Review ArticleR

Endometrial Cancer Stem Cells: A New Target for Cancer Therapy

KIYOKO KATO
Anticancer Research June 2012, 32 (6) 2283-2293;

Abstract

Adult stem cells have recently been identified in several types of mature tissue and it has also been suggested that stem-like cells exist in cancerous tissues. In this regard, stem-like cell subpopulations, referred to as side-population (SP) cells, have been identified in several tissue and tumor types, based on their ability to remove intracellular Hoechst 33342, a fluorescent dye. We have isolated and characterized SP cells from normal human endometrium and an endometrial cancer cell line. Endometrial SP cells can function as progenitor cells. Endometrial cancer SP cells possess the following characteristics: i) reduced expression levels of differentiation markers, ii) long-term repopulating properties, iii) self-renewal capacity, iv) enhanced migration and podia formation, v) enhanced tumorigenicity, and vi) bi-potential development (tumor cells and stroma-like cells), suggesting that they have cancer stem-like cell features. Recently, we demonstrated that sodium butyrate, a histone deacetylase (HDAC) inhibitor, inhibited the self-renewal capacity of endometrial cancer SP cells by inducing a DNA damage response. Here, we review recent articles that show the presence of stem cells in normal endometrium and endometrial cancer and introduce the results of our own recent studies.

A stem cell is an undifferentiated cell that is defined by its ability to self-renew and to produce mature progeny cells (1). Adult stem cells have been identified in several types of mature tissue, such as the adult intestine (2), skin (3), muscle (4), blood (5), and the nervous system (6-8). Recent evidence also suggests that stem-like cells, so-called cancer stem cells (CSCs), exist in several types of malignant tumors, such as leukemia (9, 10), breast cancer (11) and brain tumors (12). CSCs are defined as a subset of tumor cells that have the ability to self-renew and generate the diverse cells that comprise the bulk of the tumor (1, 13).

It is believed that many CSCs up-regulate the expression of drug transporters, allowing them to efficiently pump antitumor agents out of the cells. CSCs reside in a quiescent state, making them resistant to chemotherapeutic agents that target rapidly cycling cells. Finally, they are endowed with a more invasive and metastatic phenotype (Figure 1).

Methods for Identification of CSCs

There are several methods for the identification of CSCs. For example, they are often detected by CSC markers. Several markers, such as CD133, CD44 and aldehyde dehydrogenase 1 (ALDH1) are commonly used for the enrichment of CSCs. CSCs in several types of malignant tumors express surface markers similar to those expressed by normal stem cells in the respective tissue (9, 14). Other functional analyses include clonogenicity or sphere-forming assays that evaluate the frequency with which these prospectively isolated cells form clones, and in vivo tumor-forming assays using immunodeficient mice. In addition, retention of the DNA synthesis label bromo-deoxyuridine (BrdU) for identification of label-retaining cells and the assessment of ALDH1 activity using the commercial reagent ALDEFLUOR (STEM-CELL Technologies Inc) have been used for identification and isolation of CSCs. Side-population (SP) cells, identified based on their ability to remove intracellular Hoechst 33342, a fluorescent dye, are stem cell-enriched subpopulations (15). The SP phenotype is associated with a high expression level of the ATP-binding cassette transporter protein ABCG2/BCRP1 (16). Most recently, established malignant cell lines maintained for many years in culture, have also been shown to contain SP cells as a minor subpopulation (17).

Figure 1.
Figure 1.

Concept of cancer stem cells.

Stem Cells in Endometrium

The human endometrium is a highly dynamic tissue undergoing cycles of growth, differentiation, shedding and regeneration throughout the reproductive life of women. Endometrial adult stem/progenitor cells are likely to be responsible for endometrial regeneration (18). The human endometrium contains rare populations of epithelial and stromal colony-forming cells (19). We have isolated SP cells from normal human endometrium and characterized their properties for the first time (20). Isolated SP cells in the human endometrium have a long proliferating potential and produce gland- or stroma-like cells. They can function as progenitor cells. Tsuji et al. demonstrated that ABCG2 was strongly expressed in the vascular endometrium (21). Several other groups have isolated SP cells (22-24) and shown that endometrial SP cells have multilineage developmental potential. That is, they have the potential to generate glandular cells and stromal cells (20), endothelial cells (22), adipose cells and osteocyte (24).

