Clinical significance of myosin in colorectal cancerLucía González, MSca, Noemí Eiró, MSca, Salomé González-Reyes, PhDa,
Alejandro Andicoechea, MD, PhDa,b, Luis O. González, MDa,c,José Luis García-Muñiz, MDa,d, Francisco J. Vizoso, PhD, MDa,b,⁎
aUnidad de Investigación, Fundación Hospital de Jove, 33290 Gijón, SpainbServicio de Cirugía General, Fundación Hospital de Jove, 33290 Gijón, Spain
cServicio de Anatomía Patológica, Fundación Hospital de Jove, 33290 Gijón, SpaindServicio de Cirugía General, Hospital Central de Asturias, 33290 Oviedo, Spain
Abstract Myosin has raised an interest in cancer research because of its role in tumor progression. The aim of
⁎ Corresponding a33290 Gijón, Asturias
E-mail address: in
1092-9134/$ – see frodoi:10.1016/j.anndiag
this study was to investigate the expression and clinical relevance of myosin in colorectal cancer(CC). Myosin was detected in CC tumors with recurrence using matrix-assisted laser desorption/ionization time-of-flight analysis. An immunohistochemical study was performed using tissue arraysand specific antibodies against myosin heavy chain. Determinations on cancer specimens from 91patients with resectable CCs were performed. The minimum follow-up period was of 12.5 years forthese patients without tumor recurrence. Western blot and real-time polymerase chain reactionanalysis were also performed. Samples of carcinomas with recurrence showed an increasedexpression of myosin. Tumors with high myosin expression by tumor cell were significantlyassociated with higher probability of metastasis. Our results suggest that myosin expression in CCs isassociated with tumor progression and metastasis development. Therefore, myosin tumor expressionmay contribute to an improved prognostic evaluation in patients with CC.
Colorectal carcinoma (CC) is the fourth most commoncancer in men and the third most common in women,accounting for approximately 1 million new cases per year inthe world [1]. Early detection, adequate surgical excision,and optimal adjuvant treatment are of critical importance foroutcome. However, it is difficult to predict the prognosisbecause CC is a heterogeneous disease. For all these reasons,new prognostic factors are indispensable to improve theclassic risk classification in CC.
Tumor invasion and metastasis development are theprimary determinants of patient outcome, and accordingly,molecules involved in these processes are obvious candidatesto be identified as new prognostic markers in CC. Metastasis
uthor. Servicio de Cirugía General, Hospital de Jove,. Tel.: +34 985320050; fax: +34 [email protected] (F.J. Vizoso).
formation is a complex process, involving invasion, transport,arrest, adherence, extravasation, and tumor cell proliferation.Fundamentally, this process involves the movement of cellsfrom one site to another. A molecular depiction of cellmigration in vitro models has emerged, which involvesdynamic cytoskeletal changes, cell-matrix interactions, local-ized proteolysis, actin-myosin contractions, and focal contactdisassembly [2]. Proteins implicated in cell migration play acritical role in these processes. The cancer cells can use 2distinct and interchangeable modes of motility referred to asmesenchymal and amoeboid migration [3].
Myosins are a large family of molecular motor proteinsimplicated in amoeboid motility, and their immunoex-pression has previously been demonstrated in variety ofepithelial cancer cells [4,5]. The well-characterized biologicfunction of myosins is their ability to use the energy ofadenosine triphosphate hydrolysis to move actin filamentsand produce muscle force. Thus, myosins play fundamentalroles in many forms of eukaryotic motility such as cellcrawling, cytokinesis, phagocytosis, growth cone extension,
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maintenance of cell shape, and organelle/particle trafficking[6]. Nevertheless, there are also data indicating that myosinsare implicated also in a variety of other cellular functionsthat are relevant for cancer formation, such as proliferationand migration of cancer cells [7-9]. In this sense, it wasreported that MYH11 (encodes the smooth-muscle myosinheavy chain) mutations appear to contribute also to humanCC formation. Unregulated MYH11 may affect the cellularenergy balance or disturb cell lineage decisions in tumorprogenitor cells [10,11]. Therefore, these data change theview on MYH11 as a passive differentiation markerfunctioning in muscle contraction and add to our under-standing of intestinal neoplasia.
The purpose of the present study was to investigatethe expression of myosin in CC as well as its relation-ship with prognosis. To address these questions, we usedthe Western blot, real-time polymerase chain reaction(PCR), tissue microarrays technology, and immunohisto-chemical techniques.
