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[Cancer Research 66, 5143-5150, May 15, 2006]
© 2006 American Association for Cancer Research

Cell, Tumor, and Stem Cell Biology

Mirk/Dyrk1b Mediates Cell Survival in Rhabdomyosarcomas

Stephen E. Mercer, Daina Z. Ewton, Sejal Shah, Asghar Naqvi and Eileen Friedman

Department of Pathology, Upstate Medical University, State University of New York, Syracuse, New York

Requests for reprints: Eileen A. Friedman Pathology Department, Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210. Phone: 315-464-7138; Fax: 315-464-8419; E-mail: friedmae{at}upstate.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Rhabdomyosarcoma is the most common sarcoma in children and is difficult to treat if the primary tumor is nonresectable or if the disease presents with metastases. The function of the serine/threonine kinase Mirk was investigated in this cancer. Mirk has both growth arrest and survival functions in terminally differentiating skeletal myoblasts. Maintenance of Mirk growth arrest properties would cause down-regulation of Mirk in transformed myoblasts. Alternatively, Mirk expression would be retained if rhabdomyosarcoma cells used Mirk survival capability. Mirk expression was significant in 12 of 16 clinical cases of rhabdomyosarcoma. Mirk was detected in each rhabdomyosarcoma cell line examined. Mirk was a functional kinase in each of three rhabdomyosarcoma cell lines, where it proved to be more active than in C2C12 skeletal myoblasts. Mirk mediated survival of the majority of clonogenic rhabdomyosarcoma cells. Knockdown of Mirk by RNA interference reduced the fraction of RD and of Rh30 rhabdomyosarcoma cells capable of colony formation 3- to 4-fold in multiple experiments. Depletion of Mirk induced cell death by apoptosis, as shown by increased numbers of terminal deoxynucleotidyl transferase–mediated nick-end labeling–positive cells and by increased binding of Annexin V. Mirk is a stress-activated kinase that mediates expression of contractile proteins in differentiating myoblasts, but Mirk is not essential for muscle formation in the embryo. It is likely that Mirk also facilitates survival of satellite cell–derived rhabdomyoblasts in regenerating skeletal muscle and aids their differentiation. This survival function is maintained in rhabdomyosarcoma, where Mirk may be a novel therapeutic target. (Cancer Res 2006; 66(10): 5143-50)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mirk/Dyrk1B is a serine/threonine kinase that is up-regulated in many solid tumors and mediates cell survival in colon carcinomas (1), the tumor from which our group originally cloned Mirk (2). Mirk is expressed at low levels in most normal tissues but is up-regulated and activated in skeletal muscle; thus, the function of Mirk in nontransformed cells was defined in the physiologically relevant C2C12 myoblast system. Three major functions were observed. First, Mirk functions as a checkpoint kinase. Mirk is up-regulated and activated in myoblasts arrested in G0 when they initiate differentiation (3) and in NIH3T3 cells arrested in G0 by serum starvation (4). Enrichment of Mirk in G0 is a result of dual effects on its transcription. Mirk transcription is induced by RhoA and Cdc42 but is down-regulated in other phases of the cell cycle by mitogenic activation of the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase/ERK system, which initiates movement into G1 (3). Mirk aids in the maintenance of G0 arrest of differentiating nontransformed myoblasts, NIH3T3 cells, and Mv1Lu epithelial cells by posttranslational mechanisms. Mirk destabilizes cyclin D1 by phosphorylation at T288 (5) and stabilizes the cyclin-dependent kinase (CDK) inhibitor p27 by phosphorylation at S10 in G0 (4). These phosphorylations have biological consequences, as depletion of Mirk by RNA interference (RNAi) enables G0 arrested NIH3T3 cells and C2C12 myoblasts to enter the cell cycle, whereas transient overexpression of Mirk blocks cycling cells in G0 (3–5).

Second, Mirk is a stress-activated kinase (6) that mediates expression of contractile proteins in differentiating myoblasts (3) through its effects on the myogenic regulatory factors myogenin and MEF2 (7). Mirk is activated by phosphorylation (8) by the stress-activated MAP kinase kinase MKK3 (6). The induction and activation of Mirk under stress conditions suggests that Mirk may play a role in the response to cellular injury. Skeletal muscle is regenerated after injury by activation of quiescent satellite cells, which enter the cell cycle and then differentiate and fuse with uninjured muscle fibers to repair the damage. Mirk is expressed at low levels in skeletal muscle satellite cells, and Mirk expression is increased when these cells are activated to enter the cell cycle (3). Mirk is less likely to play a significant a role in embryonic muscle development because a Mirk/Dyrk1B knockout mouse survived to 18 days after conception, during which time skeletal muscles were developed (9). Thus, Mirk seems to function during the repair of normal skeletal muscle.

