Article

Lu WJ, Lee NP, Kaul SC, Lan F, Poon RT, Wadhwa R et al.. Induction of mutant p53-dependent apoptosis in human hepatocellular carcinoma by targeting stress protein mortalin. Int J Cancer 129: 1806-1814

Department of Surgery, The University of Hong Kong, Pokfulam, Hong Kong, China.
International Journal of Cancer (Impact Factor: 5.09). 10/2011; 129(8):1806-14. DOI: 10.1002/ijc.25857
Source: PubMed
ABSTRACT
Stress protein mortalin (mtHSP70) is highly expressed in cancer cells. It was shown to contribute to carcinogenesis by sequestrating the wild type p53, a key tumor suppressor protein, in the cytoplasm resulting in an abrogation of its transcriptional activation function. We have found that the level of mortalin expression has significant correlation with human hepatocellular carcinoma (HCC) malignancy and therefore investigated whether it interacts with and influences the activities of mutant p53, frequently associated with HCC development. We have detected mortalin-p53 interactions in liver tumor and five HCC cell lines that harbored mutant p53. The data was in contrast to the normal liver and immortalized normal hepatocytes that lacked mortalin-p53 interaction. Furthermore, we have found that the shRNA-mediated mortalin silencing could induce mutant p53-mediated tumor-specific apoptosis in HCC. Such allotment of apoptotic function to mutant p53 by targeting mortalin-p53 interaction in cancer cells is a promising strategy for HCC therapy.

Full-text

Available from: Wen jing Lu, Jul 16, 2014
Induction of mutant p53-dependent apoptosis in human
hepatocellular carcinoma by targeting stress protein mortalin
Wen-Jing Lu
1
, Nikki P. Lee
1
, Sunil C. Kaul
2
, Feng Lan
3
, Ronnie T.P. Poon
1
, Renu Wadhwa
2
and John M. Luk
1,4,5,6
1
Department of Surgery, The University of Hong Kong, Pokfulam, Hong Kong, China
2
National Institute of Advanced Industrial Science & Technology (AIST), Tsukuba, Ibaraki, Japan
3
Department of Medicine, Division of Cardiology, Stanford University School of Medicine, Stanford, CA
4
Department of Pharmacology, National University Health System, Singapore
5
Department of Surgery, National University Health System, Singapore
6
Cancer Science Institute, National University of Singapore, Singapore
Stress protein mortalin (mtHSP70) is highly expressed in cancer cells. It was shown to contribute to carcinogenesis by
sequestrating the wild type p53, a key tumor suppressor protein, in the cytoplasm resulting in an abrogation of its
transcriptional activation function. We have found that the level of mortalin expression has significant correlation with human
hepatocellular carcinoma (HCC) malignancy and therefore investigated whether it interacts with and influences the activities of
mutant p53, frequently associated with HCC development. We have detected mortalin–p53 interactions in liver tumor and five
HCC cell lines that harbored mutant p53. The data was in contrast to the normal liver and immortalized normal hepatocytes
that lacked mortalin–p53 interaction. Furthermore, we have found that the shRNA-mediated mortalin silencing could induce
mutant p53-mediated tumor-specific apoptosis in HCC. Such allotment of apoptotic function to mutant p53 by targeting
mortalin–p53 interaction in cancer cells is a promising strategy for HCC therapy.
Hepatocellular carcinoma (HCC) is a lethal malignancy asso-
ciated with poor prognosis and the fifth most common can-
cer worldwide. Effective HCC therapeutics still await molecu-
lar understanding of the mechanisms and development of
effective reagents that could selectively kill cancer cells. Like
any other tumors, HCC has high rate of mutations in tumor
suppressor protein p53, and is afflicted in p53-mediated func-
tions including cell cycle arrest and apoptosis.
1
Recently, sev-
eral studies have identified that the mutant p53 executes
transcription and nontranscription activities.
2,3
Several p53
mutants with a low level of transcriptional activation function
were also shown to induce apoptosis in cancer cells demon-
strating nontranscriptional apoptotic ability of mutant p53.
4,5
However, in cancer cells such apoptotic function of p53 is of-
ten deregulated by multiple pathways including its binding
with other proteins.
6,7
Restoration of p53-mediated apoptosis
is an attractive strategy for cancer therapy.
Mortalin/mthsp70/GRP75/PBP74 is a member of the heat
shock protein 70 family and has been shown to have multiple
functions including chaperoning, mitochondrial import, energy
generation and intracellular trafficking.
8,9
It exists in multiple
subcellular sites and possesses multiple binding partners.
8,9
Sev-
eral studies have shown that mortalin is frequently upregulated
in cancers and contributes to carcinogenesis.
