Suppression of GATA6 leads to nuclear morphological deformation and aneuploidy in ovarian surface epithelial cells.
GATA6 was previously found to be lost in the majority of ovarian cancers (8
). To determine the consequence of GATA6 elimination in cell culture, we suppressed GATA6 expression by siRNA or shRNA in primary HOSE and nontumorigenic HIO cells (8
). The GATA6 siRNA treatment caused nearly complete suppression of GATA6 protein over the 2- to 5-day period, as reported previously (8
). Within 2 to 3 days after targeting GATA6 with siRNA, we observed a dramatic occurrence of cells with large and irregularly shaped nuclei (Fig. ). In several experiments with multiple HOSE cell preparations, GATA6 downregulation consistently resulted in nearly 65% of the cells having a large and atypical nuclear morphology. Conversely, cells transfected with control siRNA exhibited smooth, round or oval nuclei with a background of cells having atypical nuclei of less than 4%. The effect of GATA6 downregulation on the cell size and morphology of the whole population was also revealed by flow cytometric analysis (Fig. ). Presumably, the size and shape of cells reflect the changes in size and shape of the nucleus. GATA6 downregulation caused a shift of the cell population toward larger and irregular shapes, as indicated by forward (FSC-H) and sideward (SSC-H) light scattering, respectively. Additionally, the GATA6-suppressed cells were aneuploid or polyploid, as determined by cell cycle flow cytometry (Fig. ). In this experiment, the 4n cell-containing P3 fractions were collected by flow cytometry with Vybrant violet dye as a DNA content indicator in both vector control (16% in P3) and shRNA GATA6-suppressed (36% in P3) cells. After culturing for an additional 7 weeks, the entire cell population was again analyzed by flow cytometry. In control shRNA-treated cells, the majority of the cells redistributed back to 2n stages (22% in P3), indicating that the previously collected P3 fraction of the 4n cells was in the G2
/M phase (Fig. ). In contrast, the GATA6-suppressed cells in the P3 fraction largely remained in 4n and also 8n stages (86% in P3) following prolonged culturing (7 weeks), suggesting that the collected 4n cells were mostly tetraploid (Fig. ). Thus, most of the GATA6-suppressed cells with larger than 2n DNA content were not proliferating S-phase or M-phase cells but rather were aneuploid or polyploid cells.
FIG. 1. Suppression of GATA6 in HOSE cells leads to nuclear defects and polyploidy. GATA6 expression was suppressed by either an shRNA expression vector or siRNA oligonucleotides in HOSE and HIO cells. (A) Nuclear morphology was examined following DAPI staining (more ...)
The formation of polyploid and aneuploid cells was also demonstrated by cytogenetic analysis. At day 3 following transfection with the GATA6 siRNA vector (with a 20% transfection efficiency, as indicated by cotransfected GFP expression vectors), a significant percentage of metaphase chromosome spreads were found to be aneuploid and polyploid. In representative metaphase spreads of HOSE cells from a consecutive counting, 7 (14%) of 50 spreads were nearly tetraploid (84 to 92 chromosomes). By day 7, the presence of aneuploid cells increased further; 2 tetraploid spreads and 5 aneuploid spreads with chromosome counts of 26 to 45 were found among the 50 spreads counted, representing 16% aneuploid or tetraploid spreads. Representative metaphase spreads are shown in Fig. . In 50 metaphases from the cells transfected with the control siRNA vector, only one tetraploid spread was counted at day 3 and one nearly diploid spread (45 chromosomes) was found at day 7, and all other metaphase spreads had normal diploid counts. Thus, GATA6 suppression causes a dramatic increase in the formation of polyploid and aneuploid cells. The extent of aneuploidy in metaphase spreads observed likely was an underestimation due to limited transfection efficiency and the fact that the cytogenetic analysis is limited to dividing cells. Indeed, we found significantly fewer metaphase chromosome spreads in GATA6-suppressed than control cultures.
In experiments completed over an 18-month period, the effects of GATA6 suppression on nuclear morphology were replicated in 10 different HOSE cell preparations. Additionally, similar and/or more dramatic effects of GATA6 downregulation were observed in HIO-80, HIO-105, and HIO-114 cells, which are ovarian surface epithelial cells transfected with simian virus 40 T antigen to prolong the life span of the cells in culture (8
). The HIO cells were suitable to be used for transfection with shRNA vector and selection of stable clones of GATA6 suppression. Thus, the development of aneuploidy/polyploidy and nuclear morphology abnormalities in primary HOSE and HIO cells following GATA6 suppression is reproducible and robust.
