Discovery of Potent, Selective, and In Vivo Efficacious AKT Kinase Protein Degraders via Structure-Activity Relationship Studies.

We recently reported a potent, selective, and in vivo efficacious AKT degrader, MS21, which is a von Hippel-Lindau (VHL)-recruiting proteolysis targeting chimera (PROTAC) based on the AKT inhibitor AZD5363. However, no structure-activity relationship (SAR) studies that resulted in this discovery have been reported. Herein, we present our SAR studies that led to the discovery of MS21, another VHL-recruiting AKT degrader, MS143 (compound 20) with similar potency as MS21, and a novel cereblon (CRBN)-recruiting PROTAC, MS5033 (compound 35). Compounds 20 and 35 induced rapid and robust AKT degradation in a concentration- and time-dependent manner via hijacking the ubiquitin-proteasome system. Compound 20 suppressed cell growth more effectively than AZD5363 in multiple cancer cell lines. Furthermore, 20 and 35 displayed good plasma exposure levels in mice and are suitable for in vivo efficacy studies. Lastly, compound 20 effectively suppressed tumor growth in vivo in a xenograft model without apparent toxicity.

Novel Aurora A and Protein Kinase C (α, ß1, ß2, and θ) Multitarget Inhibitors: Impact of Selenium Atoms on the Potency and Selectivity.

Aurora kinases and protein kinase C (PKC) have been shown to be involved in different aspects of cancer progression. To date, no dual Aurora/PKC inhibitor with clinical efficacy and low toxicity is available. Here, we report the identification of compound 2e as a potent small molecule capable of selectively inhibiting Aurora A kinase and PKC isoforms α, ß1, ß2 and θ. Compound 2e demonstrated significant inhibition of the colony forming ability of metastatic breast cancer cells in vitro and metastasis development in vivo. In vitro kinase screening and molecular modeling studies revealed the critical role of the selenium-containing side chains within 2e, where selenium atoms were shown to significantly improve its selectivity and potency by forming additional interactions and modulating the protein dynamics. In comparison to other H-bonding heteroatoms such as sulfur, our studies suggested that these selenium atoms also confer more favorable PK properties.

Discovery of a Potent and Selective ATAD2 Bromodomain Inhibitor with Antiproliferative Activity in Breast Cancer Models.

ATAD2 is an epigenetic bromodomain-containing target which is overexpressed in many cancers and has been suggested as a potential oncology target. While several small molecule inhibitors have been described in the literature, their cellular activity has proved to be underwhelming. In this work, we describe the identification of a novel series of ATAD2 inhibitors by high throughput screening, confirmation of the bromodomain region as the site of action, and the optimization campaign undertaken to improve the potency, selectivity, and permeability of the initial hit. The result is compound 5 (AZ13824374), a highly potent and selective ATAD2 inhibitor which shows cellular target engagement and antiproliferative activity in a range of breast cancer models.

Numb inhibits migration and promotes proliferation of colon cancer cells via RhoA/ROCK signaling pathway repression.

Numb regulates cell proliferation and differentiation through endocytosis and ubiquitination of signaling molecules. Besides, Numb controls the migration of epithelial cells by regulating intercellular junctions. Studies have shown that Numb promotes or inhibits tumor progression in different tumors. However, its role and mechanism in colorectal cancer remain unclear. We found that the expression level of Numb in colon tumor tissues has a great variety in different patients. Numb expression was negatively correlated with TNM stage and lymph node metastasis but positively correlated with tumor size. Elevated expression of Numb was associated with a good prognosis. Inhibiting Numb expression promoted the migration and invasion of colon cancer cells induced by TGF-ß, up-regulated the expression of EMT-related molecule Snail, and prevented the expression of E-cadherin. We also found that Numb promoted the proliferation and clones formation while inhibiting colon cancer cells' late apoptosis. In addition, Numb inhibited the RhoA activation and ROCK inhibitor Y-27632 or interfered with ROCK expression, partially inhibiting Numb-regulated cell proliferation and migration. In vivo tumorigenesis assay in nude mice also found that Numb promoted the proliferation of colon cancer cells, inhibited the expression of E-cadherin, and strengthened the expression of Snail. In conclusion, our study found that Numb plays multiple roles in the occurrence and progression of colon cancer by regulating the RhoA/ROCK signaling pathway, which provides a new theoretical molecular basis for the pathogenesis of colon cancer.

