Association of FTH1-Expressing Circulating Tumor Cells With Efficacy of Neoadjuvant Chemotherapy for Patients With Breast Cancer: A Prospective Cohort Study.

BACKGROUND: The association between different phenotypes and genotypes of circulating tumor cells (CTCs) and efficacy of neoadjuvant chemotherapy (NAC) remains uncertain. This study was conducted to evaluate the relationship of FTH1 gene-associated CTCs (F-CTC) with/without epithelial-mesenchymal transition (EMT) markers, or their dynamic changes with the efficacy of NAC in patients with non-metastatic breast cancer. PATIENTS AND METHODS: This study enrolled 120 patients with non-metastatic breast cancer who planned to undergo NAC. The FTH1 gene and EMT markers in CTCs were detected before NAC (T0), after 2 cycles of chemotherapy (T1), and before surgery (T2). The associations of these different types of CTCs with rates of pathological complete response (pCR) and breast-conserving surgery (BCS) were evaluated using the binary logistic regression analysis. RESULTS: F-CTC in peripheral blood ≥1 at T0 was an independent factor for pCR rate in patients with HER2-positive (odds ratio [OR]=0.08, 95% confidence interval [CI], 0.01-0.98, P = .048). The reduction in the number of F-CTC at T2 was an independent factor for BCS rate (OR = 4.54, 95% CI, 1.14-18.08, P = .03). CONCLUSIONS: The number of F-CTC prior to NAC was related to poor response to NAC. Monitoring of F-CTC may help clinicians formulate personalized NAC regimens and implement BCS for patients with non-metastatic breast cancer.

Glucagon Enhances Chemotherapy Efficacy By Inhibition of Tumor Vessels in Colorectal Cancer.

Chemotherapy is widely used to treat colorectal cancer (CRC). Despite its substantial benefits, the development of drug resistance and adverse effects remain challenging. This study aimed to elucidate a novel role of glucagon in anti-cancer therapy. In a series of in vitro experiments, glucagon inhibited cell migration and tube formation in both endothelial and tumor cells. In vivo studies demonstrated decreased tumor blood vessels and fewer pseudo-vessels in mice treated with glucagon. The combination of glucagon and chemotherapy exhibited enhanced tumor inhibition. Mechanistic studies demonstrated that glucagon increased the permeability of blood vessels, leading to a pronounced disruption of vessel morphology. Signaling pathway analysis identified a VEGF/VEGFR-dependent mechanism whereby glucagon attenuated angiogenesis through its receptor. Clinical data analysis revealed a positive correlation between elevated glucagon expression and chemotherapy response. This is the first study to reveal a role for glucagon in inhibiting angiogenesis and vascular mimicry. Additionally, the delivery of glucagon-encapsulated PEGylated liposomes to tumor-bearing mice amplified the inhibition of angiogenesis and vascular mimicry, consequently reinforcing chemotherapy efficacy. Collectively, the findings demonstrate the role of glucagon in inhibiting tumor vessel network and suggest the potential utility of glucagon as a promising predictive marker for patients with CRC receiving chemotherapy.

Vascular endothelial growth factor B regulates insulin secretion in ß cells of type 2 diabetes mellitus mice via PLCγ and the IP3R­evoked Ca2+/CaMK2 signaling pathway.

Vascular endothelial growth factor B (VEGFB) plays a crucial role in glucolipid metabolism and is highly associated with type 2 diabetes mellitus (T2DM). The role of VEGFB in the insulin secretion of ß cells remains unverified. Thus, the present study aimed to discuss the effect of VEGFB on regulating insulin secretion in T2DM development, and its underlying mechanism. A high­fat diet and streptozocin (STZ) were used for inducing T2DM in mice model, and VEGFB gene in islet cells of T2DM mice was knocked out by CRISPR Cas9 and overexpressed by adeno­Associated Virus (AAV) injection. The effect of VEGFB and its underlying mechanism was assessed by light microscopy, electron microscopy and fluorescence confocal microscopy, enzyme­linked immunosorbent assay, mass spectrometer and western blot analysis. The decrement of insulin secretion in islet ß cell of T2DM mice were aggravated and blood glucose remained at a high level after VEGFB knockout (KO). However, glucose tolerance and insulin sensitivity of T2DM mice were improved after the AAV­VEGFB186 injection. VEGFB KO or overexpression can inhibit or activate PLCγ/IP3R in a VEGFR1­dependent manner. Then, the change of PLCγ/IP3R caused by VEGFB/VEGFR1 will alter the expression of key factors on the Ca2+/CaMK2 signaling pathway such as PPP3CA. Moreover, VEGFB can cause altered insulin secretion by changing the calcium concentration in ß cells of T2DM mice. These findings indicated that VEGFB activated the Ca2+/CaMK2 pathway via VEGFR1­PLCγ and IP3R pathway to regulate insulin secretion, which provides new insight into the regulatory mechanism of abnormal insulin secretion in T2DM.

