Abstract
Minibrain-related kinase (Mirk) is a member of the dual specificity tyrosine-phosphorylation-regulated kinase (Dyrk)/minibrain family of dual-specificity protein kinases and is identical to Dyrk1B. Mirk/Dyrk1B is a serine/threonine kinase that has been found to be upregulated in solid tumors and mediates cell survival in colon cancer, pancreatic ductal adenocarcinoma and rhabdomyosarcomas. There is little known about Mirk in lung cancer. In the present study, we showed that Mirk protein was widely overexpressed in 13 of 19 NSCLC cell lines. Mirk immunoprecipitation coupled with anti-phosphotyrosine western blotting confirmed tyrosine phosphorylation of Mirk in NSCLC cells. Mirk knockdown by small interfering RNA induced cell apoptosis concomitant with upregulation of Bak, a Bcl-2 family member, and downregulation of signal transducers and activators of transcription 3 (STAT3) tyrosine phosphorylation. Mirk knockdown led to decreased cell colony formation in vitro as well as delayed tumor growth in an orthotopic mouse model and sensitized cells to cisplatin-induced apoptosis. Using automated quantitative determination of the Mirk protein in tumor specimens of patients with early-stage lung cancer, overexpression of Mirk was found in nearly 90% of tumor specimens in both the cytoplasm and nucleus. These results suggest that Mirk is overexpressed in lung cancer, acts as a survival factor in lung cancer cells and may be a novel therapeutic target.
Keywords: Mirk/Dyrk1B, STAT3, Bcl-2, siRNA, apoptosis, lung cancer
Introduction
Mirk, also known as Dyrk1B, was cloned from a colon carcinoma cell line1 and from human and murine testes2 in the late 1990s by two separate groups. Mirk/Dyrk1B is a member of the dual-specificity tyrosine-regulated kinase (Dyrk)/minibrain family of dual-specificity protein kinases.3 Dyrk kinases have the ability to autophosphorylate themselves on tyrosine during their translation, and then can phosphorylate other substrates on serine and threonine;4 therefore, they are categorized as dual function kinases. Mirk/Dyrk1B exhibits restricted mRNA in normal tissue, with low levels of expression in normal colon and normal lung tissues; levels of expression in skeletal muscle, testes, heart and brain, however, are high.1 Thus, to date, most of the studies of Mirk have been conducted using myogenesis as a model system in which Mirk has been demonstrated to play a critical role in muscle differentiation by regulatory effects on motility, transcription, cell cycle progression and cell survival.5,6 Emerging data from tumor biology studies have indicated that Mirk is expressed in solid tumors and that it mediates tumor cell survival in several different types of human cancer, including colon cancer cells,1 pancreatic ductal adenocarcinoma cells,7 rhabdomyosarcoma cells8 and HeLa cervical carcinoma cells.9 Knockdown of Mirk does not cause embryonic lethality, indicating that Mirk is not essential for normal cell growth and may be a novel therapeutic target7.
Lung cancer is the leading cause of cancer death in both women and men in the United States, with an estimated 215,020 new cases and 161,840 deaths attributed to lung cancer in 2008.10 Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, making up nearly 80% of all cases. The distinct histologies of lung cancer usually require multiple signaling pathways, including phos-phoinositide 3-kinase (PI3K)/Akt and Janus kinase/signal transducer and activator of transcription 3 (STAT3) for growth and survival, and they exhibit generalized resistance to apoptosis induced by chemotherapeutics.11-14 Recent studies have shown that a subset of patients with NSCLC have tumors driven by genomic alterations in the epidermal growth factor receptor (EGFR) resulting in sensitivity to small-molecule inhibitors of the tyrosine kinase domain, such as gefitinib and erlotinib. However, because only 10% of patients with lung cancer in North America and Western Europe exhibit an EGFR mutation, use of these inhibitors has been limited.15 In addition, constitutive activation of the serine/threonine kinase Akt has been identified in 90% of NSCLC cells lines, and it has been demonstrated that activated Akt promotes cell survival.16 Also, STAT3 has been found to be a critical mediator of the oncogenetic effects in NSCLC.11,17,18 This inherent antiapoptotic property of lung cancer could be ascribed in part to the activation of various survival signals, suggesting that additional antiapoptotic pathways must function in lung cancer cells; therefore, targeting inhibition of multiple pathways may be more effective than targeting a single pathway.