Design, Synthesis, and Biological Evaluation of 1-(Indolizin-3-yl)ethan-1-ones as CBP Bromodomain Inhibitors for the Treatment of Prostate Cancer.

CREB (cyclic-AMP responsive element binding protein) binding protein (CBP) is a potential target for prostate cancer treatment. Herein, we report the structural optimization of a series of 1-(indolizin-3-yl)ethan-1-one compounds as new selective CBP bromodomain inhibitors, aiming to improve cellular potency and metabolic stability. This process led to compound 9g (Y08284), which possesses good liver microsomal stability and pharmacokinetic properties (F = 25.9%). Furthermore, the compound is able to inhibit CBP bromodomain as well as the proliferation, colony formation, and migration of prostate cancer cells. Additionally, the new inhibitor shows promising antitumor efficacy in a 22Rv1 xenograft model (TGI = 88%). This study provides new lead compounds for further development of drugs for the treatment of prostate cancer.

Circ_0006948 Contributes to Cell Growth, Migration, Invasion and Epithelial-Mesenchymal Transition in Esophageal Carcinoma.

BACKGROUND: Circular RNAs (circRNAs) can act as promoters or inhibitors in cancer progression. Has_circ_0006948 (circ_0006948) was reported to aggravate the malignant behaviors of esophageal carcinoma (EC). AIMS: This study focused on investigating the molecular mechanism of circ_0006948 in EC progression. METHODS: The quantitative real-time polymerase chain reaction was performed to detect the expression of circ_0006948, microRNA-4262 (miR-4262) and fibronectin type III domain containing 3B (FNDC3B). Cell growth analysis was conducted by Cell Counting Kit-8 and colony formation assays. Cell migration and invasion were assessed by transwell assay. Epithelial-mesenchymal transition (EMT)-associated proteins and FNDC3B protein expression were assayed using western blot. Dual-luciferase reporter and RNA pull-down assays were performed to validate the target combination. Xenograft tumor assay was used for investigating the role of circ_0006948 in vivo. RESULTS: Circ_0006948 was upregulated in EC tissues and cells. Downregulating the expression of circ_0006948 or FNDC3B repressed cell growth, migration, invasion and EMT in EC cells. Target analysis indicated that miR-4262 was a target for circ_0006948 and FNDC3B was a downstream gene for miR-4262. Moreover, circ_0006948 could affect the expression of FNDC3B via sponging miR-4262. The effects of si-circ_0006948#1 on EC cells were partly restored by miR-4262 inhibition or FNDC3B overexpression. In addition, circ_0006948 also facilitated EC tumorigenesis in vivo by targeting the miR-4262/FNDC3B axis. CONCLUSION: Taken together, circ_0006948 functioned as an oncogenic factor in EC by the miR-4262-mediated FNDC3B expression regulation.

MKL1 regulates hepatocellular carcinoma cell proliferation, migration and apoptosis via the COMPASS complex and NF-κB signaling.

