Positioning determines function: Wandering PKM2 performs different roles in tumor cells

Short for pyruvate kinase M2 subtype, PKM2 can be said of all‐round player that is notoriously known for its metabolic involvement in glycolysis. Holding a dural role as a metabolic or non‐metabolic (kinase) enzyme, PKM2 has drawn extensive attention over its biological roles implicated in tumor cells, including proliferation, migration, invasion, metabolism, and so on. wandering PKM2 can be transboundary both intracellularly and extracellularly. Specifically, PKM2 can be nuclear, cytoplasmic, mitochondrial, exosomal, or even circulate within the body. Importantly, PKM2 can function as an RNA‐binding protein (RBP) to self‐support its metabolic function. Despite extensive investigations or reviews available surrounding the biological roles of PKM2 from different angles in tumor cells, little has been described regarding some novel role of PKM2 that has been recently found, including, for example, acting as RNA‐binding protein, protection of Golgi apparatus, and remodeling of microenvironment, and so forth. Given these findings, in this review, we summarize the recent advancements made in PKM2 research, mainly from non‐metabolic respects. By the way, PKM1, another paralog of PKM2, seems to have been overlooked or under‐investigated since its discovery. Some recent discoveries made about PKM1 are also preliminarily mentioned and discussed.

distinctively different from these earlier reviews, whatever content or angle from which PKM2 is narrated.We lay out the content mainly from the aspect of the wandering PKM2 can be transboundary both intracellularly and intracellularly, to elaborate on wandering PKM2 plays different roles to suit the ficke microenvironment, which appears to have been seldom brought up by those earlier review papers published.Some similar content published already elsewhere will not be recapitulated here any longer.An overview outlining all the possible positions PKM2 can reach intra-or extracellularly based on literatures available was provided here (Figure 1) to serve as a big picture for readers to better aid in understanding the role of wandering PKM2 in tumor cells.

| RNA-BINDING PROTEIN (RBP)
As its name suggests, RBP is a kind of protein that binds with RNA.
Having a variety of regulatory, structural, and catalytic roles, functional RNA interacts with proteins to conduct functions in the cell, such as processing, transport, translation, RNA stabilization, modification, and localization (Hashimoto & Kishimoto, 2022;Qi et al., 2022;Smith & Costa, 2022).Examples supporting these abound.One of these is that RNA interacts directly with proteins to form ribonucleoprotein particles (RNPs) (Faoro & Ataide, 2014) in the posttranscriptional regulation of gene expression.Required for recognition of specific sequence elements present in RNA to control the function played by RNA, RNPs whose biogenesis (Woodson et al., 2018) must be carried out with high fidelity in that incorrect formation of RNP or abnormal expression of RBP can bring about genetic disorders that may give rise to disease, including cancers (Kashikar et al., 2022;Lu et al., 2022).Consequently, RBPs are pivotal for the regulation of RNA in the pathogenesis of cancer.In turn, some F I G U R E 1 Wandering PKM2 can be nuclear, cytoplasmic, mitochondrial, and exosomal.Cytoplasmic PKM2 is always tetrameric, whereas nuclear PKM2 is dimeric.Dimeric PKM2 has a protein kinase effect, while tetramic PKM2 is metabolic and participates in aerobic glycosis.

Highlights
Collating all the literatures related to PKM2, we found the following points that have rarely been summarized: • PKM2 can be cytoplasmic, nuclear, mitochondrial, exosomal even circulating.
• PKM2 can protect the Golgi apparatus.
• PKM2 can modulate the immune checkpoint expression.functional RNAs are also indispensable for the role, stability, and maintenance of RBPs.Studies performed on cytoplasmic PKM2 revealed that PKM2 is an RBP whose metabolic function requires the interaction and engagement of some RNAs, which will be illustrated in the following section.

