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Genomic Landscape Survey Identifies SRSF1 as a Key Oncodriver in Small Cell Lung Cancer.

Jiang L, Huang J, Higgs BW, Hu Z, Xiao Z, Yao X, Conley S, Zhong H, Liu Z, Brohawn P, Shen D, Wu S, Ge X, Jiang Y, Zhao Y, Lou Y, Morehouse C, Zhu W, Sebastian Y, Czapiga M, Oganesyan V, Fu H, Niu Y, Zhang W, Streicher K, Tice D, Zhao H, Zhu M, Xu L, Herbst R, Su X, Gu Y, Li S, Huang L, Gu J, Han B, Jallal B, Shen H, Yao Y - PLoS Genet. (2016)

Bottom Line: A few sequencing studies performed on limited number of samples have revealed potential disease-driving genes in SCLC, however, much still remains unknown, particularly in the Asian patient population.Here we conducted whole exome sequencing (WES) and transcriptomic sequencing of primary tumors from 99 Chinese SCLC patients.Additionally, Serine/Arginine Splicing Factor 1 (SRSF1) DNA copy number gain and mRNA over-expression was strongly associated with poor survival using both discovery and validation patient cohorts.

View Article: PubMed Central - PubMed

Affiliation: Department of Pulmonary, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai, China.

ABSTRACT
Small cell lung cancer (SCLC) is an aggressive disease with poor survival. A few sequencing studies performed on limited number of samples have revealed potential disease-driving genes in SCLC, however, much still remains unknown, particularly in the Asian patient population. Here we conducted whole exome sequencing (WES) and transcriptomic sequencing of primary tumors from 99 Chinese SCLC patients. Dysregulation of tumor suppressor genes TP53 and RB1 was observed in 82% and 62% of SCLC patients, respectively, and more than half of the SCLC patients (62%) harbored TP53 and RB1 mutation and/or copy number loss. Additionally, Serine/Arginine Splicing Factor 1 (SRSF1) DNA copy number gain and mRNA over-expression was strongly associated with poor survival using both discovery and validation patient cohorts. Functional studies in vitro and in vivo demonstrate that SRSF1 is important for tumorigenicity of SCLC and may play a key role in DNA repair and chemo-sensitivity. These results strongly support SRSF1 as a prognostic biomarker in SCLC and provide a rationale for personalized therapy in SCLC.

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SRSF1 is required for tumorigenecity of SCLC.(a) and (b): DMS114 cells were transfected with non-targeting control or SRSF1-directed siRNAs for 48 hrs, then treated with cisplatin (2.5ug/ml) or topotecan (2.5ug/ml) for 24 hrs. Cell growth (a) and Caspase-3/7 activities (b) were assessed and normalized against non-targeting ctrl siRNA-transfected cells as 100% control. (c): DMS114 cells were transfected with non-targeting and SRSF1 siRNAs for 48 hrs and then seeded in sphere forming media and allowed to grow for 4 days. Phase-contrast images of the sphere formation under each condition were captured and viable cell mass quantitated by CTG assay. (d): Reconstitution of SRSF1 expression using a siRNA-resistant Flag-tagged SRSF1 expression construct was carried out in SRSF1 siRNA transfected cells. Impact on sphere growth rate was assessed by CTG assay, and successful SRSF1 protein re-expression was confirmed using either anti-SRSF1 antibody or anti-Flag antibody. (e) DMS114 cells transfected with non-targeting control siRNA or SRSF1 siRNA were implanted into immunocompromised mice and tumor formation rates were monitored and measured as described in Materials and Methods.
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pgen.1005895.g004: SRSF1 is required for tumorigenecity of SCLC.(a) and (b): DMS114 cells were transfected with non-targeting control or SRSF1-directed siRNAs for 48 hrs, then treated with cisplatin (2.5ug/ml) or topotecan (2.5ug/ml) for 24 hrs. Cell growth (a) and Caspase-3/7 activities (b) were assessed and normalized against non-targeting ctrl siRNA-transfected cells as 100% control. (c): DMS114 cells were transfected with non-targeting and SRSF1 siRNAs for 48 hrs and then seeded in sphere forming media and allowed to grow for 4 days. Phase-contrast images of the sphere formation under each condition were captured and viable cell mass quantitated by CTG assay. (d): Reconstitution of SRSF1 expression using a siRNA-resistant Flag-tagged SRSF1 expression construct was carried out in SRSF1 siRNA transfected cells. Impact on sphere growth rate was assessed by CTG assay, and successful SRSF1 protein re-expression was confirmed using either anti-SRSF1 antibody or anti-Flag antibody. (e) DMS114 cells transfected with non-targeting control siRNA or SRSF1 siRNA were implanted into immunocompromised mice and tumor formation rates were monitored and measured as described in Materials and Methods.

Mentions: We next evaluated SRSF1 as a potential tumor driver in SCLC. We first screened SRSF1 DNA CNs in 13 SCLC cell lines using TaqMan assays. Five of thirteen had SRSF1 CN> = 3: Four including NCI-H82 had 3 copies, and DMS114 had 4 copies. These cell lines also expressed high levels of SRSF1 protein (S4 Fig). SRSF1 siRNA was transfected into DMS114, and the growth effect of SRSF1 ablation in two dimensional cell culture either alone or in conjunction with a sub-lethal dose of cisplatin or topotecan (two of the most common standard of care treatments in SCLC), was evaluated (Fig 4A). SRSF1 knockdown alone caused a 35% decrease in the proliferation rate. Treatment with a low dose of cisplatin or topotecan only induced a modest decrease of cell growth. However, combination with SRSF1 siRNA significantly enhanced the overall growth inhibition effect.