Although Mirk expression has been found to be very low in normal lung tissue,1 little is known about the function of Mirk in human lung cancers and the mechanisms involved. In this study, we found that Mirk is overexpressed in a wide spectrum of NSCLC cell lines and human lung cancer specimens. Knockdown of Mirk led to NSCLC cell apoptosis, which is associated with increased protein levels of the Bcl-2 family member Bak and decreased activation of STAT3 tyrosine phosphorylation.
Results
Mirk is overexpressed in a wide spectrum of NSCLC cells and is enhanced by serum starvation
As part of an ongoing phospho-proteomic screen using anti-phosphotyrosine antibodies coupled with liquid chromatography and mass spectrometry, we identified a IYQyIQSR peptide corresponding to Dyrk1 where y indicates tyrosine phosphorylation.19 Similar results have been previously published in lung cancer cell lines and tumors.20 To examine this in more detail, we evaluated protein expression of Mirk in 19 human NSCLC cell lines (Fig. 1A). We observed high levels of Mirk protein in 13 cell lines. With prolonged exposure of the film, we only observed detectable levels of Mirk proteins in HCC827, PC-9, H441, H460, H157 and H322 cell lines. To confirm that Mirk is tyrosine phosphoryalted and therefore activated in these cells, we examined tyrosine phosphorylation of Mirk. Cell protein extracts from H292, H358, A549 and H1299 cells were immunoprecipitated with Mirk antibody and immunoblotted by pY 100 and Mirk antibodies. The corresponding Mirk pY bands were found in all of the four cell lines (Fig. 1B). As a control, there was no obvious band in immunoprecipitates prepared with IgG (Fig. 1B). There seemed to be positive correlation between the expression of Mirk protein and the pY abundance of Mirk in NSCLC cells (Fig. 1A and B). We also examined if serum conditions could affect Mirk expression based on studies linking it to survival. As shown in Figure 1C, Mirk protein levels were upregulated in cells cultured in the medium with 0% FBS compared with that with 10% FBS, suggesting that the overexpressed Mirk may be required to mediate NSCLC cell survival.
Figure 1.
Mirk is overexpressed in a wide variety of NSCLC cell lines. (A) Mirk expression was determined in 19 human NSCLC cell lines by western blot analyses. Equal loading and transfer were shown by repeat probing with β-actin. (B) cell protein extracts from H292, H358, A549 and H1299 cells were immunoprecipitated with Mirk antibody and normal rabbit IgG and immunoblotted by pY 100 and Mirk antibodies. (C) Mirk expression was determined in cells after ~24–48 h of serum starvation by western blot analyses. Equal loading and transfer were shown by repeat probing with β-actin.
Knockdown of Mirk inhibits cell growth and induces apoptosis in vitro
We next examined the consequence of Mirk knockdown using a pool of four individual siRNA targeting Mirk. We found concentration-and target-dependent effects on Mirk protein and apoptosis occurred in A549 cells induced by Mirk siRNA pool (~5–20 nM) (Fig. 2A, left) and the corresponding individual siRNAs (20 nM) (Fig. 2A, right), of which the 20 nM siRNA pool was used for further characterization. To further explore the consequence of overexpressed Mirk in various NSCLC cells, we exposed NSCLC cell lines to Mirk siRNA for 120 h followed by assessment of cell growth. As shown in Figure 2B, cell growth in all 19 cell lines was inhibited (~40.9–94.3% of control) by Mirk siRNA treatment compared to cells treated with non-targeting siRNA. We next determined whether changes in cell growth reflect changes in cell cycle progression and/or apoptosis. NSCLC cells treated with Mirk siRNA for 72 h demonstrated no obvious inhibition of the cell cycle profile compared to control siRNA-treated cells (data not shown). However, in these 19 cell lines, Mirk knockdown by siRNA resulted in cellular apoptosis (~1.3- to 3.6-fold of control), as evidenced by more Mirk siRNA-treated cells staining with cleaved caspase-3 (Fig. 2C). These results indicate that apoptosis contributes to the inhibitory effects of Mirk siRNA treatment in NSCLC cells.
Figure 2.
