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.

Loss of EMP2 Inhibits Melanogenesis of MNT1 Melanoma Cells via Regulation of TRP-2

Melanogenesis is the production of melanin from tyrosine by a series of enzyme-catalyzed reactions, in which tyrosinase and DOPA oxidase play key roles. The melanin content in the skin determines skin pigmentation. Abnormalities in skin pigmentation lead to various skin pigmentation disorders. Recent research has shown that the expression of EMP2 is much lower in melanoma than in normal melanocytes, but its role in melanogenesis has not yet been elucidated. Therefore, we investigated the role of EMP2 in the melanogenesis of MNT1 human melanoma cells. We examined TRP-1, TRP-2, and TYR expression levels during melanogenesis in MNT1 melanoma cells by gene silencing of EMP2. Western blot and RT-PCR results confirmed that the expression levels of TYR and TRP-2 were decreased when EMP2 expression was knocked down by EMP2 siRNA in MNT1 cells, and these changes were reversed when EMP2 was overexpressed. We verified the EMP2 gene was knocked out of the cell line (EMP2 CRISPR/Cas9) by using a CRISPR/Cas9 system and found that the expression levels of TRP-2 and TYR were significantly lower in the EMP2 CRISPR/Cas9 cell lines. Loss of EMP2 also reduced migration and invasion of MNT1 melanoma cells. In addition, the melanosome transfer from the melanocytes to keratinocytes in the EMP2 KO cells cocultured with keratinocytes was reduced compared to the cells in the control coculture group. In conclusion, these results suggest that EMP2 is involved in melanogenesis via the regulation of TRP-2 expression.

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.

Suppressor of Variegation 3–9 Homolog 2, a Novel Binding Protein of Translationally Controlled Tumor Protein, Regulates Cancer Cell Proliferation

Suppressor of Variegation 3–9 Homolog 2 (SUV39H2) methylates the lysine 9 residue of histone H3 and induces heterochromatin formation, resulting in transcriptional repression or silencing of target genes. SUV39H1 and SUV39H2 have a role in embryonic development, and SUV39H1 was shown to suppress cell cycle progression associated with Rb. However, the function of human SUV39H2 has not been extensively studied. We observed that forced expression of SUV39H2 decreased cell proliferation by inducing G1 cell cycle arrest. In addition, SUV39H2 was degraded through the ubiquitin-proteasomal pathway. Using yeast two-hybrid screening to address the degradation mechanism and function of SUV39H2, we identified translationally controlled tumor protein (TCTP) as an SUV39H2-interacting molecule. Mapping of the interacting regions indicated that the N-terminal 60 amino acids (aa) of full-length SUV39H2 and the C-terminus of TCTP (120–172 aa) were critical for binding. The interaction of SUV39H2 and TCTP was further confirmed by co-immunoprecipitation and immunofluorescence staining for colocalization. Moreover, depletion of TCTP by RNAi led to up-regulation of SUV39H2 protein, while TCTP overexpression reduced SUV39H2 protein level. The half-life of SUV39H2 protein was significantly extended upon TCTP depletion. These results clearly indicate that TCTP negatively regulates the expression of SUV39H2 post-translationally. Furthermore, SUV39H2 induced apoptotic cell death in TCTP-knockdown cells. Taken together, we identified SUV39H2, as a novel target protein of TCTP and demonstrated that SUV39H2 regulates cell proliferation of lung cancer cells.