Aberrant cancer metabolism in epithelial–mesenchymal transition and cancer metastasis: Mechanisms in cancer progression

Accepted Manuscript Title: Aberrant cancer metabolism in epithelial–mesenchymal transition and cancer metastasis: Mechanisms in cancer progression Authors: Run Huang, Xiangyun Zong PII: DOI: Reference:

S1040-8428(16)30226-8 http://dx.doi.org/doi:10.1016/j.critrevonc.2017.04.005 ONCH 2375

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Critical Reviews in Oncology/Hematology

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20-9-2016 24-3-2017 10-4-2017

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Aberrant Cancer Metabolism in Epithelial–Mesenchymal Transition and Cancer Metastasis: Mechanisms in Cancer Progression Run Huang1, Xiangyun Zong1 1

Department of Breast Surgery, Shanghai Jiao Tong University Affiliated Sixth

People’s Hospital. 600 Yishan Road, Shanghai, China. 200233. Correspondence: Xiangyun Zong, MD,PhD. Department of Breast Surgery, Shanghai Jiao Tong University Affiliated Shanghai Sixth People’s Hospital, 600 Yishan Road, Shanghai 200233, China. TEL: +86(21) 24058549, Fax: +86(21) 24058549. E-mail: [email protected]

Abstract Cancer metastasis is a prominent feature of cancer cells, which is responsible for most cancer -associated mortality. Epithelial–mesenchymal transition (EMT) plays an essential role in the initiation and development of cancer metastasis. Studies have shown that EMT can induce cancer metastasis by promoting tumor malignances, reprograming cancer metabolism, and disrupting extracellular matrix. Accumulating evidence has demonstrated that aberrant cancer metabolism can induce EMT through multiple pathological pathways. Conversely, EMT exacerbates dysregulation of glucose metabolism by EMT transcriptional factors such as Snail and Twist. Furthermore, the crosstalk network between aberrant glucose metabolism and EMT synergistically triggers cancer metastasis. Therefore, aberrant cancer metabolism can be used for the prediction, diagnosis, monitoring, and intervention of EMT, as well as cancer metastasis. To conclude, aberrant cancer metabolism plays a key role in the development of EMT and cancer metastasis, suggesting its clinical promise for the management of EMT and cancer metastasis.

Keywords: Cancer metabolism; ; ; , Epithelial–mesenchymal transition, Aerobic glycolysis, Cancer metastasis

1. Introduction Cancer metabolism has been widely regarded as a general hallmark of cancer (Cantor and Sabatini, 2012; Hanahan and Weinberg, 2011). In recent years, mounting evidence has revealed that cancer cells would rewire their metabolism to an anomalous state which favors their survival, progression and metastasis. A rather heterogeneous metabolic profile can be observed between normal cells and cancer cells. And this metabolic heterogeneity may help explain why cancerous cells behave so much distinctively from normal cells phenotypically. In normal cells, when oxygen is sufficient, the Pasteur effect is a predominant feature that almost all of the final products of glycolysis pathway pyruvate flows into the tricarboxylic acid cycle (TCA cycle) and are completely oxidised to carbon dioxide in the mitochondria through oxidative phosphorylation (OXPHOS). Otherwise, under hypoxic circumstances, anaerobic glycolysis will take the place to redirect the glycolytic pyruvate to generating lactate (Tennant et al., 2010). Moreover, in cancer cells, even under normoxia, metabolism is reprogrammed to consume more glucose and produce more lactate, which is well known as the Warburg effect or aerobic glycolysis (Dang, 2007; Vander Heiden et al., 2009). Consequently, an acidic microenvironment is generated around the cancer cells, contributing to tumor growth, invasion and immune evasion (Gatenby and Gillies, 2004; Kraus and Wolf, 1996; Parks et al., 2011). These findings have led to questions about the usefulness of targeting individual signaling molecules as a practical therapeutic strategy. However, it is becoming clear that many key oncogenic signaling pathways converge to adapt tumor cell metabolism in order to support their growth and survival. Furthermore, some of these metabolic alterations seem to be absolutely required for malignant transformation. Cancer metastasis is a prominent feature of cancer cells, which is responsible for most cancer -associated mortality. The phenomenon of epithelial cancer cells losing epithelial characteristics and acquiring mesenchymal characteristics is well defined as epithelial–mesenchymal transition (EMT), which has been regarded as the initiation step of cancer metastasis. During the process of EMT, cancer cells lose their cell-to-cell junctions and cellular polarity under the modulation of multiple signaling pathways, thus attaining increased motilities and invasive phenotype (MartinBelmonte and Perez-Moreno, 2012). Accumulating evidence indicated that this EMT-related program rises as a central driver of tumor malignancy (1). Particularly, cancer stem cells (CSCs) display EMT characteristics, which might favor successful tumor colonization at distant sites, where cancer cells would undergo a reversed process, mesenchymal-epithelial transition (MET). It is noteworthy that dysregulated cellular metabolism would provide support for the basic needs for dividing cancer cells: rapid ATP generation; increased biosynthesis of macromolecules; and tightened maintenance of appropriate cellular redox status (Newsholme et al., 1985; Vander Heiden et al., 2009). To meet these needs, cancer cells therefore alter cellular metabolites ultimately to direct available nutrients into the synthesis of new biomass while maintaining adequate levels of ATP for cell survival. Many similar alterations are also observed in rapidly proliferating normal cells, in which they represent appropriate responses to physiological growth signals as opposed to constitutive cell autonomous adaptations (Benjamin et al., 2012; Levine and Puzio-Kuter, 2010). In the case of cancer cells, these adaptations must be implemented in the stressful and dynamic microenvironment of the solid tumor, where concentrations of crucial

