minutes, washed CA). The Robust Multichip Average (RMA) signal was com- in PBS (three times, 5 minutes each), and then incubated with puted for exon and gene-level probeset summaries by perform- a streptavidin-horseradish peroxidase-conjugated solution for 30 ing the RMA-sketch analysis for CORE probesets in Affymetrix minutes. After washing, samples were incubated with the diamin- Expression Console Version 1.1 (http://www.affymetrix.com). obenzidine reagent until color production developed. Finally, the Log2 RMA expression values were exported as a text file and slides were rinsed with tap water and counter-stained with hema- additional calculations were performed in Matlab 2009a (The toxylin, dehydrated, and mounted with coverslips. Importantly, MathWorks, Natick, MA; www.mathworks.com) and Microsoft critical negative controls were performed in parallel for all of the Excel (Microsoft, Redmond, WA; www.microsoft.com). The dif- immunohistochemical studies. ference between average expression in knockout samples and the Genome-wide transcriptional profiling. Total RNA was average expression in wild type samples was computed as well as isolated from WT and Cav-1 (-/-) stomal cells using RNAeasy the fold-change between the two sample groups. Results from one mini columns (Qiagen). RNA was prepared from three wild- exon for each gene analyzed were selected for inclusion in Tables type and three Cav-1 (-/-) stromal cell isolates. DNase-treated 3–5. A p-value of <0.05 was considered statistically significant.

3998 Cell Cycle Volume 8 Issue 23 Figure 9. The Reverse Warburg Effect: Aerobic glycolysis in cancer associated fibroblasts (CAFs) and the tumor stroma. Our hypothesis is that epithelial cancer cells induce the Warburg effect (aerobic glycolysis) in neighboring stromal fibroblasts. These cancer-associated fibroblasts, then undergo myo-fibroblastic differentiation, and secrete lactate and pyruvate (energy metabolites resulting from aerobic glycolysis). Epithelial cancer cells could then take up these energy-rich metabolites and use them in the mitochondrial TCA cycle, thereby promoting efficient energy production (ATP generation via oxidative phosphorylation), resulting in a higher proliferative capacity. (A and B) provide complementary views of this model. Transfer of pyruvate/lactate from myo-fibroblasts to epithelial cancer cells and endothelia would occur via a mono-carboxylate transporter (MCT), such as MCT1/4. Thus, CAFs and the tumor epithelial cells would be metabolically coupled.

Acknowledgements the W.W. Smith Charitable Trust, and a Career Catalyst Award M.P.L. and his laboratory were supported by grants from the from the Susan G. Komen Breast Cancer Foundation. R.G.P. NIH/NCI (R01-CA-80250; R01-CA-098779; R01-CA-120876; was supported by grants from the NIH/NCI (R01-CA-70896, R01-AR-055660), the Susan G. Komen Breast Cancer Foundation, R01-CA-75503, R01-CA-86072, and R01-CA-107382) and the and the Department of Defense-Breast Cancer Research Program Dr. Ralph and Marian C. Falk Medical Research Trust. The (Synergistic Idea Award). A.K.W. was supported by a Young Kimmel Cancer Center was supported by the NIH/NCI Cancer Investigator Award from Breast Cancer Alliance, Inc., and a Center Core grant P30-CA-56036 (to R.G.P.). Funds were Susan G. Komen Career Catalyst Grant. F.S. was supported by also contributed by the Margaret Q. Landenberger Research grants from the W.W. Smith Charitable Trust, the Breast Cancer Foundation (to M.P.L.). Alliance (BCA), and a Research Scholar Grant from the American This project is funded, in part, under a grant with the Cancer Society (ACS). P.G.F. was supported by a grant from Pennsylvania Department of Health (to M.P.L.). The Department

www.landesbioscience.com Cell Cycle 3999 specifically disclaims responsibility for any analyses, interpreta- Note tions or conclusions. Supplementary materials can be found at: www.landesbioscience.com/supplement/PavlidesCC8-23-Sup.pdf

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