Suppression of GATA6 in ovarian surface epithelial cells leads to mitotic failure that is the cause of the formation of tetraploidy and aneuploidy.
Next, we cotransfected the shRNA-GATA6 suppression vector with a histone H2B-GFP expression construct (23
) to label chromosomal DNA and examined the cells over a 24-h period by time-lapse video microscopy to observe the causes of abnormal nuclear morphology in HIO cells. In more than 20 videos made of four independent transfections, we observed that in every instance when a cell was cotransfected with shRNA-GATA6 and histone H2B-GFP, the GFP-expressing cell failed to divide in mitosis and acquired an enlarged and aberrant nuclear morphology. GFP-expressing cells cotransfected with the control siRNA vector, however, completed cytokinesis and mitosis (Fig. ). Three typical phenotypes were observed in GATA6 siRNA-transfected cells (Fig. ; for time-lapse videos, see the supplemental material). As shown in Fig. , a transfected cell (arrow) progressed to metaphase/anaphase morphology by 6 h in the video. However, the dividing DNA failed to resolve the chromatin bridge, which persisted for 12 more h. At 18 h, the furrow regressed, and by 21 h, the nucleus fractured to form micronuclei. In a second example (Fig. ), a dividing nucleus (arrow) regressed by 7 h, attempted to segregate again at 12 h, and persisted until 24 h. In a third example (Fig. ), several cells showed the process of failed cytokinesis. A dividing nucleus regressed to form a 4n nucleus (red arrow), another dividing nucleus condensed but then broke down to form micronuclei (red arrowhead), or the last dividing cell formed two nuclei linked by a chromatin bridge (yellow arrow). In all, time-lapse analysis indicated that abnormally large and irregular nuclei formed due to failure of cytokinesis or mitosis following GATA6 suppression. Thus, we conclude that mitotic defects and failure of cytokinesis are the causes of the formation of tetraploid cells and deformed nuclei following suppression of GATA6.
FIG. 2. Suppression of GATA6 leads to mitotic failure in HIO and HOSE cells. An shRNA-GATA6 suppression vector and a histone H2B-GFP expression construct were cotransfected into HOSE or HIO-80 cells. Time-lapse video microscopy was used to monitor the mitotic (more ...) Loss of GATA6 and emerin and nuclear deformation in ovarian cancer.
We further examined the relationship between GATA6 expression and nuclear morphology in ovarian cancer tissues. In all of 16 cases of ovarian carcinomas in which monolayers of ovarian surface epithelial cells linked contiguously with malignant cells were present (53
), we found that loss of GATA6 expression is associated with loss of Dab2 protein and the morphological transformation of monolayer ovarian surface epithelial cells (arrowhead) to carcinoma cells (arrow) (an example is shown in Fig. ). Additionally, loss of GATA6 also correlates with enlarged and misshapen nuclei in malignant ovarian cancer cells (Fig. , arrows), consistent with the above observation in culture cells.
FIG. 3. Nuclear defect and loss of GATA6 in ovarian cancer cells. (A) Loss of GATA6 is associated with enlargement and misshaping of the nucleus in malignant ovarian cancer cells. Adjacent sections of ovarian carcinomas were immunostained with GATA6 and its transcription (more ...)
Because of the unique nuclear deformation of the GATA6-suppressed HOSE cells and GATA6-negative cancer cells, we examined the nuclear envelope integrity of ovarian cancer cells. Several nuclear envelope proteins are known to be critical for the maintenance of nuclear shape (26
). We reasoned that the loss of a nuclear envelope protein following loss of GATA6 might be the cause of the nuclear phenotypes observed and examined the expression of several nuclear envelope proteins in ovarian epithelial and cancer cells (Fig. ). In a panel of ovarian surface epithelial and cancer cells examined by Western blotting, GATA6 was often lost, correlating with loss of expression of its transcription target, Dab2 (8
) (Fig. ). Among several nuclear envelope proteins examined, emerin, which was initially identified as the product of an X-linked gene responsible for Emery-Dreifuss muscular dystrophy (2
), was absent or greatly reduced in many cancer cell lines, although a smaller amount of emerin was detectable in a darker exposure of the Western blot in several cancer cell lines (Fig. ). A darker exposure of the Western blot for GATA6 also showed the presence of some GATA6 protein in the corresponding cell lines (Fig. ). In HIO-118 cells (Fig. ) and emerin-positive NIHOVCAR10 (OV10) ovarian cancer cells (Fig. ), emerin immunofluorescence staining showed a nuclear envelope localization and a smooth, oval-shaped nucleus. Moreover, emerin staining overlapped with that of Baf (barrier to autointegration factor), an emerin-binding nuclear envelope protein (47
). The nuclei of OV10 cells are abnormal in a unique manner in that two enveloped nuclei are often closely associated (Fig. ). In emerin-negative NIHOVCAR5 (OV5) ovarian cancer cells, the absence of emerin was associated with a high percentage (approximately 20%) of cells possessing large and irregularly shaped nuclei (Fig. ). Loss of emerin is known to cause nuclear morphological deformation (27
), suggesting that absence of emerin may be the cause of nuclear deformation in ovarian cancer cells.