nutrients such as glucose, glutamine and oxygen are spatially and temporally heterogeneous (Tatum et al., 2006). The link between altered metabolism and cancer metastasis is not new, as many observations made during the early period of cancer biology research identified several transcriptional factors as co-activators in both processes. In this review, we propose the idea that there exists a reciprocal effect on cancer metabolism and EMT. Cancer metabolism drives EMT and conversely, EMT reprograms cancer metabolic profile. Both biological phenomena are closely associated with malignant cancer behaviors such as growth advantage and invasive ability.

2. Acidic Microenvironment Contributes to Cancer Metastasis A prominent feature of tumor microenvironment is extracellular acidosis, which results from the terminal glycolytic metabolite lactate and a series of proton exchangers anchoring on the cell membrane. Mounting evidence has revealed that acidic microenvironment confers a growth advantage and metastatic advantage to cancer cells. As cancer cells have a higher glycolytic rate than normal cells, an elevated glucose influx is necessary for cancer cells to product excessive lactate (Mookerjee et al., 2015). Thus, aberrant glycolytic metabolism imposes an increased acid load on tumor cells. Moreover, hypoxia-inducible factor 1α (HIF1α) and Myc cooperate to transcriptionally promote the expression of multiple glycolytic enzymes, all of which synergize to drive glucose influx and lactate production (Gordan et al., 2007; Semenza, 2010). On the other side, the acid clearance rate outside tumor cells is at a low level (Dhup et al., 2012; Spugnini et al., 2015), thus generating the low pH tumor microenvironment. Lactate is produced in the intracellular plasma and lactate deriving from glycolysis is exported to the extracellular matrix (ECM) by the proton-linked monocarboxylate transporter 4 (MCT4) in highly glycolytic cells (Dimmer et al., 2000; Manning Fox et al., 2000; Ullah et al., 2006). Moreover, MCT4 is a target gene of HIF1α (Semenza, 2010; Ullah et al., 2006). As tumor cells usually locate in a hypoxic environment, the hypoxia-induced HIF1α will transcriptionally upregulate the expression of MCT4, which is favorable to the output of intracellular lactate to the ECM. As extracellular lactate accumulates, microenvironment acidosis forms. In addition to the lactate transporter, multiple proton exchangers, such as sodium-proton exchanger 1 (NHE1, a member of SLC9 gene family of Na+/H+ antiporters) (Donowitz et al., 2013; Lee et al., 2010), anion exchanger 2 (AE2, a member of SLC4 gene family of Na+-independent chloride–bicarbonate exchangers) (Alper et al., 2002) and sodium bicarbonate transporter 1, function as extracellular acidosis drivers to decrease extracellular pH (pHe). The first process of tumor cell metastasis is morphological alteration (the initial step of EMT). During this process, cancer cells are compressed and elongated to form lamellipodia, where these pHe regulators preferentially expressed (Klein et al., 2000; Lagana et al., 2000). This phenomenon partially suggests that low pH might be required for cancer cell metastasis. Solid tumors usually develop an acidic environment, where cancer cells adapt to survive in this radical circumstance whereas periphery normal cells vanish. As discussed above, extracellular acidosis is a result of aberrant cancer metabolism. Conversely, acid load would induce a series of alterations of cancer cells to promote cancer progression. Low pHe has been found to be favorable