10–14
It was shown
to cause cytoplasmic sequestration of wild type p53 resulting in
inhibition of its transcriptional activation and control of centro-
some duplication functions, both commonly associated with
cancers.
15–17
Furthermore, mortalin binding p53 peptides and
cationic inhibitor of mortalin (MKT-077) were shown to (i)
release p53 from mortalin–p53 complexes, (ii) cause nuclear
translocation of p53 and (iii) cause growth arrest of cancer cells
that contained wild type p53.
16,18
Mortalin was identified as a
marker for HCC metastasis and recurrence by proteomics anal-
ysis of matched tumor and nontumor tissues.
11
In our study,
we have investigated whether mortalin interacts with mutant
p53 (characteristic of cancers), affecting the function of p53.
We have found that (i) mortalin interact with mutant p53 in
all the five HCC-derived cell lines examined and (ii)mortalin
shRNA could reactivate p53-mediated-apoptosis. These findings
demonstrate that mortalin is an attractive target to induce mu-
tant p53-dependent apoptosis in HCC treatment.
Key words: human hepatocellular carcinoma, mortalin, RNAi, mutant
p53-dependent apoptosis, therapy
Additional Supporting Information may be found in the online
version of this article.
Grant sponsors: The HKU CRCG seed fund, New Energy &
Industrial Technology Development Organization (NEDO) and
Grant-in-Aid for Japan Society for the Promotion of Science (JSPS),
Japan
DOI: 10.1002/ijc.25857
History: Received 2 Oct 2010; Accepted 8 Nov 2010; Online 16 Dec
2010
Correspondence to: Renu Wadhwa, National Institute of Advanced
Industrial Science & Technology (AIST), Central 4, 1-1-1 Higashi,
Tsukuba, Ibaraki 305 8562, Japan, Tel.: þ81-29-861-9464, Fax:
þ81-29-861-2900, E-mail: renu-wadhwa@aist.go.jp; or John M. Luk,
Department of Pharmacology, National University of Singapore,
MD-11 #05-09, 10 Medical Drive, 117597, Singapore, Tel.:
þ65-65164516, Fax þ65-68737690, E-mail: jmluk@nus.edu.sg
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Materials and methods
Cell lines and culture
Human HCC cell lines, MHCC97H (97H), MHCC97L (97L),
H2M, H2P, PLC/PRF/5 (PLC), Hep3B and a immortalized
human hepatocyte liver cell line (MIHA), were cultured as
described
19
in Dulbecco’s modified Eagle’s minimal essential
medium (DMEM) (Gibco BRL, Grand Island, NY) containing
10% heat-inactivated fetal bovine serum (Gibco BRL, Grand
Island, NY) in a 5% CO
2
humidified atmosphere at 37
C.
Patient samples and statistical analyses
Human HCC and their adjacent nontumor liver tissue samples
were obtained during routine surgical procedures performed at
Queen Mary Hospital, the University of Hong Kong, from
1993 to 2008.
20
The clinical–pathological correlation of HCC
patients was analyzed by SPSS for Windows 16.0 (SPSS, Chi-
cago, IL). Categorical data were analyzed via Fishers exact test,
whereas an independent t test was used for continuous data.
Test results were considered significance at p < 0.05. All clini-
cal studies were conducted at the Department of Surgery, The
University of Hong Kong following the institutional
regulations.
Mortalin shRNA plasmid construction and transfections
The following DNA template oligonucleotides corresponding
to HSPA9 gene (GenBank NM_004134) were synthesized: 5
0
-
ACT CTA GGA GGT GTC TTT ATT CAA GAG ATA AAG
ACA CCT CCT AGA GTT TTT TTG GAA A-3
0
(1,451); 5
0
-
GCC AGA AGG ACA ACA TAT GTT CAA GAG ACA
TAT GTT GTC CTT CTG GCT TTT TTG GAA A-3
0
(2,166); 5
0
-ACC ATC TCG CAC ACA GCA ATT CAA GAG
ATT GCT GTG TGC GAG ATG GTT TTT TTG GAA A-3
0
(2,466). The above sequences were inserted into the BamHI
and HindIII sites in a pSilencer 2.1-U6 neo vector (Ambion,
Austin, TX) and were verified by DNA sequencing. For all
the experiments, 30 10
4
cells were transfected with shRNA
(2 lg for single or 1 lg for combinatorial transfection) using
the Lipofectamine
TM
2000 (Invitrogen, Carlsbad, CA).
21
Treatment of transfected cells with drugs
Cells were treated with different concentrations of Pi fithrin-a
(Sigma, St. Louis, MO) (PFT-a; 0.5, 2.5 and 5 lM) and Pifi-
thrin-l (Merck Chemicals Ltd., Nottingham, UK) (PFT-l;
0.25, 0.5 and 1 l M) for 72 hr post-transfection.