Reducing VEGFB expression regulates the balance of glucose and lipid metabolism in mice via VEGFR1.

In recent years, studies have demonstrated that vascular endothelial growth factor B (VEGFB) can affect the metabolism of fatty acids and glucose, and it is expected to become a target for the diagnosis and treatment of metabolic diseases such as obesity and diabetes. At present, the specific mechanism that VEGFB regulates lipid and glucose metabolism balance is not completely understood. The present study used systemic VEGFB gene­knockout mice to investigate the effects of downregulation of the VEGFB gene on lipid metabolism and insulin secretion, and to explore the mechanism of the VEGFB pathway involved in the regulation of glucose and lipid metabolism. The morphological changes in the liver and pancreas of mice after VEGFB gene deletion were observed under a light microscope and a scanning electron microscope, and the effects of VEGFB gene deletion on lipid metabolism and blood glucose balance were detected by a serological technique. The detection indexes included total cholesterol (TC), triglyceride (TG), low­density lipoprotein cholesterol (LDL­C) and high­density lipoprotein cholesterol. Simultaneously, fasting blood glucose, glycosylated hemoglobin A1c (HbA1c), fasting insulin and glucagon were measured. Insulin sensitivity was assessed by using the insulin tolerance tests and glucose tolerance tests, and function of ß­cell islets was evaluated by using the insulin resistance index (HOMA­IR) and pancreatic ß­cell secretion index (HOMA­ß). Τhe protein expression changes of vascular endothelial growth factor receptor 1 (VEGFR1) and vascular endothelial growth factor receptor 2 (VEGFR2) in mouse islets were detected by western blotting and reverse transcription­quantitative polymerase chain reaction (RT­qPCR) after the VEGFB gene was knocked down to analyze the mechanism of VEGFB that may be involved in glucose and lipid metabolism. It was observed that after VEGFB was knocked down, mouse hepatocytes exhibited steatosis and increased secretory vesicles in islet cells. The lipid metabolism indexes such as TG, TC and LDL increased significantly; however, the levels of FBS, postprandial blood glucose and HbA1c decreased, whereas the glucose tolerance increased. Serum insulin secretion increased and HOMA­IR decreased since VEGFB was knocked down. Western blotting and RT­qPCR results revealed that the expression levels of VEGFR1 and neuropilin­1 decreased after the VEGFB gene was knocked down, while the expression levels of VEGFA and VEGFR2 increased. The absence of VEGFB may be involved in the regulation of glucose and lipid metabolism in mice by activating the VEGFA/VEGFR2 signaling pathway. VEGFB is expected to become a new target for the treatment of metabolic diseases such as obesity and diabetes. At present, the mechanism of VEGFB involved in regulating lipid metabolism and glucose metabolism is not completely clear. It was identified that downregulating VEGFB improved lipid metabolism and insulin resistance. The role of VEGFB/VEGFR1 pathway and other family members in regulating glucose and lipid metabolism was detected, which provided a theoretical and experimental basis for VEGFB to affect the regulation of glucose and lipid metabolism balance.

A Biodegradable High-Efficiency Magnetic Nanoliposome Promotes Tumor Microenvironment-Responsive Multimodal Tumor Therapy Along with Switchable T2 Magnetic Resonance Imaging.