Third, studies of Mirk in differentiating C2C12 myoblasts and other cells strongly suggest that Mirk functions as a survival factor, particularly during skeletal muscle regeneration. Mirk has antiapoptotic functions in both differentiating myoblasts and cancer cells. Overexpression of Mirk in each of two colon carcinoma cell lines increased their survival capacity under stress conditions (1), whereas depletion of endogenous Mirk by RNAi reduced their viability.1 The antiapoptotic properties of Mirk are also seen in the normal cell type in which Mirk is most abundant, skeletal myoblasts. A large fraction of cycling myoblasts (20-30%) are not able to differentiate and undergo apoptosis when deprived of mitogens. Depletion of Mirk by RNAi blocked myoblast survival and increased the activation of caspase-3 (10). Moreover, overexpression of wild-type Mirk depressed apoptosis during muscle differentiation, whereas overexpression of kinase-inactive mutant Mirk had no antiapoptotic activity (10). Mirk/Dyrk1B was also shown to have survival functions in HeLa cervical carcinoma cells in high-throughput screening of the human kinome by RNAi (11). These observations led us to speculate that Mirk is also likely to mediate cell survival in rhabdomyosarcomas.

Mirk has both growth arrest and prosurvival functions in normal cells. Because malignant cells are characterized by unregulated growth, the growth arrest properties of Mirk must somehow be abrogated to enable these cancers to continue to express Mirk protein. Results from the current study suggest that the kinase Mirk functions as a survival factor in rhabdomyosarcoma and, as such, may present a novel therapeutic target.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines. The alveolar rhabdomyosarcoma cell line Rh41 was the kind gift of Dr. Peter Houghton (St. Jude's, Memphis, TN), whereas the embryonal rhabdomyosarcoma cell line RD, the alveolar rhabdomyosarcoma cell line Rh30, and C2C12 mouse skeletal myoblasts were obtained from the American Type Culture Collection (Rockville, MD). C2C12 cells were maintained in growth medium [DMEM, 4 mmol/L L-glutamine, 4.5 g/L glucose, containing 20% fetal bovine serum (FBS)] and induced to undergo differentiation by switching to differentiation medium (DMEM containing 2% horse serum). Rhabdomyosarcoma cells were maintained in DMEM, 4 mmol/L L-glutamine, 4.5g/L glucose, containing 10% FBS. Cells were used for experiments only from passages 3 to 10 from our frozen stocks.

Immunodetection, band analysis, and Northern blotting. Northern analysis was done (3), and immunoblots were made and scanned as previously described (4).

Mirk activity. Mirk kinase activity was determined by immune complex kinase assays of immunoprecipitated Mirk on myelin basic protein normalized to the amount of immunoprecipitated Mirk as determined by Western blots of the immunoprecipitates. Aliquots of 300 to 500 µg of total lysate were immunoprecipitated with 3 µg of either Mirk NH2-terminal directed or Mirk COOH-terminal directed anti-peptide affinity-purified rabbit polyclonal antibody overnight at 4°C. The complexes were collected by addition of 20 µL protein A-agarose and incubation for either 2 or 24 hours at 4°C, as noted, then washed thrice with immunoprecipitation buffer [50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1.0% NP40, 10% glycerol, and a tablet of protease inhibitor (Roche, Indianapolis, IN)]. The immunoprecipitates were then washed thrice with kinase buffer [50 mmol/L Tris-HCl (pH 7.5), 10 mmol/L MgCl2, 1.0 mmol/L DTT] and incubated for 15 minutes at 30°C with 20 µL of kinase buffer containing 50 µmol/L cold ATP plus 5 µCi of [32P-{gamma}]ATP and 2 µg of purified recombinant myelin basic protein as substrate, then analyzed by PAGE and autoradiography.

Colony formation assay. RH30 or RD rhabdomyosarcoma cells were plated overnight at 1.5 to 2 x 106 per T25 flask and transfected (using 20 µL of PLUS reagent and 20 µL or LipofectAMINE 2000) with 1 µg of pCDNA3.1 encoding the neomycin resistance gene and 9 µg of either pSilencer carrying an RNAi construct for Mirk (GACCTACAAGCACATCATT) or a mutant Si construct (CAGAGCCTACCGATACAG). Cells were transfected in serum-free DMEM for 4 hours, and then FBS was added to 10%. Forty-eight hours after transfection, cells were plated in triplicate at 25,000 to 100,000 per 100-mm dish. Transfected cells were then selected in 600 µg/mL G418-containing medium for 10 to 14 days until discrete colonies were visible.

