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Research Papers:

The tumor suppressor capability of p53 is dependent on non-muscle myosin IIA function in head and neck cancer

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Oncotarget. 2017; 8:22991-23007. https://doi.org/10.18632/oncotarget.14967

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Sonya D. Coaxum, Jessica Tiedeken, Elizabeth Garrett-Mayer, Jeffrey Myers, Steven A. Rosenzweig and David M. Neskey _

Abstract

Sonya D. Coaxum1, Jessica Tiedeken2, Elizabeth Garrett-Mayer3, Jeffrey Myers4, Steven A. Rosenzweig2, David M. Neskey1,2

1Department of Otolaryngology, Head and Neck Surgery, Medical University of South Carolina, Charleston, SC, USA

2Department of Cell and Molecular Pharmacology & Experimental Therapeutics, Medical University of South Carolina, Charleston, SC, USA

3Department of Public Health Sciences and Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, USA

4Department of Head & Neck Surgery, M.D. Anderson Medical Center, Houston, TX, USA

Correspondence to:

David M. Neskey, email: Neskey@musc.edu

Keywords: head and neck squamous cell carcinoma, TP53, non-muscle myosin IIA, tumor suppressor

Received: July 18, 2016    Accepted: January 10, 2017    Published: February 01, 2017

ABSTRACT

Over 300,000 patients develop squamous cell carcinoma of the head and neck (HNSCC) worldwide with 25-30% of patients ultimately dying from their disease. Currently, molecular biomarkers are not used in HNSCC but several genes have been identified including mutant TP53 (mutp53). Our recent work has identified an approach to stratify patients with tumors harboring high or low risk TP53 mutations. Non-muscle Myosin IIA (NMIIA) was recently identified as a tumor suppressor in HNSCC. We now demonstrate that low MYH9 expression is associated with decreased survival in patients with head and neck cancer harboring low-risk mutp53 but not high-risk mutp53. Furthermore, inhibition of NMIIA leads to increased invasion in cells harboring wildtype p53 (wtp53), which was not observed in high-risk mutp53 cells. This increased invasiveness of wtp53 following NMIIA inhibition was associated with reduced p53 target gene expression and was absent in cells expressing mutp53. This reduced expression may be due, in part, to a decrease in nuclear localization of wtp53. These findings suggest that the tumor suppressor capability of wtp53 is dependent upon functional NMIIA and that the invasive phenotype of high-risk mutp53 is independent of NMIIA.


INTRODUCTION

Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide and affects over 60,000 patients annually in the US [1]. Treatment of advanced HNSCC requires complex, multimodality therapy, employing either definitive radiation with or without chemotherapy or surgical resection and post-operative radiation, with chemotherapy for patients with high-risk of recurrence [2, 3]. Currently, there are no molecular biomarkers to guide these management decisions. Multiple studies have demonstrated TP53 mutations are prognostic for poor outcomes in HNSCC, yet molecular testing for TP53 alterations has not become routine [4-8]. Our previous work developed and validated a novel method, EAp53, which can stratify patients with tumors harboring TP53 mutations as low or high risk which is an extension of the Evolutionary Trace (ET) approach, an extensively validated method to identify key functional or structural residues in proteins [9]. In an effort to predict which TP53 mutations are highly deleterious every sequence position is assigned a grade of functional sensitivity to sequence variations, defined by whether its evolutionary substitutions correlate with larger or smaller phylogenetic divergences. Residues with large ET grades typically cluster structurally into evolutionary ‘hot-spots’ that overlap and predict functional sites [10].

We have demonstrated that the ET method could assess the impact of TP53 missense mutations. The impact was shown to be greater when the mutated residues were more evolutionarily sensitive to sequence variations, i.e. have a larger ET grade, and also when the amino acid change was least conservative, so the mutational impact is the largest. These two components were computed and combined into a single score, called Evolutionary Action EA [11]. To apply this Evolutionary Action to TP53 mutations in HNSCC, we further developed a scoring system (EAp53) to stratify TP53 missense mutations into high and low risk. The subset of oncogenic or high-risk p53 mutations was associated with decreased survival in patients with HNSCC and increased cellular invasion and tumorigenicity [12]. In contrast, low-risk p53 mutations appeared to have retained some p53 function since patients with HNSCC containing these alterations had similar survival outcomes to wildtype p53 and cells had an intermediate level of invasiveness and tumorigenicity [12].

Class 2 myosins include a family of three nonmuscle myosins that are implicated in force generation and cell migration [13, 14]. Class 2 non-muscle myosins are hexameric molecules, comprised of a pair of heavy chains, a pair of essential light chains, and a pair of regulatory light chains (RLCs). The distinction between the three myosin II molecules is their unique heavy chain isoforms but each functions through the binding and contracting of F-actin in an ATP-dependent manner. MYH9 encodes the heavy chain of nonmuscle myosin IIA protein (NMIIA). Depletion or inactivation of NMIIA consistently leads to an increase in polarized lamellipodia formation and migration (wound healing) with a concomitant decrease in non-polarized, blunt, cylindrical protrusions or lobopodia (cellular protrusions that share functional attributes with lamellipodia and membrane blebs) formation and focal adhesions [15]. This increase in cell migration following suppression or loss of NMIIA function appears to be due to microtubule stabilization and expansion into lamellae, which can be detected by increased acetylation of α-tubulin in epithelial cells [16]. In NMIIA depleted cells, stabilized microtubules within lamellae may be driving migration through activation of Rac1 leading to enhanced actin polymerization at the leading edge [16]. This mechanism of increased migration through NMIIA suppression can be translated clinically as patients with decreased MYH9 expression have an associated decrease in overall survival [17]. Therefore, further investigation of NMIIA’s role in microtubule regulation will be significant by providing the foundation for treatment strategies targeting actively migrating cells.

