Clinical Chemistry 55:10
1834–1842 (2009)
Cancer Diagnostics
Increased Tissue Factor Expression Is Associated with
Reduced Survival in Non–Small Cell Lung Cancer and with
Mutations of TP53 and PTEN
Sandra Regina,1,2 Jean-Baptiste Valentin,1 Sébastien Lachot,1 Etienne Lemarié,2,3 Jérôme Rollin,1,2
and Yves Gruel1,2*
BACKGROUND: Tissue factor (TF), the main initiator of
blood coagulation, is also a signaling protein that regulates cancer progression. TF synthesis was recently
shown to be affected by tumor suppressor genes
(TSGs) in tumor cell lines. We therefore studied TF
gene (F3) expression and the status of genes coding for
tumor protein p53 (TP53), phosphatase and tensin
homolog (PTEN), and serine/threonine kinase 11
(STK11) in non–small cell lung cancer (NSCLC).
Heparanase (HPSE) gene expression was also measured because this endo-␤-D-glucuronidase was recently shown to enhance TF gene expression.
CONCLUSIONS: These results provide clear evidence that
combined oncogene events affecting TSG dramatically
increase TF gene expression in lung tumors. Moreover,
this study suggests that TF gene expression could be
used as a prognostic marker in NSCLC.
© 2009 American Association for Clinical Chemistry
TF mRNA levels were significantly higher in
T3–T4 tumors (P ⫽ 0.04) and in stages III–IV of
NSCLC (P ⫽ 0.03). Mutations of TP53, STK11, and
PTEN were identified in 20 (37.7%), 21 (39%), and 20
(37.7%) of tumors, respectively. TF expression was
higher in mutated TP53 (TP53Mut) (P ⫽ 0.02) and
PTENMut (P ⫽ 0.03) samples. Moreover, TF mRNA
increased from 2700 copies (no mutation) to 11 6415
when 3 TSG were mutated. Heparanase gene expression did not differ according to TF gene (F3) expression or TSG mutation. The median survival time was
shorter in patients with tumor TF mRNA levels above
median values (relative risk 2.2; P ⫽ 0.03, multivariate
analysis) and when TP53 was mutated (relative risk 1.8;
P ⫽ 0.02).
Lung cancer is the most common malignant disease
worldwide and a major cause of cancer-related death,
particularly among men, with 1 200 000 new cases diagnosed each year and only 10% of patients surviving
the disease (1 ). Lung cancers are almost exclusively
carcinomas that originate in the epithelia of the trachea, bronchi, or lungs. Several histological types have
been identified and they are divided into 2 groups,
small cell lung cancer (SCLC)4 (20%) and non-SCLC
(NSCLC) (80%).
Tissue factor (TF), a 47-kDa transmembrane protein, is the essential receptor for factor VII/VIIa and the
physiological trigger of blood coagulation (2 ). TF synthesized by cancerous cells or by the tumor microenvironment probably contributes to the hypercoagulable
state that occurs in patients with cancer. In addition,
TF plays a direct role in tumor evolution by promoting
tumor growth, angiogenesis, cell migration, and development of metastases (3 ).
In NSCLC, we recently demonstrated that tumor
TF gene (F3) expression varied between patients but
was significantly higher in patients with advanced
stages of cancer (4 ). Moreover, our study also showed a
relationship between overexpression of tumor TF and
the presence of a mutation of codon 12 of v-Ki-ras2
Kirsten rat sarcoma viral oncogene homolog (KRAS).5
1
4
METHODS:
TF and heparanase mRNA expression was
measured by real-time PCR in 53 NSCLC tumors. Exons 5– 8 of TP53 were sequenced from genomic DNA.
Mutations of PTEN and STK11 were screened by multiplex ligation-dependent probe amplification.
RESULTS:
Department of Hematology-Hemostasis, Trousseau Hospital and François Rabelais University, Tours, France; 2 INSERM, Tours; 3 Department of Pneumology,
Bretonneau Hospital and François Rabelais University, Tours, France.
* Address correspondence to this author at: Professor Yves Gruel, INSERM U 618,
“Protéases et Vectorisation Pulmonaires,” Faculté de Médecine, 10 bis Bd
Tonnellé, 37032 Tours Cedex, France. Fax ⫹33-2-47-36-60-46; e-mail
[email protected].
