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Nuclear envelope structural defects cause chromosomal numerical instability and aneuploidy in ovarian cancer
© Capo-chichi et al; licensee BioMed Central Ltd. 2011
Received: 28 January 2011
Accepted: 26 March 2011
Published: 26 March 2011
Despite our substantial understanding of molecular mechanisms and gene mutations involved in cancer, the technical approaches for diagnosis and prognosis of cancer are limited. In routine clinical diagnosis of cancer, the procedure is very basic: nuclear morphology is used as a common assessment of the degree of malignancy, and hence acts as a prognostic and predictive indicator of the disease. Furthermore, though the atypical nuclear morphology of cancer cells is believed to be a consequence of oncogenic signaling, the molecular basis remains unclear. Another common characteristic of human cancer is aneuploidy, but the causes and its role in carcinogenesis are not well established.
We investigated the expression of the nuclear envelope proteins lamin A/C in ovarian cancer by immunohistochemistry and studied the consequence of lamin A/C suppression using siRNA in primary human ovarian surface epithelial cells in culture. We used immunofluorescence microscopy to analyze nuclear morphology, flow cytometry to analyze cellular DNA content, and fluorescence in situ hybridization to examine cell ploidy of the lamin A/C-suppressed cells.
We found that nuclear lamina proteins lamin A/C are often absent (47%) in ovarian cancer cells and tissues. Even in lamin A/C-positive ovarian cancer, the expression is heterogeneous within the population of tumor cells. In most cancer cell lines, a significant fraction of the lamin A/C-negative population was observed to intermix with the lamin A/C-positive cells. Down regulation of lamin A/C in non-cancerous primary ovarian surface epithelial cells led to morphological deformation and development of aneuploidy. The aneuploid cells became growth retarded due to a p53-dependent induction of the cell cycle inhibitor p21.
We conclude that the loss of nuclear envelope structural proteins, such as lamin A/C, may underlie two of the hallmarks of cancer - aberrations in nuclear morphology and aneuploidy.
A deformed and enlarged nuclear morphology is a common characteristic of cancer cells, and the "roundness" of the nucleus is a good indicator to distinguish benign, low grade, and malignant cells [1, 2]. In the clinical setting, the morphology of the nucleus is used universally for diagnostic and prognostic prediction of malignancies of tumor cells, referred to as "nuclear grade" [1, 2]. The most well known diagnostic test based on cell and nuclear morphology is the cervical Papanicolaou (PAP) smear test . In PAP smears, cells collected from a swab of the cervix are examined under microscope to determine the presence of large and atypical nuclei, which serves as an initial diagnosis of cervical or uterine cancer. The simple procedure was invented in the 1930s, widely implemented by the 1960s, still universally practiced worldwide today, and is credited for saving millions of lives.
In the last 5 decades, much research has been devoted to understand the molecular basis for the atypical and enlarged nucleus that accompanies malignancy. In ovarian cancer, nuclear size and morphology correlate with the degree of genetic changes and can be used to distinguish low- from high-grade serous cancer, as well as to predict outcome [4–6]. Molecular changes in the nuclear matrix and/or nuclear envelope have been postulated, and deformation of nuclear morphology was shown to associate with oncogenic signaling [7–9], but no definite conclusions have been reached regarding the molecular basis of nuclear deformation in malignant cells [1, 10].
Another hallmark of cancer cells, first recognized over one hundred years ago by Boveri [11, 12] is aneuploidy, or an abnormal and unbalanced number of chromosomes compared to diploid normal cells. The majority (around 90%) of human ovarian cancers are aneuploid and possess a hyperdiploid (> 46) to subtetraploid (< 96) chromosome number http://www.ncbi.nlm.nih.gov/sky/skyweb.cgi?form_type=submitters. Cancer cells within one tumor or cell line are not uniform in chromosomal number, indicating the presence of chromosomal numerical instability in cancer cells . Although an association between aneuploidy and malignancy has been well recognized, the causes and significance of aneuploidy in cancer remain unsettled [14–17]. Formation of aneuploid cells was found to be an early event in the development of ovarian cancer, suggesting aneuploidy contributed to cancer initiation . Mitotic failure, tetraploid intermediates, and subsequent unbalanced cytokineses are the most common cause of aneuploidy [19–21]. Frequently, tetraploid cells are the products of mitotic regression after failed cytokinesis and the intermediates that produce aneuploid cells in subsequent mitotic events . Nevertheless, in ovarian cancer, the molecular basis for the causes of aneuploidy is largely unknown.
