Tumor Biol.
DOI 10.1007/s13277-016-5229-1
ORIGINAL ARTICLE
Prognostic value of mixed lineage kinase domain-like
protein expression in the survival of patients with gastric caner
Zhai Ertao 1 & Chen Jianhui 1 & Wang Kang 1 & Ye Zhijun 1 & Wu Hui 1 & Chen Chuangqi 1 &
Qin Changjiang 1 & Chen Sile 1 & He Yulong 1 & Cai Shirong 1
Received: 25 March 2016 / Accepted: 15 July 2016
# International Society of Oncology and BioMarkers (ISOBM) 2016
Abstract The aim of this study was to detect mixed lineage
kinase domain-like protein (MLKL) expression in gastric can-
cer (GC) and to analyze its association with the prognosis of
GC patients. Immunohistochemical staining, Western blot-
ting, and quantitative reverse-transcriptase polymerase chain
reaction were performed to detect MLKL tissue expression in
117 GC patients. Clinicopathological characteristics and sur-
vival data were retrospectively analyzed to discover the clin-
ical importance of MLKL expression. The chi-square test was
used to analyze the relationship between MLKL expression
and the clinicopathological characteristics. Survival curves
were plotted by using the Kaplan–Meier method and com-
pared using the log-rank test. Survival data were evaluated
using univariate and multivariate Cox regression analyses.
The expression of MLKL mRNA was significantly higher in
adjacent normal samples than in the tumor tissues (P = 0.003).
Clinicopathological analysis showed that MLKL expression
was significantly correlated with age (P = 0.013), histologic
type (P = 0.049), differentiation grade (P < 0.001), depth of
invasion (P = 0.022), and lymph node metastasis (P =
0.003). Low MLKL expression was significantly associated
with decreased overall survival (median 29 months vs. 56
months, P < 0.001). Multivariate analysis suggested that
MLKL expression might be an independent prognostic
Zhai Ertao and Chen Jianhui contributed equally to this work.
* Wu Hui
Docwuhui@126.com
* Cai Shirong
Caisrteam@163.com
1
Department of Gastrointestinal Surgery, The First Affiliated Hospital
of Sun Yat-sen University, 58 Zhongshan 2nd road,
Guangzhou 510080, China
indicator (HR = 0.645, 95 % CI, 0.446–1.165, P = 0.002) for
GC patients. In conclusion, our findings provide evidence that
MLKL might serve as a candidate tumor suppressor and a
potential prognostic biomarker for GC.
Keywords MLKL . Gastric cancer . Overall survival .
Prognosis
Introduction
Gastric cancer (GC) is one of the most common carcinoma in
worldwide [1]. GC is the second leading cause of cancer-
related morbidity and mortality in China [2]. It was estimated
that there are approximately 679,100 new cases in China an-
nually [2]. Despite the development in chemotherapy, radio-
therapy, and surgery, the prognosis of patients with advanced
GC remains poor [3]. The 5-year survival rate is only 4 % for
stage IV GC [4]. Thus, identifying novel molecular bio-
markers with prognostic value is important for improving
therapeutic methods and extending survival of GC patients.
Necrosis is a form of cell death characterized morphologi-
cally by cell volume increase, nuclear membrane dilation,
chromatin condensation, cell rounding, disruption of the cyto-
plasmic membrane, organelle swelling, and a lack of caspase
activation [5–10]. Necrosis was initially considered a passive
form of cell death; for instance, resulting from extreme cellu-
lar stress, it has become clear that necrosis can be induced in a
regulated manner by certain physiological or pathophysiolog-
ical stimuli, and viral or chemical inhibitors of caspases cause
an increase in necrotic cell death [5, 6]. This form of pro-
grammed necrotic death is named necroptosis. Necroptosis
requires receptor-interacting kinase 1 (RIP1) and RIP1-
related kinase RIP3 [11]. During necroptosis, RIP3 interacts
with RIP1 to recruit the downstream effector MLKL protein to

