Overactivated MAP and PI3 kinase pathways drive a Warburg effect

Both mutations and oncogene-driven overexpression of GFRs contribute to STS development. Gain-of-function mutations of the GFR KIT or PDGF-Rα drive gastrointestinal stromal tumor (GIST) progression [93]. In the translocation-associated Ewing’s sarcoma (EWS), myxoid liposarcomas (MLS) or alveolar rhabdomyosarcoma (ARMS), the fusion proteins EF, FUS-DDIT3 or PAX3-FOXO1, respectively, enhance IGF1R expression, a major driver of RAS/AKT/mTOR activation [37]. Hyperactivation of the RAS pathway is predictive of a high risk of disease recurrence and impaired overall survival in 30% undifferentiated pleomorphic sarcoma (UPS), a common adult STS [33, 34]. Similarly, the loss of the phosphatase PTEN induces growth-factor independent PI3K/AKT activation that sustains autonomous nutrient uptake in some LMS or MPNST [94]. Accordingly, sarcoma incidence increases in hereditary neurofibromatosis patients with carrying deletions of the RAS negative regulator genes NF1 or NF2 [49, 95]. To investigate the mechanisms of tumor progression in a mouse model, whole-exome sequencing was performed on STS induced by either KrasG12D activation/p53 deletion, 3-methylcholanthrene (MCA) or ionizing radiation [96]. Whereas CNVs were very frequent in radiation-induced STS, MCA-induced tumors showed a high mutational burden, combined with high genomic instability in the absence of p53. Candidate oncogenic drivers affecting MAPK signaling were identified either as mutations of Kras, Nf1 and Hippo effectors (Fat1/4), or as amplification of Kras and Myc in p53 deficient mice, or Met and Yap1 in radiation-induced STS. Mutations in the RAS pathway influence the prognosis of human STS such as DDLPS or pediatric embryonic rhabdomyosarcoma (ERMS) [97]. In the latter, the overactivation of p38 MAPK induces high levels of reactive oxygen species (ROS) that increase the mutation rate [97] and may sensitize tumors to therapies enhancing oxidative stress [98, 99].

RAS- or PI3K/AKT-driven activation increases the expression of glucose importers (GLUT) and of the upstream ATP-consuming glycolytic enzymes hexokinase (HK) and phosphofructokinase (PFK), recently shown to control the glycolytic flow quantitatively [100]. The oncogenic KRAS4A isoform and to lesser extent other RAS isoforms were shown to interact with HK1 on mitochondria (Fig. 3), preventing its allosteric inhibition by glucose-6-phosphate (G6P), thereby enhancing glycolysis [101]. Hypoxia or mutations affecting the RAS pathway modulated the activity of PKM2 or PGK1, two ATP-generating enzymes in the last steps of glycolysis, thus providing them with non-metabolic pro-oncogenic functions [102]. Thus, an increase in the proportion of PKM2 dimers, lacking pyruvate kinase activity, drives PKM2 nuclear translocation where it participates in STAT3 phosphorylation and mek5 gene transcription, driving cell growth [103]. Another study showed that activated ERK phosphorylates PGK1, promoting its association with PIN1 and its translocation into the mitochondria. There, it phosphorylates and activates pyruvate dehydrogenase kinase 1 (PDK1), an inhibitor of pyruvate dehydrogenase (PDH), the checkpoint of pyruvate entry in the TCA cycle [104] (Fig. 3). Globally, these RAS-driven effects reinforce glycolysis over mitochondrial respiration and favor glucose- and glutamine-dependent anabolism as shown in a pancreatic ductal adenocarcinoma (PDAC) model [105, 106]. Several clinical trials are currently based on drugs inhibiting PI3K, AKT, mTOR and ERK signaling in STS (Fig. ​(Fig.44 and Table ​Table22).

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Fig. 4

Integrated view of cues and pathways amenable to pharmacological modulation in STS. This diagram places the different existing therapies in sarcoma according to their therapeutic targets. Panel (A) stratify therapeutic option according to main cellular pathways and table B index the current clinical trial and biomarker available in sarcoma disease

