Systematic interrogation of 3q26 identifies TLOC1 and SKIL as cancer drivers

Abstract

3q26 is frequently amplified in several cancer types with a common amplified region containing 20 genes. To identify cancer driver genes in this region, we interrogated the function of each of these genes by loss- and gain-of-function genetic screens. Specifically, we found that TLOC1 (SEC62) was selectively required for the proliferation of cell lines with 3q26 amplification. Increased TLOC1 expression induced anchorage-independent growth, and a second 3q26 gene, SKIL (SNON), facilitated cell invasion in immortalized human mammary epithelial cells. Expression of both TLOC1 and SKIL induced subcutaneous tumor growth. Proteomic studies showed that TLOC1 binds to DDX3X, which is essential for TLOC1-induced transformation and affected protein translation. SKIL induced invasion through upregulation of SLUG (SNAI2) expression. Together, these studies identify TLOC1 and SKIL as driver genes at 3q26 and more broadly suggest that cooperating genes may be coamplified in other regions with somatic copy number gain.

Significance: These studies identify TLOC1 and SKIL as driver genes in 3q26. These observations provide evidence that regions of somatic copy number gain may harbor cooperating genes of different but complementary functions.

Figure 1. 3q26 is frequently amplified in ovarian, breast and lung non-small cell cancer

(A) Copy number plots of the samples in Tumorscape for the q arm of chromosome 3. A vertical line represents each sample where red represents a high chromosomal copy number ratio, blue low and white neutral. Chromosomal bands are shown to the left. Two horizontal blue lines indicate the minimal common amplified region and the genes are listed to the right. (B) Illustration of samples with 3q26 amplification. Samples that did not exhibit copy number gain of the PIK3CA, SOX2 or TP63 locus are shown at the top. The 3q distal regions have been magnified to show the position of these in relation to each other. (C) A summary of the screens performed to identify cancer driver genes residing in the minimal common amplified region of 3q26. (D) Cell lines with normal or amplified levels of 3q26 used for the proliferation screen. The amplification data of each cell line is illustrated as above for panel A. The arrow marks the 3q26 region.

Figure 2. Identification of TLOC1 and SKIL as tumor driver genes in 3q26

(A) RIGER analysis. Differential proliferation scores for each shRNA as compared between cell lines with normal or amplified 3q26 levels are represented by blue lines. Survival scores less than one indicate a relative reduced proliferation in cells with 3q26 amplification. The normalized enrichment score (NES) is represented by a red line and is calculated for each gene based upon the proliferative effect of all shRNAs against that gene. False discovery rates (FDRs) are listed below. All the proliferation effects were normalized against 10 different GFP targeting shRNAs. (B) Anchorage independent growth for HMLE-MEKDD cells expressing each gene from the minimal common region. Each bar shows average number of colonies. Myr-AKT1 was used a positive control. The bottom and top dotted horizontal lines indicate the median and median + S.D. colony number for the tested genes. Representative images of formed colonies are shown below the graph. (C) Effect of TLOC1-specific shRNAs on the proliferation of 4 cell lines with 3q26 amplification and 2 cell lines with diploid 3q26 level. (D) Matrigel induced invasion in HMLE-MEKDD cells overexpressing genes from the common amplified region. The bars indicate the number of invaded cells. The lower dotted horizontal line represents median number of invaded cell and the upper dotted line the median + 2 S.D. (E) Immunoblots for Flag-epitope tagged SKIL immune complexes (IPs) from cells expressing SKIL used (D). (F) Tumor formation of NIH3T3 cells stably expressing control vector (hcRED), TLOC1, SKIL or IKBKE as assessed at 8 wks. (G) Immunoblots for Flag-epitope tagged TLOC1 in Flag-bead immune complexes (IPs) from TLOC1 overexpressing cells used (D). Arrows indicate specific bands. (H) V5-immunoblots for V5-tagged 399 amino acids or 220 amino acids splice variants of TLOC1 and hcRED in HMLE-MEKDD cells. Arrows indicate specific bands. (I) The 220 and 399 amino acids splice variant of TLOC1 significantly induced anchorage independent growth in HMLE-MEKDD as compared to hcRED control (p<0.01, student’s t-test). Bars indicate average fold colony number. (J) Expression levels of endogenous 399 and 220 amino acid forms of TLOC1 in a panel of cell lines. Cell lines with 3q26 amplification are indicated with red text and a (+) sign and cells with normal 3q26 status with black text and a (−) sign. Cell lines that were determined by quantitative real time PCR on genomic DNA to harbor moderately increased copy number of 3q26 are indicated with a (+/−) sign. HMEC cells expressing the short form of TLOC1 were loaded in parallel as immunoblot controls. (K) Comparison in expression levels of the 399 or 220 amino acid forms of TLOC1, or SKIL in cell lines with normal or increased 3q26 copy number (p=0.02, p=0.04, p=0.41 student’s t-test). Plots showing relative expression levels between cell lines with normal or increased 3q26 copy number. The longer parallel bar represent the mean expression and the whiskers S.D. Quantification of TLOC1 and SKIL expression levels relative to actin are from the previous panel. The values are standardized to expression levels in the HMEC cells. n.s. = not significant. Error bars = 1 S.D.