2.1. Patient selection, patient characteristics, and tissuespecimen handling
Tumor samples were obtained, at the time of surgerybetween 1980 and 1992, from 91 consecutive patients withresectable CC (R0 according to recommendations of theUnion International Contra la Cancrum [12]), whose clinicalfeatures are listed in Table 1. Tumors were staged accord-ing to Dukes' classification [13]. Adjuvant therapy with5-fluoruracil and levamisole was given to patients with DukesC tumors, and locoregional radiotherapy was also given tothose with rectal tumors. All patients were followed up fordisease status by clinical and biologic studies every 3 monthsfor the first 2 years and annually thereafter. Radiologic studieswere performed annually or when considered necessary.
Of these patients with resectable CCs, 40 developed tumorrecurrence (24with distant metastases, 7 with local recurrence,and 9 with both types of tumor recurrence), and 34 of themdied of recurrence. The median follow-up period in patientswithout tumor recurrencewas 152.9months and 40.58monthsin patients with tumor recurrence. The study adhered tonational regulations and was approved by the hospital ethicsand investigation committee. Tissue samples were obtainedwith prior informed consent from the patients.
2.2. Preparation of cell and membrane extracts
Human colorectal tissue was homogenized with a Douncehomogenizer in hypotonic homogenization buffer containing25 mmol/L HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; pH 7.4), 4 mmol/L EDTA, 250mmol/L sucrose, and a protease inhibitor cocktail (RocheDiagnostics, Basel, Switzerland). Intact cells and nuclei in theresulting extract were sedimented by centrifugation at 4°C for
5 minutes at 6000g (Allegra 64R Centrifuge; BeckmanCoulter, Brea, CA, USA). The membranes were sedimentedfrom the supernatant by a further spin at 20.000g for 30minutes at 4°C and resuspended in homogenization buffer.Protein concentration was measured by the Bradford assayusing bovine serum albumin (BSA) as a standard. All extractswere stored at −20°C until use.
2.3. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gelelectrophoresis) and protein electrotransfer
Samples were separated by SDS-PAGE using 10%polyacrylamide gels and run at a constant 120 V (Mini-Protean 3; Bio-Rad, Hercules, CA, USA). A Semi-PhorTE70 apparatus (Hoefer Scientific Instruments, San Fran-cisco, CA, USA) was used to electrotransfer proteins tonitrocellulose membranes at 160 mA for 1 hour in transferbuffer (0.248 mol/L Tris/HCl [pH 8.8], 1.92 mol/L glycine,and 20% methanol).
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2.4. In-gel digestion protein and analysis matrix-assistedlaser desorption/ionization time-of-flight
Protein bands were excised manually and digestedautomatically using a Proteineer DP protein digestion station(Bruker-Daltonics, Billerica, MA, USA). The digestionprotocol used was that of Shevchenko et al [14] withminor variations: gel plugs were reduced with 10 mmol/Ldithiothreitol (Amersham Biosciences, Piscataway, NJ,USA) in 50 mmol/L ammonium bicarbonate (99.5% purity;Sigma Chemical Co, St. Louis, MO, USA) and alkylatedwith 55 mmol/L iodoacetamide (Sigma Chemical Co) in 50mmol/L ammonium bicarbonate. Matrix-assisted laserdesorption/ionization (MALDI) mass spectrometry (MS) orMS/MS data were obtained in an automated analysis loopusing an Ultraflex time-of-flight (TOF) mass spectrometer(Bruker-Daltonics) equipped with a LIFT-MS/MS device[15]. Spectra were acquired in the positive-ion mode at 50Hz laser frequency, and 100 to 1000 individual spectra wereaveraged. For fragment ion analysis in the TOF/TOF mode,precursors were accelerated to 8 kV and selected in a timedion gate. Fragment ions generated by laser-induced decom-position of the precursor were further accelerated by 19 kVin the LIFT cell, and their masses were analyzed afterpassing the ion reflector. Automated analysis of mass datawas performed using Flex Analysis software (Bruker-Daltonics). Matrix-assisted laser desorption/ionization–MSand MS/MS data were combined through the BioToolsprogram (Bruker-Daltonics) to search a nonredundantprotein database (NCBInr: approximately 4.86106 entries,National Center for Biotechnology Information; or Swiss-Prot: approximately 2.66105 entries, Swiss Institute forBioinformatics) using Mascot software (Matrix Science,London, UK). Matrix-assisted laser desorption/ionization–MS (/MS) spectra and database search results were inspectedmanually in detail using the above programs as well as usingsoftware produced in house [16].
