The RNA binding protein FXR1 is a new driver in the 3q26-29 amplicon and predicts poor prognosis in human cancers

Abstract

Aberrant expression of RNA-binding proteins has profound implications for cellular physiology and the pathogenesis of human diseases such as cancer. We previously identified the Fragile X-Related 1 gene (FXR1) as one amplified candidate driver gene at 3q26-29 in lung squamous cell carcinoma (SCC). FXR1 is an autosomal paralog of Fragile X mental retardation 1 and has not been directly linked to human cancers. Here we demonstrate that FXR1 is a key regulator of tumor progression and its overexpression is critical for nonsmall cell lung cancer (NSCLC) cell growth in vitro and in vivo. We identified the mechanisms by which FXR1 executes its regulatory function by forming a novel complex with two other oncogenes, protein kinase C, iota and epithelial cell transforming 2, located in the same amplicon via distinct binding mechanisms. FXR1 expression is a candidate biomarker predictive of poor survival in multiple solid tumors including NSCLCs. Because FXR1 is overexpressed and associated with poor clinical outcomes in multiple cancers, these results have implications for other solid malignancies.

Keywords: 3q amplification; FXR1; RNA binding protein; non-small cell lung cancer.

Fig. 1.

(A) Copy number alteration of FXR1, ECT2, and PRKCI in 343 TCGA lung SCCs (only 182 samples harboring 3q amplicon were shown). “2” is a high-level amplification (log2 ratio > 0.8), “1” indicates a low-level gain (log2 ratio > 0.3), “0” is diploid, “−1” is a single-copy loss (heterozygous deletion). Frequencies of high-level amplification are shown as a percentage of all cases. (B) Representative dual color FISH of the FXR1 gene (3q26.3, green spots) and a chromosome 3 centromeric probe (red spots) in lung SCCs. (C) Lung SCCs (T) express higher FXR1, ECT2, and PRKCI mRNA levels than matched normal tissues (N) in TCGA dataset (n = 50). ***P < 0.0001. (D) Immunoblot analysis of ten primary SCCs and matched normal lung tissue for FXR1, ECT2, PRKCI, and β-actin.

Fig. 2.

FXR1 regulates lung cancer cell growth in vitro and in vivo. (A) Immunoblot analysis of a panel of NSCLC cell lines for FXR1 and β-actin. Relative FXR1 protein level was normalized to β-actin. (B) Effect of FXR1-specific siRNA on viable cells over time. Cells were measured 1, 2, 4, and 6 d after plating using the Cyquant assay. *P < 0.05; **P < 0.01. (C) Down-regulation of FXR1 protein in H520 and HCC95 cells treated with siRNA against FXR1 after 48 h. SC: scrambled siRNA control. si#1, si#2: two individual FXR1 siRNAs. (D) Activated Caspase 3 and PARP in H520 cells treated with pooled FXR1-siRNA after 72 h. SC: scrambled siRNA control. (E) Effect of FXR1-shRNA on H520 anchorage-independent soft agar colony formation. H520 cells were infected with three lentiviruses containing shRNA targeting FXR1 or a nontarget (NT) sequence. Two stable FXR1 knockdown (KD) cell lines are shown. H520/FXR1 KD cells grow significantly slower than H520/NT cells (*P < 0.05, **P < 0.01). (F) Effect of FXR1 overexpression on BEAS-2B anchorage independent cell growth compared with vector control. pBabe-V, BEAS-2B-vector group; pBabe-FXR1, BEAS-2B cells stably-transfected with pBabe-Flag-FXR1. (G) Effects of FXR1 knockdown on tumorigenicity in nude mice. H520 NT and H520 FXR1 KD cells were injected s.c. into the flanks of nude mice (n = 10). Tumor volume was measured twice a week in all experiments by caliper and calculated by the formula: V = 3.14 (smaller diameter)²(larger diameter)/6. The quantification of tumor volume changes over a 4-wk period is shown; **P < 0.01. (H) Representative H&E staining, immunohistochemical staining of FXR1 and Ki67 in tumors formed by H520 NT and H520 KD in one mouse. Scale bars, 50 μm. (I) Quantification of FXR1 and Ki67 immunohistochemical staining shown by the percentage of positively stained cells compared with total number of cells per field. The P value (Student’s t test) relative to NT is shown. **P < 0.01. Data are mean ± SEM.

Fig. 3.

FXR1 regulates ECT2 and PRKCI expression via distinct mechanisms. (A) Down-regulation of ECT2 and phospho-ERK1/2 in FXR1 knockdown NSCLC cells. (B) Immunoprecipitation (IP) of FXR1 reveals that FXR1 binds to PRKCI protein but not to ECT2 in H520 cells. (C) HEK293T cells were cotransfected with Flag-tagged pCMV-Tag4A-FXR1 and either HA-tagged pHACE-PRKCI-WT or pHACE-PRKCI-DN for 48h. Cell lysates were then subjected to IP with anti-HA, anti-Flag, anti-FXR1 or anti-PRKCI. The IP experiments reveal that FXR1 binds to active PRKCI but not the PRKCI DN mutant. NS, nonspecific band. (D) Reduced phosphorylated PRKCI T555 is observed in FXR1 knockdown H520 cells. (E) IP using either anti-FXR1 or normal IgG in H520 under conditions that preserve the association of RNA-binding proteins with target mRNAs. IP was followed by RT-PCR analysis to detect endogenous ECT2 mRNA; PCR products were resolved by electrophoresis in 1.5% agarose gels stained with ethidium bromide.

Fig. 4.

FXR1 overexpression is associated with poor overall survival in NSCLC. (A) Representative immunohistochemical staining of FXR1 protein expression in sections of formalin-fixed paraffin-embedded normal lung tissue, lung ADC, and SCCs. (B) Kaplan–Meier plots of overall survival of stage I NSCLC patients (n = 161) or SCC (n = 78) stratified by FXR1 protein expression. The log-rank P values are shown. (C) FXR1 mRNA level is associated with poor overall survival in stage I lung ADC patients indicated by a meta-analysis in six independent cohorts (SI Appendix, Table S9). Patients who received adjuvant chemotherapy were excluded.

Fig. 5.

FXR1 is associated with poor outcome in multiple human cancers. (A) FXR1 overexpression is associated with worse disease specific free survival in METABRIC breast cancer cohort, worse relapse-free survival in early stage of ovarian cancer (combined GSE9891 and TCGA) and worse overall survival in TCGA HNSC cohort. The log-rank P values are shown. The datasets were described in detail in SI Appendix, Tables S17, S27, and S29. (B) Down-regulation of FXR1 protein in BT549 and SCC12 cells treated with shRNA against FXR1. NT: nontarget shRNA control. KD: shRNA FXR1 knockdown. (C) Effect of FXR1-shRNA on BT549 and SCC12 cell invasion. Significant differences between NT and FXR1 KD cells are indicated; *P < 0.05.