Mirk functions to drive cell survival in NSCLC cells. (A) A549 cells were treated with Mirk siRNA pool in dose gradient (left) and the corresponding individual Mirk siRNAs #1-#4 (right) for 72 h, and the effects on Mirk and PARP cleavage were analyzed by western blot. Equal loading and transfer were shown by repeat probing with β-actin. The cell apoptosis (fold of control), evidenced by positive cells of active caspase-3 in the cells, was assessed by flow cytometry analysis. (B) cells were exposed to siRNAs (20 nM) for 120 h, and cell growth were measured by MTT assay. *p < 0.05; **p < 0.001 compared with control. (C) cells were exposed to siRNAs (20 nM) for 72 h, and percentage of cell apoptosis evidenced by positive cells of active caspase-3 in the cells was assessed by flow cytometry analysis. *p < 0.05; **p < 0.001 compared with control. (D) cells were exposed to siRNAs (20 nM) for 72 h followed by measurement of PARP cleavage, Bcl-2 family members, and phosphorylated/total STAT3 proteins by western blot analysis. Equal loading and transfer were shown by repeat probing with β-actin.
Mirk knockdown affects Bak and activated STAT3
It has been reported that both STAT3 and the BH3-interacting domain death agonist Bid may contain a canonical Mirk phosphorylation site.7,21 To investigate the mechanisms involved in apoptosis induced by Mirk siRNA, the Bcl-2 family members and STAT3 phosphorylation status were determined in a panel of six NSCLC cell lines by western blot analysis. Of six cell lines, both H1650 and H1975 cells harbored activating EGFR mutations.12 As shown in Figure 2D, exposure of these cell lines to Mirk siRNA for 72 h was associated with knockdown of Mirk and PARP cleavage, compared with that shown with control siRNA, and resulted in upregulation of pro-apoptotic Bak in all cell lines but no alterations of Bcl-2, Bax and Bid. Activated STAT3 at both Tyr705 and Ser727 sites are crucial for NSCLC cell survival. In this study, we tried to link Mirk to the both sites and found that knockdown of Mirk by siRNA induced downregulation of phosphorylated Tyr705 but not Ser727 of STAT3 in H292, H358, H1650 and H1975 cells; no changes in phosphorylation of either site was observed in A549 and H1299 cells. There was no change in total STAT3 by siRNA in all cell lines (Fig. 2D). It is interesting to note that the lack of changes in STAT3 phosphorylation induced by Mirk knockdown in A549 and H1299 cells parallel the less obvious apoptosis in these cells. This could suggest that STAT3 may be a predominant downstream signaling pathway of Mirk for NSCLC cell survival. Overall, these results suggest that Mirk mediates NSCLC cell survival through alterations in Bak and STAT3 signaling.
Knockdown of Mirk results in decreased colony formation in vitro and delayed tumor growth in an orthotopic mouse model
We further determined the effects of Mirk knock-down using colony formation assay in vitro. As shown in Figure 3A, Mirk siRNA-treated A549 cells demonstrated nearly 55% reduction in colony formation versus control siRNA treated cells. Despite the transient transfection, Mirk expression was reduced 7–10 d following the 72 h siRNA treatment (Fig. 3B).
Figure 3.
Mirk knockdown inhibits cell colony formation in vitro and orthotopic tumor growth in CD-1 nude mice. (A) A549 cells treated with siRNAs (20 nM) were grown in RPMI 1640 medium supplied with 10% FBS and colonies were counted after 14 d. *p < 0.05 compared with control. (B) A549 cells treated with siRNAs (20 nM) for 72 h were split and grown, and Mirk protein expression was determined at indicated time points by western blot analysis. Equal loading and transfer were shown by repeat probing with β-actin. (C) orthotopic tumor growth was evaluated by Xenogen 200 imaging system. (D) Seven mice in each group were euthanized 7 w after tumor injection, when control mice became moribund. §, weight designates lung and tumor weight; #, number of positive mice/number of treated mice; *p < 0.05 compared with control siRNA group.
To examine this effect on in vivo tumor growth, A549 cells stably expressing luciferase were exposed to control siRNA or Mirk siRNA for 72 h and the same amount of viable cells were injected directly into mouse lungs to generate orthotopic mouse lung cancer models. We employed Xenogen Imaging System 200 to monitor and measure tumor growth in the orthotopic mouse models based on bioluminescence output detected every other week. Results from 3 w after injection demonstrated differences between control and Mirk knockdown groups (data not shown). After 7 w of injection when control mice became moribund, orthotopic tumor growth measured in the Mirk knockdown group was shown in Figure 3C and significantly delayed compared with the control group. Analysis of lung weight was performed as a surrogate of tumor mass, and in agreement with the bioluminescence data, we found significantly decreased lung weight in Mirk siRNA-treated cells versus controls (Fig. 3D). Together, our results in vitro and in an orthotopic mouse model demonstrated Mirk functions in NSCLC cell survival.