Received January 9, 2009; accepted July 9, 2009.
Previously published online at DOI: 10.1373/clinchem.2009.123695
1834
Nonstandard abbreviations: SCLC, small cell lung cancer; NSCLC, non-SCLC; TF,
tissue factor; TSG, tumor suppressor genes; MLPA, multiplex ligation-dependent
probe amplification; Mut, mutated; WT, wild type; mTOR, mammalian target of
rapamycin.
5
Human genes: F3, coagulation factor III (thromboplastin, tissue factor); KRAS,
v-KI-ras 2 Kirsten rat sarcoma viral oncogene homolog; TP53, tumor protein p53;
PTEN, phosphatase and tensin homolog; STK11, serine/threonine kinase 11; HPSE,
heparanase; EGR1, early growth response 1; EGFR, epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian).
TF Expression and Oncogene Events in NSCLC
The involvement of tumor suppressor genes (TSG) or
oncogenes other than KRAS in TF gene expression has
recently been reported with cell lines and animal models in colorectal cancer with tumor protein p53 (TP53)
(also called p53) (5 ) and in glioma with phosphatase
and tensin homolog (PTEN) (6 ) but has never been
shown in samples from patients with cancer. Therefore
in this study we investigated the presence of lesions in
the TP53 and PTEN genes in lung tumors of patients
with NSCLC and the possible association with increased TF gene expression. We also looked for mutations of serine/threonine kinase 11 (STK11) (also called
LKB1), which have also been frequently detected in primary human lung adenocarcinoma (7 ), because it was
recently shown that STK11 can regulate lung cancer
differentiation and metastasis in combination with
KRAS (8 ).
TF gene (F3) expression can also be regulated in
some cancers by heparanase, a mammalian endo-␤-Dglucuronidase cleaving heparan sulfate chains in the
extracellular matrix and on cell surfaces (9 ). In physiological conditions, the heparanase gene (HPSE) is
preferentially expressed by cells of the immune system,
platelets, and placenta cells. However, recent studies
have also shown that heparanase protein is synthesized
by tumor cells (10 ) with an expression possibly regulated by p53 (11 ) and EGR1 (early growth response 1)
(12 ). On the other hand, heparanase can induce TF
synthesis in endothelial cells and cancer cells (13 ) and
can be involved in tumor angiogenesis and metastasis
(14, 15 ).
Therefore in the present study we looked for a relationship between F3 and HPSE expression in NSCLC
tumors and investigated whether TF and heparanase
are regulated by TP53, PTEN, and STK11.
of malignant tumors (17 ). All patients gave informed
consent for genetic studies as recommended by French
legislation and in accordance with the current revision
of the Helsinki declaration.
RNA EXTRACTION AND REAL-TIME PCR FOR HEPARANASE
AND TF
As previously described, we extracted total RNA from
the 53 tumors by using the Perfect RNA Eukaryotic
Mini Kit (Eppendorf) according to the manufacturer’s
instructions (18 ). The number of heparanase transcripts within lung tumors was assessed by a real-time
PCR method adapted from Uno et al. (19 ). The results
were expressed as the number of heparanase mRNA
copies for 108 copies of 18S RNA.
Tumor TF gene (F3) expression was analyzed by
real-time PCR as previously described (4 ).
Results obtained were compared with TF protein
analyzed by use of a tissue microarray technique that
allowed tumors to be designated as either “low TF”
(⬍33% of positive cells) or “high TF” (⬎33% of positive cells), as previously described (4 ).
TP53 GENE MUTATION ANALYSIS
We prepared genomic DNA from lung tumors by using
a DNA purification kit (Qiagen) as previously described (18 ). Sequencing of exons 5– 8 of the TP53 gene
was performed on DNA extracted from 53 tumor samples. Primers were chosen with the assistance of the
Primer3 computer program, and their sequences are
presented in Table 2. We used 50 ng of DNA for PCR
amplification. PCR products were cycle-sequenced using the Big Dye Terminator V3.1 cycle sequencing kit
(Applera) and an ABI PRISM 3130 Genetic Analyzer
(Applied Biosystems).
Materials and Methods
DNA ANALYSIS FOR PTEN AND STK11 MUTATIONS BY
PATIENTS
We analyzed tumor samples from 53 white patients
with NSCLC who had undergone complete surgical resection as initial treatment (i.e., without prior radiotherapy or chemotherapy) between January 2002 and
February 2005 in our hospital (CHU Trousseau, Tours,
France) (Table 1).