form the necrosome, within which MLKL becomes phosphor-
ylated [12, 13]. MLKL depletion in cancer cells also leads to
the spontaneous phosphorylation of H2AX, an early marker
for DNA damage, suggesting that MLKL may play a critical
role in the response to DNA damage [14]. Collectively, these
findings suggest that MLKL may be an important determinant
of cancer cell death, response to therapy, and outcome in pa-
tients with cancer.
Recent studies demonstrated that MLKL expression could
serve as a potential prognostic biomarker for patients with
carcinoma [15–17]. However, the clinicopathological features
of MLKL in GC remains unknown. In the present study, we
therefore assessed the expression of MLKL in a series of GC
specimens and investigated the association with clinicopatho-
logic parameters and overall survival in patients with GC.
Materials and methods
Patient samples
Ethical approval for human subjects and the use of tissue from
these subjects was obtained from the Institutional Review
Board of the First Affiliated Hospital of Sun Yat-sen
University (FAHSYSU), and written consent was obtained
from each patient. Samples from 117 GC patients, who
underwent surgery at FAHSYSU between 2004 and 2005,
were collected, confirmed to be GC, and then made available
for this study. Follow-ups were performed until December
2013.
Fresh tumor samples were obtained from the resection
specimens of primary GC patients treated by gastric surgery
at FAHSYSU in 2015 without prior radiotherapy or chemo-
therapy (N = 30). All excised tissues were frozen immediately
in liquid nitrogen and then stored at −80 °C until use.
Immunohistochemical staining
Immunohistochemistry (IHC) was conducted as described
previously [18]. Paraffin-embedded GC tissues were obtained
from the Department of Pathology. For immunohistochemis-
try, sections were taken, deparaffinized, and pretreated with
10-mM sodium citrate buffer for antigen unmasking (pH 6.0,
boiling temperature, 30 min). Then, they were blocked in
normal serum (Vectastain ABC kit, Vector Laboratories, Inc.
Burlingame, CA), incubated with primary antibodies at 4 °C
overnight, rinsed, and incubated with the secondary antibody
(Vectastain ABC kit). Signals were amplified using the
Vectastain ABC kit as per manufacturer’s instructions. The
target protein was visualized using diaminobenzidine as the
substrate.
The results were interpreted by two independent patholo-
gists who were blinded to the specific diagnosis and prognosis
Tumor Biol.
for each case and were scored using a semi-quantitative meth-
od where the staining of more than 10 % of the tumor cells
was considered positive. The staining intensity was scored as
Bnegative^ (immunohistochemical [IHC] score 0), Bweak^
(IHC score 1+), Bmoderate^ (IHC score 2+), and Bstrong^
(IHC score 3+) staining. According to MLKL expression,
GC patients were divided into the Blow MLKL expression^
and Bhigh MLKL expression^ groups.
Western blot
Western blot was conducted as described previously [19].
Total protein was extracted with cell lysis buffer and the pro-
tein concentration was quantified using an enhanced BCA
Protein Assay Kit. Proteins were separated using 8–10 % so-
dium dodecyl sulfate polyacrylamide gel electrophoresis and
electrotransferred onto polyvinylidene difluoride membrane
(Millipore, Billerica, MA, USA). The membrane was blocked
for 1 h with 5 % bovine serum albumin in tris-buffered saline-
Tween 20, probed with the relevant primary antibodies over-
night at 4 °C, and then incubated with rabbit and mouse horse-
radish peroxidase-coupled secondary antibodies for 1 h.
Specific bands were detected using the enhanced chemilumi-
nescence reagent (Millipore, Billerica, MA, USA) on autora-
diographic film. The following primary antibodies were used:
anti-MLKL (Abcam, USA) and anti-GAPDH (Proteintech,
Wuhan, China).
Quantitative reverse-transcriptase PCR
qRT-PCR was conducted as described previously [20]. Total
RNA was isolated using the RNA plus reagent (TaKaRa,
Japan). Complementary DNA was prepared using oligo-dT
primers according to the protocol supplied with the Primer
Script TM RT Reagent (TaKaRa, Japan). Expression levels
of MLKL mRNA were determined by quantitative reverse
transcriptase PCR using Power SYBR green PCR master
mix (Applied Biosystems).
Statistical analysis
SPSS version 18.0 (SPSS Inc., Chicago, IL) was used for data
analysis. The relationships between MLKL expression and
clinicopathological characteristics were analyzed using
the chi-square test. Kaplan–Meier survival curves were
constructed and the log-rank test was performed for uni-