2.5. Western blot
Colorectal samples were separated by SDS-PAGE using10% polyacrylamide gels and run at constant 120 V (Mini-Protean Tetra Electrophoresis System; Bio-Rad). Thetetraprotean transference kit was used to electrotransferproteins to nitrocellulose membranes at 160 mA for 1 hourin transfer buffer (0.248 mol/L Tris pH 8.8, 1.92 mol/Lglycine, and 20% methanol). The nitrocellulose membranescontaining the transferred proteins were blocked with 1%nonfat dry milk in Tris-buffered saline (TBS) for 1 hourand then rinsed 3 times in TBS. The membranes wereincubated for 2 hours at room temperature with 1 of thesemonoclonal antibodies: antimyosin (IS-066) (Dako,Glostrup, Denmark) and anti–β-actin (sc-47778) (SantaCruz Biotechnology, Santa Cruz, CA, USA) diluted in TBScontaining 1% nonfat dry milk. The blots were thenwashed with TBS and incubated with protein A peroxidase,and the reactive protein bands were visualized by
chemiluminiscence (Pierce ECL Western Blotting Substrate,Rockford, Ill).
2.6. Real-time PCR
Total RNA was isolated from 5 colorectal tissue samplesof patients who had recurrence and 5 colorectal cancer tissuesamples from patients who did not have recurrence usingthe NucleoSpin FFPE RNA (Macherey-Nagel, Düren,Germany), including DNase treatment. The integrity of theeluted total RNAwas checked by agarose gel electrophoresis,and the RNA concentration was determined spectrophoto-metrically. The ratio of absorbance at 260 and 280 nm,measured in a NanoDrop ND-1000 spectrophotometer(Thermo Scientific, Bremen, Germany), was used as aparameter that allowed us to quantify and evaluate the qualityof the total RNA extracted (values ranging from 2.07 to 2.35).First-strand complementary DNA (cDNA) was made usingthe High Capacity cDNA Reverse Transcription kit (AppliedBiosystems, Cheshire, UK) following the manufacturer'sinstructions. The reverse transcription step was carried usingthe following program: 25°C for 10 minutes, 37°C for 120minutes, and 85°C for 5 seconds. Expression of myosin andβ-actin expression levels were assessed by real-time PCRusing ABI Prism 7900 HT thermocycler (Applied Biosys-tems) and the Fast SYBR Green Master Mix (AppliedBiosystems) with the following cycling conditions: 95°C for20 seconds, 40 cycles of 95°C for 1 second, and 60°C for20 seconds. The primers used were 5′-GGAGGATAGATCCTGGTCA-3′(forward) and 5′-TTAGCCGCACTTCCAGTTCT-3′(reverse) and 5′-GGCACCCAGCACAATGAAG-3′ (forward) and 5′-CCGATCCACACGGAGTACTTG-3′(reverse) for β-actin. All real-time PCRs were performed intriplicate, and the amplification signal from the target wasnormalized using β-actin as control. SDS RQ ManagerProgram (Applied Biosystems) was used to analyze theresults. The PCR products were separated on 2% agarosegels containing ethidium bromide (0.5 μg/mL).
2.7. Immunohistochemistry
Colon carcinoma tissue samples were obtained at the timeof surgery. All specimens were routinely fixed in 10%neutral-buffered formalin and embedded in paraffin at roomtemperature. Histopathologic representative tumor areaswere defined on hematoxylin and eosin–stained sectionsand marked on the slide.
Serial 5-μm sections were consecutively cut with amicrotome (Leica Microsystems GmbH, Wetzlar, Germany)and transferred to adhesive-coated slides. Tissue sectionswere deparaffinized in xylene and then rehydrated in gradedconcentrations of ethyl alcohol (100%, 96%, 70%) andwater. Immunohistochemistry was done using a TechMateTM50 autostainer (Dako, Glostrup, Denmark). Monoclonalantibody for Myosin (IS-066) was obtained from Dako readyto use (no dilution). Endogenous peroxidase activity wasblocked by incubating the slides in peroxidase-blocking
Fig. 1. Matrix-assisted laser desorption/ionization–MS result. Homogenates of human colorectal tissue were separated by SDS-PAGE; differential protein bandswere analyzed by MALDI-MS. (A) Myosin was detected as a differential band of 200 kd. (B) Representative Western blots of immunoreactive myosin ofcarcinoma with (R) and without recurrence (NR). (C) Myosin gene expression measured by semiquantitative real-time PCR in 10 CCs. The graphic shows thepercentage of myosin expression in recurrence samples (R) and no recurrence ones (NR). The housekeeping used was β-actin. Data represent the mean ± SD of 3independent experiments.