BACKGROUND: Histone modification plays essential roles in hepatocellular carcinoma (HCC) pathogenesis, but the regulatory mechanisms remain poorly understood. In this study, we aimed to analyze the roles of Megakaryoblastic leukemia 1 (MKL1) and its regulation of COMPASS (complex of proteins associated with Set1) in HCC cells. METHODS: MKL1 expression in clinical tissues and cell lines were detected by bioinformatics, qRT-PCR and western blot. MKL1 expression in HCC cells were silenced with siRNA, followed by cell proliferation evaluation via Edu staining and colony formation, migration and invasion using the Transwell system, and apoptosis by Hoechst staining. HCC cell tumorigenesis was assessed by cancer cell line-based xenograft model, combined with H&E staining and IHC assays. RESULTS: MKL1 expression was elevated in HCC cells and clinical tissues which was correlated with poor prognosis. MKL1 silencing significantly repressed proliferation, migration, invasion and colony formation but enhanced apoptosis in HepG2 and Huh-7 cells. MKL1 silencing also inhibited COMPASS components and p65 protein expression in HepG2 and Huh-7 cells. HepG2 cell tumorigenesis in nude mice was severely impaired by MKL1 knockdown, resulted into suppressed Ki67 expression and cell proliferation. CONCLUSION: MKL1 promotes HCC pathogenesis by regulating hepatic cell proliferation, migration and apoptosis via the COMPASS complex and NF-κB signaling.

Inhibition of matrix stiffness relating integrin ß1 signaling pathway inhibits tumor growth in vitro and in hepatocellular cancer xenografts.

BACKGROUND: Cancer development is strictly correlated to composition and physical properties of the extracellular matrix. Particularly, a higher matrix stiffness has been demonstrated to promote tumor sustained growth. Our purpose was to explore the role of matrix stiffness in liver cancer development. METHODS: The matrix stiffness of tumor tissues was determined by atomic force microscopy (AFM) analysis. In vitro, we used a tunable Polyacrylamide (PA) hydrogels culture system for liver cancer cells culture. The expression level of integrin ß1, phosphorylated FAK, ERK1/2, and NF-κB in SMMC-7721 cells was measured by western blotting analysis. We performed MTT, colony formation and transwell assay to examine the tumorigenic and metastatic potential of SMMC-7721 cells cultured on the tunable PA hydrogels. SMMC-7721 cancer xenografts were established to explore the anticancer effects of integrin inhibitors. RESULTS: Our study provided evidence that liver tumor tissues from metastatic patients possessed a higher matrix stiffness, when compared to the non-metastatic group. Liver cancer cells cultured on high stiffness PA hydrogels displayed enhanced tumorigenic potential and migrative properties. Mechanistically, activation of integrin ß1/FAK/ ERK1/2/NF-κB signaling pathway was observed in SMMC-7721 cells cultured on high stiffness PA hydrogels. Inhibition of ERK1/2, FAK, and NF-κB signaling suppressed the pro-tumor effects induced by matrix stiffness. Combination of chemotherapy and integrin ß1 inhibitor suppressed the tumor growth and prolonged survival time in hepatocellular cancer xenografts. CONCLUSION: A higher matrix stiffness equipped tumor cells with enhanced stemness and proliferative characteristics, which was dependent on the activation of integrin ß1/FAK/ERK1/2/NF-κB signaling pathway. Blockade of integrin signals efficiently improved the outcome of chemotherapy, which described an innovative approach for liver cancer treatment.

The endocytic pathway of Pt nanoclusters and their induced apoptosis of A549 and A549/Cis cells through c-Myc/p53 and Bcl-2/caspase-3 signaling pathways.

In recent years, multifunctional platinum nanoclusters (Pt-NCs) as new Pt-based anti-cancer drugs exhibit a promising therapeutic efficiency for several cancer diseases, especially for human pulmonary carcinoma. However, the endocytosis behaviors (like uptake pathway, etc.) and induced apoptosis mechanism of Pt-NCs for drug-resistant non-small cell lung cancer (NSCLC), are still inconclusive. In this research, we explored the endocytic pathway of Pt-NCs in both typical NSCLC A549 cells and cisplatin-resistant A549/Cis cells through qualitative confocal laser scanning microscope (CLSM) measurement and quantitative flow cytometry (FCM) and inductive coupled plasma-optical emission spectroscopy (ICP-OES) analysis, by the means of introducing the specific inhibitors which impede the classical ways of endocytosis. It was found that Pt-NCs dominatingly entered A549 cells via caveolin-mediated endocytosis as well as A549/Cis cells through micropinocytosis approach. Pt-NCs possessed an excellent inhibitory effect on the cell proliferation, migration and invasion, which the cell activity of A549 cells reduced to 14% and that of A549/Cis cells went down about four fifths. Moreover, Pt-NCs treatment increased caspase-3 protein levels and downregulated the expression of c-Myc and Bcl-2, proving the Pt-NCs-induced apoptosis of NSCLC cells was related to c-Myc/p53 and Bcl-2/caspase-3 signal pathways. These results demonstrate the explicit uptake pathway and apoptotic signaling pathway of Pt-NCs for NSCLC, which provides an in-depth and reasonable theoretical basis for the development of new Pt-NCs-based chemotherapeutics in future clinical practice.

