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Department of Oncology & Cancer Institute, Sichuan Academy of Medical Sciences, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu, ChinaDepartment of Laboratory Medicine and Sichuan Provincial Key Laboratory for Human Disease Gene Study, Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China, Chengdu, ChinaSichuan Cancer Hospital & Institute, Sichuan Cancer Center, University of Electronic Science and Technology of China, Chengdu, China
Department of Oncology & Cancer Institute, Sichuan Academy of Medical Sciences, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu, ChinaSichuan Cancer Hospital & Institute, Sichuan Cancer Center, University of Electronic Science and Technology of China, Chengdu, China
Department of Oncology & Cancer Institute, Sichuan Academy of Medical Sciences, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu, ChinaDepartment of Laboratory Medicine and Sichuan Provincial Key Laboratory for Human Disease Gene Study, Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China, Chengdu, ChinaSichuan Cancer Hospital & Institute, Sichuan Cancer Center, University of Electronic Science and Technology of China, Chengdu, China
Department of Oncology & Cancer Institute, Sichuan Academy of Medical Sciences, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu, ChinaDepartment of Laboratory Medicine and Sichuan Provincial Key Laboratory for Human Disease Gene Study, Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China, Chengdu, ChinaDepartment of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA
Liver kinase B1 (LKB1) is a serine-threonine kinase that participates in multiple cellular and biological processes, including energy metabolism, cell polarity, cell proliferation, cell migration, and many others. LKB1 is initially identified as a germline-mutated causative gene in Peutz-Jeghers syndrome (PJS) and is commonly regarded as a tumor suppressor due to frequent inactivation in a variety of cancers. LKB1 directly binds and activates its downstream kinases including the AMP-activated protein kinase (AMPK) and AMPK-related kinases by phosphorylation, which has been intensively investigated for the past decades. An increasing number of studies has uncovered the posttranslational modifications (PTMs) of LKB1 and consequent changes in its localization, activity, and interaction with substrates. The alteration in LKB1 function as a consequence of genetic mutations and aberrant upstream signaling regulation leads to tumor development and progression. Here, we review current knowledge about the mechanism of LKB1 in cancer and the contributions of PTMs, such as phosphorylation, ubiquitination, SUMOylation, acetylation, prenylation, and others, to the regulation of LKB1 function, offering new insights into the therapeutic strategies in cancer.
Liver kinase B1 (LKB1), also known as serine-threonine kinase 11 (STK11), is the gene encoding an evolutionarily conserved serine/threonine kinase belonging to the calcium/calmodulin regulated kinase-like family (
). LKB1 was initially identified as a causative gene with loss of function mutations responsible for the Peutz-Jeghers syndrome (PJS), a rare hereditary disease and the first cancer-susceptibility syndrome characterized by the predisposition of benign and malignant tumors of many organ systems (
) with approximately 5-30% and 20% cases of LKB1 mutants, respectively. Studies have also reported the infrequently aberrant expression levels of LKB1 caused by somatic mutation or epigenetic alteration in several malignancies, including melanoma (
), etc. In vivo studies using conditionally Lkb1-deleted mouse models and cancer cell lines have implicated the tumor suppressive role of LKB1 in different types of cancers, such as hepatocellular carcinoma (HCC) (
). Additionally, loss of LKB1 is associated with poor prognosis in cancer patients. Remarkably, concomitant LKB1 deficiency with KRAS or PTEN mutation accelerates cancer progression and induces immunosuppressive tumor microenvironment (
Generally, LKB1 forms a heterotrimeric complex with the pseudokinase Ste20-related adaptor (STRAD) and the scaffolding mouse protein 25 (MO25; also known as calcium-binding protein 39, CAB39), which promotes the nucleocytoplasmic shuttling of LKB1 and its activation (
). Upon activation, LKB1 functions as a master kinase that modulates a plethora of cellular processes through the phosphorylation of AMP-activated protein kinase (AMPK) and other 13 AMPK-related kinases, including brain-specific kinases (BRSK)-1/2 (also referred to as SAD-B/A) (
), thereby regulating tumor initiation and progression.
Besides the downstream signaling, a myriad of studies has been conducted to understand the impact of posttranslational modifications (PTMs) of LKB1 on its function. Overwhelming data have demonstrated that PTMs, including phosphorylation, ubiquitination, neddylation, SUMOylation, prenylation, and others, alone or cooperatively modulate the stability, localization, and function of LKB1. In this review, we will review recent discoveries of the mechanisms underlying LKB1-mediated cancer progression, summarize recent advances in PTMs of LKB1, and propose the therapeutic implications in cancer.