Although co-expression of CD146 and platelet-derived growth factor receptor (PDGFR) β identifies a population of mesenchymal stem-like cells from human endometrium (25), specific stem cell markers of endometrium remain unclear. Recently, Gotte et al. demonstrated that the adult stem cell marker, Musashi-1, was co-expressed with Notch-1 in a subpopulation of endometrial cells (26). Furthermore, they showed that telomerase- and Musashi-1-expressing cells were significantly increased in proliferative endometrium, endometriosis and endometrial carcinoma tissue compared to secretory endometrium, suggesting a stem cell origin for endometriosis and endometrial carcinoma.

CSCs in Endometrial Cancer

Endometrial cancer (EC) is the most common gynecological malignancy in the industrialized world. Two different clinicopathological types can be distinguished. Estrogen-related ECs (type I) develop in both pre-and postmenopausal women, and include endometrioid type and low cellular grade. In type I EC, estrogen receptor (ER, especially ERα) is expressed. This type of EC is frequently preceded by endometrial hyperplasia and carries a good prognosis. Type II non-estrogen-related ECs occur in postmenopausal women. They are non-endometrioid types (mainly papillary serous or clear cell carcinomas), without associated hyperplasia. Type II ECs are negative for ER and progesterone receptor (PR) and have high cellular grade and poor prognosis. The most frequent genetic alteration in type I EC is phosphatase and tensin homolog (PTEN) inactivation, followed by microsatellite instability and mutations of KRAS and β-catenin. In type II EC, p53 mutation is the most frequent genetic alteration, followed by amplification of human epidermal growth factor receptor 2 (HER2). Some of these pathways are important determinants of stem cell activity (Wnt, β-catenin and PTEN) (27-29). These suggest a stem cell contribution to endometrial cancer development.

Figure 2.
Figure 2.

Isolation of side population (SP) cells from human endometrial cancer cells. Hec1 cells and RK12V cells were stained with Hoechst 33342 and analyzed by flow cytometry. SP cells were present in Hec1 cells and RK12V cells. Verapamil treatment blocked dye efflux, increased staining, and rendered the SP cells undetectable by fluorescence. [adapted with alterations from Kato et al. (38)]).

The existence of CSCs in EC has been reported by several groups. Gorai et al. demonstrated that colony-initiating cells derived from a uterine carcinosarcoma cell line grew for more than 50 serial passages and were composed of cells similar to those found in the parent cell line (30). They hypothesized the existence of stem cells in their uterine carcinosarcoma cell line. Friel et al. showed that SP cells were present in two EC cell lines (AN3CA and Ishikawa) (31). AN3CA had features of CSCs, including low proliferative activity during nine days of cultivation, chemoresistance and enhanced tumorigenicity. Hubbard et al. demonstrated that a small population of clonogenic cells from EC tissues possessed self-renewal, differentiation and tumorigenic properties (32). Gotte et al. demonstrated that siRNA depletion of Musashi-1, an adult stem cell marker enriched in the SP cells from the EC cell line Ishikawa, leads to interference with the notch signalling pathway and p21 expression, resulting in an antiproliferative effect and induction of apoptosis (33).

Recent evidence suggests that CD133 expression is associated with CSC properties. Rutella et al. isolated CD133-expressing cells from primary endometrial tumors. They determined that CD133-expressing cells manifested CSC characteristics, including the ability to self-renew in culture, to differentiate into cells that recapitulated the original tumor, and to form tumors when transplanted into non-obese diabetic (NOD)/severe-combined immunodeficient (SCID) mice. CD133-positive cells co-expressed the Tn-MUC1 glycoform, CD44, chemokine (C-X-C motif) receptor (CXCR) 4 and interleukin (IL)-8. CD133-positive cells, but not CD133-negative cells, expanded in response to exogenously added estradiol. CD133-positive cells were relatively resistant to cisplatin and paclitaxel (34).

Figure 3.
Figure 3.

SP cells demonstrate the features of cancer stem cells. A: Hec1-SP cells have self-renewal capacity. The cloning plates were stained with crystal violet solution. Secondary and tertiary cloning plates are shown. Hec1-SP cells, but not NSP cells formed well-separated colonies. An average of 520 secondary colonies were formed per 2×104 seeded single cells (2.6%), and an average of 390 tertiary colonies were formed per 2×104 seeded single cells from the secondary colonies (1.9%). The number of colonies were counted in triplicate. Errors bar represent the standard error of the mean (SEM) from the number of colonies in three independent experiments. (The number of colonies per 2×104 seeded single cells: primary colonies, 7.43±3.09: secondary colonies, 518±60.4: tertiary colonies, 393±60.7). B: Tumorigenicity was enhanced in Hec1-SP cells. SP cells or NSP cells were inoculated into the subcutaneous tissues of nude mice. There was a trend for an increased size of tumor from inoculation with SP cells compared to NSP cells. C: Hec1-SP cells exhibited podia formation and prominent migration. In time-lapse video imaging studies, the frequency of cell division was higher in Hec1-SP cells than in Hec1-NSP cells. Hec1-SP cells formed podia (lamellipodia at the leading edge and uropodia at the trailing edge). Hec1-NSP cells did not form podia. [adapted with alterations from Kato et al. (38)].

