Histone Methyltransferase G9a Drives Chemotherapy Resistance by Regulating the Glutamate-Cysteine Ligase Catalytic Subunit in Head and Neck Squamous Cell Carcinoma.

Transient chemotherapeutic response is a major obstacle to treating head and neck squamous cell carcinomas (HNSCC). Histone methyltransferase G9a has recently been shown to be abundantly expressed in HNSCC, and is required to maintain the malignant phenotype. In this study, we found that high G9a expression is significantly associated with poor chemotherapeutic response and disease-free survival in HNSCC patients. Similarly, G9a expression and enzymatic activity were elevated in cisplatin-resistant HNSCC cells. Genetic or pharmacologic inhibition of G9a sensitized the resistant cells to cisplatin, increasing cellular apoptosis. Mechanistic investigations indicated that G9a contributes to transcriptional activation of the glutamate-cysteine ligase catalytic subunit (GCLC), which results in upregulation of cellular glutathione (GSH) and drug resistance. In addition, we observed a significant positive correlation between G9a and GCLC expression in tumors of HNSCC patients. Taken together, our findings provide evidence that G9a protects HNSCC cells against chemotherapy by increasing the synthesis of GSH, and imply G9a as a promising target for overcoming cisplatin resistance in HNSCC. Mol Cancer Ther; 16(7); 1421-34. ©2017 AACR.

HMGCS2 enhances invasion and metastasis via direct interaction with PPARα to activate Src signaling in colorectal cancer and oral cancer.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS2) is the rate-limiting enzyme of ketogenesis. Growing evidence indicates that HMGCS2 may be involved in cancer progression, but its exact role is largely unknown. In this study, we demonstrate that HMGCS2 mRNA expression is associated with poor clinical prognosis and outcomes in patients with colorectal cancer (CRC) and oral squamous cell carcinoma (OSCC). In vitro, ectopic expression of HMGCS2 enhanced cancer cell motility in a ketogenesis-independent manner. Moreover, HMGCS2 promoted Src activity by directly binding to peroxisome proliferator-activated receptor alpha (PPARα), a transcriptional activator of Src. Taken together, these results suggest that HMGCS2 may serve as a useful prognostic marker and vital target for future therapeutic strategies against advanced cancer.

CCN Detection of Cancer Tissues by Immunohistochemistry Staining.

CCN family members are involved in many physiologic and pathological functions, and be detected in many different cancer types. Immunohistochemistry (IHC) is an important diagnostic pathology tool to demonstrate protein expression in clinical and research fields. Here, we explain the preparation of sample slides, staining procedure, and the problems that might be met during the time. The differential staining of CCN proteins is shown in breast cancer, oral cancer, lung cancer, colorectal cancer, and gastric cancer.

Glutaminase 2 stabilizes Dicer to repress Snail and metastasis in hepatocellular carcinoma cells.

Glutaminolysis that catabolizes glutamine to glutamate plays a critical role in cancer progression. Glutaminase 2 (GLS2) has been reported as a tumor suppressor. Recent studies implied that GLS2 may display its multifunction besides classical metabolic feature by different localizations and potential protein binding domains. Here, we showed that GLS2 expression correlates inversely with stage, vascular invasion, tumor size and poor prognosis in human hepatocellular carcinoma (HCC) tissues. We found that GLS2 significantly represses cell migration, invasion and metastasis of HCC through downregulation of Snail in vitro and in vivo. Moreover, our results demonstrated that GLS2 interacts with Dicer and stabilizes Dicer protein to facilitate miR-34a maturation and subsequently represses Snail expression in a glutaminase activity independent manner. Our findings indicate that non-glutaminolysis function of GLS2 inhibits migration and invasion of HCC cells by repressing the epithelial-mesenchymal transition via the Dicer-miR-34a-Snail axis.

G9a/RelB regulates self-renewal and function of colon-cancer-initiating cells by silencing Let-7b and activating the K-RAS/ß-catenin pathway.

Epigenetic reprogramming has been associated with the functional plasticity of cancer-initiating cells (CICs); however, the regulatory pathway has yet to be elucidated. A siRNA screen targeting known epigenetic genes revealed that G9a profoundly impairs the chemo-resistance, self-renewal and metastasis of CICs obtained from patients with colorectal cancer (CRC). Patients with elevated G9a were shown to face a high risk of relapse and poor survival rates. From a mechanistic perspective, G9a binds with and stabilizes RelB, thereby recruiting DNA methyltransferase 3 on the Let-7b promoter and repressing its expression. This leads to the activation of the K-RAS/ß-catenin pathway and regulates self-renewal and function of CICs. These findings indicate that the G9a/RelB/Let-7b axis acts as a critical regulator in the maintenance of CIC phenotypes and is strongly associated with negative clinical outcomes. Thus, these findings may have diagnostic as well as therapeutic implications for the treatment of chemotherapy-resistant or metastatic CRC.