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solution (Dako) for 5 minutes. The EnVision Detection Kit(Dako) was used as the staining detection system. Sectionswere counterstained with hematoxylin, dehydrated withethanol, and permanently coverslipped.
For each preparation studied, the location of immunore-activity for myosin, percentage of stained cells, and intensitywere determined. All the cases were semiquantified for eachprotein-stained area. An image analysis system with theOlympus BX51 microscope and analysis soft (analysis; Softimaging system, Münster, Germany) was used as follows:tumor sections were stained with the antibody according tothe method explained above and counterstained withhematoxylin. There are different optical thresholds for bothstains. Each preparation was scanned with a 400× powerobjective in 4 fields. Fields were selected searching for theprotein-stained areas. The computer program selects andtraces a line around antibody-stained areas (higher opticalthreshold: red spots), with the remaining, nonstained areas(hematoxylin-stained tissue with lower optical threshold)standing out as a blue background. Any field has an arearatio of stained [17] vs nonstained areas (blue). A final arearatio was obtained after averaging 4 fields. To evaluateimmunostaining intensity, we used a numeric score rangingfrom 0 to 3, reflecting the intensity as follows: 0, no staining;
1, weak staining; 2, moderate staining; and 3, intensestaining. Using an Excel spreadsheet (Microsoft Corp,Kansas City, MO, USA), the mean score was obtained bymultiplying the intensity score (I) by the percentage ofstained cells (PC), and the results were added together (totalscore: I × PC). This overall score was then averaged with thenumber of fields that were done for each patient.
2.8. Data analysis and statistical methods
Differences in percentages were calculated with the χ2
test. Comparison of immunostaining values between groupswas made with the Mann-Whitney U or Kruskal-Wallis test.For metastasis-free survival analysis, we used the Coxunivariate method. Cox regression model was used toexamine interactions of different prognostic factors in amultivariate analysis. The PASW Statistics 18.0 program(International Business Machines Corp, Armonk, NY, USA)was used for all calculations.
3. Results
To search differences between CCs that develop tumorrecurrence and those ones without tumor recurrence, we
Fig. 2. Example of immunohistochemical staining for myosin in CC sample(original magnification ×200).
Table 2Relationship between myosin expression and clinicopathologic characteristicsin 91 patients with colorectal cancer
Clinicopathologic characteristics No. of cases Myosin, mean(median) [range]
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prepared extract of proteins. Homogenates of 5 humancolorectal tumors were separated by SDS-PAGE. Gel slicescontaining the reacting proteins were digested with trypsin,and the resulting peptides were subjected to MS analysis.The peptide mass fingerprints were then used to search thedatabases to identify the corresponding proteins. Mass listwas submitted for a search against the NCBI protein databaseusing the Mascot search engine (Perkins Engines, Cam-bridgeshire, UK). The sample containing the 200-kd reactingprotein was characterized as myosin (Fig. 1A). Thespecificity of this reaction was also supported by the resultsobtained in a parallel Western blot analysis using antimyosinantibody (Fig. 1B), which reacted specifically with a 200-kdprotein present in the membrane fraction, and real-time PCRand showed the percentage of myosin cDNA expression insamples obtained from patients with CC with and withouttumor recurrence (Fig. 1C).
Oncemyosinwas identified, from protein extracts of tumors,a study was conducted to check the expression of this protein intumors from 91 patients with CC. Fig. 2 shows a cytoplasmicimmunostaining of myosin in positive cancerous cells from 36CCs (39.5%). Myosin immunostaining score values rangedwidely among tumors (median, 0; range, 0-122.36).
We evaluated the possible relationship between the myosinexpression and the clinicopathologic factors of CC includingage, sex, tumor location, tumor stage, and histologic grade.Wefound no significant associations ofmyosin expressionwith allof these factors (Table 2). However, myosin expression wassignificantly associated with a high rate of tumor recurrence(Table 2). In addition, univariate analysis demonstrated thatmyosin expression was significantly associated with ashortened overall survival in patients (Fig. 3).
Fig. 3. Probability of overall survival as function of myosin median(P = .024).