to mutagenesis, chromosomal abnormalities and genome instability (Yuan and Glazer, 1998; Yuan et al., 2000). Moreover, it has been reported that low pH triggers cell apoptosis through increased caspase activity (Park et al., 1999) or p53-dependent pathway (Williams et al., 1999), thus leaving the apoptosis-resistant cancer cells to form clones and primary lumps. Besides, drug resistance might also be associated with low pHe (Wojtkowiak et al., 2011). ECM is a vital histological barrier preventing cancer cells from metastasizing to a distant region. Transforming and remodelling of ECM are necessary for tumor cell invasion. In this context, the ECM components tend to be transformed and degraded to facilitate cancer invasion. Low pHe plays a critical role in activating cancer cells to secrete cysteine proteases such as Cathepsins B and L (Buck et al., 1992; Maciewicz et al., 1990), matrix metalloproteinases (MMPs) (Kato et al., 2005; Kato et al., 2007) and glycosidases (Bourguignon et al., 2004). All of these enzymes cooperate to degrade collagens, laminins, fibronectins or proteoglycan in the ECM, creating a loose microenvironment surrounding cancer cells in favor of their invasion and metastasis. Except for the ECM degradation, angiogenesis is also promoted by a low pHe environment. As neo-formed blood vessels extend into the neoplasm, detached tumor cells acquire an increased chance of intravasation and metastasizing. This process, which is mainly through the activation and release of vascular endothelial growth factor (VEGF)-A (Fukumura et al., 2001; Shi et al., 2001; Xu et al., 2002) and interleukin 8 (IL-8) (Shi et al., 2000; Xu and Fidler, 2000), has been proved in various cancer cell lines. Furthermore, extracellular acid burden has been found to repress lymphocyte activity and proliferation, thereby promoting the immune evasion of tumor cells (Bosticardo et al., 2001; Kellum et al., 2004; Lardner, 2001). Moreover, previous acidic priming in cancer cells has been reported to enhance their capability of successful colonizing in the lungs of nude mice, which suggests a potential effect of acidosis on a macroscopic metastasis (Riemann et al., 2014; Rofstad et al., 2006). As discussed above, acidosis is taking effect in the whole process of metastatic dissemination from phenotypic alterations of primary tumor to successful colonization of secondary tumor. All of these biological processes synergize to exert metastatic advantage on cancer cells. However, acid accumulation is the result of abnormal cancer metabolism. Due to the predominant role of aerobic glycolysis in cancer cells, excessive lactate is produced and then exported to the ECM by the transporter MCT4, contributing to the acidosis formation. Targeting MCT4 has been proved in multiple cancer types to be an efficient strategy of inhibiting tumor migration and metastasis (Gallagher et al., 2007; Izumi et al., 2011). This prompts us to propose the idea that Warburg effect might be the primary motivation of metastasis due to the low pHe (Fig. 1).

3. Aberrant Cancer Metabolism Promotes EMT In general, reprogrammed cancer metabolism is indispensable for the malignant biological processes of cancerous cells. Multiple studies have revealed that aberrant cancer metabolism, particularly, the enhanced aerobic glycolysis, not only provides tumor cells with ample energy and intermediate glycolytic metabolites for rapid tumor growth, but it also imparts cancer cells with anoikis resistance and metastatic advantage. In solid tumors, because of poor vascularization,

