LACTB, a Metabolic Therapeutic Target in Clinical Cancer Application

Serine beta-lactamase-like protein (LACTB) is the only mammalian mitochondrial homolog evolved from penicillin-binding proteins and β-lactamases (PBP-βLs) in bacteria. LACTB, an active-site serine protease, polymerizes into stable filaments, which are localized to the intermembrane space (IMS) of mitochondrion and involved in the submitochondrial organization, modulating mitochondrial lipid metabolism. Cancer pathogenesis and progression are relevant to the alterations in mitochondrial metabolism. Metabolic reprogramming contributes to cancer cell behavior. This article (1) evidences the clinical implications of LACTB on neoplastic cell proliferation and migration and tumor growth and metastasis as well as LACTB’s involvement in chemotherapeutic and immunotherapeutic responses; (2) sketches the structural basis for LACTB activity and function; and (3) highlights the relevant regulatory mechanisms to LACTB. The abnormal expression of LACTB has been associated with clinicopathological features of cancer tissues and outcomes of anticancer therapies. With the current pioneer researches on the tumor-suppressed function, structural basis, and regulatory mechanism of LACTB, the perspective hints at a great appeal of enzymic property, polymerization, mutation, and epigenetic and post-translational modifications in investigating LACTB’s role in cancer pathogenesis. This perspective provides novel insights for LACTB as a metabolic regulator with potential to develop targeted cancer therapies or neoadjuvant therapeutic interventions.


Introduction
The shared biochemical features between mitochondria and Gram-negative bacteria, including DNA organization, core metabolism, and double-membrane architecture, are elucidated by the endosymbiont hypothesis [1]. Serine beta-lactamase-like protein (LACTB) is the exclusive mammalian mitochondrial homolog evolved from penicillin-binding proteins and β-lactamases (PBP-βLs) in bacteria [2]. The role of LACTB, the bacterial legacy, has consequently been switched from synthesizing the peptidoglycan layer to mediating lipid metabolism and tumorigenicity in mammalians [2][3][4]. This perspective provides novel insights on LACTB in cancer progression and clinical prognosis and developing targeted cancer therapies or neoadjuvant therapeutic interventions via elucidating LACTB's influence on diverse cancers and involvement in the anticancer chemotherapeutic and immunotherapeutic responses; performing a molecular dissection of LACTB to streamline its role on reprogramming lipid metabolism, a hallmark in cancer [5]; and revealing relevant regulatory mechanisms (Figure 1).  [6]. hLACTB, human LACTB; PTMs, post-translational modifications.

Potential Prospects of LACTB in Cancer Applications
Rogue cells ready the body for tumorigenesis. The bona fide tumor suppressor gene makes a difference. In most conditions, LACTB is negatively associated with the growth of rogue cells while having a minimal effect on non-tumorigenic cells, and a low LACTB expression predicts poor prognosis. However, in nasopharyngeal carcinoma (NPC) and pancreatic adenocarcinoma (PAAD), LACTB is highly expressed and correlated with poor patient survival [7,8]. The relationship between the LACTB content and cancer development is reviewed in Table 1.  [6]. hLACTB, human LACTB; PTMs, post-translational modifications.

Potential Prospects of LACTB in Cancer Applications
Rogue cells ready the body for tumorigenesis. The bona fide tumor suppressor gene makes a difference. In most conditions, LACTB is negatively associated with the growth of rogue cells while having a minimal effect on non-tumorigenic cells, and a low LACTB expression predicts poor prognosis. However, in nasopharyngeal carcinoma (NPC) and pancreatic adenocarcinoma (PAAD), LACTB is highly expressed and correlated with poor patient survival [7,8]. The relationship between the LACTB content and cancer development is reviewed in Table 1. The downregulated expression of LACTB is due to promoter methylation and histone deacetylation.
[10] LACTB regulates the activity of phosphoinositide-3-kinase regulatory subunit 3, influences the level of phosphoinositide 3-kinases, and promotes autophagy via the protein kinase B/rapamycin signaling pathway. [11] Colorectal cancer LACTB expression is lower in colorectal cancer tissue than that in the adjacent tissue; LACTB is associated with clinical stage in colorectal cancer; LACTB expression is lower in the colorectal cancer patients with lymph node metastasis than those without lymph node metastasis; Low LACTB expression is associated with a lower 5-year survival rate in colorectal cancer patients compared to those with high LACTB expression.
Not mentioned. [12] Colorectal cancer Silencing LACTB promotes the viability, migration, and invasiveness in colorectal cancer cells; Silencing LACTB partially abolishes the apoptotic effect of pinocembrin on colorectal cancer cells.
Silencing LACTB enhances the expression of Matrix metalloproteinase-2 and N-cadherin but inhibits that of E-cadherin. [13] Colon cancer LACTB mRNA expression is lower in colon cancer than in normal tissue; Overexpressing miR-1276 in colon cancer cells increases proliferation, migration, invasiveness, and epithelial-to-mesenchymal transition and decreases apoptosis, while supplementing LACTB suppresses these effects of miR-1276.
LACTB is inhibited miR-1276 inhibits in colon cancer cells, which inhibits autophagy. [14] Gastric Glioma LACTB expression is decreased in glioma; Low LACTB expression is correlated with a poor prognosis of glioma patients; Overexpressing LACTB suppresses the proliferation, invasion, and angiogenesis of glioma cells.

