Introduction: Glioblastoma (GBM) is the most common and lethal of the central nervous system (CNS) malignancies. The initiation, progression, and infiltration ability of GBMs are attributed in part to the dysregulation of microRNAs (miRNAs). Thus, targeting dysregulated miRNAs with RNA oligonucleotides (RNA interference, RNAi) has been proposed for GBM treatment. Despite promising results in the laboratory, RNA oligonucleotides have clinical limitations that include poor RNA stability and off-target effects. RNAi therapies against GBM confront an additional obstacle, as they need to cross the blood-brain barrier (BBB). Methods: Here, we developed gold-liposome nanoparticles conjugated with the brain targeting peptides apolipoprotein E (ApoE) and rabies virus glycoprotein (RVG). First, we functionalized gold nanoparticles with oligonucleotide miRNA inhibitors (OMIs), creating spherical nucleic acids (SNAs). Next, we encapsulated SNAs into ApoE, or RVG-conjugated liposomes, to obtain SNA-Liposome-ApoE and SNA-Liposome-RVG, respectively. We characterized each nanoparticle in terms of their size, charge, encapsulation efficiency, and delivery efficiency into U87 GBM cells in vitro. Then, they were administered intravenously (iv) in GBM syngeneic mice to evaluate their delivery efficiency to brain tumor tissue. Results: SNA-Liposomes of about 30-50 nm in diameter internalized U87 GBM cells and inhibited the expression of miRNA-92b, an aberrantly overexpressed miRNA in GBM cell lines and GBM tumors. Conjugating SNA-Liposomes with ApoE or RVG peptides increased their systemic delivery to the brain tumors of GBM syngeneic mice. SNA-Liposome-ApoE demonstrated to accumulate at higher extension in brain tumor tissues, when compared with non-treated controls, SNA-Liposomes, or SNA-Liposome-RVG. Discussion: SNA-Liposome-ApoE has the potential to advance the translation of miRNAbased therapies for GBM as well as other CNS disorders.

Glioblastoma multiforme (GBM) is the most common and deadliest type of primary brain tumor with a prognosis of 14 months after diagnosis. Current treatment for GBM patients includes “total” tumor resection, temozolomide-based chemotherapy, radiotherapy or a combination of these options. Although, several targeted therapies, gene therapy, and immunotherapy are currently in the clinic and/or in clinical trials, the overall survival of GBM patients has hardly improved over the last two decades. Therefore, novel multitarget modalities are urgently needed. Recently, RNA interference (RNAi) has emerged as a novel strategy for the treatment of most cancers, including GBM. RNAi-based therapies consist of using small RNA oligonucleotides to regulate protein expression at the post-transcriptional level. Despite the therapeutic potential of RNAi molecules, systemic limitations including short circulatory stability and low release into the tumor tissue have halted their progress to the clinic. The effective delivery of RNAi molecules through the blood-brain barrier (BBB) represents an additional challenge. This review focuses on connecting the translational process of RNAi-based therapies from in vitro evidence to pre-clinical studies. We delineate the effect of RNAi in GBM cell lines, describe their effectiveness in glioma mouse models, and compare the proposed drug carriers for the effective transport of RNAi molecules through the BBB to reach the tumor in the brain. Furthermore, we summarize the most important obstacles to overcome before RNAi-based therapy becomes a reality for GBM treatment.

Glioblastoma (GBM) is the most common and aggressive of all brain tumors, with a median survival of only 14 months after initial diagnosis. Novel therapeutic approaches are an unmet need for GBM treatment. MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression at the post-transcriptional level. Several dysregulated miRNAs have been identified in all cancer types including GBM. In this study, we aimed to uncover the role of miR-143 in GBM cell lines, patient samples, and mouse models. Quantitative real-time RT-PCR of RNA extracted from formalin-fixed paraffin-embedded (FFPE) samples showed that the relative expression of miR-143 was higher in GBM patients compared to control individuals. Transient transfection of GBM cells with a miR-143 oligonucleotide inhibitor (miR-143-inh) resulted in reduced cell proliferation, increased apoptosis, and cell cycle arrest. SLC30A8, a glucose metabolism-related protein, was identified as a direct target of miR-143 in GBM cells. Moreover, multiple injections of GBM tumor-bearing mice with a miR-143-inh-liposomal formulation significantly reduced tumor growth compared to control mice. The reduced in vitro cell growth and in vivo tumor growth following miRNA-143 inhibition suggests that miR-143 is a potential therapeutic target for GBM therapy.