Annexin assay. RH30 or RD rhabdomyosarcoma cells were plated overnight in Labtek two-well chamber slides (3 x 105 per well) and then transfected (using 8 µL LipofectAMINE 2000 per well) with 1 µg green fluorescent protein (GFP)-vector and 3 µg of either pSilencer vector, pSilencer carrying an RNAi construct for Mirk, or a mutant Si construct. Cells were transfected in 10% FBS/DMEM overnight, and then medium was replaced with differentiation medium (2% HS/DMEM) for 24 hours. Cells were incubated with 2 µL of Annexin V-Alexa 568 stock solution (Roche) in 1 mL differentiation medium per well for 30 minutes. Cells were washed twice with PBS with Ca2+ and Mg2+ (because Annexin binding is calcium dependent) and fixed for 30 minutes with 3% paraformaldehyde (4% PFA/PBS diluted with PBS/Ca2+/Mg2+); washed twice with PBS, once in distilled water then mounted with BioMedia GelMount. At least 300 GFP-expressing cells were observed in each of four separate preparations, and the number of cells labeled for both GFP and Annexin was determined using a green/orange V2 filter set (Chroma) that allowed simultaneous visualization of both fluorophores. Labeling of pyknotic nuclei with 4',6-diamidino-2-phenylindole was used to confirm the apoptotic phenotype. Combined counts were analyzed by the {chi}2 test to determine the significance of differences between the RNAi constructs. Efficiency of cotransfection was determined to be >85% in parallel experiments using a combination of GFP and dsRed. Overall transfection efficiency in the Rh30 and RD cell lines was 15% to 20%.

Terminal deoxynucleotidyl transferase–mediated nick-end labeling assay. RD rhabdomyosarcoma cells were plated overnight in LabTek two-well chamber slides (3 x 105 per well) and then cotransfected with 0.5 µg phospho-enhanced GFP (pEGFP) and 1.5 µg of pSilencer vector, RNAi to Mirk or mutant RNAi (4 µL PLUS and 4 µL LipofectAMINE per well). Cells were transfected in serum-free media for 4 hours, and then an equal volume of 20% FBS/DMEM was added. Following 24 hours of expression, cells were incubated with differentiation medium (2% horse serum/DMEM) for 24 hours. After 24 hours in differentiation medium, DNA breaks in apoptotic cells were labeled with tetramethyl-rhodamine-dUTP by terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL) using the Roche In situ Cell Death Detection kit. At least 300 GFP-expressing cells were observed in each of four separate preparations. The number of GFP expressing cells labeled with the TUNEL marker was determined using a green/orange V2 filter set (Chroma) that allowed simultaneous visualization of both fluorophores. Nuclear morphology was used to confirm the apoptotic phenotype. Combined counts were analyzed by the {chi}2 test to determine the significance of differences between the RNAi constructs. Efficiency of cotransfection was determined to be >85% in parallel experiments using a combination of GFP and DsRed.

Immunohistochemistry. Sixteen archived blocks containing formalin-fixed, paraffin-embedded rhabdomyosarcomas were obtained from the Department of Pathology, State University of New York (SUNY) Upstate Medical University Hospital in accordance with institutional review procedures for clinical specimen use. A section of skeletal muscle was placed adjacent to each tumor section to serve as an internal positive control. Immunohistochemical stains were done on citrate-treated, 3-µm-thick, paraffin-embedded sections on a Ventana ES automated immunostainer using the streptavidin-biotin-peroxidase method. Rabbit polyclonal antibody to the COOH terminus of Mirk was used at a dilution of 1:500. Nonspecific rabbit IgG diluted to an equivalent mass/concentration was used as a negative control. Antibody complexes were detected with 3,3-diaminobenzidine tetrahydrochloride. Mirk protein abundance was quantitated on images of representative areas of each tumor using the IP Lab Gel program, with a mean of six assay points per tumor. For quantitation of Mirk expression in cultured rhabdomyosarcoma cells by immunohistochemistry, RD and Rh30 cells were plated on glass chamber slides (LabTek) for 24 hours in growth medium, fixed for 10 minutes with 4% paraformaldehyde/PBS, and immunostained for Mirk using the same protocol outlined above for paraffin-embedded tissues.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mirk is widely expressed in rhabdomyosarcomas. Mirk expression was assessed in resected human rhabdomyosarcomas by immunohistochemistry. Sixteen cases of rhabdomyosarcoma seen in SUNY Upstate University Hospital were examined for expression of Mirk by immunohistochemistry (Table 1 ). Although Mirk was not present in all of the tumor cells, Mirk was detected in each case. The abundance of Mirk protein identified by immunohistochemistry was quantified by assaying a mean of six determinations per tumor compared with the positive control of skeletal muscle placed adjacent to each tumor section on each slide, to give an intensity score (Table 1). Tumors were judged to have significant expression of Mirk if the intensity of staining was ++ to ++++, and Mirk expression was seen in >25% of tumor cells. By this standard, tumors with significant expression of Mirk in this study included three of four embryonal rhabdomyosarcomas, each of four pleomorphic rhabdomyosarcomas, and five of eight alveolar rhabdomyosarcomas (Table 1; Fig. 1 ). In one additional alveolar rhabdomyosarcoma, Mirk exhibited strong (+++) focal expression in <10% of the tumor cells.


