Liver Kinase B1 Mediates Its Anti-Tumor Function by Binding to the N-Terminus of Malic Enzyme 3

Liver kinase B1 (LKB1) is a crucial tumor suppressor involved in various cellular processes, including embryonic development, tumor initiation and progression, cell adhesion, apoptosis, and metabolism. However, the precise mechanisms underlying its functions remain elusive. In this study, we demonstrate that LKB1 interacts directly with malic enzyme 3 (ME3) through the N-terminus of the enzyme and identified the binding regions necessary for this interaction. The binding activity was confirmed to promote the expression of ME3 in an LKB1-dependent manner and was also shown to induce apoptosis activity. Furthermore, LKB1 and ME3 overexpression upregulated the expression of tumour suppressor proteins (p53 and p21) and downregulated the expression of antiapoptotic proteins (nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and B-cell lymphoma 2 (Bcl-2)). Additionally, LKB1 and ME3 enhanced the transcription of p21 and p53 and inhibited the transcription of NF-κB. Moreover, LKB1 and ME3 suppressed the phosphorylation of various components of the phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B signaling pathway. Overall, these results suggest that LKB1 promotes pro-apoptotic activities by inducing ME3 expression.

LKB1/STK11 Tumor Suppressor Reduces Angiogenesis by Directly Interacting with VEGFR2 in Tumorigenesis

Cervical tumors represent a prevalent form of cancer affecting women worldwide; current treatment options involve surgery, radiotherapy, and chemotherapy. Angiogenesis, the process of new blood vessel formation, is a crucial factor in cervical tumor growth. The molecular mechanisms underlying the effects of the liver kinase B1 (LKB1/STK11) tumor suppressor protein on tumor angiogenesis have not been elucidated. Therefore, we investigated the role of LKB1 in cervical tumor angiogenesis both in vitro and in vivo in this study. Our results demonstrated that LKB1 inhibited cervical tumor angiogenesis by suppressing the expression of angiogenesis-related factors such as vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1α. LKB1 directly affected both carcinoma and vascular endothelial cells, resulting in a significant reduction in tumor growth and angiogenesis. Furthermore, LKB1 was found to bind to VEGF receptor 2 (VEGFR-2) and target the VEGFR-2-mediated protein kinase B/ mechanistic target of rapamycin signaling pathway in endothelial cells, thereby reducing cervical tumor growth and angiogenesis.Our study provides new insights into the molecular mechanisms underlying the anti-tumor and anti-angiogenic effects of LKB1 in cervical cancer. These findings will help develop new therapeutic strategies for cervical cancer.

Snail Promotes Cancer Cell Proliferation via Its Interaction with the BIRC3

Snail is implicated in tumour growth and metastasis and is up-regulated in various human tumours. Although the role of Snails in epithelial-mesenchymal transition, which is particularly important in cancer metastasis, is well known, how they regulate tumour growth is poorly described. In this study, the possible molecular mechanisms of Snail in tumour growth were explored. Baculoviral inhibitor of apoptosis protein (IAP) repeat-containing protein 3 (BIRC3), a co-activator of cell proliferation during tumourigenesis, was identified as a Snail-binding protein via a yeast two-hybrid system. Since BIRC3 is important for cell survival, the effect of BIRC3 binding partner Snail on cell survival was investigated in ovarian cancer cell lines. Results revealed that Bax expression was activated, while the expression levels of anti-apoptotic proteins were markedly decreased by small interfering RNA (siRNA) specific for Snail (siSnail). siSnail, the binding partner of siBIRC3, activated the tumour suppressor function of p53 by promoting p53 protein stability. Conversely, BIRC3 could interact with Snail, for this reason, the possibility of BIRC3 involvement in EMT was investigated. BIRC3 overexpression resulted in a decreased expression of the epithelial marker and an increased expression of the mesenchymal markers. siSnail or siBIRC3 reduced the mRNA levels of matrix metalloproteinase (MMP)-2 and MMP-9. These results provide evidence that Snail promotes cell proliferation by interacting with BIRC3 and that BIRC3 might be involved in EMT via binding to Snail in ovarian cancer cells. Therefore, our results suggested the novel relevance of BIRC3, the binding partner of Snail, in ovarian cancer development.