1. Structure of LKB1
The human LKB1/STK11 gene is located on the short arm of chromosome 19 (19p13.3) and encodes three protein isoforms through alternative transcription of the LKB1 mRNA, including the classical full-length LKB1 (FL-LKB1, 50 kDa), a 3’ alternative spliced isoform (S-LKB1, 48 kDa) (
) (Fig.1). In 2008, Mhairi C.T. et al. identified two distinct variants of LKB1 mRNA generated by alternative splicing in exon 9A, 9B and 10, and the two variants are correspondingly translated into FL-LKB1 and S-LKB1 (
). FL-LKB1 contains three major domains, including the N-terminal regulatory domain (NRD), the catalytical kinase domain (CKD), and the C-terminal regulatory domain (CRD). Two nuclear localization signals (NLSs) and the farnesylation motif, which are essential for the subcellular localization of LKB1, lie in NRD and CRD, respectively (
). Compared to FL-LKB1, S-LKB1 lacks the critical serine phosphorylation site (Ser428 in human and Ser431 in mouse) and the cysteine farnesylation site (Cys430 in human and Cys433 in mouse) at the C-terminus. Both two isoforms are ubiquitously expressed in human and rodent tissues, whereas FL-LKB1 and S-LKB1 are more predominantly expressed in brain and testis, respectively (
). Further functional studies have revealed that both FL-LKB1 and S-LKB1 can equivalently be activated and activate downstream targets. The subcellular localization of both isoforms is only affected by the association with STRAD and MO25 (
). Dahmani R. and colleagues later identified a novel isoform ΔN-LKB1 mostly expressed in mouse heart and skeletal muscle, as well as human lung cancer cell lines (
). ΔN-LKB1 is the product of alternative transcription and internal initiation of translation of LKB1 mRNA in exon3. Although catalytically inactive, ΔN-LKB1 has a strong association with the regulatory autoinhibitory domain of AMPK, which changes the conformation of the heterotrimeric AMPK complex, thereby elevating the phosphorylation of AMPKα at Thr172 and enhancing AMPK activity. Further mechanical studies in the mouse model and cell lines have uncovered the paradoxical functions of ΔN-LKB1. Unlike the classical LKB1, ΔN-LKB1 reduces cell polarity enhanced by FL-LKB1, restrains the cell growth inhibition regulated by FL-LKB1 in cancer cell lines, and confers tumor growth in subcutaneously implanted tumor mouse models, indicative of pro-tumor role of ΔN-LKB1 (
Figure 1The structure of LKB1 complex. A. Diagram of the exon structure of the LKB1 mRNA. LKB1 gene consists of 10 exons and three isoforms are translated through alternative splicing. The start codon (ATG) of both FL-LKB1 and S-LKB1 is in the Exon 2 and the translation initiation codon of ΔN-LKB1 is in Exon 3. The stop codons of all three isoforms are in the Exon9. B. Structure of human FL-LKB1, S-LKB1, ΔN-LKB1, and the other two components of LKB1 complex, STRAD and MO25. The nuclear localization signals are localized in the N-terminal regulatory domain, while the farnesylation motif is localized in the C-terminal regulatory domain. C430 is the farnesylation residue. C, cysteine; 5’UTR, 5’ untranslated region; 3’UTR, 3’ untranslated region; NRD, N-terminal regulatory domain; CKD, catalytic kinase domain; CRD, C-terminal regulatory domain. Created with BioRender.com
). The analysis of the LKB1/STRAD/MO25 complex structure by Zeqiraj E. et al. reveals that STRAD adopts a closed conformation maintained through its cooperative binding to adenosine triphosphate (ATP) and MO25. STRAD subsequently binds to the kinase domain of LKB1 through its pseudokinase domain, which promotes the interaction between MO25 and LKB1 and the shuttling of LKB1 from the nucleus to the cytoplasm. The interaction with STRAD/MO25 also triggers the conformational change and the activation of LKB1. Meanwhile, MO25 stabilizes the activation loop of LKB1 in a conformation required for phosphorylating its substrates (
). In gastric cancer cells, LKB1 is also found in complexes with STRAD and MO25-like protein, the paralog of MO25, which induces the activation of AMPK and the expression of peroxisome proliferator-activated receptor-gamma coactivator 1 α (PGC1α)-mediated genes involved in mitochondrial respiration complexes (
). In another study of the mechanism underlying nuclear-cytoplasm shuttling of LKB1 in hepatocyte and diabetes mouse models, the orphan nuclear receptor Nur77 is reported to interact with LKB1 through its ligand binding domain and retain LKB1 in the nucleus (
). Upon activation in the cytosol, LKB1 generally binds to and phosphorylates the downstream targets. Of note is that the study by Shengcai Lin group has demonstrated that glucose starvation triggers the formation of v-ATPase-Ragulator complex on the lipid raft region of late endosome/lysosome and subsequently promotes the translocation of LKB1 to lysosome, ultimately leading to AMPK activation and switching from anabolism to catabolism in cells (
). Cumulatively, the subcellular localization of LKB1 is crucial for its activation and interaction with downstream substrates. In this section, we will review recent studies that have uncovered both tumor-suppressive and tumor promoting roles of LKB1 by rewiring cell metabolism, disrupting cell polarity, abrogating genomic integrity, and suppressing immune response.