In addition to NMIIA’s role in cell migration, it has also been identified as a tumor suppressor that can modulate wildtype p53 (wtp53) expression. The inhibition or suppression of NMIIA leads to decreased p53 nuclear accumulation and subsequent decreases in expression of downstream target genes [17]. To date, whether the tumor suppressor capability of p53 is dependent on the function of NMIIA remains unknown. Furthermore, the tumor suppressor characteristics of NMIIA in the context of mutated p53 have yet to be studied. The phenotypic similarities between high-risk mutp53 and NMIIA depleted cells suggests their common oncogenic phenotype may be due, in part, to loss of NMIIA’s tumor suppressor function. Therefore, the goal of this study was to determine whether loss of NMIIA function in wtp53 harboring cells reduces its tumor suppressor capability, leading to invasive cell behavior similar to that seen in high-risk mutp53.

RESULTS

MYH9 expression correlates with increased survival in patients with HNSCC having functional p53

Our previous work demonstrated in two cohorts totaling 264 patients, the novel EAp53 classification could identify high-risk p53 mutations associated with decreased survival in patients with head and neck cancer [12]. Furthermore, EAp53 identified low-risk p53 mutations that were similar to wildtype p53 and associated with improved survival outcomes and appear to retain some residual p53 function [12]. EAp53 was applied to the p53 sequence data and subsequently integrated with the MYH9 RNAseq expression data from The Cancer Genome Atlas Network Head and Neck Project (Table 1) [18]. This analysis revealed patients with low-risk mutp53 and low MYH9 expression (n=75) had decreased survival outcomes relative to patients with low-risk mutp53 and high MYH9 expression, p=.020 (n=27) (Figure 1A). High (n=70) or low (n=20) MYH9 expression was not prognostic in patients with high-risk p53 mutations (Figure 1B).

Table 1: TP53 mutations scored and stratified by EAp53 with MYH9 expression data from The Cancer Genome Atlas HNSCC Project