An altered HIPPO pathway induces aerobic glycolysis in STS

Several sarcoma histiotypes re-express genes involved in developmental pathways [107–110], such as HIPPO that controls organ size. Its engagement in intercellular adhesion complexes activates the MST and LATS kinases that phosphorylate the transcriptional factors YAP1 and TAZ, promoting their degradation. In contrast, upon nuclear translocation, YAP/TAZ cooperate with mitogenic effectors and boost proliferation (Fig. 2A). HIPPO interferes with the RAS and PI3K pathways that control cell death induction [111], acting as a tumor suppressor pathway [112]. Through cross-inhibition, MST and AKT differentially regulate the expression level of pro-apoptotic effectors (NOXA, FASL, BIM, TRAIL). In addition, ERK induces the expression of anti-apoptotic effectors (BCL2, BCL-XL, IAP, MCL1) partly via YAP/TAZ activation. Furthermore, in a PDAC model, YAP1 amplification can bypass the need for oncogenic KRAS activation [113]. The amount of translocated YAP/TAZ determines their co-activating potential for TEAD transcriptions factors and thereby the balance of proliferation versus cell death [40].

Loss of MST/LATS or YAP overexpression is pro-tumoral in mice [114, 115], and this pathway is often affected in MCA- or radiation-induced STS [96] and UPS [8]. In transgenic models, altering HIPPO effectors alone or in combination with other deficits augmented STS frequency [41]. An increased YAP/TAZ nuclear staining is predictive of poor survival in UPS, DDLPS and ERMS [42–45, 116]. Two studies investigated how fusion gene products mediate sarcomagenesis through alteration of the HIPPO pathway in mice. One study explored MLS that account for 5–10% STS, among which 90% tumors depend on the product of the FUS:DDIT3 translocation [44]. The authors performed a large-scale RNA interference screen and identified YAP1 as a non-redundant oncogenic driver. In MLS cell lines, FUS:DDIT3 led to a two to threefold increase in expression and co-transcriptional activity of YAP1. Co-immunoprecipitation and immunofluorescence studies revealed a physical interaction of YAP1 with FUS:DDIT3 in the nucleus. Pharmacological inhibition of YAP1 activity inhibited the growth of MLS xenografts. The other study, based on a new transgenic model, showed that doxycycline (DOX)-induced expression of YAP1 in the myogenic MYOD1 cell lineage provoked the development of ERMS through the transformation of activated satellite cells [45]. Retrieval of DOX released a YAP1-dependent differentiation block and reduced tumor formation. Transcriptional profiling of the tumors revealed that YAP1 induces pro-oncogenic effector genes and represses terminal differentiation of myoblasts. In line with these observations, an independent study found no evidence that mutant RAS isoforms were responsible for YAP overexpression in myoblasts [116]. In ARMS, the translocation product PAX3-FOXO1 suppresses HIPPO signaling through overexpression of RASSF4, which inhibits the MST1 kinase. Similarly, this effect is linked to PAX3-FOXO1 co-localization with YAP1 in the nuclei of cancer cells. This chimeric transcription factor cooperates with YAP1/TEAD to induce downstream effectors that trigger IGFs and NF-κB activation, and repress senescence and apoptosis in mesenchymal cells [40, 44]. Therefore, mutations of HIPPO effectors can be oncogenic in STS.

The HIPPO pathway restricts tissue growth and is connected to nutrient cues [117, 118]. Upon glucose starvation, AMPK- and LATS-kinases phosphorylate YAP resulting in its degradation [119]. Furthermore, Wang et al. showed that the phosphorylation level of YAP1 at position S61 is regulated by AMPK, itself recruited to YAP protein complexes in the cytosol of glucose-deprived cells. Addition of glucose was associated with a decrease in YAP phosphorylation and its nuclear translocation, where it interacted with TEAD transcriptional regulators to induce glycolytic genes. This glucose-sensing pathway via YAP and TAZ was required for the full deployment of glucose growth-promoting activity in breast cancer. In addition, glycolysis was required to sustain YAP/TAZ tumorigenic properties [120]. Mechanistically, phosphofructokinase (PFK1) bound to and co-activated the YAP/TAZ transcriptional cofactors TEADs (Fig. 3). In some cancers, the loss of NF2, an upstream negative regulator of HIPPO signaling, simultaneously unleashed YAP/TAZ and SMAD2/3 activation leading indirectly to the induction of aerobic glycolysis via derepression of GLUT, HK2, LDH and MCT genes [121]. Interestingly, NF2 mutations might contribute to the maintenance of rare aggressive sarcomas [122]. This hypothesis was tested in a kidney cancer cell model bearing NF2 mutations [123]. There, DOX-induced expression of shRNA downregulating YAP/TAZ expression provoked the regression of tumors in vivo. YAP/TAZ-depletion induced a substantial decrease in EGFR and AKT phosphorylation, associated with a reduction in glucose uptake, and a switch to glutamine anaplerosis that boosted mitochondrial respiration and ROS production. Under conditions of glucose or glutamine withdrawal, this metabolic shift favored cell death. Whereas restoration of AKT signaling by expression of a constitutively active form of AKT rescued cell proliferation, it did not prevent starvation-induced death. In vivo, YAP/TAZ^low tumors survived due to the engagement of a compensatory lysosome-mediated activation of MAPK signaling. By combining YAP/TAZ and MEK inhibition, tumor growth durably regressed. YAP can also induce aerobic glycolysis through a direct interaction with HIF1α in a hypoxic environment [124, 125]. Therefore, the proglycolytic effect of YAP/TAZ engagement depends on their participation in various nuclear transcriptional complexes. In a muscle-derived UPS model, a combination of epigenetic modulators suppressed YAP1 activity and reduced sarcomagenesis through regulation of metabolism. In this case, YAP1 nuclear translocation was associated with a poor prognosis [126] and its inactivation by epigenetic modulators allowed the restoration of a clock gene-mediated unfolded protein response and muscle differentiation. It also promoted a switch toward lipid catabolism and autophagy, limiting YAP-driven UPS cell growth. The YAP/TAZ inhibitor Verteporfin is currently being tested in Ewing’s sarcoma (EWS) (Fig. ​(Fig.44 and Table ​Table22).