PRR16/Largen Induces Epithelial-Mesenchymal Transition through the Interaction with ABI2 Leading to the Activation of ABL1 Kinase

Advanced or metastatic breast cancer affects multiple organs and is a leading cause of cancer-related death. Cancer metastasis is associated with epithelial-mesenchymal metastasis (EMT). However, the specific signals that induce and regulate EMT in carcinoma cells remain unclear. PRR16/Largen is a cell size regulator that is independent of mTOR and Hippo signalling pathways. However, little is known about the role PRR16 plays in the EMT process. We found that the expression of PRR16 was increased in mesenchymal breast cancer cell lines. PRR16 overexpression induced EMT in MCF7 breast cancer cells and enhances migration and invasion. To determine how PRR16 induces EMT, the binding proteins for PRR16 were screened, revealing that PRR16 binds to Abl interactor 2 (ABI2). We then investigated whether ABI2 is involved in EMT. Gene silencing of ABI2 induces EMT, leading to enhanced migration and invasion. ABI2 is a gene that codes for a protein that interacts with ABL proto-oncogene 1 (ABL1) kinase. Therefore, we investigated whether the change in ABI2 expression affected the activation of ABL1 kinase. The knockdown of ABI2 and PRR16 overexpression increased the phosphorylation of Y412 in ABL1 kinase. Our results suggest that PRR16 may be involved in EMT by binding to ABI2 and interfering with its inhibition of ABL1 kinase. This indicates that ABL1 kinase inhibitors may be potential therapeutic agents for the treatment of PRR16-related breast cancer.

Knockdown of LKB1 Sensitizes Endometrial Cancer Cells via AMPK Activation

Metformin is an anti-diabetic drug and has anticancer effects on various cancers. Several studies have suggested that metformin reduces cell proliferation and stimulates cell-cycle arrest and apoptosis. However, the definitive molecular mechanism of metformin in the pathophysiological signaling in endometrial tumorigenesis and metastasis is not clearly understood. In this study, we examined the effects of metformin on the cell viability and apoptosis of human cervical HeLa and endometrial HEC-1-A and KLE cancer cells. Metformin suppressed cell growth in a dose-dependent manner and dramatically evoked apoptosis in HeLa cervical cancer cells, while apoptotic cell death and growth inhibition were not observed in endometrial (HEC-1-A, KLE) cell lines. Accordingly, the p27 and p21 promoter activities were enhanced while Bcl-2 and IL-6 activities were significantly reduced by metformin treatment. Metformin diminished the phosphorylation of mTOR, p70S6K and 4E-BP1 by accelerating adenosine monophosphateactivated kinase (AMPK) in HeLa cancer cells, but it did not affect other cell lines. To determine why the anti-proliferative effects are observed only in HeLa cells, we examined the expression level of liver kinase B1 (LKB1) since metformin and LKB1 share the same signalling system, and we found that the LKB1 gene is not expressed only in HeLa cancer cells. Consistently, the overexpression of LKB1 in HeLa cancer cells prevented metformin-triggered apoptosis while LKB1 knockdown significantly increased apoptosis in HEC-1-A and KLE cancer cells. Taken together, these findings indicate an underlying biological/physiological molecular function specifically for metformin-triggered apoptosis dependent on the presence of the LKB1 gene in tumorigenesis.

Novel Anti-Angiogenic and Anti-Tumour Activities of the N-Terminal Domain of NOEY2 via Binding to VEGFR-2 in Ovarian Cancer

