Amplification of TRIM44: pairing a prognostic target with potential therapeutic strategy

Abstract

Background: Many prognostic biomarkers have been proposed recently. However, there is a lack of therapeutic strategies exploiting novel prognostic biomarkers. We aimed to propose therapeutic options in patients with overexpression of TRIM44, a recently identified prognostic gene.

Methods: Genomic and transcriptomic data of epithelial cancers (n = 1932), breast cancers (BCs; n = 1980) and esophago-gastric cancers (EGCs; n = 163) were used to identify genomic aberrations driving TRIM44 overexpression. The driver gene status of TRIM44 was determined using a small interfering RNA (siRNA) screen of the 11p13 amplicon. Integrative analysis was applied across multiple datasets to identify pathway activation and potential therapeutic strategies. Validation of the in silico findings were performed using in vitro assays, xenografts, and patient samples (n = 160).

Results: TRIM44 overexpression results from genomic amplification in 16.1% of epithelial cancers, including 8.1% of EGCs and 6.1% of BCs. This was confirmed using fluorescent in situ hybridization. The siRNA screen confirmed TRIM44 to be a driver of the amplicon. In silico analysis revealed an association between TRIM44 and mTOR signalling, supported by a decrease in mTOR signalling after siRNA knockdown of TRIM44 in cell lines and colocalization of TRIM44 and p-mTOR in patient samples. In vitro inhibition studies using an mTOR inhibitor (everolimus) decreased cell viability in two TRIM44-amplified cells lines by 88% and 70% compared with 35% in the control cell line. These findings were recapitulated in xenograft models.

Conclusions: Genomic amplification drives TRIM44 overexpression in EGCs and BCs. Targeting the mTOR pathway provides a potential therapeutic option for TRIM44-amplified tumors.

Figure 1.

TRIM44 expression in esophago-gastric cancers (EGCs). A) Percentage of patients with TRIM44 overexpression in different stages of the pathogenesis of EGC. P value computed by two-sided χ² test. Percentage of TRIM44 overexpression in EGC samples was extracted from our previous publication (12) (n = 349). BE = Barret’s esophagus; LGD = low-grade dysplasia; HGD = high-grade dysplasia; NS = not statistically significant. B) Representative immunohistochemistry pictures of one case with adjacent Barrett’s esophagus (BE) with no dysplasia, HGD, and EGC. C) Kaplan–Meier curve comparing survival of patients with TRIM44-positive cancer cells in metastatic lymph nodes with patients with TRIM44-negative cancer cells in the metastatic lymph nodes (P value computed by two-sided log-rank test).

Figure 2.

Correlation of TRIM44 expression, copy number, and survival. A) Expression of TRIM44 in 75 esophago-gastric cancers (EGCs) plotted on a log10 scale. Dotted red line and solid red line segregate tumors with more than 1 standard deviation and more than 2 standard deviations of TRIM44 expression relative to the rest of the tumors. B) Heat map showing the copy number status of 1932 tumors on Tumorscape (various different tumor types). Blue arrow shows the location of TRIM44 on chromosome 11. C) Representative fluorescent in situ hybridization images performed on tissue microarrays of EGCs. The individual green and red signals indicate TRIM44 and chromosome 11 centromeric probes, respectively. D) Box and whisker plots correlating expression and copy number status of TRIM44 in the METABRIC cohort (21) consisting of 1980 breast tumors. P value computed by one-sided Jonckheere–Terpstra test. AMP = amplifications; GAIN = gain in copy numbers; HETD = heterozygously deleted; HOMD = homozygously deleted; NEUT = neutral. E) Kaplan–Meier curve comparing survival of patients with gains and amplifications of TRIM44 to patients with loss or normal copy number of TRIM44 (n = 1980; P value computed by two-sided log rank test). The survival information was not available for 16 patients.

Figure 3.

Minimal common region of amplification and effect of gene-specific knockdown across the amplicon. A) Minimal common region analysis of the 11p13 amplicon. Arrow indicates the position of TRIM44. B) Representative images of fluorescent in situ hybridization performed on metaphase preparations of HSC-39. The individual green and red signals indicate TRIM44 and chromosome 11 centromeric probes, respectively. The white arrows point to the centromeric probe. C) siRNA screen performed for eight genes present in the double minute amplifications in HSC39. Red squares highlight the three genes (CD44, FJX1, and TRIM44) that showed more than 50% decrease in proliferation of cells relative to treatment with control siRNA with at least three siRNAs. The screen was performed with three technical triplicates and three biological repeats for each siRNA transfection.

