The nuclear export receptor, Crm1 (exportin 1), is involved in the nuclear translocation of proteins and certain RNAs from the nucleus to the cytoplasm and is thus crucial for the correct localisation of cellular components. Crm1 has recently been reported to be highly expressed in certain types of cancers, yet its expression in oesophageal cancer has not been investigated to date. We investigated the expression of Crm1 in normal and tumour tissues derived from 56 patients with human oesophageal squamous cell carcinoma and its functional significance in oesophageal cancer cell line models. Immunohistochemistry revealed that Crm1 expression was significantly elevated in oesophageal tumour tissues compared to normal tissues and its localisation shifted from predominantly nuclear to nuclear and cytoplasmic. Real-time RT-PCR revealed that Crm1 expression was elevated at the mRNA level. To determine the functional significance of elevated Crm1 expression in oesophageal cancer, its expression was inhibited using siRNA, and a significant decrease in cell proliferation was observed associated with G1 cell cycle arrest and the induction of apoptosis. Similarly, leptomycin B (LMB) treatment resulted in the effective killing of oesophageal cancer cells at nanomolar concentrations. Normal oesophageal epithelial cells, however, were much less sensitive to Crm1 inhibition with siRNA and LMB. Together, this study reveals that Crm1 expression is increased in oesophageal cancer and is required for the proliferation and survival of oesophageal cancer cells.
Oesophageal cancer is the eighth most common cancer worldwide and the sixth most common cause of death from cancer (
Crm1 (the chromosome region maintenance 1 protein or exportin 1) is a protein that has recently been identified as being highly expressed and playing a functional role in several types of cancers. Crm1 is a member of the karyopherin β protein family and the major nuclear export receptor in the cell (
Recent studies have reported that the expression of Crm1 is altered in cancer, with elevated Crm1 levels reported in cervical cancer (
In the present study, we examined the expression and functional relevance of Crm1 expression in oesophageal squamous cell carcinoma. We found that Crm1 levels are elevated in oesophageal tumours compared to normal epithelium and that its cellular localisation is also altered. We also found that Crm1 is required for the proliferation and survival of oesophageal cancer cells, supporting its use as a potential target for anticancer drugs.
Immunohistochemical examination of Crm1 was performed on archived paraffin-embedded tissue sections of matched normal and cancer tissues obtained from 56 patients with oesophageal squamous cell carcinoma (Groote Schuur Hospital, Cape Town, South Africa). Immunohistochemistry was performed using standard procedures. Briefly, slides were heat-fixed for 10 min, deparaffinised and rehydrated, followed by antigen retrieval by incubation in 10 mM EDTA in a pressure cooker for 2 min. Sections were blocked for endogenous peroxidase activity by submerging the slides in hydrogen peroxide for 20 min and were then incubated at room temperature with a 1:20 dilution of goat serum (Dako, Glostrup, Denmark) for 30 min. Slides were next incubated with a 1:500 dilution of Crm1 primary antibody (sc-5595; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. A negative control was included where no primary antibody was used. The Dako REAL™ EnVision™/HRP rabbit/mouse detection system was subsequently used to detect Crm1 expression, according to the manufacturer’s instructions. The intensity and localisation of Crm1 staining were evaluated and scored independently by a pathologist. Crm1 expression was noted in nuclear and cytoplasmic cellular compartments and thus a separate scoring for Crm1 expression was performed in each case. For nuclear staining, the Allred system for oestrogen receptor was used (
For the analysis of Crm1 mRNA expression, RNA was isolated from matched normal and tumour tissue biopsies obtained from 22 patients with oesophageal squamous cell carcinoma (Groote Schuur Hospital and Tygerberg Hospital, Cape Town, South Africa). For cDNA synthesis, 1 μg RNA was reverse transcribed using ImProm-II™ Reverse Transcriptase (Promega, Madison, WI, USA). Quantitative real-time PCR was performed using the KAPA SYBR qPCR kit (KAPA Biosystems, Cape Town, South Africa) using the following primers: Crm1 (F 5′-GCA CCT CTT GGA CTG AAT CG-3′ and R 5′-AAG CGA CAG CAC ACA CAC AC-3′), β-glucuronidase (F 5′-CTC ATT TGG AAT TTT GCC GAT T-3′ and R 5′-CCG AGT GAA GAT CCC CTT TTT A-3′) and cyclophilin D (F 5′-TGA GAC AGC AGA TAG AGC CAA GC-3′ and R 5′-TCC CTG CCA ATT TGA CAT CTT C-3′), where β-glucuronidase and cyclophilin D were used to normalise for Crm1 expression. The StepOne Real-time PCR System (Applied Biosystems, USA) was used. The comparative threshold cycle (CT) method (
Oesophageal carcinoma cell lines, WHCO1, WHCO5 and WHCO6, were originally established from South African patients with oesophageal squamous cell carcinoma and were provided by Dr R. Veale (
For the inhibition of gene expression, Crm1 siRNA was used (sc-35116; Santa Cruz Biotechnology). Control siRNA-A consisting of a scrambled sequence (sc-37007; Santa Cruz Biotechnology) was used as a non-silencing control. Leptomycin B (LMB), an inhibitor of Crm1 activity, was obtained from Sigma (St. Louis, MO, USA) and stored as a 10.2 μM stock in methanol.