Corosolic Acid Attenuates the Invasiveness of Glioblastoma Cells by Promoting CHIP-Mediated AXL Degradation and Inhibiting GAS6/AXL/JAK Axis.

Corosolic acid (CA), a bioactive compound obtained from Actinidia chinensis, has potential anti-cancer activities. Glioblastoma (GBM) is a malignant brain tumor and whether CA exerts anti-cancer activity on GBM remains unclear. This study was aimed to explore the anticancer activity and its underlying mechanism of CA in GBM cells. Our findings showed that CA ≤ 20 µM did not affect cell viability and cell proliferative rate of normal astrocyte and four GBM cells. Notably, 10 or 20 µM CA significantly inhibited cell migration and invasion of three GBM cells, decreased the protein level of F-actin and disrupted F-actin polymerization in these GBM cells. Further investigation revealed that CA decreased AXL level by promoting ubiquitin-mediated proteasome degradation and upregulating the carboxyl terminus of Hsc70-interacting protein (CHIP), an inducer of AXL polyubiquitination. CHIP knock-down restored the CA-reduced AXL and invasiveness of GBM cells. Additionally, we observed that CA-reduced Growth arrest-specific protein 6 (GAS6) and inhibited JAK2/MEK/ERK activation, and GAS6 pre-treatment restored attenuated JAK2/MEK/ERK activation and invasiveness of GBM cells. Furthermore, molecular docking analysis revealed that CA might bind to GAS6 and AXL. These findings collectively indicate that CA attenuates the invasiveness of GBM cells, attributing to CHIP upregulation and binding to GAS6 and AXL and subsequently promoting AXL degradation and downregulating GAS6-mediated JAK2/MEK/ERK cascade. Conclusively, this suggests that CA has potential anti-metastatic activity on GBM cells by targeting the CHIP/GAS6/AXL axis.

Dnmt3a is downregulated by Stat5a and mediates G0/G1 arrest by suppressing the miR-17-5p/Cdkn1a axis in Jak2V617F cells.

BACKGROUND: Despite of the frequently reported Dnmt3a abormality in classical myeloproliferative neoplasms (cMPNs) patients, few research explores how the Dnmt3a is regulated by Jak2V617F mutation. In this study, we have investigated how the Dnmt3a is regulated by Jak2V617F mutation and its effects on downstream signaling pathways in cMPNs. METHODS: Specimens of Jak2V617F positive cMPN patients and normal controls were collected. Murine BaF3 cell line was used to construct cell models. Dual-Glo luciferase assays and chromatin immunoprecipitation (ChIP)-qPCR were performed to detect the impact of Stat5a on transcription activity of Dnmt3a. Soft agar colony formation assay and cell counting assay were performed to detect cell proliferation. BrdU staining and flow cytometry were used to investigate cell cycle distribution. Western blotting and quantitative reverse-transcription PCR (qPCR) were performed to detect the expression levels of genes. RESULTS: Firstly, the results of western blotting and qPCR revealed that compared with the control samples, Dnmt3a is downregulated in Jak2V617F positive samples. Then we explored the mechanism behind it and found that Dnmt3a is a downstream target of Stat5a, the transcription and translation of Dnmt3a is suppressed by the binding of aberrantly activated Stat5a with Dnmt3a promoter in Jak2V617F positive samples. We further revealed the region approximately 800 bp upstream of the first exon of the Dnmt3a promoter, which includes a gamma-activated sequence (GAS) motif of Stat5a, is the specific site that Stat5a binds to. Soft agar colony formation assay, cell counting assay, and BrdU staining and flow cytometry assay found that Dnmt3a in Jak2V617F-BaF3 cells significantly affected the cell proliferation capacity and cell cycle distribution by suppressing Cdkn1a via miR-17-5p/Cdkn1a axis and mediated G0/G1 arrest. CONCLUSIONS: Transcription and translation of Dnmt3a is downregulated by the binding of Stat5a with Dnmt3a promoter in Jak2V617F cells. The GAS motif at promoter of Dnmt3a is the exact site where the Stat5a binds to. Dnmt3a conducted G0/G1 arrest through regulating miR-17-5p/Cdkn1a axis. The axis of Stat5a/Dnmt3a/miR-17-5p/Cdkn1a potentially provides a treatment target for cMPNs.