No.a

TCGA
IDb

P53
statusc

Mutationd

EA Scoree

EA Riskf

MYH9
expressiong

Lower 25th
percentileh

1

7250

Wildtype

NA

0

Low

19059.1436

Yes

2

6441

Wildtype

NA

0

Low

21185.7003

Yes

3

6871

Wildtype

NA

0

Low

22738.9068

Yes

4

4730

Wildtype

NA

0

Low

23025.1362

Yes

5

6939

Wildtype

NA

0

Low

25580.2255

Yes

6

4228

Wildtype

NA

0

Low

26217.3401

Yes

7

6938

Wildtype

NA

0

Low

30394.2155

Yes

8

7406

Wildtype

NA

0

Low

30611.831

Yes

9

7440

Wildtype

NA

0

Low

32681.4044

Yes

10

7631

Wildtype

NA

0

Low

32909.1267

Yes

11

7250

Wildtype

NA

0

Low

34016.2272

Yes

12

7261

Wildtype

NA

0

Low

34079.456

Yes

13

7068

Wildtype

NA

0

Low

36307.9777

Yes

14

7406

Wildtype

NA

0

Low

38976.1036

No

15

6954

Wildtype

NA

0

Low

39143.3037

No

16

6492

Wildtype

NA

0

Low

39459.7833

No

17

5243

Wildtype

NA

0

Low

40087.7311

No

18

6939

Wildtype

NA

0

Low

41136.5435

No

19

7632

Wildtype

NA

0

Low

41442.4973

No

20

7410

Wildtype

NA

0

Low

41670.6444

No

21

6938

Wildtype

NA

0

Low

42522.8748

No

22

5247

Wildtype

NA

0

Low

42815.4169

No

23

5625

Wildtype

NA

0

Low

42879.4466

No

24

7774

Wildtype

NA

0

Low

43144.2177

No

25

6955

Wildtype

NA

0

Low

44674.5453

No

26

5325

Wildtype

NA

0

Low

45864.7399

No

27

5355

Wildtype

NA

0

Low

49493.0351

No

28

5149

Wildtype

NA

0

Low

50893.2238

No

29

6227

Wildtype

NA

0

Low

52429.5174

No

30

7429

Wildtype

NA

0

Low

52733.2198

No

31

7373

Wildtype

NA

0

Low

53396.9971

No

32

7261

Wildtype

NA

0

Low

54162.1353

No

33

6010

Wildtype

NA

0

Low

54341.1575

No

34

7407

Wildtype

NA

0

Low

54573.2791

No

35

7392

Wildtype

NA

0

Low

55223.3527

No

36

7832

Wildtype

NA

0

Low

56462.9173

No

37

7427

Wildtype

NA

0

Low

61582.7367

No

38

7367

Wildtype

NA

0

Low

61906.8592

No

39

7395

Wildtype

NA

0

Low

63655.5958

No

40

5369

Wildtype

NA

0

Low

64139.3305

No

41

55565369

Wildtype

NA

0

Low

65945.6815

No

42

74405556

Wildtype

NA

0

Low

66238.6672

No

43

70857440

Wildtype

NA

0

Low

73182.8255

No

44

71837085

Wildtype

NA

0

Low

77566.3146

No

45

71837183

Wildtype

NA

0

Low

77566.3146

No

46

74117183

Wildtype

NA

0

Low

85911.796

No

47

60037411

Wildtype

NA

0

Low

91717.6666

No

48

73976003

Wildtype

NA

0

Low

94650.3973

No

49

74017397

Wildtype

NA

0

Low

102414.7452

No

50

40747401

Mutant

p.E258D

57.73

Low

14600.1858

Yes

51

6962

Mutant

p.Y236C

62.93

Low

20903.5941

Yes

52

5332

Mutant

p.P151S

64.12

Low

20955.9877

Yes

53

6225

Mutant

p.E224D

39.02

Low

21525.3994

Yes

54

4076

Mutant

p.Q136P

71.29

Low

22118.8508

Yes

55

5329

Mutant

p.R273H

66.12

Low

22833.8745

Yes

56

7245

Mutant

p.R282W

73.21

Low

24809.4695

Yes

57

5973

Mutant

p.R282W

73.21

Low

25385.2785

Yes

58

7424

Mutant

p.E271V

74.39

Low

25942.2319

Yes

59

7437

Mutant

p.S106R

21.82

Low

27579.5418

Yes

60

7423

Mutant

p.A159V

62.4

Low

27604.2023

Yes

61

6436

Mutant

p.R273H

66.12

Low

31219.2933

Yes

62

6962

Mutant

p.Y236C

62.93

Low

35420.671

Yes

63

6951

Mutant

p.Y234C

62.94

Low

35732.5631

Yes

64

7398

Mutant

p.R337L

61.55

Low

36115.016

Yes

65

4736

Mutant

p.I195T

72.13

Low

37253.7068

No

66

4740

Mutant

p.M237I

63.68

Low

37284.3268

No

67

6933

Mutant

p.P151H

71.97

Low

38325.6981

No

68

7238

Mutant

p.R273H

66.12

Low

39422.9096

No

69

7592

Mutant

p.Y220C, p.R110L

72.52, 28.14

Low

39492.7307

No

70

7630

Mutant

p.139_142KTCP>T, p.M1V

43.89

Low

40807.4754

No

71

5430

Mutant

p.R282W, p.P89fs

73.21,

Low

41157.1652

No

72

7424

Mutant

p.E271V

74.39

Low

41401.4767

No

73

7437

Mutant

p.S106R

21.82

Low

43367.2117

No

74

4739

Mutant

p.R337C

63.66

Low

44781.6908

No

75

5151

Mutant

p.V143M

51.72

Low

44811.7412

No

76

4217

Mutant

p.R158L

57.61

Low

44929.7646

No

77

7238

Mutant

p.R273H

66.12

Low

44945.2333

No

78

5978

Mutant

p.V172F

65.55

Low

45299.2832

No

79

7414

Mutant

p.E285K

69.87

Low

47003.5151

No

80

7235

Mutant

p.F270C, p.T211I

66.32, 68.48

Low

47573.0464

No

81

6013

Mutant

p.R282W

73.21

Low

48053.8936

No

82

7374

Mutant

p.R273H

66.12

Low

49671.3671

No

83

7099

Mutant

p.E285K

69.87

Low

49963.8243

No

84

5434

Mutant

p.Y236C, p.R213*

62.93

Low

53941.7304

No

85

4737

Mutant

p.H168L

62.62

Low

54291.7232

No

86

7235

Mutant

p.F270C, p.T211I

66.32, 68.48

Low

54443.1599

No

87

5334

Mutant

p.S166*, p.R158H

43.94

Low

55111.0587

No

88

7089

Mutant

p.Y163C

70

Low

60442.0202

No

89

6933

Mutant

p.P151H

71.97

Low

62108.8343

No

90

7394

Mutant

p.R273H

66.12

Low

64876.995

No

91

5629

Mutant

p.V157F

55.26

Low

66941.442

No

92

7423

Mutant

p.A159V

62.4

Low

67549.236

No

93

5366

Mutant

p.P151T

70.26

Low

71424.9734

No

94

7435

Mutant

p.Y220C

72.52

Low

72632.5124

No

95

7380

Mutant

p.R282W

73.21

Low

77702.8389

No

96

7588

Mutant

p.L137Q

64.66

Low

80069.0459

No

97

4733

Mutant

p.R273H

66.12

Low

80139.2593

No

98

7236

Mutant

p.V143M

51.72

Low

85991.952

No

99

7365

Mutant

p.V216M

73.3

Low

87008.6792

No

100

6221

Mutant

p.V272M

63.49

Low

98089.8266

No

101

7245

Mutant

p.R282W

73.21

Low

112385.1618

No

102

7090

Mutant

p.R273H

66.12

Low

128727.6925

No

103

5370

Mutant

p.R175H, p.Y126_splice

78.51,

High

17991.2352

Yes

104

6023

Mutant

p.G245S

86.45

High

20435.1948

Yes

105

7178

Mutant

p.C176Y, p.R110L

93.11, 28.14

High

22902.4691

Yes

106

5152

Mutant

p.G245S

86.45

High

24189.1074

Yes

107

6943

Mutant

p.R248W

84.11

High

24203.8835

Yes

108

7065

Mutant

p.H179P

98.89

High

24548.4018

Yes

109

6934

Mutant

p.Y205C

77.88

High

24737.1678

Yes

110

7242

Mutant

p.V173M

75.53

High

25359.3433

Yes

111

7254

Mutant

p.E258A

93.29

High

26404.9051

Yes

112

6959

Mutant

p.R248W

84.11

High

26977.7238

Yes

113

7418