HIF1α is stabilized and accumulated, which upregulates the expression of a series of glycolytic enzymes (Gordan et al., 2007). Consequently, in the meantime of promoting aerobic glycolysis, EMT phenotype is induced and metastatic capability is heightened (Fig. 2). Here in this review, we are going to make a short review of several critical EMT-inducing glycolytic enzymes. 3.1. Aldolase A Aldolase A (ALDOA) is a glycolytic enzyme which catalyzing of reversible conversion of fructose-1,6-bisphosphate (F1,6BP) to glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). It has been demonstrated that ALDOA is associated with the metastatic phenotype in multiple cancer types, such as lung squamous cell carcinoma (Du et al., 2014), hepatocellular carcinoma (HCC) (Coulouarn et al., 2009), and pancreatic carcinoma (Ji et al., 2016). In the work of Coulouarn et al. (Coulouarn et al., 2009), they revealed that ALDOA’s expression was negatively correlated with miR-122’s expression and positively correlated with invasive phenotype in HCC. Besides, other markers (HIF1α, VEGFA and Vimentin) implicated in cancer metastasis were also upregulated due to the loss of miR-122. Nevertheless, how those EMT markers and ALDOA interact with each other was not explained in the article. Recently, Ji et al. (Ji et al., 2016) provided us evidence that ALDOA acting as an inducer of EMT with upregulating Ncadherin and Vimentin and downregulating E-cadherin. However, the mechanisms of how ALDOA regulates those EMT markers still need further investigations. 3.2. Pyruvate Kinase-2 Pyruvate Kinase-2 (PKM2) is the final rate-limiting enzyme of glycolysis, which has been intensely studied, catalyzing the last irreversible biochemical reaction of phosphoenolpyruvate (PEP) to pyruvate. PKM2 is reported to be required for aerobic glycolysis and tumor growth advantage (Christofk et al., 2008). Overexpressed PKM2 accompanies by the aggressive clinicopathological features and adverse patient prognosis of various types of carcinomas originated from, for instance, tongue (Yuan et al., 2014), esophagus (Zhang et al., 2013), liver (Chen et al., 2015; Liu et al., 2015) and cervix (Zhao et al., 2015). Apart from the metabolic function of PKM2, it can translocate into the nucleus in response to variable signals, of which, EGF (epithelial growth factor)-ERK (extracellular signal-regulated kinase) pathway is the best studied (Fan et al., 2014; Yang et al., 2011; Yang et al., 2012). Translocated PKM2 is required for the EGFinduced EMT during which PKM2 regulates the transcriptional activity of β-catenin promoting increased expression of SNAIL and Vimentin, and decreased expression of E-cadherin (Fan et al., 2014). Besides, PKM2 can interact with TGIF2 (TGFβ–induced factor homeobox 2), which recruits the HDAC3 (histone deacetylase 3) to the promoter of E-cadherin and represses its expression (Hamabe et al., 2014). Moreover, STAT3, a member of signal transducer and activator of transcription (STAT) protein family, was reported to be phosphorylated by PKM2 and downstream genes relating to EMT phenotype were consequently modulated promoting cancer cell migration and motility (Gao et al., 2012; Yang et al., 2014). All of these studies strongly proved PKM2 as a potent EMT inducer with its nonenzymatic activity. And targeting PKM2 using pharmacological and RNA interfering technology has also been found to reprogram cancer cell metabolism towards OXPHOS and suppress tumor invasion and metastasis (Giannoni et al., 2015; Sun et al., 2015).

3.3. Lactate Dehydrogenase A Lactate dehydrogenase A (LDHA) is responsible for the final process of conversing pyruvate to lactate. Because of the truncated glucose flux from aerobic glycolysis to TCA cycle, plenty of pyruvates tend to convert to lactate due to the enzymatic activity of LDHA. As discussed previously, accumulated lactate is secreted into the ECM and generates an acidic microenvironment, which is favorable to tumor growth and metastasis. Plenty of studies demonstrated that attenuation of LDHA expression represses the invasive and metastatic ability of cancer cells (Arseneault et al., 2013; Sheng et al., 2012; Xian et al., 2015). Interestingly, LDHA knockdown could induce increased E-cadherin expression and decreased focal adhesion kinase (FAK), MMP2 and VEGF, which are vital alterations for EMT (Arseneault et al., 2013). Despite the increased pHe due to LDHA attenuation, glucose flux is redirected to TCA cycle for OXPHOS, which stimulates mitochondrial respiration and induces reactive oxygen species (ROS) generation (Arseneault et al., 2013; Fantin et al., 2006; Le et al., 2010; Sheng et al., 2012). Increased ROS exerts an inhibitive effect on tropomyosin-mediated cell migration and cytoskeletal remodelling, which is adverse to the EMT phenotype and metastasis (Arseneault et al., 2013). All of these indicate that LDHA is critical for cancer progression and metastasizing, and that LDHA might be a potential and potent therapeutic target for cancer patients. 3.4. Pyruvate Dehydrogenase Kinase-1 Pyruvate dehydrogenase kinase-1 (PDK1) functions as an inhibitor of pyruvate dehydrogenase (PDH), a key rate-limiting enzyme for pyruvate conversion to acetyl-coA. PDH, which diverts glucose flux from glycolysis to OXPHOS, plays a vital linking role between these two biological processes. In a study of metastatic breast cancer, PDK1 was reported to be required for successful liver metastasis in which conversion of pyruvate to lactate is increased and oxidative metabolism is concomitantly reduced (Dupuy et al., 2015). Suppressed mitochondrial metabolism can lead to decreased production of ROS, which is responsible for apoptosis resistance of cancer cells (Sutendra et al., 2013). Sutendra et al. (Sutendra et al., 2013) revealed that HIF1α signaling and angiogenesis, both of which are critical malignant processes of cancer metastasis, can be repressed by mitochondrial activation due to PDK1 inhibition, conversely confirming the fatal role PDK1 in tumor progression. Except for the discussed enzymes above, many other glycolytic enzymes such as alphaenolase (ENO1) have also been studied. Attenuation of ENO1 would suppress the expression of mesenchymal cell markers, such as Vimentin, Snail, and N-Cadherin by inactivating PI3K/AKT signaling pathway, thus reverse the EMT phenotype of cancer cells (Fu et al., 2015; Song et al., 2014). Aberrant cancer metabolism possesses altered enzyme expression and activity, which provides cancer cells sufficient energy and metabolites for rapid biosynthesis (DeBerardinis et al., 2008; Vander Heiden et al., 2009). With the abnormal metabolic profile, a malignant phenotype of elevated motility, invasion and metastasis ability is concomitantly induced. In spite of the research above of exploring the kinship between the metabolic profile and EMT phenotype, the mechanisms of cancer metabolism regulating EMT still remains to be fully investigated.