Silencing FYVE, RhoGEF, and PH domain containing 1 (FGD1) suppresses melanoma progression by inhibiting PI3K/AKT signaling pathway.

Cutaneous melanoma is the leading cause of death among skin cancers despite the availability of diverse treatments. FGD1 plays an important role in multiple cancers, but how it works in cutaneous melanoma has not been illustrated. Thus, this study was intended to investigate the roles of FGD1 and its underlying mechanisms in cutaneous melanoma. Bioinformatics tools and quantitative real-time polymerase chain reaction (qRT-PCR) were used to analyze the expression of FGD1 in cutaneous melanoma. After the knockdown of FGD1 in melanoma cells, the proliferation, migration, and invasion of cells were analyzed by cell counting kit-8 (CCK8) assay, colony formation assays and transwell assays. Western blot was used to check the expression of key factors in PI3K/AKT pathway. In addition, nude mice models were used to study the role of FGD1 in melanoma development and metastasis in vivo. The data demonstrated that FGD1 was up-regulated and predicted a poor clinical outcome for cutaneous melanoma patients. Knockdown of FGD1 inhibited melanoma cell proliferation, migration, and invasion. The expressions of p-PI3K and p-AKT were significantly decreased, while the expressions of PI3K and AKT showed no marked difference in the knockdown group. Meanwhile, knockdown of FGD1 suppressed the development of melanoma in vivo. This study suggested that knockdown of FGD1 could block melanoma formation and proliferation by inhibiting PI3K/AKT signaling pathway. FGD1 might be a promising therapeutic target for melanoma.

CCNE1 copy number is a biomarker for response to combination WEE1-ATR inhibition in ovarian and endometrial cancer models.

CCNE1-amplified ovarian cancers (OVCAs) and endometrial cancers (EMCAs) are associated with platinum resistance and poor survival, representing a clinically unmet need. We hypothesized that dysregulated cell-cycle progression promoted by CCNE1 overexpression would lead to increased sensitivity to low-dose WEE1 inhibition and ataxia telangiectasia and Rad3-related (ATR) inhibition (WEE1i-ATRi), thereby optimizing efficacy and tolerability. The addition of ATRi to WEE1i is required to block feedback activation of ATR signaling mediated by WEE1i. Low-dose WEE1i-ATRi synergistically decreases viability and colony formation and increases replication fork collapse and double-strand breaks (DSBs) in a CCNE1 copy number (CN)-dependent manner. Only upon CCNE1 induction does WEE1i perturb DNA synthesis at S-phase entry, and addition of ATRi increases DSBs during DNA synthesis. Inherent resistance to WEE1i is overcome with WEE1i-ATRi, with notable durable tumor regressions and improved survival in patient-derived xenograft (PDX) models in a CCNE1-level-dependent manner. These studies demonstrate that CCNE1 CN is a clinically tractable biomarker predicting responsiveness to low-dose WEE1i-ATRi for aggressive subsets of OVCAs/EMCAs.