Mutant

p.H179Y

77.78

High

29714.3994

Yes

114

7370

Mutant

p.C238S

86.53

High

31748.1805

Yes

115

6869

Mutant

p.C238F, p.R156P

96.54, 42.93

High

31750.7152

Yes

116

7399

Mutant

p.P278S, p.R213L

84.34, 90.71

High

33442.556

Yes

117

7082

Mutant

p.R248W

84.11

High

34283.7838

Yes

118

6936

Mutant

p.V173L

82.64

High

34347.8836

Yes

119

6935

Mutant

p.C242S

86.74

High

34914.732

Yes

120

7848

Mutant

p.E286V, p.P58fs

94.09

High

35968.0861

Yes

121

7413

Mutant

p.G105C

90.8

High

36065.8762

Yes

122

7263

Mutant

p.Y126C

81.09

High

36794.0111

Yes

123

5558

Mutant

p.R282W, p.R175H

73.21, 78.51

High

39755.88

No

124

6992

Mutant

p.Q331H, p.R249M, p.G245D

9.79, 95.41, 89.56

High

40108.2414

No

125

6870

Mutant

p.C242Y

93.46

High

40158.9134

No

126

5444

Mutant

p.R248Q, p.G245S

78.95, 86.45

High

40765.04

No

127

6936

Mutant

p.V173L

82.64

High

41410.0741

No

128

7242

Mutant

p.V173M

75.53

High

41666.9217

No

129

7248

Mutant

p.C242F

97.04

High

43282.1983

No

130

6945

Mutant

p.H193L

95.4

High

43425.0326

No

131

5431

Mutant

p.H193P, p.H179Y

92.46, 77.78

High

43788.9126

No

132

4725

Mutant

p.C275F

97.06

High

44576.836

No

133

6872

Mutant

p.R175H

78.51

High

45312.0393

No

134

6493

Mutant

p.C229fs, p.S127Y

87.62

High

45516.3524

No

135

7371

Mutant

p.R175H

78.51

High

45522.7596

No

136

5373

Mutant

p.G245V

98.74

High

45758.6207

No

137

7402

Mutant

p.R267P

88.48

High

45787.4794

No

138

6824

Mutant

p.K132N

92.16

High

45810.6559

No

139

6478

Mutant

p.H179R

81.91

High

45909.8192

No

140

7368

Mutant

p.R248Q

78.95

High

46323.4255

No

141

6935

Mutant

p.C242S

86.74

High

46893.4093

No

142

5331

Mutant

p.A307_splice, p.R280T

96.08

High

47291.6078

No

143

7416

Mutant

p.R248Q

78.95

High

47571.3318

No

144

7415

Mutant

p.M133K

93.62

High

47578.0949

No

145

4729

Mutant

p.H179R, p.V157F

81.91, 55.26

High

48687.3857

No

146

5966

Mutant

p.V173M

75.53

High

49092.3772

No

147

6218

Mutant

p.V218G, p.L194fs

89.92

High

49304.6974

No

148

7388

Mutant

p.R273C

84.52

High

49483.4242

No

149

7379

Mutant

p.G262V, p.Q136H

88.02, 47.50

High

49736.8282

No

150

6952

Mutant

p.C275F

97.06

High

49779.8418

No

151

5631

Mutant

p.E336*, p.G245S

86.45

High

50265.4975

No

152

6012

Mutant

p.Y126S

94.81

High

50330.1475

No

153

6020

Mutant

p.C176S

86.9

High

51044.6781

No

154

4723

Mutant

p.C242F

97.04

High

52741.6378

No

155

7376

Mutant

p.R280S, p.L32_splice

94.74

High

52830.2812

No

156

5436

Mutant

p.G266E, p.E56*

93.08

High

53609.38

No

157

6024

Mutant

p.L265R

84.18

High

54654.3939

No

158

7416

Mutant

p.R248Q

78.95

High

55950.4367

No

159

7372

Mutant

p.R248W

84.11

High

56103.6943

No

160

7219

Mutant

p.R196P

95.55

High

59325.7308

No

161

6011

Mutant

p.P278S, p.Y205fs

84.34

High

59562.9966

No

162

5365

Mutant

p.H193L

95.4

High

59633.7932

No

163

6491

Mutant

p.M237V, p.H179R

75.79, 81.91

High

61669.9501

No

164

6516

Mutant

p.G262V

88.02

High

61964.9147

No

165

6022

Mutant

p.S261_splice, p.R248W

84.11

High

62102.963

No

166

4738

Mutant

p.Q331*, p.H179Y

77.78

High

62189.1787

No

167

6220

Mutant

p.R280G

95.71

High

62341.3455

No

168

5367

Mutant

p.R273C, p.A161T

84.52, 58.51

High

62735.4238

No

169

7178

Mutant

p.C176Y, p.R110L

93.11, 28.14

High

64539.6273

No

170

7229

Mutant

p.R249S

93.65

High

65436.9925

No

171

5976

Mutant

p.Y236D

92.17

High

70703.2873

No

172

6018

Mutant

p.R248W

84.11

High

70937.0085

No

173

5970

Mutant

p.R248Q

78.95

High

75136.8374

No

174

5330

Mutant

p.G266R

91.41

High

77750.4254

No

175

6517

Mutant

p.S127F

88.07

High

83366.1651

No

176

6943

Mutant

p.R248W

84.11

High

83366.6878

No

177

7102

Mutant

p.G266E

93.08

High

84155.9398

No

178

7421

Mutant

p.R175H

78.51

High

84446.3616

No

179

5979

Mutant

p.R248Q

78.95

High

84769.3762

No

180

6994

Mutant

p.R283P, p.R175H

75.75, 78.51

High

85123.8984

No

181

6934

Mutant

p.Y205C

77.88

High

85532.0304

No

182

6224

Mutant

p.R175H

78.51

High

86128.7828

No

183

6959

Mutant

p.R248W

84.11

High

86287.8985

No

184

6873

Mutant

p.H193L, p.PHHERC177del

95.4

High

87361.6071

No

185

6016

Mutant

p.G245S

86.45

High

89802.686

No

186

6826

Mutant

p.V173G

93.47

High

95987.892

No

187

6868

Mutant

p.L194P

79.72

High

98740.1575

No

188

5555

Mutant

p.H193R

85.96

High

103716.6397

No

189

7389

Mutant

p.P278S

84.34

High

104961.0553

No

190

5326

Mutant

p.R249S, p.L32_splice

93.65

High

107124.1051

No

191

7753

Mutant

p.E286K

76.21

High

118776.7221

No

192

6474

Mutant

p.G245V

98.74

High

152088.3031

No

a. The number of patients included in the analysis

b. The short ID extracted from The Cancer Genome Atlas Head and Neck Project

c. P53 status delineated as either wildtype or mutant

d. Denotes the specific mutation for each patient, wildtype is delineated as NA

e. Evolutionary Action score from 0-100 with higher scores representing more deleterious mutations. Wildtype p53 (wtp53) sequences were scored as zero since this is the normally functioning protein.

f. Evolutionary Action Risk was determined as previously described but a score greater than 77.78 was consider high-risk [12].

g. Level of MYH9 expression extracted from The Cancer Genome Atlas Head and Neck Project RNA seq data

h. Low MYH9 expression was defined as the lower 25th percentile while high MYH9 expression was defined as greater the 25th percentile.

Impact of MYH9 expression and p53 mutational status.

Figure 1: Impact of MYH9 expression and p53 mutational status. A. Patients with low-risk (functional) p53 mutations and MYH9 expression in the lower quartile (<25%) have decreased survival relative to patients with high MYH9 expression (>25%). B. The expression level of MYH9 did not impact the survival of patients with high-risk (oncogenic) p53 mutations.