In mammalian cells, loss or mutation of nuclear envelope structural proteins such as emerin  or lamin A/C  produces nuclear morphological deformation. Lamin A and lamin C are differentially spliced forms of the same gene and differ only at the C-terminal domain . Lamin A/C expression is absent in embryonic stem cells and early embryos, and is progressively expressed in nearly all tissues at later developmental stages . The initiation of lamin A/C expression is associated with cell differentiation, which suggests that lamin A/C expression may serve as a limit on the plasticity of cells for further developmental events [25, 26]. Additionally, the cell types that seem to lack lamin A/C, such as embryonic carcinoma cells and some cells of the spleen, thymus, bone marrow and intestine in the adult mouse may fall into the "stem cell" category, but the correlation will need to be carefully tested [25, 26]. Loss or mutation of nuclear envelope proteins such as lamin A/C  or emerin  causes muscular dystrophy, severe premature aging (progeria) , and several additional diseases catalogued as laminopathies , but a link between the nuclear envelope protein deficiency with the deformed nuclear morphology in cancer cells has not been established.
Loss of lamin A/C expression is often found in cancer cells [26, 31], including leukemia and lymphoma [32, 33], colon cancer [34, 35], prostatic cancer , lung cancer , and gastric cancer . One study reported the identification of high lamin A/C expression in a fraction of ovarian cancer cases using high-density protein microarrays . However, normal ovarian surface epithelial cells were also found to be high in lamin A/C staining . The observation may be explained by the low fraction of epithelial cells relative to the total ovarian tissue masses used for protein array analysis.
Recently, we found that the nuclear envelope protein emerin is lost in a fraction of ovarian cancer cells . Here, we further explored the expression of additional nuclear envelope proteins in ovarian cancer and studied the phenotypes for the loss of lamin A/C in ovarian epithelial cells.
Materials and methods
Tumor specimens and immunohistochemistry
The current study utilized archived tumor tissues and cell lines prepared from human ovaries obtained from prophylactic surgeries. The tissues and cells lines were obtained from Fox Chase Cancer Center tumor bank. These tissues are designed for research only and contain no link to personal information of the donors. The study was reviewed by the Institutional Review Board (IRB) of both Fox Chase Cancer Center and the University of Miami Miller School of Medicine, and was approved and judged as non-human subject research.
The three ovarian tumor tissue microarrays (duplicate core of 120 tumor tissues and 5 controls) and 20 prophylactic oophorectomies were provided by the Tumor Bank of Fox Chase Cancer Center. These ovarian tissues and tumor specimens were described in more detail previously [40, 41]. Immunostaining was performed using the mouse DAKO Envision TM+ System and the Peroxidase (DAB) Kit (Dako Carpinteria, CA) as previously described [24–26]. Negative controls were performed by replacing the primary antibodies with non-immunized IgG.
Human ovarian surface epithelial and cancer cell cultures
Human ovarian surface epithelial (HOSE) cells were established from ovaries obtained from prophylactic oophorectomies . The donors were usually individuals who were either BRCA1/2 carriers or had a family history of breast or ovarian cancer but there was no indication of cancer at the time of surgery. Each fresh specimen of intact whole ovary from surgeries was immersed in medium and the ovarian surface was gently scraped with a rubber policeman to collect cells. The ovarian tissues were then subjected to histology analysis by pathologists to confirm the absence of microscopic tumors. HOSE cells were transfected with SV40 T-antigen to prepare human "immortalized" ovarian (HIO) cells. These HIO cells have a longer lifespan in culture and can be cultured for 20 to 50 passages before undergoing senescence, compared to HOSE cells that can only be maintained for 5 to 7 passages [42, 43].
Ovarian epithelial cancer cell lines were cultured in (D)MEM with 10% FBS as previously reported [42, 43]. The OVCAR lines were previously established by Thomas Hamilton  and the others (A1847, A2780, ES2, and SKOV-3) were obtained from American Type Culture Collection.