We explored the catalytic activity and magnetic resonance imaging (MRI) capacity of Cu-doped ultrasmall iron oxides with different doping ratios. Then, we screened a highly efficient ultrasmall active catalyst (UAC). Subsequently, a biodegradable magnetic nanoliposome was developed for multimodal cancer theranostics through pH-sensitive liposome coating of these UACs. Upon entering the body, the magnetic nanoliposomes significantly prolonged the metabolic time of UACs and promoted their accumulation in tumors. Then, the strong photothermal (PT) effect of the magnetic nanoliposome quickly ablated the tumor, showing promising PT therapy. Upon entering tumor cells, the magnetic nanoliposome rapidly degraded into many UACs and released chemotherapeutic drugs, contributing to chemotherapy. In addition, UACs not only catalyzed Fenton-type reaction to produce excessive reactive oxygen species (ROS) but also inhibited the synthesis of endogenous GSH by inactivating glutamyl cysteine ligase, contributing to cancer ferroptosis. Furthermore, the assembly-dissociation process of UACs showed the function of magnetic relaxation switches, significantly enhancing tumor MRI signal change, achieving a more accurate diagnosis of the tumor. Therefore, this magnetic nanoliposome splits into many UACs upon drug release and regulates the tumor microenvironment to overproduce ROS for enhanced synergistic tumor theranostics, which provides a strategy for developing next-generation magnetic catalysts with biodegradability and multimodal antitumor theranostics.

Targeting Hyaluronic Acid and Peritoneal Dissemination in Colorectal Cancer.

Peritoneal metastasis (PM) from colorectal cancer (CRC) carries a significant mortality rate for patients and treatment is challenging. The development of PM is a multistep process involving detachment, adhesion, invasion and colonization of the peritoneal cavity. Cytoreductive surgery and HIPEC (hyperthermic intraperitoneal chemotherapy) for PM from CRC has some benefit but overall survival is poor and recurrence rates are high. Treatments to prevent the development of peritoneal metastasis could have the potential to improve CRC survival and disease-free outcomes. The ability of cancer cells to invade the peritoneum and become established as metastatic tumors is influenced by a multifactorial process. Hyaluronic acid (HA) has been shown to coat the mesothelial cells of the peritoneum and has been demonstrated to be utilized in various malignancies as part of the metastatic process in peritoneal dissemination. CD44, RHAMM (CD168) and ICAM-1 have all been shown to be binding partners for HA. Targeting HA-mediated binding may prevent adhesion to distant sites within the peritoneum through suppression of interaction of these molecules. Here we review the current literature and discuss key molecules involved with PM dissemination, with the potential to target these mechanisms in the delivery of future treatments.

Silencing CTNND1 Mediates Triple-Negative Breast Cancer Bone Metastasis via Upregulating CXCR4/CXCL12 Axis and Neutrophils Infiltration in Bone.

Bone metastasis from triple-negative breast cancer (TNBC) frequently results in poorer prognosis than other types of breast cancer due to the delay in diagnosis and intervention, lack of effective treatments and more skeletal-related complications. In the present study, we identified CTNND1 as a most reduced molecule in metastatic bone lesion from TNBC by way of high throughput sequencing of TNBC samples. In vivo experiments revealed that knockdown of CTNND1 enhanced tumor cells metastasis to bones and also increased neutrophils infiltration in bones. In vitro, we demonstrated that knockdown of CTNND1 accelerated epithelial-mesenchymal transformation (EMT) of tumor cells and their recruitment to bones. The involvement by CTNND1 in EMT and bone homing was achieved by upregulating CXCR4 via activating the PI3K/AKT/HIF-1αpathway. Moreover, TNBC cells with reduced expression of CTNND1 elicited cytotoxic T-cells responses through accelerating neutrophils infiltration by secreting more GM-CSF and IL-8. Clinically, patients with triple-negative breast cancer and lower level of CTNND1 had shorter overall survival (OS) and distant metastasis-free survival (DMFS). It was concluded that downregulation of CTNND1 played a critical role in facilitating bone metastasis of TNBC and that CTNND1 might be a potential biomarker for predicting the risk of bone metastases in TNBC.

Vascular endothelial growth factor B inhibits insulin secretion in MIN6 cells and reduces Ca2+ and cyclic adenosine monophosphate levels through PI3K/AKT pathway.