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  		Table 1. Immunohistochemical analysis of Mirk expression

 

Figure 1
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  		Figure 1. Localization of Mirk in tissues by immunohistochemistry. Clinical cases of the three major histologic classes of rhabdomyosarcoma (alveolar, embryonal, and pleomorphic), showing Mirk expression primarily in the cytoplasm. Inset, lower magnification of a sequential section from the same tumor stained with H&E to show tumor cell morphology. Skeletal muscle, cytoplasmic restriction of Mirk seen in adult human muscle. Note that Mirk is found in only a subset of fibers, where it is localized exclusively in the cytoplasm and often enriched in the perinuclear region. Original magnification, x400.

 
As an additional control, cultures of RD and Rh30 rhabdomyosarcoma cells were examined for Mirk expression by immunohistochemistry following culture in serum-containing growth medium or low serum differentiation medium to induce expression of Mirk. Parallel cultures were analyzed for Mirk protein expression by Western blotting and for Mirk mRNA levels by Northern blotting (Fig. 2 ). Mirk expression was lowest in RD cells cultured in growth medium, whereas 6- to 7-fold higher expression of Mirk was seen in Rh30 cells cultured in growth medium as analyzed by Western blotting (Fig. 2A), by Northern blotting (Fig. 2B), and by immunohistochemistry (Fig. 2C). Thus, the Northern analysis of Mirk mRNA levels and the measurement of Mirk protein levels by Western blotting confirmed the measurement of Mirk protein by immunohistochemistry.


Figure 2
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  		Figure 2. Correlation of Mirk levels by immunohistochemistry with Mirk levels as determined by Western blotting and by Northern analysis. RD and Rh30 cells were cultured for 1 day in either serum-containing growth medium (GM or G) or in low serum differentiation medium (DM or D), and parallel cultures were examined for Mirk protein expression by Western blotting (A), Mirk mRNA levels by Northern analysis (B). C, cells cultured in growth medium are shown immunostained for Mirk using the same protocol used for paraffin-embedded tissues. A, control is tubulin, and the ratio of Mirk to tubulin is shown under each lane. B, ribosomal 18S and 28S RNAs are shown by ethidium bromide staining, and the ratio of Mirk mRNA to 28S rRNA is shown below each lane.

 
Endogenous Mirk is predominately cytoplasmic in clinical cases of rhabdomyosarcoma and in normal skeletal muscle. The localization of a kinase can profoundly affect its activity. Mirk is distributed predominately within the nucleus in undifferentiated NIH3T3 cells, in which Mirk was previously shown to mediate arrest in G0 (4). In contrast, in most clinical cases of rhabdomyosarcoma, Mirk was predominately cytoplasmic (Fig. 1). Mirk had both cytoplasmic and nuclear localization in three cases. In the latter tumors, Mirk expression was elevated (+++/++++), and Mirk was found in 50% to 100% of the cells (embryonal tumors 11 and 12 and pleomorphic tumor 16 in Table 1). Mirk was predominately cytoplasmic in the more aggressive variant, alveolar rhabdomyosarcoma. Traditional immunohistochemistry was done on formalin-fixed paraffin sections of adult human muscle to determine if this distribution of Mirk occurred in mature myofibers. Mirk was localized to the cytoplasm of a subset of adult muscle fibers (Fig. 1). This pattern is likely to coincide with a specific type of fiber or it may possibly denote a subset of fibers that have undergone recent repair. No staining was noted in control sections treated with nonspecific rabbit IgG instead of primary antibody (data not shown). Identical patterns of localization were observed using our two distinct anti-Mirk antibodies in immunofluorescence analysis of frozen sections of adult human muscle (data not shown), thus showing that the findings did not occur due to a nonspecific binding of either antibody. Mirk localization in the cytoplasm in normal skeletal muscle and in rhabdomyosarcoma tumors may afford Mirk different properties than the growth arrest properties observed when Mirk had a nuclear localization (4).