P53 function is dependent upon a functional NMIIA

Using the isogenic HNSCC cell lines, HN30 and HN31, which endogenously express either wtp53 (HN30) or missense p53 mutations, C176F and A161S, (HN31), HN30 was shown to upregulate expression of downstream p53 targets CDKN1A (p21) and MDM2 following treatment with nutlin-3; which inhibits the interaction between mdm2 and wild type p53, therefore stabilizing and leading to increased levels of the p53 protein. This target gene upregulation is not observed with the mutp53 cell line, HN31 (Figure 2A). NMIIA has been shown to be essential for nuclear retention of activated p53 therefore to determine the impact of NMIIA function on the upregulation of target gene expression observed in the wtp53 cells, the selective, small molecule NMIIA ATPase inhibitor, blebbistatin was applied prior to activation of p53 with nutlin-3. NMIIA inhibition led to a significant reduction in expression of target genes p21 (p=.02) and MDM2 (p=.04) in wtp53, HN30 cells, which was not observed in HN31 cells harboring high-risk mutations (Figure 2A). Inhibiting the nuclear export transporter Crm1 restored target gene expression in wtp53 expressing cells, which was not observed in high-risk mutp53 (Figure 2B). Taken together this data implies with NMIIA is defective, wtp53 cannot activate target genes because of an inability to accumulate within the nucleus.

NMIIA is necessary for wtp53 function, which is lost in high-risk mutp53.

Figure 2: NMIIA is necessary for wtp53 function, which is lost in high-risk mutp53. A. After treatment with DMSO, blebbistatin, or a combination of DMSO + nutlin (D+N) or blebbistatin + nutlin (B+N), qRT-PCR revealed the induction of p53 target genes p21 (p = 0.02) and MDM2 (p = 0.04) were significantly reduced following blebbistatin treatment in HN30 cells but not in HN31 cells. B. Inhibition of Crm1 nuclear exporter with Leptomycin B rescued p53 target gene expression in HN30 cells. Data expressed as means ± standard deviation; n=3. * p<0.05 reduction in p21 and MDM2 expression following blebbistatin treatment.

In an effort to directly assess the impact of NMIIA function on cell invasion, a CMV-GFP-NMII-A plasmid was stably overexpressed in HN30 and HN31 cell lines resulting in a ~50% increase in NMIIA expression in both cell lines (Figure 3) [19]. Even this modest (<2 fold) NMIIA overexpression preferentially decreased invasion in cells harboring wtp53 (p=.02) which was not observed in the mutp53 cells (Figure 4A). In contrast, inhibition of NMIIA led to an increase in cellular invasion in wtp53 expressing HN30 cells (p=.001) but not high-risk mutp53 HN31 cells (Figure 4B). Taken together this data suggests the function of wildtype p53 as a transcription factor and regulating cell invasion is dependent on a functional NMIIA.

Western blot of cell lines stably expressing EGFP-NMIIA construct.

Figure 3: Western blot of cell lines stably expressing EGFP-NMIIA construct. The histogram represents average relative density of NMIIA protein expression compared to actin loading controls and is the results of three independent experiments. EV:empty vector; M9:EGFP-NMIIA vector.

Modulation of NMIIA expression or function alters wtp53 expressing cell invasion.

Figure 4: Modulation of NMIIA expression or function alters wtp53 expressing cell invasion. A. Forced NMIIA expression significantly reduced invasion in HN30 (wtp53) but not HN31 (high-risk mutp53) cells relative to vector controls, p=0.02.EV: empty vector control. B. NMIIA inhibition significantly increased invasion in HN30 but not HN31 cells, p=0.001.

Inhibition of NMIIA alters wtp53 but not mutp53 function and cellular localization

Differences in NMIIA’s effect on wtp53 vs. mutp53 remain unknown [17]. To determine if the selective effect of NMIIA on wtp53 is due to its role in nuclear retention of activated wtp53 but not mutp53, cell fractionation was utilized. The initial fractionation experiment isolated insoluble cellular components (nuclear and cytoskeletal) from soluble cellular components (cytosol). As shown in Figure 5A (red boxed lane) following a dual nutlin-3 / blebbistatin treatment a decrease in nuclear / cytoskeletal expression of wtp53 and reduced induction of p21 was observed. The same treatment in mutp53 cells had no effect on the nuclear / cytoskeletal fraction of p53 or target gene induction (Figure 5). To assess if NMIIA specifically effects the nuclear retention of wtp53, the nuclear export receptor Crm1 was inhibited which resulted in the restoration of p53 nuclear accumulation (Figure 5B, boxed blue lane). To validate these findings a second fractionation protocol was utilized that specifically extracts the nuclear fraction from the cytoskeletal and cytoplasmic fractions. As seen in Supplementary Figure 1, inhibition of NMIIA following nutlin treatment significantly reduced the nuclear accumulation of wtp53 and p21 induction (Supplementary Figure 1). Furthermore, this decrease in nuclear p21 induction following combined nutlin-3 / blebbistatin treatement inhibition was associated with an significant increase in cytosolic p21 induction (Supplementary Figure 1). To confirm these findings immunofluorescent staining of intact cells following nutlin-3 treatment was performed. As shown in Figure 6, we observed a significant increase in co-localization of wtp53 and NMIIA in HN30 cells following nutlin treatment (p<.001) as determined by Pearson’s correlation coefficient (Figure 6B) and depicted by the yellow staining in the representative confocal images of nutlin treated HN30 cells (Figure 6A). Furthermore, in blebbistatin treated cells co-localization of wtp53 and NMIIA was attenuated. To determine if the wtp53 / NMIIA co-localization was occurring within the nucleus, the relative fluorescence for individual cells was determined and the average fluorescence for p53 and NMIIA was quantified in the cytoplasm and nucleus (2 and 7 microns from the cell membrane edge respectively (Figure 6A). Additionally orthogonal images were constructed from Z stack image capture through the depth of each cell. These analyses revealed that following nutlin-3 treatment, wtp53 and NMIIA appear to co-localize within the nucleus, which is attenuated following blebbistatin treatment supporting the finding that nuclear retention of wtp53 requires a functional NMIIA (Figure 6A and 6B column B+N). While co-localizatiohn of NMIIA and mutp53 was also observed it appeared to be independent of p53 and NMIIA activity given that treatment with either nutlin-3 or blebbistatin did not alter their co-localization.