Although both CD133-positive and -negative tumor-derived cells were capable of initiating tumor growth in vivo, the percentage of CD133-positive cells in secondary transplants was higher compared with the primary xenograft. In addition, Nakamura et al. conducted immunohistological analyses of CD133 expression in 62 surgical specimens of EC (35) and showed that high CD133 expression was an independent prognostic factor (35). Friel et al. demonstrated that regions of the CD133 promoter were hypomethylated in malignant endometrial tissue, relative to benign control endometrial tissues. Moreover, methylation of the CD133 promoter decreased during serial transplantation of an endometrial tumor xenograft (36). Rahadiani et al. examined ALDH1 expression in EC cell lines and uterine endometrioid adenocarcinoma (37); authors found that ALDH1-expressing cells were more tumorigenic, resistant to anticancer agents and more invasive than cells expressing ALDH1 at low levels. Clinically, patients with high ALDH1 expression had a poorer prognosis compared to those with low expression. High ALDH1 expression was an independent factor for poor prognosis.

Figure 4.
Figure 4.

Side population (SP) cells differentiated to α-smooth muscle actin (SMA) expressing cells. Hec1-SP and NSP cells (1×105) were cultured in Matrigel with a mesenchymal stem cell maintenance medium (MF medium), or a standard growth medium [Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS)] or a smooth muscle differentiation medium. After eight weeks, the level of α-SMA expression was analyzed in each Matrigel sample. SP cells cultured with either the standard growth medium or the smooth muscle differentiation medium expressed α-SMA. Hec1-NSP cells did not express α-SMA in any culture condition. [adapted with alterations from Kato et al. (38)].

Figure 5.
Figure 5.

RK12V-SP cells display higher resistance to conventional chemotherapeutic drugs than to RK12V-NSP cells. RK12V-SP cells and -NSP cells (2×104) were plated and cultured with Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) in the presence or absence of 1 μM cisplatin (CDDP), 10 nM paclitaxel (PTX) and 1 μM doxorubicin (DOX) for 96 h. The proportion of viable cells at day 4 relative to that of the control after incubation is shown. Inhibition (relative to control) was 61% in 1 μM cisplatin (CDDP), 51% in 10 nM paclitaxel (PTX) and 56% in 1 μM doxorubicin (DOX). All of these drugs inhibited the proliferation of RK12V-NSP cells significantly compared with the control (p<0.001). In contrast, none of these drugs had an inhibitory effect on the growth of RK12V-SP cells. Error bars represent the standard error of the mean (SEM) in ten independent experiments. [adapted with alterations from Kato et al. (55)].

Figure 6.
Figure 6.

Treatment with sodium butyrate (NaB) inhibits the self-renewal capacity of RK12V-SP cells. A: RK12V-SP cells and -NSP cells (2 x 104) were plated and cultured with Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) in the presence or absence of 2 mM or 5 mM NaB for 96 h. The proportion of viable cells relative to control after incubation is shown. Treatment with 2 or 5mM NaB for 96 h significantly inhibited cell proliferation of both RK12V-SP and RK12V-NSP cells (p<0.01). Error bars represent the standard error of the mean (SEM) in three independent experiments. B: RK12V-SP cells were re-analyzed by flow cytometry two weeks after culture in the presence or absence of NaB. Treatment with NaB for 24 h significantly inhibited the proportion of SP cells reproduced (the control, 15%; 2 mM NaB, 1.5%, p<0.02; 5 mM NaB, 0.023%, p<0.01). Error bars represent the SEM in four independent experiments. C: RK12V-SP cells were plated in collagen-coated dishes (10 cells/cm2) and cultured with mesenchymal stem cell maintenance medium (MF medium) in the presence or absence of 2 mM NaB for seven days. Treatment with 2 mM NaB completely suppressed primary colony-forming potential of RK12V-SP cells. D: Treatment with NaB completely suppressed colony formation of RK12V-SP cells in soft agar cultures. [adapted with alterations from Kato et al. (55)].

Figure 7.
Figure 7.