| CYTOPLASMIC PKM2 MOONLIGHTED AS AN RBP
Mounting evidence has been presented revealing that PKM2 can moonlight as an RBP, playing a certain role in tumor cells.Collated the current literature available regarding RNAs that were able to bind directly with PKM2, the majority of which were identified as long noncoding RNAs (lncRNAs).In terms of function, these lncRNAs tend to either work as scalfolds to stabilize PKM2 or as PKM2 splicing regulators that mediate the switching between PKM1 and PKM2 splicing.For instance, in a very recent article identifying PKM1/2 as an RNA-binding protein (Lan et al., 2020), using mass spectrometry and parallel reaction monitoring (PRM) assays, the authors identified PKM1/2 as an RBP that can directly bind with lncRNA GACAT2.
Subsequent functional experiments demonstrated that upregulated GACAT2 led to the increased expression of PKM2, which eventually resulted in enhanced pyruvate kinase activity and increased translocation of PKM2 into mitochondria.lncRNA CCAT1 is another long noncoding RNA identified that can directly interact with PKM2 in the context of osteosarcoma (Pu et al., 2022).In addition, another recent study conducted in the setting of hepatocellular carcinoma unveiled that lncRNA MNX1-AS1 served as a scaffold to buttress the interactions between PKM2 and importin α5 (Wu, Wang, Yao, et al., 2022), hence promoting the translocation of PKM2 into the nucleus to fuel the Warburg effect.As an aside, PKM2 mRNA was also subject to RNA modification.A case in point is that 5-methylcytidine (m5C) modification can occur on PKM2 mRNA.Once m5C took place, ALYREF (Wang, Zhu, et al., 2021) was shown to be able to stabilize PKM2 mRNA and bound to m5C sites in the 3'-untranslated regions of PKM2.Similar examples also include LncRNA-FEZF1-AS1 (Bian et al., 2018), lncRNA UCA1a (Yu et al., 2023), and lncRNA HITT (Zhao, Wang, Zhao, et al., 2022), whose experimental details, including molecular mechanisms and experimental approaches, will not be dwelled upon here.Instead, in a recent review article made by Zhu, Guo, et al. (2021), in which the authors conducted a comprehensive review centering upon molecular mechanism of PKM2-related lncRNAs.Other than this piece of review, Interested readers can also refer to a couple of similar review articles published previously (Puckett et al., 2021;Tao et al., 2019) point came from human glioma.Mukherjee J and associates (Mukherjee et al., 2016) discovered that PKM2 can bind directly with the RNA-binding protein HuR in the nucleus of glioma cells, which modulates HuR sub-cellular localization, p27 levels, cell cycle progression and glioma cell growth.From this single piece of evidence, one can see that PKM2 could have a bearing on the intracellular distribution of RBPs.More investigations obviously will be needed to consolidate this finding.

| NUCLEAR PKM2 CAMEOED AS TRANSCRIPTION FACTOR
Generally, nuclear PKM2 acts as a transcription factor that complexes or interacts with histone H3 (Dai et al., 2020), HIF-1α (Wang et al., 2014) or NF-κB p65 (Gu et al., 2021) to enhance the transcription of genes engaging in glycolysis.A direct consequence is that glycolysis was naturally and markedly boosted with PKM2 translocating into the nucleus (Wu, Wang, Zeng, et al., 2022).The underlying mechanism by which PKM2 translocates into the nucleus can be summarized into the three following aspects: first, phosphorylated ERK1/2 is dependent or independent; second, it depends on JMJD5, which can change the homo/hetero-oligomeric restructure of PKM2; third, in response to metabolic stresses, PKM2 can actively enter into the nucleus to facilitate tumor cell survival in an adverse environment.The first evidence observing that nuclear translocation of PKM2 occurred after PKM2 phosphorylation was dependent on ERK1/2 phosphorylation came from Lu Zhimin's group (Yang et al., 2012).Working as a coactivator of β-catenin to induce c-Myc expression, nuclear PKM2 was found to lead to the upregulation of GLUT1 and LDHA in a positive feedback loop.The seminal study underscored the pivotal role of nuclear PKM2 in anaerobic glycolysis and tumorigenesis.Subsequently, in another separate study (Dai et al., 2020), the authors completely reproduced the key findings that ERK1/2 phosphorylation promoted the nuclear translocation of PKM2.However, there was also an indication (Zhao, Yuan, et al., 2022) that nuclear PKM2 was independent of phosphorylated ERK1/ 2. DDX39B was shown to recruit importin α5 to accelerate the nuclear translocation of PKM2 independent of ERK1/2-mediated phosphorylation of PKM2.
Aside from DDX39B, one can assume that there bound to be other molecules mediating the nuclear translocation of PKM2.JMJD5 is a good example.Identified as a novel interplaying partner for PKM2 in another tumor study (Wang et al., 2014), JMJD5 was shown to have a significant impact on the nuclear translocation of PKM2 into the nucleus in that JMJD5 physically hinders PKM2 tetrameric assembly, whereby preventing PKM2 from translocating into the nucleus.Once entering the nucleus, nuclear PKM2 was proven to be complexed with JMJD5 and HIF-1α, thereby specifically enhancing HIF-1α binding to the LDHA locus.Reviewing the abovementioned important literatures performed in the tumor setting, one can see that phosphorylation at Ser37 of PKM2 (Yang et al., 2012) was required for nuclear translocation of PKM2 (Wang et al., 2014).
In addition to Ser37, Yu et al. (2016) in their study supplemented another novel phosphorylation site at Thr454 that was proven to be essential for PKM2 nuclear translocation.Note should be taken that, apart from phosphorylation of certain amino acids of PKM2, acetylation also seems to be involved in the nuclear translocation of PKM2 into the nucleus (Lei et al., 2022).A piece of report from acute myeloid leukemia (AML) deeply revealed that decreasing acetylation of PKM2 at K433 induced by JOSD2 was capable of suppressing the nuclear localization of PKM2, explicitly indicating that acetylation of PKM2 can also promote nuclear translocation.
Intriguingly, going without phosphorylation mediated by ERK1/2, PKM2 can actively translocate into the nucleus under metabolic stresses such as hypoxia and nutrient constraints to operate as a transcription factor to induce the expression of cancer stemnessrelated genes, thereby helping tumor cells survive the adverse pathophysiological environment (Yang et al., 2018).Whether nuclear PKM2 participates in other biological activities remains unknown and remains to be further discovered.