BACKGROUND: Type 2 diabetes (T2D) is characterized by insufficient insulin secretion caused by defective pancreatic ß-cell function or insulin resistance, resulting in an increase in blood glucose. However, the mechanism involved in this lack of insulin secretion is unclear. The level of vascular endothelial growth factor B (VEGF-B) is significantly increased in T2D patients. The inactivation of VEGF-B could restore insulin sensitivity in db/db mice by reducing fatty acid accumulation. It is speculated that VEGF-B is related to pancreatic ß-cell dysfunction and is an important factor affecting ß-cell secretion of insulin. As an in vitro model of normal pancreatic ß-cells, the MIN6 cell line can be used to analyze the mechanism of insulin secretion and related biological effects. AIM: To study the role of VEGF-B in the insulin secretion signaling pathway in MIN6 cells and explore the effect of VEGF-B on blood glucose regulation. METHODS: The MIN6 mouse pancreatic islet ß-cell line was used as the model system. By administering exogenous VEGF-B protein or knocking down VEGF-B expression in MIN6 cells, we examined the effects of VEGF-B on insulin secretion, Ca2+ and cyclic adenosine monophosphate (cAMP) levels, and the insulin secretion signaling pathway. RESULTS: Exogenous VEGF-B inhibited the secretion of insulin and simultaneously reduced the levels of Ca2+ and cAMP in MIN6 cells. Exogenous VEGF-B also reduced the expression of phospholipase C gamma 1 (PLCγ1), phosphatidylinositol 3-kinase (PI3K), serine/threonine kinase (AKT), and other proteins in the insulin secretion pathway. Upon knockdown of VEGF-B, MIN6 cells exhibited increased insulin secretion and Ca2+ and cAMP levels and upregulated expression of PLCγ1, PI3K, AKT, and other proteins. CONCLUSION: VEGF-B can regulate insulin secretion by modulating the levels of Ca2+ and cAMP. VEGF-B involvement in insulin secretion is related to the expression of PLCγ1, PI3K, AKT, and other signaling proteins. These results provide theoretical support and an experimental basis for the study of VEGF-B in the pathogenesis of T2D.

SIPA1 Is a Modulator of HGF/MET Induced Tumour Metastasis via the Regulation of Tight Junction-Based Cell to Cell Barrier Function.

BACKGROUND: Lung cancer is the leading cause of cancer death. SIPA1 is a mitogen induced GTPase activating protein (GAP) and may hamper cell cycle progression. SIPA1 has been shown to be involved in MET signaling and may contribute to tight junction (TJ) function and cancer metastasis. METHODS: Human lung tumour cohorts were analyzed. In vitro cell function assays were performed after knock down of SIPA1 in lung cancer cells with/without treatment. Quantitative polymerase chain reaction (qPCR) and western blotting were performed to analyze expression of HGF (hepatocyte growth factor), MET, and their downstream markers. Immunohistochemistry (IHC) and immunofluorescence (IFC) staining were performed. RESULTS: Higher expression of SIPA1 in lung tumours was associated with a poorer prognosis. Knockdown of SIPA1 decreased invasiveness and proliferation of in vitro cell lines, and the SIPA1 knockdown cells demonstrated leaky barriers. Knockdown of SIPA1 decreased tight junction-based barrier function by downregulating MET at the protein but not the transcript level, through silencing of Grb2, SOCS, and PKCµ (Protein kinase Cµ), reducing the internalization and recycling of MET. Elevated levels of SIPA1 protein are correlated with receptor tyrosine kinases (RTKs), especially HGF/MET and TJs. The regulation of HGF on barrier function and invasion required the presence of SIPA1. CONCLUSIONS: SIPA1 plays an essential role in lung tumourigenesis and metastasis. SIPA1 may be a diagnostic and prognostic predictive biomarker. SIPA1 may also be a potential therapeutic target for non-small cell lung cancer (NSCLC) patients with aberrant MET expression and drug resistance.

Tim-3 promotes cell aggressiveness and paclitaxel resistance through NF-κB/STAT3 signalling pathway in breast cancer cells.