Tumor samples were selected by a pathologist
from fresh surgical samples, and a section was snap
frozen in liquid nitrogen and immediately stored at
⫺80 °C until DNA extraction. In addition, another
part of the tumor biopsy sample was immediately
stored in RNA-later (Ambion) until RNA extraction.
Histological diagnosis and grade of differentiation
were assigned according to the WHO criteria for lung
tumors (16 ), and pathological staging was based on the
revised TNM (tumor-node-metastasis) classification
MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION
STK11 and PTEN mutations were screened by use of
multiplex ligation-dependent probe amplification
(MLPA) using the SALSA-MLPA Kits P101 and P225,
respectively (MRC-Holland). The principle of this
analysis is based on sequence-specific probe hybridization to genomic DNA, followed by amplification of the
hybridized probe, and semiquantitative analysis of the
resulting PCR products. The relative peak height and
band intensity for each target indicate their initial concentration. For both genes, 70 ng of genomic DNA was
incubated at 98 °C for 5 min. After the preparation had
cooled to 21 °C, 1 ␮L probe mix and 1 ␮L SALSA hybridization buffer were added, and the solution was
denatured at 95 °C for 2 min and hybridized at 60 °C
for at least 16 h. Hybridized probes were next ligated
at 54 °C for 15 min with 21 ␮L of ligation mixture,
Clinical Chemistry 55:10 (2009) 1835
Table 1. Clinicopathological features of patients with NSCLC, status of codon12 of the KRAS gene (WT or
mutated), and relationship with mutations of TP53, STK11, and PTEN tumor suppressor genes (Fisher exact test).
TP53
Patient features
Median age (range), y
STK11
All
patients, n
(%)
WT
(n ⴝ 33)
Mutated
(n ⴝ 20)
66 (44–83)
67 (45–80)
65 (44–83)
P
PTEN
WT
(n ⴝ 32)
Mutated
(n ⴝ 21)
65 (44–79)
68 (45–83)
P
WT
(n ⴝ 29)
Mutated
(n ⴝ 20)
67 (45–83)
65 (44–79)
P
Sex
Female
7 (13)
5
2
46 (87)
28
18
5 (10)
3
2
48 (90)
30
18
T1–T2
46 (87)
29
17
T3–T4
7 (13)
4
3
Male
0.62
5
2
27
19
3
2
29
19
27
19
5
2
0.52
5
1
24
19
2
2
27
18
23
18
6
2
19
10
10
10
27
15
2
5
15
10
14
10
16
13
9
5
4
2
25
16
4
4
0.37
Smoking status
Nonsmokers
Smokers
1
0.98
0.98
Tumor status
0.99
0.52
0.44
Node status
N0
29 (55)
18
11
N⫹
24 (45)
15
9
M0
44 (83)
29
15
M⫹
9 (17)
4
5
I–II
28 (53)
18
10
III–IV
25 (47)
15
10
Adenocarcinoma
32 (60)
22
10
Carcinoma epidermitis
13 (24)
7
6
8 (16)
4
4
45 (85)
28
17
8 (15)
5
3
1
16
11
16
10
25
19
7
2
15
13
17
8
21
11
5
8
6
2
29
16
3
5
0.86
0.93
Metastasis status
0.27
0.24
0.1
Stage
0.78
0.28
0.93
Histological type
Others
0.51
0.9
0.77
Codon 12 KRAS
WT
Mutated
followed by an inactivation step of 5 min at 95 °C. Finally, 7.5 ␮L of ligation product was mixed with the
SALSA-PCR mixture and subjected to amplification
for 30 cycles according to the manufacturer’s recommendations. Amplification products were diluted at 1
0.98
0.15
part to 9 parts in Hi-DI formamide (Applied Biosystems) containing 500-LIZ size standard (Applied Biosystems) and then separated by size on an ABI 3130
Genetic Analyzer (ABI). The data obtained were analyzed as previously described (20 ).
Table 2. Oligonucleotide primers used in sequencing analysis of TP53.