| PKM2 AS CHECKPOINT MODULATOR
A growing body of literature has been published surrounding PKM2 playing as an RBP.However, little attention has been paid to PKM2 as a checkpoint modulator that can regulate immune checkpoints, such as PD-L1.Although there has been a review (Chen et

| EXOSOMAL PKM2
Said of being ubiquitous, PKM2 can be nuclear (Yang et al., 2011), cytoplasmic, and exosomal (Hou et al., 2020), which means that PKM2 can also be released intracellularly, performing different roles.Containing DNA, RNA, protein, lipids and metabolites of producing cells, exosomes are released into the extracellular space under both physiological and pathological circumstances.Recently, the effects of exosomes have been extensively investigated in several pathophysiological conditions, such as cancer and cardiovascular disease.The first evidence reporting that PKM2 can be contained within exosomes was provided by Buschow et al. (2010), who, using quantitative MS, identified a small subset of proteins that were specifically coimmunoprecipitated with MHC II from exosomes derived from B cells.
Among these proteins, PKM2 was identified.Subsequent studies analyzed the biological roles of exosomal PKM2, revealing that exosomal PKM2 derived from cancer cells can facilitate the premetastatic niche, thereby promoting the metastasis of cancer cells (Dai et al., 2019).In addition to promoting metastasis, there was solid demonstration (Hou et al., 2020) that exosomal PKM2 can reshape the tumor microenvironment by skewing monocytes toward macrophage differentiation in a hepatocellular carcinoma setting.Furthermore, exosomal PKM2 was shown to be heavily engaged in cisplatin resistance in nonsmall cell lung cancer (Wang, Zhao, et al., 2021).In our own study (Yang, Zheng, et al., 2021) from esophageal squamous cell carcinoma (ESCC), PKM2 was also identified to be included in exosomes derived from the peripheral blood of patients with ESCC (Yang, Salai, et al., 2022).We found that exosomal PKM2 accelerates the proliferation, migration, and invasion of ESCC cells in vitro.Mechanistically, exosomal PKM2 can activate the STAT3 signaling pathway in ESCC cells, which was implicated in the mechanism by which exosomal PKM2 operates in ESCC.Another seminal study (Wei et al., 2017) has to be brought up here in that tumor cell exosome secretion was shown to be controlled by PKM2 itself.This study has important implications for understanding the physical formation of exosomes derived from tumor cells.Not only can PKM2 be secreted via exosomes from tumor cells but also in turn can regulate the physical formation of exosomes.Reviewing the literature described above, one may be tempting to conclude the oncogenic traits of exosomal PKM2 from tumor cells in outline; however, many biochemical details are still missing regarding how exosomal PKM2 operates in promoting the malignant behaviors of tumor cells.