). Other earlier studies have reported that LKB1 acts through AMPK/mTOR axis to modulate multiple metabolic pathways, including glycolysis, hexosamine biosynthesis pathway (HBP), pentose phosphate pathway (PPP), tricarboxylic acid cycle (TCA), Urea cycle, autophagosome, ferroptosis, as well as asparagine (Asn)- aspartate (Asp) homeostasis (
Figure 2LKB1 regulates diverse metabolic pathways in cancer cells. Generally, LKB1 regulates metabolic signaling mainly through AMPK/mTORC1 axis. AMPK inhibits key enzymes from hexosamine biosynthesis pathway (HBP), fatty acid synthesis, and urea cycle in direct or indirect manner. AMPK also promotes autophagy through direct phosphorylation of p27 and ULK1. In addition, AMPK inhibits mTORC1 leading to inhibiting autophagy, glycolysis, nucleotide synthesis, and protein synthesis. LKB1 also shows to regulate autophagy by promoting the translocation of ATG2/WIPI4 from ATG2/WIPI4/AMPK/ULK1 complex to autophagosome. LKB1 can directly interact with Ru5P and asparagine (Asn) to inhibit the activation of LKB1. Additionally, aspartate (Asp) directly binds to LKB1 and regulates its phosphorylation and activation. The arrowed line and arrowed line with fading tail represent promotion and translocation, respectively. The red stop line represents inhibition. The solid line and dashed line represent that regulation is in a direct or indirectly manner, respectively. Created with BioRender.com
In KRAS-mutant NSCLC, glucose and glutamine metabolism is usually enhanced through the induction of metabolism-associated enzymes or transporter. Loss of LKB1 forces the flux to the HBP by relieving the LKB1/AMPK-mediated suppression of glutamine-fructose-6-phosphate transaminase [Isomerizing] 2 (GFPT2), thus increasing protein glycosylation and promoting cell proliferation and survival (
). In KRAS/LKB1 co-mutant lung cancer, the urea cycle enzyme carbamoyl phosphate synthetase-1 (CPS1), which might be indirectly inhibited by LKB1/AMPK, is highly expressed. CPS1 promotes the non-canonical pathway of nitrogen flow from ammonia into pyrimidines by catalyzing the production of carbamoyl phosphate from ammonia and bicarbonate in the mitochondria, maintains the nucleotide homeostasis, and enables lung cancer cell survival and proliferation (
). The deficiency of both LKB1 and PTEN in cells hyperactivates phosphoinositide 3-kinase (PI3K)/Akt signaling. LKB1 deficiency also promotes the expression and activation of β-catenin, and the expression of glycogen synthase kinase 3 β (GSK3β) but inhibits the phosphorylation of GSK3β at Ser9 (
). PTEN is a well-characterized tumor suppressor, whose subcellular localization is vital to its functions. PTEN is localized in the nucleus and exported from the nucleus into the cytoplasm upon the activation of PI3K/Akt/mammalian target of rapamycin (mTOR)/S6 kinase (S6K) cascade during the G1/S transition (
). In LKB1-null A549 cells, PTEN is restrained in the nucleus upon the treatment of ATP or a calmodulin-dependent protein kinase kinase (CaMKK) activator but not AMPK activator metformin. Reintroduction of LKB1 in LKB1-null A549 cells recovers the metformin-regulated blockade of the nuclear-cytoplasm trafficking, suggesting that LKB1 might regulate the nuclear-cytoplasm shuttling of PTEN through AMPK or non-AMPK signaling (
). Collectively, these studies suggest that LKB1 deficiency results in metabolic alterations to favor tumor growth.
The canonical target of LKB1 is the metabolic AMPK, which is a key regulator of energy metabolism and metabolic homeostasis in eukaryotes. Under energy stress, AMPK coordinates glucose and lipid metabolism to maintain the intracellular ATP level (
). Under the stimulation with metabolic signals and stressors, the cytosolic activated-LKB1 phosphorylates AMPKα at Thr172 leading to AMPK activation and inhibiting multiple downstream signaling, such as the mammalian target of rapamycin complex 1 (mTORC1), which restrains the anabolic processes, especially lipid metabolism (
). In the absence of LKB1 or AMPK, cancer cells exhibit hyperactivated mTOR signaling and upregulated hypoxia inducible factor 1 α (HIF1α), as well as increased glucose utilization (
). Silencing LKB1 attenuated the suppression of mTOR without altering mTOR expression, which leads to aberrant proliferating immature chondrocytes and the formation of enchondroma-like tumors (
). In mouse embryonic fibroblasts (MEFs), the introduction of high-risk human papillomavirus (HPV) 16 E6/E7 promotes cell proliferation and elevates glucose metabolism by inducing c-Myc–mediated expression of hexokinase 2 (HK2). Ectopic expression of LKB1 inhibits the activation of mTOR in an AMPK-dependent manner and the expression of c-Myc and its downstream HK2, resulting in retarding the HPV16-driven tumor progression (
). Therefore, cancer cells deficient for LKB1 typically exhibit decreased AMPK activity and increased mTOR activity, while restoration of LKB1 could restrict tumor progression.
LKB1 has been reported to regulate autophagy directly or indirectly. LKB1 promotes autophagy by boosting AMPK-driven phosphorylation of Unc-51 like autophagy activating kinase 1 (ULK1) at Ser317 and Ser777 and diminishing mTOR-mediated inhibitory phosphorylation of ULK1 at Ser 757 (
). Under the condition of energy stress in response to cancer therapies, such as chemotherapy, the activated LKB1/AMPK phosphorylates p27 at Thr198 leading to p27 stabilization and sustaining cell survival through the induction of autophagy (
). Upon amino-acid starvation, LKB1 mediates the dissociation of WD repeat protein interacting with phosphoinositide 4 (WIPI4)-autophagy-related 2 (ATG2) from the WIPI4-ATG2/AMPK-ULK1 complex and promotes the translocation of WIPI4-ATG2 to nascent autophagosomes, which constrains the size of autophagosomes (
). Ferroptosis characterized by accumulating reactive oxygen species (ROS) and lipid peroxides is shown to suppress tumor growth but also enhance tumor growth by promoting immunosuppression in the tumor microenvironment (
). Ferroptosis induced by energy stress activates LKB1-AMPK signaling, which results in the inhibition of lipid synthesis through inhibitory phosphorylation of acetyl coenzyme A (acetyl-CoA) carboxylase 1 (ACC1) and successively confers the resistance to ferroptosis (
Interestingly, metabolites have been recently demonstrated to coordinate the activation of LKB1 through direct binding. In tumors with upregulated 6-phosphogluconate dehydrogenase (6PGD), the third enzyme in the oxidative PPP, 6-phosphogluconate (G6P) is rapidly converted to ribulose-5-phosphate (Ru5P). Ru5P binds to LKB1 and disrupts the formation of LKB1/STRAD/MO25 complex without altering its protein levels. This process subsequently inhibits the AMPK/ACC1-mediated lipogenesis and eventually promotes cancer cell proliferation and tumor growth (
). A high level of intracellular Asp directly binds to LKB1, leading to phosphorylation and activation of LKB1. The activated LKB1 activates AMPK/p53 cascade and thus suppresses the expression of asparagine synthetase (ASNS) expression, which eventually promotes tumor cell growth by hampering the catalyzed conversion from Asp to Asn. However, Asn can also directly bind to LKB1 and interfere with the activation of LKB1 and its downstream signaling activation (
). Interestingly, LKB1 can regulate mitochondrial TCA cycle and subsequent cytokine Interleukin 17 (IL17) expression to control the pool of T helper 17 (TH17) cells (
). Taken together, the tumor suppressor LKB1 plays a central role in regulating metabolic signaling mainly through both AMPK-dependent and independent mechanisms.