Inhibition of VEGF165/VEGFR2-dependent signaling by LECT2 suppresses hepatocellular carcinoma angiogenesis.

Hepatocellular carcinoma (HCC) relies on angiogenesis for growth and metastasis. Leukocyte cell-derived chemotaxin 2 (LECT2) is a cytokine and preferentially expressed in the liver. Previous studies have found that LECT2 targets to both immune and tumor cells to suppress HCC development and vascular invasion. Although LECT2 did not affect HCC cells growth in vitro, it still suppressed HCC xenografts growth in immune-deficient mice, suggesting other cells such as stroma cells may also be targeted by LECT2. Here, we sought to determine the role of LECT2 in tumor angiogenesis in HCC patients. We found that LECT2 expression inhibited tumor growth via angiogenesis in the HCC xenograft model. Specifically, we demonstrated that recombinant human LECT2 protein selectively suppressed vascular endothelial growth factor (VEGF)165-induced endothelial cell proliferation, migration, and tube formation in vitro and in vivo. Mechanistically, LECT2 reduced VEGF receptor 2 tyrosine phosphorylation and its downstream extracellular signal-regulated kinase and AKT phosphorylation. Furthermore, LECT2 gene expression correlated negatively with angiogenesis in HCC patients. Taken together, our findings demonstrate that LECT2 inhibits VEGF165-induced HCC angiogenesis through directly binding to VEGFR2 and has broad applications in treating VEGF-mediated solid tumors.

GIT1 promotes lung cancer cell metastasis through modulating Rac1/Cdc42 activity and is associated with poor prognosis.

G-protein-coupled receptor kinase interacting protein 1 (GIT1) is participated in cell movement activation, which is a fundamental process during tissue development and cancer progression. GIT1/PIX forming a functional protein complex that contributes to Rac1/Cdc42 activation, resulting in increasing cell mobility. Although the importance of Rac1/Cdc42 activation is well documented in cancer aggressiveness, the clinical importance of GIT1 remains largely unknown. Here, we investigated the clinical significance of GIT1 expression in non-small-cell lung cancer (NSCLC) and also verified the importance of GIT1-Rac1/Cdc42 axis in stimulating NSCLC cell mobility. The result indicated higher GIT1 expression patients had significantly poorer prognoses in disease-free survival (DFS) and overall survival (OS) compared with lower GIT1 expression patients. Higher GIT1 expression was an independent prognostic factor by multivariate analysis and associated with migration/invasion of NSCLC cells in transwell assay. In vivo studies indicated that GIT1 promotes metastasis of NSCLC cells. Finally, GIT1 was found to stimulate migration/invasion by altering the activity of Rac1/Cdc42 in NSCLC cells. Together, the GIT1 expression is associated with poor prognosis in patients with NSCLC. GIT1 is critical for the invasiveness of NSCLC cells through stimulating the activity of Rac1/Cdc42.

CCM-AMI, a Polyethylene Glycol Micelle with Amifostine, as an Acute Radiation Syndrome Protectant in C57BL/6 Mice.

Acute radiation syndrome results from radiation exposure, such as in accidental nuclear disasters. Safe and effective radioprotectants, mitigators, and treatment drugs must be developed as medical countermeasures against radiation exposure. Here, the authors evaluated CCM-Ami, a novel polyethylene glycol micelle encapsulated with amifostine, for its radioprotective properties after total-body irradiation from a 60Co source. Male C57BL/6 mice (6-8 wk old) were intravenously injected with 45 mg kg(-1) of CCM-Ami 90 min before exposure to 7.2 and 8.5 Gy irradiation at a dose rate of 0.04 Gy min(-1). Both survival benefit and hematopoietic protection were observed after prophylactic CCM-Ami administration when compared with the effects measured in excipient control and amifostine groups. Pharmacokinetic results showed that after the intravenous injection, the plasma concentration of WR-1065, the active form of amifostine, was higher in CCM-Ami-treated mice than in amifostine-treated mice. These findings suggest that CCM-Ami-mediated hematopoietic protection plays a key role in enhancing survival of mice exposed to radiation toxicity and thus indicate that CCM-Ami is a radioprotectant that can be used safely and effectively in nuclear disasters.

Macrophage Infiltration Induces Gastric Cancer Invasiveness by Activating the ß-Catenin Pathway.