| MITOCHONDRIAL PKM2
Mitochondria are another place PKM2 loves to go, with the exception of the nucleus and extra cell.The original report establishing the relationship between PKM2 expression and mitochondria was made by Wu et al. (2016), who showed that elevated PKM2 led to mitochondrial fusion.Only exploring the pilot correlation between PKM2 and mitochondrial fusion, this investigation did not give much biochemical details concerning how PKM2 leads to mitochondrial fusion.This question seems to be remedied by another study (Li et al., 2019)   of PKM2 in the modulation of mitochondrial function, we will not discuss here.Instead, we refer readers to the comprehensive review paper published already (Gao et al., 2022).

| GOLGI PKM2
To date, there has not been any indication that PKM2 can sojourn in the Golgi apparatus.Therefore, the subtitle "Golgi PKM2" formulated here is somewhat less than pertinent.To come off as consistent with the preceding sections, we tentatively set the subtitle as Golgi PKM2 here.However, again, there was an indication (Taguchi et al., 2023) that PKM2 can maintain the integrity of the Golgi apparatus within tumor cells.In a novel study made by Taguchi et al. (2023), in which the authors, utilizing HeLa and ME-180 cervical cancer cells, serendipitously found that silencing PKM2 can lead to downregulation of three key proteins that were required in the maintenance of function and architecture of Golgi apparatus, including GM130, giantin and p115 proteins.To elucidate the regulatory mechanism, the transcription factor TFE3 was identified as a regulator responsible for the expression of GM130, giantin, and p115 proteins.PKM2 happened to bind with the 5′ untranslated regions on TFE3 mRNA, thereby promoting translation.What the study teaches us is that whether PKM2 sojourn in the Golgi apparatus remains completely unknown, but one point the authors made is clear that PKM2 was endowed with a novel function of maintaining the integrity of the Golgi apparatus in tumor cells.

| PLASMA PKM2
Existing as an active tetramer and inactive dimer, PKM2 can circulate.
Circulating PKM2 is always dimeric.Dimeric PKM2 is predominantly expressed in various solid tumor cells and is released into the peripheral blood, therefore theoretically serving as a biomarker for tumor load in patients with cancer.Earlier research exploring dimeric PKM2 as a biomarker was from melanoma (Ugurel et al., 2005).The authors showed that, compared with healthy control, concentrations of dimeric PKM2 were significantly increased in melanoma patients and markedly associated with tumor load and stage.In addition, the concentration of dimeric PKM2 was an independent predictor of overall survival in melanoma.However, the sensitivity and specificity of dimeric PKM2 seem to be not as good as expected.Basically, in support of this viewpoint, with the intention of validating dimeric PKM2 as a biomarker for tumors, Staib et al. (2006) demonstrated that the specificity and sensitivity of dimeric PKM2 were 59% and 51%, respectively.Particular care should be taken that 37% of healthy controls and 44% of patients with nonmalignant disease, especially those with only acute inflammatory reactions (as high as 67%), were displayed to have elevated dimeric PKM2 in plasma.
Considering the analyses, the authors pointed out that plasma PKM2 should not be used as a tumor marker for hematological malignancies and solid tumors.Partly consistent with the point made by Staib et al. (2006) that plasma PKM2 was not a useful biomarker for both hematological and solid malignancies, Goonetilleke et al. (2007) evaluated its potential utility as a biomarker in periampullary cancer, revealing that plasma PKM2 alone as a biomarker predictive of diagnosis was limited but could be used as a prognostic predictor for periampullary cancer.Analyzing the concentration of plasma PKM2 in different types of cancer, Munoz-Colmenero et al. (2015) confirmed that plasma PKM2 was not a specific marker for tumors.

| URINE PKM2
The majority of studies concerning urine PKM2 have been carried out in nontumor nephropathy, such as acute kidney injury, nephrotoxicity, and diabetic nephropathy.Here, discussion will not be made about urine PKM2 in these nontumor diease.A piece of research (Liu et al., 2019) performed in bladder cancer argued that dimeric PKM2 in urine could serve as a urinary marker for bladder cancer.
However, this study suffers from one fact that the authors did not analyze the specificity and sensitivity of dimeric PKM2 in urine collected from patients with bladder cancer.Instead, these authors just simply compared and analyzed the significant difference of concentration of PKM2 in urine.In addition, no additional studies are available regarding dimeric PKM2 in urine acting as a biomarker for tumors.