CircCDC45 promotes the malignant progression of glioblastoma by modulating the miR-485-5p/CSF-1 axis.

BACKGROUND: Glioblastoma (GBM) is characterized by progressive growth and metastasis. Numerous studies claim that the deregulation of circular RNAs (circRNAs) is associated with cancer progression. However, the role of circRNAs in GBM is largely limited. The purpose of this study was to investigate the functions of circCDC45 in GBM and provide a feasible functional mechanism to support its role. METHODS: The expression of circCDC45, miR-485-5p and colony-stimulating factor 1 (CSF-1) mRNA was examined using quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was assessed using cell counting kit - 8 (CCK-8) assay and colony formation assay. Cell migration and cell invasion were monitored using transwell assay. The protein levels of proliferation-related markers and CSF-1 were determined using western blot. The target relationship was predicted using bioinformatics tools and validated using dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Animal models were constructed to verify the role of circCDC45 in vivo. RESULTS: The expression of circCDC45 and CSF-1 was elevated in GBM tissues and cells, while the expression of miR-485-5p was declined. Downregulation of circCDC45 or CSF-1 blocked GBM cell proliferation, invasion and migration as well as tumor growth in vivo. In mechanism, circCDC45 positively regulated the expression of CSF-1 by targeting miR-485-5p. Inhibition of miR-485-5p reversed the biological effects caused by circCDC45 downregulation in GBM cells. CONCLUSION: CircCDC45 promoted the progression of GBM by mediating the miR-485-5p/CSF-1 axis, and circCDC45 might be a promising plasmatic biomarker for GBM diagnosis and treatment.

Blast cells surviving acute myeloid leukemia induction therapy are in cycle with a signature of FOXM1 activity.

BACKGROUND: Disease relapse remains common following treatment of acute myeloid leukemia (AML) and is due to chemoresistance of leukemia cells with disease repopulating potential. To date, attempts to define the characteristics of in vivo resistant blasts have focused on comparisons between leukemic cells at presentation and relapse. However, further treatment responses are often seen following relapse, suggesting that most blasts remain chemosensitive. We sought to characterise in vivo chemoresistant blasts by studying the transcriptional and genetic features of blasts from before and shortly after induction chemotherapy using paired samples from six patients with primary refractory AML. METHODS: Leukemic blasts were isolated by fluorescence-activated cell sorting. Fluorescence in situ hybridization (FISH), targeted genetic sequencing and detailed immunophenotypic analysis were used to confirm that sorted cells were leukemic. Sorted blasts were subjected to RNA sequencing. Lentiviral vectors expressing short hairpin RNAs were used to assess the effect of FOXM1 knockdown on colony forming capacity, proliferative capacity and apoptosis in cell lines, primary AML cells and CD34+ cells from healthy donors. RESULTS: Molecular genetic analysis revealed early clonal selection occurring after induction chemotherapy. Immunophenotypic characterisation found leukemia-associated immunophenotypes in all cases that persisted following treatment. Despite the genetic heterogeneity of the leukemias studied, transcriptional analysis found concerted changes in gene expression in resistant blasts. Remarkably, the gene expression signature suggested that post-chemotherapy blasts were more proliferative than those at presentation. Resistant blasts also appeared less differentiated and expressed leukemia stem cell (LSC) maintenance genes. However, the proportion of immunophenotypically defined LSCs appeared to decrease following treatment, with implications for the targeting of these cells on the basis of cell surface antigen expression. The refractory gene signature was highly enriched with targets of the transcription factor FOXM1. shRNA knockdown experiments demonstrated that the viability of primary AML cells, but not normal CD34+ cells, depended on FOXM1 expression. CONCLUSIONS: We found that chemorefractory blasts from leukemias with varied genetic backgrounds expressed a common transcriptional program. In contrast to the notion that LSC quiescence confers resistance to chemotherapy we find that refractory blasts are both actively proliferating and enriched with LSC maintenance genes. Using primary patient material from a relevant clinical context we also provide further support for the role of FOXM1 in chemotherapy resistance, proliferation and stem cell function in AML.