FIG. 4. Loss of emerin expression in ovarian cancer cells. (A) Immunostaining of emerin in human ovary tissue. Representative emerin staining of normal human ovary tissue is shown. Emerin staining is intensively positive around the nuclear envelope of ovarian (more ...)
It was noticed that in HIO-118 cells, GATA6 expression is very low, although emerin expression is present, indicating that an exception to the correlation between GATA6 and emerin expression exists (Fig. ).
In human ovarian tissues, ovarian surface epithelial cells are intensely stained with emerin around the nuclei (Fig. ). We found that emerin immunostaining was completely absent in 32 (38%) of the 84 ovarian adenocarcinomas examined in tissue microarrays (Fig. ). Additionally, most of the emerin-positive tumors showed abnormal emerin expression patterns, such as heterogeneous staining of cells or staining that was not localized to the nuclear envelope but instead distributed in the cytoplasm (Fig. ). Thus, the loss or abnormal distribution of emerin occurs in a significant percentage of human ovarian cancers.
Furthermore, in another 12 cases of ovarian cancer in which contiguous epithelia linking morphologically normal monolayer ovarian surface epithelial cells and multilayer cancer cells are present, a close correlation between the loss of GATA6 and the loss of emerin was observed, as shown in an example in Fig. . In regions containing contiguous epithelia linking benign and neoplastic cells, the loss of GATA6 correlates well with the loss of emerin and epithelial and nuclear morphological transformation (Fig. ).
We conclude that the loss of emerin correlates well with the loss of GATA6 in ovarian cancer, and the results support the idea that loss of GATA6 results in loss of the nuclear envelope protein emerin.
Suppression of GATA6 leads to reduction of the nuclear envelope protein emerin.
Next, we examined if suppression of GATA6 in HOSE or HIO cells might lead to the downregulation of emerin (Fig. ). Indeed, suppression of GATA6 by the shRNA approach resulted in the reduction of Dab2, emerin, and, to a lesser degree, lamin B and lamin A/C (Fig. ). The loss of emerin as a consequence of GATA6 suppression was also observed by immunofluorescence microscopy in double staining experiments in which cells with large and morphologically atypical nuclei also lacked GATA6 and emerin expression (Fig. ). Although nuclear deformation indicated by DAPI staining was obvious, a continuous envelope covering the nuclear DNA was present, as shown by positive staining for lamin A/C (Fig. ).
FIG. 5. Loss of emerin following GATA6 suppression by siRNA in HOSE cells. siRNA oligonucleotides targeting GATA6 were transfected into HOSE or HIO cells. (A) Three days following siRNA suppression of GATA6, HIO-80 cells were analyzed by Western blotting for (more ...)
Initially, we reasoned that emerin, like Dab2, might be one of the potentially many GATA6 transcription targets. Thus, we determined the alteration of the mRNA levels following GATA6 suppression in HOSE and HIO cells by RT-PCR. Surprisingly, in both of the HOSE lines and the one HIO line tested, the emerin mRNA level was not reduced following GATA6 suppression, although the Dab2 mRNA level was reduced (Fig. ). Thus, it appears that the loss of GATA6 affects emerin at the protein level rather than at the mRNA level. Indeed, when emerin or GFP-emerin was transfected and expressed in HIO cells, the exogenous emerin or GFP-emerin protein rapidly diminished upon suppression of GATA6 by siRNA. Thus, emerin loss following GATA6 suppression is likely the consequence of an increase in emerin degradation.
FIG. 6. Regulation of emerin mRNA and protein levels. (A) siRNA oligonucleotides targeting GATA6 were transfected into HIO cells. The mRNA levels of Baf1, Dab2, emerin, and lamin A/C were measured by real-time RT-PCR. The results are expressed as n2 for the ratio (more ...)