2.2 LKB1 deficiency promotes cancer metastasis.
Metastasis leading to cancer recurrence is the leading cause of cancer mortality. The metastasis cascade comprises diverse biological processes including invasion, intravasation, circulation, extravasation, formation of pre-metastatic niche, micro-metastasis, and metastatic colonization (
). An organized polarized epithelial structure, which is strictly regulated by cell polarity, preserves structural integrity and homeostasis, and obstructs unrestrained proliferation and metastasis. Deficiency of epithelial integrity is one of the hallmarks in the advanced cancer (
). In Caenorhabditis Elegans (C. Elegans), partitioning-defective 4 (Par-4), the ortholog of LKB1, has been associated with embryonic asymmetry through the regulation of cell polarity (
In the 3D organotypic model of mammary acini, chronic activation of c-Myc causes hyperproliferation and transformed acinar morphology, whereas acute activation of c-Myc in mature organoids with established epithelial architecture confers no alteration in cell cycle and morphology. However, silencing LKB1 synergizes with acute activation of c-Myc to disrupt the cell polarity and epithelial structure and enables aberrant proliferation of epithelial cells (
). Loss of LKB1 also promotes c-Myc transcription by upregulating myeloid zinc finger 1 (MZF1) expression, which facilitates lung tumor growth, migration, and invasion (
) (Fig. 3). Disrupted basal polarity and aberrant proliferation of transformed fallopian tube epithelial cells are also detected in LKB1/p53 co-mutant tumor tissues (
). A further mechanistic study has revealed that LKB1 deficiency might drive the reallocation of a type Ⅱ transmembrane serine protease Hepsin from the cell surface to the cytoplasm, resulting in subsequently epithelial disorganization (
). Meanwhile, LKB1 deficiency in stromal cells, such as mesenchymal cells, reduces the production of tumor growth factor β (TGF-β) and contributes to gastrointestinal polyposis by expanding epithelial cells (
Figure 3The pathways are involved in LKB1-modulated metastasis. In the absence of LKB1, cells exhibit high level of Sox17 and MZF1 and hyperactivation of NF-κB. Sox17 enters the nucleus and binds to the chromatin, thereby enhances the gene accessibility and the transcription activity of metastasis-associated genes. MZF1 induces the expression of c-Myc that promotes the transcription of NEDD9, VEGFC, CD24, and MMP2. Hyperactivation of NF-κB impedes the production of ROS. Additionally, loss of LKB1 facilitates the translocation of Hepsin from Desmosome on the membrane to the cytoplasm. ROS also activates LKB1 and its downstream MKK3/6-p38/AFT2 axis. Interestingly, LKB1 also modulates anoikis, formation of actomyosin, activity of YAP, HIF1α/LOX/β-integrin-mediated collagen deposition, and expression and degradation of Snail. Thus, loss LKB1 promotes anoikis resistance and snail-mediated metastasis. Created with BioRender.com
To migrate to the distant sites, cancer cells first detach from the primary sites into circulation. Anchorage-independent growth activates LKB1/SIK1 axis and induces the association of SIK1 with its substrates. SIK1 regulates the phosphorylation and abundance of p53, thereby stimulating anoikis to restrict metastasis (
). Anoikis, a form of cell death due to the detachment from the matrix and excessive reactive oxygen species (ROS), is one of the main barriers to metastasis (
). In LKB-null tumor cells, it has also been reported that α-ketoglutarate (α-KG) binds to CaMKK2 and recruits AMPK to CaMKK2, which promotes AMPK-dependent anoikis resistance and tumor metastasis (
). Additionally, the expression of lysyl oxidase (LOX) is increased via activated mTOR/HIF1α signaling leading to enhancing collagen deposition and promoting anchorage-independent tumor growth and migration in LKB1-deficient NSCLC by activating β1 integrin signaling (
). Increased ROS level promotes the binding of LKB1 to the Cdc42/p21-activating kinases 1 (PAK1) complex, which triggers the activation of mitogen-activated protein kinase-kinase (MKK) 3/6-p38-activating transcription factor 2 (ATF2) cascade and the expression of anti-oxidative enzymes to protect cells against oxidative damage (
). In LKB1/NUAK1-deficient epithelial ovarian cancer (EOC) cells and the spheroid model of high-grade serous ovarian cancer, the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) signaling pathway is hyperactivated to suppress ROS production and maintain cell survival (
). Upon cell detachment, LKB1-activated NUAK1 interacts with protein phosphatase 1β (PP1β)/phosphorylates the myosin phosphatase targeting 1 (MYPT1) complex and phosphorylates MYPT1 at Ser445, Ser472, and Ser90, which stimulates 14-3-3 binding to MYPT1 and blocks its interaction with myosin, thereby suppressing the dephosphorylation of myosin light chain-2 (MLC2) at Ser19 and Thr18 by PP1β/MYPT1 complex. Inhibition of the LKB1/NUAK1 axis impairs the formation of the actomyosin “contractile ring” and promotes cell adhesion, in turn facilitating metastatic colonization (
Moreover, previous studies have reported the abundance of metastasis- and angiogenesis-related proteins in LKB1-deficient tumor cells. LKB1-null tumor cells express a high level of metastasis-related Snail (