Knockdown of Mirk sensitizes NSCLC cells to cisplatin-induced apoptosis
To investigate the effects of constitutively overexpressed Mirk on sensitivity of NSCLC cells to conventional chemotherapeutics, Mirk siRNA-treated A549, H1299 and H1650 cells were exposed to increasing doses of cisplatin for cell viability and apoptosis assays. As shown in Figure 4A–C, Mirk siRNA-treated A549, H1299 and H1650 cells exposed to cisplatin showed decreased cell growth and IC50 of cisplatin versus that shown in control siRNA-treated cells. Similarly, Mirk siRNA treatment and exposure to cisplatin in these cells resulted in increased apoptosis (measured in fold) compared with cells treated with control siRNA by caspase-3 assay (Fig. 4D) as well as by Annexin V assay (data not shown), indicating that knockdown of Mirk sensitizes NSCLC cells to chemotherapy-induced apoptosis.
Figure 4.
Mirk knockdown sensitizes NSCLC cells to cisplatin-induced apoptosis. A549 (A), H1299 (B) and H1650 (C) cells were treated with siRNAs (20 nM) for 72 h, collected, and plated in 96-well plates at 8,000 cells per well; cells were allowed to grow for ~18–24 h before 48-h exposure to cisplatin in dose gradient. Percentage of cell growth treated with siRNA alone was qualified by MTT assay. IC50 of cisplatin was calculated using Matlab software. □, Control siRNA + CDDP; □, MUC1 siRNA + CDDP. (D) cells treated with siRNAs (20 nM) for 72 h were collected and plated in 6-well plates and allowed to grow for ~18–24 h before 48 h exposure to 10 μM cisplatin. The cell apoptosis (fold of samples by siRNA alone), evidenced by positive cells of active caspase-3 in the cells, was assessed by flow cytometry analysis. *p < 0.05; **p < 0.001 compared with control.
Mirk is overexpressed in tumor specimens from clinical NSCLC cases
We examined expression patterns of Mirk in early stage NSCLC. Table 1 summarizes pertinent clinical information. Using AQUA to quantify Mirk expression, we assessed Mirk expression in NSCLC tumor specimens from 187 patients. In all tumor specimens, Mirk was located in both the nucleus and cytoplasm (Fig. 5A–D). We further found that levels of Mirk expression in the cytoplasm and nucleus were highly correlated (r = 0.92, p = 0.0001). Importantly, Mirk levels were higher in tumors compared to corresponding adjacent tissues from 36 tumor specimens (Table 1). In tumor specimens, the AQUA scores ranged from 78 to 1761 (median, 659; mean, 679) in the nucleus and from 76 to 2010. (median, 573; mean, 596) in the cytoplasm (Table 1). In tumor-adjacent tissues, Mirk was also detected in both the nucleus and cytoplasm. AQUA scores ranged from 141 to 682 (median, 307; mean, 327) in the nucleus and from 115 to 477 (median, 259; mean, 278) in the cytoplasm (Table 1). A previous study demonstrated that Mirk was weakly expressed in normal lung tissue.1 Based on the AQUA scores, in this study we identified that Mirk expression levels were weaker in less than 10% of tumor specimens (Fig. 5F), similar to that shown in tumor-adjacent tissues (Fig. 5E). In most of the samples (~90%), Mirk expression was moderate (Fig. 5G) and higher (Fig. 5H) compared with that in adjacent tissue. Interestingly, overexpressed Mirk observed in tumor specimens was predominantly enriched in the perinuclear region (Fig. 5D, G and H). In a multivariate analysis, there was no significant association between Mirk expression and tumor stage, histological type, age, sex, smoking status or overall survival (Table 1).
Table 1. Characteristics of the 187 patients.
Characteristic | No. of patients (%) | p value* |
---|---|---|
Total | 187 | |
Age, years† | 0.05 | |
<71 years | 94 (50) | |
≥71 years | 93 (50) | |
Sex | 0.87 | |
Male | 101 (54) | |
Female | 86 (46) | |
Pathologic stage | 0.76 | |
1A | 85 (45) | |
1B | 102 (55) | |
Histology | 0.77 | |
Adenocarcinoma | 96 (51) | |
Squamous cell carcinoma | 68 (36) | |
Large-cell carcinoma | 23 (12) | |
Median overall survival, months | 77.7 (58.9 to ∞)‡ | |
Mirk expression, nucleus/cytoplasm§ | ||
Tumor | 679.56 (291.44)#/ 596.02 (267.37)# |
|
Adjacent tissue | 327.49 (121.23)**/ 278.19 (99.16)** |
Comparison of cytoplasmic Mirk expression between subgroups.