LncRNA LINC00342 promotes gastric cancer progression by targeting the miR-545-5p/CNPY2 axis.

BACKGROUND: This study aimed to explore the role and underlying molecular mechanisms of long non-coding RNA (lncRNA) LINC00342 in gastric cancer (GC). METHODS: The expression of LINC00342 in GC tissues was evaluated by Quantitative reverse transcription polymerase chain reaction (qRT-PCR). Silencing of LINC00342 was conducted to investigate the effect of LINC00342 in vitro and in vivo. The underlying molecular mechanisms of LINC00342 were determined by dual luciferase reporter assay, Western blotting analysis and rescue experiments. Biological functions of LINC00342 were evaluated by cell counting kit-8 (CCK-8) assay, colony formation assay, wound healing assay and Transwell assays. In addition, a tumor model was used to verify the effect of LINC00342 in tumorigenesis in vivo. RESULTS: LINC00342 was significantly upregulated in GC tissues and cell lines. Silencing of LINC00342 efficiently inhibited proliferation, migration and invasion of AGS cells in vitro, and also suppressed the tumorigenesis of GC in vivo. Functional experiments showed that LINC00342 regulated the expression of canopy fibroblast growth factor signaling regulator 2 (CNPY2) by competitively sponging miR-545-5p. Rescue experiments showed that inhibition of miR-545-5p and overexpression of CNPY2 significantly reversed cell phenotypes caused by silencing of LINC00342. CONCLUSION: LINC00342 plays a potential oncogenic role in GC by targeting the miR545-5p/CNPY2 axis, and might act as a novel therapeutic target for GC.

Transmembrane protein 97 exhibits oncogenic properties via enhancing LRP6-mediated Wnt signaling in breast cancer.

Upregulation of transmembrane protein 97 (TMEM97) has been associated with progression and poor outcome in multiple human cancers, including breast cancer. Recent studies suggest that TMEM97 may be involved in the activation of the Wnt/ß-catenin pathway. However, the molecular mechanism of TMEM97 action on Wnt/ß-catenin signaling is completely unclear. In the current study, TMEM97 was identified as an LRP6-interacting protein. TMEM97 could interact with LRP6 intracellular domain and enhance LRP6-mediated Wnt signaling in a CK1δ/ε-dependent manner. The binding of TMEM97 to LRP6 facilitated the recruitment of CK1δ/ε to LRP6 complex, resulting in LRP6 phosphorylation at Ser 1490 and the stabilization of ß-catenin. In breast cancer cells, knockout of TMEM97 attenuated the Wnt/ß-catenin signaling cascade via regulating LRP6 phosphorylation, leading to a decrease in the expression of Wnt target genes AXIN2, LEF1, and survivin. TMEM97 deficiency also suppressed cell viability, proliferation, colony formation, migration, invasion, and stemness properties in breast cancer cells. Importantly, TMEM97 knockout suppressed tumor growth through downregulating the Wnt/ß-catenin signaling pathway in a breast cancer xenograft model. Taken together, our results revealed that TMEM97 is a positive modulator of canonical Wnt signaling. TMEM97-mediated Wnt signaling is implicated in the tumorigenesis of breast cancer, and its targeted inhibition may be a promising therapeutic strategy for breast cancer.

RANBP10 promotes glioblastoma progression by regulating the FBXW7/c-Myc pathway.