Forward primer, 5ⴕ33ⴕ
Reverse primer, 5ⴕ33ⴕ
PCR
product, bp
Exon 5
CTCTTCCTACAGTACTCCCCTGC
CCAAGCTGCTCACCATCGCTA
Exon 6
GATTGCTCTTAGGTCTGGCCCCTC
GGCCACTGACAACCACCCTTACC
182
Exon 7
GCTTGCCACAGGTCTCCCCAAG
AGGGTGGCAAGTGGCTCCTGAC
192
Exon 8
TGGTAATCTACTGGGACGGA
GCTTAGTGCTCCCTGGGGGC
134
1836 Clinical Chemistry 55:10 (2009)
0.77
214
TF Expression and Oncogene Events in NSCLC
Table 3. Relationship between tumor TF or heparanase gene expression and mutations of TP53, PTEN, and
STK11 tumor suppressor genes in NSCLC tumors (Mann–Whitney U-test).
Tissue factor
Gene
TP53
Status (n)
WT (33)
Mutated (20)
STK11
WT (32)
Mutated (21)
PTEN
WT (29)
Mutated (20)
a
Median gene
expression (range)
6225 (10–137 720)
15 655 (110–309 660)
8800 (100–309 660)
15 810 (10–154 800)
8530 (10–309 660)
17 370 (110–154 800)
Heparanase
P
0.02a
0.43
0.03a
Median gene
expression (range)
1848 (80–113 000)
3311 (530–39 750)
3700 (225–19 500)
1990 (80–113 000)
3310 (80–39 750)
1690 (90–25 541)
P
0.09
0.42
0.38
P ⬍ 0.05.
DNA extracted from NL20, a normal pulmonary
cell line, was systematically tested as a negative control
in each experiment. In addition, DNA samples from 3
NSCLC cell lines (Calu-1 mutated for PTEN, A549 and
H23 mutated for STK11) were also tested as positive
controls (21, 22 ).
STATISTICAL ANALYSIS
A Mann–Whitney U-test was used to analyze the continuous variables, i.e., TF and heparanase gene expressions according to the status of TP53, PTEN, and
STK11 (i.e., mutated or not) and the clinicopathological features. Spearman rank correlation coefficients
were used in analysis of the coexpression of TF and
heparanase genes in NSCLC tumors. The Fisher exact
test was used in assessing the association between clinicopathological factors and TSG mutations. A P value
ⱕ0.05 was considered to be statistically significant.
The postoperative survival rate was analyzed by the
Kaplan–Meier method, and the differences in survival
rates were assessed by the log-rank test. Multivariate
analysis of prognostic factors was performed using Cox
regression. Variables included in the analysis were TF
and heparanase gene expressions (i.e., above or below
the median value of measured transcripts), age,
NSCLC stage, and TP53 status (i.e., mutated or not).
Results
TUMOR EXPRESSION OF TF AND HEPARANASE IN NSCLC AND
RELATIONSHIP WITH TP53, PTEN, AND STK11 STATUS
The expression of TF and heparanase genes (F3 and
HPSE) was evaluated by specific real-time PCR in lung
tumors surgically removed from the 53 patients.
TF gene expression was variable from one tumor
to another with a median number of transcripts equal
to 11540 per 108 copies of 18S RNA. TF protein expression was also variable, as evidenced by tissue microar-
ray analysis results, with 39 NSCLC tumors classified in
the low-TF group and 14 samples identified as high TF.
As expected, median TF mRNA levels were lower in
samples from low-TF tumors than in samples from
high-TF tumors (7670 vs 89 590 copies/108 18S RNA).
Heparanase mRNA levels were lower in lung tumors (median ⫽ 2922 transcripts/108 copies of 18S
RNA) and also were variable but without any correlation with TF gene expression (Spearman rank correlation, r ⫽ 0.216; P ⫽ 0.2).
The status (i.e., mutated or not mutated) of the
TP53, STK11, and PTEN genes was studied on genomic
DNA extracted from NSCLC biopsy samples.
Exons 5– 8 of TP53 were analyzed by genomic PCR
and direct sequencing. We identified 17 mutations in
20 of 53 tumors (37.7%) located in exon 5 (n ⫽ 7),
exon 6 (n ⫽ 5), or exon 7 (n ⫽ 8). All mutations
found had been reported in the UMD TP53 mutation
database (23 ), but 5 had never been associated with
NSCLC (479T⬎A, 645T⬎A, 685T⬎A, 686G⬎C, and
706T⬎G).