Median age was 71 years (range = 46–84).
Association with cytoplasmic Mirk expression (p = 0.53).
Average AQUA score (standard deviation).
In ~90% tumor samples, Mirk expression was moderate and higher compared with the lower level of that in adjacent tissue.
p < 0.001 compared with corresponding tumor tissues (total of 36 cases).
Figure 5.
Confocal microscopy of Mirk expression in NSCLC specimens and adjacent tissue. Top panels show multitarget immunofluorescence labeling formaldehyde-fixed and paraffin-embedded histologic sections of NSCLC specimens. (A) nuclei were labeled with 4′,6-diamidine-2-phenylindole (DAPI, blue). (B) Mirk was visualized with the use of Alexa 488 (green). (C) cytokeratin was visualized with the use of Alexa 594 (red). (D) merge of DAPI, Mirk and cytokeratin shows DAPI is only nuclear, Mirk is both nuclear and cytoplasmic, and cytokeratin is cytoplasmic. Bottom panels show the merge of DAPI, Mirk and cytokeratin. The merge was in adjacent tissue (E) and in an NSCLC specimen, which shows that Mirk expression is weaker (F), moderate (G) and higher (H).
Discussion
Our study demonstrates that Mirk is overexpressed in a variety of NSCLC cells and clinical specimens. Knockdown of Mirk induced apoptosis of NSCLC cells in vitro, delayed tumor growth in an orthotopic mouse model, and sensitized NSCLC cells to chemotherapeutics. Furthermore, Mirk-mediated NSCLC cell survival involves a Bcl-2 family member Bak and affects signaling through STAT3. To the best of our knowledge, our study is the first to show an effect of Mirk on NSCLC cells. These results are consistent with previous studies1,7,8 on other types of human cancers, indicating that Mirk may be a novel therapeutic target for NSCLC treatment.
It has been reported that Mirk has limited expression in normal tissue but has high levels of expression in skeletal muscle, heart, testes and brain.1,6 Although Mirk usually tends to be enriched in the nucleus to aid in the maintenance of G0-G1 arrest of differentiating myoblasts, it also exhibits considerable cytoplasmic location, which is cell type-dependent. Cytoplasmic restriction of Mirk has been detected in fetal myoblasts, differentiating human muscle, and mature adult human muscle, as well as in some types of human tumors.1 Overall, these findings suggest that Mirk localization in the cytoplasm in normal skeletal muscle and in tumor cells may afford Mirk different properties than the growth arrest properties observed when Mirk had a nuclear localization.22 Our results show that Mirk is overexpressed in both the nucleus and cytoplasm and is predominantly enriched in the perinuclear regions of NSCLC specimens, which is similar to that found in adult human muscle.8 Moreover, Mirk knockdown induced apoptosis, which was accompanied by a reduction in cell numbers. Thus, we conclude that the major function of Mirk in NSCLC cells is to mediate cell survival, which outweighs the potential of altered growth arrest induced by knockdown of nucleus-localized Mirk.
It has been known that tumors have high metabolic demands due to their rapid rate of division; they frequently outgrow their blood supply and become necrotic in the center. Our results of increased Mirk expression by serum deprivation in vitro suggest that tumor cells may be dependent on Mirk kinase for survival under stress, such as during serum deprivation. This dependence may be even greater in vivo because of stresses unique to tumor tissue (such as hypoxia, acidity, abnormal vascularization). In this study, we found that Mirk was predominantly overexpressed in the cytoplasm of some cancer cells, with morphological changes likely induced by nutrient depletion (data not shown). Therefore, our results and those from previous studies1,23 suggest that Mirk can be activated by various stressors in tumor cells and then act as a checkpoint kinase to arrest damaged tumor cells and to allow cellular repair in a quiescent state, to ultimately maintain cell survival. Our study, along with others,7,8 suggests that pharma-cological inhibition of Mirk may enhance the antitumor effect of chemotherapeutic drugs.
We found that Mirk is widely overxpressed in NSCLC cell lines and more than 90% of the tumor specimens from clinical cases that we tested, suggesting that Mirk may be important in these tumors. We found Mirk to be expressed in all histological subtypes and no differences in age or smoking status were found.