Background Even though targeted therapies are available for cancers expressing oncogenic epidermal growth receptor (EGFR) and (or) human EGFR2 (HER2), acquired or intrinsic resistance often confounds therapy success. Common mechanisms of therapy resistance involve activating receptor point mutations and (or) upregulation of signaling downstream of EGFR/HER2 to Akt and (or) mitogen activated protein kinase (MAPK) pathways. However, additional pathways of resistance may exist thus, confounding successful therapy. Methods To determine novel mechanisms of EGFR/HER2 therapy resistance in breast cancer, gefitinib or lapatinib resistant variants were created from SKBR3 breast cancer cells. Syngenic therapy sensitive and resistant SKBR3 variants were characterized for mechanisms of resistance by mammosphere assays, viability assays, and western blotting for total and phospho proteins. Results Gefitinib and lapatinib treatments reduced mammosphere formation in the sensitive cells, but not in the therapy resistant variants, indicating enhanced mesenchymal and cancer stem cell-like characteristics in therapy resistant cells. The therapy resistant variants did not show significant changes in known therapy resistant pathways of AKT and MAPK activities downstream of EGFR/HER2. However, these cells exhibited elevated expression and activation of the small GTPase Rac, which is a pivotal intermediate of GFR signaling in EMT and metastasis. Therefore, the potential of the Rac inhibitors EHop-016 and MBQ-167 to overcome therapy resistance was tested, and found to inhibit viability and induce apoptosis of therapy resistant cells. Conclusions Rac inhibition may represent a viable strategy for treatment of EGFR/HER2 targeted therapy resistant breast cancer.

Triple negative breast cancer (TNBC) is an aggressive variant of breast cancer that lacks the expression of estrogen, progesterone, and human epithelial growth factor receptors. Due to the scarce availability of receptor‐targeted or hormonal treatments, TNBC patients are submitted to taxane‐based chemotherapies like paclitaxel. Patients initially respond to paclitaxel, but 4 out of 10 patients may develop tumor recurrence along with high metastasis and mortality rates. Several studies demonstrate that paclitaxel may induce chemoresistance and metastasis through the activation of Toll‐Like Receptor 4 (TLR4)/NFKB signaling pathway, a known cancer prognostic marker in breast cancer. Hence, new treatments are needed to improve TNBC prognosis in patients. Recently, our laboratory and collaborators developed MBQ‐167, a potent Rac and Cdc42 inhibitor that decreases tumor burden, cell proliferation, and metastasis in TNBC mouse models. In preliminary studies with TNBC mice, we observed that MBQ‐167/Paclitaxel combination treatment prevented metastasis progression compared to paclitaxel treatment alone. This led us to hypothesize that MBQ‐167 chemosensitizes aggressive breast cancer cells and reduces metastasis by blocking Rac/Cdc4, hence decreasing their downstream target NfkB activity. This research project aims to evaluate cellular and molecular events that explain how MBQ‐167 prevents paclitaxel‐induced metastasis. Here, we generated TLR4‐knockdowns (TLR4‐KD) in MDA‐MB‐231 TNBC cell lines using lentiviral particles with shRNAs against the TLR4 mRNA. After knockdown validation with western blot analysis (WB), Wildtype (MDA‐MB‐231), Scramble control knockdowns (Sc‐KD), and TLR4‐KD cells were treated with different concentrations of MBQ‐167, paclitaxel, doxorubicin, or combinations. After treatments, we evaluated the effect on cell viability from MTT assays at 96 and 120 hours, cell apoptosis through Caspase‐Glo 3/7 Assays at 48 hours, and cellular migration through scratch wound healing assays. Our results demonstrate that partial inhibition of TLR4 decreases the cell viability of cells, increases apoptosis, and decreases cell migration after treatment with MBQ‐167 (250 nM and 500 nM), Paclitaxel (5 nM and 10 nM), or Doxorubicin (250 nM and 500 nM). We also observed an additive response upon combinatorial treatments with MBQ‐167/Paclitaxel and MBQ‐167/Doxorubicin when TLR4‐KD cells were compared to wild‐type and Sc‐KD cells. Altogether, these results suggest that TLR4‐KD chemosensitizes cells to Paclitaxel and Doxorubicin chemotherapies. In addition, TLR4 inhibition improves cellular response to Rac/Cdc42 inhibitor, MBQ‐167. Therefore, TLR4‐KD may reduce Rac and NfkB signaling, improving treatment response to MBQ‐167, MBQ‐167/Paclitaxel, and MBQ‐167/Doxorubicin. Other cellular studies, including Rac and NfkB activation assays, are being evaluated in our laboratory.