Probes for Northern blot and antibodies for Western blot
Probes for lamin A/C, emerin, and lamin B1 were obtained by reverse transcriptase-polymerase chain reaction (PCR) using total RNA extracts from HOSE cells at passage 2 as a template. The ascension number, sense primer (SP), and anti-sense primer (ASP) are as following: lamin B1 (BC078178): cggtg tacat cgaca aggtg (SP), cagct gttgc tgcat ttgat (ASP); BAF (BC005942); gaacc gttac gggaa ctgaa (SP), tcaaa cggat tgaaa gtgag g (ASP); and lamin A/C (NM_005572): tctgc tgaga ggaac agcaa (SP), ggtga tggag caggt catct (ASP). The PCR products were sequenced to confirm the identity.
Mouse anti-emerin, rabbit anti-p21, and goat anti-lamin B antibodies were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA). Anti-p53 mouse IgGs were purchased from Dako (Carpinteria, CA) and mouse anti-beta-tubulin from Sigma St. Louis, MO.
Small interfering RNA (shRNA) transfection
siRNA oligos and shRNA plasmids directed against human lamin A/C were purchased from Santa Cruz Biotechnology and were transfected into cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Cells were retrieved 72 hours after transfection for analysis by Western blot, Northern blot, or immunofluorescence microscopy.
Flow cytometry analysis
For sorting of live cells, 0.5 μM vybrant violet dye (Molecular Probe) was added and incubated for 30 minutes at 37°C. For fixed cell analysis, the trypsinized cells were resuspended in ice-cold ethanol/PBS (70% v/v) and kept at -20°C until ready to use. Before flow cytometry analysis, the fixed cells were resuspended in 0.5 μM vybrant violet dye or propidium iodide and RNase A solution. Cell sorting and flow analyses were performed on a LSR Fortessa driven by BD's FACS Diva software, version 6.1.3 (Becton Dickinson, San Jose, CA).
Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 5 minutes, blocked with 3% BSA in PBS containing 0.1% Tween-20 for 30 minutes, incubated for 1 hour at 37°C with primary antibodies, and subsequently with secondary antibodies conjugated with AlexaFluor 488 (green fluorescence) or AlexaFluor 594 (red fluorescence) (Molecular Probes/Invitrogen, Carlsbad, CA). The nuclei were stained with Hoechst 33342 (Molecular Probes). Cells were mounted/sealed in anti-fade reagent containing 0.1 M n-propyl gallate (pH 7.4) and 90% glycerol in PBS. A Zeiss microscope and AxioCam Camera and AxioVision software 4.8 were used for image acquisition.
Chromosome number counting and fluorescence in situ hybridization (FISH) analysis were performed by the University of Miami Cytogenetic Core Facility. Briefly, the cells were exposed to colcemid (demecolcine) (0.015 mg/ml) for 12 hours, resuspended in 10 ml hypotonic buffer (75 mM KCl) for 20 minutes, and then kept in 1 ml of chilled fixative (methanol/acetic acid, 3/1). The cell suspension was dropped on wet slides and heated (70°C) overnight, dyed with Wright stain, and analyzed to count 50 metaphases for each sample in duplicate. For FISH analysis, an alpha-satellite DNA probe for chromosome X centromeres (Xp11.1-q11.1) and a probe for chromosome Y satalite III (Yq12) were used in interphase cells. The pre-labeled FISH probes were purchased from Abbott Molecular (Abbott Park, Illinois, USA).
Lamin A/C expression is frequently lost in ovarian cancer cells and tissues
Most ovarian cancer cells and tissues have a heterogeneous Lamin A/C protein expression pattern
Furthermore, the tumors grouped as lamin A/C-positive usually also contain a fraction of lamin A/C-negative cells, ranging from 10% to 40% [see Additional file 1: Table S1]. In contrast, all ovarian epithelial cells in normal tissues have strong lamin A/C staining around the nuclear envelope (Figure 2A). Thus, nearly all ovarian cancer cases have some degrees of nuclear envelope structural defect due to the loss of lamin A/C (reported here) or emerin  protein in a fraction to all of the tumor cell population.