Inhibition of NMIIA alters wtp53 but not high-risk mutp53 cellular localization.

Figure 5: Inhibition of NMIIA alters wtp53 but not high-risk mutp53 cellular localization. A. Nutlin-induced nuclear / cytoskeletal p53 and p21 was detected in HN30 (wtp53) cells. Blebbistatin treatment attenuated the effect of nutlin on nuclear p53 and p21 induction (red box). B. Average relative density in the nuclear / cytoskeletal and cytosolic fractions normalized the level of p53 and p21 to Lamin B and BCAR3 respectively. This revealed a significant increase in p53 and p21 after nutlin treatment relative to control (* p=.010), along with a significant decrease in p53 and p21 after blebbistatin relative to control (§ p=.032). C. Nuclear accumulation of p53 was restored by leptomycin B (Lept B) treatment. D. Average relative density in the nuclear / cytoskeletal and cytosolic fractions normalized the level of p53 to Lamin B and BCAR3 respectively. The expression levels of p53 in HN31 (mutp53) cells was unaffected by nutlin, blebbistatin, or leptomycin B treatment. The histograms represent the cumulative results of three independent experiments.

NMIIA co-localization with wtp53 is attenuated following NMIIA inhibition.

Figure 6: NMIIA co-localization with wtp53 is attenuated following NMIIA inhibition. A. Representative confocal fluorescence images including Z stack generated orthogonal views (xzy) showed the colocalization of wtp53 and NMIIA in HN30 cells not seen in high-risk HN31 cells (mutp53). The relative immunofluorescence profile revealed a significant increase in nuclear colocalization of p53 / NMIIA following nutlin treatment (p<0.001) which is attenuated following NMIIA inhibition. B. Data summary shows colocalization efficiency of NMIIA and p53. Nutlin treatment (D+N) caused a significant increase in colocalization in HN30 (p<0.001) but not HN31 (p=0.179) cells. There was a significant reduction in colocalization in HN30 cells following blebbistatin treatment (B+N) not observed in HN31 cells (p=0.019 vs .25). Data expressed as means +/- SEM; n=3. * p<.05 versus DMSO control group; § p<0.05 (DMSO + nutlin versus blebbistatin + nutlin). D+N, DMSO +nutlin; B+N, blebbistatin + nutlin.

To confirm the nuclear co-localization of wtp53 and NMIIA, cell fractionation followed by direct co-immunoprecipitation from these fractions was performed. This approach revealed an increase in wtp53/NMIIA association in the nuclear / cytoskeletal fraction along with a concomitant decrease in cytosolic interaction following nutlin treatment (Figure 7 blue box lanes). The nuclear / cytoskeletal association of wtp53 / NMIIA was reduced in cells treated with blebbistatin (Figure 7 red boxed lane). As observed by immunofluorescence microscopy, there appears to be an association of mutp53 / NMIIA based on co-immunoprecipitation, but this interaction remained at basal levels following addition of nutlin and/or combined treatment with blebbistatin.

NMIIA exhibits increased interaction with wtp53 in the nucleus.

Figure 7: NMIIA exhibits increased interaction with wtp53 in the nucleus. HN30 and HN31 cells were treated with blebbistatin (Blebb) or control (PBS) followed by nutlin for 8 h. Cells were fractionated followed by co-immunoprecipitation:immunoblot analysis of NMIIA and p53. A. Following p53 activation with nutlin there was a significant increase in association between wtp53 / NMIIA in the nuclear / cytoskeletal fraction of HN30 cells and a concomitant decrease in association in the cytosolic fraction, p<.001 and 0.01, respectively (Blue highlight). The increased association in the nuclear / cytoskeletal fraction was significantly reduced by blebbistatin, p=0.02 (Red highlight). Neither nutlin or blebbistatin treatment had an effect on the nuclear / cytoskeletal or cytosolic mutp53/NMIIA interaction. B. Average relative density normalizes the level p53 to NMIIA. The histograms represent the results of three independent experiments. * Significant change in interaction after nutlin treatment relative to control. § Significant decrease in p53/NMIIA interaction after blebbistatin relative to nutlin.

DISCUSSION

TP53 is the most frequently mutated gene in HNSCC occurring in more than 70% of cases that are non-human papilloma virus related [18, 20, 21]. Whereas most alterations involving tumor suppressor genes render them nonfunctional through truncation or deletions, p53 is unique in that there is a strong selection bias for missense mutations, particularly within its DNA-binding domain. P53 mutation can result in loss of wild type functions (LOF), which are considered low-risk, through loss of DNA-binding activity to p53 responsive elements or a dominant negative effect where the mutated allele binds and inhibits the remaining functional wild-type allele [22]. Moreover, some mutp53 display oncogenic properties, termed “gain of function” (GOF) or high-risk mutations, which are independent of the loss of wild-type p53 function [23]. Accordingly, GOF p53 mutants can enhance cell transformation, increase tumor formation in mice and confer cellular resistance to chemotherapy [24, 25]. We previously developed and validated a novel method, EAp53 that stratifies patients with tumors harboring TP53 mutations as high or low risk. Although the underlying mechanisms responsible for high-risk mutp53 remain unresolved, a potential mechanism involves interaction with NMIIA. In addition to the critical role NMIIA has in cell contractility and migration, it also functions as a tumor suppressor through regulation of p53 stability and nuclear retention [15-17, 26]. Despite this novel finding, there continues to be a significant gap in the understanding of the impact of NMIIA on mutp53 and its ability to function as a tumor suppressor and/or contribute to the oncogenic phenotype of p53. Given this lack of understanding, the objective of this study was to correlate the tumor suppressor effects of p53 with NMIIA function and demonstrate NMIIA dysfunction in cells harboring wildtype p53 results in characteristics resembling high-risk mutp53 including increased invasion. We hypothesized that the tumor suppressor capability of p53 is dependent on NMIIA function, which when abrogated leads to an oncogenic phenotype of p53 that is similar to high-risk mutp53.