Mirk is an active kinase in myoblasts and in rhabdomyosarcomas. Because Mirk expression was maintained in rhabdomyosarcomas, it was necessary to determine whether Mirk was an active kinase in this tumor. Furthermore, if Mirk activity was increased in rhabdomyosarcomas compared with nontransformed C2C12 skeletal myoblasts, it would be consistent with a role for Mirk in tumor evolution or survival. Mirk was immunoprecipitated from C2C12 myoblasts (C2), Rh30 (RH), and Rh41 (R41) alveolar rhabdomyosarcoma cells and RD embryonal rhabdomyosarcoma cells. Cells were cultured in either low serum differentiation medium or in growth medium, with preimmune serum (IgG) serving as the control. The kinase activity of immunoprecipitated Mirk was determined by an immune complex kinase reaction on MBP (Fig. 3A and B, top ) and normalized to the amount of immunoprecipitated Mirk. Mirk was an active kinase in each of the rhabdomyosarcoma cell lines, regardless of the growth conditions, as shown by its ability to phosphorylate exogenous MBP (Fig. 3A and B). In addition, the rhabdomyosarcoma cell lines exhibited more Mirk kinase activity than the nontransformed C2C12 line under both low serum and high serum growth conditions (Fig. 3D and E). These data show that Mirk is an active kinase in nontransformed skeletal myoblasts, and that Mirk kinase activity is increased in embryonal and alveolar rhabdomyosarcoma cells.


Figure 3
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  		Figure 3. Mirk is an active kinase in both nontransformed C2C12 myoblasts and rhabdomyosarcoma cell lines. A, using a Mirk NH2-terminal directed affinity-purified antibody (left) or a Mirk COOH-terminal directed affinity-purified antibody (right), Mirk was immunoprecipitated overnight from C2C12 myoblasts (C2), Rh30 (RH), and Rh41 (R41) alveolar rhabdomyosarcoma cells and RD embryonic rhabdomyosarcoma cells cultured in either growth medium (GM) or in low serum differentiation medium (DM) for 2 days. Control immunoprecipitations from C2C12 cells were done with purified rabbit IgG. The kinase activity of immunoprecipitated Mirk, after collection with protein A-agarose beads for 24 hours (left) or 2 hours (right) was determined by an immune complex kinase reaction on MBP, which was analyzed by SDS-PAGE followed by autoradiography (top). The amount of Mirk in each immunoprecipitate was determined by Western blotting. The total amount of Mirk, myogenin, and ß-tubulin in each lysate was evaluated by Western blotting (bottom). B, a similar analysis of Mirk kinase activity as in (A), but the Mirk COOH-terminal directed antibody was used to immunoprecipitate Mirk overnight before a 2-hour collection with protein A-agarose beads. The cells were cultured in growth medium or in differentiation medium before assay, as noted. The total amounts of Mirk and myogenin in each lysate were evaluated by Western blotting (bottom). C, the amount of Mirk in lysates of Rh30 rhabdomyosarcoma cells after knockdown by RNAi (Si), treatment with a mutant Si sequence (Mt) or vector control (V), each cotransfected with a neomycin resistance marker and cultured in G418, as analyzed by Western blotting with ß-tubulin as control (bottom). Parallel cultures were analyzed 3 days after transfection when there was a background of untransfected cells and 16 days after transfection when most of the nontransfected cells had been lost to selection in G418. D, the kinase activity of Mirk in low serum differentiation medium was determined by laser densitometry from the autoradiograms in (A) and (B) and other data (data not shown) and normalized to the amount of Mirk that had been immunoprecipitated. E, the kinase activity of Mirk in growth medium was determined by laser densitometry from the autoradiograms in (A) and (B) and other data (data not shown) and normalized to the amount of Mirk that had been immunoprecipitated.

 
Depletion of endogenous Mirk in C2C12 myoblasts by RNAi blocked the expression of many contractile proteins (3) because Mirk mediates the expression of the muscle regulatory transcription factor myogenin (7). Myogenin expression was detected in each rhabdomyosarcoma line and in C2C12 myoblasts (Fig. 3, lysates). Myogenin is often found in rhabdomyosarcomas in vivo (12). This suggests that Mirk retains at least some of its transcriptional activation function in rhabdomyosarcomas.