Hepatocellular carcinoma
The mRNA and protein expressions of LACTB are down-regulated in hepatocellular carcinoma; Low LACTB expression is associated with TNM stage, histologic grade, and overall survival of patients; Overexpressing LACTB inhibits proliferation, invasion, and migration in vitro and decreases tumor growth in vivo.
Leukemia Total LACTB is highly expressed in HL60 cells and low expressed in Raji cells; LACTB has multiple splicing isomers in leukemia cell lines, including XR1, V1, V2, and V3; The expression of V1 is high in U937 cells and low in Raji cells, that of V2 is high in HL60 cells and low in Raji cells, and V3 is poorly expressed in THP-1 cells.
Not mentioned. [19] Lung cancer LACTB expression is downregulated in lung cancer tissues, while its methylation level is increased; Patients with high LACTB level exhibit improved survival; Overexpressing LACTB inhibits cell migration and invasion and induces apoptosis in H1299 and H1975 cells, while knocking down LACTB has opposite effects; LACTB negatively regulates the epithelial-to-mesenchymal transition process in lung cancer; LACTB strengthens the anticancer role of docetaxel in lung cancer; i.e., LACTB combined with docetaxel leads to a higher apoptotic rate and more potent inhibitory effects on H1299 and H1975 cells compared to docetaxel.

Knockdown of LACTB decreases the expression of E-cadherin (an epithelial cell-derived marker) and increases that of N-cadherin and vimentin (stromal cell-derived markers);
Overexpressing LACTB increases the expression of E-cadherin and decreases that of N-cadherin and vimentin. [20] Melanoma LACTB expression is low in melanoma tissues and cell lines; Low LACTB expression is correlated with short survival time in melanoma patients; Overexpressing LACTB suppresses the proliferation, migration, and invasion of melanoma cells in vitro; Overexpressing LACTB induces cell apoptosis and cell cycle arrest at the G2/M phase; Overexpressing LACTB suppresses the tumorigenicity and lung metastasis of MUM2B uveal melanoma cells in vivo.
Sex-determining region Y (SRY)-related HMG box-containing factor 10 binds to the promoter of LACTB and negatively regulates its transcription; Overexpressing LACTB prevents Yes-associated protein from translocating to the nucleus; LACTB directly binds to protein phosphatase-1, attenuates the interaction between protein phosphatase-1 and Yes-associated protein, and decreases the consequent dephosphorylation of Yes-associated protein. [21] Melanoma LACTB expression is downregulated in melanoma tissues compared to the normal epithelium; Decreased LACTB expression predicts poor prognosis; iDPP/LACTB nanocomplexes promote cell apoptosis and block the cell cycle in vitro; iDPP/LACTB nanocomplexes inhibit tumor proliferation and induce cell apoptosis, consequently inhibiting melanoma growth in the subcutaneous B16-F10 melanoma model.
iDPP/LACTB nanocomplexes upregulates the p53 pathway and increases the mRNA expression of apoptosis-and cell cycle-related genes, including p21, BCL2-associated X, BH3-interacting domain death agonist, p53-induced death domain protein 1, and apoptosis regulatory protein Siva.
[22] LACTB activates Erb-B2 receptor tyrosine kinase 3/Epidermal growth factor receptor-extracellular signal-regulated kinase signaling, promoting the metastasis of nasopharyngeal carcinoma; Suppressing LACTB reduced nasopharyngeal carcinoma cellular motility by enhancing histone H3 stability and its acetylation. [8] Pancreatic adenocarcinoma The mRNA and protein expressions of LACTB are high in pancreatic adenocarcinoma; Elevated LACTB mRNA expression in pancreatic adenocarcinoma patients is associated with a poor survival rate.
High LACTB expression is correlated with cell cycle-related genes and multiple immune marker sets. [7]