variate analysis. Multivariate analysis was performed
using Cox’s proportional hazards model. A P value of
0.05 was considered statistically significant for all
analyses.
Tumor Biol.
Results
MLKL expression is downregulated in primary GC
We first determined the MLKL protein and mRNA expression
status in GC tissues by Western blot (Fig. 1a) and qRT-PCR
(Fig. 1b) in a cohort of 30 patients. The protein level of MLKL
expression was constantly with mRNA level of MLKL. The
rate of low MLKL expression was 60 % (18/30) in tumor
tissues. MLKL mRNA levels were significantly higher in ad-
jacent normal tissues (1.117 ± 0.047) than that in tumor tissues
(0.858 ± 0.053) (P = 0.003). In this part, we demonstrated that
MLKL expression is downregulated in primary GC.
MLKL protein staining in GC tissues
The expression of MLKL protein in archived GC tissue sam-
ples was analyzed by immunohistochemistry (IHC). We chose
117 patients with GC for IHC staining. The MLKL expression
is mainly located in cytoplasm, partially in the nucleus
(Fig. 2). Moreover, we confirmed that negative staining
(IHC score 0), weak staining (IHC score: 1+), moderate stain-
ing (IHC score: 2+), and strong staining (IHC score: 3+) were
found in 21.37 % (25/117), 35.90 % (42/117), 25.64 % (30/
117), and 17.09 % (20/117) of patients, respectively. In con-
clusion, 42.74 % (50/117) of GC tissues exhibited high
MLKL staining (moderate and strong staining) and 57.27 %
(67/117) of GC tissues presented low MLKL staining (nega-
tive and weak staining).
Relationship of MLKL protein expression
with clinicopathological features of GC
The association between MLKL protein expression and clin-
icopathological characteristics of GC was explored by the chi-
square test. As shown in Table 1, low MLKL expression in
GC was significantly correlated with older age (P = 0.013),
histologic type (P = 0.049), poor differentiation grade
(P < 0.001), advanced depth of invasion (P = 0.022), and
lymph node metastasis (P = 0.003). However, there were no
statistically significant relationships between MLKL
Fig. 1 MLKL protein and mRNA Levels are decreased in gastric cancer
tissue. a Expression of the MLKL protein was analyzed by Western blot
in tumor (T) and normal (N) gastric tissues in three typical samples. b
expression and other clinicopathological variables, such as
sex (P = 0.201), tumor location (P = 0.368), tumor size (P =
0.890), Bormann classification (P = 0.376), distant metastasis
(P = 0.741), and tumor-node-metastasis staging (P = 0.143).
Low MLKL expression correlated with poor patient
survival
For all the study subjects, the follow-up period ranged from 3
to 114 months, with a mean survival time of 48 months. The
5-year overall survival (OS) rate was 25.64 % for all patients
with GC. We found that patients with low MLKL expression
had poor prognosis (Fig. 3a, P < 0.001). The median survival
time was 29 and 56 months in the low MLKL expression and
high MLKL expression groups, respectively. The 5-year OS
rate was 11.94 % in the former and 44 % in the latter, respec-
tively. Furthermore, we also detected the prognosis of MLKL
expression in different TNM staging, including stages I and II
and stages III and IV. There were 22 and 95 cases in stages I
and II and stages III and IV, respectively. In stages I and II of
patients with gastric cancer, the median survival time was
30 months in low MLKL expression group and 85 months
in high MLKL expression group. The 5-year OS rate was
9.09 % in the former and 50.00 % in the latter, respectively.
In stages III and IV, the median survival time in low MLKL
expression group was 29 months and the 5-year OS rate was
10.91 %; in high MLKL expression group, the median surviv-
al time was 50 months and the 5-year OS rate was 42.50 %. In
stages I and II and stages III and IV, low MLKL expression
was significantly correlated with poor prognosis (stages I and
II, P = 0.035; and stages III and IV, P = 0.001; Fig. 3b, c).
Association of MLKL expression with OS in GC patients
We found that low MLKL expression patients exhibited sig-
nificantly reduced survival than high MLKL expression pa-
tients. To determine whether MLKL expression is an indepen-
dent prognostic predictor for GC patients, univariate and mul-
tivariate analyses were performed to compare the impact of
MLKL expression and other clinicopathological factors on the
prognosis of GC patients. Univariate Cox regression analysis
Expression of STIP1 mRNA was analyzed by quantitative reverse-
transcriptase polymerase chain reaction in tumor and normal gastric tis-
sues (N = 30)
 Tumor Biol.