To date, the downstream signals of Mirk remain unclear. Given the main function of Mirk in NSCLC cells in mediating cell survival observed in this study, the mechanisms involved may include Bcl-2 family member(s) and other antiapoptotic proteins, such as STAT3, which are constitutively activated in NSCLC cells.16,24 It has been reported both STAT3 and the BH3-interacting domain death agonist Bid may contain a canonical Mirk phosphorylation site.7,21 In addition, the mitochondrial death program is activated by Bax or Bak, and so far the most effective way of blocking apoptosis other than Bcl-2 or Bcl-xl is deletion of Bax and Bak.25,26 Our results demonstrated that Mirk knockdown in various NSCLC cell lines resulted in upregulation of Bak, but no change of Bcl-2, Bax and Bid, indicating a possible role of Bak in Mirk-mediated NSCLC cell survival. Moreover, Mirk knockdown-induced cell apoptosis in NSCLC cells in this study is associated with downregulated activity of STAT3 tyrosine phosphorylation but not serine phosphorylation in a cell type-dependent manner. Dyrk family members, as serine/threonine kinases, were suggested to directly phosphorylate STAT3 Ser727 in mammalian cells.21 Decreased STAT3 tyrosine phosphorylation by Mirk knockdown was a surpising result, suggesting possible crosstalk of Mirk and tyrosine kinase signaling pathways known to affect STAT3 such as Janus kinase (JAK), Src and EGFR. Therefore, Mirk mediates NSCLC cell survival through multiple pathways, including the Bcl-2 family member Bak and STAT3 signaling.
Taken together, Mirk/Dyrk1B is overexpressed in a wide spectrum of NSCLC cell lines and human NSCLC specimens. Mirk/Dyrk1B mediates cell survival in NSCLC cells via a Bcl-2 family member Bak and STAT3 phosphorylation. Therefore, Mirk/Dyrk1B may be a novel target for treatment of lung cancer.
Materials and Methods
Antibodies
The rabbit polyclonal Mirk antibody1 was purified and supplied by Dr. Eileen Friedman’s laboratory (Upstate Medical University, State University of New York, Syracuse, NY). Anti-total STAT3, anti-Bcl-2, and goat anti-mouse IgG horseradish peroxidase (HRP)-conjugated secondary antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphorylated STAT3 (Tyr705), anti-Bax, anti-phosphotyrosine (pY100), and anti-poly (ADP-ribose) polymerase (PARP) were purchased; and anti-phosphorylated STAT3 (Ser727) and anti-Bid were gifts from Cell Signaling Technology (Danvers, MA). Anti-Bak and normal rabbit IgG were purchased from Upstate (Lake Placid, NY). Anti-cytokeratin (clones AE1/AE3) was purchased from Dako (Carpinteria, CA). Alexa Fluor 488 F(ab’) fragment of goat anti-rabbit IgG and Alexa Fluor 594 F(ab’) fragment of goat anti-mouse IgG were purchased from Invitrogen (Eugene, OR). Anti-β-actin and donkey anti-rabbit IgG HRP-conjugated secondary antibody were purchased from Sigma (St. Louis, MO) and Amersham Biosciences (Piscataway, NJ), respectively.
Cell lines and cell culture
Human NSCLC cells were maintained in RPMI 1640 supplemented with 5% heat-inactivated (56°C, 30 min) fetal bovine serum (FBS; Invitrogen, Grand Island, NY). HCC827 and H4006 cells were provided by Dr. Jonathan M. Kurie (University of Texas M.D. Anderson Cancer Center, Houston, TX). H226, H1648, H157, H2122, H820 and H2279 cells were provided by Dr. John D. Minna (University of Texas Southwestern Medical Center, Dallas, TX). H322 cells were provided by Dr. Paul A. Bunn (University of Corolado Health Sciences Center, Denver, CO). PC-9 cells were provided by Dr. Matthew Lazzara (Massachusetts Institute of Technology, Boston, MA). The stable luciferase A549 cell line (A549-Luc-C8) was purchased from Caliper Life Science (Hopkinton, MA). The other cell lines were purchased from American Type Culture Collection (Manassas, VA). Monolayer cultures were incubated at 37°C in a 95% humidified atmosphere air containing 5% CO2.