). LKB1-activated MARK1/4 phosphorylate Dishevelled-Axin domain containing 1 (DIXDC1) at Ser592, which facilitates the proper localization of DIXDC1 to focal adhesions and represses Snail expression in a FAK-mediated manner (
). In pancreatic cancer, LKB1 promotes the interaction of Snail and E3 ligase F-box and leucine-rich repeat protein 14 (FBXL14), which results in the ubiquitin-mediated degradation of Snail (
). Loss of LKB1 also promotes the expression of genes involved in angiogenesis and metastasis in lung adenocarcinomas, such as neural precursor cell expressed developmentally down-regulated protein 9 (NEDD9), vascular endothelial growth factor C (VEGFC), CD24, and matrix metallopeptidase 2 (MMP2) (
). Pierce et al. reported increased levels of Sox17 specifically in LKB1-deficiency metastatic tumors but not in primary tumors through CRIPSR-based mutagenesis screen. Further investigation identified a small population of cells in LKB1 deficient tumors with upregulated Sox17 in NSCLC. Mechanistically, Sox17 promotes chromatin accessibility and successively induces genotype-specific epigenic program, thereby enabling metastasis (
). For its role in YAP signaling, LKB1 promotes the localization of YAP in the cytoplasm and a remarkable reduction of YAP/TEAD transcriptional activity by inducing the phosphorylation of MST1/2 at Thr183/Thr180 and LATS1/2 at Thr1079 through MARK1/2/3/4. In LKB1-deficient tumors, the phosphorylation of YAP is repressed leading to YAP activation and upregulated downstream targets of YAP, such as angiomotin-like protein 2 (Amotl2) and Cysteine-rich angiogenic inducer 61 (Cyr61), which promote tumor growth and invasion (
2.3 LKB1 regulates genomic integrity and cell fate.
Accumulating studies have also indicated the role of LKB1 in regulating genome integrity and cell fate (Fig.4). LKB1 maintains Breast cancer type 1 (BRCA1) mRNA stability by retaining an RNA binding protein Hu Antigen R (HuR) inside the nucleus dependent on AMPK, which maintains genome integrity (
). In the UV-induced cancer model, UVB irradiation promotes both LKB1 and NUAK1 binding to and phosphorylating CDKN1A at Thr80 and Thr146, respectively, leading to the degradation of CDKN1A and DNA damage response. In the absence of LKB1, cells exhibit accumulated CDKN1A, elevated DNA double-strand breaks (DSBs), impaired homology-directed DNA repair, and resistance to apoptosis (
). In the LKB1-deficient NSCLC, cAMP response element-binding protein (CREB) regulated transcription coactivator 2 (CRTC2) remains dephosphorylated and persistently activated in the nucleus to promote the tumor growth and the apoptotic resistance via the induction of CREB target genes, such as the inhibitor of DNA binding 1 (ID1) (
). By generating a conditionally inactivated and restored Lkb1XTR allele, Christopher W.M. and colleagues demonstrated that LKB1 inactivation at the initial stage of tumorigenesis increases lung tumor burden and leads to the transformation of alveolar type Ⅱ cell into progenitor-like cells, which can be reversed by LKB1 restoration (
), indicative of the critical role of LKB1 in cell fate control.
Figure 4LKB1 regulates genomic integrity and cell fate. UVB stimulates NUAK1 and LKB1 bind to and phosphorylate CDKN1A, thus activating DNA repair. The cytosolic HuR binds to BRCA mRNA and induces its degradation. LKB1/AMPK inhibits the translocation of HuR from the nucleus to the cytoplasm, which protects cells from genotoxic stress. LKB1 inactivates CRTC2 through direct phosphorylation, leading to suppressing the transcription of ID1. LKB1 regulates stem-like phenotype and transdifferentiation by inhibiting the transcription of stem-associated genes and mTOR/HIF1α/LOX/β-integrin axis, respectively. Created with BioRender.com
Loss of LKB1 induces expression of stem cell-like genes, such as POU class 5 homeobox 1 (Pou5f1), Nanog, and Sox2, and promotes the formation of mammosphere in breast cancer (
). Recent studies in signatures of cancers with LKB1 mutation alone and in the combination of other gene mutations implicate the role of LKB1 in regulating transdifferentiation. One study demonstrates that LKB1 inactivation specifically drives KRAS-mutant lung adenocarcinoma with polycomb repressive complex 2 (PRC2) loss to squamous cell carcinoma (
). PRC2 is composed of enhancer of zeste homolog 2 (EZH2), embryonic ectoderm development (EED), and suppressor of zeste 12 (SUZ12) and catalyzes the methylation of H3 on lysine27 (
). Loss of PRC2 activates the transcription of squamous-associated genes, including nerve growth factor receptor (Ngfr), Sox2, DNp63, and keratin 5/6 (Krt5/6) (
). Inhibition of the elevated Lox in Lkb1-deficient lung adenocarcinoma provokes extracellular matrix remodeling and induces DNp63 expression, thereby promoting the transdifferentiation of lung adenocarcinoma into lung squamous cell carcinoma (
). Further mechanistic study reveals that activated YAP translocates into the nucleus and promotes zinc finger E-box binding homeobox (ZEB2) transcription by binding to the ZBE2 promoter region. Increased ZEB2 then represses DNp63 transcription to inhibit the transdifferentiation of lung adenocarcinoma to lung squamous cell carcinoma (
). Although the critical role of LKB1 inactivation in genomic integrity and cell fate control presented above, the detailed mechanisms by which LKB1 deficiency orchestrates these processes remain to be elucidated.
