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

Abstract

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.

GASC1 promotes glioma progression by enhancing NOTCH1 signaling

“…Besides NOTCH1, the current data revealed that GASC1 also affects the Shh and Wnt signaling pathways. In support of this notion, previous studies reported that GASC1 can regulate numerous other molecules, such as MYC, NOTCH1, SOX2, MDM2, JAK2, peroxisome proliferator activated receptor gamma, Nanog homeobox and HIF-1α in cancer cells (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)35,51). All the data suggested that GASC1 may regulate NOTCH1 and other signaling pathways to control the viability, migration and stemness of cancer cells.…”

“…H3K9me3/2 mark is associated with transcriptional repression and the formation of heterochromatin, while H3K36me3/2 is involved in the suppression of incorrect transcription (34). Since GASC1 can either remove the repressive H3K9me3/2 or the active H3K36me3/2 factor in modifying the H3 process, GASC1 has been reported to promote proliferation and survival in various cancer cells by activating several classical oncogenes, such as MYC, NOTCH1, SOX2 and MDM2 proto-oncogene (MDM2) (7)(8)(9)(10)(11)(12)(13)(14). For example, GASC1 overexpression in the non-tumorigenic breast cell line MCF10A induces transformed phenotypes (6,7), while GASC1-knockdown in skin squamous cell carcinoma and breast cancer cells inhibits proliferation (6,7,9,11).…”

“…at corresponding promoter regions (6). Previous studies have revealed that GASC1 is widely expressed and serves as a transforming oncogene in several types of cancer, including lymphoma, medulloblastoma, lung, prostate and breast cancer (7)(8)(9)(10)(11)(12)(13)(14). Moreover, GASC1 was found to be highly expressed in the brain astrocytes, and the brain of GASC1-hypomorphic mutant mice displayed an increased number of astrocytes (12), suggesting a possible role of GASC1 in the development of glioma.…”

The JmjN domain as a dimerization interface and a targeted inhibitor of KDM4 demethylase activity

“…Recent studies reported deleterious effects upon inhibition of KDM4 subfamily members in cancer cells [ 33 , 35 , 36 ]. In this respect, silencing KDM4C expression by siRNA led to abnormal mitotic cells [ 50 ], as well as inhibition of cancer cell growth [ 25 , 51 ] and cell death in KYSE150 and U2OS cells [ 39 ], rendering these enzymes an attractive druggable target both for research as well as for anti-cancer therapeutics. Since the catalytic JmjC domain is present in many different histone lysine demethylases, designing specific competitive inhibitors has proven to be very challenging [ 22 , 38 , 52 – 54 ].…”

Targeted Delivery of siRNA to Transferrin Receptor Overexpressing Tumor Cells via Peptide Modified Polyethylenimine

“…The selective and efficient delivery of siRNA to tumors is hindered by many biological barriers [2]. Numerous modifications of polymers have been discovered to target tumor cells including folic acid [14,15,16], transferrin [17], epidermal growth factor (EGF) [18,19] and the tripeptide arginine-glycine-aspartate RGD [20]. In this study, two human TfR binding peptides, HAIYPRH (HAI peptide) and THRPPMWSPVWP (THR peptide), were tested for gene delivery in TfR positive cells.…”