The imprinted tumour suppressor NOEY2 is downregulated in various cancer types, including ovarian cancers. Recent data suggest that NOEY2 plays an essential role in regulating the cell cycle, angiogenesis and autophagy in tumorigenesis. However, its detailed molecular function and mechanisms in ovarian tumours remain unclear. In this report, we initially demonstrated the inhibitory effect of NOEY2 on tumour growth by utilising a xenograft tumour model. NOEY2 attenuated the cell growth approximately fourfold and significantly reduced tumour vascularity. NOEY2 inhibited the phosphorylation of the signalling components downstream of phosphatidylinositol-3’-kinase (PI3K), including phosphoinositide-dependent protein kinase 1 (PDK-1), tuberous sclerosis complex 2 (TSC-2) and p70 ribosomal protein S6 kinase (p70S6K), during ovarian tumour progression via direct binding to vascular endothelial growth factor receptor-2 (VEGFR-2). Particularly, the N-terminal domain of NOEY2 (NOEY2-N) had a potent anti-angiogenic activity and dramatically downregulated VEGF and hypoxia-inducible factor-1α (HIF-1α), key regulators of angiogenesis. Since no X-ray or nuclear magnetic resonance structures is available for NOEY2, we constructed the threedimensional structure of this protein via molecular modelling methods, such as homology modelling and molecular dynamic simulations. Thereby, Lys15 and Arg16 appeared as key residues in the N-terminal domain. We also found that NOEY2-N acts as a potent inhibitor of tumorigenesis and angiogenesis. These findings provide convincing evidence that NOEY2-N regulates endothelial cell function and angiogenesis by interrupting the VEGFR-2/PDK-1/GSK-3β signal transduction and thus strongly suggest that NOEY2-N might serve as a novel anti-tumour and anti-angiogenic agent against many diseases, including ovarian cancer.

Novel Anti-Angiogenic and Anti-Tumour Activities of the N-Terminal Domain of NOEY2 via Binding to VEGFR-2 in Ovarian Cancer

The imprinted tumour suppressor NOEY2 is downregulated in various cancer types, including ovarian cancers. Recent data suggest that NOEY2 plays an essential role in regulating the cell cycle, angiogenesis and autophagy in tumorigenesis. However, its detailed molecular function and mechanisms in ovarian tumours remain unclear. In this report, we initially demonstrated the inhibitory effect of NOEY2 on tumour growth by utilising a xenograft tumour model. NOEY2 attenuated the cell growth approximately fourfold and significantly reduced tumour vascularity. NOEY2 inhibited the phosphorylation of the signalling components downstream of phosphatidylinositol-3’-kinase (PI3K), including phosphoinositide-dependent protein kinase 1 (PDK-1), tuberous sclerosis complex 2 (TSC-2) and p70 ribosomal protein S6 kinase (p70S6K), during ovarian tumour progression via direct binding to vascular endothelial growth factor receptor-2 (VEGFR-2). Particularly, the N-terminal domain of NOEY2 (NOEY2-N) had a potent anti-angiogenic activity and dramatically downregulated VEGF and hypoxia-inducible factor-1α (HIF-1α), key regulators of angiogenesis. Since no X-ray or nuclear magnetic resonance structures is available for NOEY2, we constructed the threedimensional structure of this protein via molecular modelling methods, such as homology modelling and molecular dynamic simulations. Thereby, Lys15 and Arg16 appeared as key residues in the N-terminal domain. We also found that NOEY2-N acts as a potent inhibitor of tumorigenesis and angiogenesis. These findings provide convincing evidence that NOEY2-N regulates endothelial cell function and angiogenesis by interrupting the VEGFR-2/PDK-1/GSK-3β signal transduction and thus strongly suggest that NOEY2-N might serve as a novel anti-tumour and anti-angiogenic agent against many diseases, including ovarian cancer.

Cardamonin Suppresses TGF-beta1-Induced Epithelial Mesenchymal Transition via Restoring Protein Phosphatase 2A Expression

Epithelial mesenchymal transition (EMT) is the first step in metastasis and implicated in the phenotype of cancer stem cells. Therefore, understanding and controlling EMT, are essential to the prevention and cure of metastasis. In the present study, we examined, by Western blot, reverse transcription polymerase chain reaction (RT-PCR), and confocal microscopy, the effects of cardamonin (CDN) on transforming growth factor-beta1 (TGF-beta1)-induced EMT of A549 lung adenocarcinoma cell lines. TGF-beta1 induced expression of N-cadherin and decreased expression of E-cadherin. CDN suppressed N-cadherin expression and restored E-cadherin expression. Further, TGF-beta1 induced migration and invasion of A549 cancer cells, which was suppressed by CDN. TGF-beta1 induced c-Jun N-terminal kinase (JNK) activation during EMT, but CDN blocked it. Protein serine/threonine phosphatase 2A (PP2A) expression in A549 cancer cells was reduced by TGF-beta1 but CDN restored it. The overall data suggested that CDN suppresses TGF-beta1-induced EMT via PP2A restoration, making it a potential new drug candidate that controls metastasis.