Immunoblotting
Cell lysates were prepared in RIPA (150 mM NaCl, 50 mM
Tris-HCl pH 7.5, 0.5mM EDTA, 0.5% sodium deoxycholate,
0.1% SDS, 1% NP40, 1 mM PMSF) buffer, electrophoretically
separated on 10% SDS-PAGE gels, electrotransferred to a nylon
membrane and probed with indicated antibodies, followed by
incubation with horseradish peroxidase-conjugated secondary
antibody and Enhanced Chemi Luminescence (ECL) detection
reagents, GE Healthcare, Piscataway, NJ.
Determination of cell viability
Cells were transfected with different vectors as indicated.
Twelve hours post-transfection, each group was reseeded
with equal density of 4,000 cells per well in 96-well plate. For
viability assay, 10 ll of MTT (5 mg/ml) was added, and
plates were placed at 37
C for 2 hr. Dimethylsulfoxide (100
ll) was added to each well for cell lysis. Absorbance was
measured at 570 nm with the reference filter (655 nm). All
the experiments were done in triplicates and three times.
TUNEL assay
Cells were grown on coverslips and fixed with 4% parafor-
maldehyde in phosphate-buffered saline (PBS; pH 7.4). The
terminal deoxynucleotidyl transferase-mediated dUTP nick
end labeling (TUNEL) assay was carried out following the
manufacturer’s instructions (11684817910; Roche, Indianapo-
lis, IN).
22
Hematoxylin was used as a counter stain.
In vitro co-immunoprecipitation
Cell lysates (500 lg protein in 400 ll RIPA lysis buffer) were
precleared with protein A/G PLUS-Agarose beads (sc-2003,
Santa Cruz Biotechnology, Santa C ruz, CA) and then incu-
bated with anti-mortalin antibody (H-155, sc-13967; Santa
Cruz Biotechnology) at 4
C overnight. Immunocomplexes
separated by incubation with protein A/G Agarose beads
were resolved on SDS-PAGE. Immunoblotting was performed
with anti-p53 antibody (DO-7; Dako, Carpinteria, CA, USA).
Immunofluorescence staining
Cells were fixed with 4% paraformaldehyde in PBS, permeab-
ilized with 0.1% Triton X-100 for 15 min, immunostained for
mortalin and p53 as described
16
and visualized using an
Eclipse E600 image analysis system (Nikon, Tokyo, Japan).
Luciferase reporter assay
Cells grown in a 24-well tissue culture plate were co-trans-
fected with the wild type p53 driven fire fly luciferase expres-
sion vector (PG13-Luc; 250 ng) and herpes simplex virus thy-
midine kinase promoter driven Renilla luciferase expression
vector (pRL-TK; 20 ng) as internal control reporter using
Lipofectamine
TM
2000 reagent (Invitrogen, Carlsbad, CA).
After 24 hr, the promoter activity was assessed using Dual-
Luciferase reporter assay system (Promega, Madison, WI) by
measuring the intensity of chemiluminescence in a luminom-
eter (Thermo Fisher Scientific, Waltham, MA). The experi-
ments were performed in triplicates and repeated, at least,
three times.
Results
Overexpression of mortalin and its interaction with p53
associate with HCC malignancy
Immunohistochemical (IHC) examination of mortalin protein
in 100 pairs (tumor and adjacent nontumor tissue) of clinica l
HCC samp les revealed its high expression in 61% cases. It
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correlated with the tumor aggressiveness; there was a marked
increase in mortalin level from the early (stage I and stage II)
to late (stage III and stage IV) stages (Fig. 1a). Quantitative
analysis in 49 pairs of clinical HCC samples (20 of these
were included in IHC group and 29 were randomly selected)
revealed higher level of expression in aggressive stage IV
tumors (Figs. 1b and 1c). Based on the clinical–pathological
analysis, mortalin expression was significantly associated
with HCC recurrence (p ¼ 0.005) and the microsatellite for-
mation (p ¼ 0.028), a crucial feature of intrahepatic metas-
tasis (Supporting Information Table 1). The data suggested
that the overexpression of mortalin has functional correla-
tion with HCC development and progression. To address
the correlation between mortalin and p53 expression
(mutated and stabilized in large majority of tumors
3,23,24
),
we first examined the expression of p53 in 29 pairs of
human HCC tissues as well as the corresponding adjacent
normal hepatic tissues by Western blotting. Compared to
the adjacent nontu mor tissue, tumor tissue showed upregu-
lation of p53 in 9/29 (31%) samples (Fig. 1d). More
importantly, seven of these nine (77.8%) samples also
showed mortalin overexpression. The data suggested that
Figure 1. Overexpression of mortalin and its interaction with p53 in HCC clinical samples. Immunoreactivity of mortalin was shown by
immunohistochemical staining (a) and Western blot analysis (b) of the representative pathological samples of hepatocellular carcinoma
(HCC). The relative level of mortalin expression in tumor (T) and adjacent nontumor (NT) clinical liver samples was semiquantitated by
comparison with an internal control (actin) (c). Western blot analysis of p53, the representative tumor (T) and adjacent nontumor (NT)
clinical liver samples are shown (d). Co-immunoprecipitaion of mortalin and p53 in liver tumor, but not in the normal liver tissue, is shown.
