Misbehaviour of XIST RNA in breast cancer cells - PubMed (original) (raw)

Misbehaviour of XIST RNA in breast cancer cells

Silvia M Sirchia et al. PLoS One. 2009.

Abstract

A role of X chromosome inactivation process in the development of breast cancer have been suggested. In particular, the relationship between the breast cancer predisposing gene BRCA1 and XIST, the main mediator of X chromosome inactivation, has been intensely investigated, but still remains controversial. We investigated this topic by assessing XIST behaviour in different groups of breast carcinomas and in a panel of breast cancer cell lines both BRCA1 mutant and wild type. In addition, we evaluated the occurrence of broader defects of heterochromatin in relation to BRCA1 status in breast cancer cells. We provide evidence that in breast cancer cells BRCA1 is involved in XIST regulation on the active X chromosome, but not in its localization as previously suggested, and that XIST can be unusually expressed by an active X and can decorate it. This indicates that the detection of XIST cloud in cancer cell is not synonymous of the presence of an inactive X chromosome. Moreover, we show that global heterochromatin defects observed in breast tumor cells are independent of BRCA1 status. Our observations sheds light on a possible previously uncharacterized mechanism of breast carcinogenesis mediated by XIST misbehaviour, particularly in BRCA1-related cancers. Moreover, the significant higher levels of XIST-RNA detected in BRCA1-associated respect to sporadic basal-like cancers, opens the possibility to use XIST expression as a marker to discriminate between the two groups of tumors.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. XIST expression and status of X chromosomes and BRCA1 in HMEC and breast cancer cell lines, and evaluation of XIST levels in different groups of breast carcinomas.

A) Classification of HMEC and breast cancer cell lines according to XCI type, based on the indicated X chromosome related features. BRCA1 status is also reported. B) Box-plots of the log2-transformed amounts of XIST RNA measured by quantitative real-time RT-PCR in the indicated groups of primary human breast cancers. Each box-plot represents the first quartile (lower edge of the box), median value (bar inside the box), third quartile (upper edge of the box), and minimum and maximum values (horizontal lines). Points at a distance from the quartiles >1.5 times the inter-quartile range are plotted individually. Statistically significant p values between groups are reported (Kruskal-Wallis Rank Sum test).

Figure 2

Figure 2. Effects of BRCA1 RNAi on XIST expression in cells with different XCI status.

HMEC (XCI type 0), MCF7 (XCI type 1) and T47D (XCI type 2) were transfected with a mix of two _BRCA1_-specific siRNAs, mapping to exons 12 and 24, or a control siRNA. After 72 hrs, cells were processed for BRCA1 immunofluorescence and RNA purification. In all panels BRCA1 is immunostained in green and nuclei are marked with DAPI. The histogram represents quantitative RT-PCR analysis performed on cDNAs of the indicated cell lines, before and after BRCA1 silencing, using primers specific for spliced and unspliced XIST RNA. XIST RNA levels are expressed as a ratio to GAPDH mRNA levels, after subtraction of the background signal from cDNA synthesis reactions lacking reverse transcriptase. To facilitate comparison between cell lines with different XCI status, the XIST/GAPDH transcript ratio was normalised relative to HMEC. Error bars represent standard deviation and the asterisks indicate statistically significant differences (p<0.05, Student's test).

Figure 3

Figure 3. XIST RNA behaviour in HMEC and in MCF7 breast cancer cell line.

A) Localisation of XIST RNA (red signal) revealed by RNA-FISH on HMEC and MCF7 nuclei (DAPI staining). The percentages of XIST-positive (middle) and XIST-negative (bottom) MCF7 nuclei are reported. Positive cells show different XIST distribution, clustered clouds (full arrows) or dispersed signals (empty arrows). B–D) Localisation of XIST RNA (red) respect to nuclear X chromosome territories (blue) revealed by FISH analysis in HMEC and MCF7 nuclei. MCF7 XIST-positive nuclei always show three X chromosome domains. In MCF7 nuclei the overlap between XIST and X chromosome territory appears more limited (panel C), respect to HMEC (panel B) and very often spreads outside the X chromosome domain (panel D).