OBJECTIVE: Although T-cell immunoglobulin and mucin-domain containing molecule-3 (Tim-3) has been recognized as a promising target for cancer immunotherapy, its exact role in breast cancer has not been fully elucidated. METHODS: Tim-3 gene expression in breast cancer and its prognostic significance were analyzed. Associated mechanisms were then explored in vitro by establishing Tim-3-overexpressing breast cancer cells. RESULTS: In a pooled analysis of The Cancer Genome Atlas (TCGA) database, Tim-3 gene expression levels were significantly higher (P<0.001) in breast cancer tissue, compared with normal tissues. Tim-3 was a prognosis indicator in breast cancer patients [relapse-free survival (RFS), P=0.004; overall survival (OS), P=0.099]. Tim-3 overexpression in Tim-3low breast cancer cells promoted aggressiveness of breast cancer cells, as evidenced by enhanced proliferation, migration, invasion, tight junction deterioration and tumor-associated tubal formation. Tim-3 also enhanced cellular resistance to paclitaxel. Furthermore, Tim-3 exerted its function by activating the NF-κB/STAT3 signalling pathway and by regulating gene expression [cyclin D1 (CCND1), C-Myc, matrix metalloproteinase-1(MMP1), TWIST, vascular endothelial growth factor (VEGF) upregulation, concomitant with E-cadherin downregulation). Lastly, Tim-3 downregulated tight junction-associated molecules zona occludens (ZO)-2, ZO-1 and occludin, which may further facilitate tumor progression. CONCLUSIONS: Tim-3 plays an oncogenic role in breast cancer and may represent a potential target for antitumor therapy.

Tim-3 promotes tube formation and decreases tight junction formation in vascular endothelial cells.

As a negative immune checkpoint molecule, T-cell immunoglobulin domain and mucin domain containing molecule-3 (Tim-3) has been found to serve a crucial role in immune escape and tumour progression. Previous studies have reported that Tim-3 is important to endothelial cells and it has also been demonstrated to be involved in numerous types of human diseases, including melanoma, lymphoma, rickettsial infection and atherosclerosis; however, its exact mechanism of action remains largely unknown. In the present study, Tim-3 was overexpressed in vascular endothelial human lung microvascular endothelial cells (HMVECs) and human umbilical vein endothelial cells (HUVECs), and in vitro assays were used to determine that Tim-3 promoted cell proliferation, migration, invasion and tube formation through activating cyclin D1 (CCND1), Ras homolog gene family member A and vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2). Additionally, Tim-3 decreased tight junction (TJ) formation and the transepithelial resistance (TER) of endothelial cells by decreasing the expression levels of TJ protein 2, Occludin and claudin 1 (CLND1). In conclusion, these findings suggested that Tim-3 may exert a positive role in angiogenesis and a negative role in TJ formation in vascular endothelial cells, which may provide novel strategies for the treatment of Tim-3-associated diseases.

System biology analysis reveals the role of voltage-dependent anion channel in mitochondrial dysfunction during non-alcoholic fatty liver disease progression into hepatocellular carcinoma.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common causes of hepatocellular carcinoma (HCC), but the underlying mechanisms behind the correlation of NAFLD with HCC are unclear. We aimed to uncover the genes and potential mechanisms that drive this progression. This study uncovered the genes and potential mechanisms through a multiple 'omics integration approach. Quantitative proteomics combined with phenotype-association analysis was performed. To investigate the potential mechanisms, a comprehensive transcriptome/lipidome/phenome-wide association analysis was performed in genetic reference panel BXD mice strains. The quantitative proteomics combined with phenotype-association results showed that VDAC1 was significantly increased in tumor tissues and correlated with NAFLD-related traits. Gene co-expression network analysis indicated that VDAC1 is involved in mitochondria dysfunction in the tumorigenic/tumor progression. The association between VDAC1 and mitochondria dysfunction can be explained by the fact that VDAC1 was associated with mitochondria membrane lipids cardiolipin (CL) composition shift. VDAC1 was correlated with the suppression of mature specie CL(LLLL) and elevation level of nascent CL species. Such profiling shift was supported by the significant positive correlation between VDAC1 and PTPMT1, as well as negative correlation with CL remodeling enzyme Tafazzin (TAZ). This study confirmed that the expression of VADC1 was dysregulated in NAFLD-driven HCC and associated with NAFLD progression. The VDAC1-driven mitochondria dysfunction is associated with cardiolipin composition shift, which causes alteration of mitochondria membrane properties.

TfR1 binding with H-ferritin nanocarrier achieves prognostic diagnosis and enhances the therapeutic efficacy in clinical gastric cancer.