Fig. 2 Immunohistochemical
staining of MLKL protein in
gastric cancer and adjacent
normal tissues. Negative, weak,
moderate, and strong MLKL
staining in gastric cancer tissues.
Original magnifications of ×200
and ×400 were shown

revealed that clinical variables including gender (hazard ratio
[HR] = 0.398, 95 % confidence interval [CI], 0.233–0.680,
P = 0.001), age (HR = 1.794, 95 % CI, 1.112–2.893, P =
0.017), Bormann classification (HR = 1.468, 95 % CI,
1.065–2.025, P = 0.019), depth of invasion (HR = 4.411,
95 % CI, 1.808–10.757, P = 0.001), lymph node metastasis
(HR = 2.003, 95 % CI, 1.227–3.270, P = 0.005), and MLKL
expression (HR = 0.965, 95 % CI, 0.888–1.565, P = 0.003)
were significantly associated with OS (Table 2).
Furthermore, multivariate Cox regression analysis was
also performed to evaluate the potential of MLKL ex-
pression as an independent predictor of OS in GC pa-
tients. Besides gender (HR = 0.551, 95 % CI, 0.354–
0.857, P = 0.008), depth of invasion (HR = 2.170, 95 %
CI, 1.290–3.651, P = 0.003), and lymph node metastasis
(HR = 1.487, 95 % CI, 1.184–1.867, P = 0.001), MLKL
low expression (HR = 0.645, 95 % CI, 0.446–1.165,
P = 0.002) was also an independent prognostic predictor
for GC patients (Table 2).
Discussion
Necroptosis is a caspase-independent form of regulated cell
death executed by the receptor-interacting protein kinase 1
(RIP1), RIP3, and mixed lineage kinase domain-like protein
(MLKL) [21, 22]. MLKL has been described as a key medi-
ator that participated in TNF-induced necroptosis and MLKL
could regulate necrotic plasma membrane permeabilization
[23–24]. Programmed necrosis is activated in response to
Tumor Biol.

Table 1 Relationship between

MLKL expression and Characteristic N MLKL expression X2 value P value
clinicopathological characteristics
in gastric cancer (N = 117) Low (N = 67) High (N = 50)

