Identification of ASF/SF2 as a critical, allele-specific effector of the cyclin D1b oncogene.

The cyclin D1b oncogene arises from alternative splicing of the CCND1 transcript, and harbors markedly enhanced oncogenic functions not shared by full-length cyclin D1 (cyclin D1a). Recent studies showed that cyclin D1b is selectively induced in a subset of tissues as a function of tumorigenesis; however, the underlying mechanism(s) that control tumor-specific cyclin D1b induction remain unsolved. Here, we identify the RNA-binding protein ASF/SF2 as a critical, allele-specific, disease-relevant effector of cyclin D1b production. Initially, it was observed that SF2 associates with cyclin D1b mRNA (transcript-b) in minigene analyses and with endogenous transcript in prostate cancer (PCa) cells. SF2 association was altered by the CCND1 G/A870 polymorphism, which resides in the splice donor site controlling transcript-b production. This finding was significant, as the A870 allele promotes cyclin D1b in benign prostate tissue, but in primary PCa, cyclin D1b production is independent of A870 status. Data herein provide a basis for this disparity, as tumor-associated induction of SF2 predominantly results in binding to and accumulation of G870-derived transcript-b. Finally, the relevance of SF2 function was established, as SF2 strongly correlated with cyclin D1b (but not cyclin D1a) in human PCa. Together, these studies identify a novel mechanism by which cyclin D1b is induced in cancer, and reveal significant evidence of a factor that cooperates with a risk-associated polymorphism to alter cyclin D1 isoform production. Identification of SF2 as a disease-relevant effector of cyclin D1b provides a basis for future studies designed to suppress the oncogenic alternative splicing event.

Distinct roles for RB loss on cell cycle control, cisplatin response, and immortalization in Schwann cells.

Schwann cells play a critical role in peripheral nerve function. Regulated proliferation of Schwann cells is an important facet of the response to nerve injury; however, aberrant proliferation can give rise to Schwann cell tumors such as malignant peripheral nerve sheath tumors (MPNST). These tumors exhibit a range of genetic lesions that include loss of the retinoblastoma tumor suppressor (RB) pathway. RB plays a critical role in the regulation of cellular proliferation and its loss is a common event in human cancers. Here, the specific action of RB loss on Schwann cell proliferation and response to therapeutic intervention was explored. In primary mouse Schwann cells, conditional RB loss led to increased levels of critical cell cycle regulatory gene products, yet provided only a modest influence on proliferation. However, RB-deficient Schwann cells efficiently bypassed the cell cycle inhibitory response to the chemotherapeutic agent cisplatin, which is used in the treatment of MPNST and other glial tumors. Surprisingly, RB loss did not facilitate Schwann cell immortalization; and RB-deficient cells actually were less prone to immortalization than cells containing RB. Furthermore, RB-deficient cells that ultimately re-entered the cell cycle had lost both Schwann cell morphology and markers. Since, RB loss is likely a late event in Schwann cell tumor progression, the action of acute RB loss in immortalized Schwann cells was investigated. In this context, loss of RB had a profound effect on expression of target genes and the response to cisplatin. Thus, the loss of RB in both primary and immortal Schwann cells disrupted the response to anti-mitogenic signals and has implications for therapeutic intervention.

An intact intermediate filament network is required for collateral sprouting of small diameter nerve fibers.

Expression of the intermediate filament (IF) protein peripherin is initiated during development at the time of axonal extension and increases during regeneration of nerve fibers. To test whether the IF network is essential for neuron process outgrowth in the mature organism in vivo, we disrupted endogenous peripherin IF in small-sized dorsal root ganglion (DRG) neurons in transgenic mice via expression of a mutant peripherin transgene under control of peripherin gene regulatory sequences. Anatomical and functional analyses showed that these neurons send peripheral and central axonal projections to correct targets, express correct neuropeptides, and mediate acute pain responses normally. However, disruption of IF significantly impaired the ability of uninjured small-sized DRG neurons to sprout collateral axons into adjacent denervated skin, indicating a critical role for intact IF in plasticity, specifically in compensatory nociceptive nerve sprouting.

Brain lipid binding protein in axon-Schwann cell interactions and peripheral nerve tumorigenesis.

Loss of axonal contact characterizes Schwann cells in benign and malignant peripheral nerve sheath tumors (MPNST) from neurofibromatosis type 1 (NF1) patients. Tumor Schwann cells demonstrate NF1 mutations, elevated Ras activity, and aberrant epidermal growth factor receptor (EGFR) expression. Using cDNA microarrays, we found that brain lipid binding protein (BLBP) is elevated in an EGFR-positive subpopulation of Nf1 mutant mouse Schwann cells (Nf1(-/-) TXF) that grows away from axons; BLBP expression was not affected by farnesyltransferase inhibitor, an inhibitor of H-Ras. BLBP was also detected in EGFR-positive cell lines derived from Nf1:p53 double mutant mice and human MPNST. BLBP expression was induced in normal Schwann cells following transfection with EGFR but not H-Ras12V. Furthermore, EGFR-mediated BLBP expression was not inhibited by dominant-negative H-Ras, indicating that BLBP expression is downstream of Ras-independent EGFR signaling. BLBP-blocking antibodies enabled process outgrowth from Nf1(-/-) TXF cells and restored interaction with axons, without affecting cell proliferation or migration. Following injury, BLBP expression was induced in normal sciatic nerves when nonmyelinating Schwann cells remodeled their processes. These data suggest that BLBP, stimulated by Ras-independent pathways, regulates Schwann cell-axon interactions in normal peripheral nerve and peripheral nerve tumors.

Intron 1 is required for cell type-specific, but not injury-responsive, peripherin gene expression.

The "primitive" neurons of the peripheral nervous system (PNS) have the remarkable ability to regenerate new fibers. This regenerative process requires a sequence of gene activation and repression that is poorly understood. One gene that is almost exclusively expressed in neurons of the PNS and is activated after nerve injury is the peripherin intermediate filament gene, but little is known about the genomic elements that control either its restricted expression or its response to nerve injury in adult mice. Previous studies suggested that both 5' flanking sequence and intragenic regions were required for cell type-specific and injury-specific expression. To determine which intragenic regions were critical, mice were generated that expressed peripherin transgenes lacking different introns. Analyses of these mice revealed that deletion of introns 2-8 had no effect on either the cell type-specific or injury-specific expression of the peripherin gene; however, the remaining intron, intron 1, differentially bound Sp1 transcription-related proteins/protein complexes in extracts from peripherin-expressing and nonexpressing tissues. Furthermore, a transgene that lacked intron 1 was not expressed in many neurons that contain endogenous peripherin but was activated after injury. Thus, accurate cell type-specific peripherin gene expression in the PNS depends on elements within intron 1, but other sequences, most likely in the 5'flanking region, are required for activating the peripherin gene in response to nerve injury.