TF gene expression was significantly higher in mutated TP53 (TP53Mut) tumors than in TP53 wild type
(TP53WT) tumors (median 15655 vs 6225 transcripts/
108 copies of 18S RNA, P ⫽ 0.02). In contrast, heparanase mRNA levels appeared higher in TP53Mut tumors
than in TP53WT tumors, but the difference was not
statistically significant (median 3311 vs 1848 transcripts per 108 copies of 18S RNA; P ⫽ 0.09).
The 2 other TSG, STK11 and PTEN, were analyzed
using the MLPA method. For STK11, sequence alterations were detected in 21 samples (39%) and were
mainly deletions (n ⫽ 15), most of them being localized in exon 1 (n ⫽ 14) and the others in exon 3 (n ⫽ 5)
and in exon 8 (n ⫽ 2). However, no relationship was
found between STK11 status and TF or heparanase
gene expression (Table 3).
Clinical Chemistry 55:10 (2009) 1837
P = 0.0 1
105
104
103
102
10
Median 2700
14
n*
0
C 106
Number of copies /108 copies of 18S RNA
B
TF Gene Expression
106
13 700
16 500
116 415
17
14
8
1
2
3
Number of copies /108 copies of 18S RNA
Number of copies /108 copies of 18S RNA
A
Heparanase Gene Expression
106
P = 0.2 1
105
104
103
102
10
Median
6460
4480
1780
14
17
14
8
0
1
2
3
n*
Number of mutated genes (TP53, PTEN, STK11)
Number of mutated genes (TP53, PTEN, STK11)
D
P = 0.001
TF Gene Expression
105
Number of
mutated genes
n*
0
12
1
16
104
2
103
18
Mutated genes
(n*)
102
3
6
10
1500
11 045
12
16
0
1
16 135
87 765
309 550
18
6
1
2
3
4
4
1
Median
number of TF
mRNA copies
1500
TP53 (3)
2030
PTEN (5)
13 710
STK11 (8)
11 045
STK11 + PTEN (2)
TP53 + STK11 (4)
Median
n*
2920
17 370
6305
STK11 + KRAS (5)
19 150
TP53 + PTEN (7)
16 500
TP53 + STK11 + KRAS (1)
78 000
PTEN + STK11 + KRAS (1)
97 530
TP53 + PTEN + STK11 (2)
106 405
TP53 + PTEN + KRAS (2)
74 485
All
309 550
Number of mutated genes (TP53, PTEN, STK11, KRAS)
Fig. 1. TF (A,C) and heparanase (B) gene expression in NSCLC according to the number of mutations affecting TP53,
PTEN, STK11 (A,B) and KRAS (C).
(D), all combinations of mutations identified and the median number of TF mRNA copies measured in corresponding lung
tumors; *n, number of lung tumors.
PTEN status was studied in all tumors by MLPA
but could not be defined in 4 samples, owing to insufficient DNA amplification. Lesions in PTEN were
detected in 20 samples and deletions/mutations were
the most frequently evidenced (n ⫽ 18/20). A single
abnormality was found in 7 tumors, and several
deletions/mutations were found in the other 13. The
TF gene was overexpressed in mutated tumors
(PTENMut) compared to PTENWT samples, and statistical analysis showed a significant relationship between TF expression and PTEN status (median 17 370
vs 8530 transcripts/108 copies of 18S RNA P ⫽ 0.03)
(Table 3). In contrast, no relationship between tumor
heparanase mRNA levels and PTEN status was
evidenced.
Importantly, in lung tumors TF gene expression
was also shown to increase progressively with the number of mutated genes. The median number of mRNA
1838 Clinical Chemistry 55:10 (2009)
transcripts (per 108 copies of 18S RNA) varied from
2700, for which no mutation was detected, to 13 700,
16 500, and 116 415 transcripts, for which 1, 2, and 3
TSG were mutated (P ⫽ 0.01) (Fig. 1A). In addition,
codon 12 of KRAS was mutated in 8 tumors (Table 1),
and this event in combination with other mutations
also appeared to increase TF gene expression in cancerous tissues (Fig. 1, C and D). No relationship was
detected between heparanase mRNA levels and the
number of oncogene mutations evidenced in lung tumors (Fig. 1B).
