BackgroundThe MCF7 (ER+/HER2-), T47D (ER+/HER2-), BT474 (ER+/HER2+) and SKBR3 (ER-/HER2+) breast cancer cell lines are widely used in breast cancer research as paradigms of the luminal and HER2 phenotypes. Although they have been subjected to cytogenetic analysis, their chromosomal abnormalities have not been carefully characterized, and their differential cytogenetic profiles have not yet been established. In addition, techniques such as comparative genomic hybridization (CGH), microarray-based CGH and multiplex ligation-dependent probe amplification (MLPA) have described specific regions of gains, losses and amplifications of these cell lines; however, these techniques cannot detect balanced chromosomal rearrangements (e.g., translocations or inversions) or low frequency mosaicism.ResultsA range of 19 to 26 metaphases of the MCF7, T47D, BT474 and SKBR3 cell lines was studied using conventional (G-banding) and molecular cytogenetic techniques (multi-color fluorescence in situ hybridization, M-FISH). We detected previously unreported chromosomal changes and determined the content and frequency of chromosomal markers. MCF7 and T47D (ER+/HER2-) cells showed a less complex chromosomal make up, with more numerical than structural alterations, compared to BT474 and SKBR3 (HER2+) cells, which harbored the highest frequency of numerical and structural aberrations. Karyotype heterogeneity and clonality were determined by comparing all metaphases within and between the four cell lines by hierarchical clustering. The latter analysis identified five main clusters. One of these clusters was characterized by numerical chromosomal abnormalities common to all cell lines, and the other four clusters encompassed cell-specific chromosomal abnormalities. T47D and BT474 cells shared the most chromosomal abnormalities, some of which were shared with SKBR3 cells. MCF7 cells showed a chromosomal pattern that was markedly different from those of the other cell lines.ConclusionsOur study provides a comprehensive and specific characterization of complex chromosomal aberrations of MCF7, T47D, BT474 and SKBR3 cell lines.The chromosomal pattern of ER+/HER2- cells is less complex than that of ER+/HER2+ and ER-/HER2+ cells. These chromosomal abnormalities could influence the biologic and pharmacologic response of cells. Finally, although gene expression profiling and aCGH studies have classified these four cell lines as luminal, our results suggest that they are heterogeneous at the cytogenetic level.
Non-coding RNAs are a complex class of nucleic acids, with growing evidence supporting regulatory roles in gene expression. Here we identify a non-coding RNA located head-to-head with the gene encoding the Glioma-associated oncogene 1 (GLI1), a transcriptional effector of multiple cancer-associated signaling pathways. The expression of this three-exon GLI1 antisense (GLI1AS) RNA in cancer cells was concordant with GLI1 levels. siRNAs knockdown of GLI1AS up-regulated GLI1 and increased cellular proliferation and tumor growth in a xenograft model system. Conversely, GLI1AS overexpression decreased the levels of GLI1, its target genes PTCH1 and PTCH2, and cellular proliferation. Additionally, we demonstrate that GLI1 knockdown reduced GLI1AS, while GLI1 overexpression increased GLI1AS, supporting the role of GLI1AS as a target gene of the GLI1 transcription factor. Activation of TGFβ and Hedgehog signaling, two known regulators of GLI1 expression, conferred a concordant up-regulation of GLI1 and GLI1AS in cancer cells. Finally, analysis of the mechanism underlying the interplay between GLI1 and GLI1AS indicates that the non-coding RNA elicits a local alteration of chromatin structure by increasing the silencing mark H3K27me3 and decreasing the recruitment of RNA polymerase II to this locus. Taken together, the data demonstrate the existence of a novel non-coding RNA-based negative feedback loop controlling GLI1 levels, thus expanding the repertoire of mechanisms regulating the expression of this oncogenic transcription factor.
SETDB1 is a key histone lysine methyltransferase involved in gene silencing. The SETDB1 gene is amplified in human lung cancer, where the protein plays a driver role. Here, we investigated the clinical significance of SETDB1 expression in the two major forms of human non-small cell lung carcinoma (NSCLC), i.e., adenocarcinoma (ADC) and squamous cell carcinoma (SCC), by combining a meta-analysis of transcriptomic datasets and a systematic review of the literature. A total of 1140 NSCLC patients and 952 controls were included in the association analyses. Our data revealed higher levels of SETDB1 mRNA in ADC (standardized mean difference, SMD: 0.88; 95% confidence interval, CI: 0.73–1.02; p < 0.001) and SCC (SMD: 0.40; 95% CI: 0.13–0.66; p = 0.003) compared to non-cancerous tissues. For clinicopathological analyses, 2533 ADC and 903 SCC patients were included. Interestingly, SETDB1 mRNA level was increased in NSCLC patients who were current smokers compared to non-smokers (SMD: 0.26; 95% CI: 0.08–0.44; p = 0.004), and when comparing former smokers and non-smokers (p = 0.009). Furthermore, the area under the curve (AUC) given by the summary receiver operator characteristic curve (sROC) was 0.774 (Q = 0.713). Together, our findings suggest a strong foundation for further research to evaluate SETDB1 as a diagnostic biomarker and/or its potential use as a therapeutic target in NSCLC.
BackgroundIn diagnostic pathology, HER2 status is determined in interphase nuclei by fluorescence in situ hybridization (FISH) with probes for the HER2 gene and for the chromosome 17 centromere (CEP17). The latter probe is used as a surrogate for chromosome 17 copies, however chromosome 17 (Chr17) is frequently rearranged. The frequency and type of specific structural Chr17 alterations in breast cancer have been studied by using comparative genomic hybridization and spectral karyotyping, but not fully detailed. Actually, balanced chromosome rearrangements (e.g. translocations or inversions) and low frequency mosaicisms are assessable on metaphases using G-banding karyotype and multicolor FISH (M-FISH) only.MethodsWe sought to elucidate the CEP17 and HER2 FISH patterns of interphase nuclei by evaluating Chr17 rearrangements in metaphases of 9 breast cancer cell lines and a primary culture from a triple negative breast carcinoma by using G-banding, FISH and M-FISH.ResultsThirty-nine rearranged chromosomes containing a portion of Chr17 were observed. Chromosomes 8 and 11 were the most frequent partners of Chr17 translocations. The lowest frequency of Chr17 abnormalities was observed in the HER2-negative cell lines, while the highest was observed in the HER2-positive SKBR3 cells. The MDA-MB231 triple negative cell line was the sole to show only non-altered copies of Chr17, while the SKBR3, MDA-MB361 and JIMT-1 HER2-positive cells carried no normal Chr17 copies. True polysomy was observed in MDA-MB231 as the only Chr17 alteration. In BT474 cells polysomy was associated to Chr17 structural alterations. By comparing M-FISH and FISH data, in 8 out of 39 rearranged chromosomes only CEP17 signals were detectable, whereas in 14 rearranged chromosomes HER2 and STARD3 genes were present without CEP17 signals. HER2 and STARD3 always co-localized on the same chromosomes and were always co-amplified, whereas TOP2A also mapped to different derivatives and was co-amplified with HER2 and STARD3 on SKBR3 cells only.ConclusionThe high frequency of complex Chr17 abnormalities suggests that the interpretation of FISH results on interphase nuclei using a dual probe assay to assess gene amplification should be performed “with caution”, given that CEP17 signals are not always indicative of normal unaltered or rearranged copies of Chr17.
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