Mirk mediates survival of the majority of clonogenic rhabdomyosarcoma cells. The biological result of Mirk knockdown was next determined following depletion of Mirk by RNAi using the pSilencer expression plasmid. The rhabdomyosarcoma cell lines Rh30 and RD were cotransfected with pCDNA3.1 encoding the neomycin resistance gene and either the pSilencer plasmid encoding RNAi to Mirk or the pSilencer plasmid encoding a mutant RNAi. After 2 days of treatment, Si1 depleted Mirk protein levels to 25% of controls treated with mutant RNAi (data not shown but similar to Fig. 3C). Parallel cultures of Mirk-depleted cells and controls were then plated at single-cell density and cultured for 10 to 14 days in G418-containing medium to select for the cotransfected neomycin resistance plasmid (Fig. 4A ). In this colony-forming assay, only about 0.1% of plated cells gave rise to colonies. Any partially differentiated cells with low growth potential would not be expected to be able to grow to form colonies from single cells. Thus, this assay measures the effect of Mirk depletion on a more aggressive subpopulation within the culture. Knockdown of Mirk reduced the viability of RD embryonal rhabdomyosarcoma cells 3- to 4-fold compared with mutant RNAi, with results highly statistically significant (Fig. 4A, duplicate experiments shown). Similarly, knockdown of Mirk in Rh30 alveolar rhabdomyosarcoma cells reduced their viability 3- to 4-fold compared with mutant RNAi (Fig. 4A, three independent experiments shown). For each experiment, P was <0.001 by Student's t test. In a similar experiment, parallel cultures were analyzed 3 days after transfection when there was a background of untransfected cells and 16 days after transfection when most of the nontransfected cells had been lost to selection in G418 (Fig. 3C, one of duplicate experiments shown). In the cells selected by the cotransfected neomycin resistance gene, Mirk levels were reduced >5-fold. This decrease was enough to substantially reduce the viability of the more aggressive rhabdomyosarcoma cells, those able to grow to colonies from single cells. Thus, Mirk is a survival factor for these tumor cells.


Figure 4
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  		Figure 4. Mirk depletion causes death of rhabdomyosarcoma cells by inducing apoptosis. A, knockdown of Mirk by RNAi strongly inhibits the survival of RD and Rh30 rhabdomyosarcoma cells. Mirk protein levels were reduced 4-fold by RNAi to Mirk (Si Mirk) compared with cells treated with a mutant RNAi (Si Mutant). Cells were cotransfected with a neomycin resistance plasmid and selected in G418 containing medium 10 to 14 days until colony formation. Columns, mean of triplicate measurements from five independent experiments; bars, SE. P = 0.0003, for all measurements comparing Mirk si versus mutant si (Student's t test). B, knockdown of Mirk increases apoptosis in RD cells cultured under restricted mitogen conditions. Cells were cotransfected with an expression plasmid for EGFP together with either RNAi to Mirk in the pSilencer plasmid (Si-Mirk), a mutant RNAi (Mutant), or the pSilencer vector (Vec). After 24 hours of expression, cells were cultured in low serum differentiation medium to induce differentiation, the DNA breaks in the apoptotic cells were labeled by the TUNEL reaction, and the GFP-expressing cells that were also labeled with the TUNEL marker were simultaneously visualized by fluorescence microscopy. The number of cells scored for each transfection condition is listed. Combined counts were analyzed by the {chi}2 test to determine the significance of differences between the RNAi constructs.

 
Apoptosis was increased in rhabdomyosarcomas by knockdown of endogenous Mirk. The loss of survival capability in these cells following depletion of Mirk was caused by the loss of the antiapoptotic functions of Mirk. Apoptosis accompanies differentiation of myoblasts, resulting in a loss of about 20% to 30% of cells when myoblasts are transferred to differentiation medium (13). Mirk mediates myoblast cell survival, at least in part, through localization of p21cip1 to the cytoplasm where it can block proapoptotic effectors, including procaspase-3 (10). To assess whether Mirk functions to block apoptosis in rhabdomyosarcomas, rhabdomyosarcoma cell lines Rh30 and RD were cotransfected with an expression plasmid for GFP together with either RNAi to Mirk in the pSilencer plasmid, a mutant RNAi, or the pSilencer vector. Apoptosis was induced by switching cells to low serum differentiation medium. Analysis of Mirk levels in Rh30 cells by Western blotting showed that Mirk knockdown was >50%. The transfected cells were identified by coexpressed GFP, and the subpopulation of these cells undergoing apoptosis was detected by fluorescence microscopy using an Alexa 568–coupled Annexin V conjugate (Roche). A green/orange V2 filter set (Chroma) was used to visualize both fluorochromes simutaneously. In parallel studies, the DsRed plasmid cotransfected into the same cell with the EGFP plasmid >90% of the time.