H-ferritin (HFn) nanocarrier is emerging as a promising theranostic platform for tumor diagnosis and therapy, which can specifically target tumor cells via binding transferrin receptor 1 (TfR1). This led us to investigate the therapeutic function of TfR1 in GC. The clinical significance of TfR1 was assessed in 178 GC tissues by using a magneto-HFn nanoparticle-based immunohistochemistry method. The therapeutic effects of doxorubicin-loaded HFn nanocarriers (HFn-Dox) were evaluated on TfR1-positive GC patient-derived xenograft (GC-PDX) models. The biological function of TfR1 was investigated through in vitro and in vivo assays. TfR1 was upregulated (73.03%) in GC tissues, and reversely correlated with patient outcome. TfR1-negative sorted cells exhibited tumor-initiating features, which enhanced tumor formation and migration/invasion, whereas TfR1-positive sorted cells showed significant proliferation ability. Knockout of TfR1 in GC cells also enhanced cell invasion. TfR1-deficient cells displayed immune escape by upregulating PD-L1, CXCL9, and CXCL10, when disposed with IFN-γ. Western blot results demonstrated that TfR1-knockout GC cells upregulated Akt and STAT3 signaling. Moreover, in TfR1-positive GC-PDX models, the HFn-Dox group significantly inhibited tumor growth, and increased mouse survival, compared with that of free-Dox group. TfR1 could be a potential prognostic and therapeutic biomarker for GC: (i) TfR1 reversely correlated with patient outcome, and its negative cells possessed tumor-aggressive features; (ii) TfR1-positive cells can be killed by HFn drug nanocarrier. Given the heterogeneity of GC, HFn drug nanocarrier combined with other therapies toward TfR1-negative cells (such as small molecules or immunotherapy) will be a new option for GC treatment.

MiR-221 Is Specifically Elevated in PC3 Cells and its Deletion Reduces Adhesion, Motility and Growth.

BACKGROUND/AIM: MiR-221, often described both as an oncogenic microRNA and as a tumour suppressor, targets mRNAs involved in carcinogenesis. While other oncogenic microRNAs showed correlations with prostate cancer cell lines' aggressiveness, miR-221 showed an unusual overexpression in PC3. MATERIALS AND METHODS: CRISPR was used to delete miR-221 from PC3 cells. Analysing the characteristics of PC3miR-221del cells, a reduced growth rate and expression of cell-cycle genes was observed. In global gene expression/ontology analysis of PC3miR-221del cells, cell-cell and cell-substrate adhesion pathways were found to be greatly affected. In addition, reduced levels of adhesion, invasion and motility for PC3miR-221del cells, a change in F-actin localisation and a reduction of EMT markers were observed. RESULTS: The tumour suppressor gene, DIRAS3, was a predicted target of miR-221. In PC3miR-221del cells DIRAS3 was up-regulated at the gene and protein level. Ectopic expression of DIRAS3 in PC3wt cells recapitulated the cellular morphology changes seen in PC3miR-221del cells. DIRAS3 3'UTR was more stable in PC3miR-221del cells, as measured by semi-quantitative PCR and luciferase fusion reporter assays. CONCLUSION: MiR-221 promotes aggressiveness of PC3 cells by down-regulating DIRAS3, and promoting epithelial-to-mesenchymal transition.

Identification and functional characterization of Lys-trimethylation of lactate dehydrogenase A.

Background: Trimethylation of histones has been extensively studied, where histone methyltransferases catalyze the transfer of methyl groups from S-adenosyl methionine. Thus far, there have been no researches on the trimethylation of non-histone proteins. The precise mechanisms by which trimethylation affects cell progress and the related protein functions remain unclear. Purpose: The objective of this study was to identify the Lys-trimethylated proteins in kidney-derived cells and tissues, as well as to better understand the mechanisms underlying Lys-trimethylation-mediated cell metabolism. Methods: The levels of Lys-trimethylation in kidney-derived cells and tissues were assayed by Western blotting. Additionally, high-resolution mass spectrometry was used to analyze kidney-derived cells and tissues, and the eukaryotic expression vectors that led to the mutations of lysine were constructed and transfected into HEK293T cells. The LDHA activity of HEK293T cells was detected under conditions of Lys-trimethylation inhibition, and the proliferation of HEK293T cells was measured using EdU and Western blotting analyses. Results: The different proteins in kidney-derived cells and tissues showed different levels of Lys-trimethylation. In particular, lactate dehydrogenase A (LDHA) was Lys-trimethylated on lysine (K5). Inhibition of the Lys-trimethylation in LDHA increased the LDH activity of HEK293T cells and upregulated their proliferation. Conclusion: We suggested that LDHA affects the metabolism and proliferation of cells via a Lys-trimethylation-mediated mechanism; Lys-trimethylation might be a potential target for therapeutic research or used as a prognostic and treatment biomarker of several diseases.

