Genetic Engineering & Biotechnology News

DEC 2017

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10 | DECEMBER 2017 | GENengnews.com | Genetic Engineering & Biotechnology News At the Annual Summit on Cell Therapy and Molecular Medicine, a conference held in September 2017 in Chicago, Dr. Kazansky described how alternative splicing could be used to inhibit the expression of STAT5B, a proto-oncogene involved in prostate cancer progression, and simultaneously increase the expression of its naturally truncated isoform, STAT5∆B, which acts as a tumor suppres- sor gene. The truncated STAT5∆B isoform results from the insertion of an alternatively spliced exon that introduces a premature stop codon in STAT5B. STAT proteins are cytoplasmic transcrip- tion factors critical for cellular processes, and mammalian cells contain at least seven types of STAT protein. One of the challenges in targeting STAT proteins is that existing approaches indiscriminately suppress both their proto-oncogene and tumor suppressor gene functions. The approach used by Dr. Kazansky and colleagues is based on the finding that under acidic conditions, such as the ones encountered in cancer cells, a water-soluble peptide forms an alpha-helix across membrane bilayers. "We generated a conjugate of our switch- ing oligonucleotide, which was connected to the C-terminus of a peptide molecule," de- tails Dr. Kazansky. "This allows the peptide to work as a nanosyringe." Once the conjugate is inside the cell, it encounters a reducing environment, which breaks the disulfide bond that conjugates the insertion peptide to the switching oligonucle- otide. Breaking this bond releases the oligo- nucleotide and allows it to perform its action. "In mice that carried xenografts, we de- ployed oligonucleotide conjugates that were labeled with a near-infrared dye," states Dr. Kazansky, "and we demonstrated that the conjugates were delivered to the cancer." Fusion Genes "In our studies, which combined transcrip- tome and whole-genome sequence analyses, we validated several fusion genes that occur in prostate cancer," says Jianhua Luo, M.D., Ph.D., professor of pathology and UPMC Endowed Chair of Molecular Carcinogen- esis at the University of Pittsburgh Medical Center. This work with fusion genes, Dr. Luo suggests, could illuminate the molecular and genomic changes associated with aggressive behavior in prostate cancer. Previously, Dr. Luo and colleagues com- pared whole-genome DNA methylation pat- terns in prostate cancer cells, benign cells adjacent to the tumor, and cells from healthy prostate tissue. The investigators revealed that CpG island methylation did not clearly correlate with RNA expression and could not explain, by itself, the suppression of gene expression. Subsequent efforts in Dr. Luo's labora- tory combined whole-genome and transcrip- tome sequencing, and identified several fu- sion transcripts that were present in cancer cells. Eight of the fusion constructs that were found were validated using reverse transcrip- tion polymerase chain reaction (RT–PCR), Sanger sequencing, and fluorescence in situ hybridization. Most patients harboring one of these fusion proteins showed a higher risk of recurrences, metastases, or prostate can- cer-specific death after surgery. One of these fusion constructs, MAN2A1- FER, is a fusion between MAN2A1, which encodes a Golgi enzyme involved in convert- ing mannose to complex N-glycan molecules for the glycosylation of membrane proteins, and FER, an oncogene that encodes a tyro- sine kinase. The fusion construct harbors a deletion of the 441 C-terminal amino acids from MAN2A1 and of the 571 N-terminal amino acids from FER. Dr. Luo and col- leagues identified the same fusion protein in a cancer cell line and in six different human cancers, including ovarian cancer, liver can- cer, and glioblastoma multiforme. Co-immunostaining revealed that MA- N2A1-FER localizes to the Golgi apparatus. To test whether this fusion protein retained its tyrosine kinase activity, Dr. Luo and colleagues expressed and purified the construct from bac- terial cells for in vitro enzymatic studies. "We found that the fusion construct had a four-fold higher kinase activity," reports Dr. Luo. This finding suggested that loss of the SH2 domain from FER, which is not part of the fusion protein, could increase its tyrosine kinase activity. Subsequent analyses revealed that the fu- sion construct phosphorylates the N-terminus of EGFR. "We identified the phosphylation site as tyrosine 88, which is not supposed to be phosphorylated or contribute to any kind of kinase activity, because that domain is ex- posed extracellularly," notes Dr. Luo. This tyrosine is situated at the interface between domains I and II of EGFR. "Phos- phorylation of tyrosine 88 is essential for the MAN2A1-FER-mediated activation and for dimerization of EGFR," adds Dr. Luo. Mutagenesis of this tyrosine residue into alanine, which eliminates the phosphoryla- tion site, abrogated EGFR activation in cell lines containing the fusion protein. This is the first example of a constitutively active ty- rosine kinase that is translocated to the Golgi apparatus and leads to the oncogenic activa- tion of the EGFR pathways. "This tyrosine residue is present only in cancer cells, and it is exposed," Dr. Luo points out. "Eventually it could become a valuable cellular target." References available online. 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