4. EMT conversely Regulates Cancer Metabolism EMT program occurs in normal embryogenesis and wound healing, allowing epithelial cells convert into mesenchymal derivatives to gain the ability of migration and invasion (De Craene and Berx, 2013; Eastham et al., 2007; Gupta and Maitra, 2016). Accumulating evidence indicated that this EMT-related program rises as a central driver of tumor malignancy (De Craene and Berx, 2013). This physiological phenotypic shift is orchestrated by a cohort of transcriptional factors (EMT-TFs), among which the most important ones are the Snail, Twist, Zeb, and Fox family (Ye and Weinberg, 2015). Many researches have been focusing EMT on cancer metastases and its crucial role in generating cancer stem-like cells since it was first defined in 1982 (Thiery, 2002). Intriguingly, several recent studies have connected activation of EMT program to malignant cancer metabolism in various cancers (Fig. 3). 4.1. Snail The transcriptional factor Snail is a central driver of EMT and its expression correlates with metastasis and poor clinical prognosis of many malignancies, by directly binding to the promoter of E-Cadherin (Batlle et al., 2000). Wnt/Snail signal was found to inhibit mitochondrial respiration through repressing the activity of COX (cytochrome C oxidase), the terminal enzyme of the mitochondrial respiratory chain, and consequently induce the glycolytic switch (Lee et al., 2012). Moreover, Snail was recently discovered to regulate glucose metabolism. Fructose-1,6bisphosphatase (FBP1) is a rate-limiting enzyme in gluconeogenesis, which catalyzes the splitting of F1,6BP into fructose 6-phosphate (F6P) and inorganic phosphate. FBP1 is downregulated in cancer cells and restoration of FBP1 suppresses glucose uptake, glycolysis and lactate generation, and meanwhile increases mitochondrial oxygen consumption as well as ROS production (Liu et al., 2010). Dong et al. identified FBP1 as a direct target of Snail. Snail-G9a-Dnmts (DNA methyltransferases) recruits a series of chromatin-modifying enzymes to the FBP1 promoter, along with a dramatic increase of H3K9me2 (dimethylation at lysine 9 of histone 3) and decrease of H3K9ac (acetylation at lysine 9 of histone 3), in turn causing CpG methylation of the promoter and formation of a constitutive heterochromatin that is resistant to transcription activation (Dong et al., 2013). Loss of FBP1 triggers glycolytic reprogramming, with increased lactate generation and reduced oxygen consumption. Taken together, Snail facilitates the metabolic shift towards glycolysis (Dong et al., 2013). 4.2. Twist Twist is a highly conserved transcription factor that belongs to the basic helix-loop-helix (bHLH) family of proteins (Yang et al., 2008). It acts as a regulator of various processes including early development, apoptosis of cancer, and osteoblast differentiation as well as inducing EMT. Suppression of Twist in a highly metastatic mammary carcinoma cell line reduced the frequency of lung metastases in a mouse model of breast cancer (Yang et al., 2004). Twist silencing also reduced the number of circulating tumor cells in this model without affecting anchorageindependent growth and survival, indicating that Twist has critical roles in cancer cell invasion and intravasation that may be exploited therapeutically (Yang et al., 2004). Moreover, researchers revealed that Twist is a direct target of HIF1α, which bounds to the hypoxia-response element

(HRE) in the Twist proximal promoter (Yang et al., 2008). Yang L et al. showed that enzymes that modulate metabolism reprogramming, like LDHA, PKM2, HK2, G6PD, are upregulated in Twistoverexpressing cancer cells, indicating that Twist could trigger energy metabolism reprogramming in cancer cells (Yang et al., 2015). Given the pleiotropic functions of EMT activation in driving cancer progression and the growing reports of its association with various metabolic types, it is plausible that the two hallmarks of cancer may work coordinately to promote tumor progression.