RK12V-SP cells are susceptible to sodium butyrate (NaB)-induced DNA damage. RK12V-SP cells and-NSP cells were cultured in mesenchymal stem cell maintenance medium (MF medium) in the presence or absence of 5 mM NaB for 6 or 12 h. Protein expression was analyzed by western blot and the ratio relative to that of the control is shown. The level of phosphorylated histoneH2A (γH2AX) proteins was markedly increased in RK12V-SP cells (44-fold) compared with that in RK12V-NSP cells (two-fold). Expression levels of p21, p27, and phospho-p38 mitogen-activated protein kinase (MAPK) were also enhanced more in RK12V-SP cells than in RK12V-NSP cells. Glyceraldehyde3-phosphate dehydrogenase (GAPDH) was the internal control. Representative data are shown. Similar results were obtained three times. [adapted with alterations from Kato et al. (55)].

Figure 8.
Figure 8.

Targeted drugs for endometrial cancer stem cells.

Characterization of SP Cells in EC

We have demonstrated that endometrial cancer SP cells show CSC features, marked migratory capacity, and the potential to differentiate into the mesenchymal cell lineage (38). We isolated and characterized SP cells from human EC cells (Hec1 cells) and rat endometrial cells expressing oncogenic human K-Ras protein (RK12V cells) (Figure 2). SP cells exhibited a reduction in the expression levels of differentiation markers, long-term proliferative capacity in cell culture, self-renewal capacity in vitro, enhanced migration, formation of lamellipodia and uropodia, and enhanced tumorigenicity (Figure 3). When SP cells and non-SP (NSP) cells formed in nude mice, they differed in their biological properties. Tumors generated from SP cells (but not NSP cells) consisted of tumor tissues and an extracellular matrix (ECM) enriched with stromal-like components. Evidence exists that stromal cells, such as inflammatory cells, vascular cells, and fibroblasts from the bone marrow give rise to a tumor matrix in response to growth factors or cytokines secreted from tumor cells or activated fibroblasts (39, 40). Alternatively, stromal cells may be derived from tumor cells that have undergone an epithelial-mesenchymal transition (EMT) (41, 42). We showed that stroma-like tissues stained positively for vimentin, CD13, and α smooth muscle actin (SMA), and contained human KRAS DNA sequences. FISH studies demonstrated that both human genomic and mouse genomic signals were detected in the stroma-like tissues with enriched ECM (human 76%, mouse 24%), showing that most of these stromal-like cells were derived from the inoculated SP cells. Additionally, Hec1-SP cells had the potential to differentiate into αSMA-expressing cells when seeded into Matrigel and incubated with a differentiation medium (Figure 4). These results suggest that EC SP cells are capable of undergoing EMT. This feature of endometrial SP cells is putatively involved in the development of endometrial stromal sarcoma or carcinosarcoma of the uterus.

We investigated the effect of cisplatin, paclitaxel and doxorubicin (clinically used for chemotherapy of EC) on the proliferation of RK12V both SP and NSP cells (Figure 5). Incubation of RK12V-NSP cells with medium containing these chemotherapeutic drugs for 96 h-inhibited proliferation compared to untreated controls. Relative to the controls, the extent of inhibition was 61% in 1 μM cisplatin, 51% in10 nM paclitaxel and 56% in 1 μM doxorubicin. All drugs inhibited the proliferation of RK12V-NSP cells significantly compared to the control (p<0.001). In contrast, none of these drugs had an inhibitory effect on the growth of RK12V-SP cells. These results clearly demonstrate that RK12V-SP cells have a higher resistance to conventional chemotherapeutic drugs, indicating a requirement for new targets for the treatment of CSCs. To develop new approaches in molecular cancer therapy, we performed microarray assays to identify overexpressed genes in RK12V-SP cells compared to those in RK12V-NSP cells. The expression of a number of genes including cytokines and growth factors was enhanced in RK12V-SP cells, suggesting that multiple signaling pathways maintain the phenotype of SP cells. It would be difficult to identify a single selective molecular target for SP cells.

Sodium Butyrate (NaB), an Inhibitor of Histone Deacetylase (HDAC), Alters the Properties of Endometrial SP Cells

HDAC inhibitors have multiple biological effects, including growth arrest, apoptosis, senescence, reactive oxygen-species facilitated cell death, mitotic cell death and antiangiogenesis (43). The regulation of histone acetylation is a vital mechanism controlling cellular differentiation and the biological phenotype of cancer cells (43). HDACs and histone acetyl transferases are enzymes that ensure a proper level of histone acetylation. Dysregulated HDAC activity has been found in certain types of human cancer (44-47). Several studies have demonstrated the antiproliferative or the proapoptotic effects of HDAC inhibitors on EC cells (48-50). HDAC inhibitors include short-chain fatty acids [e.g. butyrates and valproic acid (VPA)], organic hydroxamic acids [trichostatin A(TSA) and suberoyl anilide bis-hydroxamine (SAHA)], cyclic tetra peptides (e.g. Trapoxin), and benzamides (e.g. MS-275). TSA, NaB, VPA and SAHA can inhibit malignant cells in vitro and in vivo (51-53). We have previously demonstrated that NaB induces p21 expression, resulting in growth arrest and cell death (54).