Figure 4.

Association of TRIM44 overexpression and mTOR activity. A) Identification of pathway signatures enriched in esophago-gastric cancer (EGC) patients with TRIM44 overexpression (ranking genes by correlation with TRIM44 expression in two publicly available independent EGC gene expression datasets). Pathways are ranked by the P value of enrichment. *Statistically enriched pathways enriched in the Greenawalt dataset (26). †Statistically enriched pathways in the Kim dataset (25). Normalized enrichment score indicates strength of enrichment. Scores greater than 0 indicate overenrichment and pathway activation, and scores less than 0 indicate underenrichment and pathway suppression. B) Validation of enriched pathway signatures in the METABRIC cohort (ranking genes by correlation with TRIM44 expression in the METABRIC cohort). Pathways are ranked by the P value of enrichment. *Statistically significantly enriched by nominal P value. †Statistically significantly enriched by false discovery rate–corrected Q value. Normalized enrichment score indicates strength of enrichment. Scores greater than 0 indicate overenrichment and pathway activation, and scores less than 0 indicate underenrichment and pathway suppression. P values obtained using gene set enrichment analysis (GSEA), which is based on an ad hoc modification of the two-sided KS test. C) Heatmap for expression of genes in the mTOR signature upon small interfering RNA (siRNA) knockdown in the TRIM44-amplified cell line HSC39. The samples included HSC39 cells treated with two independent TRIM44 siRNAs (in duplicate) and HSC39 treated with All Stars Negative siRNA (in quadruplicate), making a total of eight samples. The bars represent the scaled Z score values (using median and median of absolute deviation) of gene expression of the 48 most highly enriched genes involved in the mTOR signature (genes in the leading edge analysis of the GSEA and with a P value less than .01 in the original paper generating the signature, Molecular signatures database (MSigDB) Broad Institute, http://www.broadinstitute.org/gsea/msigdb/index.jsp, [also Ref. 18]). Gold represents high expression; purple represents low expression. D) Immunoblot of TRIM44-amplified cell lines (HSC-39 and SNU16) after siRNA knockdown for TRIM44 and components of the AKT and mTOR signaling pathway.

Figure 5.

Comparison of TRIM44 and p-mTOR staining in patient samples. A) Percentage of patients with p-mTOR positivity stratified by TRIM44 amplification and gain status. P value computed by two-sided Fisher exact test. B) Immunofluorescence staining of TRIM44 and p-mTOR in patient 3875. C) Immunohistochemistry performed on patient 8312 for TRIM44 p-mTOR, p-P70S6K, and p-4EBP1 staining in the primary tumor. Red arrows show cells with high expression of TRIM44 and high mTOR activity, whereas the green arrows show the cells with low TRIM44 expression and low mTOR activity. All statistical tests were two-sided.

Figure 6.

Treatment of TRIM44-amplified cells with mTOR inhibitors in vitro and in vivo. A) Effect of everolimus (mTOR inhibitor) on two amplified cell lines (HSC39 and SNU-16) and a low TRIM44-expressing cell line (OE19). The dotted lines represent the maximum inhibition of the cells relative to dimethyl sulfoxide-treated cells. All experiments were repeated at least three times. B) Comparison of HSC39 xenograft size in mice treated with everolimus or control (assessed using calliper measurements). Error bars represent ± standard deviation; n = 3. C) Magnetic resonance imaging of HSC39 xenografts in nude mice. Left panel shows the size of xenografts treated with vehicle control, and right panel shows the size of xenografts treated with everolimus. The circles in the figures mark out the xenografts on magnetic resonance imaging. D) HSC39 xenografts from everolimus-treated mice and control mice were harvested and stained for p-P70S6K. E) Effect of everolimus treatment on OE19 xenografts in mice. P value computed by two-sided Student t test. Error bars represent ± standard deviation; n = 5 in each group.