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Multivariate analysis according to Cox model demon-strated that lymph node involvement (positive: relative riskconfidence interval, 2.6 [1.3-5.1]; P = .006) and histologicgrade (II, 2.29 [0.68-7.72]; III, 7.12 [1.52-33.3]; P = .038)were significantly and independently associated with overallsurvival. However, this same analysis also demonstrated thatmyosin expression was an independent factor associatedwith overall survival (score, myosin N median: 2.61 [1.29-5.25]; P = .007).
4. Discussion
To our knowledge, this is the first study that analyzes theexpression of myosin in primary CC as well as its prognosticsignificance in patients with CC. The results demonstrate anassociation between myosin expression by tumors and pooroutcome from patients with CC.
Our results, showing high messenger RNA and proteinexpression in a significant percentage of CCs, are inaccordance with several studies indicating overexpression ofmyosins in human cancer cells of different origins, suchas prostate cancer [18], melanoma [19], breast cancer [20],pancreatic cancer [8], gastric cancer [21], or colorectal cancer[22]. On the other hand, our data, showing a positiveassociation between myosin expression and tumor recurrencein patients with CC, are in accordance with these results ofexperimental studies indicating that myosin expressions areassociated with different aspects related with tumor aggres-siveness, such as motility and adhesion or proliferation.
Invasion and metastasis of colorectal cancer require cellmotility and adhesion, which depend on the activity ofcytoskeleton. There is now increasing evidence that myosinmotor proteins, together with the dynamic actin filamentmachinery and associated adhesion proteins, play crucialroles in the events leading to motility at leading edge ofmigrating cells [23].
Myosins exist as a large superfamily of diverse adenosinetriphosphate–dependent motors. In the present study, wefound the clinical interest of the determination of the totalintratumoral myosins. Nevertheless, it is of note that thereare several reports indicating a role of different myosins intumor progression and thereby as new potential therapeutictarget in several tumors. Thus, it has been shown thatdepletion of myosin II decreased cell migration and invasionin melanoma cells [19] and pancreatic cancer cells [8] aswell as in colonic epithelial cells [24]. Myosin light-chainkinase contributes to the proliferation and migration ofbreast cancer cells through cross-talk with activated ERK1/2[9]. In addition, based on experimental studies, it has beenreported that myosin VI is a potential therapeutic target forprostate cancer because it could be used as a modulator ofandrogen receptor-dependent gene expression [18].
With regard to studies with colorectal cancer cells, thereare also data supporting our clinical findings. Thus, themessenger RNA expression of myosin Va was found
increased in several highly metastatic cancer cell lines andmetastatic colorectal cancer tissues, and the expression ofmyosin Va by lentivirus-based RNA interference inhighly metastatic cancer cells impeded their migration andmetastasis capabilities both in vitro and in vivo [22]. Inthis latter study, the levels of myosin Va in cancer celllines were positively correlated with the expression ofSnail, a transcriptional repressor that triggers epithelial-mesenchymal transition. Repression or overexpression ofSnail in cancer cells caused reduced or elevated levels ofmyosin Va, respectively. Likewise, it has also beenreported that nm23-H1, a gene known as a potentialmetastasis suppressor gene in various types of carcinomas,reduces in vitro cell migration and the liver metastaticpotential of colon cancer cells by regulating myosin light-chain phosphorylation [25].
Another interesting aspect related with the role ofmyosins favoring tumor aggressiveness is related withtheir potential role in cellular proliferation. It is known thatcancer cells often have unstable genomes and increasedcentrosome and chromosome numbers, which are animportant part of malignant transformation in the mostrecent model of tumorigenesis. However, very little is knownabout divisional failures in cancer cells that may lead tochromosomal and centrosomal amplifications. Recently, ithas been shown that deficiency in myosin light-chainphosphorylation causes cytokinesis failure and multipolarityin cancer cells [26]. In addition, it was reported thatmyosin light-chain kinase is responsible for high prolifera-tive ability of breast cancer cells via antiapoptosis involvingp38 pathway [20].
Our results suggest that up-regulation of myosin expres-sion is associated with tumor progression in CC, which is inaccordance with recent data of experimental studies showingthat myosins are associated with tumor growth. Therefore,myosin tumor expression may contribute to an improvedprognostic evaluation, and it might be useful as a prognosticindex in CC.
Acknowledgments
This work was supported by grants from Fondo deInvestigación Sanitaria del Instituto Carlos III (FIS-PI070306) and FICEMU. The authors declare that theyhave no conflict of interest.
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