Lamin A/C suppression in human ovarian epithelial cells causes nuclear morphological deformation
The nuclear sizes of the cell population also changed upon lamin A/C suppression (Figure 4E), as determined using computer software (AxioVision software 4). The diameter of the nucleus had a range of 13 to 29 μm in HOSE cells and shifted up to 16 to 38 μm in the lamin A/C-suppressed cells (Figure 4E). The average size increased from 18 to 29 μm upon lamin A/C suppression, and the change was found statistically significant (P < 0.005). Following lamin A/C suppression in HOSE cells, the nuclear size distribution of the cell population resembles OVCAR5 ovarian cancer cells (Figure 4E). The variation in nuclear size may be caused by chromatin organization, condensation, and DNA content. OVCAR5 has 51 to 57 chromosomes per cell http://www.ncbi.nlm.nih.gov/sky/skyweb.cgi?form_type=submitters. Thus, the experiments showed that the loss of lamin A/C leads to atypical nuclear morphology and increased nuclear size in HOSE cells, resembling those of cancer cells. Similar to OVCAR5 cells, the increased nuclear size distribution in lamin A/C-suppressed HOSE cells was speculated to be caused by increased DNA content and changes in chromatin organization. These experiments have been repeated three times over a one-year period using three independent preparations of HOSE cells, and the reproducibility of the results was very good.
Suppression of Lamin A/C leads to polyploidy and aneuploidy in ovarian surface epithelial cells
These cells were further analyzed by FISH using an X centromere probe. Typically, the control cells showed two X-chromosome markers per cell, indicating a diploid ploidy (Figure 5B). Rare (less than one per 100 nucleus examined) tetraploid cells were observed, as shown in an example (Figure 5B). In contrast, three to four X-chromosomes per nucleus were detected in 37% of the lamin A/C-suppressed HOSE cells, as shown in examples from six images (Figure 5C). Thus, suppression of lamin A/C leads to the formation of tetraploid and aneuploid ovarian epithelial cells. The wide distribution of nuclear sizes and the broad range of DNA contents in the lamin A/C-suppressed cells is consistent with a variation of chromosomal numbers and aneuploidy of the cells; however, we were not able to determine the exact chromosomal number in each cell due to difficulty in obtaining a sufficient number of mitotic cells (discussed below).
Suppression of Lamin A/C leads to reduced mitosis and retarded cell growth
Although the primary HOSE cells proliferated slowly in culture (compared to cancer cells), we observed that about 12% of the control cells in culture were undergoing mitosis using beta-tubulin staining to mark the mitotic spindles (Figure 6C). In contrast, mitotic spindles were rare (< 1%) in lamin A/C-suppressed cells (Figure 6D), indicating a reduced cell division following the loss of lamin A/C and aneuploidy. It is known that the p53/p21 pathway is activated to mediate growth arrest of aneuploid cells . Indeed, we found that p53 and p21 proteins were increased in lamin A/C-suppressed, aneuploid cells (Figure 6E). The induced cell cycle inhibitor p21 following lamin A/C-suppression is p53-dependent, since simultaneous suppression of both lamin A/C and p53 did not stimulate an increased p21 expression. It was reasoned that the lamin A/C-suppressed and aneuploid cells might overcome growth arrest once p53 function is lost, and future experiments are to determine the long-term consequence of lamin A/C suppression in the absence of p53.
A significant understanding of ovarian cancer biology and genetics has been achieved [46, 47], though the causes and importance of chromosomal instability as well as nuclear atypia are still topics of research. We found that lamin A/C is absent in a significant fraction of ovarian (47%) cancers. Intriguingly, most ovarian carcinoma tissues and cancer cell lines exhibit a heterogeneous pattern of lamin A/C expression in the population of cancer cells, which may account for the variation of nuclear shapes within an ovarian tumor. Lamin A/C expression is regulated during cell differentiation and embryonic development, but the expression is not known to be regulated in a cell cycle-dependent manner [25, 26]. Thus the heterogeneous lamin A/C expression in cancer cells may link to heterogeneity of tumor cells.
Despite its being such a prominent feature of malignancy, what role a deformed nucleus may play in the development of cancer is unclear. The identification of the loss of nuclear envelope structural proteins emerin and lamin A/C as the molecular basis of nuclear deformation in cancer cells may provide some explanation. We found that suppression of emerin  or lamin A/C (current study) lead to mitotic failure and the formation of polypoid and subsequent aneuploid cells. The essential role of lamin A/C in mitosis and the formation of the envelope to house the newly formed nuclei in dividing cells have been shown in Caenorhabditis elegans [48, 49], which has only one lamin gene. Lamin suppression results in the formation of chromatin bridges, large and deformed nuclei, and ultimately developmental failure . Emerin suppression only slightly affects cell division and its role is redundant with MAN gene in C. elegans .