Input lanes show expression of p53 in 20 lg (1:10 used for immunoprecipitation) of the lysate (e).
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many tumors with mutant p53 (as indicated by its increased
stability) have overexpression of mortalin. Furthermore, by
mortalin–p53 co-immunoprecipitation assay, we detected
the interaction of mortalin and p53 in the tumor, but not in
the normal, liver tissue (Fig. 1e). These data demonstrated
that mortalin is enriched in HCC tumors and it interacts
with p53 in tumor, but not in normal liver tissues.
Mortalin interacts with mutant p53 and its knockdown
induces apoptosis in HCC-derived cells and not in
normal cells
To investigate whether the upregulation of mortalin in HCC
has any functional impact on the mutant p53 function, we
first investigated their interaction by co-immunoprecipitation
in HCC-derived five mutant p53 cell lines. As shown in Fig-
ure 2a, p53 was pulled-down along with mortalin in 97H,
97L, H2M, H2P and PLC cells (HCC cells harboring mutant
p53), but not in MIHA (immortalized normal hepatocytes
harboring wild type p53). To examine the functional signifi-
cance of mortalin, we performed its knockdown by shRNA
expression plasmids. Three mortalin-specific shRNA vectors
(1,451, 2,166 and 2,466, indicating the nucleotide targeting
site on mortalin cDNA) were generated. HCC-derived cells
PLC (mutant p53 249
ser
mutation, the most common p53
mutation site in HCC with G: C to T: A transversion) when
transfected with mortalin shRNA vectors showed significantly
reduced expression of mortalin. ShRNA-2166 was the strong-
est and caused 70% knockdown at 72 hr post-transfection
(Figs. 2b and 2c). Similar to PLC, immortalized human hepa-
tocytes (MIHA) transfected with shRNA-2166 showed 70%
knockdown of mortalin expression as compared to the con-
trol (untransfected-M and scramble shRNA-S vector trans-
fected) cells (Fig. 2c). Whereas shRNA-2166 caused a dra-
matic reduction (almost 50%) in the viability of PLC cells, it
had no effect on the MIHA cells (Fig. 2d). Based on the cell
morphology and a rapid decline in cell viability of PLC cells,
it appeared that the shRNA-2166 caused apoptosis that was
further confirmed by TUNEL staining (Fig. 2e). Of note, con-
sistent with the cell viability data (Fig. 2d), no apoptosis was
observed in MIHA cells (Fig. 2e).
Mortalin shRNA-induced mutant p53-dependent apoptosis
in HCC
We next investigated the molecular markers of apoptosis in
control and mortalin shRNA-tra nsfected cells. Whereas mor-
talin-compromised PLC cells exhibited increase in proapop-
totic protein Bax, cleavage of caspase 3 and PARP, there was
decrease in antiapoptotic protein Bcl-xL (Fig. 3a). Of note,
consistent with the absence of apoptosis, these molecular
events were not detected in MIHA cells subsequent to mor-
talin knockdown (Fig. 3a). Further, we used p53 inhibitors,
PFT-a and PFT-l, to competitively block its transcriptional
(by nuclear accumulation) and apoptotic (by mitochondrial
accumulation) activities, respectively. As shown in Figure 3b,
both p53 inhibitors reversed mortalin knockdown-induced
apoptosis: Bax, cleaved caspa se 3 and cleaved PARP expres-
sion were decreased, and Bcl-xL expression was increased in
a dose-dependent manner. These data demonstrated that
mortalin shRNA-in duced apoptosis was (i) p53-dependent
and (ii) conditional to both nuclear and mitochondrial func-
tions of p53. We further investigated the effect of mortalin
knockdown in four other HCC cells (97H, 97L, H2M and
H2P) with mutant p53 (249
ser
mutation). Interestingly,
cleaved caspase 3 and apoptosis were detected in all the four
cell lines that underwent apoptosis. Hep3B cells that lacked
mutant p53 did not show caspase cleavage or apoptosis sub-
sequent to mortalin knockdown (Figs. 3c–3e). Taken to-
gether, these data demonstrated that mortalin knockdown
caused mutant p53-dependent apoptosis in HCC.
Carboxyl-terminal region of p53 containing the nuclear
localization signals (also functional in the mutant p53) was
shown to interact with mortalin resulting in its cytoplasmic
retension.