Cells in culture were grown to 80% confluency and lysed on ice in RIPA buffer (10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1% deoxycholate, 0.1% SDS, 1% Triton X-100 and 1X Complete Protease Inhibitor Cocktail (Roche, Basel, Switzerland). Western blot analyses were performed using the rabbit anti-Crm1 (H-300) (sc-5595) and rabbit anti-β-tubulin (H-235 (sc-9104) antibodies (Santa Cruz Biotechnology).
For immunofluorescence analysis of Crm1, cells were plated on coverslips and fixed with 4% paraformaldehyde. After fixing, cells were permeabilised in 0.1% Triton X-100 in PBS, followed by quenching in 50 mM NH4Cl in PBS. Cells were blocked in 0.2% gelatin for 30 min and subsequently incubated with an α-Crm1 primary antibody (1:100 dilution, sc-5595; Santa Cruz Biotechnology) for 45 min in a humidified chamber. After washing in PBS, Cy3-conjugated goat anti-rabbit antibody (1:300; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was applied for a further 45 min. Cell nuclei were stained with DAPI (100 ng/ml) and coverslips were mounted in Mowiol. Fluorescence was visualised using standard fluorescence microscopy.
To examine the effect of Crm1 siRNA or LMB on cell viability, the MTT assay was used. For the analysis of the effect of Crm1 siRNA on cell viability, 2.0×103 cells were seeded into each well of a 96-well plate and transfected with 20 nM siRNA on the following day, whereas for treatment with LMB, 1.0×104 cells were seeded into each well of a 96-well plate and treated with varying concentrations of LMB on the following day. Cell proliferation was measured five days after siRNA transfection and two days after LMB treatment by the addition of MTT reagent (Sigma), and crystals were subsequently solubilized using solubilization buffer. Absorbance was measured at an OD of 595 nM using a microplate reader.
For analysis of the effect of Crm1 inhibition on the cell cycle, 1.8×105 KYSE30 and WHCO5 cells were plated on 60-mm dishes and transfected with 20 nM control or Crm1 siRNA for 72 h. Subsequently, cells were harvested and fixed in 95% ethanol, after which the cells were stained with propidium iodide and the cell cycle profiles were analyzed using the BD FACSVerse™ flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Quantification of the percentage of cells at different stages of the cell cycle was performed using Modfit 3.3 software.
To assay for caspase-3/7 activity, the Caspase-Glo™ 3/7 assay (Promega) was performed, according to the manufacturer’s instructions. Briefly, 2.0×103 cells were plated/well in 96-well plates and transfected with 20 nM control or Crm1 siRNA. Caspase-3/7 activity was measured 48 h after siRNA transfection, and luminescence was monitored using the Veritas™ microplate luminometer (Promega). MTT assays were performed concurrently and caspase-3/7 luminescence values were normalised to OD595 values to control for any differences in cell number.
The statistical significance of the means was calculated using the Student’s t-test. Fisher’s exact tests were used to compare the expression of Crm1 in the different groups. Survival analysis was carried out using the Kaplan-Meier method. A p-value of <0.05 was required for statistical significance.