| PKM2 SPLICING
Almost always overexpressed in cancer, PKM2 expediates aerobic glycolysis.Unlike PKM2, PKM1, the adult isoform of PKM2, promotes oxidative phosphorylation (Taniguchi et al., 2016).In effect, resulting from mutually exclusive alternative splicing of the PKM2 pre-mRNA, the two isoforms are expressed from the same gene under the control of two different promoters (David et al., 2010).Produced by alternative splicing of transcripts of the PKM gene, which is to say, exon 9 gave birth to PKM1 while exon 10 yielded PKM2, PKM1, and PKM2 mRNAs differ only by inclusion of one or another of two mutually exclusive exons, so understanding the regulation of switching between PKM1 and PKM2 pre-mRNA splicing is of great importance for tumor metabolic regulation (Chen et al., 2010).Nevertheless, this viewpoint seems to have been challenged by Bluemlein et al. (2011)'s report that no evidence was observed of a shift in pyruvate kinase PKM1 to PKM2 expression during tumorigenesis; nor did the data they presented support conclusions that PKM2 is specific for proliferating, whereas PKM1 for non-proliferating tissue.To date, this controversy remains unresolved.
It has been well established that, often determined by cisregulatory elements, whether an alternative exon is included in an mRNA will be recognized by transacting regulatory proteins.Hence, pivotal is searching for proteins that bind to the two alternative exons and/or their flanking regions.Following this line of thought, David et al. (2010) investigated the mechanism of PKM alternative splicing, finding that heterogeneous nuclear ribonucleoprotein (hnRNP) protein family members hnRNP A1, hnRNP A2 and polypyrimidine tract binding protein (PTB) were found to bind to intronic sequences flanking exon 9 (contained in PKM1) but not exon 10 (contained in PKM2), indicating that the expression levels of hnRNP A1 (Zhu, Li, et al., 2021), hnRNP A2 and PTB are critical for PKM2 expression in cancer cells.This pioneering work was a milestone that opened up a new research area for PKM2 splicing.Subsequently, a proliferation of studies sprung up surrounding the searching for proteins that bind to the two alternative exons and/or their flanking regions of PKM2.For example, PPP1R26 (Yang, Ren, et al., 2022), SAM68 (Zhu, Chen, et al., 2021), SNHG6 (Lan et al., 2020), RBM4 (Su et al., 2017), and PTBP1 (Bielli & Sette, 2017;Jiang et al., 2017;Zhang et al., 2023)  chemoresistance.Actually, regarding the involvement of PKM2 in tumorigenesis, there has been much controversy over its (Hillis et al., 2018;Israelsen et al., 2013;Lau et al., 2017;Xia, Wang, et al., 2022) in different cancer types.Therefore, attempting to remedy the controversy, Morita et al. (2018), using a transgenic mouse model and solid data, demonstrated that the expression of PKM1, instead of PKM2, is sufficient to activate glucose metabolism and is required to promote small-cell lung cancer (SCLC) cell proliferation in a Kras LSL- G12D mouse model and in KRAS G12V mouse embryonic fibroblasts.
Quite different from or even contrary to this study, utilizing a transgenic mouse model of prostate cancer, Davidson et al. (2022) chiefly evaluated the role of PKM1 in terms of tumor growth, demonstrating that PKM1 hinders the development of prostate cancer.Metabolically, another report (Park et al., 2022) demonstrated that PKM1 inhibits glycolysis and hypoxia-inducible factor-1 alpha (HIF-1α).Reflected from these scattered evidence available, the function of PKM1 implicated in tumor growth is still controversial and appears to be tissue specific.Anyway, these articles bring some evidence to bear on our current understanding of PKM1 in cancer.In view of the literatures concerned with PKM1 in the context of cancer is scarce, the biological and pathological roles of PKM1 remain elusive, that is, left to be further investigated.