The 24-month observational multicentric study from 159 NSCLC patients, who have received chemotherapy or immunotherapy, has demonstrated that the median overall survival (OS) of patients harboring LKB1 mutation is significantly shorter than those with wild-type LKB1 (4.7 months versus 16.2 months, P < 0.001). The first-line treatment failure is also remarkedly shorter in LKB1-mutant NSCLC patients (
). In this section, we will summarize recent discoveries of the mechanisms underlying resistance to tyrosine kinase inhibitors (TKIs) and immunotherapy regulated by LKB1 deficiency.
2.4.1 LKB1 deficiency regulates resistance to TKIs
TKIs for epithelial growth factor receptor (EGFR) have been proven to treat tumor patients with EFGR mutations, especially EGFR mutant NSCLC. EGFR TKIs also show certain efficacy in NSCLC with wild-type EGFR by activating the intrinsic apoptosis pathway. LKB1 deficiency causes impaired AMPK activity and activated mTOR signaling, which can be selectively blocked by Erlotinib in LKB1-deficient NSCLC cells, which suggests that NSCLC cells with LKB1 deficiency might be more sensitive to EGFR TKIs, PI3K inhibitor, and rapamycin (
). However, another study in cigarette smoke-induced NSCLC revealed that LKB1 deficiency might contribute to resistance to EGFR TKIs. Upon treatment with cigarette smoke extract, all six regions of the LKB1 promoter are hypermethylated and thereby reducing the expression of LKB1, which contributes to the resistance to EGFR TKIs (Erlotinib and Gefitinib) via attenuating AMPK-mediated inhibition of mTOR. The result indicates that LKB1 expression is a critical determinant for the sensitivity to EGFR TKI in NSCLC with EGFR WT (
). Whether the LKB1 expression could be a potential biomarker for TKI treatment varies from distinct contexts and requires further investigation.
2.4.2 LKB1 deficiency regulates resistance to immune checkpoint inhibitors (ICIs)
The emergence of immunotherapy, especially ICIs, changes the treatment paradigm for solid cancers including NSCLC. Numerous clinical trials have demonstrated that the OS or progression-free survival (PFS) of NSCLC patients who received ICIs is remarkably prolonged compared to those who receive chemotherapies (
Pembrolizumab versus chemotherapy for previously untreated, PD-L1-expressing, locally advanced or metastatic non-small-cell lung cancer (KEYNOTE-042): a randomised, open-label, controlled, phase 3 trial.
Updated Analysis From KEYNOTE-189: Pembrolizumab or Placebo Plus Pemetrexed and Platinum for Previously Untreated Metastatic Nonsquamous Non-Small-Cell Lung Cancer.
). Skoulidis F. et al. have reported that LKB1 alteration is the key cause of primary resistance to anti-programmed death-1 (PD-1) immunotherapy in KRAS-mutant NSCLC by analyzing online datasets and utilizing Kras-mutant murine lung adenocarcinoma models (
). Retrospective analysis of phase Ⅰ/Ⅱ clinical studies of NSCLC patients treated with anti-programmed death-ligand 1 (PD-L1) or anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) immunotherapy shows that LKB1 mutation is significantly associated with poor OS and poor objective response to ICIs (NCT01693562, NCT02087423, and NCT02000947) (
Resistance to Durvalumab and Durvalumab plus Tremelimumab Is Associated with Functional STK11 Mutations in Patients with Non-Small Cell Lung Cancer and Is Reversed by STAT3 Knockdown.
). Increasing evidence has implicated the impact of LKB1 alteration on the tumor microenvironment (TME), which confers resistance to the immunotherapy (
To analyze the immune and metabolic signatures in LKB1-deficient tumors, the mice with homozygous deletion of Lkb1, Pten, or p53 as well as the Kras mutation developed lung cancer recapitulate human lung cancer. Specifically, Lkb1-/-/Pten-/- mice develop lung squamous carcinoma expressing a high level of CD274 (gene encoding PD-L1). A stem-like subpopulation expressing stem cell antigen-1 (SCA1) and NGFR shows more abundant PD-L1. The high PD-L1 in tumor cells might induce the filtration of regulatory T (Treg) cells expressing PD-1 (
). Foxp3-expressing Treg cells along with Th are a subset of CD4+ T cells that were originally found to prevent autoimmune disease and maintain tolerance of self-antigens (
). Studies have indicated that Treg suppresses anti-tumor immunity and that Treg cell infiltration is negatively associated with prognosis in cancer patients (
). Additionally, high tumor-associated neutrophils (TANs) (CD45+CD11b+Ly6G+) and low tumor-associated macrophages (TAMs) (CD45+CD11c+CD11b-CD103-) infiltration have been detected in the Lkb1-/-/Pten-/- tumors, which might be regulated by elevating C-X-C motif chemokine ligand (CXCL) 1, CXCL2, CXCL5 and CXCL7 in bronchoalveolar lavage fluid (
). A similar population of TANs and TAMs is also detected in the KrasG12D/+/Lkb1-/- mouse tumor model. In KrasG12D/+/Lkb1-/- tumor cells, Lkb1 loss induces the production of IL-1α. In turn, IL-1α activates the production of IL-6, CXCL7, and granulocyte-colony stimulating factor (G-CSF) in a signal transducer and activator of transcription 3 (STAT3)-dependent manner, thereby recruiting TANs and subsequently leading to a reduction in total CD8+ T cells and impaired proliferation and function of CD8+ T cells (
STK11/LKB1 Deficiency Promotes Neutrophil Recruitment and Proinflammatory Cytokine Production to Suppress T-cell Activity in the Lung Tumor Microenvironment.
