The Karyopherin proteins, Crm1 and Karyopherin beta1, are overexpressed in cervical cancer and are critical for cancer cell survival and proliferation

Abstract

The Karyopherin proteins are involved in nucleo-cytoplasmic trafficking and are critical for protein and RNA subcellular localization. Recent studies suggest they are important in nuclear envelope component assembly, mitosis and replication. Since these are all critical cellular functions, alterations in the expression of the Karyopherins may have an impact on the biology of cancer cells. In this study, we examined the expression of the Karyopherins, Crm1, Karyopherin beta1 (Kpnbeta1) and Karyopherin alpha2 (Kpnalpha2), in cervical tissue and cell lines. The functional significance of these proteins to cancer cells was investigated using individual siRNAs to inhibit their expression. Microarrays, quantitative RT-PCR and immunofluorescence revealed significantly higher expression of Crm1, Kpnbeta1 and Kpnalpha2 in cervical cancer compared to normal tissue. Expression levels were similarly elevated in cervical cancer cell lines compared to normal cells, and in transformed epithelial and fibroblast cells. Inhibition of Crm1 and Kpnbeta1 in cancer cells significantly reduced cell proliferation, while Kpnalpha2 inhibition had no effect. Noncancer cells were unaffected by the inhibition of Crm1 and Kpnbeta1. The reduction in proliferation of cancer cells was associated with an increase in a subG1 population by cell cycle analysis and Caspase-3/7 assays revealed increased apoptosis. Crm1 and Kpnbeta1 siRNA-induced apoptosis was accompanied by an increase in the levels of growth inhibitory proteins, p53, p27, p21 and p18. Our results demonstrate that Crm1, Kpnbeta1 and Kpnalpha2 are overexpressed in cervical cancer and that inhibiting the expression of Crm1 and Kpnbeta1, not Kpnalpha2, induces cancer cell death, making Crm1 and Kpnbeta1 promising candidates as both biomarkers and potential anticancer therapeutic targets.

Figures

Figure 1 –

Expression of Crm1, Kpnβ1 and Kpnα2 in normal and cervical cancer tissues. (a) Gene expression of crm1, kpnβ1 and kpna2 in normal and cervical cancer biopsies, analyzed by microarray technologies [normal (n = 8), cancer (n = 16), *p < 0.0005]. (b) Real-time RT-PCR analysis confirming upregulation of crm1, kpnβ1 and kpna2 in cervical cancer biopsies compared to normal [normal (n = 9), cancer (n = 13), *p < 0.0005]. Results shown are the mean ± SE. (c–e) Quantification of Crm1 (c), Kpnβ1 (d) and Kpnα2 (e) immunofluorescence in 10 normal and sixteen cervical cancer tissue sections. Fluorescence was quantified using AxioVision, Release 4.5 software. A total of 6 views per slide were used to calculate the average fluorescence intensity per sample. Box-and-whisker plots comparing Crm1 (c), Kpnβ1 (d) and Kpnα2 (e) expression in normal and cancer tissue sections were generated using Graphpad Prism (*p < 0.05). (f) Representative fluorescence images showing hematoxylin and eosin (H&E) staining, Crm1, Kpnβ1 and Kpnα2 expression, and nuclear staining (DAPI) in normal (GSH7) and cervical cancer (GSH28) sections. All images were collected at identical exposure settings and magnification (E: epithelium; S: stroma). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 2 –

Expression of Crm1, Kpnβ1 and Kpnα2 in cells grown in culture. (a) Western blot analyses of Crm1, Kpnβ1 and Kpnα2 expression in normal primary cervical epithelial cells (HCX), HPV E6/E7-transformed cells (HCX-E6/E7) and cervical cancer cell lines HeLa, SiHa, CaSki, ME180, Ms751 and C33A. β-tubulin was used as a control for protein loading. Results shown are representative of Western blots performed in triplicate. (b) Western blot analyses of Crm1, Kpnβ1 and Kpnα2 expression in normal lung fibroblasts, WI38, compared to transformed fibroblasts, SVWI38 and CT1.

Figure 3 –

Effect of Leptomycin B (LMB) treatment on cell viability. (a) CaSki cervical cancer cells, HCX-E6/E7-transformed cervical cells and HCX primary normal cervical cells were treated with LMB at increasing nanomolar concentrations and cell viability assayed 48 hr after treatment using MTT reagent. Results shown are the percentage of viable cells after LMB treatment relative to the untreated controls. Experiments were performed in quadruplicate and repeated at least 2 times. (b) CaSki cell proliferation in the absence and presence of 10 nM LMB. Cell proliferation was measured using MTT after a 48-hr incubation in the absence or presence of LMB. Cell proliferation in LMB-treated cells is expressed relative to that in untreated cells (*p < 0.005). (c) Effect of LMB on cell cycle progression. CaSki cells were left untreated or treated with 10 nM LMB for 48 hr, and harvested for cell cycle analysis using flow cytometry. LMB-treated cells showed an increased subG1 population. Experiments were performed in triplicate. (d) Quantitation of the cell cycle data showing the significant increase in the subG1 cell population of cells treated with LMB (*p < 0.01). (e) Caspase-3/7 activity in LMB-treated cells. Cells were treated with 10 nM LMB for 24 hr and Caspase-3/7 activity analyzed as a measure of apoptosis (*p < 0.001).