Small interfering RNA treatment
Cells were reverse transfected with small interfering RNAs (siRNAs) using lipofectamine RNAiMAX transfection reagent (Invitrogen) according to the manufacturer’s instructions. The Mirk siRNA (On-Target plus SMART pool) and the corresponding individual Mirk siRNAs #1-#4 as well as nonspecific control siRNA (On-Target plus Non-Targeting pool) were supplied by Dharmacon. Of these individual Mirk siRNAs, #1: 5′-GAG AUG AAG UAC UAU AUA GUU-3′ (sense) and 5′-PCU AUA UAG UAC UUC AUC UCU U-3′ (antisense) was for target sequence: 5′-GAG AUG AAG UAC UAU AUA G-3′; #2: 5′-CGA AAG AAC UCA GGA AGG AUU-3′ (sense) and 5′-PUC CUU CCU GAG UUC UUU CGU U-3′ (antisense) was for target sequence: 5′-CGA AAG AAC UCA GGA AGG A-3′; #3: 5′-GGU GAA AGC CUA UGA UCA UUU-3′ (sense) and 5′-PAU GAU CAU AGG CUU UCA CCU U-3′ (antisense) was for target sequence: 5′-GGU GAA AGC CUA UGA UCA U-3′; #4: 5′-GGA CCU ACC GCU ACA GCA AUU-3′ (sense) and 5′-PUU GCU GUA GCG GUA GGU CCU U-3′ (antisense) was for target sequence: 5′-GGA CCU ACC GCU ACA GCA A-3′. After a 72 h incubation or at indicated time points, cells were harvested or trypsinized and replated for subsequent experiments.
Cell growth assay
Cells were plated in 96-well plates, and siRNA transfection was performed for 120 h as described above. Cell growth was measured by [3-(4,5)-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) analysis. Briefly, after cells were washed with PBS, they were incubated in MTT solution for 4 h and then supplemented with 100 μl of dissolving solution (10% SDS in 0.01 M HCl). The absorbance (optical density units) was measured using a microplate spectrophotometer (Bio-Rad Laboratories, Hercules, CA) with Microplate Manager 5.1 software at wavelengths of 590 nm and 660 nm. Each assay was performed in quadruplicate. Viability of siRNA-transfected cells after 48-hour exposure to the chemotherapeutic agent cisplatin (CDDP) was also evaluated by MTT assay.
Flow cytometry analysis
After 72 h treatment with siRNAs, cells were subjected to flow cytometry analyses of apoptosis and cell cycle alterations. Apoptosis was assayed using Pharmingen (San Diego, CA) PE-conjugated monoclonal active caspase-3 anti-body and FITC Annexin V apoptosis kits without modification. We determined the percentage of cells in G1, S and G2/M phase by propidium iodide staining as described previously.18 A total of 10,000 cells per experimental condition were used for processing and analysis of fluorescence on Becton-Dickinson FACScan (BD, Franklin Lakes, NJ) using CellQuest software. Apoptosis of siRNA-transfected cells after 48 h exposure to the chemotherapeutic agent CDDP was also detected by flow cytometry analysis.
Colony formation assay
After 72 h treatment with siRNAs, cells were collected and mixed (1,000/ml) with RPMI 1640 culture medium containing 10% FBS and added to 12-well plates (1 ml/well). Colonies per well were stained with the HEMA3 stain set (Fisher Scientific, Middletown, VA) and counted using a Nikon inverted microscope after cells were plated for 14 d and numbers were averaged. Each assay was performed in triplicate.
Western blot and immunoprecipitation analyses
Cells were washed twice with cold PBS and lysed with buffer A [10 mM Tris-HCl (pH 7.4), 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10 μ/ml leupeptin, 5 μg/ml aprotinin]. After incubation for 30 min on ice, the suspensions were centrifuged (15,000 g for 30 min). The supernatants were removed and stored at −80°C until analysis using gel electrophoresis. The protein concentration was determined by Bio-Rad protein estimation assay according to the manufacturer’s instructions. For western blot analysis, ~60–100 μg of whole cell proteins were separated using 10% or 12% SDS-PAGE and transferred to nitrocellulose membranes. After blocking of the membranes with 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.1% Tween 20 containing 5% nonfat dry milk at room temperature for 60 min, the membranes were incubated with indicated antibodies at 4°C overnight and then with the HRP-conjugated secondary anti-rabbit or anti-mouse antibodies at room temperature for 60 min. Each protein was detected using the enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ) system. β-actin was used as an internal control.