Our results show patients stratified by EAp53 with low-risk mutp53 had a decreased overall survival with low MYH9 expression relative to those patients with low-risk mutp53 and high MYH9 expression. In contrast, the relative expression of MYH9 did not impact survival in patients with high-risk mutp53. Our previous work demonstrated low-risk TP53 mutations appear to retain some residual wildtype TP53 function as demonstrated by an intermediate level of activation of downstream p53 target genes following treatment with cisplatin [27]. Furthermore, this intermediate activation was associated with decreased cell migration and tumor growth in animal models [12]. Taken together, these data indicate that the tumor suppressive capability of NMIIA appears to be confined to tumor cells with functional TP53.

In addition to identifying the potential prognostic significance of MYH9 expression in low-risk mutp53 disease, we demonstrated inhibition of NMIIA leads to increased invasion in wtp53 expressing cells but not in high-risk mutp53 expressing cells. Furthermore, overexpression of NMIIA reduced invasion only in cells expressing wildtype p53. These findings corroborate a previous study and support our hypothesis that the tumor suppressor capability of p53 is dependent on NMIIA function [17]. This hypothesis is further supported by the finding of reduced p53 target gene expression in wildtype p53 cells following NMIIA inhibition, which was not observed in high-risk mutp53. The ability of wildtype p53 to activate downstream target genes appears to be dependent on nuclear localization of p53 as the reduction of target gene expression following NMIIA inhibition could be reversed with nuclear export inhibition. Furthermore, cell fractionation studies revealed induction of p53 and p21 in the nuclear fraction by nutlin treatment of wtp53 cells can be attenuated with blebbistatin treatment. The decrease in nuclear p21 induction was associated with a concomitant increase in the cytosolic p21 level which has been associated with increased cell survival and proliferation [28] This reduction in p53 induction in wtp53 cells with inhibition of the NMIIA ATPase can be reversed with Crm1 inhibition, which supports published data [17]. In contrast, inhibition of NMIIA did not alter expression of p21 or MDM2 in mutp53 cells or retention of mutp53 within the nucleus.

These findings are supported by immunofluorescence microscopy demonstrating an increase in the co-localization of wtp53 and NMIIA following nutlin treatment, which was subsequently reduced by NMIIA inhibition. Colocalization was predominantly nuclear as demonstrated by the Z stack generated orthogonal views and the relative cellular immunofluorescence, which was confirmed by cell fractionation:immmunoprecipitation findings. Although mutp53 and NMIIA appeared to co-localize, this was independent of NMIIA ATPase activity and was observed diffusely throughout the cell based on microscopy and supported by cell fractionation data.

In conclusion, the current findings indicate that cells expressing wtp53 are dependent on NMIIA inhibition to become pro-invasive secondary to decreased nuclear accumulation of wtp53 and subsequent reduction in target gene expression. In contrast, cells harboring high-risk mutp53 attain an invasive phenotype independent of NMIIA.

MATERIALS AND METHODS

Patient data

Patient dataset from The Cancer Genome Atlas HNSCC Project that had human papilloma virus (HPV)-negative tumors (n=192) were identified and EAp53 was applied to the p53 sequence data [12, 18]. MYH9 RNAseq expression data from The Cancer Genome Atlas Network Head and Neck Project was subsequently integrated with the p53 sequence data. MYH9 expression less than or equal to the lower quartile (≤25 percentile) for the entire cohort was considered to be low expression while expression greater than the lower quartile (>25 quartile) was considered to be high expression (Table 1). Overall survival data was extracted from TCGA HNSCC Supplementary Data. Curves describing overall survival were generated by the Kaplan-Meier method. The statistical significance of differences between the actuarial curves were assessed by the log rank test. Overall survival was measured from the date of diagnosis of recurrent disease to the date of death or last contact. Statistical analyses were performed using GraphPad Prism 7.0e (GraphPad Software, Inc., La Jolla, CA) statistical software.

Cell culture

The isogenic HNSCC cell lines HN30 and HN31 (provided by Dr. John Ensley; Wayne State University) were chosen as they were derived from a pharyngeal primary tumor and lymph node from the same patient. HN30 harbours a wtp53 while HN31 harbours two p53 mutations, C176F (high-risk) and A161S (low-risk). HN30 and HN31 cells were grown in DMEM with high glucose containing 10% FBS, 0.5% penicillin and streptomycin, 2 mM L-glutamine, 1 mM Sodium pyruvate, 85 mg/mL NaCl, 1 mg/mL D-calcium pantothenate, 1 mg/mL choline chloride, 1 mg/mL folic acid, 2 mg/mL i-inositol, 1 mg/mL niacinamide, 1 mg/mL pyridoxine- HCl, 0.1 mg/mL riboflavin, 1 mg/mL thiamine - HCl and non essential amino acids including 0.1 mM glycine, 0.1 mM alanine, 0.1 mM asparagine, 0.1 mM aspartic acid, 0.1 mM glutamic acid, 0.1 mM proline, 0.1 mM serine. The cells were maintained in a 37°C incubator with 95% air and 5% CO2

Drug incubation

HN30 and HN31 cells were growth arrested in serum-free medium for 24 hrs prior to drug treatment. Cells were pretreated with DMSO or 25 μM blebbistatin (Cayman Chemicals 674289-55-5) for 30 min prior to 8 hrs of treatment with 5 μM of nutlin-3 (Sigma-Aldrich N6287). For some Western blot analyses, (where indicated), 20 nM of leptomycin (Cayman Chemical 87081-35-4) was added 30 min prior to pretreatment of cells with DMSO or blebbistatin.

qRT-PCR

RNA was prepared from HN30 and HN31 cells using High Pure RNA Isolation Kit (Roche Diagnostics). cDNA was synthesized from RNA using iScript cDNA Syntheis Kit (Bio-Rad). The amplified cDNA was used in quantitative real-time PCR using SYBR Green PCR Master Mix (Applied Biosytems). The primer pairs used for analyzing p21, MDM2, and GAPDH were previously published [27]. The primer pairs used were as followed: p21 forward 5′-CGCTAATGGCGGGCTG-3′, reverse 5′-CGGTGACAAAGTCGAAGTTCC-3′; MDM2 forward 5′-ACCTCACAGATTCCAGCTTCG-3′, reverse 5′-TTTCATAGTATAAGTGTCTTTTT-3′; GAPDH forward 5’-TGATGGTACATGACAAGGTGC-3’, GA PDH reverse 5’-ACAGTCCATGCCATCACTGC-3’.