Depletion of Mirk caused a slow, asynchronous loss of cells from the culture that took >48 hours (data not shown); thus, the Annexin V assay would only show the cells dying over a small portion of this ongoing process. Pilot experiments showed that after 48 hours of serum deprivation, the large number of detached, apoptotic cells prevented an accurate assessment of the proportion of GFP/RNAi–transfected and apoptotic cells. Therefore, subsequent trials focused on the 24-hour time point, where the limited number of apoptotic cells permitted a more accurate analysis. However, in both lines, about twice as many cells expressing RNAi to Mirk (Si-Mirk) were undergoing apoptosis and thus positive for Annexin V as were cells expressing mutant RNAi (Si-Mut) or vector control (Table 2 ). The ratio of apoptotic cells after Mirk knockdown compared with treatment with mutant RNAi was 31% to 15% for RD cells and 28% to 14% for RH30 cells. Because data were collected as counts, the {chi}2 test was used to determine the significance of observed differences between the RNAi constructs. In both lines, the doubling of the amount of apoptotic cells with Mirk knockdown was highly significant (P < 0.0001).


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  		Table 2. Apoptosis in cells cotransfected with Mirk RNAi + GFP

 
Similar experiments were done using the TUNEL assay to measure the antiapoptotic function of Mirk in rhabdomyosarcoma cells. The RD embryonal rhabdomyosarcoma cell line was cotransfected with an expression plasmid for GFP together with either RNAi to Mirk in the pSilencer plasmid (Si-Mirk), a mutant RNAi (Mutant), or the pSilencer vector (Vec; Fig. 4B). After 24 hours of expression, cells were cultured in low serum differentiation medium, and the DNA breaks in the apoptotic cells were labeled by the TUNEL reaction. Fluorescence microscopy was used to simultaneously determine the number of GFP-expressing cells, which were also labeled with the TUNEL marker. Depletion of Mirk significantly increased the fraction of transfected RD cells undergoing apoptosis by 60% (Fig. 4B, P < 0.0001).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Rhabdomyosarcoma is the most common soft tissue sarcoma in children and is difficult to treat if the primary tumor is nonresectable or if the disease presents with metastases (12). There are two major histologic types: embryonal and alveolar. Alveolar histology is associated with a significantly worse prognosis with a 5-year survival rate of <30%. The precise etiology of rhabdomyosarcoma is unknown, but it has been suggested to arise in "satellite" cells, the committed skeletal muscle precursor cells (12). The serine/threonine kinase Mirk/Dyrk1B was expressed to some extent in each clinical case of human rhabdomyosarcoma examined (Table 1) and in myoblast satellite cells (3). Furthermore, Mirk was found to be an active kinase in each rhabdomyosarcoma cell line tested (Fig. 3). Some insight into the possible role of Mirk in rhabdomyosarcoma can be derived from our earlier studies of Mirk in skeletal muscle myoblasts. Mirk is expressed in skeletal muscle satellite cells in primary culture and is up-regulated about 10-fold when the satellite cells are induced to differentiate, whereas knockdown of endogenous Mirk by RNAi blocks myoblast differentiation (3). Mirk is activated by the stress-activated MAP kinase kinase MKK3 (6). These results together imply a role for Mirk in the response to cellular injury. Skeletal muscle is regenerated after injury by activation of quiescent satellite cells, which enter the cell cycle and then differentiate and fuse with uninjured muscle fibers to repair the damage. Mirk is likely to play a key role in muscle regeneration because Mirk is a stress-activated kinase that modulates the activation of the myogenic regulatory factors MEF2 and myogenin, which subsequently mediate myoblast differentiation (7). Mirk is less likely to play as significant a role in embryonic muscle development because a Mirk/Dyrk1B knockout mouse survived to 18 days after conception, during which time skeletal muscles were developed (9). Thus, Mirk/Dyrk1B may be most critical as a survival factor in skeletal myoblasts undergoing repair.

This study has shown that Mirk also functions as a survival factor in rhabdomyosarcomas. Depletion of endogenous Mirk levels by RNAi reduced the clonogenicity of RD embryonal rhabodmyosarcoma cells and Rh30 alveolar rhabdomyosarcoma cells 3- to 4-fold in multiple colony formation experiments. These reductions in viability of the cells most capable of proliferation, the colony-forming cells, indicate that Mirk is a survival factor in these tumors. Mirk/Dyrk1B was recently identified as a survival-mediating kinase in HeLa cervical carcinoma cells (11). These investigators used an RNAi screen against all the known and predicted kinases in the human genome. Mirk and its family member Dyrk3 were detected in this screen. Depletion of these kinases in HeLa cells increased cell death by apoptosis.