Immunoprecipitations were performed with 500 μg of whole cell protein lysates, using protein A-agarose (Roche, Indianapolis, IN). Briefly, equal amounts of protein lysates were incubated with Mirk antibody, and normal rabbit IgG was used as negative control. After overnight incubation at 4°C, the immune complexes were precipitated with protein A-agarose. The immunoprecipitates were washed with lysis buffer according to the manufacturer’s instructions, separated by SDS-PAGE, and then transferred to nitrocellulose membranes followed by incubation of pY100 or Mirk antibodies for western blot analysis as described above.
Orthotopic lung tumor mouse model
Female athymic CD-1 nude mice, 8 weeks old, purchased from Charles River Laboratories, were anesthetized in an acrylic chamber using 2.5% isoflurane-air mixture and placed in the right lateral decubitus position. Equal amounts of control siRNA- and Mirk siRNA-transfected A549-Luc-C8 cells (1.5 × 106 viable cells) in 50 μl of PBS containing 50 μg of growth factor-reduced Matrigel (BD Biosciences, Bedford, MA) were injected into the left lateral thorax of the mice at the lateral dorsal axillary line as described previously.27 The mice (7 per group) were housed and maintained in specific pathogen-free conditions. Experimental procedures were approved by the Institutional Animal Care and Use Committee and conducted in accordance with the standards established by the H. Lee Moffitt Cancer Center and Research Institute.
After injection, all mice were euthanized and autopsied when control mice became moribund. Relative tumor weight (total tumor-bearing lung weight), pleural effusion, and metastasis were evaluated. Orthotopic tumor growth was also evaluated before death by in vivo bioluminescence assay using Xenogen Imaging System 200, for which luciferin (150 mg/kg body weight) was injected intraperitoneally 30 min before imaging.
Patients and tumor specimens
The tumor specimens were obtained from 187 patients with stage I NSCLC.28 The patients were a subgroup of all patients who underwent thoracotomy for resection of a primary lung cancer at the H. Lee Moffitt Cancer Center and Research Institute between 1991 and 2001. Patients were eligible for inclusion in the study if they had an adenocarcinoma, squamous cell carcinoma, or large cell carcinoma; had undergone a complete resection of the tumor (R0 resection); and had stage I disease by pathological staging. All patients did not have any chemotherapy or radiation prior to resection. The study was approved by the Institutional Review Board of the H. Lee Moffitt Cancer Center and Research Institute.
In situ detection and quantification of Mirk protein expression
A tissue microarray was constructed as described previously.28 Namely, immunofluorescence combined with automated quantitative analysis (AQUA) was used to assess in situ expression of the target molecules.29 After antigen retrieval with citrate, the endogeneous peroxidase activity was blocked by incubation with 0.3% hydrogen peroxide. Slides were incubated for 1 hour with primary antibodies. Optimal concentrations of antibodies were used to detect Mirk7 and cytokeratin,28 which were visualized with the use of fluorochrome-labeled second antibodies. The final slides were scanned with SpotGrabber (HistoRx), and image data were analyzed with AQUA (PM-2000, HistoRx).
Statistical analysis
Each experiment was repeated two or three times. Data are presented as means ± SD. Statistical comparisons between control and experimental groups were performed using χ2 test (for incidence only) and Student’s t-test. The associations between Mirk expression and tumor stage, histological type, age and sex were assessed using Student’s t-test and ANOVA, and Mirk expression with overall survival was analyzed by univariate Cox regression. Differences were considered to be statistically significant when p was less than 0.05.
Acknowledgements
We thank Dr. Eileen Friedman (Upstate Medical University, State University of New York, Syracuse, NY) for providing Mirk antibody and helpful discussions; Dr. Jonathan M. Kurie (UT M.D. Anderson Cancer Center, Houston, TX), Dr. John D. Minna (UT Southwestern Medical Center, Dallas, TX), Dr. Paul A. Bunn (University of Corolado Health Sciences Center, Denver, CO), Dr. Matthew Lazzara (Massachusetts Institute of Technology, Boston, MA) for providing cells; Rasa G. Hamilton for editorial assistance; and Patricia A. Johnston for administrative assistance. This work was supported in part by NCI 1R01 CA121182-01A1 (E.B.H.), the Molecular Imaging Core, Mouse Models Core, and the Flow Cytometry Core at the H. Lee Moffitt Cancer Center and Research Institute.
Footnotes
The authors declare no conflicts of interest.
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