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

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.

Fig 1. Mutations in CDH10 associate with poor survival in Chinese SCLC patients.

a) Schematic representation of amino acid consequences from mutations identified in SCLC patients in human CDH10 protein b) Kaplan-Meier (KM) curves comparing survival between patients harboring at least one nonsilent mutation in CDH10 (n = 12) and those not (n = 84).p* = log-rank test; p = Cox PH regression model; HR = hazard ratio

Fig 2. Top mutated DNA polymerases and mutation prevalence in Fanconi anemia pathway genes in SCLC.

a) Schematic representation of amino acid changes in human POLG, POLD1, POLQ proteins; b) the amino acid alterations in human POLG catalytic domain. Mutations were mapped onto the structure of human POLG using PDB Id entry 3IKM as template [6]. c) Relevant amino acid alterations in POLD1. Mutations in human POLD1 gene were mapped onto structure of the yeast DNA polymerase subunit δ using PDB entry 3IAY Orange colored ribbon represents exonuclease domain, blue colored ribbon corresponds to polymerase domain, and the green ribbon represents the N-terminal portion of the protein [27]. The mutations in both structures are shown in red spheres. d) Mutation prevalence in Fanconi anemia pathway genes.

Fig 3. SRSF1 CN gain and mRNA expression correlates with survival.

A: The time-to-event analysis schema with available patient specimens. In the time-to-event analyses, 96 Chinese primary SCLC patients with clinical outcome were divided into training and test cohorts according to the availabilities of matched normal, RNAseq and survival outcome information. The training set includes 22 patients with each patient having tumor and normal WES data and survival outcome. The test set includes 74 patient tumors only. Each patient has WES data from tumor and survival outcome. Among those patients, 48 patients have WES, RNAseq data, and survival outcome. b) SRSF1 mRNA expression in CN gain group and no CN gain group (p = Welch’s t-test). c) Kaplan-Meier (KM) curves comparing survival between SRSF1 low and high mRNA expression groups (n = 48). Similarly, KM curves used to evaluate the difference of survival between different SRSF1 CN statuses in d) discovery set (n = 22), e) validation set (n = 74), and f) combination of discovery set and validation set (n = 96). p* = log-rank test; p = Cox PH regression model; HR = hazard ratio.

Fig 4. 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.

Fig 5. Mechanism of action for SRSF1 in SCLC.

a) SRSF1 prevents DNA-damage. DMS114 cells were transfected with control or SRSF1 siRNA and then treated with topotecan or Cisplatin for the indicated times. SRSF1, phosphor-H2AX and phosphor-Chk2 were probed with their corresponding antibodies. b) c) SRSF1 mediates the activation of AKT and ERK pathways. DMS114 cells transfected with ctrl or SRSF1 siRNAs were lysed and applied to the phospho-kinase array as detailed in Materials and Methods. The dot blot result was further confirmed by western blot in both DMS114 and NCI-H1048 cells.