Gender
Male 71 44 (37.61 %) 27 (23.08 %) 1.635 0.201
Female 46 23 (19.66 %) 23 (19.66 %)
Age
≤60 years 74 36 (30.77 %) 39 (33.33 %) 6.108 0.013
>60 years 43 31 (26.50 %) 12 (10.26 %)
Tumor location
Proximal 14 11 (9.40 %) 3 (2.56 %) 3.158 0.368
Middle 18 10 (8.55 %) 8 (6.84 %)
Distal 59 31 (26.50 %) 28 (23.93 %)
More than two parts 26 15 (12.82 %) 11 (9.40 %)
Tumor size
<5 cm 67 38 (32.48 %) 29 (24.79 %) 0.019 0.890
≥5 cm 50 29 (24.79 %) 21 (17.95 %)
Histologic type
Adenocarcinoma 98 60 (51.28 %) 38 (32.48 %) 2.866 0.049
Others 19 7 (5.98 %) 12 (10.26 %)
Bormann classification
1 6 5 (4.27 %) 1 (0.85 %) 3.100 0.376
2 24 13 (11.11 %) 11 (9.40 %)
3 69 41 (35.04 %) 28 (23.93 %)
4 18 8 (6.84 %) 10 (8.55 %)
Differentiation grade
Well 26 19 (16.24 %) 7 (5.98 %) 20.920 <0.001
Moderately 66 26 (22.22 %) 40 (34.19 %)
Poorly 25 22 (18.80 %) 3 (2.56 %)
Depth of invasion
T1–2 38 16 (13.68 %) 22 (18.80 %) 5.285 0.022
T3–4 79 51 (43.59 %) 28 (23.93 %)
Lymph node metastasis
N0 21 6 (5.13 %) 15 (12.82 %) 8.611 0.003
N1–3 96 61 (52.14 %) 35 (29.91 %)
Distant metastasis
M0 104 59 (50.43 %) 45 (38.46 %) 0.109 0.741
M1 13 8 (6.84 %) 5 (4.27 %)
Tumor-node-metastasis staging
I–II 36 17 (14.53 %) 19 (16.24 %) 2.143 0.143
III–IV 81 50 (42.74 %) 31 (26.50 %)

many chemotherapeutic agents and contributes to
chemotherapy-induced cell death [25]. Recently,
necroptosis-based cancer therapy has been proposed to be a
novel strategy for antitumor treatment [25]. Recent studies
indicate that MLKL maybe a potential prognostic biomarker
for patients with carcinoma, such as pancreatic cancer, ovarian
cancer, and cervical squamous cell carcinoma [15–17]. This is
the first study to explore the association of MLKL expression
with clinicopathological features in GC. In this study, we dem-
onstrated that compared to adjacent normal tissue, MLKL
protein and mRNA levels were significantly decreased in
GC tissues. In addition, low MLKL expression was signifi-
cantly correlated with older age (P = 0.013), poor tumor dif-
ferentiation grade (P < 0.001), advanced depth of invasion
(P = 0.022), and lymph node metastasis (P = 0.003).
Moreover, high MLKL expression predicted a better
 Tumor Biol.

Fig. 3 Kaplan–Meier survival

curves for gastric cancer patients
according to MLKL expression
levels. aThe cumulative 5-year
survival rate was higher in high
MLKL expression patients than in
low MLKL expression patients
(44.00 vs. 11.94 %, P < 0.001). b
The cumulative 5-year survival
rate was higher in high MLKL
expression patients than in low
MLKL expression patients in
stages I and II (50.00 vs. 9.09 %,
P = 0.035). c The cumulative 5-
year survival rate was higher in
high MLKL expression patients
than in low MLKL expression
patients in stages III–IV (42.50
vs. 10.91 %, P = 0.001)

prognosis in patient undergoing surgery for GC. These find-
ings suggested that a loss of MLKL may be involved in GC
carcinogenesis and tumor progression, and MLKL may serve
as a useful predictor of prognosis in patients with GC.
MLKL is a key mediator of necroptosis; phosphorylated
MLKL could promote cancer cell necroptosis [12]. The
function of MLKL in cancer has been studied in some
malignant tumors. In pancreatic cancer, Colbert et al. first-
ly reported that MLKL expression could be served as a
prognostic biomarker for patients with malignant tumor;
they identified that low MLKL expression was associated
with decreased overall survival (OS) and recurrence-free

Table 2 Univariate and

multivariate analysis showing the Variables Univariate analysis Multivariate analysis
overall survival rate for gastric
cancer patients [HR, hazard ratio; HR 95 % CI P HR 95 % CI P
CI, confidence interval] value value
Lower Upper Lower Upper