16
We next investigated whether mortalin would
Figure 2. Mortalin interacts with mutant p53 and its silencing
induces apoptosis in HCC-derived cells, but not in immortal normal
hepatocytes-MIHA. (a) Mortalin–p53 interaction was detected in
five HCC cells: MHCC97H, MHCC97L, H2M, H2P and PLC/PRF/5.
MIHA cells lacked this interaction. (b) Immunoblots showing the
knockdown efficiency of three different mortalin shRNA plasmids
(target sites -1451, -2166, -2466) at 72 hr post-transfection. The
knockdown efficiency was observed at 48 and 72 hr post-
transfection of shRNA-2166. (c) Reduction in mortalin (KD)
expression was obtained by shRNA-2166 both in PLC/PRF/5 and
MIHA cells. Scramble shRNA (S) and cells treated with transfection
reagent (mock, M) were included as controls. Actin was included
as a loading control. (d) Viability (MTT assay) was significantly
reduced in PLC/PRF/5 (*p < 0.05) at 72 hr post-transfection of
shRNA-2166; MIHA cells did not show any change as compared to
controls. (e) In situ TUNEL staining identified shRNA-2166-induced
apoptosis in PLC/PRF/5, but not in MIHA cells. [Color figure can be
viewed in the online issue, which is available at
wileyonlinelibrary.com.]
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cause inactivation of mutant p53 function by similar cyto-
plasmic sequestration. If so, apoptosis caused by mortalin
knockdown would be expected to involve nuclear transloca-
tion of the mutant p53. To test this hypothesis, we performed
single cell immuno-cytochemistry analysis in which mortalin
compromised cells (decrease or lack of mortalin immuno-
staining) were examined to see the presence of nuclear p53.
In all the five HCC cell lines with mutant p53, the localiza-
tion of p53 was fou nd in the cytoplasm as well as the nu-
cleus. Transfection of shRNA-2166 led to significant accumu-
lation of p53 in the nucleus (Fig. 4 and Supporting
Information Fig. 1). Hep3B (p53 null) cells exhibited no
staining with anti-p53 antibody demonstrating the specificity
of immunofluorescence. Furthermore, normal immortalized
Figure 3. Mortalin silencing-induced apoptosis in HCC cells is dependent on mutant p53. (a) Mortalin compromised PLC/PRF/5, but not
MIHA, but not MIHA, cells exhibited p53. Cells exhibited p53-mediated apoptosis (increase in Bax, cleaved caspase 3, cleaved PARP and
decrease in Bcl-xL). Scramble shRNA (S) and mock (M) were used as controls. (b) p53 inhibitors (PFT-a and PFT-l), used in combination
with mortalin shRNA-2166, caused reversal of p53-dependent apoptosis, in mortalin-knockdown (Mot KD) PLC/PRF/R cells. (c) Mortalin
shRNA-2166 induced apoptosis mediated by cleavage of caspase 3 in four HCC (MHCC97H, MHCC97L, H2M and H2P—all with mutant p53)
cells. Hep3B (p53 null) cells did not show caspase cleavage and apoptosis. (d) Cell images undergoing apoptosis in four HCC (MHCC97H,
MHCC97L, H2M and H2P) and lack of apoptosis in Hep3B are shown. (e) The cytotoxicy (MTT assay) was significantly increased in
MHCC97H, MHCC97L, H2M and H2P (*p < 0.05) at 72 hr post-transfection of shRNA-2166. Hep3B cells did not show any change as
compared to controls. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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hepatocytes (MIHA) and Hep3B (both lacked mortalin-p53
interaction) neither showed any sign of apoptosis (Fig. 3c)
nor exhibited any change in the subcellular localization of
p53 with/without shRNA-2166 (Supporting Informa tion Fig.
1).
To compare the efficiency of mortalin shRNA-induced ap-
optosis mediated by wild type or mutant p53, we introduced
either wild type or mutant (249
ser
) p53 in Hep3B (p53 null)
cells. Exogenously expressed wild type and mutant p53 inter-
acted with mortalin (Figs. 5a and 5b). Furthermore, whereas
there were no apoptotic cells in control vector transfected
cultures, cells co-transfecte d with p53 (wild type or mutant)
and mortalin shRNA showed TUNEL-positive apoptotic cells
(Fig. 5c). The quantitative analyses of apoptotic index showed
that mortalin silencing in wild type p53 harboring cells
caused 10% apoptotic cell death even at 24–48 hr post-trans-
fections. Mutant p53 harboring cells exhibited about 20% cell
death (Fig. 5d). These data clearly demonstrated that the apo-
ptotic function of mutant p53 was strongly activated by mor-
talin shRNA.