Therefore, we investigated the effect of NaB on the properties of RK12V-SP cells (55). Treatment with 2 or 5 mM NaB for 96 h significantly inhibited the proliferation of RK12V-SP cells as well as RK12V-NSP cells (Figure 6A, p<0.01). RK12V-SP cells have the potential to regenerate SP cells after incubation, which is an important characteristic of stem-like cells. Treatment with NaB for 24 h significantly inhibited the proportion of SP cells regenerated (control, 15%; 2 mM NaB, 1.5%, p<0.02; 5 mM NaB, 0.023%, p<0.01; n=4, Figure 6B). The primary colony-forming potential of RK12V-SP cells was completely suppressed by treatment with 2 mM NaB (Figure 6C). NaB treatment completely suppressed colony formation of RK12V-SP cells in soft agar cultures. These results demonstrated that treatment with NaB reduced self-renewal capacity and tumorigenicity of RK12V-SP cells.

We investigated the molecular mechanism underlying the inhibitory effect of NaB treatment of RK12V-SP cells. Recently, we reported that treatment with NaB induced cell death in several cancer cell lines mediated by enhanced reactive oxygen species (ROS) levels, DNA damage response (DDR) signals and up-regulation of p21 (56). Thus, we examined the change of these signal levels in RK12V-SP and -NSP cells.

The level of intracellular ROS was enhanced by 5 mM NaB treatment in both RK12V-SP and -NSP cells. Phosphorylated histoneH2A (γH2AX) foci, which are indicators of DNA damage, were assessed by immunohisto-chemistry. The number of γH2AX foci in both RK12V-SP and -NSP cells was increased by 5 mM NaB treatment. The level of γH2AX protein was markedly increased in RK12V-SP cells (44-fold) compared with that in RK12V-NSP cells (2-fold) (Figure 7). p21, p27, and phospho-p38 mitogen-activated protein kinase (MAPK) expression levels were also enhanced more in RK12V-SP cells compared to RK12V-NSP cells (Figure 7). These results imply that treatment with NaB induces the production of intracellular ROS and DNA damage in both RK12V-SP and -NSP cells. DNA damage response signals were elevated more in RK12V-SP cells (cancer stem-like cells) than in RK12V-NSP cells by NaB treatment. These results demonstrate that CSCs have a high susceptibility to HDAC inhibitor-induced DNA damage and suggest that HDAC inhibitors may represent an attractive antitumor therapy against CSCs.

Future Aspects

Recent evidence has demonstrated the association of EMT with properties of CSCs. Mani et al. reported a direct link between EMT and gain of epithelial stem cell properties (57). Cell-surface markers, CD44high/CD24low are associated with human breast cancer stem cells. Moreover, up-regulated expression of Snail and Twist, two EMT-inducing transcription factors, increases the size of the stem cell population and the expression levels of mesenchymal markers, fibronectin and vimentin. EMT promotes the generation of CSCs.

Salinomycin has also been identified as a selective inhibitor of CSCs (58). We have also shown that EMT may occur during tumor formation of endometrial cancer SP cells. The effect of salinomycin on Hec1-SP cells proliferation is still under investigation.

Conclusion

Since the CSC concept was first proposed for leukemia and solid tumors, studies of CSCs have increased our understanding of these cells. We have demonstrated the presence of stem-like cells both in normal endometrium and in EC. The data that CSCs play an important role in the development and progression of many types of cancers are convincing. The properties of CSCs include marked self-renewal capacity, an undifferentiated phenotype, enhanced tumorigenicity, prominent migration and resistance to radio- or chemotherapy (Figure 8). Multiple signaling pathways and factors are involved in the survival and maintenance of CSCs. Successful therapy requires the use of several drugs inhibiting different cellular properties. Future drugs may possess multiple actions. Research should focus on the identification of markers of endometrial CSCs. If cell death can be induced in CSCs by a targeted therapy at first-line treatment, the prognosis for patients with EC will definitely improve.

  • Received April 4, 2012.
  • Revision received May 15, 2012.
  • Accepted May 16, 2012.

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Endometrial Cancer Stem Cells: A New Target for Cancer Therapy
KIYOKO KATO
Anticancer Research Jun 2012, 32 (6) 2283-2293;
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