Identification and Characterization of Novel Spliced Variants of Neuregulin 4 in Prostate Cancer

Abstract

Purpose: The neuregulin (NRG) 1, 2, and 3 genes undergo extensive alternative mRNA splicing, which results in variants that show structural and functional diversity. The aims of this study were to establish whether the fourth member of this family, NRG4, is expressed in prostate cancer, if it is alternatively spliced and whether any functional differences between the variants could be observed. Experimental Design: The expression of NRG4 was determined using immunohistochemical staining of 40 cases of primary prostate cancer. Bioinformatic analysis and reverse transcription-PCR (RT-PCR) using NRG4 isotype-specific primers on a panel of normal and prostate cancer cell lines were used to identify alternatively spliced NRG4 variants. Expression of these variants was determined using isotype-specific antibodies. Transfection into Cos-7 cells of two of these green fluorescent protein-tagged variants allowed analysis of their subcellular location. Four of the variants were chemically synthesized and tested for their ability to activate the ErbB4 receptor. Results: NRG4 was variably expressed in the cytoplasm in the majority of prostate cancer cases, and in a subset of cases in the membrane, high levels were associated with advanced disease stage. Four novel NRG4 splice variants (NRGA2, NRG4 B1-3) were characterized, where each seemed to have a different subcellular location and were also expressed in the cytoplasm of the prostate tumors. NRG4 B3 was also present in endothelial cells. In transfected cells, the A type variant (NRG4 A1) was localized to the membrane, whereas the B type variant (NRG4 B1), which lacks the predicted transmembrane region, had an intracellular localization. Only the variants with an intact epidermal growth factor^like domain activated ErbB4 signaling. Conclusion: NRG4 overexpression is associated with advanced-stage prostate cancer. The alternative splice variants may have different roles in cell signaling, some acting as classic receptor ligands and some with as-yet unknown functions.

Alternative Splicing in Prostate and Breast Cancer

“…The NRG4-B1-3 isoforms are cytoplasmic and cannot activate ErbB4. The NRG4-A1 and NRG-B1-3 isoforms are expressed in PrC tissue and PrC cell lines; however, NRG4-A2 is expressed only in metastatic PrC cell lines [54].…”

“…NRG4 is expressed in PrC and over-expression is associated with advanced-stage PrC. AS of NRG4 results in several variants with different roles in cell signaling [54]. NRG4-A1 and NRG4-A2 retain the transmembrane and EGF-like domains, are capable of activating ErbB4, and are expressed on the membrane.…”

The complete family of epidermal growth factor receptors and their ligands are co-ordinately expressed in breast cancer

“…Here two factors emerged, but further test datasets 240 would be required to determine whether this was general-241 isable. The Neuregulins are produced as multiple splice 242 variants for instance, five have so far been identified as 243 products of the NRG4 gene [18] and these have very dif-244 ferent destinations within or without the cell and as such 245 may also have different functions. The antibodies used here 246 to the ligands (where known) are directed to the EGF-like 247 sequence which is shared by all the so far reported splice 248 variants and should thus detect the sum of the expressed 249 gene products.…”

Expression of neuregulin 4 splice variants in normal human tissues and prostate cancer and their effects on cell motility

“…The NRG2 and NRG3 gene products have been less well studied, but at least six alternative splice variants have been described for NRG2 and two for NRG3 . Recently, five gene products have been described for the NRG4 gene, which all share the same translation initiation site (Hayes et al 2007). Two of these variants, NRG4A1 and NRG4A2, have a transmembrane region and encode a shared biologically active 8 kDa fragment that includes an epidermal growth factor (EGF) motif .…”

“…A chemically synthesised and refolded EGF domain of NRG4 binds directly to ErbB4 and stimulates its phosphorylation (Harari et al 1999, Hayes et al 2007. Four human ErbB4 isoforms have been identified, two of which permit proteolytic cleavage and translocation to the nucleus of the subsequently released cytoplasmic domain (Carpenter & Liao 2009).…”