12-O-Tetradecanoylphorbol-13-Acetate Induces Keratin 8 Phosphorylation and Reorganization via Expression of Transglutaminase-2

The stiffness of cancer cells is attributable to intermediate filaments such as keratin. Perinuclear reorganization via phosphorylation of specific serine residue in keratin is implicated in the deformability of metastatic cancer cells including the human pancreatic carcinoma cell line (PANC-1). 12-O-Tetradecanoylphorbol-13-acetate (TPA) is a potent tumor promoter and protein kinase C (PKC) activator. However, its effects on phosphorylation and reorganization of keratin 8 (K8) are not well known. Therefore, we examined the underlying mechanism and effect of TPA on K8 phosphorylation and reorganization. TPA induced phosphorylation and reorganization of K8 and transglutaminase-2 (Tgase-2) expression in a time- and dose-dependent manner in PANC-1 cells. These effects peaked after 45 min and 100 nM of TPA treatment. We next investigated, using cystamine (CTM), Tgase inhibitor, and Tgase-2 gene silencing, Tgase-2's possible involvement in TPA-induced K8 phosphorylation and reorganization. We found that Tgase-2 gene silencing inhibited K8 phosphorylation and reorganization in PANC-1 cells. Tgase-2 gene silencing, we additionally discovered, suppressed TPA-induced migration of PANC-1 cells and Tgase-2 overexpression induced migration of PANC-1 cells. Overall, these results suggested that TPA induced K8 phosphorylation and reorganization via Tgase-2 expression in PANC-1 cells.

Involvement of Transglutaminase-2 in alpha-MSH-Induced Melanogenesis in SK-MEL-2 Human Melanoma Cells

Skin hyperpigmentation is one of the most common skin disorders caused by abnormal melanogenesis. The mechanism and key factors at play are not fully understood. Previous reports have indicated that cystamine (CTM) inhibits melanin synthesis, though its molecular mechanism in melanogenesis remains unclear. In the present study, we investigated the effect of CTM on melanin production using ELISA reader and the expression of proteins involved in melanogenesis by Western blotting, and examined the involvement of transglutaminase-2 (Tgase-2) in SK-MEL-2 human melanoma cells by gene silencing. In the results, CTM dose-dependently suppressed melanin production and dendrite extension in alpha-MSH-induced melanogenesis of SK-MEL-2 human melanoma cells. CTM also suppressed alpha-MSH-induced chemotactic migration as well as the expressions of melanogenesis factors TRP-1, TRP-2 and MITF in alpha-MSH-treated SK-MEL-2 cells. Meanwhile, gene silencing of Tgase-2 suppressed dendrite extension and the expressions of TRP-1 and TRP-2 in alpha-MSH-treated SK-MEL-2 cells. Overall, these findings suggested that CTM suppresses alpha-MSH-induced melanogenesis via Tgase-2 inhibition and that therefore, Tgase-2 might be a new target in hyperpigmentation disorder therapy.

Ethacrynic Acid Inhibits Sphingosylphosphorylcholine-Induced Keratin 8 Phosphorylation and Reorganization via Transglutaminase-2 Inhibition

Sphingosylphosphorylcholine (SPC) is significantly increased in the malicious ascites of tumor patients and induces perinuclear reorganization of keratin 8 (K8) filaments in PANC-1 cells. The reorganization contributes to the viscoelasticity of metastatic cancer cells resulting in increased migration. Recently, we reported that transglutaminase-2 (Tgase-2) is involved in SPC-induced K8 phosphorylation and reorganization. However, effects of Tgase-2 inhibitors on SPC-induced K8 phosphorylation and reorganization were not clearly studied. We found that ethacrynic acid (ECA) concentration-dependently inhibited Tgase-2. Therefore, we examined the effects of ECA on SPC-induced K8 phosphorylation and reorganization. ECA concentration-dependently suppressed the SPC-induced phosphorylation and perinuclear reorganization of K8. ECA also suppressed the SPC-induced migration and invasion. SPC induced JNK activation through Tgase-2 expression and ECA suppressed the activation and expression of JNK in PANC-1 cells. These results suggested that ECA might be useful to control Tgase-2 dependent metastasis of cancer cells such as pancreatic cancer and lung cancers.