Glutamine and arginine metabolic pathways contribute to STS growth

In a UPS mouse model harboring Kras mutations and p53 deletion in the muscle [59], tumors developed in the hindlimb and metastasized in the lung, as in the human disease. Additional deletion of HIF-2α or its binding partner aryl hydrocarbon receptor nuclear translocator (ARNT) enhanced tumor development. Use of an unbiased pan metabolomics strategy combining LC–MS and stable isotope metabolite tracing revealed a reliance on glutaminolysis for tumors, unlike muscle cells. Accordingly, glutaminase (GLS) inhibitors blocked UPS tumor growth in vivo and are the object of clinical trials in humans (Fig. ​(Fig.44 and Table ​Table2).2). Mechanistically, GLS hydrolyses glutamine to glutamate, which is then dehydrogenated to alpha-ketoglutarate (αKG) by glutamate dehydrogenase GLUD or an aminotransferase (such as PSAT), boosting mitochondrial anaplerosis [127]. In this UPS model, tracing using C¹³- or N¹⁵-labeled glutamine demonstrated that glutamine is a carbon donor for the TCA cycle and a nitrogen donor for aspartate production from oxaloacetate. Aspartate is a crucial carbon source for purine and pyrimidine synthesis and sustains cell growth [128, 129]. Aspartate is also required for the conversion of citrullin into arginine through the activity of the rate-limiting argininosuccinate synthase 1 (ASS1) that initiates the urea cycle. This reaction generates arginine and contributes to the clearance of nitrogenous wastes. ASS1 deficiency has been observed in various cancers, including MFS, due to epigenetic silencing of its promoter [61]. Reexpression of ASS1 inhibited tumor growth and metastases. To investigate how arginine auxotrophy induced by ASS1 deficiency contributed to the progression of tumors, another study used a pegylated arginine deiminase (ADI-PEG20) to deplete arginine pharmacologically [89]. A short-term treatment by ADI-PEG20 applied to LMS cell lines, immediately induced cell proliferation arrest and autophagy. Upon prolonged therapy, cell lines became resistant to ADI-PEG20 due to the reexpression of ASS1 that regenerated arginine. A metabolomic profiling of treated cell lines revealed a reduction in PKM2 levels. In addition, U¹³C glucose tracing studies showed that carbons were shifted away from lactate and citrate production, and reoriented toward serine/glycine synthesis. Analysis of metabolic requirements for growth showed a reduced reliance on glucose and a reinforcement in OXPHOS and glutaminolysis, as an alternative source of TCA cycle intermediates via anaplerosis. In STS, a clinical trial using ADI-PEG20 in combination with Gemcitabine and Docetaxel has been launched (Fig. ​(Fig.44 and Table ​Table2).2). Similarly, targeting glutamine metabolism through GLS inhibition could provoke the lethality of ASS1-deficient cancers and is currently being evaluated in GIST and NF1-mutated cancers (Fig. ​(Fig.44 and Table ​Table22).