To investigate whether such activation of mutant p53 was
due to assignment of wild type p53 activity to the mutant
p53, we performed wild type p53-specific reporter assay in
Hep3B cells that were compromised for mortalin under wild
type p53 and mutant p53 backgrounds. As shown in Fig. 5e,
mortalin silencing caused increase in the transcription activ-
ity of wild type p53 only. Hep3B cells expressing mutant p53
did not show transcriptional activation function in control or
mortalin shRNA-transfected cells (Fig. 5e) suggesting that the
apoptosis caused by mortalin knockdown involved activation
of mutant p53 functions that are independent of the wild
type p53 transcriptional activation function.
We also investigated whether similar activation of apopto-
tic function of p53 could be achieved by targeting mortalin–
mutant p53 interaction by other small molecules including
chemical MKT-077 and p53 (312–352) peptide that were
shown to bind to mortalin and abrogate mortalin–p53 inter-
action.
16,18
As shown in Figure 6, we found that the PLC/
PRF/5 cells (mutant p53) when treated with either MKT-077
or p53 (312–352) peptide showed strong nuclear transloca-
tion of p53 (Figs. 6a and 6c) and activation of mutant p53-
mediated apoptosis (Figs. 6b and 6d). Taken together, the
data demonstrated that (i) apoptotic activity of p53 is sup-
pressed in cancer cells by mortalin–p53 interaction and (ii)
targeting this interaction instigates p53-mediated apoptosis
that could serve as an effective anticancer therapeutics.
Discussion
Tumor suppressor protein p53 is regarded as a central player
in tumor suppression. It controls apoptosis and cellular se-
nescence directly or indirectly at multiple levels of the tumor
suppression network by invoking a myriad of mechanisms
that involves its transcription-dependent and transcription-
independent functions.
23,24
In vast majority of tumors, loss of
p53 function attained by mutations is required for tumor
maintenance in which deregulated apoptosis (tumor clearance
mechanism) serves as a major cancer-sustaining mechanism.
Reactivation of mutant p53 in tumors has been expected to
Figure 4. Mortalin silencing-induced apoptosis is mediated by nuclear translocation of mutant p53. Double immunostaining images of PLC/
PRF/5 cells show mortalin knockdown-induced nuclear translocation of p53 (mortalin, green; p53, red).
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induce apoptosis and eliminate the tumor cells. Hence the
therapeutic strategies aimed at reactivation of apoptotic activ-
ities of mutant p53 in tumors are emerging as a promising
cancer therapeutic approach. It was shown that the transcrip-
tion-incompetent mutant p53 could induce apoptosis in can-
cer cells
5
suggesting that there are transcription-independent
mechanisms that deregulate such mutant p53 activities in
cancer cells.
In contrast t o t he thymus- and spleen-derived cells that
undergo p53-mediated apoptosis in response to genotoxic
stress, liver cancer cells tend to be resistant
25–27
suggesting
that the HCC may have mechanism(s) leading to inactiva-
tion of p53-mediated apoptosis. In our earlier proteomic
studies,
11,28,29
we identified members of the heat shock fam-
ily proteins t hat are often upregulated in HCC and associ-
ated prognostic outcomes, and mortalin as a marker for
predicting HCC metastasis and recurrence. In our study, we
demonstrated the interaction of mortalin and p53 in liver
tumors and HCC-derived cell lines that harbored mutant
p53. Based on the existing literature and our present find-
ings, we speculated that mortalin could be responsible for
inactivation of p53 function and tumor sustainability. To
Figure 5. Mortalin silencing induced apoptosis in p53 null Hep3B HCC after reintroduction of either wild type or mutant p53. (a) The
double immunofluresence staining showing the exogenous expression of wild type or mutant (249
ser
) p53 (red staining) in Hep3B cells. (b)
Mortalin and p53 interactions were detected by co-immunoprecipitation assay in Hep3B cells after transfection with either wild type or
mutant p53. A polyclonal anti-mortalin antibody was applied to pull-down the complex that was examined for the presence of p53 by
Western blotting with a monoclonal anti-p53 antibody. Both wild type and mutant p53 were co-immunoprecipitated with mortalin. Rabbit
normal IgG was used as pull down control. (c) In situ TUNEL staining identified apoptosis in Hep3B cells co-transfected with wild type or
mutant p53 and mortalin shRNA, but not in cells either transfected with the vector control or transfected with wild type or mutant p53
alone. (d) Quantitation of the apoptotic cells is shown (*p < 0.05). (e) Wild type p53-dependent luciferase reporter assay showing the
reactivation of wild type p53 transcriptional activity in mortalin knockdown cells; mutant p53 did not show any effect in the transcriptional
activation assays (*p < 0.05).