Altered Crm1 expression has been described in certain cancer types. In the present study we investigated Crm1 expression in oesophageal squamous cell carcinoma (OSCC). Crm1 expression was investigated in formalin-fixed tissue sections of matched tumour and normal stratified squamous epithelium, obtained from 56 South African patients with oesophageal cancer. The basic patient demographics and pathological characteristics are documented in
Immunohistochemical analysis, followed by independent scoring by a pathologist, revealed that most normal sections had weak nuclear staining for Crm1, with no observable cytoplasmic staining (
It was next determined whether any correlation exists between the expression of Crm1 and tumour stage. Correlation analyses were performed and demonstrated that the expression of Crm1 (where nuclear, cytoplasmic and nuclear membrane staining patterns were taken into account) did not significantly associate with tumour stage (
Since Crm1 levels were found to be significantly elevated in oesophageal tumour tissues in both the cytoplasmic and nuclear cellular components, a Kaplan-Meier survival analysis was performed to determine whether Crm1 expression predicts patient survival. A trend, where high cytoplasmic Crm1 expression appeared to be associated with poor overall survival, was observed (data not shown). There was no correlation between nuclear Crm1 expression and overall survival. These findings were likely influenced by the limited data and tissue specimens available for early stage oesophageal cancer patients. Access to early stage patient material may have a significant effect on the interpretation of overall survival analyses.
As an increase in Crm1 protein expression was observed in oesophageal cancer patient specimens, it was next investigated whether Crm1 was similarly upregulated at the mRNA level. Crm1 mRNA levels were thus examined by quantitative real-time RT-PCR using RNA obtained from matched normal and cancer tissue biopsies obtained from 22 patients with OSCC. A significant increase in Crm1 mRNA expression was observed in the oesophageal tumour specimens compared to the normal tissues (
As elevated Crm1 expression was observed in oesophageal cancer tissues compared to that in the normal tissues, Crm1 expresssion was next evaluated in 9 oesophageal cancer cell lines (from South African and Japanese origin) and compared to the expression in hTERT-immortalised EPC2 cells derived from normal oesophageal epithelium. Western blot analysis revealed that Crm1 protein expression was high in all the oesophageal cancer cell lines (WHCO1, WHCO5, WHCO6, KYSE30, KYSE70, KYSE150, KYSE180, KYSE420, KYSE450), while lowest expression was observed in the normal epithelial cell line, EPC2 (
To evaluate the functional significance of elevated Crm1 expression in oesophageal cancer, Crm1 siRNA was used to inhibit its expression. A control siRNA with no known silencing effect was used to control for the non-specific effects of siRNA transfection. WHCO5 and KYSE30 oesophageal cancer cells and EPC2 normal oesophageal cells were transfected with 20 nM siRNA, and protein knockdown was confirmed by western blot analysis (
To corroborate the effect of Crm1 siRNA on oesophageal cancer cell proliferation, the commercially available Crm1 inhibitor, leptomycin B (LMB), was used. Cells were treated with varying concentrations of LMB, and cell proliferation was determined 48 h later. In line with the siRNA data, WHCO5 and KYSE30 cancer cells were significantly more sensitive to LMB treatment (70–80% decrease in cell number at 100 nM LMB) when compared to the normal EPC2 cells, which showed little cytotoxicity, even at high LMB concentrations (30% decrease in cell number at 100 nM LMB) (
These results suggest that Crm1 expression is upregulated in oesophageal cancer cell lines and that these cells are highly dependent on its expression for cell survival and proliferation, whereas non-cancer oesophageal cells appear less reliant on its expression.