RNA interference is a therapeutic modality in which RNA molecules, such as small interference RNAs (siRNAs) and microRNAs (miRNAs) (inhibitors and mimics), are used to regulate the expression of target mRNAs. MiRNAs are multitargeting RNA molecules that are involved in the regulation of several targets and can therefore influence multiple pathways when deregulated. On the other hand, siRNAs repress a specific gene by sequence complementarity to the target mRNA. Although both miRNAs and siRNAs have an increasing therapeutic potential, systemic delivery of RNA‐based technologies is limited by the poor stability of RNA molecules in circulation. Hence, our main goal is to develop a carrier for the efficient and targeted delivery of RNA molecules to ovarian cancer cells. To achieve this, we propose a nanocarrier formulation of gold nanoparticle (AuNP)‐RNA conjugates encapsulated on folate receptor targeted liposomes that could achieve a double protection layer for the RNA while increasing delivery to ovarian cancer cells that overexpress the folate receptor on their surface. We prepared carriers for siRNAs and miRNA oligonucleotide mimics by conjugation of the RNA molecules to the surface of 15 nm diameter AuNPs via thiol linkages. We performed this conjugation in presence of polyethylene glycol (PEG‐2000) to avoid gold precipitation. Physical characterization of the carriers was conducted by dynamic light scattering (DLS) and quantification of conjugated RNA was assessed by fluorescence. The conjugation resulted in approximately 47 to 75 RNA molecules linked per nanoparticle and conjugates of 20 to 25 nm diameter with a slightly negative charge. In addition, encapsulation of the AuNP‐RNA conjugates into liposomes was attained with more than 70% encapsulation efficiency. These results indicate the feasibility of the development and production of our nanocarrier. As a proof of principle we are using this nanoparticle conjugate to target Integrin‐linked kinase (ILK). ILK is a pseudokinase that has been found to be aberrantly expressed in ovarian tumors. Further experiments of internalization, stability and toxicity demonstrate the efficacy of this nanocarrier as a delivery system for miRNA mimics and siRNAs to ovarian cancer cells. We conclude that siRNA‐based technologies are an alternative strategy to target ILK and other target proteins lacking specific chemical inhibitors.Support or Funding InformationRCMI: U54 MD007600This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glioblastoma is the most aggressive type of primary brain tumor with an overall survival of 14.6 months with standard care treatment (surgery, radiotherapy, and Temozolomide chemotherapy). About 90% of patients develop recurrent tumors due to acquired resistance to temozolomide. Therefore, new multimodal treatments that target more than one molecular pathway are needed. MicroRNAs (miRNAs) are small (18–22 nucleotides) non‐coding RNAs that regulate more than 60% of protein coding genes post‐transcriptionally. Their dysregulation is implicated in many diseases including diabetes, heart disease, and cancer. MicroRNA‐92b (miR92b) is highly upregulated in glioblastoma tumors and its expression has an inverse correlation with patient overall survival. This study aims to determine the biological effect of targeting miR92b in glioblastoma. We hypothesized that its inhibition would lead to cell death and decreased proliferation by allowing the expression of messenger RNA (mRNA) targets needed for normal cell function. To determine miR92b targeting effect in vitro, we transfected glioblastoma cell lines with an anti‐miR92b and evaluated cellular apoptosis, clonogenicity, and proliferation. Results showed that by inhibiting miR92b there was an increase in apoptotic cells and reduction of cell proliferation. We next evaluated the miR92b targeting effect in vivo, administering intraperitoneal injections of anti‐miR92b encapsulated liposomes to glioblastoma‐xenograft mice. Inhibiting miR92b in mice xenografts led to a significant decrease in tumor volume and tumor weight. Furthermore, we performed bioinformatic analysis to identify mRNA potential targets of miR92b. We both inhibited and overexpressed miR92b in glioblastoma cells (U87 and CRL1620 respectively) and determined the expression of more than 90 potential mRNA targets by quantitative PCR. This analysis lead to the identification of five potential miR92b mRNA targets‐ASB5, TEF, KIAA1024, ZNF776, FBXW7 and BAZ2‐ which are currently under further validation using Western blot analysis. Our results show that miR92b is a good therapeutic target, since its inhibition leads to increased cell death and decreased cellular proliferation both in vitro and in vivo. MiR92b is a potential target for glioblastoma patients and its targeting could lead to the regulation of tumor suppressive protein coding genes.Support or Funding InformationNIGMS‐RISE Grant Number R25‐GM061838, RCMI grant U54 MD007600 (National Institute of Health and Health Disparities), and UPR Comprehensive Center Seed Funds.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Background Even though targeted therapies are available for cancers expressing oncogenic epidermal growth receptor (EGFR) and (or) human EGFR2 (HER2), acquired or intrinsic resistance often confounds therapy success. Common mechanisms of therapy resistance involve activating receptor point mutations and (or) upregulation of signaling downstream of EGFR/HER2 to Akt and (or) mitogen activated protein kinase (MAPK) pathways. However, additional pathways of resistance may exist thus, confounding successful therapy. Methods To determine novel mechanisms of EGFR/HER2 therapy resistance in breast cancer, gefitinib or lapatinib resistant variants were created from SKBR3 breast cancer cells. Syngenic therapy sensitive and resistant SKBR3 variants were characterized for mechanisms of resistance by mammosphere assays, viability assays, and western blotting for total and phospho proteins. Results Gefitinib and lapatinib treatments reduced mammosphere formation in the parental cells, but not in the therapy resistant variants, indicating enhanced cancer stem cell-like and epithelial to mesenchymal transition (EMT) characteristics in therapy resistant cells. The therapy resistant variants did not show significant changes in established therapy resistant pathways of Akt and MAPK activities downstream of EGFR/HER2. However, these cells exhibited elevated expression and activation of the small GTPase Rac, which is a pivotal intermediate of GFR signaling in EMT and metastasis. Therefore, the potential of the Rac inhibitors EHop-016 and MBQ-167 to overcome therapy resistance was tested and found to inhibit viability and induce apoptosis of therapy resistant cells. Conclusions Rac inhibition may represent a viable strategy for treatment of EGFR/HER2 targeted therapy resistant breast cancer.