). In the Lenti-Sox2-cre-infected KrasG12D/+/p53-/- lung cancer mouse model, highly expressed Sox2 suppresses NK2 homebox 1 (NKX2-1) activity and induces CXCL5 expression. The Lenti-CXCL5-cre-infected KrasG12D/+/p53-/- lung cancer mouse model displays a high expression of CXCL5 and a remarkable accumulation of neutrophils, further confirming that CXCL5 promotes the accumulation of tumor-derived neutrophils and subsequently accelerates tumorigenesis (
). Elevated TGF-β and IL-6, which have been shown to promote tumor growth and induce immunosuppressive TME, are also increased in bronchoalveolar lavage fluid (
STK11/LKB1 Deficiency Promotes Neutrophil Recruitment and Proinflammatory Cytokine Production to Suppress T-cell Activity in the Lung Tumor Microenvironment.
STK11/LKB1 Deficiency Promotes Neutrophil Recruitment and Proinflammatory Cytokine Production to Suppress T-cell Activity in the Lung Tumor Microenvironment.
). In the KrasG12D/Lkb1-/- mutant NSCLC mouse model, combined therapy of radiotherapy and anti-PD1 immunotherapy increases the infiltration of CD8+ T cells expressing increased inhibitory lymphocyte activation gene 3 (LAG3) and T-cell immunoglobulin and mucin-domain containing-3 (TIM3), thus contributing to poor therapeutic efficacy (
). Knocking down LKB1 in normal bronchial epithelial cells and Kras-mutant NSCLC cells upregulates a panel of the CXC chemokines with an NH2-terminal Glu-Leu-Arg motif, including CXCL1, CXCL2, CXCL3, CXCL5, and CXCL8. In the KrasG12D/Lkb1-/- or KrasG12D/Lkb1-/-/p53-/- mouse model, the CXC chemokines are also elevated, accompanied by increased accumulation of granulocytic myeloid-derived suppressor cells (G-MDSC) in both TME and peripheral blood and spleen. Depletion of G-MDSC sensitizes LKB1-deficient tumors to anti-PD-1 therapy by boosting the proliferation and function of tumor-infiltrating lymphocytes (
). LKB1-deficient NSCLC tumors also show defective TCF1+CD8+ T cells and mouse KrasG12D/Lkb1-/-/p53-/- tumor exhibits poor response to anti-PD-1 immunotherapy. Inhibition of Axl by Bemcentinib sensitizes LKB1-deficient tumors to anti-PD-1 immunotherapy by inducing the secretion of type I interferon by activated dendritic cells (DCs) and the expansion of TCF1+CD8+ T cells (
In the retrospective study of immune phenotype in NSCLC patients with LKB1 mutation, the immune signatures are not completely concordant with those in mouse models. The LKB1-mutant tumors are characterized by the increased genes involved in the regulation of cytokine production by macrophages/neutrophils and TH17 cells, as well as the decreased baseline and posttreatment quantities of NK cells, CD4+ effector memory, CD4+ HLA-DR+, CD8+ effector memory, and CD8+ HLA-DR+ T cells. The single-cell RNA sequencing and flow cytometry results reveal the elevating intratumoral Ly6G+ granulocytes and CD163+ TAMs in Lkb1-KO tumors, as well as the upregulation of immunosuppressive cytokines and chemokines and low expression of PD-L1 in CD45- tumor cells, which might contribute to insensitivity to ICIs treatment. Further analysis of proteomics data from the TCGA database reveals the positive association between phosphor-STAT3 and LKB1 mutation. Inhibition of STAT3 potentiates Lkb1-mutant tumors to ICIs in a mouse model by enhancing the function of antigen-presenting cells (
Resistance to Durvalumab and Durvalumab plus Tremelimumab Is Associated with Functional STK11 Mutations in Patients with Non-Small Cell Lung Cancer and Is Reversed by STAT3 Knockdown.
). It has also been reported that administration of anti-PD-1 immunotherapy in combination with CCL7 could promote the DCs and CD8+ T cell recruitment in KrasG12D/Lkb1-/- mouse lung cancer resulting in inhibiting the cancer development and prolonging the mice survival (
Deng J. and colleagues reported higher tumor mutational burden (TMB) resulting from defects in DSBs repair regulated by LKB deficiency in LKB1-mutant NSCLC from nonsmoker and genetically engineered mouse models (
). However, NSCLC patients with KRAS/LKB1 co-mutation show poor response to immunotherapy, and tumors with KRAS/LKB1 co-mutation also exhibit reduced MHC Ⅰ on cell surface (
). Further mechanistic study implicates that LKB deficiency promotes autophagy and suppresses proteasomal degradation of antigenic peptides, which compromises antigen processing and presentation by MHC Ⅰ and ultimately immune evasion in KRAS/LKB1 co-mutant NSCLC (
). STING has been reported to regulate the innate immune response by sensing the abnormal cytosolic double-stranded DNA (dsDNA) via cyclic GMP-AMP synthase (cGAS) and therefore inducing type Ⅰ interferons and chemokines that recruit T cells (
). In particular, DNA (cytosine-5)-methyltransferase (DNMT1) and EZH2 regulate the methylation of STING promoter and impede its transcription, which fails to sense the cytosolic dsDNA from defective mitochondria for subsequent STAT1 signaling activation, eventually leading to impaired intratumoral T cell recruitment (
The metabolic signature has also been analyzed among KrasG12D/+, KrasG12D/+/Keap1-/-, KrasG12D/+/Lkb1-/-, and KrasG12D/+/Keap1-/-/Lkb1-/- mouse lung tumors. KrasG12D/+/Lkb1-/- tumor nodules specifically show increased glutaminase (Gls1) mRNA, rapid conversion from glutamine to glutamate, and significantly accumulated glutamate (
). Although studies have implicated that inhibition of glutamine metabolism in T cells might enhance the cytotoxic effector function of CD8+ T cells and shortly induce the exhaustion of cytotoxic CD8+ T cells, glutaminase inhibition fails to boost the efficacy of anti-PD-1 immunotherapy in KrasG12D/+/Lkb1-/- mouse lung cancer model (
Collectively, LKB1 deficiency in tumor cells alters the sensitivity of tumors to either targeted or immunotherapy by regulating immunomodulators and reprogramming metabolic networks inside tumor cells and in the TME, indicating that the impact of LKB1 deficiency on therapeutic resistance results from not only the monologue of tumor cells but the dialogue among tumor cells and stromal cells.