| SUMMARY AND OUTLOOK
Among the molecules implicated in carcinogenesis, PKM2 can be said of absolutely all-round player that engages in many pivotal activities essential for the development of tumor cells.holding dual roles as metabolic enzyme (tetramer) and protein kinase (dimer), wandering PKM2 can be transboundary both intracellularly and extracellularly.
Specifically, PKM2 can be nuclear, cytoplasmic, mitochondrial, or exosomal and even circulating within the body.Nuclear PKM2 acts as a transcription factor that can regulate the transcription of some al., 2021)    to which we refer readers that comprehensively summarized the possible mechanisms of PKM2 mediating cancer immunity and metabolism in the tumor microenvironment, there is still a need to give a brief account of the role PKM2 plays as a checkpoint regulator in this section.The original evidence uncovering that PKM2 was indispensable for the expression of PD-L1 in both tumor and immune cells was contributed byPalsson-McDermott et al. (2017).In this study, the underlying molecular mechanism by which PKM2 regulates PD-L1 is that PKM2 and HIF-1α can directly bind with the hypoxia response element on the promoter of PD-L1.This observation was influential that enriched our understanding and changes our settled opinion of PKM2 as a rate-limiting enzyme involved in glycolysis.This finding also leads us to reason that PKM2 can act as an inflammation or immune regulator in the tumor microenvironment.Based on this literature, one subsequent work(Deng et al., 2018) performed in the setting of sepsis confirmed that PKM2 expression was required for the expression of PD-L1 in a STAT1-dependent fashion.Entirely consistent with the working mechanism digged out in sepsis, another recent study(Xia, Jia, et al., 2022) from pancreatic ductal adenocarcinoma fully replicated the molecular mechanism by which PKM2 significantly leads to PD-L1 overexpression through activation of STAT1.Now that PKM2 can have a direct bearing upon PD-L1 expression, a hypothesis naturally proposed here that PKM2 may also affect the infiltration of cytotoxic T cells, such as CD8+ T cells, into tumor tissues.Supporting this hypothesis, Li et al. (2020) established a statistically and inversely correlation between PKM2 expression and lymphocyte infiltration in hepatocellular carcinoma.Similar HE ET AL. et al. (2020) in lung adenocarcinoma and Mohammad et al. (2016) in pancreatic cancer.The molecular mechanism by which PKM2 regulates the infiltration of CD8+ cells into tumor tissues remains largely unknown; however, signal transduction and the activator of transcription (STAT) pathway appear to be heavily involved.So far, there has been little discussion about the regulatory mechanism by which PKM2 modulates the infiltration of CD8+ cells in cancer; more studies are therefore needed.
interacts with and phosphorylates Bcl2 at threonine 69 in the mitochondria.This phosphorylation stabilizes Bcl2, thereby preventing the apoptosis of glioma cells.This finding was completely supported by the investigation ofZhao, Wang, Han, et al. (2022) concerning the mediation of mitochondrial PKM2 in the lung ischemia/reperfusion injury setting.Implicating mitochondrial PKM2 in the regulation of antiapoptosis in cancer cells, this study(Liang et al., 2017) was instrumental in better understanding the biological role of mitochondrial PKM2.That said, the study did not offer any mechanistic data regarding how PKM2 translocates into mitochondria.To address this question,Qi et al. (2019), through delicate design and sophisticated techniques, discovered that succinylation mediated mitochondrial translocation of PKM2 under glucose starvation from the angle of posttranslational modification.the literature exploring the molecular mechanism by which PKM2 translocates into mitochondria has been scant.With regard to the detailed implications important genes related to metabolites and the cell cycle.Importantly, cytoplasmic PKM2 cameoed as RNA-binding protein (RNP), which can bind with different long-non-coding RNAs, thereby strengthening itself as a metabolic enzyme.Mitochondrial PKM2 bears directly on the mitochondria fusion, therefore participating in the antiapoptosis of tumor cells.Noticeably, PKM2 can safeguard the integrity and structure of the Golgi in tumor cells.To tersely summarize the various functions of PKM2 we reviewed in tumor cells, a schematic diagram was offered here for the readers' convenience (Figure 2).When necessary, exosomal PKM2 even performs remote assistance to remodel the microenvironment where F I G U R E 2 Schematic diagram summarizing the multifaceted roles of PKM2 described in the main text.