COMPARATIVE ANALYSIS OF BIOLOGICAL DATA WITH
CLINICOPATHOLOGICAL FEATURES AND SURVIVAL OF PATIENTS
WITH NSCLC
As previously reported (4 ), TF gene expression was significantly higher in T3–T4 tumors (median 19 150
transcripts/108 copies of 18S RNA vs 8530 transcripts
TF Expression and Oncogene Events in NSCLC
Survival probability
HPSE
1
TF Gene (F3)
1
TF < Median
0.5
0.5
P = 0.29
TF > Median
P = 0.02
0
0
10
20
HPSE > Median
30
40
50
60
0
70
0
10
20
Months
1
PTEN
WT TP53
0.5
P = 0.05
40 50
Months
60
70
70
WT STK11
0.5
P = 0.87
Mut STK11
0
0
30
60
Mut PTEN
Mut TP53
20
50
STK11
P = 0.83
0
10
40
1
WT PTEN
0.5
0
30
Months
1
TP53
HPSE < Median
0
10
20
30 40
4
50
Months
60
70
0
10
20
30 40 50
Months
60
70
Fig. 2. Kaplan–Meier curves according to TF (F3) and HPSE gene expression in NSCLC tumors and to the status (WT
or Mut) of TP53, PTEN, and STK11 TSG.
in T1–T2 tumors; P ⫽ 0.04) and in samples from patients with stage III–IV (median 17 710 copies/108 copies of 18S RNA vs 9780 copies in stage I–II; P ⫽ 0.03),
but no correlation with age was evidenced (P ⫽ 0.16,
Mann–Whitney U-test).
On the other hand, no relationship was evidenced
between tumor heparanase mRNA levels and characteristics such as tumor size, stage of tumor, tumor differentiation, node involvement. and metastasis status
(data not shown). In addition, none of the clinicopathological features analyzed was different in patients according to the status (i.e., mutated or not mutated) in
cancerous tissues of the 3 TSG (Table 1).
Survival curves were then analyzed according to
median TF and heparanase mRNA levels measured in
tumors and to TP53, STK11, and PTEN status
(Kaplan–Meier analysis) (Fig. 2). The median time of
follow-up was 35 months at the time of data analysis,
and overexpression of the TF gene in lung tumors or
the mutated status of TP53 was associated with a significantly shorter survival rate. Indeed, the median survival time was 26 months for patients with high tumor
TF gene expression (above median) compared to 66
months for those in whom lower TF mRNA levels were
measured in NSCLC tumors (hazard ratio 1.96, 95% CI
1.01–3.8; P ⫽ 0.02) (Fig. 2). In addition, the median
survival time was 33 months for patients in whom
TP53 was mutated in tumors, compared to 43 months
for those without TP53 mutation (hazard ratio 1.5,
95% CI 1.02–2.5; P ⫽ 0.05). In contrast, no significant
difference in survival was found according to age,
HPSE gene expression, or STK11 or PTEN gene status.
Importantly, Cox regression analysis also demonstrated that high TF gene expression (above median)
and TP53 mutation were independent risk factors for
shortened survival time in patients with NSCLC (Table
4).
Discussion
TF, the well-known receptor for factor VIIa, is mainly
involved in triggering the coagulation cascade, but it is
also a signaling protein for the regulation of tumor cell
movement, angiogenesis, and metastasis (24 ). TF is
synthesized by a variety of cell types, including tumor
cells, and is in part responsible for the prothrombotic
state associated with many cancers. Several experimental models have provided evidence that genetic lesions
affecting various oncogenes and TSG are associated
with upregulation of TF, which in turn induces protumorigenic effects, independently of hemostasis activation (25 ).
Clinical Chemistry 55:10 (2009) 1839
Table 4. Overall survival with univariate analysis (Kaplan–Meier method) or multivariate analysis
(Cox regression model).
