Post-Transcriptional Regulation of the GASC1 Oncogene with Active Tumor-Targeted siRNA-Nanoparticles.

Basal-like breast cancer (BLBC) accounts for the most aggressive types of breast cancer, marked by high rates of relapse and poor prognoses and with no effective clinical therapy yet. Therefore, investigation of new targets and treatment strategies is more than necessary. Here, we identified a receptor that can be targeted in BLBC for efficient and specific siRNA mediated gene knockdown of therapeutically relevant genes such as the histone demethylase GASC1, which is involved in multiple signaling pathways leading to tumorigenesis. Breast cancer and healthy breast cell lines were compared regarding transferrin receptor (TfR) expression via flow cytometry and transferrin binding assays. Nanobioconjugates made of low molecular weight polyethylenimine (LMW-PEI) and transferrin (Tf) were synthesized to contain a bioreducible disulfide bond. siRNA complexation was characterized by condensation assays and dynamic light scattering. Cytotoxicity, transfection efficiency, and the targeting specificity of the conjugates were investigated in TfR positive and negative healthy breast and breast cancer cell lines by flow cytometry, confocal microscopy, RT-PCR, and Western blot. Breast cancer cell lines revealed a significantly higher TfR expression than healthy breast cells. The conjugates efficiently condensed siRNA into particles with 45 nm size at low polymer concentrations, showed no apparent toxicity on different breast cancer cell lines, and had significantly greater transfection and gene knockdown activity on mRNA and protein levels than PEI/siRNA leading to targeted and therapeutic growth inhibition post GASC1 knockdown. The synthesized nanobioconjugates improved the efficiency of gene transfer and targeting specificity in transferrin receptor positive cells but not in cells with basal receptor expression. Therefore, these materials in combination with our newly identified siRNA sequences are promising candidates for therapeutic targeting of hard-to-treat BLBC and are currently further investigated regarding in vivo targeting efficacy and biocompatibility.

Histone Demethylase GASC1 Inhibitor Targeted GASC1 Gene to Inhibit the Malignant Transformation of Esophageal Cancer through the NOTCH-MAPK Signaling Pathway.

OBJECTIVE: Esophageal cancer is a common gastrointestinal tumor, with high incidence in our country. Histone demethylase 4 plays an important role in chromosome structural modification and gene expression regulation, becoming a new target for tumor treatment. GASC1 is an important member of the KDM4 family, closely related to the malignancy of tumors. METHODS: Constructing the short hairpin interfering RNA plasmid and blank control plasmid of gene KDM4C (also known as GASC1), transfecting them into human esophageal squamous cell carcinoma cell lines (KYSE-150 and KYSE-30, respectively), and screening the best treatment concentration based on cell viability. Cell cloning experiments analyzed the proliferation characteristics of each group of cells. Cell migration and scratch healing experiments analyzed the tumor's malignant metastasis and invasion capabilities. Immunofluorescence analysis was used to test the expression characteristics of protein GASC1. Western blot was used to analyze protein Notch1, HIF1A, Flt-1, c-myc, c-fos expression in each group of cell lines. RESULTS: In this experiment, caffeic acid and interfering RNA plasmids were added to regulate the expression level of GASC1 protein in each group. After that, a series of characterization methods were used to determine the positive correlation of the metastasis and proliferation ability of esophageal cancer cells with the expression level of GASC1 protein. The regulation of GASC1 protein was further proved by measuring the expression of each cancer-related protein. CONCLUSION: GASC1 gene plays a crucial role in the progression of esophageal cancer. By inhibiting the expression of GASC1 gene, pathways closely related to cancer development such as NOTCH and MAPK will also be inhibited, which may ultimately control the malignant development.

Genetic alterations of KDM4 subfamily and therapeutic effect of novel demethylase inhibitor in breast cancer.