LACTB Drives a Quiescence-like Tumor Suppressive State
A rogue cell escapes a normal behavioral control and acquires progenitor properties, sharing its abnormal behaviors or capabilities. Metabolic reprogramming is the driving force for cancer transformation and drug resistance. LACTB alters the phospholipid composition via functionally connected with phosphatidylserine decarboxylase (PISD), which converts phosphatidylserine (PS) to phosphatidylethanolamine (PE) and regulates the quantity of their lyso-forms [4]. Lyso-phosphatidylethanolamine (Lyso-PE, LPE) modulates the differentiation of neuronal PC12 [23] and cardiac cells [24]. Silencing LACTB increases the quantity of PE/LPE in mitochondrial membranes, which facilitates cellular proliferation and upregulates CD44 (cancer stem cell maker), while restoring LACTB expression reduces the PISD activity as well as the accumulation of PE/LPE, leading to a quiescence-like tumor suppressive state [4,25].

LACTB Mediates Neoplastic Proliferation and Apoptosis
The rogue cells have a high replication rate, which requires a high metabolism and a great deal of subtracts for DNA double helixes and phospholipid bilayers. LACTB directly binds to the C-terminal of p53, preventing p53 from degradation and consequentially impacting DNA-break repair and promoting apoptosis in colorectal cancer (CRC) [10]. Parallelly, melanoma-targeted nanoparticle iDPP-delivered LACTB upregulates p53 signaling pathway, promotes cell apoptosis, and blocks the cell cycle [22]. p53 exerts control over glucose and amino acid metabolism as well as lipid droplet formation [26]. The reprogrammed lipid metabolism has been widely recognized as an emerging hallmark of malignant cells in addition to Warburg effect [5]. Moreover, LACTB has exhibited an inhibitory role in breast cancer (BRCA) cellular proliferation via regulating the accumulation of the PE/LPE lipid species [4].

LACTB Mediates Epithelial-Mesenchymal Transition and Metastasis
Epithelial-mesenchymal transition (EMT) is a key step in carcinogenesis and confers metastasis by enhancing mobility and invasion [27]. EMT is regulated by lipid metabolism, and intervening cholesterol metabolism overcomes EMT-associated drug resistance [28]. Moreover, knockdown of LACTB decreases the expression of the epithelial cell-derived markers and increases the expression of stromal cell-derived markers in lung cancer, while overexpressing LACTB inhibits the migration and invasion of H1299 and H1975 cells, suppressing carcinogenesis [20]. However, LACTB expression is elevated in the NPC tissue compared to the non-cancer nasopharyngeal tissue, which is correlated with malignant behaviors and poorer survival in NPC [8]. Overexpressing LACTB promotes metastasis, and knockdown of LACTB reduces the metastasis of NPC cells [8]. The inconsistency of the LACTB's roles between lung cancer and NPC may be related to the enzymic properties of LACTB, involving the polymerization dynamics (discussed in Section 3.2) and posttranslational modifications (PTMs, discussed in Section 4.3) of LACTB.

LACTB Predicts Chemotherapeutic Responses
Chemotherapy is a predominant anticancer therapy. However, some cancer patients develop resistance after an initial treatment [29]. Identifying genes involved in chemotherapeutic responses is crucial for predicting antitumor response and treating drug-resistant cancer patients. Death receptor 4 (DR4) is palmitoylated and translocated into lipid rafts in circulating tumor cells, which is associated with increased oxaliplatin resistance and elevated efficacy of TRAIL liposomes [30]. LACTB has been associated with oxaliplatinresistance [15] and oxaliplatin plus S-1 neoadjuvant chemotherapeutic response [16]. Moreover, it regulates the efficiency of pinocembrin [13] and strengthens the effect of docetaxel [20]. For instance, LACTB inhibits cell migration and invasion and suppresses EMT under the administration of docetaxel in lung cancer patients [20]. Hence, targeting lipid metabolism by combing traditional chemotherapeutic drugs is a promising strategy to overcome drug resistance in cancer, and LACTB is an effective lipid metabolic switch in chemotherapeutic responses.