Univariate analysis
Multivariate analysis
Hazard ratio
95% CI
P
Relative risk
95% CI
Age
0.9
0.4–1.8
0.76
1.15
0.96–1.3
0.32
Stage
3.1
1.4–5.7
0.005
2.8
1.6–5.9
0.006
TF gene expression
1.96
1.01–3.8
0.02
2.2
1.1–4.2
0.03
Heparanase gene expression
1.6
0.32–1.34
0.29
1.1
0.7–1.7
0.75
TP53 status
1.5
1.02–2.5
0.05
1.8
1.2–4.5
0.02
In the present study, we looked for changes in 3
TSG in patients with NSCLC, and we demonstrated
that mutations in TP53 and PTEN genes are associated with significant increase in TF gene expression
in lung tumors. TF is frequently upregulated in cancer cells, particularly in the later stages of disease
progression (26 ), and we recently reported that TF
gene expression was higher in advanced stages of
NSCLC and when codon 12 of the KRAS gene was
mutated (4 ). Both TF expression and TF procoagulant and proangiogenic activity have previously been
shown to be regulated by mutations of the KRAS
oncogene and TP53 tumor suppressor gene in human colorectal cell lines (5 ). In addition, these oncogenic events were associated with increased release
of TF-bearing microvesicules, which contributes to
the high risk of thrombosis associated with colorectal cancer (27 ). TP53 mutations are the most frequent gene abnormalities identified in 50% to 70%
of NSCLC, with variations related in part to smoking
profiles of affected patients (28 ). In this study, we
sequenced exons 5– 8 of the TP53 gene, a mutational
hot-spot region, and we identified mutations in
37.7% of lung tumors, results that are in agreement
with previous reports (29 ). Importantly, a significant shortening of survival in patients with TP53
mutations was also evidenced compared to cases
without mutation (relative risk 1.8; 95% CI 1.2– 4.5;
P ⫽ 0.02, multivariate analysis), as previously shown
(29, 30 ). Shorter survival time was also associated
with high tumor TF expression (relative risk 2.2,
95% CI 1.1– 4.2; P ⫽ 0.03), and these results strongly
support a pathogenic relationship between TP53 and
TF in lung tumorigenesis in vivo.
Apart from the role of KRAS, it has also been
demonstrated that inactivation of PTEN in human
glioma cells also resulted in upregulation of TF, particularly under hypoxic conditions that potentially
have a critical role in lung cancer (6 ). The changes in
PTEN identified in lung tumors in our study were
mainly deletions/mutations present in 37.7% of pa1840 Clinical Chemistry 55:10 (2009)
P
tients. This frequency appears higher than previously reported (31 ), but we used the recently developed MLPA technique that is very sensitive to a
variety of gene changes, including deletions and mutations (32, 33 ). On the other hand, PTEN protein
synthesis was previously found to be reduced in most
lung cancers, although hypermethylation of the
PTEN promoter was evidenced in 26% of tumors
and loss of heterozygosity was rare, affecting ⬍20%
of samples (34 ). Mechanisms other than epigenetic
silencing and deletions are therefore probably involved in reducing PTEN protein production. PTEN
inactivation is a crucial event in the progression of
lung tumorigenesis initiated by KRAS (35 ), and our
results also suggest that it may affect TF synthesis in
NSCLC tumors. Other oncogenic events may also be
involved in regulating TF gene expression, however,
and we therefore looked for mutations in STK11, a
recently identified TSG that is important in NSCLC
(36 ). This enzyme has a major role in various cellular pathways (37 ), and inactivation of somatic mutations of STK11 has been reported in primary lung
carcinomas and in cell lines (38 ). STK11 status was
analyzed in our study by MLPA, as recently described (8 ), and gene changes were found in 39% of
tumors, including all histologic subtypes of NSCLC.
These results are in accordance with those of previous studies (7, 8 ), although STK11 modifications
were not more frequent in lung tumors with KRAS
mutations, as was previously found with cell lines
(7 ), but the number of samples studied was possibly
not sufficient to address this issue. Ji et al. recently
developed a KRAS-driven model of mouse lung cancer (8 ), and they showed a strong cooperation between KRAS mutation and loss of p53 upon pulmonary tumorigenesis in the presence of homozygous
inactivation of STK11. According to our results, somatic STK11 lesions did not affect TF gene expression in human lung tumors when present alone.
However, an effect of mutated STK11 cannot be excluded, because a progressive increase in TF mRNA
TF Expression and Oncogene Events in NSCLC
levels was found when STK11 was associated with
mutations of TP53 or PTEN in cancerous tissues.
Importantly, this increase in TF gene expression associated with combined TSG mutations (P ⫽ 0.01,
Fig. 1A) appeared to be amplified when codon 12 of
KRAS was also mutated (P ⬍ 0.001, Fig. 1D). However, no effect on tumor TF gene expression of one
particular combination could be evidenced (Fig. 1D)
owing to the limited number of samples studied.