Complex metabolic rewiring in Kaposi’s sarcoma

Kaposi’s sarcoma (KS) is caused by a lytic oncogenic herpes virus (KSHV/HHV8), infecting endothelial cell precursors in immunosuppressed individuals. Infection, which is necessary but not sufficient for the growth of KS lesions, leads to the development of a vascular neoplasm associated with cytokine dysregulation driven by the virally encoded G protein coupled receptor (vGPCR). In lytically infected cells, vGPCR induced Rac1/NOX-dependent production of ROS that activated the redox sensitive STAT3 and HIF pathways [130]. Infected cells had increased lactate production and decreased mitochondrial respiration, a phenotype in part attributable to HIF1α activation [131]. Indeed, aerobic glycolysis favored by PKM2 induction sustains the maintenance of KS cells [132] (Fig. 3). Infected cells also exert a paracrine effect on neighboring endothelial cells. Indeed, PKM2 acts as a coactivator of HIF1α reinforcing the production of angiogenic cytokines [131]. Among them, PDGFRA, found phosphorylated in KS-biopsies [133], plays a major oncogenic role. HIF1α also participates in the reactivation of latently infected cells [134]. Upon transformation by KHSV, however, endothelial cells depended on glutamine for proliferation. KHSV also provoked an increase in ASS1 expression, in part through the action of KHSV-encoded miRNAs [62], leading to increased arginine production. Knockdown of ASS1 inhibited cell proliferation and iNOS-dependent, arginine-derived NO production. Treatment of KS cells with a NO donor-activated STAT3 without affecting ROS cell levels. A recent article questioned the relevance of these metabolic changes by comparing 2D versus 3D cultures of KHSV-infected cells [135]. An unbiased metabolomics analysis revealed significant changes in the levels of various non-essential amino acids in 3D cultures. GST-pull down studies showed that the viral K1 protein physically interacted with and activated the pyrroline-5-carboxylate reductase PYCR leading to increased proline production. This phenotype, abrogated by PYCR depletion, promoted 3D spheroid culture and tumorigenesis in nude mice. These results highlight the complex metabolic rewiring that occurs during infection and transformation by KHSV but also the need for appropriate in vitro culture systems to evaluate metabolic adaptation.

One carbon metabolism is overactive in aggressive STS

In Ewing’s sarcoma (EWS), the fusion protein resulting from a single translocation event between the regulatory domain of EWS and the DNA-binding domain of FLI1 behaves as a chimeric transcription factor called EF that enhances IGFR1 activation (Fig. 2A). Transcriptomic studies [60, 64] identified EF as an upstream regulator of PHGDH, PSAT, PSPH and SHMT1/2 genes involved in serine-glycine biosynthesis as well as SLC1A4/5 glutamine transporter genes (Fig. 3). Accordingly, knockdown of EF reduced the proportion of glucose-derived 3-phosphoglycerate reoriented toward serine and glycine synthesis; EWS cell lines were highly dependent on glutamine for growth and survival. Earlier work using a metabolomics approach with isotope labeling had already shown that a large proportion of glycolytic carbon was diverted into serine and glycine metabolism in melanoma; this was due to the amplification of the PHGDH gene [136], also elevated in high-risk EWS patients [100]. Serine or glycine can provide one carbon to tetrahydrofolate initiating the folate cycle. The enhancement of one-carbon metabolism, considered as an integrator of nutrient status [97], boosts the interconnected folate and methionine cycles leading to enhanced NADPH and nucleotide synthesis. NADPH regulates ROS-dependent death and methyl transfer contributes to epigenetic modifications. Knockdown of PHGDH recapitulated the effect of anti-metabolite chemotherapies and had a major effect on cell growth and epigenetic control. Two studies investigated the sensitizing effect of methionine restriction on chemo- or radio resistant models of RAS-driven colorectal cancer and STS, respectively [137, 138]. In the FSF Kras^G12D/+; Tp53−/− STS mouse model, tumor development was triggered by the intramuscular injection of an adenovirus carrying the FlpO recombinase. In these aggressive tumors, only the combination of diet and radiation delayed tumor growth. By combining tumor metabolomics and metabolite tracing with a time-course analysis of data, alterations were observed for nucleotide and redox metabolisms. Interestingly, the consequences of methionine restriction could be detected at the metabolic level when applied to healthy individuals. These results indicated that a targeted dietary manipulation could improve tumor response to therapies.