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test this hypothesis, we ex ploited mortalin shRNA to knock-
down mortalin and expected it to discharge p53 from mor-
talin–p53 complex resulting in its functional activation. As
showninthedata,wefoundthattheknockdownofmor-
talin induc ed apoptosis in all the H CC c ell lines that har-
bored mutant p53. Immortalized normal liver cells (wild
type p53) and Hep3B (p53 null) c ells that lacked mortalin–
p53 interactions did not show apoptosis and the associated
molecularevents(nucleartranslocationofp53andcaspase
activation; Figs. 2, 3 and 4). These data strongly suggested
that mortalin shRNA resulted in mutant p5 3-mediated apo-
ptosis in cancer cells only and hence could be used to selec-
tively kill cancer cells.
The majority of p53 mutations observed in human cancers
are missense mutations in its DNA binding domain that
abrogate its sequence-specific binding to the wild type p53
responsive element making it transcriptionally inactive.
2
However, mutant p53 has been reported to have functions
(growth arrest
30
and induction of apoptosis
31
) independent
to its transcriptional activation function. It was also shown
that the p53-mediated apoptosis could occur in the presence
of p53-DNA binding inhibitors, like PIASc, suggesting that
sequence-specific transactivation is not essential for p53-de-
pendent apoptosis.
31–33
In our study, we demonstrated that
the mutant p53 binds to mortalin in tumor cells and causes
inactivation of its apoptotic function. Silencing of mortalin
was found to activate p53-mediated apoptosis in all the cell
lines tested. To determine whether such p53-dependent apo-
ptosis involved restoration of the wild type p53 transactiva-
tion in the mutant p53, we performed wild type p53-depend-
ent luciferase reporter assays in Hep3B (p53-null) cells with
exogenous expression of either the wild type or the mutant
p53. We found that mortalin knockdown enhanced transacti-
vation ability of the wild type p53. In the same assays, mu-
tant p53 did not show any transcriptional activation function
in control or mortalin shRNA-transfected cells (Fig. 5e), sug-
gesting that the apoptosis caused by mortalin knockdown
involved activation of mutant p53 functions, independent of
the wild type p53 transactivation. Nevertheless, the apoptotic
ability of mutant p53 was stronger than the wild type p53
suggesting that mortalin shRNA is a good candidate for HCC
therapy as HCC is frequently associated with p53 mutations.
Transcription-independent apoptotic ability of mutant p53
has been reported in several studies.
31–33
It was shown that
the p53-mediated apoptosis involves (i) activation of Bax
dimer formation and changes in the mitochondrial mem-
brane permeabilization
34
and (ii) formation of nuclear Bax/
p53 complexes
35
and their crosstalk with nuclear chaperone,
nucleophosmin.
36
We found that in mutant p53 harboring
cells, mortalin knockdown resulted in the nuclear accumula-
tion of p53, increased levels of Bax (Fig. 3) and increased
level of mutant p53–bax interaction (data not shown) sug-
gesting that mortalin silencing caused activation of p53–Bax
apoptosis pathway.
The identification of nontoxic chemicals and small mole-
cules capable of restoring the tumor suppression and apopto-
tic function of mutant p53 is an exciting prospective for
future cancer therapy. In our study, we demonstrated that
mortalin silencing reactivates p53 activities selectively in can-
cer cells. Based on these data, we anticipated that mortalin–
p53 interaction could serve as a target for selective cancer cell
killing. The data was also validated by using cationic dye
(MKT-077) and p53 carboxyl-terminal peptide (p53
312–352
)
that were previously shown to bind to mortalin and release
p53 from mortalin–p53 complex leading to growth arrest of
cells.
16,18
As shown in Figure 6, we found that mortalin bind-
ing chemical (MKT-077) and short peptide trigger the nuclear
translocation of mutant p53 in PLC/PRF/5 cells. The data
demonstrated that the interaction of mortalin and p53 is
unique to cancer cells, and this interaction causes inactivation
of mutant p53 functions and hence constitutes a selective tar-
get to reactivate apoptotic activities of p53 in cancer cells.
Furthermore, as more than half of the cancers possess mutant
p53, such assignment of apoptotic function to mutant p53 is
expected to be of great advantage in cancer therapeutics. Nor-
mal cells lack mortalin–p53 interaction and hence such thera-
peutic strategy will be highly selective for cancer cells.
Acknowledgements
We thank S.Y. Yik, Department of Surgery, The University of Hong Kong
for preparing the clinical samples.
Figure 6. Mortalin binding chemical (MKT-077) and peptide trigger
the nuclear translocation of mutant p53 in PLC/PRF/5 cells. (a)
and (c) Double immunostaining showed the nuclear translocation
of mutant p53 with mortalin binding chemical (MKT-077) and
peptide (p53
312–352
) in PLC/PRF/5 cells (mortalin, green staining;
p53, red staining). Of note, in (b) and (d) nuclear translocation of
mutant p53 either by MKT-077 or by peptide was associated with
induction of apoptosis as detected by TUNEL staining.