Incorporating MicroRNA into Molecular Phenotypes of Circulating Tumor Cells Enhances the Prognostic Accuracy for Patients with Metastatic Breast Cancer.

BACKGROUND: The molecular phenotype of circulating tumor cells (CTCs) was associated with clinical outcome of patients with breast cancer. CTCs isolated from patients with metastatic breast cancer (MBC) display a unique microRNA (miRNA) expression profile. The aim of this study was to enhance the prognostic accuracy of the CTC phenotype in patients with MBC, by incorporating miRNA into a combined prediction model. SUBJECTS, MATERIALS, AND METHODS: CTCs were detected by CellSearch and enriched by magnetic cell sorting. miRNA deep sequencing and quantitative polymerase chain reaction were used to screen and verify potentially CTC-specific miRNA candidates. Patients with MBC were enrolled from two independent cohorts, and overall survival (OS) and chemotherapy response were analyzed. RESULTS: We screened and identified that miR-106b was an upregulated molecule in patients with MBC with CTC ≥5/7.5 mL (n = 16) compared with patients with CTC = 0/7.5 mL (n = 16) and healthy donors (n = 8). The expression of CTC-specific miR-106b correlated with vimentin and E-cadherin in CTC and acted as an independent factor for predicting OS (hazard ratio 2.157, 95% confidence interval [CI] 1.098-4.239, p = .026). Although CTC-specific miR-106b, E-cadherin, and vimentin showed a prognostic potential independently, the prognostic performance for OS based on the combination of three markers was significantly enhanced in Cohort 1 (area under the curve [AUC] 0.752, 95% CI 0.658-0.847, n = 128) and further validated in Cohort 2 (AUC 0.726, 95% CI 0.595-0.856, n = 91). Besides, a combined model incorporating miR-106b was associated with therapy response. CONCLUSION: The phenotypic assemblies of CTC incorporating miR-106b show enhanced prognostic accuracy of overall survival in patients with MBC. IMPLICATIONS FOR PRACTICE: In order to enhance the prognostic accuracy of the circulating tumor cell (CTC) phenotype in patients with metastatic breast cancer (MBC), this study screened and identified a CTC-specific microRNA (miRNA), miR-106b, as an upregulated molecule based on the comparison of miRNA profile between CTCs, primary tumors, and healthy blood donors. By incorporating miR-106b into a combined prediction model, the prognostic accuracy of the CTC phenotype for patients with MBC was greatly improved in both the training and validation cohorts. This work provides clinical evidence supporting the prognostic potential of CTC-specific miRNA for patients with MBC. These results indicate that developing CTC-specific miRNAs as new biomarkers will help to further optimize personalized therapy.

Identification of prothymosin alpha (PTMA) as a biomarker for esophageal squamous cell carcinoma (ESCC) by label-free quantitative proteomics and Quantitative Dot Blot (QDB).

BACKGROUND: Esophageal cancer (EC) is one of the malignant tumors with a poor prognosis. The early stage of EC is asymptomatic, so identification of cancer biomarkers is important for early detection and clinical practice. METHODS: In this study, we compared the protein expression profiles in esophageal squamous cell carcinoma (ESCC) tissues and adjacent normal esophageal tissues from five patients through high-resolution label-free mass spectrometry. Through bioinformatics analysis, we found the differentially expressed proteins of ESCC. To perform the rapid identification of biomarkers, we adopted a high-throughput protein identification technique of Quantitative Dot Blot (QDB). Meanwhile, the QDB results were verified by classical immunohistochemistry. RESULTS: In total 2297 proteins were identified, out of which 308 proteins were differentially expressed between ESCC tissues and normal tissues. By bioinformatics analysis, the four up-regulated proteins (PTMA, PAK2, PPP1CA, HMGB2) and the five down-regulated proteins (Caveolin, Integrin beta-1, Collagen alpha-2(VI), Leiomodin-1 and Vinculin) were selected and validated in ESCC by Western Blot. Furthermore, we performed the QDB and IHC analysis in 64 patients and 117 patients, respectively. The PTMA expression was up-regulated gradually along the progression of ESCC, and the PTMA expression ratio between tumor and adjacent normal tissue was significantly increased along with the progression. Therefore, we suggest that PTMA might be a potential candidate biomarker for ESCC. CONCLUSION: In this study, label-free quantitative proteomics combined with QDB revealed that PTMA expression was up-regulated in ESCC tissues, and PTMA might be a potential candidate for ESCC. Since Western Blot cannot achieve rapid and high-throughput screening of mass spectrometry results, the emergence of QDB meets this demand and provides an effective method for the identification of biomarkers.