5. Crosstalk Network between EMT and Cancer Metabolism EMT is a highly conserved program initially characterized in embryonic development necessary for orchestrating distant cell migration (Eastham et al., 2007). A great deal of researches in cancer has demonstrated a critical role for EMT in the initial stages of tumorigenesis and later during tumor invasion. EMT transcription factors such as Snail, Twist, and Zeb are master regulators aberrantly overexpressed in many malignancies(De Craene and Berx, 2013). Recent evidence proposes that EMT-related transcriptomic alterations correlate with metabolic reprograming in cancer. Metabolic alterations may allow cancer to adapt to environmental stressors, supporting the irregular molecular demand of rapid proliferation of cancer cells (Cairns et al., 2011). Moreover, it is demonstrated that the expressions of key signaling pathways in EMT are also main regulator in dysregulated metabolic types (Friedl and Alexander, 2011). Here, we will highlight three most important signaling pathways involved in EMT and metabolism (Fig. 4). 5.1. HIF1α Most solid human tumors contain regions of hypoxia or anoxia that may lead to a negative clinical prognosis for the cancer patient owing to local relapse and systemic metastases (Tatum et al., 2006). Normally hypoxia occurs as a result of unstable tumor vasculature (Blais et al., 2006). It is a common concept that hypoxia can drive and maintain genetic instability. The most aggressive manifestation of tumor progression is the development of distant metastases. In most cancer types, patients with hypoxic primary tumors are more likely to relapse locally as well as at distant sites, regardless of their initial treatment. Cells reprogram metabolism towards increased glycolysis and suppressed oxidative phosphorylation at the circumstances of hypoxia. HIF1α stands as the prominent determinant of this metabolic shift and regulates nearly the expression of all the enzymes in the glycolytic pathway (Chi et al., 2006; van der Mijn et al., 2016; Vengellur et al., 2003), as well as the glucose transporters (GLUTs) (Chen et al., 2001), which are responsible for glucose uptake in cancer cells. As increased levels of pyruvate in hypoxia tumor cells, the enzyme PDK1 guided pyruvate to be excluded from mitochondria. Recently PDK1 is determined as a direct transcriptional target of HIF1α. PDK1 phosphorylate and inactivate PDH, the mitochondria enzyme pyruvate dehydrogenase, prevent pyruvate from fueling the mitochondrial tricarboxylic acid cycle, hence reduce mitochondrial oxygen consumption and prevent the excessive production of ROS (Lu et al., 2008). LDHA converts pyruvate to lactate to further glycolysis, which is also a direct target of HIF1α (Firth et al., 1995).