There is a growing awareness that survival pathways are essential for tumor viability and aggressiveness. Recent work analyzed the successful treatment of non–small cell lung cancer targeted by small-molecule inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase activity. Multiple signaling pathways are activated by the EGFR. However, the drug gefitinib did not block the signaling pathways, which initiated tumor cell growth, but instead targeted tumor cell survival pathways mediated by the kinase Akt and the transcriptional activator STAT3 (signal transducers and activators of transcription 3) (14). It has also recently been shown that prostate cancer cells develop redundancy in downstream signaling pathways mediating cell survival compared with normal cells (15). Normal prostate cells required only simultaneous ERK and Janus-activated kinase (JNK) signaling for survival, whereas malignant cells used ERK-, JNK-, p38-, and Akt-initiated pathways for survival. The Mirk-induced survival pathway may provide a strong selective pressure to maintain expression of Mirk in rhabdomyosarcoma and complement other survival pathways activated by growth factors, such as fibroblast growth factors.

Mirk mediates cell survival in rhabdomyosarcomas through its antiapoptotic function, as shown by both the TUNEL assay and Annexin V labeling of exposed phosphatidylserine. Mirk mediates cell survival in nontransformed myoblasts (10) and colon carcinoma cells (1). Mirk helps myoblasts to survive through localization of its substrate p21cip1 to the cytoplasm, where p21 can block various proapoptotic proteins. Possibly, Mirk and p21cip1 function cooperatively in adult skeletal muscle in some stress signaling pathway, perhaps by limiting the effects of normal exercise induced changes in tissue osmolality, pH, oxygenation, and/or nutrient balance. This antiapoptotic capacity is retained by Mirk expressed in rhabdomyosarcomas, although Mirk may target other proteins in these tumors.

Many genetic lesions have been associated with rhabdomyosarcoma, including deletion of the CDK inhibitor p16ink4a, mutations in p53, overexpression of Met, and translocations which create Pax:Forkhead (Fkht) chimeric transcription factors (12). Recent studies have evaluated these lesions in murine models of rhabdomyosarcoma. Alveolar rhabdomyosarcoma is characterized by 2:13 or 1:13 chromosomal translocations, which juxtapose Pax3 or Pax7 with Fkht to create chimeric, highly active transcriptional activators (12). However, a Pax3:Fkht knock-in allele targeted to terminally differentiating Myf6-expressing skeletal muscle led to alveolar rhabdomyosarcoma only at a very low frequency (1 in 228 animals at 12 months; ref. 16). A much higher incidence of alveolar rhabdomyosarcoma was seen in compound mutant mice bearing homozygous Pax3:Fkht and either mutant p53 or conditional knockout of Ink4a/Arf (16), the gene which encodes the p16ink4a CDK inhibitor and p19arf. The p19arf knockout mouse does not give rise to rhabdomyosarcoma (17), indicating that the function of p16ink4a, not p19Arf, must be altered for the induction of rhabdomyosarcoma. These studies show that the Pax3:Fkht chimera is a weak oncogene by itself and must cooperate with either p16ink4a or with p53 to cause the onset of rhabdomyosarcoma.

The major role for Pax3:Fkht in rhabdomyosarcoma may be to induce transcription of Met. The Met proto-oncogene is often amplified or overexpressed in human rhabdomyosarcoma (12). Met is the receptor for hepatocyte growth factor, also called scatter factor (HGF/SF). Expression of the HGF/SF transgene in the Ink4a/Arf knockout mouse led to rhabdomyosarcoma with an extremely high penetrance and short latency (18). Virtually all of the mice developed rhabdomyosarcoma within 3 months. This striking observation, together with the necessity for loss of the Ink4a/Arf locus for efficient rhabdomyosarcoma induction in the Pax3:Fkht knock-in mouse model, point to the p16inka CDK inhibitor as a critical target for oncogenesis in rhabdomyosarcoma. Before it differentiates, the skeletal myoblast undergoes a cell cycle arrest in G0, which is mediated by the CDK inhibitor p21 and the retinoblastoma protein (19). Abrogation of this arrest in G0 by deletion of the p16Ink4a/Arf gene seems to be a necessary precondition for induction of rhabdomyosarcoma. Mirk aids in the maintenance of G0 arrest of differentiating nontransformed myoblasts by destabilizing cyclin D1 by phosphorylation at T288 (5), a site conserved in all cyclin D isoforms, and by stabilizing the CDK inhibitor p27 by phosphorylation at S10 (4). Deletion of the p16Ink4a/Arf gene, whose protein product inhibits cyclin D/CDK complexes, would abrogate Mirk's destabilization of cyclin D and enable rhabdomyosarcomas to maintain Mirk expression to use the survival function of Mirk.


   Acknowledgments
 
Grant support: NIH grant RO1 CA67405 (E. Friedman).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
1 X. Deng and E. Friedman, in preparation. Back

Received 5/ 3/05. Revised 1/ 3/06. Accepted 2/28/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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