2.5 Oncogenic role of LKB1
Although dozens of studies have generally identified LKB1 as a tumor suppressor, multiple studies have reported the oncogenic role of LKB1 in tumorigenesis. One study in acute myeloid leukemia (AML) shows the pro-oncogenic role of LKB1. Tarumoto et al. reported that LKB1 phosphorylates the T-loop of SIK3 at Thr221 and activates SIK3. The activated SIK3 phosphorylates and prevents the histone deacetylase 4 (HDAC4)-mediated inhibition of myocyte enhancer factor 2C (MEF2C), which promotes AML proliferation (
). In HCC cells, LKB1 overexpression resulting in AMPK signaling activation to prevent energy stress-induced cell death enhances liver cancer development (
). Additionally, LKB1 is essential for the phosphorylation of Akt downstream targets in the context of constitutively activating Akt, which hampers apoptosis and favors tumor cell survival (
). Collectively, LKB1 appears to be an oncogenic player in certain contexts, and more investigations are required to comprehensively understand the functional role of LKB1 both in cancer cells and stromal cells and its underlying mechanisms in cancer regulation.
3. Posttranslational modifications of LKB1
Increasing evidence has suggested that the inactivation or deletion of both two alleles might be required for the recessive function of the tumor suppressor gene in the tumorigenesis (
). The LKB1’s mutation types, especially in NSCLC, include deep deletion, truncation mutation, splice mutation, missense mutation, inframe mutation, and structural variant, which might cause alterations in copy number (
). Further analysis of different mutations of LKB1, missense mutation accounts for 30% of the total and mainly causes loss-of-function of LKB1 protein by disrupting its kinase activity or localization (
). PTM is a biologically catalyzed process of covalently adding small chemical molecular groups, peptides, or complex molecules to the amino-acid sidechain of proteins, which often alters the conformation, properties, and localization of proteins and/or increases the diversity of proteome (
). A panel of PTMs have been identified in managing activity, cellular distribution, and stability of LKB1 in the past decades, ultimately orchestrating tumorigenesis. In this section, we will summarize and discuss the PTMs of LKB1, including phosphorylation, ubiquitination, SUMOylation, neddylation, prenylation, acetylation, ADP-ribosylation, S-nitrosylation, and HNE-adduction (Fig. 5 and Table 1).
Figure 5The regulators involved in PTM of LKB1A. the schematic figure of PTMs residues in LKB1 domains. B. Modification by varied enzymes regulates LKB1 function in biological processes. Enzymes known to modify LKB1 modulate its PTM state, thus in turn affecting various cellular functions as indicated. Solid arrows indicate promotion, dashed arrows indicate transferring, red stop lines indicate inhibition. Yellow balls indicate phosphorylation, red ball indicates ADP-ribosylation, purple balls indicate ubiquitination, orange balls indicate acetylation, grey ball indicates 4-HNE adduction, lavender ball indicates SUMOylation, and green ball indicates S-nitrosylation. SNO, S-nitrosylation; SUMO, SUMOylation; S, Ser, serine; K, Lys, lysine; E, Glu, glutamic acid; C, Cys, cysteine; T, Thr, threonine. Created with BioRender.com.
Identification and characterization of four novel phosphorylation sites (Ser31, Ser325, Thr336 and Thr366) on LKB1/STK11, the protein kinase mutated in Peutz-Jeghers cancer syndrome.
Identification of the serine 307 of LKB1 as a novel phosphorylation site essential for its nucleocytoplasmic transport and endothelial cell angiogenesis.
Identification and characterization of four novel phosphorylation sites (Ser31, Ser325, Thr336 and Thr366) on LKB1/STK11, the protein kinase mutated in Peutz-Jeghers cancer syndrome.
Identification and characterization of four novel phosphorylation sites (Ser31, Ser325, Thr336 and Thr366) on LKB1/STK11, the protein kinase mutated in Peutz-Jeghers cancer syndrome.
Phosphorylation of serine 399 in LKB1 protein short form by protein kinase Czeta is required for its nucleocytoplasmic transport and consequent AMP-activated protein kinase (AMPK) activation.
Phosphorylation of LKB1 at serine 428 by protein kinase C-zeta is required for metformin-enhanced activation of the AMP-activated protein kinase in endothelial cells.
Phosphorylation of the protein kinase mutated in Peutz-Jeghers cancer syndrome, LKB1/STK11, at Ser431 by p90(RSK) and cAMP-dependent protein kinase, but not its farnesylation at Cys(433), is essential for LKB1 to suppress cell vrowth.
Phosphorylation of the protein kinase mutated in Peutz-Jeghers cancer syndrome, LKB1/STK11, at Ser431 by p90(RSK) and cAMP-dependent protein kinase, but not its farnesylation at Cys(433), is essential for LKB1 to suppress cell vrowth.
Investigation of LKB1 Ser431 phosphorylation and Cys433 farnesylation using mouse knockin analysis reveals an unexpected role of prenylation in regulating AMPK activity.
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