Generation of stable cell lines

For stable transfections, HN30 and HN31 cells were cultured in 6-well plates until they reached 70-80% confluency. The cells were transfected with CMV-GFP-NMHC II-A (Addgene plasmid # 11347) using 6 μg of using NanoJuice Transfection Reagent in serum-free medium (Novagen) according to the manufacturer’s protocol [19]. HN30 and HN31 cells were cultured for 7-14 days in 400 μg/ml of geneticin before being sorted for selection of stable clones.

Invasion assay

After stably transfecting HN30 and HN31 cells with vectors, invasion studies were conducted using Corning BioCoat Matrigel Invasion Chambers as described by the manufacturer (Corning). Cells were seeded in Matrigel Basement Membrane Matrix inserts in 24-well plates at a density of 2.5x104 cells per well. After 22 hr in a 37°C incubator, cells were fixed with 3% formalin and stained with silver stain. Membranes were washed and allowed to dry before an image was obtained and the number of invaded cells quantified. For studies that involved drug incubation, HN30 and HN31 cells were plated at a density of 2.5x104 cells in medium containing either DMSO or 25 μM of Blebbistatin.

Cell fractionation and western blot analysis

For Western blot analysis, HN30 and HN31 cells were grown in 100 mm tissue culture dishes. After treatment with the various drugs described, cells were rinsed and then lysed in cytosolic fractionation buffer (5 mM of EDTA, 1 mM of dithiothreitol, 1% Triton X-100 in PBS) supplemented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM NaF, 1 mM Na3VO3, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 1 μg/ml pepstatin). After brief centrifugation the supernatants were collected as the cytosolic extract and the pellets were washed and resuspended in nuclear extraction buffer (20 mM of TRIS-HCl, 1% SDS, 5 mM EGTA, 0.5% Triton X-100, 150 mM of NaCl) supplemented with protease inhibitors.

An alternative fractionation protocol was used where after treatment with the various drugs as described, nuclear and cytoplasmic extracts were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit from ThermoFisher Scientific (Catalog #: 78833). Equal amounts of protein sample were loaded per lane on Mini-PROTEAN TGX Precast Gels (Bio-RAD) which were transferred to nitrocellulose membranes following electrophoresis. Blots were incubated with primary antibodies to p53 (Santa Cruz Biotechnology sc-126), p21 (EMD Millipore OP64), NMIIA (Santa Cruz Biotechnology sc-98978), BCAR3 (Bethly Laboratories A301-671A), Lamin B (Santa Cruz sc-6216), GFP (Cell Signaling 2956S), actin (Millipore MAB1501) and subsequently reacted with the corresponding secondary antibodies. All secondary antibodies were horseradish peroxidase conjugates. Blots were developed by Enhanced Chemiluminescence Kit (Thermo Scientific) before exposure to X-ray film. Densitometry was performed using FIJI/Image J software and paired t-tests compared the relative intensities using Microsoft Excel (Microsoft Corp, Redmond, WA) [29].

Immunoprecipitation

For NMIIA immunoprecipitation, HN30 and HN31 cells grown in 100 mm tissue culture dishes were fractionated as described above equal amounts of nuclear and cytoplasmic proteins were pre-cleared by incubation with protein A/G Sepharose beads for 30 min at 4°C. After brief centrifugation, supernatants were removed and incubated with anti-Myosin 9 antibody (Santa Cruz Biotechnologies sc-98978) overnight. Immunoprecipitates were captured with 60 μl of protein A/G beads at 4°C for 3 hr. Samples were centrifuged and washed three-fold with PBS and proteins were eluted from the beads using 2x Laemmli buffer, boiled for 5 min, and resolved by SDS-PAGE and subsequent immunoblot analysis with mouse monoclonal antibodies for p53 (Santa Cruz Biotechnologies sc-126) and Myosin 9 (Millipore MABT164). Densitometry and statistical analysis were performed as described above.

Immunofluorescence microscopy

Following the drug treatments described above, cells were fixed with 4% paraformaldehyde for 10 min at room temperature, permeabilized with 0.5% Triton X-100 (Sigma) in PBS for 5 min and non-specific binding sites were blocked with 1% BSA in PBS for 1h. Primary and secondary antibodies were diluted in blocking solution as directed by the manufacturer. Antibodies employed were anti-p53 (1:500; Santa Cruz Biotechnology), anti-NMIIA (1:500; Santa Cruz Biotechnology), anti-rabbit Alexa Flour 488 (Life Technologies) anti-mouse Alexa Fluor 546 (Molecular Probes). Confocal microscopy was performed using an Olympus FV10i laser scanning confocal microscope (Olympus, Tokyo, Japan). Colocalization of NMIIA and p53 was analyzed by FIJI/Image J software with the coloq2 plugin [29]. The plot profile for 8 cells per condition was determined and the mean was relative immunofluorescence was calculated. The average cell diameter was estimated to be 14 microns, there the relative immunofluorescence within the cytoplasm and nucleus was measured 2 and 7 microns from the cell membrane respectively. The average relative immunofluorescence following various treatments within the nucleus and cytoplasm was compared using a paired T-test. The summarized colocalization efficiency data were expressed as Pearson correlation coefficients as previously described [30].

ACKNOWLEDGMENTS

We wish to acknowledge the services of the Cell and Molecular Imaging Shared Resource - with a special thanks to Jennifer Bestman for immunofluorescence image acquisition and data analyses, Biostatistics Shared Resource and the Flow Cytometry and Cell Sorting Shared Resource, Hollings Cancer Center (supported by P30 CA 138313).

CONFLICTS OF INTEREST

None.

FUNDING

This work was supported by the K12 Paul Calebresi Clinical and Translational Oncology Training Program K12 CA157688 (DMN), NIDCR K08 DE026542-01 (DMN), the American Cancer Society Institutional Research Grant IRG-97-291-14 (DMN), and NCI R01 CA 134845 (SAR).

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