Figure 4

Figure 4. X chromosomes characterization of MCF7 cell line.

A) FISH analysis on metaphases using X painting probe. The percentages of MCF7 cells with two (left) or three (right) X chromosomes are indicated. B) Barr body staining. The percentages of HMEC, used as positive control, with (right) or without (left) a detectable Barr body (arrow) are indicated. No positive cell was observed in MCF7. C) High level of homozygosity (87%) detected by genotyping of the indicated panel of STRs. D) DNA FISH using a mix of telomeric (red, green and orange) and X alpha-satellite (blue) probes. The orange spots decorate the Xq telomeric region: two on X chromosomes, positive for X-alpha satellite probe (white arrows) and one on a rearranged chromosome, negative for X-alpha-satellite probe (red arrow). Telomeric red and green probes were used as hybridization control. E) Methylation analysis of ZMYM3 gene subjected to XCI (DXS6673E locus). Electropherograms show the allelic patterns obtained by PCR of HMEC and MCF7 DNAs undigested (top) and digested with methylation-sensitive enzymes (bottom). After digestion, HMEC show a methylated allele, whereas in MCF7 the locus is completely demethylated, resulting in the lack of amplification.

Figure 5

Figure 5. XIST RNA cloud paints an active X chromosome in MCF7 nuclei.

A) DNA FISH using the single Xq22.3 locus RP11-349A16 probe (red), and X alpha-satellite probe (green). Cell populations with three Xs display two different hybridization patterns: two Xs with a single red spot (white arrows) and a chromosome with two red signals (red arrow). B) Simultaneous detection of XIST RNA (green), X chromosome territory (blue) and Xq22.3 locus (red). The X chromosome expressing XIST has one copy of the RP11-349A16 region (merge). C) Replication timing analysis. MCF7 and HMEC were briefly labelled with BrdU and analyzed by DNA FISH using the single Xq22.3 locus RP11-349A16 probe and by BrdU immunofluorescence. The pattern of FISH staining seen in BrdU-positive cells was scored for at least 300 nuclei of each cell type: nuclei with only “singlets” are those in which no Xs has yet replicated; nuclei with “singlet/s+doublet/s” pattern contain unreplicated and replicated Xs; nuclei with only “doublets” have all replicated Xs. Both MCF7 subpopulations with two and three Xs display a synchronous replication timing. D) Characterisation of the chromatin signatures of XIST-positive X chromosome in MCF7 and HMEC, by simultaneous FISH detection of XIST RNA (red) and Cot-1 RNA (green); DAPI nuclear staining is in blue. A line scan of fluorescence intensity (white bars) is shown for both cell types. In HMEC, the scan plot revealed overlap of the DAPI and XIST RNA signals, whereas the Cot-1 RNA signal is depleted, as expected for an inactive X chromosome. On the contrary, in MCF7 cells the line scan through the XIST-positive territory shows high intensity of the Cot-1 RNA signal combined with low DAPI intensity, typical signs of euchromatin.

Figure 6

Figure 6. XIST RNA staining patterns and global defects of heterochromatin are independent of BRCA1 status.

A) Percentages of nuclei positive for XIST RNA signal before and after BRCA1 silencing in HMEC and in MCF7. Specific signal morphology was evaluated, distinguishing clustered and dispersed signals. HMEC always display clustered staining only. In both cell types no relevant variation was observed after siRNA treatment. The results were reproducible in independent experiments. B) Distribution of MCF7 cell populations respect to X chromosome numbering before and after BRCA1 knockdown. No relevant variation in the relative content of the different populations was observed after siRNA treatment. The results were reproducible in independent experiments. C) Analysis of heterochromatin status in BRCA1 normal and mutant cells by FISH analysis with Cot-1 probe (green) on DAPI-stained nuclei. A line scan of fluorescence intensity (white bars) is shown for each cell type. Shaded areas indicating regions of peripheral heterochromatin are evident in the scan plot relative to HMEC, but not in those of MCF7 (BRCA1wt) and HCC1937 (BRCA1−/−) breast cancer cell lines.

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