Small cell lung cancer (SCLC) is a highly malignant form of cancer, which originates from primitive neuroendocrine cells in the lung. SCLC cells express several autocrine neurotransmitters/neuropeptides and their respective receptors. Expression of these neuronal markers is frequently regulated by RE1-silencing transcription factor (REST). In SCLC cells, an SCLC-specific isoform of REST (sREST) is highly expressed, whereas REST expression is undetectable, suggesting that the expression of sREST correlates with the pathogenesis of SCLC. Expression of sREST, which is derived through alternative splicing of REST, is abnormally regulated in SCLC cells, but the mechanism is unknown. Most recently, nSR100 (SRRM4) was described as an activator of REST alternative splicing. We now show that nSR100 is highly expressed in SCLC cells correlating with high sREST and low REST expression. Adhesion to the extracellular matrix (ECM) is thought to enhance tumorigenicity and confer resistance to apoptosis. Interestingly, nSR100 expression is enhanced in cells grown with ECM. Overexpression of REST caused repression of sREST and nSR100, the latter containing RE1 element controlled by REST. Culturing the SCLC cell line NCI-N417 cells with ECM also upregulated RE1-containing gene, the voltage-gated calcium channel subunit. Inhibition of the PI3K/Akt/mTOR pathway by LY294002 induced nSR100 expression, whereas the specific MEK/ERK inhibitor U0126 inhibited nSR100 expression. Repressing nSR100 by siRNA effectively repressed sREST, and conversely increased REST in NCI-N417 cells. Taken together, this report clarifies the ECM-dependent signaling pathway that impacts nSR100 expression and its regulation of alternative splicing in SCLC.

Small cell lung cancer (SCLC) is the most aggressive neuroendocrine phenotype of the deadliest human lung cancers. However the therapeutic landscape for SCLC has not changed in over 30 years. Effective treatment and prognosis are needed to combat this aggressive cancer. Herein we report that Ser/Arg repetitive matrix 4 (SRRM4), a splicing activator, is abnormally expressed at high levels in SCLC and thus is a potential therapeutic target. We screened an effective gapmer antisense oligonucleotide (gASO) targeting SRRM4 in vitro which led to cell death of SCLC. Our gASO, which is stabilized by containing artificial nucleotides, effectively represses SRRM4 mRNA. We found that our gASO repressed SRRM4 synthesis leading to a dramatic tumor reduction in a lung cancer mouse model. We also analyzed miRNA microarray and found that the miR-4516 is abnormally increased in exosomes in the blood of SCLC patients. Treating with gASO suppressed tumors in the SCLC model mouse concurrently reduced plasma miR-4516. In conclusion this study reports that administration of an SRRM4-targeted gASO coupled with a novel miRNA diagnostic methodology represents a potential breakthrough in the therapeutic treatment of high mortality SCLC.

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<div>Abstract<p>Small cell lung cancer (SCLC) is a highly malignant form of cancer, which originates from primitive neuroendocrine cells in the lung. SCLC cells express several autocrine neurotransmitters/neuropeptides and their respective receptors. Expression of these neuronal markers is frequently regulated by RE1-silencing transcription factor (REST). In SCLC cells, an SCLC-specific isoform of REST (sREST) is highly expressed, whereas REST expression is undetectable, suggesting that the expression of sREST correlates with the pathogenesis of SCLC. Expression of sREST, which is derived through alternative splicing of REST, is abnormally regulated in SCLC cells, but the mechanism is unknown. Most recently, nSR100 (SRRM4) was described as an activator of REST alternative splicing. We now show that nSR100 is highly expressed in SCLC cells correlating with high sREST and low REST expression. Adhesion to the extracellular matrix (ECM) is thought to enhance tumorigenicity and confer resistance to apoptosis. Interestingly, nSR100 expression is enhanced in cells grown with ECM. Overexpression of REST caused repression of sREST and nSR100, the latter containing RE1 element controlled by REST. Culturing the SCLC cell line NCI-N417 cells with ECM also upregulated RE1-containing gene, the voltage-gated calcium channel subunit. Inhibition of the PI3K/Akt/mTOR pathway by LY294002 induced nSR100 expression, whereas the specific MEK/ERK inhibitor U0126 inhibited nSR100 expression. Repressing nSR100 by siRNA effectively repressed sREST, and conversely increased REST in NCI-N417 cells. Taken together, this report clarifies the ECM-dependent signaling pathway that impacts nSR100 expression and its regulation of alternative splicing in SCLC.</p><p><b>Implications:</b> The splicing factor nSR100 may be novel SCLC-specific biomarker, as well as a therapeutic target. <i>Mol Cancer Res; 11(10); 1258–68. ©2013 AACR</i>.</p></div>

<p>PDF - 202KB, Supplementary Figure 1: Growth curve for human SCLC cell lines growing on plastic or ECL extracellular matrix coated plastic dishes. Supplementary Table 1: Primers used for gene expression analysis. Supplementary Table 2: Comprehensive list of SCLC-associated autoantigens and autoantibodies.</p>