Effects of Lidocaine and Ropivacaine on Gastric Cancer Cells Through Down-regulation of ERK1/2 Phosphorylation In Vitro.

BACKGROUND: Lidocaine and ropivacaine have been widely used in gastric cancer surgery. In recent years, lidocaine and ropivacaine have attracted increasing attention in cancer research, whilst effects of lidocaine and ropivacaine on gastric cancer cells have not been investigated before. This study explored the effect of lidocaine and ropivacaine on AGS and HGC-27 gastric cancer cells. MATERIALS AND METHODS: AGS and HGC-27 cells were incubated with lidocaine or ropivacaine at concentrations of 10, 100 and 1 mM. At 24, 48 and 72 h after treatment, proliferation and invasion were evaluated by crystal violet assay and transwell invasion assay. Electric cell-substrate impedance sensing was applied to measure the migration of cancer cells. Phosphorylation status of extracellular-regulated protein kinases (ERK1/2) was evaluated by western blot analysis. RESULTS: Lidocaine (1 mM) and ropivacaine (1 mM) significantly inhibited the proliferation of AGS and HG-27 cells, but had no significant effects on invasion. Lidocaine (1 mM) and ropivacaine (1 mM) also inhibited the migration of AGS and HGC-27cells. After treatment with lidocaine (1 mM) or ropivacaine (1 mM) for 24 h, the phosphorylation of ERK1/2 in AGS and HGC-27 cells was reduced. CONCLUSION: Lidocaine at a clinically relevant concentration (10 µM), and ropivacaine at 1 mM inhibited the proliferation of gastric cancer cell lines by down-regulation of p-ERK1/2. The migration of HGC-27 cells, rather than AGS cells was more obviously inhibited by lidocaine (1 mM) and ropivacaine (1 mM). This research is easy to implement, and lays a foundation for the future research of local anaesthetics in cancer.

Novel Trifluoromethylated Enobosarm Analogues with Potent Antiandrogenic Activity In Vitro and Tissue Selectivity In Vivo.

Prostate cancer often develops antiandrogen resistance, possibly via androgen receptor (AR) mutations, which change antagonists to agonists. Novel therapies with increased anticancer activity, while overcoming current drug resistance are urgently needed. Enobosarm has anabolic effects on muscle and bone while having no effect on the prostate. Here, we describe the activity of novel chemically modified enobosarm analogues. The rational addition of bis-trifluoromethyl groups into ring B of enobosarm, profoundly modified their activity, pharmacokinetic and tissue distribution profiles. These chemical structural modifications resulted in an improved AR binding affinity-by increasing the molecular occupational volume near helix 12 of AR. In vitro, the analogues SK33 and SK51 showed very potent antiandrogenic activity, monitored using LNCaP/AR-Luciferase cells where growth, PSA and luciferase activity were used as AR activity measurements. These compounds were 10-fold more potent than bicalutamide and 100-fold more potent than enobosarm within the LNCaP model. These compounds were also active in LNCaP/BicR cells with acquired bicalutamide resistance. In vivo, using the AR-Luc reporter mice, these drugs showed potent AR inhibitory activity in the prostate and other AR-expressing tissues, e.g., testes, seminal vesicles, and brain. These compounds do not inhibit AR activity in the skeletal muscle, and spleen, thus indicating a selective tissue inhibitory profile. These compounds were also active in vivo in the Pb-Pten deletion model. SK33 and SK51 have significantly different and enhanced activity profiles compared with enobosarm and are ideal candidates for further development for prostate cancer therapy with potentially fewer side effects. Mol Cancer Ther; 17(9); 1846-58. ©2018 AACR.