LACTB Is Potentially Involved in Immunoregulation in Cancer
Immunotherapy enhances the body's immune response and inhibits immune escape [31]. Targeting macrophages holds great promise for cancer immunotherapy since macrophages account for 30% to 50% of the infiltrating immune cells in the tumor microenvironment (TME) compared to the low infiltration of T cells [32]. During apoptosis, PS is translated to the outer leaflet of plasma membrane, activating phagocytes and provoking apoptotic cell clearance, while their disruption results in secondary necrosis and immune activating danger signal release [33]. In the TME, PS is significantly increased, sharing innate immunosuppressive properties and facilitating tumorigenesis and progression [34]. The serine protease domain of LACTB has been proposed to participate in proteolytic signaling cascades, such as mitochondria-mediated apoptosis [3]; furthermore, LACTB participates in the PS metabolism [4], manifesting its involvement in immunoregulation in cancer. Moreover, in atherosclerotic plaques, LACTB expression is upregulated [35]; on the other hand, inhibiting LACTB attenuates the secretion of monocyte chemoattractant protein-1 (MCP-1) in lipopolysaccharide (LPS)-stimulated THP-1 macrophages [35], a crucial chemokine upregulated in inflammatory diseases [36]. Hence, LACTB may exhibit a pro-inflammatory effect in the macrophage-based anticancer therapy, and the correlations of LACTB expression with macrophage infiltration level are visualized in various cancers ( Figure 2).

Structural Basis for the Enzymatic Properties of LACTB
The emerging roles of mammalian LACTB in metabolic signaling [37][38][39] prompt the explorations regarding the structural basis of its functionality, i.e., the enzymic properties. Cancer pathogenesis and progression have been relevant to the alterations in mitochondrial metabolism [40]. Recently, the cryo-electron microscopy (Cryo-EM) structure of LACTB filaments was reviewed by Cascone et al. [41]. Herein, this article focuses on the comprehensive impacts of LACTB on cancers and attends to undermine the structural basis for the catalytic activity of LACTB to elucidate its role in cancer events.  [2]. LACTB is of a moderate sequence similarity (16-27%) with bacterial class B low-molecular-weight PBP-βLs (LPBP-B) [2], which is the first reported vertebrate example of microbial peptidase family [42]. In addition, the phylogenetic analysis indicates that the LACTB orthologs cluster with LPBP-B genes from free-living α-proteobacteria [42]. PBP-βLs function in synthesizing peptidoglycan, the major cell wall constituent in most bacteria [2]. β-lactamases governs penicillin metabolism, converting penicillin to penicilloate, while the LACTB single-nucleotide polymorphism (SNP) rs2729835 has been marginally associated with self-reported penicillin allergy in patients [43]. Hence, during eukaryote evolution, the prokaryotic PBP-βLs are conferred to the higher metazoan species and endowed with novel biochemical properties [2], i.e., the mammalian homolog LACTB in the mitochondrial intermembrane space (IMS) [44]. LACTB harbors a N-terminal region with a length of 50-100 amino acids and of no sequence similarity to the bacterial PBP-βLs, where a predicted mitochondrial import sequence starts [2], protecting LACTB from autoproteolysis [3]. In mitochondria, LACTB is polymerized into stable filaments with a length extending more than a hundred nanometers [44], promoting intramitochondrial membrane organization and micro-compartmentalization and therefore affecting mitochondrial metabolon organization.

Tertiary Structure of LACTB Underlies Its Enzymic Properties
The causative link between LACTB and obesity has been detected by data integration [45] and validated in the transgenic mice; i.e., LACTB overexpression results in obesity phenotype [46]. However, endurance exercise training downregulates LACTB in highmolecular-mass complexes (>600 kDa) without affecting the LACTB expression in the total proteome of skeletal muscle [47], which suggests the LACTB polymers (filaments) rather than total LACTB content within mitochondria may have conferred the metabolic advantages [48]. In this way, the human LACTB (hLACTB) filament formation may have played a critical role in cancer development and progression as well as anticancer therapeutic responses.
The tertiary structure of LACTB is intimately associated with its catalytic activity. It has been revealed by the high-resolution structure of a wild-type (WT) hLACTB filament, a middle region deletion mutant (in the absence of the catalytically important region), and an inhibitor peptide-bound filament [48]. Briefly, the middle region of the filament is required to perform the hydrolysis function, and hLACTB polymerizes via three distinct interfaces. The cleavage activity and polymerization dynamics of hLACTB are influenced by the pH value in IMS (ranged pH < 6-pH 7.4 in normal physiological conditions): the cleavage activity peaks at pH 8.6 and decreases along with pH, while the polymerization decreases at pH 6.0 compared to that at pH 7.4. Moreover, the alkaline pH, one of the unique mitochondrial characteristics in pathological cancers [49], may have facilitated the cleavage activity of LACTB. In this way, the increased/decreased total LACTB expression may not justify the altered behavior and enzymatic activity of LACTB in multi-cancerous events.