Metabolic imbalances in STS

Through their evolution, most tumors tend to acquire metabolic features including an increase in nucleotide synthesis [168]. Investigations using PET-FDG uptake in STS patients confirmed the strong glycolytic bias documented in metastasized and poor prognosis STS [169, 170] such as ARMS [171] or ES [172]. However, these studies also revealed the considerable heterogeneity within a given tumor and between different tumor types, suggesting that the Warburg phenotype might be unstable and amenable to pharmacologic control [173]. Whereas the level of oxidative phosphorylation (OXPHOS) varies between tumors (Fig. 1), there is a general correlation between reduced mitochondrial activity, an epithelial-to-mesenchymal transition (EMT) gene signature and a poor prognosis [168]. Some STS tend to exhibit high levels of mitochondrial respiration compared to carcinomas (Fig. 1) [55, 174]. In vitro analysis of OS and RMS cell lines showed differences in the reliance on glycolysis versus respiration of tumors, with ARMS being in general less oxidative than OS or ERMS [175]. The equilibrium between glycolysis and mitochondrial respiration can be affected by various oncogenic alterations and/or metabolic requirements. Accordingly, the receptor tyrosine kinase Her4/ErbB4, an EGFR family member, is upregulated in several cancers including OS [176]. Exploration of xenograft models using untargeted metabolomics and 18F-FDG microPET/CT scan approaches showed that Her4 overexpression boosted glycolysis, glutaminolysis and OXPHOS in tumors. This hypermetabolic phenotype contributed to sustained growth and ATP production while conferring chemoresistance, as also shown in PDAC [177].

The crosstalk between metabolic pathways can also be altered in cancer as discussed in the following examples. Firstly, the upstream reaction committing glucose to glycolysis is catalyzed by phosphofructokinase-1 (PFK-1), itself allosterically inhibited by high ATP levels [178] (Fig. 3). Cancer cells express various PFK isoenzymes [179] such as the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) that produces F2,6-BP, thereby overriding ATP-dependent inhibition of PFK1 [180, 181] (Fig. 3). Reciprocally, an activation of a F1,6-biphosphatase such as FBP1 enhances the gluconeogenic flow and restrains glycolysis [174]. The FBP2 isoform is frequently lost in STS including LPS, FS, LMS and UPS and lower FBP2 mRNA levels correlated with poor survival in LPS [55]. In the latter study, increasing FBP2 expression impaired sarcoma cell growth, through glycolysis inhibition and induction of mitochondrial biogenesis. The latter effect was due to FBP2 nuclear translocation where, independently of its enzymatic activity, it interacted with and inhibited c-Myc-driven transcriptional activation of TFAM, an inducer of mitochondrial biogenesis [182].

Secondly, the maintenance of glycolytic flow requires the regeneration of NAD+ which originates from cytosolic lactate dehydrogenase (LDH) activity and from the malate aspartate shuttle between mitochondria and cytosol [183] (Fig. 2). By regulating NAD+ levels, mitochondrial activity limits glycolysis and consequently the Warburg effect [184]. This suggests that the persistence of mitochondrial activity can be beneficial to tumors. In addition, an LDH activity has been identified in the mitochondria where it catalyzes the aerobic oxidation of lactate into pyruvate. It is thought to contribute to the maintenance or enhancement of OXPHOS in glycolytic cells [185, 186]. Pyruvate oxidation in the mitochondria depends on PDH activity, itself inhibited by PDK. The inhibition of PDK by dichloroacetate (DCA) shifts metabolism from glycolysis to glucose oxidation and boosts ROS production as well as mitochondria-dependent apoptosis in tumors [187]. This effect is exploited in EWS and other tumors where DCA synergizes with apoptosis-inducing drugs such as cisplatin. Manipulating ROS levels appears to be a promising therapeutic approach [188]. Indeed, scavenging mitochondrial ROS (mtROS) induces p53, reduces the cell transforming potential of oncogenic RAS and in some fibrosarcoma (FS) and RMS model cell lines suppresses tumor growth [17, 189].

Thirdly, the level of mitochondrial activity depends on the availability of coenzyme A (CoA) and the acetylated form, AcCoA. CoA synthesis requires the intracellular phosphorylation of pantothenate (or vitamin B5) by pantothenate kinases [190]. Reciprocally, pantothenate derives from the recycling of food-derived or cellular CoA through an extracellular degradative process involving the vanin pantetheinases [191, 192]. Interestingly, a high vanin1 (VNN1) level correlates with a better prognosis in STS patients [66]. Lack of Vnn1 in CDKN2A deficient mice enhanced the proportion of fibrosarcomas compared to that of other cancers. In RAS-driven mouse STS lines, Vnn1 exerted an anti-Warburg effect by enhancing CoA levels and mitochondrial activity to the detriment of glycolysis, and by maintaining cell differentiation.