In mammals, neither lamin A/C  nor emerin [52, 53] is essential for cell division and development as shown by gene knockout in mice. However, lamin A/C-deficient cells exhibit a phenotype of chromosomal numerical instability, though the mechanism was not analyzed . Mutations in the lamin A/C gene have also been shown to affect cell cycle and mitosis in human cells [55, 56]. We also report an increased mitotic failure and the formation of polyploidy and aneuploidy in HOSE cells when emerin expression is suppressed . In the current study, we conclude that though lamin A/C is not essential for the initial division of ovarian epithelial cells in culture, nevertheless, the loss of lamin A/C increases mitotic failure and the development of polyploidy and subsequent aneuploidy. We further speculate that nuclear structural defects as a consequence of the loss of lamin A/C (and also loss of emerin) may be a principal mechanism for the chromosomal numerical instability, and the underlying cause of aneuploidy in ovarian cancer. Since lamin A/C expression has been found to be lost in several cancer types , loss of lamin A/C expression may be the possible common cause of nuclear atypia and chromosomal numerical instability observed in many cancer types.
A characteristic of laminopathies (disorders caused by mutated and defective lamin A/C) is genomic instability [30, 54], which is shared by cancer. However, laminopathies are not linked to a predisposition to cancer. In fact, we observed that suppression of lamin A/C results in a lowered rate of mitosis and cell growth, coincident with polyploidy and aneuploidy. We reasoned that loss of lamin A/C and subsequent aneuploidy by itself is not oncogenic, but actually reduces the efficiency in cell division and proliferation. Aneuploidy is not tolerated in development, has severe consequences on cell proliferation, and can be detrimental to individual normal cells [57, 58]. However, with additional oncogenic changes (such as p53 mutation) to bypass cell growth regulation and cellular stress, chromosomal instability brought by the loss of nuclear structural integrity may enable the cells to undergo oncogenic evolution and development of malignant tumors [59, 60]. Thus, neoplastic cells may acquire a nuclear structural defect and trade cell growth efficiency for versatility and agility in chromosomal instability. Indeed, ovarian cancer accumulates vast amounts of seemingly random genomic changes [13, 61], indicative of chromosomal numerical instability. Both p53 mutation and acquisition of aneuploidy are closely associated early events [18, 62], and p53 mutation is common in high-grade ovarian cancer [46, 47, 63, 64], allowing the survival and proliferation of aneuploid cells.
Aneuploidy activates the p53/p21 pathway . Indeed we found that p53 and p21 are increased in lamin A/C-suppressed cells, presumably mediating the observed cell growth arrest. In our experiments, unfortunately, the siRNA-suppression of p53 is short-lived, not permitting our study to determine if loss of both of p53 and lamin A/C might allow survival of aneuploid cells and the selection of clones with a chromosomal composition presenting a neoplastic phenotype. Proper experimental systems (mutant mice) are currently under development to test this hypothesis.
In summary, we report the finding of the frequent loss and aberrant expression of lamin A/C in ovarian cancer. The experimental results support that a nuclear envelope structural defect caused by the loss of lamin A/C may be the molecular basis of two of the prominent hallmarks of human cancer, nuclear morphological deformation and aneuploidy.
These studies were supported by funds from concept awards BC097189 and BC076832 from Department of Defense (USA). Grants R01 CA095071, R01 CA099471, and CA79716 to X.X. Xu from NCI, NIH also contributed to the studies. The early stage of this work was also supported by Ovarian Cancer SPORE P50 CA83638 (PI: RF Ozols).
We acknowledge the excellent technical assistance of Carolyn Slater, Jennifer Smedberg, and Lisa Vanderveer at Fox Chase Cancer Center, and Shannon Opiela, Jay Enten, and James Phillips (Flow Cytometry Core), and Carmen Casas and Ana Prada (Cytogenetic Core) at the University of Miami. We thank our colleague, Dr. Elizabeth Smith, for reading, suggestions, and editing during the course of the experiments and the preparation of the manuscript.
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