BACKGROUND: Despite evidence that activated macrophages act in an inflammatory microenvironment to promote gastric tumorigenesis via ß-catenin signaling, the effects of ß-catenin signaling on gastric cancer cell metastasis and the relationship of these cells with surrounding tumor associated macrophages have not been directly studied. METHODS: Immunohistochemical staining was employed to analyze 103 patients. An invasion assay was used to evaluate the relationship between macrophages and gastric cancer cells. ß-catenin gain-of-function and loss-of-function approaches were performed. To assess the ß-catenin regulation mechanism in gastric cancer cells, Western blotting and reverse-transcription polymerase chain reaction were used. RESULTS: Increased density of macrophages was associated with advanced stage and poor survival. Gastric cancer cell lines co-cultured with macrophages conditioned medium showed increased nuclear accumulation of ß-catenin and increased invading ability. AKT but not ERK regulated ß-catenin translocation. MMP7 and CD44, both ß-catenin downstream genes, were involved in macrophage-activated gastric cancer cell invasion. CONCLUSION(S): Collectively, the clinical data suggest that macrophage infiltration is correlated with increased grade and poor prognosis for gastric cancer patients who underwent radical resection. Macrophages may induce invasiveness by activating the ß-catenin pathway.

Keap1-Nrf2 Interaction Suppresses Cell Motility in Lung Adenocarcinomas by Targeting the S100P Protein.

PURPOSE: Kelch-like ECH-associated protein 1 (Keap1) is an E3 ligase participated in the cellular defense response against oxidative stress through nuclear factor erythroid-2-related factor 2 (Nrf2). However, the role of Keap1 in regulating cancer motility is still controversial. We investigated the contribution of the Keap1-Nrf2 axis in the progression of non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: The expression of Keap1 and Nrf2 was examined via immunohistochemistry, real-time PCR, and Western blot analysis in a cohort of NSCLC tissues and cells. A series of in vivo and in vitro assays was performed to elucidate the contribution of the Keap1-Nrf2 axis in lung cancer mobility and progression. RESULTS: Keap1 expression was decreased in specimens from NSCLC patients with lymph node metastasis compared with patients without metastasis. Higher Keap1 expression levels were correlated with the survival of NSCLC patients. Moreover, manipulation of Keap1 expression affected cell migration/invasion abilities. Depletion of Nrf2 relieved the migration promotion imposed by Keap1 suppression. Mechanistic investigations found that S100P was downregulated in both Keap1-overexpressing and Nrf2-knockdown NSCLC cells. Overexpression of Keap1 and knockdown of Nrf2 both suppressed S100P expression in NSCLC cells. Knockdown of S100P inhibited cell migration in highly invasive NSCLC cells and also relieved the migration promotion imposed by Keap1 suppression in weakly invasive NSCLC cells. CONCLUSIONS: Our findings suggest that Keap1 functions as a suppressor of tumor metastasis by targeting the Nrf2/S100P pathway in NSCLC cells. In addition, overexpression of Keap1 may be a novel NSCLC treatment strategy and/or useful biomarker for predicting NSCLC progression.

Prognostic Significance of CDCP1 Expression in Colorectal Cancer and Effect of Its Inhibition on Invasion and Migration.

BACKGROUND: To assess the correlations and functions of complement C1r/C1s, Uegf, Bmp1 domain-containing protein-1 (CDCP1) in identifying colorectal cancer (CRC) patients who are at high risk for metastasis. METHODS: Tumor specimens from 101 patients were analyzed by real-time polymerase chain reaction to detect CDCP1 expression. CDCP1 expression plasmids and shRNA were used to knock down CDCP1 expression in this study to investigate migratory and invasive abilities by Boyden chambers. The mRNA expression profiles in shCDCP1 transfectants were compared to those in control cells by conducting microarray analysis. Its downstream effectors were also invested in this study. RESULTS: CRC patients with a high CDCP1 expression had a statistically significant lower overall survival and disease-free survival compared to those exhibiting low CDCP1 expression. In vitro, knock-down CDCP1 expression significantly decreased migratory and invasive abilities in HCT116. Aberrant expression of CDCP1 increased cancer cell migration and invasion. By using integrated genomics, we identified ROCK1 (rho-associated, coiled-coil-containing protein kinase 1 pseudogene 1) as a downstream effector in CDCP1-mediated migration and as an invasion mediator. Clinically, ROCK1 and CDCP1 mRNA expression exhibited a strong positive correlation in CRC patient samples. CONCLUSIONS: Our results implicated CDCP1 as a key regulator of CRC migration and invasion, and suggest that it is a useful prognostic factor for patients with CRC. Improved identification of a high-risk subset of early metastatic patients may guide indications of individualized treatment in clinical practice.