The key EMT transcription factors, including Snail, Twist, Zeb1, have been demonstrated to be directly regulated by HIF1α (Peinado et al., 2007; Yang and Wu, 2008). Subsequently, these transcription factors bind to the promoters of E-cadherin to modulate metastasis. HIF1α is also shown to mediate the expression of MMPs to induce tumor metastasis (Lin et al., 2008). Evidence from several studies indicates that HIF1α regulates angiogenesis by directly targeting VEGF, which plays a crucial role in tumor vascularization (Colombo et al., 2016; Coulon et al., 2010; Polet and Feron, 2013). Given that hypoxia arises in most tumors, HIF1α adapts cells to low oxygen environment by activating genes involved in glycolytic metabolism, cell proliferation, metastasis, angiogenesis, and cell apoptosis. It would be a therapeutic benefit to characterize the cellular response to hypoxia, and HIF1α would be a promising target for cancer treatment. 5.2. MYC The proto-oncogene Myc lies at the crossroads of many growth-promoting signal transduction pathways involved in the regulation of cell proliferation, apoptosis, differentiation, cell adhesion and energy metabolism (Adhikary et al., 2005; Dang, 2012). Recent study shows that epigenetic modifier Jumonji domain-containing protein 6 (JMJD6) cooperates with Myc to enhance cellular transformation and tumor metastasis (Aprelikova et al., 2016). The other one demonstrates that Myc interacts with Sterol regulatory element-binding protein-2 (SREBP-2) transcription factor to promote stem cell-like properties and metastasis in prostate cancer (Li et al., 2016). Further studies revealed interactions between Myc and HIF1α in controlling tumor metastasis and metabolism (Doe et al., 2012). Overexpression of Myc stabilizes HIF1α and enhances HIF1α accumulation under hypoxic conditions. Myc activates glycolysis through transcriptional induction of various glycolytic enzymes (He et al., 2015; Wahlstrom and Henriksson, 2015). He at al. provided evidence from clinical and mechanistic researches that the Myc-LDHA axis is responsible for aerobic glycolysis and tumor progression in pancreatic cancer (He et al., 2015). Recent experiments using double transgenic mice expressing Myc in a doxycycline-dependent manner specifically in hepatocytes revealed that overexpression of a multitude of glycolysis genes in tumors was abrogated by switching off Myc expression (Carroll et al., 2015). Myc not only induces genes that domain the glucose to lactate shift, but also links EMT via activating miR-9 (Khew-Goodall and Goodall, 2010; Ma et al., 2010). Liu M et al. discovered that Myc raised a repressed E-cadherin and an increased Vimentin expression at the posttranscriptional level through miR-9 (Liu et al., 2013). Myc is also shown to contribute to neoplastic transformation of susceptible cell lines by down-regulating genes that control cytoskeleton and cell adhesion (Levens, 2003). 5.3. FOXOM1 Forkhead box protein M1 (FOXM1) is an oncogenic transcription factor belonging to the Forkhead transcription factor superfamily. An increasing number of studies have revealed deregulation of FOX family members is associated with progression of malignant transformation. FOXM1 plays essential roles in the regulation of cell differentiation, angiogenesis and metastasis

by directly promoting its target gene expression and networking with other factors (Koo et al., 2012). Several studies have revealed that FOXM1 regulated the activity of MMP2 and MMP9, which are consistent with tumor invasion, metastasis and angiogenesis, in many malignancies (Ahmad et al., 2010; Dai et al., 2007; Uddin et al., 2011). MMP2 is a direct target of FOXM1, while MMP9 is indirectly targeted via its downstream target JNK1 (c-jun N-terminal kinase 1) (Wang et al., 2008). Moreover, FOXM1 was shown to directly bind to the promoter of VEGF to govern the angiogenic switch (Karadedou et al., 2012). Researchers also found evidence that FOXM1 participates in the regulation of metabolism. In leptin-knockout mice, overexpression of FOXM1 was related to elevated consumption of glucose (Davis et al., 2010). Cui et al. found that FOXM1 directly bound to the promoter region of LDHA, and thus promoted the aerobic glycolysis and metastasis in pancreatic cancer cells (Cui et al., 2014). Another study showed that FOXM1-LDHA axis signaling converted gastric cancer into glycolytic phenotype and promoted its progression (Jiang et al., 2015). FOXM1 regulates the expression of glycolytic enzymes at the transcriptional level and reprogrammed glucose metabolism towards aerobic glycolysis (Wang et al., 2016). Collectively, there is overwhelming evidence that FOXM1 transcription factor plays a central role in cell invasion, metastasis, metabolism reprogramming, which are essential in tumorigenesis. Therefore, FOXM1 would be a promising target for the treatment of cancer patients.

6. Concluding Remarks The collection of advances made in the understanding of tumor metabolism and EMT in recent years has not only afforded us a better understanding of tumor biology, but as well, has provided further comprehension in cancer metastasis. Moreover, numerous studies have illustrated that there exists a reciprocal effect between metabolic adaptations and morphological alterations in cancer progression. Anomalous cancer metabolism adapts to adverse microenvironment with increased glucose uptake and excessive lactate production, thus generating the low pHe environment. Lactate is the final product of aerobic glycolysis, which involves in nearly each critical step of tumor metastasis. Nevertheless, metastatic advantage is imparted not only by abundant lactate, but also by the process of aerobic glycolysis, during which glycolytic enzymes, through their metabolic or nonenzymatic activities, mediate the EMT formation of cancerous cells. Accordingly, we propose that there is no sequencing between acidosis and glycolytic enzyme activity, whereas they synergize with each other to induce the EMT phenotype and distant metastases. As discussed above, aberrant cancer metabolism promotes EMT and metastatic ability. Recent studies have revealed that EMT is able to conversely modulate cancer metabolism. Typically, HIF1α, MYC and FOXM1 act as linking molecules regulating both biological processes, concomitantly conferring cancer cells the malignant phenotype. In this review, we firstly propose the mutual effect between cancer metabolism and EMT, which drives the invasive capability of malignant tumors. Deciphering the interplay between cancer metabolism and EMT that together contribute to the malignant phenotype in a given setting may serve as the critical factor in

determining therapeutic targets that enable maximal drug efficacy with minimal deleterious effect on normal cells. Although various research has been implicated in the exploration of metabolic profile and invasive phenotype, further studies still need to be performed to decipher the underlying mechanisms which drive these two features of cancer.

