1. Identification of TLOC1 and SKIL as tumor driver genes in 3q26.
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 1 indicate a relative reduced proliferation in cells with 3q26 amplification. The NES is represented by a red line and is calculated for each gene based on the proliferative effect of all shRNAs against that gene. 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. Myristoylated AKT1 (Myr-AKT1) was used a positive control. The bottom and top dotted horizontal lines indicate the median and median + SD 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 four cell lines with 3q26 amplification and two 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 cells and the upper dotted line the median + 2 SD. E, immunoblots for Flag-epitope–tagged SKIL immune complexes (IP) from cells expressing SKIL used in D. F, tumor formation of NIH3T3 cells stably expressing control vector (hcRED), TLOC1, SKIL, or IKBKE as assessed at 8 weeks. G, immunoblots for Flag-epitope–tagged TLOC1 in Flag-bead immune complexes (IP) from TLOC1-overexpressing cells used in D. Arrows indicate specific bands. H, V5-immunoblots for V5-tagged 399– 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 variants of TLOC1 significantly induced anchorage-independent growth in HMLE–MEKDD as compared with hcRED control (P < 0.01, Student t test). Bars indicate average fold colony number. J, expression levels of endogenous 399- and 220-amino acid forms of TLOC1 (top) and SKIL (bottom) 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 qRT-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, 0.04, and 0.41, Student t test). Plots showing relative expression levels between cell lines with normal or increased 3q26 copy number. The longer parallel bar represents the mean expression and the whiskers SD. 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 SD. hcRED, Heteractis crispa Red; IP, immunoprecipitation; IB, immunoblot.
Daniel Hagerstrand et al. Cancer Discovery 2013;3:
©2013 by American Association for Cancer Research