Genomic Landscape Survey Identifies SRSF1 as a Key Oncodriver in Small Cell Lung Cancer.

Jiang L, Huang J, Higgs BW, Hu Z, Xiao Z, Yao X, Conley S, Zhong H, Liu Z, Brohawn P, Shen D, Wu S, Ge X, Jiang Y, Zhao Y, Lou Y, Morehouse C, Zhu W, Sebastian Y, Czapiga M, Oganesyan V, Fu H, Niu Y, Zhang W, Streicher K, Tice D, Zhao H, Zhu M, Xu L, Herbst R, Su X, Gu Y, Li S, Huang L, Gu J, Han B, Jallal B, Shen H, Yao Y - PLoS Genet. (2016)

SRSF1 is required for tumorigenecity of SCLC.(a) and (b): DMS114 cells were transfected with non-targeting control or SRSF1-directed siRNAs for 48 hrs, then treated with cisplatin (2.5ug/ml) or topotecan (2.5ug/ml) for 24 hrs. Cell growth (a) and Caspase-3/7 activities (b) were assessed and normalized against non-targeting ctrl siRNA-transfected cells as 100% control. (c): DMS114 cells were transfected with non-targeting and SRSF1 siRNAs for 48 hrs and then seeded in sphere forming media and allowed to grow for 4 days. Phase-contrast images of the sphere formation under each condition were captured and viable cell mass quantitated by CTG assay. (d): Reconstitution of SRSF1 expression using a siRNA-resistant Flag-tagged SRSF1 expression construct was carried out in SRSF1 siRNA transfected cells. Impact on sphere growth rate was assessed by CTG assay, and successful SRSF1 protein re-expression was confirmed using either anti-SRSF1 antibody or anti-Flag antibody. (e) DMS114 cells transfected with non-targeting control siRNA or SRSF1 siRNA were implanted into immunocompromised mice and tumor formation rates were monitored and measured as described in Materials and Methods.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4836692&req=5

pgen.1005895.g004: SRSF1 is required for tumorigenecity of SCLC.(a) and (b): DMS114 cells were transfected with non-targeting control or SRSF1-directed siRNAs for 48 hrs, then treated with cisplatin (2.5ug/ml) or topotecan (2.5ug/ml) for 24 hrs. Cell growth (a) and Caspase-3/7 activities (b) were assessed and normalized against non-targeting ctrl siRNA-transfected cells as 100% control. (c): DMS114 cells were transfected with non-targeting and SRSF1 siRNAs for 48 hrs and then seeded in sphere forming media and allowed to grow for 4 days. Phase-contrast images of the sphere formation under each condition were captured and viable cell mass quantitated by CTG assay. (d): Reconstitution of SRSF1 expression using a siRNA-resistant Flag-tagged SRSF1 expression construct was carried out in SRSF1 siRNA transfected cells. Impact on sphere growth rate was assessed by CTG assay, and successful SRSF1 protein re-expression was confirmed using either anti-SRSF1 antibody or anti-Flag antibody. (e) DMS114 cells transfected with non-targeting control siRNA or SRSF1 siRNA were implanted into immunocompromised mice and tumor formation rates were monitored and measured as described in Materials and Methods.
Mentions: We next evaluated SRSF1 as a potential tumor driver in SCLC. We first screened SRSF1 DNA CNs in 13 SCLC cell lines using TaqMan assays. Five of thirteen had SRSF1 CN> = 3: Four including NCI-H82 had 3 copies, and DMS114 had 4 copies. These cell lines also expressed high levels of SRSF1 protein (S4 Fig). SRSF1 siRNA was transfected into DMS114, and the growth effect of SRSF1 ablation in two dimensional cell culture either alone or in conjunction with a sub-lethal dose of cisplatin or topotecan (two of the most common standard of care treatments in SCLC), was evaluated (Fig 4A). SRSF1 knockdown alone caused a 35% decrease in the proliferation rate. Treatment with a low dose of cisplatin or topotecan only induced a modest decrease of cell growth. However, combination with SRSF1 siRNA significantly enhanced the overall growth inhibition effect.

Bottom Line: A few sequencing studies performed on limited number of samples have revealed potential disease-driving genes in SCLC, however, much still remains unknown, particularly in the Asian patient population.Here we conducted whole exome sequencing (WES) and transcriptomic sequencing of primary tumors from 99 Chinese SCLC patients.Additionally, Serine/Arginine Splicing Factor 1 (SRSF1) DNA copy number gain and mRNA over-expression was strongly associated with poor survival using both discovery and validation patient cohorts.

View Article: PubMed Central - PubMed

Affiliation: Department of Pulmonary, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai, China.

ABSTRACT
Small cell lung cancer (SCLC) is an aggressive disease with poor survival. A few sequencing studies performed on limited number of samples have revealed potential disease-driving genes in SCLC, however, much still remains unknown, particularly in the Asian patient population. Here we conducted whole exome sequencing (WES) and transcriptomic sequencing of primary tumors from 99 Chinese SCLC patients. Dysregulation of tumor suppressor genes TP53 and RB1 was observed in 82% and 62% of SCLC patients, respectively, and more than half of the SCLC patients (62%) harbored TP53 and RB1 mutation and/or copy number loss. Additionally, Serine/Arginine Splicing Factor 1 (SRSF1) DNA copy number gain and mRNA over-expression was strongly associated with poor survival using both discovery and validation patient cohorts. Functional studies in vitro and in vivo demonstrate that SRSF1 is important for tumorigenicity of SCLC and may play a key role in DNA repair and chemo-sensitivity. These results strongly support SRSF1 as a prognostic biomarker in SCLC and provide a rationale for personalized therapy in SCLC.

Show MeSH
Related in: MedlinePlus