This result supports the important concept that sequential changes in several oncogenes and TSG are
often necessary for alteration of the cancer cell phenotype and progression of malignant tumors.
We also studied HPSE expression, which was
found to be comparable in lung tumors and in nonaffected lungs. In addition, heparanase mRNA levels did
not correlate with TF gene expression. Heparanase is
constitutively synthesized in various normal cells and
tissues such as the placenta, keratinocytes, platelets and
cells of the immune system, but the HPSE gene has also
been shown to be upregulated in cancer as well in inflammation and wound healing (10 ). HPSE gene transcription can be regulated by promoter methylation,
early growth response 1 (EGR1), and TP53 in human
tumors, but these are unlikely to be involved in NSCLC
because mRNA levels measured in lung tumors were
not higher than those of noncancerous lungs. The first
study that focused on heparanase in NSCLC was performed in 76 Japanese patients and showed that
heparanase activity was significantly higher in lung tumors than in nonaffected tissues (39 ). More importantly, the disease-free survival was significantly reduced in patients with increased heparanase activity.
Recently, another study also revealed that heparanase
protein measured by immunohistochemistry was increased in 75% of patients with lung cancer and correlated with lymph node invasion and metastasis (40 ).
Results from these 2 studies therefore support indications that molecular techniques have potential limitations in investigating the role of heparanase in cancer
progression compared to methods measuring protein
levels. Indeed, in our study mRNA levels were relatively
low in lung tumors, and posttranslational events regulating heparanase processing and cellular localization
and secretion are thus probably other key mechanisms
that are involved in NSCLC and explain the increased
protein that has been previously found. In this regard,
Cohen et al. also showed that the cellular localization of
heparanase in cancerous cells was critical in patients
with lung tumors, a nuclear localization of the enzyme
being associated with a more favorable outcome (40 ).
As with many cancers, in NSCLC the survival of
patients is related to tumor growth and metastasis,
and TF overexpression has been shown to influence
these processes in various human malignancies (26 ).
Our results are the first to demonstrate a link between TF gene expression and the status of TP53
and PTEN TSG in NSCLC, and to establish a relationship between TF expression and the survival of
affected patients. Such a relationship has been previously evidenced with cell lines and animal models
in colorectal cancer (5 ) and glioma (6 ). Because
TP53, together with PTEN and STK11, regulate the
mammalian target of rapamycin (mTOR) pathway,
we can hypothesize that upregulation of TF gene expression in NSCLC results from an impairment of
this effect. The formation of TF/FVIIa complexes activates the mTOR pathway (41 ), which consists of
PI3K and Akt, and in turn may influence proliferation, invasiveness, metastasis spread, and tumor angiogenesis in lung carcinogenesis (28 ). In addition,
the mTOR pathway is also activated by the binding
of epidermal growth factor to epidermal growth factor receptor in lung cancer cells (28 ), and mutations
of epidermal growth factor receptor (erythroblastic
leukemia viral (v-erb-b) oncogene homolog, avian)
(EGFR) and PTEN genes have been recently identified as being responsible for TF upregulation in
glioblastoma (42 ). TF may therefore contribute to
lung tumor development, and the possibility of targeting this receptor in the treatment of NSCLC to
enhance the efficacy of inhibitors of mTOR or epidermal growth factor receptor warrants further
investigation.
Author Contributions: All authors confirmed they have contributed to
the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design,
acquisition of data, or analysis and interpretation of data; (b) drafting
or revising the article for intellectual content; and (c) final approval of
the published article.
Authors’ Disclosures of Potential Conflicts of Interest: Upon
manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: Institut pour la Recherche sur la Thrombose et
l’Hémostase.
Expert Testimony: None declared.
Role of Sponsor: The funding organizations played no role in the
design of study, choice of enrolled patients, review and interpretation
of data, or preparation or approval of manuscript.
Acknowledgments: We thank Prof. S. Guyetant, Dr. C. Bléchet (Department of Pathology), and Prof. P. Dumont (Department of thoracic surgery, Tours Hospital, France) for their help. We also thank
Dr. B. Giraudeau for helping with the statistical analysis and D. Raine
for editing the English.
Clinical Chemistry 55:10 (2009) 1841
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