Gender 0.398 0.233 0.680 0.001 0.551 0.354 0.857 0.008

Age 1.794 1.112 2.893 0.017
Tumor location 1.182 0.905 1.544 0.219
Tumor size 0.913 0.565 1.474 0.709
Histologic type 0.661 0.337 1.299 0.230
Bormann classification 1.468 1.065 2.025 0.019
Differentiation grade 0.833 0.510 1.361 0.466
Depth of invasion 4.411 1.808 10.757 0.001 2.170 1.290 3.651 0.003
Lymph node metastasis 2.003 1.227 3.270 0.005 1.487 1.184 1.867 0.001
Distant metastasis 2.147 0.516 8.930 0.293
Tumor-node-metastasis 0.362 0.072 1.817 0.217
staging
MLKL low expression 0.965 0.888 1.565 0.003 0.645 0.446 1.165 0.002
Tumor Biol.
survival (RFS) in patients receiving adjuvant chemotherpy
for early-stage resected pancreatic adenocarcinoma [17].
He et al. demonstrated that high MLKL expression was
significantly associated with increased disease-free sur-
vival (DFS) and decreased OS; they also identified that
low MLKL expression level was an independent factor for
the poor prognosis of patients with ovarian cancer [16].
Similarly with pancreatic cancer and ovarian cancer, Ruan
et al. showed that MLKL expression was obviously higher
that that in normal cervical tissues; its significantly corre-
lated histological grade and lymphatic metastasis; and low
MLKL expression was associated with a poor prognosis
of patients with cervical squamous cell carcinoma [15].
This study firstly demonstrated that MLKL expression
level was lower in GC tissues than that in adjacent normal
tissues; low MLKL expression predicted a worse progno-
sis for patients with GC.
In conclusion, our study revealed that MLKL is an inde-
pendent prognostic factor for overall survival in patients with
GC. High expression of MLKL in tumor is strongly correlated
with older age, poor tumor differentiation grade, advanced
depth of invasion, and lymph node metastasis. Our findings
suggest that MLKL might be used as a new biomarker and a
potential therapeutic target for GC. However, since relatively
little is known about the detailed role of MLKL in necroptosis
or in other signaling pathways, future studies need to elucidate
the molecular mechanisms of MLKL in GC cell death with
chemotherapy.
Acknowledgments This study was supported by the National Natural
Science Foundation of China (Grant Nos. 81372341 and 30972883) and
B3 & 3^ project of The First Affiliated Hospital of Sun Yat-sen University
(He Yulong).
Compliance with ethical standards
Ethical approval All procedures performed in studies involving hu-
man participants were in accordance with the ethical standards of the
institutional and/or national research committee and with the 1964
Helsinki declaration and its later amendments or comparable ethical
standards.
Conflicts of interest None
References
1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A.
Global cancer statistics, 2012. CA Cancer J Clin. 2015;65:87–108.
2. Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, et al. Cancer
statistics in China, 2015. CA Cancer J Clin. 2016;66:115–32.
3. Ueda T, Volinia S, Okumura H, Shimizu M, Taccioli C, Rossi S,
et al. Relation between microRNA expression and progression and
prognosis of gastric cancer: a microRNA expression analysis.
Lancet Oncol. 2010;11:136–46.
4. Jung H, Lee HH, Song KY, Jeon HM, Park CH. Validation of the
seventh edition of the American Joint Committee on Cancer TNM
staging system for gastric cancer. Cancer-Am Cancer Soc.
2011;117:2371–8.
5. Vanlangenakker N, Vanden BT, Vandenabeele P. Many stimuli pull
