Determination of cell uptake pathways for tumor inhibitor lysyl oxidase propeptide.

The lysyl oxidase propeptide (LOX-PP) is derived from pro-lysyl oxidase (Pro-LOX) by extracellular biosynthetic proteolysis. LOX-PP inhibits breast and prostate cancer xenograft tumor growth and has tumor suppressor activity. Although, several intracellular targets and molecular mechanisms of action of LOX-PP have been identified, LOX-PP uptake pathways have not been reported. Here we demonstrate that the major uptake pathway for recombinant LOX-PP (rLOX-PP) is PI3K-dependent macropinocytosis in PWR-1E, PC3, SCC9, MDA-MB-231 cell lines. A secondary pathway appears to be dynamin- and caveola dependent. The ionic properties of highly basic rLOX-PP provide buffering capacity at both high and low pHs. We suggest that the buffering capacity of rLOX-PP, which serves to limit endosomal acidification, sustains PI3K-dependent macropinocytosis in endosomes which in turn is likely to facilitate LOX-PP endosomal escape into the cytoplasm and its observed interactions with cytoplasmic targets and nuclear uptake.

Genetically engineered microvesicles carrying suicide mRNA/protein inhibit schwannoma tumor growth.

Microvesicles (MVs) play an important role in intercellular communication by carrying mRNAs, microRNAs (miRNAs), non-coding RNAs, proteins, and DNA from cell to cell. To our knowledge, this is the first report of delivery of a therapeutic mRNA/protein via MVs for treatment of cancer. We first generated genetically engineered MVs by expressing high levels of the suicide gene mRNA and protein-cytosine deaminase (CD) fused to uracil phosphoribosyltransferase (UPRT) in MV donor cells. MVs were isolated from these cells and used to treat pre-established nerve sheath tumors (schwannomas) in an orthotopic mouse model. We demonstrated that MV-mediated delivery of CD-UPRT mRNA/protein by direct injection into schwannomas led to regression of these tumors upon systemic treatment with the prodrug (5-fluorocytosine (5-FC)), which is converted within tumor cells to 5-fluorouracil (5-FU)-an anticancer agent. Taken together, these studies suggest that MVs can serve as novel cell-derived "liposomes" to effectively deliver therapeutic mRNA/proteins to treatment of diseases.

miR-1289 and "Zipcode"-like Sequence Enrich mRNAs in Microvesicles.

Despite intensive studies, the molecular mechanisms by which the genetic materials are uploaded into microvesicles (MVs) are still unknown. This is the first study describing a zipcode-like 25 nucleotide (nt) sequence in the 3'-untranslated region (3'UTR) of mRNAs, with variants of this sequence present in many mRNAs enriched in MVs, as compared to their glioblastoma cells of origin. When this sequence was incorporated into the 3'UTR of a reporter message and expressed in a different cell type, it led to enrichment of the reporter mRNA in MVs. Critical features of this sequence are both a CUGCC core presented on a stem-loop structure and a miRNA-binding site, with increased levels of the corresponding miRNA in cells further increasing levels of mRNAs in MVs.

miRNA-7 attenuation in Schwannoma tumors stimulates growth by upregulating three oncogenic signaling pathways.

Micro RNAs (miRNA) negatively regulate protein-coding genes at the posttranscriptional level and are critical in tumorigenesis. Schwannomas develop from proliferation of dedifferentiated Schwann cells, which normally wrap nerve fibers to help support and insulate nerves. In this study, we carried out high-throughput miRNA expression profiling of human vestibular schwannomas by using an array representing 407 known miRNAs to explore the role of miRNAs in tumor growth. Twelve miRNAs were found to be significantly deregulated in tumor samples as compared with control nerve tissue, defining a schwannoma-typical signature. Among these miRNAs, we focused on miR-7, which was one of the most downregulated in these tumors and has several known oncogene targets, including mRNAs for epidermal growth factor receptor (EGFR) and p21-activated kinase 1 (Pak1). We found that overexpression of miR-7 inhibited schwannoma cell growth both in culture and in xenograft tumor models in vivo, which correlated with downregulation of these signaling pathways. Furthermore, we identified a novel direct target of miR-7, the mRNA for associated cdc42 kinase 1 (Ack1), with the expression levels of miR-7 and Ack1 being inversely correlated in human schwannoma samples. These results represent the first miRNA profiling of schwannomas and the first report of a tumor suppressor function for miR-7 in these tumors that is mediated by targeting the EGFR, Pak1, and Ack1 oncogenes. Our findings suggest miR-7 as a potential therapeutic molecule for schwannoma treatment, and they prompt clinical evaluation of drugs that can inhibit the EGFR, Pak1, and Ack1 signaling pathways to treat this tumor type.

A novel imaging-compatible sciatic nerve schwannoma model.

Benign schwannomas are common tumors of the cranial and peripheral nerves, causing pain and loss of function. The development of effective therapy for these tumors has been hampered by the lack of relevant experimental in vivo models for convenient testing. Here, we describe a novel schwannoma model in which an immortalized human schwannoma cell line, HEI-193, established from an neurofibromatosis type 2 patient, has been stably transduced with fluorescent protein and luciferase reporters and implanted within the sciatic nerve of nude mice. These cells reliably formed a tumor within several weeks which had pathologic characteristics of schwannoma tumors. This model system will be useful for investigation of schwannoma biology and for preclinical assessment of therapeutic agents.