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.

Ethacrynic Acid: A Promising Candidate for Drug Repurposing as an Anticancer Agent.

Ethacrynic acid (ECA) is a diuretic that inhibits Na-K-2Cl cotransporter (NKCC2) present in the thick ascending loop of Henle and muculo dens and is clinically used for the treatment of edema caused by excessive body fluid. However, its clinical use is limited due to its low bioavailability and side effects, such as liver damage and hearing loss at high doses. Despite this, ECA has recently emerged as a potential anticancer agent through the approach of drug repositioning, with a novel mechanism of action. ECA has been shown to regulate cancer hallmark processes such as proliferation, apoptosis, migration and invasion, angiogenesis, inflammation, energy metabolism, and the increase of inhibitory growth factors through various mechanisms. Additionally, ECA has been used as a scaffold for synthesizing a new material, and various derivatives have been synthesized. This review explores the potential of ECA and its derivatives as anticancer agents, both alone and in combination with adjuvants, by examining their effects on ten hallmarks of cancer and neuronal contribution to cancer. Furthermore, we investigated the trend of synthesis research of a series of ECA derivatives to improve the bioavailability of ECA. This review highlights the importance of ECA research and its potential to provide a cost-effective alternative to new drug discovery and development for cancer treatment.

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.

HPV-18 E7 Interacts with Elk-1 Leading to Elevation of the Transcriptional Activity of Elk-1 in Cervical Cancer.

The human papillomavirus (HPV)-18 E7 (E7) oncoprotein is a major transforming protein that is thought to be involved in the development of cervical cancer. It is well-known that E7 stimulates tumour development by inactivating pRb. However, this alone cannot explain the various characteristics acquired by HPV infection. Therefore, we examined other molecules that could help explain the acquired cancer properties during E7-induced cancer development. Using the yeast two-hybrid (Y2H) method, we found that the Elk-1 factor, which is crucial for cell proliferation, invasion, cell survival, anti-apoptotic activity, and cancer development, binds to the E7. By determining which part of E7 binds to which domain of Elk-1 using the Y2H method, it was found that CR2 and CR3 of the E7 and parts 1-206, including the ETS-DNA domain of Elk-1, interact with each other. As a result of their interaction, the transcriptional activity of Elk-1 was increased, thereby increasing the expression of target genes EGR-1, c-fos, and E2F. Additionally, the colony forming assay revealed that overexpression of Elk-1 and E7 promotes C33A cell proliferation. We expect that the discovery of a novel E7 function as an Elk-1 activator could help explain whether the E7 has novel oncogenic activities in addition to p53 inactivation. We also expect that it will offer new methods for developing improved strategies for cervical cancer treatment.

Hypoxia-induced ELF3 promotes tumor angiogenesis through IGF1/IGF1R.

Epithelial ovarian cancer (EOC) is one of the most lethal gynecological cancers despite a relatively low incidence. Angiogenesis, one of the hallmarks of cancer, is essential for the pathogenesis of EOC, which is related to the induction of angiogenic factors. We found that ELF3 was highly expressed in EOCs under hypoxia and functioned as a transcription factor for IGF1. The ELF3-mediated increase in the secretion of IGF1 and VEGF promoted endothelial cell proliferation, migration, and EOC angiogenesis. Although this situation was much exaggerated under hypoxia, ELF3 silencing under hypoxia significantly attenuated angiogenic activity in endothelial cells by reducing the expression and secretion of IGF1 and VEGF. ELF3 silencing attenuated angiogenesis and tumorigenesis in ex vivo and xenograft mouse models. Consequently, ELF3 plays an important role in the induction of angiogenesis and tumorigenesis in EOC as a transcription factor of IGF1. A detailed understanding of the biological mechanism of ELF3 may both improve current antiangiogenic therapies and have anticancer effects for EOC.

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.

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.

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.

Stem Cell Factor SOX9 Interacts with a Cell Death Regulator RIPK1 and Results in Escape of Cancer Stem Cell Death.