The histone lysine demethylase KDM4 subfamily, comprised of four members (A, B, C, and D), play critical roles in controlling transcription, chromatin architecture and cellular differentiation. We previously demonstrated that KDM4C is significantly amplified and overexpressed in aggressive basal-like breast cancers and functions as a transforming oncogene. However, information regarding the genomic and transcriptomic alterations of the KDM4 subfamily in different subtypes of breast cancer remains largely incomplete. Here, we conducted a meta-analysis of KDM4A, B, C and D in breast cancer and identified associations among recurrent copy number alterations, gene expression and breast cancer subtypes. We demonstrated that KDM4A and D are also significantly overexpressed in basal-like breast cancer, whereas KDM4B overexpression is more dominant in estrogen-receptor-positive, luminal breast cancer. Next, we investigated the therapeutic potential of a novel histone demethylase inhibitor, NCDM-32B, in breast cancer. The treatment of basal breast cancer cell lines with NCDM-32B resulted in the decrease of cell viability and anchorage independent growth in soft agar. Furthermore, we found that NCDM-32B impaired several critical pathways that drive cellular proliferation and transformation in breast cancer. Our findings demonstrate genetic amplification and overexpression of the KDM4 demethylases in different subtypes of breast cancer. Furthermore, histone methylation is reversible and KDM4 demethylases are druggable targets. Thus, KDM4 inhibitors may serve as a novel therapeutic approach for a subset of aggressive breast cancer.

A High-Throughput Mass Spectrometry Assay Coupled with Redox Activity Testing Reduces Artifacts and False Positives in Lysine Demethylase Screening.

Demethylation of histones by lysine demethylases (KDMs) plays a critical role in controlling gene transcription. Aberrant demethylation may play a causal role in diseases such as cancer. Despite the biological significance of these enzymes, there are limited assay technologies for study of KDMs and few quality chemical probes available to interrogate their biology. In this report, we demonstrate the utility of self-assembled monolayer desorption/ionization (SAMDI) mass spectrometry for the investigation of quantitative KDM enzyme kinetics and for high-throughput screening for KDM inhibitors. SAMDI can be performed in 384-well format and rapidly allows reaction components to be purified prior to injection into a mass spectrometer, without a throughput-limiting liquid chromatography step. We developed sensitive and robust assays for KDM1A (LSD1, AOF2) and KDM4C (JMJD2C, GASC1) and screened 13,824 compounds against each enzyme. Hits were rapidly triaged using a redox assay to identify compounds that interfered with the catalytic oxidation chemistry used by the KDMs for the demethylation reaction. We find that overall this high-throughput mass spectrometry platform coupled with the elimination of redox active compounds leads to a hit rate that is manageable for follow-up work.

Histone demethylase GASC1, a potential prognostic and predictive marker in esophageal squamous cell carcinoma.

Gene amplified in squamous cell carcinoma 1 (GASC1) is a member of Jumonji C-domain containing histone demethylases that play an essential role in affecting chromatin architecture and gene expression. The purpose of this study was to determine the expression features and the clinical significance of GASC1 in esophageal squamous cell carcinoma (ESCC). GASC1 expression was detected on tissue microarrays of ESCC samples in 185 cases using immunohistochemical staining. Strong nuclear staining for GASC1 was observed in a subset of ESCC samples. The nuclear expression of GASC1 was significantly associated with lymph node metastasis (P=0.030) and tumor-node metastasis stages (P=0.013). Kaplan-Meier survival analysis showed a tendency that high expression of GASC1 in the nucleus was associated with poor survival of ESCC patients, with a 5-year survival rate of 26.5%, as compared to 43.7% for patients with GASC1-negative/low expression. Furthermore, multivariate analysis revealed that high expression of GASC1 likely acts as a predictive factor for overall survival of ESCC patients, despite the P-value failing to reach significance (P=0.059). The findings indicate that histone demethylase GASC1 may play an important role in promoting cancer metastasis, and shed new light on the importance of targeting GASC1 to suppress metastatic disease in various tumor types, including ESCC.