Figure 4 –

Effect of inhibiting Crm1, Kpnβ1 and Kpnα2 expression on CaSki cell proliferation using siRNAs. (a–c) Time-course analyses of Crm1 (a), Kpnβ1 (b) and Kpnα2 (c) inhibition after transfection with siRNA. Protein was harvested at the indicated time points after transfection. Western blot analysis using antibodies specific to Crm1, Kpnβ1 and Kpnα2 was used to analyze inhibition with individual siRNAs. β-tubulin was used as a control for protein loading. (d–f) The effect of Crm1 (d), Kpnβ1 (e) and Kpnα2 (f) siRNA on cell proliferation was determined using the MTT assay. CaSki cervical cancer cells were transfected with 20 nM control siRNA or respective siRNA, and cell growth was monitored using the MTT reagent. Results shown are the mean ± SD of experiments performed in quadruplicate and repeated at least 3 times. Crm1 and Kpnβ1 siRNA resulted in a significant inhibition of cell proliferation (p < 0.00001 at 96 hr).

Figure 5 –

Effect of Crm1, Kpnβ1 and Kpnα2 inhibition on cervical cancer cell proliferation. (a–e) Cells were transfected with siRNA as described in Figure 4 and proliferation monitored 120 hr post-transfection. Proliferation is shown for each cell line relative to that of cells transfected with control siRNA. Cervical cancer cell lines, CaSki (a), HeLa (b), SiHa (c), MS751 (d) and C33A (e), showed similar sensitivities to Crm1 and Kpnβ1 inhibition, and were unaffected by Kpnα2 inhibition. Results shown are the mean ± SD of experiments performed in quadruplicate and repeated at least 2 times (*p < 0.05).

Figure 6 –

Effect of Crm1, Kpnβ1 and Kpnα2 inhibition on the proliferation of normal fibroblasts, WI38, transformed fibroblasts, SVWI38, and CaSki cervical cancer cells. (a) Cells were transfected with siRNA against Crm1 and Kpnβ1 and viable cells assayed using MTT reagent 120 hr post-transfection. Proliferation is shown for each cell line relative to that of cells transfected with the control siRNA. Results shown are the mean ± SD of experiments performed in quadruplicate and repeated at least 2 times. (b) Western blot analysis matched to proliferation assays in (a) showing Crm1 and Kpnβ1 knock-down after transfection with individual siRNAs. (c) Proliferation of normal cells, CCD-1068SK, FG0 and EPC2, 120 hr after Crm1 and Kpnβ1 inhibition. Results shown are the mean ± SD of experiments performed in quadruplicate and repeated at least 2 times (*p < 0.05).

Figure 7 –

Crm1 and Kpnβ1 inhibition induces apoptosis in CaSki cells. (a) Effect of Crm1 siRNA on cell cycle progression. CaSki cells were transfected with 20 nM control siRNA or Crm1 siRNA and harvested 96 hr later, stained with propidium iodide and respective cell cycle profiles analyzed using flow cytometry. Crm1 knock-down cells showed an increase in a subG1 population. Experiments were performed in triplicate and repeated at least 2 times. (b) Quantitation of the cell cycle data obtained from cells transfected with control, Crm1 or Kpnβ1 siRNA, showing the significant increase in the subG1 cell population of cells transected with Crm1 and Kpnβ1 siRNA (*p < 0.001) and a minimal but significant decrease in G1 (p < 0.005). (c) Caspase-3/7 activity (y-axis) for CaSki cells transfected with 20 nM control, Crm1, Kpnβ1 or Kpnα2 siRNA was measured 72 and 96 hr post-transfection (x-axis). Crm1 and Kpnβ1 inhibition caused a significant increase in Caspase-3/7 activity (*p < 0.01); however, Kpnα2 inhibition caused no change in Caspase-3/7 activity compared to control siRNA-transfected cells. Results shown are the mean ± SD of experiments performed in quadruplicate and repeated at least 2 times.

Figure 8 –

The inhibition of Crm1 and Kpnβ1 results in increased levels of p53, p21, p27 and p18. (a) CaSki cells were transfected with 20 nM control siRNA (1, 3, 5) or Crm1 siRNA (2, 4, 6) and fixed 120 hr post-transfection. Immunofluorescence analysis of Crm1 knock-down (1 vs. 2) and p53 (3 vs. 4) shows elevated nuclear p53 staining (4: marked with arrows) in cells with low Crm1 levels. Results shown are representative of experiments performed in 2 independent analyses. DAPI staining for nuclei are shown (5 and 6). (b) Western blot analysis showing p53, p21, p27, p18 and p16 levels after Crm1, Kpnβ1 and Kpnα2 knock-down. Knock-down of Crm1 and Kpnβ1 resulted in elevated p53, p21, p27 and p18 levels, while p16 remained relatively unchanged. (c) Western blot analysis showing levels of cell cycle inhibitors after Crm1, Kpnβ1 and Kpnα2 inhibition in noncancer WI38 cells. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]