Figure 3. DDX3X association with the TLOC1 is essential for TLOC1 induced transformation

(A) Analysis of co-immunoprecipitated TLOC1 associated proteins visualized by silver or SYPRO staining. The V5-immunoblot shows immunoprecipitated V5-tagged TLOC1 from 2% of the lysate. (B) 18 TLOC1 associated proteins were identified by at least 4 unique peptides. (C) Category distribution of the proteins identified in the mass spectrometry analysis. (D) The effect of suppressing TLOC1 associated proteins on TLOC1-induced anchorage independent growth in HMLE-MEKDD cells. Black bars indicate number of colonies. The upper and lower dashed horizontal lines indicate mean ± 1 S.D., respectively. (E) DDX3X and TLOC1 immunoblots for V5 and DDX3X immune complexes from V5-TLOC1 or V5-Luciferase overexpressing cells. Rabbit IgG was used as a negative control for the DDX3X immunoprecipitation and V5-tagged luciferase for the V5 experiments. (F) Co-immunoprecipitation of DDX3X with different V5-tagged TLOC1 truncation mutants TLOC1 mutants and DDX3X were detected by V5 and DDX3X immunoblotting, respectively. (G) Removal of the 81 first N-terminal amino acids of TLOC1 significantly reduced its ability to induce anchorage independent growth in HMLE-MEKDD cells (p=0.006, student’s t-test). The bars indicate average colony number. YFP and hcRED were used as negative controls. For all panels arrows indicate specific bands for the immunoblots. n.s. = not significant. Error bars = 1 S.D.

Figure 4. TLOC1 increased the cap versus IRES-dependent translation ratio by decreasing IRES-dependent translation

(A) Illustration of the bi-cistronic translation reporter used to measure translation levels. The renilla luciferase reporter is driven by cap-dependent translation and the firefly luciferase by IRES-dependent translation. (B) TLOC1 over expression significantly increased the ratio of cap-dependent versus IRES-dependent translation by inhibition of IRES-dependent translation (p=0.01, student’s t-test). The ratio of cap/IRES-dependent translation was calculated and is illustrated in the right graph. (C) Overexpression of the transforming TLOC1 truncation mutant ΔC27 significantly (p=0.03, student’s t-test) inhibited IRES-dependent translation as compared to the non-transforming variant ΔN149. (D) Suppression of DDX3X in TLOC1 overexpressing cells significantly reversed the TLOC1 induced change in translation ratio (p=0.03, student’s t-test). (E) TLOC1 and DDX3X binds to 7-methylated GTP beads, and TLOC1. (F) TLOC1 overexpression increased EIF4G protein expression. (G) TLOC1 overexpression in HMLE-MEKDD cells decreased EIF4EBP1-phosphorylation on Threonine 37.45 and Serine 65. Lysates were prepared from TLOC1 or BFP overexpressing HMLE-MEKDD cells, which had been grown with (+GFs) or without (−GFs) growth factors for 24 hrs. Error bars = 1 S.D.

Figure 5. SKIL induces SLUG, which is required for SKIL-induced invasion

(A) A Venn diagram showing the top 50 genes correlating with either high SKIL expression or low SKIL expression and the significant intersect between these two lists (p<0.001, binomial distribution test). The overlapping genes are listed below the diagram. (B) SLUG is required for SKIL induced Matrigel invasion. Suppression of SLUG significantly reduced SKIL induced invasion (p=0.03, student’s t-test). Bars indicate average number of invaded cell. (C) Suppression of SLUG in SKIL overexpressing cells had no effect on proliferation. Graph shows cell doublings over indicated amount of time. An shRNA targeting GFP was used as negative control. (D) SKIL overexpression induced SLUG expression. (E) SKIL overexpression increased gene expression of invasion and EMT related genes of which a subset could be reversed by SLUG suppression, marked as SLUG-dependent. mRNA levels were determined by quantitative real time PCR. (F) SLUG overexpression increased gene expression of the same set of genes, which were determined to be SLUG dependent in panel E. (G) Suppression of SMAD4 significantly induced Matrigel invasion (p=0.02, student’s t-test). Suppression of SLUG reversed the invasion induced by suppression of SMAD4 (p=0.05, student’s t-test). The bars represent average number of invaded cells. The experiments in this figure were performed in HMLE-MEKDD cells except panel A. Error bars = 1 S.D.

Figure 6. TLOC1 and SKIL cooperate to induce anchorage independent growth

(A) HMLE-MEKDD expressing different combinations of genes were tested for anchorage independent growth. The bars illustrate average number of colonies. A significant difference was detected between TLOC1 and control as illustrated in the graph (p=0.003, student’s t-test), and between was potentiated in combination with SKIL. (B) Growth rate of cells used in panel A showed no significant differences in growth rate. Cells were cultured, counted and reseeded at indicated time points and the cumulative increase in cell number is shown. (C) TLOC1 or SKIL display no combinatorial effect in anchorage independent growth with PIK3CA or SOX2. Bars display average colony number. Error bars = 1 S.D.