High-grade ovarian cancer (HGOC) is the most lethal gynecological cancer, with high metastasis and recurrence. Cancer stem cells (CSCs) are responsible for its apoptosis resistance, cancer metastasis, and recurrence. Thus, targeting CSCs would be a promising strategy for overcoming chemotherapy resistance and improving patient prognosis in HGOC. Among upregulated oncogenic proteins in HGOC, we found that transcription factor SOX9 showed a strong correlation with stemness-regulating ALDH1A1 and was localized predominantly in the cytoplasm of HGOC with lymph node metastasis. In order to address the role of unusual cytoplasmic SOX9 and to explore its underlying mechanism in HGOC malignancy, a Y2H assay was used to identify a necroptotic cell death-associated cytoplasmic protein, receptor-interacting serine/threonine protein kinase 1 (RIPK1), as a novel SOX9-interacting partner and further mapped their respective interacting domains. The C-terminal region containing the transactivation domain of SOX9 interacted with the death domain of R1PK1. Consistent with its stemness-promoting function, SOX9 knockdown in vitro resulted in changes in cell morphology, cell cycle, stem cell marker expression, cell invasion, and sphere formation. Furthermore, in vivo knockdown completely inhibited tumor growth in mouse xenograft model. We propose that cytoplasmic SOX9-mediated cell death suppression would contribute to cancer stem cell survival in HGOC.

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-3ß 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.

ß-TrCP1-variant 4, a novel splice variant of ß-TrCP1, is a negative regulator of ß-TrCP1-variant 1 in ß-catenin degradation.

ß-transducin repeats-containing protein-1 (ß-TrCP1) serves as the substrate recognition subunit for SCFß-TrCP E3 ubiquitin ligases, which specifically ubiquitinate phosphorylated substrates. Three variants of ß-TrCP1 are known and act as homodimer or heterodimer complexes. Here, we identified a novel full-sequenced variant, ß-TrCP1-variant 4, which harbours exon II instead of exon III of variant 1, with no change in the open reading frame. The expression of ß-TrCP1-variant 4 is lower than that of variant 1 or 2 in ovarian cancer cell lines, whereas it is abundantly expressed in normal and cancerous ovarian tissues. Moreover, ß-TrCP1-variant 2 was aberrantly expressed more than variant 1 in ovarian cancer tissues whereas variant 1 was expressed more in normal tissues. Similar to variants 1 and 2, ß-TrCP1-variant 4 directly interacts with ß-catenin, one of the substrates of SCFß-TrCP E3 ubiquitin ligase and down-regulates the transcriptional activity and protein expression of ß-catenin with a significantly weaker effect than that by variants 1 and 2. However, the co-expression of ß-TrCP1-variant 4 with variant 1 in same proportion has no effect, whereas other combinations effectively down-regulate the activity of ß-catenin, indicating that the heterodimer of variants 1 and 4 has no function. Thus, ß-TrCP1-variant 4 could play a critical role in SCFß-TrCP E3 ligase-mediated ubiquitination by acting as a negative regulator of ß-TrCP1-variant 1.

Ethacrynic acid, a loop diuretic, suppresses epithelial-mesenchymal transition of A549 lung cancer cells via blocking of NDP-induced WNT signaling.

Lung cancer is one of the leading causes of death in cancer patients. Epithelial-mesenchymal transition (EMT) plays an important role in lung cancer progression. Therefore, for lung cancer treatment, it is crucial to find substances that inhibit EMT. Ethacrynic acid (ECA) is a diuretic that inhibits cellular ion flux and exerts anticancer effects. However, the effects of ECA on EMT in lung cancer remain unclear. We examined the effects of ECA on sphingosylphosphorylcholine (SPC) or TGF-ß1-induced EMT process in A549 and H1299 cells via reverse transcription polymerase chain reaction and Western blotting. We found that ECA inhibited SPC-induced EMT and SPC-induced WNT signalling in EMT. We observed that SPC induces the expression of NDP [Norrie disease protein] and WNT-2, whereas ECA suppressed their expression. SPC-induced WNT activation, EMT, migration, and invasion were suppressed by NDP small-interfering RNA (siNDP), but NDP overexpression (pNDP) enhanced these events in A549 and H1299 cells. Accordingly, NDP expression may influence lung cancer prognosis. In summary, our results revealed that ECA inhibited SPC or TGF-ß1-induced EMT in A549 and H1299 lung cancer cells by downregulating NDP expression and inhibiting WNT activation. Therefore, ECA might be a new drug candidate for lung cancer treatment.