Since Crm1 inhibition reduced oesophageal cancer cell proliferation, we next investigated whether Crm1 inhibition associated with alterations in cell cycle distribution. Crm1 expression was inhibited in KYSE30 oesophageal cancer cells transfected with siRNA for 72 h. FACS analysis revealed that the inhibition of Crm1 resulted in a significant increase in cell in G1, associated with a significant decrease in the number of cells in the S and G2/M phases of the cell cycle (
This study investigated the expression of the nuclear transport receptor, Crm1, in oesophageal cancer. Crm1 showed weak nuclear expression patterns in normal oesophageal epithelium, while its expression was much stronger and localised to the nucleus, cytoplasm and nuclear envelope in oesophageal tumour tissues. Real-time RT-PCR analysis revealed that Crm1 was also elevated at the mRNA level. Western blot analysis confirmed the elevated expression of Crm1 in oesophageal cancer cells compared to normal oesophageal epithelial cells. Crm1 has recently been reported to be expressed at elevated levels in cervical squamous cell carcinoma, ovarian carinoma, osteosarcoma, glioma, pancreatic cancer, gastric cancer and multiple myeloma (
It is likely that the elevated levels of Crm1 in tumour tissues are derived from its cell cycle-dependent regulation. Evidence suggests that Crm1 is a cell cycle-regulated gene, as its mRNA levels have been reported to oscillate with the cell cycle, where Crm1 expression is initiated in late G1 and reaches a peak at G2/M (
We report here that the nuclear, cytoplasmic and nuclear membrane expression of Crm1 is increased in oesophageal tumours. This localisation pattern is in line with Noske
While the prognostic value of elevated Crm1 in oesophageal cancer has not yet been determined conclusively, our findings suggest that it may have value as an oesophageal cancer therapeutic target. Our results demonstrate a role for Crm1 in the biology of oesophageal cancer as its inhibition resulted in G1 cell cycle arrest and the induction of apoptosis. We similarly found that treatment with the Crm1 inhibitor, leptomycin B, resulted in oesophageal cancer cell death. Interestingly, normal oesophageal epithelial cells were less sensitive to Crm1 inhibition using siRNA and LMB, suggesting that targeting Crm1 could be an attractive therapy for oesophageal cancer. Other studies have similarly reported an increased sensitivity of cancer cells to Crm1 inhibition compared to their normal counterparts (
Overall, we demonstrated that oesophageal cancer cells contain elevated levels of Crm1 protein and are highly reliant on its expression, as its inhibition results in cancer cell death, while in normal cells its inhibition has only a minor effect. Based on this, we propose that Crm1 has potential as a diagnostic marker and/or therapeutic target for patients with oesophageal squamous cell carcinoma. Further study however is required in order to clarify its true clinical significance.
We thank Ms. Antionette Olivier for providing patient information. This study was supported by grants from the Cancer Association of South Africa (CANSA), the University of Cape Town and the Carnegie Corporation of New York.
Immunohistochemical analysis of Crm1 expression in normal and tumour tissue sections from patients with oesophageal squamous cell carcinoma. (A–C) Crm1 staining patterns in the different tumour stages are shown. Little immunoreaction of Crm1 was observed in normal oesophageal epithelium. In contrast, intense nuclear and cytoplasmic expression of Crm1 was observed in the tumour tissue (magnification, ×40). E, epithelium; S, stroma. (D–F) Elevated Crm1 expression in the tumour tissue compared to the normal tissues was observed in the nuclear (D), cytoplasmic (E) and nuclear membrane (F) cellular compartments. (G) Crm1 mRNA levels in matched normal and tumour tissues obtained from 22 patients with OSCC (*p<0.05). Results are shown as the mean ± SEM. Crm1, the chromosome region maintenance 1 protein or exportin 1.
Crm1 expression is elevated in cultured oesophageal cancer cell lines and is necessary for cancer cell survival. (A) Western blot analysis showing Crm1 protein levels in normal immortalised oesophageal epithelial EPC2 cells and in oesophageal cancer cell lines. β-tubulin was used as a loading control. (B) Immunofluorescence showing Crm1 localisation in normal EPC2 cells and representative cancer cells, WHCO5 and KYSE30. DAPI staining shows the cell nuclei. (C and D) Quantitation of Crm1 fluorescence intensity in the nucleus (C) and cytoplasm (D) of approximately 40 cells stained with the Crm1 antibody reveals a significant increase in Crm1 expression in the cancer cell lines compared to normal EPC2 cells (*p<0.05). (E) Western blot analysis showing a reduction in Crm1 protein levels after transfection of WHCO5, KYSE30 and EPC2 cell lines with Crm1 siRNA, compared to control (ctl) siRNA. (F) MTT assay showing a more pronounced decrease in proliferation of WHCO5 and KYSE30 oesophageal cancer cells after Crm1 knockdown, compared to non-cancer EPC2 cells (*p<0.05); (G) Cell viability curves showing the response of WHCO5, KYSE30 and EPC2 cells to varying concentrations of the Crm1 inhibitor, leptomycin B. Experiments were performed in triplicate and repeated at least two times. Results are shown as the mean ± SEM. Crm1, the chromosome region maintenance 1 protein or exportin 1.