Conflict of interest The authors declare that they have are no conflicts of interest.

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Figure Legends: Fig. 1 Acidic microenvironment derived from cancer cell metabolism induces the EMT phenotype and cancer metastasis. HIF1α and Myc, acting as positive regulators of cancer metabolism, promotes aerobic glycolysis, which produces plenty of lactate. Lactate is exported to the ECM by MCT4. Besides, elevated proton transporters transfer H+ to the ECM. Accumulated lactate and H+ generate an acidic microenvironment, which stimulates a series of changes of cancer cells and ECM, facilitating EMT and metastasis. Epithelial cancer cells undergo mutagenesis, chromosomal abnormalities and genome instability due to the low pHe environment, and transform into mesenchymal cancer cells. Moreover, low pHe is responsible for ECM degradation and blood vessel growth. All of these bioprocesses contribute to the intravasation into blood capillaries and successful colonization at distant sites.

Fig. 2 Enhanced aerobic glycolysis favors EMT. Aerobic glycolysis is driven by glycolytic enzymes. While regulating cancer metabolism, glycolytic enzymes promotes the mesenchymal transition of cancer cells. Some enzymes are involved in the loss of epithelial markers and gain of mesenchymal markers. Aerobic glycolysis is predominant in cancer cells, which produces excessive terminal product lactate and exerts an inhibitive effect on OXPHOS. Therefore, ROS production declines, which drives cell migration and cytoskeletal remodeling. This facilitates EMT phenotype formation and distant metastasis.

Fig. 3 The EMT transcription factors drive tumor to a mesenchymal phenotype through mediating metabolism reprogramming. Snail suppresses OXPHOS via directly binding to FBP1, leading to a decreased ROS production and EMT transition. Twist facilitates the expression of glycolytic enzymes, LDHA, HK2, PKF2, G6PD, to shift cancer cells to aerobic glycolysis, and thus promotes the EMT transition of cancer cells.

Fig. 4 HIF1α, Myc and FOXM1 signaling on aerobic glycolysis and EMT. HIF1α, Myc and FOXM1 transcriptionally activate GLUTs, PKM2, LDHA, etc. to convert oxygen phosphorylation into aerobic glycolysis. HIF1α, Myc and FOXM1 directly target EMT transcription factors, Snail, Twist, Zeb1 to facilitate distant metastasis. Enzymes that are responsible for the glycolytic phenotype can regulate EMT-TFs directly or indirectly. On the other hand, the EMT transcription factors can also effect the metabolism shift of cancer cells. All together, these two pathways interact to promote tumor progression and metastasis.

Cancer Cell

Glucose

HIF1α Glycolytic enzymes

Aerobic glycolysis↑

Myc

H+

Lactate Proton Exchanger MCT4 Lactate

H+

Colonization

Low pHe

Epithelial cancer cell

ECM

Mutagenesis Chromosomal abnormalities Genome instability Anti-apoptosis Cathepsins B and L MMPs Glycosidases

VEGFA IL-8 Cancer cell

Intravasation Degradation

Blood vessel growth

Blood vessel

Normal cell

Mesenchymal cancer cell

Aerobic Glycolysis

PKM2

Glucose

STAT3

β-catenin HDAC3

G6P

F6P ALDOA DHAP

F1,6BP

N-cadherin↑ Vimentin↑ E-cadherin↓

PI3K/AKT pathway

EMT

G3P Cell migration Cytoskeletal remodeling 2PG ENO1

PDK1

PEP PKM2 Pyruvate

PDH

Acetyl-CoA

OXPHOS

ROS

LDHA Lactate Low pHe

FBP1

EMT Snail

Twist

OXPHOS

Glycolytic enzymes

ROS

Aerobic glycolysis

HIF1α Myc FOXM1

Aerobic glycolysis

EMT

GLUTs PKM2 LDHA ...

Snail Twist Zeb1 ...

Malignant phenotypes Distant metastasis