IRF-1 Inhibits Angiogenic Activity of HPV16 E6 Oncoprotein in Cervical Cancer.

HPV16 E6 oncoprotein is a member of the human papillomavirus (HPV) family that contributes to enhanced cellular proliferation and risk of cervical cancer progression via viral infection. In this study, interferon regulatory factor-1 (IRF-1) regulates cell growth inhibition and transcription factors in immune response, and acts as an HPV16 E6-binding cellular molecule. Over-expression of HPV16 E6 elevated cell growth by attenuating IRF-1-induced apoptosis and repressing p21 and p53 expression, but activating cyclin D1 and nuclear factor kappa B (NF-κB) expression. The promoter activities of p21 and p53 were suppressed, whereas NF-κB activities were increased by HPV16 E6. Additionally, the cell viability of HPV16 E6 was diminished by IRF-1 in a dose-dependent manner. We found that HPV16 E6 activated vascular endothelial growth factor (VEGF)-induced endothelial cell migration and proliferation as well as phosphorylation of VEGFR-2 via direct interaction in vitro. HPV16 E6 exhibited potent pro-angiogenic activity and clearly enhanced the levels of hypoxia-inducible factor-1α (HIF-1α). By contrast, the loss of function of HPV16 E6 by siRNA-mediated knockdown inhibited the cellular events. These data provide direct evidence that HPV16 E6 facilitates tumour growth and angiogenesis. HPV16 E6 also activates the PI3K/mTOR signalling cascades, and IRF-1 suppresses HPV16 E6-induced tumourigenesis and angiogenesis. Collectively, these findings suggest a biological mechanism underlying the HPV16 E6-related activity in cervical tumourigenesis.

Homeoprotein Msx1-PIASy Interaction Inhibits Angiogenesis.

Previously, we demonstrated that the homeoprotein Msx1 interaction with p53 inhibited tumor growth by inducing apoptosis. However, Msx1 can exert its tumor suppressive effect through the inhibition of angiogenesis since growth of the tumor relies on sufficient blood supply from the existing vessels to provide oxygen and nutrients for tumor growth. We hypothesized that the inhibition of tumor growth by Msx1 might be due to the inhibition of angiogenesis. Here, we explored the role of Msx1 in angiogenesis. Overexpression of Msx1 in HUVECs inhibited angiogenesis, and silencing of Msx1 by siRNA abrogated its anti-angiogenic effects. Furthermore, forced expression of Msx1 in mouse muscle tissue inhibited vessel sprouting, and application of an Ad-Msx1-transfected conditioned medium onto the chicken chorioallantoic membrane (CAM) led to a significant inhibition of new vessel formation. To explore the underlying mechanism of Msx1-mediated angiogenesis, yeast two-hybrid screening was performed, and we identified PIASy (protein inhibitor of activated STAT Y) as a novel Msx1-interacting protein. We mapped the homeodomain of Msx1 and the C-terminal domain of PIASy as respective interacting domains. Consistent with its anti-angiogenic function, overexpression of Msx1 suppressed the reporter activity of VEGF. Interestingly, PIASy stabilized Msx1 protein, whereas deletion of the Msx1-interacting domain in PIASy abrogated the inhibition of tube formation and the stabilization of Msx1 protein. Our findings suggest the functional importance of PIASy-Msx1 interaction in Msx1-mediated angiogenesis inhibition.

Non-proteolytic calpain-6 interacts with VEGFA and promotes angiogenesis by increasing VEGF secretion.