the necrotic trigger, an overview. Cell Death Differ. 2012;19:75–86.
6. Tenev T, Bianchi K, Darding M, Broemer M, Langlais C, Wallberg F,
et al. The ripoptosome, a signaling platform that assembles in response
to genotoxic stress and loss of IAPs. Mol Cell. 2011;43:432–48.
7. Coupienne I, Fettweis G, Piette J. RIP3 expression induces a death
profile change in U2OS osteosarcoma cells after 5-ALA-PDT.
Lasers Surg Med. 2011;43:557–64.
8. Zhao J, Jitkaew S, Cai Z, Choksi S, Li Q, Luo J, et al. Mixed lineage
kinase domain-like is a key receptor interacting protein 3 down-
stream component of TNF-induced necrosis. Proc Natl Acad Sci
USA. 2012;109:5322–7.
9. Chu WM. Tumor necrosis factor. Cancer Lett. 2013;328:222–5.
10. Zong WX, Thompson CB. Necrotic death as a cell fate. Genes Dev.
2006;20:1–15.
11. Vandenabeele P, Galluzzi L, Vanden BT, Kroemer G. Molecular
mechanisms of necroptosis: an ordered cellular explosion. Nat
Rev Mol Cell Biol. 2010;11:700–14.
12. Sun L, Wang H, Wang Z, He S, Chen S, Liao D, et al. Mixed lineage
kinase domain-like protein mediates necrosis signaling downstream
of RIP3 kinase. Cell. 2012;148:213–27.
13. Zhou Z, Han V, Han J. New components of the necroptotic path-
way. Protein Cell. 2012;3:811–7.
14. Paulsen RD, Soni DV, Wollman R, Hahn AT, Yee MC, Guan A,
et al. A genome-wide siRNA screen reveals diverse cellular pro-
cesses and pathways that mediate genome stability. Mol Cell.
2009;35:228–39.
15. Ruan J, Mei L, Zhu Q, Shi G, Wang H. Mixed lineage kinase
domain-like protein is a prognostic biomarker for cervical squa-
mous cell cancer. Int J Clin Exp Pathol. 2015;8:15035–8.
16. He L, Peng K, Liu Y, Xiong J, Zhu FF. Low expression of mixed
lineage kinase domain-like protein is associated with poor progno-
sis in ovarian cancer patients. Onco Targets Ther. 2013;6:1539–43.
17. Colbert LE, Fisher SB, Hardy CW, Hall WA, Saka B, Shelton JW, et al.
Pronecrotic mixed lineage kinase domain-like protein expression is a
prognostic biomarker in patients with early-stage resected pancreatic
adenocarcinoma. Cancer-Am Cancer Soc. 2013;119:3148–55.
18. Ertao Z, Jianhui C, Chuangqi C, Changjiang Q, Sile C, Yulong H,
et al. Low level of FOXL1 indicates a worse prognosis for gastric
cancer patients. Tumour Biol. 2016.
19. Zhang Y, Hua W, Niu LC, Li SM, Wang YM, Shang L, et al.
Elevated growth differentiation factor 15 expression predicts poor
prognosis in epithelial ovarian cancer patients. Tumour Biol. 2016.
20. Xue WJ, Feng Y, Wang F, Li P, Liu YF, Guo YB, et al. The value of
serum RASSF10 hypermethylation as a diagnostic and prognostic
tool for gastric cancer. Tumour Biol. 2016.
21. Zhu Y, Cui H, Gan H, Xia Y, Wang L, Wang Y, et al. Necroptosis
mediated by receptor interaction protein kinase 1 and 3 aggravates
chronic kidney injury of subtotal nephrectomised rats. Biochem
Biophys Res Commun. 2015;461:575–81.
22. Moriwaki K, Chan FK. RIP3: a molecular switch for necrosis and
inflammation. Genes Dev. 2013;27:1640–9.
23. Cai Z, Jitkaew S, Zhao J, Chiang HC, Choksi S, Liu J, et al. Plasma
membrane translocation of trimerized MLKL protein is required for
TNF-induced necroptosis. Nat Cell Biol. 2014;16:55–65.
24. Galluzzi L, Kepp O, Kroemer G. MLKL regulates necrotic plasma
membrane permeabilization. Cell Res. 2014;24:139–40.
25. Liu X, Zhou M, Mei L, Ruan J, Hu Q, Peng J, Su H, Liao H, Liu S,
Liu W, Wang H, Huang Q, Li F, Li CY: Key roles of necroptotic
factors in promoting tumor growth. Oncotarget. 2016;7(16):22219–
33.