We also attempted to restore emerin expression in emerin-negative ovarian cancer cells. Upon transfection, the exogenous GFP-emerin fusion protein could be expressed in NIHOVCAR3 cells, but the level of the expressed protein declined steadily with time (Fig. ). Drug (G418)-selected clones showed neither a GFP fluorescence signal under fluorescence microscopy nor the presence of the GFP-emerin protein by Western blotting. A similar outcome was observed in OV5 ovarian cancer cells (Fig. ), although in parallel controls the fluorescence signals of the transfected GFP-histone H2B persisted over the same time course. In this experiment, we also observed the fluctuation of emerin protein levels at various times and under various culture conditions; a small amount of endogenous emerin was detected on day 1 following transfection but not on day 5 (Fig. ).
In experiments exploring potential factors regulating emerin expression levels, we found that transfection of GATA6 failed to restore emerin expression (Fig. ). Expression of lamin A (Ds-Red fusion protein) increased the emerin protein level in OV5 cells (Fig. ). Among several inhibitors tested, the caspase-6 inhibitor A3669 and the proteasome inhibitor L7035 increased emerin protein levels in OV5 cells (Fig. ). In contrast, the lamin A/C level decreased in the presence of the proteasome inhibitor (Fig. ).
We also determined if these protease inhibitors could block emerin loss upon GATA6 suppression in HIO and HOSE cells. Both A3669 and L7035 appeared to slow the decline of GFP-emerin upon GATA6 suppression in HIO cells, as observed by fluorescence microscopy. However, the toxicity of these compounds to HIO and HOSE cells in longer experimental time courses prevented us from analyzing the reversion of the phenotypes upon GATA6 suppression in detail.
These results suggest that both the interaction with nuclear lamina and the levels of cellular protease activities can influence emerin protein levels.
Suppression of emerin leads to mitotic defects and formation of aneuploid cells.
The consequence of emerin depletion was then examined in HOSE and HIO cells. Western blot analysis indicated that emerin protein was suppressed by siRNA oligonucleotides (Fig. ). Most of the cells took up siRNA oligonucleotides, as indicated by cy3-siRNA labeling, and a significant number of cells possessing aberrant nuclear morphology were evident (Fig. ). GATA6 and Dab2 were still detectable in these emerin-suppressed cells (Fig. ). As a specificity control, downregulation of another nuclear envelope structural protein, lamin B, did not lead to atypical nuclear morphology (Fig. ), consistent with the lack of a role for lamin B in maintaining the nuclear shape (26
). In three independent experiments in which random fields of about 100 cells were counted, approximately 20% of the emerin-suppressed cells had abnormal nuclei, compared to 4% of the cells treated with scrambled oligonucleotide controls. In comparison, GATA6 downregulation appeared to cause a more dramatic increase in cells with atypical nuclear morphology (Fig. ).
FIG. 7. Suppression of emerin expression leads to atypical nuclear morphology and polyploidy in HOSE cells. (A) Western blotting was used to determine emerin protein levels following the addition of siRNA oligonucleotides targeting emerin or a scrambled control (more ...)
A defect in cytokinesis similar to that produced by GATA6 suppression was observed in emerin-suppressed HOSE or HIO cells examined by time-lapse video microscopy (Fig. ). The mitotic phenotype monitored by histone H2B-GFP showed that the dividing nuclei of emerin-suppressed cells regressed to form presumably 4n nuclei (Fig. , arrows). We estimate that about one-third of the emerin-suppressed cells exhibited abnormal mitosis and failed cytokinesis. We examined the ploidy of the emerin-suppressed cells by a cytogenetic approach. Among 50 metaphase chromosome spreads counted, 6 tetraploid or nearly tetraploid spreads were found (Fig. ). Interestingly, an additional four metaphase spreads showed a signature of endoreplication in which the DNA was duplicated within each pair of parallel but separate chromosomes (Fig. ). In parallel experiments, GATA6 suppression resulted in 11 tetraploid or nearly tetraploid metaphase spreads, and 2 potential aneuploid (chromosomes 45 and 91) metaphase spreads were found in controls transfected with scrambled siRNA. Similar results were obtained with an additional independent HOSE cell preparation. It appears that emerin suppression also resulted in tetraploidy and also a high rate of endoreplication, which was also observed occasionally in GATA6-suppressed cells and in OV5 cells. Based on a relatively small number of cytogenetic analyses, we are not yet be able to conclude that endoreplication is a specific feature of emerin suppression or also a common consequence of GATA6 suppression. Nevertheless, we are able to conclude that the loss of emerin accounts for, at least in part, the mechanism of the nuclear deformation and formation of tetraploidy and aneuploidy as a consequence of GATA6 loss.