Carcinogenesis
Lu et al. 1813
Int. J. Cancer: 129, 1806–1814 (2011)
V
C
2010 UICC
Page 8
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  • Source
    • "Mortalin has been shown to inactivate p53 protein [22, 30,4344454647. In light of the above data, we next investigated the direct docking potential of embelin to mortalin and p53 proteins. "
    [Show abstract] [Hide abstract] ABSTRACT: Embelin, a natural quinone found in the fruits of Embelia ribes, is commonly used in Ayurvedic home medicine for a variety of therapeutic potentials including anti-inflammation, anti-fever, anti-bacteria and anti-cancer. Molecular mechanisms of these activities and cellular targets have not been clarified to-date. We demonstrate that the embelin inhibits mortalin-p53 interactions, and activates p53 protein in tumor cells. We provide bioinformatics, molecular docking and experimental evidence to the binding affinity of embelin with mortalin and p53. Binding of embelin with mortalin/p53 abrogates their complex resulted in nuclear translocation and transcriptional activation function of p53 causing growth arrest in cancer cells. Furthermore, analyses of growth factors and metastatic signaling using antibody membrane array revealed their downregulation in embelin-treated cells. We also found that the embelin causes transcriptional attenuation of mortalin and several other proteins involved in metastatic signaling in cancer cells. Based on these molecular dynamics and experimental data, it is concluded that the anticancer activity of embelin involves targeting of mortalin, activation of p53 and inactivation of metastatic signaling.
    Full-text · Article · Sep 2015 · PLoS ONE
  • Source
    • "Mortalin is a multifunctional stress chaperone and is present in wide subcellular localizations ranging from mitochondria, endoplasmic reticulum (ER), cytosol, cell surface, and nucleus (Ran et al. 2000; Wadhwa et al. 2002). It is enriched in a variety of cancers (Dundas et al. 2005; Lu et al. 2011a, b; 2011a; Wadhwa et al. 2006; Yi et al. 2008; Ando et al. 2014; Klaus et al. 2014; Ryu et al. 2014). Mortalin has also been detected in the serum of colorectal patients, suggesting it as a general cancer diagnostic marker (Rozenberg et al. 2011). "
    [Show abstract] [Hide abstract] ABSTRACT: Mortalin is a stress chaperone belonging to the Hsp70 family of proteins. Frequently enriched in cancers, it is a multifunctional protein and regulates cell proliferation, apoptosis, mitochondrial functions, and the activity of tumor suppressor protein p53. In the present study, we investigated circulating mortalin and its autoantibody in normal, cirrhosis, and cancerous liver. We found that although mortalin is enriched in liver cancer cells and tumors, it is not detected in the serum of either the liver cirrhosis or cancer patients. In contrast, mortalin autoantibody was detected in patients' sera and showed significant correlation with the occurrence of cirrhosis. It is suggested as a potential noninvasive marker for liver cirrhosis.
    Full-text · Article · Apr 2015 · Cell Stress and Chaperones
  • Source
    • "The mitochondrial mortalin can interact with and against Bcl-2 family proteins and p53 to mediate apoptosis on cells [25]. Mortalin is a chaperone that can negatively combine with p53 [33,36]. Then attempts were made to determine whether fucoxanthininduced apoptosis is dependent on the suppression of mortalin . "
    [Show abstract] [Hide abstract] ABSTRACT: Fucoxanthin, a natural carotenoid, has been reported to have anti-cancer activity in human colon cancer cells, human prostate cancer cells, human leukemia cells, and human epithelial cervical cancer cells. This study was undertaken to evaluate the molecular mechanisms of fucoxanthin against human bladder cancer T24 cell line. MTT analysis results showed that 5 and 10 μM fucoxanthin inhibited the proliferation of T24 cells in a dose- and time-dependent manner accompanied by the growth arrest at G0/G1 phase of cell cycle, which is mediated by the up-regulation of p21, a cyclin-dependent kinase (CDK)-inhibitory protein and the down-regulation of CDK-2, CDK-4, cyclin D1, and cyclin E. In addition, 20 and 40 μM fucoxanthin induced apoptosis of T24 cells by the abrogation of mortalin–p53 complex and the reactivation of nuclear mutant-type p53, which also had tumor suppressor function as wild-type p53. All these results demonstrated that the anti-cancer activity of fucoxanthin on T24 cells was associated with cell cycle arrest at G0/G1 phase by up-regulation of p21 at low doses and apoptosis via decrease in the expression level of mortalin, which is a stress regulator and a member of heat shock protein 70, followed by up-regulation of cleaved caspase-3 at high doses.
    Full-text · Article · Sep 2014 · Acta Biochimica et Biophysica Sinica
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