Inhibition of Crm1 expression in oesophageal cancer cells results in cell cycle arrest and the induction of apoptosis. (A) Effect of Crm1 siRNA on cell cycle progression. KYSE30 cells were transfected with 20 nM control siRNA or Crm1 siRNA and harvested 72 h later for cell cycle analysis by flow cytometry. Crm1-knockdown cells exhibited G1 cell cycle arrest and the induction of the subG1 population. Similar results were found using the WHCO5 cell line (*p<0.05). (B) Caspase-3/7 activity was measured 48 h after transfection of cells with 20 nM control or Crm1 siRNA. A significant increase in caspase-3/7 activity was observed after Crm1 knockdown in both KYSE30 and WHCO5 cell lines. Experiments were performed in triplicate and repeated at least two times (*p<0.05). Results are shown as the mean ± SEM.
Patient demographics.
Characteristics | No. of patients (%) |
---|---|
Gender | |
Male | 39 (70) |
Female | 17 (30) |
Age (years) | |
Average | 53.4 |
Range | 32–80 |
Race | |
Black | 18 (32) |
Mixed ancestry | 32 (57) |
Caucasian | 6 (11) |
Smoker | |
Yes | 48 (86) |
No | 8 (14) |
Alcohol intake | |
Heavy | 17 (30) |
Light | 15 (27) |
No | 18 (32) |
Unknown | 6 (11) |
Stage | |
I | 2 (4) |
II | 31 (55) |
III | 23 (41) |
Differentiation status | |
Well | 1 (2) |
Moderate | 40 (71) |
Poor | 13 (23) |
Unknown | 2 (4) |
Presence of keratinisation | |
Keratinisation | 32 (57) |
No keratinisation | 24 (43) |
Expression of Crm1 in normal and cancer tissue of the oesophagus.
No. of patients (%) | |||
---|---|---|---|
|
|||
Crm1 expression | Normal | Cancer | P-value |
Nuclear staining | |||
Negative | 8 (14) | 3 (5) | |
Weak | 48 (86) | 8 (14) | <0.0001 |
Strong | 0 (0) | 45 (80) | |
Cytoplasmic staining | |||
Negative | 55 (98) | 2 (4) | |
Weak | 1 (2) | 26 (46) | <0.0001 |
Strong | 0 (0) | 28 (50) | |
Nuclear membrane staining | |||
Negative | 55 (98) | 10 (18) | |
Weak | 1 (2) | 41 (73) | <0.0001 |
Strong | 0 (0) | 5 (9) |
Fisher’s exact test.
Relationship between Crm1 expression and tumour stage.
No. of patients (%) | ||||
---|---|---|---|---|
|
||||
Crm1 expression | Stage I | Stage II | Stage III | P-value |
Nuclear expression | ||||
Negative | 0 (0) | 3 (10) | 0 (0) | |
Weak | 1 (50) | 4 (13) | 4 (17) | 0.200 |
Strong | 1 (50) | 24 (77) | 19 (83) | |
Cytoplasmic expression | ||||
Negative | 0 (0) | 2 (6) | 0 (0) | |
Weak | 2 (100) | 13 (42) | 11 (48) | 0.437 |
Strong | 0 (0) | 16 (52) | 12 (52) | |
Nuclear membrane expression | ||||
Negative | 0 (0) | 7 (23) | 3 (13) | |
Weak | 2 (100) | 22 (71) | 18 (78) | 0.679 |
Strong | 0 (0) | 2 (6) | 2 (9) | |
Overall expression | ||||
Weak | 1 (50) | 6 (19) | 2 (9) | |
Moderate | 1 (50) | 15 (48) | 11 (48) | 0.184 |
Strong | 0 (0) | 10 (32) | 10 (43) |
Fisher’s exact test.