RAN binding protein 10 (RANBP10), a ubiquitously expressed and evolutionarily conserved protein, as a RAN-GTP exchange factor (GEF) to regulate several factors involved in cellular progression. Previous studies showed that RANBP10 was overexpressed in prostate cancer cells and was responsible for androgen receptor (AR) activation. However, the biological function of RANBP10 in glioblastoma (GBM) has not been studied. Here, we found that RANBP10 was overexpressed in GBM, and high RANBP10 expression was closely linked to poor survival of patients with GBM. Downregulation of RANBP10 significantly inhibited cell proliferation, migration, invasion, and tumor growth of GBM cells. In addition, we revealed that RANBP10 could suppress the promoter activity of FBXW7, and thereby increase the protein stability of c-Myc in GBM cells. Silencing of FBXW7 in RANBP10-knockdown GBM cells could partly negate the effects induced by RANBP10 downregulation. Taken together, our findings established that RANBP10 significantly promoted GBM progression by control of the FBXW7-c-Myc axis, and suggest that RANBP10 may be a potential target in GBM.

Stattic sensitizes osteosarcoma cells to epidermal growth factor receptor inhibitors via blocking the interleukin 6-induced STAT3 pathway.

Osteosarcoma (OS), the most common malignant bone tumor with high metastatic potential, frequently affects children and adolescents. Epidermal growth factor receptor (EGFR)-targeted tyrosine kinase inhibitors exhibit encouraging anti-tumor activity for patients with solid tumors, whereas their effects on OS remain controversial. In the present study, we aimed to elucidate the anti-tumor activity of gefitinib for OS, as well as to explore the underlying mechanisms. Gefitinib inhibits cell viability, tumor growth, cell migration, and invasion and promotes cell apoptosis and G1 cycle arrest in OS at a relatively high concentration via suppressing the PI3K/Akt and ERK pathways. However, gefitinib treatment results in the feedback activation of signal transducer and activator of transcription 3 (STAT3) induced by interleukin 6 (IL-6) secretion. Combined treatment with gefitinib and stattic, an inhibitor for STAT3 phosphorylation, engenders more evident inhibitory effects on cell proliferation, migration, and invasion and promotive effects on cell apoptosis and G1 phase arrest in OS, compared with the single exposure to gefitinib or stattic. Western blot analysis demonstrates that stattic treatment in gefitinib-treated OS abrogates the IL-6-induced STAT3 activation and subsequently further restrains the activities of EGFR, Akt, and ERK pathways in tumor cells. This study confirms that the EGFR inhibitor of gefitinib has moderate anti-tumor effects on OS through IL-6 secretion-mediated STAT3 activation. Additional administration of stattic in EGFR-targeted therapies may contribute to improve the efficacy for OS.

High Expression of Annexin A9 Promotes Cell Proliferation and Migration in Gastric Cancer via the TGF-ß Signaling Pathway.

Annexin A9 (ANXA9) represents an important calcium-dependent phospholipid-binding protein family member and contains a calcium-binding site that is necessary for extracellular matrix proteins. ANXA9 has a significant role in human cancers. However, there is no correlation study existing on ANXA9 in gastric cancer (GC). ANXA9 messenger RNA (mRNA) expression within patients with GC were detected with reverse transcription polymerase chain reaction and its protein expression in GC and GES-1 cells were detected through Western blotting. ANXA9 levels within normal and GC tissue samples were measured by Kaplan-Meier analysis and Oncomine. Transwell migration, colony formation, and cell cycle assay monitored the effects of ANXA9 on cell proliferation and metastasis and growth. Additionally, proteins related to epithelial-mesenchymal transition (EMT) were detected to evaluate the function of ANXA9 within GC cells. Relative to GES-1 cells, ANXA9 expression increased within GC cells. Also, ANXA9 expression increased in GC tissues and indicated an unfavorable prognosis. Furthermore, ANXA9 over-expression within HGC-27 cells increased migrated cells quantity and formed larger and more numerous cell clones; the G1 phase decreased while S and G2 phases increased; whereas ANXA9 knockdown suppressed MGC-803 cell growth and migration. Thus, ANXA9 may influence cell growth, migration and EMT through transforming growth factor ß (TGF-ß) signal transduction pathway. Immunofluorescence analyzed SMAD2/3 and p-SMAD2/3 distribution and expression when ANXA9 was overexpressed in HGC-27 cells. These results predicted that ANXA9 mediated cell migration and growth through TGF-ß signal transduction pathway within GC.