Pathologic Mutations of LACTB Dump Its Catalytic Activity
hLACTB polymers further assemble a double helix for substrate delivery and or product release, with amino acids surrounding S163, Y323, and H485, forming a positively charged plane and functioning as a catalytic center for peptide cleavage [48]. Moreover, the subtract selectivity of hLACTB was revealed by the hLACTB filament structure with an inhibitor peptide Z-AAD-CMK bound at 3.0-, 3.1-, and 2.8-Å resolution; i.e., the potential hydrogen bonds are formed between three residues in hLACTB (Y223, T325, and K394) and the aspartic acid residue in the side chain of Z-AAD-CMK [48]. H222 and G488 form potential hydrogen bonds with the backbone of Z-AAD-CMK, while F287 and L292 may have hydrophobic interactions with Z-AAD-CMK [48]. However, the LACTB mutations have been mapped in various cancers [48], e.g., E121K in BRCA, R371K in lung cancer, and E457K in urinary bladder cancer. These mutations may have a severe impact on the polymerization of hLACTB [48], which consequentially reduces its substrate affinity, dumps the V max of its cleavage activity, and reprograms cancer lipid metabolism. Hence, mutation may be another angle in studying LACTB's role in cancer.

Regulatory Mechanisms of LACTB
Given LACTB's roles in cancer pathogenesis and therapeutic responses, understanding about its regulatory mechanisms is critical for developing precise and effective LACTBtargeted cancer therapeutics. More specifically, the upstream signaling, LACTB promotor activity, alternative splicing, microRNA (miRNA) regulation, and PTMs may play decisive roles in the expression and catalytic activity of LACTB in cancer tissues.

Transcriptional Events
Sex-determining region Y (SRY)-related HMG box-containing factor 10 (SOX10) participates in melanoma genesis [50]. Ten SOX10 binding sites have been predicted in the LACTB promoter [21]. It has been evidenced that SOX10 negatively regulates LACTB's transcription in melanoma cells by binding to its promoter [21]. Moreover, LACTB responds to acute insulin signaling in skeletal muscle [38], which in turn emphasizes LACTB's role in anabolism [51].

Post-Transcriptional Regulators
Five hLACTB transcripts in the NCBI database suggest the relationship between alternative splicing and functional diversity of LACTB. When different mRNAs are assembled from a single gene, alternative splicing occurs by joining exons in different ways [53]. Splicing perturbation is common and associated with mutations in cancer [54]. Furthermore, a variety of splice isomers of LACTB have been found in the leukemia cell lines with diverse expression patterns [19].

Post-Translational Modifications
Protein PTMs, such as phosphorylation, methylation, and acetylation, affect almost all aspects of normal cell biology and pathogenesis [56]. A variety of PTMs are known to be altered in cancer development, which are potentially significant biomarkers in cancer diagnosis and prognosis [56]. LACTB has been subjected to post-translational regulation. Under general circumstances, the serine residues in LACTB are phosphorylated, which hints at LACTB's response to specific phosphoprotein phosphatases [39]. During starvation, lysine acetylation of LACTB has been detected in the liver, which suggests that LACTB is regulated by a highly conserved acetyltransferase/deacetyltransferase pathway, resembling other key enzymes in metabolism [57]. In addition, in lung cancer, the methylation level of LACTB is elevated though its total expression is decreased [20], which hints at the importance of PTMs of LACTB and its promotor in cancer research.

Summary
The mitochondrial protease LACTB regulates lipid metabolism, specifically decreasing the accumulation of PE/LPE lipid species. The effects of LACTB in cellular proliferation and EMT have been reviewed in the perspective as well as its influence in the chemotherapeutic responses and immunoregulation. In most cases, LACTB expression is negatively associated with cancer progression, but inconsistency exits. The structural basis of LACTB underlies its enzymic properties and the upstream signaling exhibits transcriptional, post-transcriptional, and post-translational regulations on the expression and activity of LACTB, which hint at a great appeal of catalytic activity and function, polymerization dynamics, mutation, and polymorphism as well as epigenetics and PTMs in studying LACTB's role in cancer pathogenesis rather than its simply total expression. This perspective provides novel insights on the LACTB's metabolic role in cancer progression and clinical prognosis for developing LACTB-targeted cancer therapies or neoadjuvant therapeutic interventions, e.g., to combine the LACTB-targeted strategy with the developing drug delivery systems, such as the transferrin/α-tocopherol modified poly(amidoamine) dendrimers to improve tumor targeting [58] or transferrin and octaarginine modified dual-functional liposomes to enhance intracellular delivery [59].