Angiogenesis is involved in both normal physiological and pathological conditions. Vascular endothelial growth factor (VEGF) is a major factor for promoting angiogenesis. The current anti-VEGF therapies have limited efficacy and significant adverse effects. To find novel targets of VEGFA for angiogenesis inhibition, we performed yeast two-hybrid screening and identified calpain-6 as a novel VEGFA-interaction partner and confirmed the endogenous VEGFA-calpain-6 interaction in mammalian placenta. A domain mapping study revealed that the Gly321-Asp500 domain in calpain-6 is required for the interaction with the C-terminus of the VEGFA protein. The functional significance of the VEGFA-calpain-6 interaction was explored by assessing its effect on angiogenesis in vitro. Whereas forced overexpression of calpain-6 increased the secretion of the VEGF protein and tube formation, knockdown of calpain-6 expression abrogated the calpain-6-mediated VEGF secretion and tube formation in HUVECs. Consistent with the domain mapping result, overexpressing calpain-6 without the VEGFA-interacting domain III (Gly321-Asp500) failed to increase the secretion of VEGF protein. Our results identify calpain-6, an unconventional non-proteolytic calpain, as a novel VEGFA-interacting protein and demonstrate that their interaction is necessary to enhance VEGF secretion. Thus, calpain-6 might be a potential molecular target for angiogenesis inhibition in many diseases.

C/EBPß Is a Transcriptional Regulator of Wee1 at the G2/M Phase of the Cell Cycle.

The CCAAT/enhancer-binding protein ß (C/EBPß) is a transcription factor that regulates cellular proliferation, differentiation, apoptosis and tumorigenesis. Although the pro-oncogenic roles of C/EBPß have been implicated in various human cancers, how it contributes to tumorigenesis or tumor progression has not been determined. Immunohistochemistry with human non-small cell lung cancer (NSCLC) tissues revealed that higher levels of C/EBPß protein were expressed compared to normal lung tissues. Knockdown of C/EBPß by siRNA reduced the proliferative capacity of NSCLC cells by delaying the G2/M transition in the cell cycle. In C/EBPß-knockdown cells, a prolonged increase in phosphorylation of cyclin dependent kinase 1 at tyrosine 15 (Y15-pCDK1) was displayed with simultaneously increased Wee1 and decreased Cdc25B expression. Chromatin immunoprecipitation (ChIP) analysis showed that C/EBPß bound to distal promoter regions of WEE1 and repressed WEE1 transcription through its interaction with histone deacetylase 2. Treatment of C/EBPß-knockdown cells with a Wee1 inhibitor induced a decrease in Y15-pCDK1 and recovered cells from G2/M arrest. In the xenograft tumors, the depletion of C/EBPß significantly reduced tumor growth. Taken together, these results indicate that Wee1 is a novel transcription target of C/EBPß that is required for the G2/M phase of cell cycle progression, ultimately regulating proliferation of NSCLC cells.

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.

TSC-22 inhibits CSF-1R function and induces apoptosis in cervical cancer.

Colony stimulating factor 1 receptor (CSF-1R) regulates the monocyte/macrophage system, which is an essential component of cancer development. Therefore, CSF-1R might be an effective target for anti-cancer therapy. The overexpression of transforming growth factor (TGF)-ß stimulated clone-22 (TSC-22) inhibits cancer cell proliferation and induces apoptosis, and TSC-22 is emerging as a key factor in tumorigenesis. In this study, we discovered CSF-1R as a new interacting partner of TSC-22 and identified its elevated expression in cervical cancer cells. In particular, we found that TSC-22 interacted with the intracellular tyrosine kinase insert domain (539-749) of CSF-1R, which activates the AKT and ERK signaling pathways. This binding blocked AKT and ERK signaling, thereby suppressing the transcriptional activity of NF-κB. The overexpression of TSC-22 significantly decreased CSF-1R protein levels, affecting their autocrine loop. TSC-22 injected into a xenograft mouse model of human cervical cancer markedly inhibited tumor growth. The reduction of CSF-1R protein significantly suppresses cervical cancer cell proliferation and motility and induces apoptotic cell death. This association between TSC-22 and CSF-1R could be used as a novel therapeutic target and prognostic marker for cervical cancer.