Introduction
Colorectal cancer (CRC) is one of the most common
human cancers and is associated with high morbidity and mortality
(1). Particularly in cases of
advanced rectal cancer, preoperative radiotherapy (PRT) and
chemoradiotherapy (PCRT) improve local control and long-term
disease-free survival, compared with surgery alone (2,3).
Furthermore, PCRT has been one of the standard therapies for rectal
cancer regarding anus preservation and is used for the prevention
of local recurrence and presurgical downstaging (4). In practice, the efficiency of PCRT
varies between individuals; pathologic complete response has been
reported in the range of 15 to 20% (5–7). Various
clinical factors have been reported as predictors of histological
regression or tumor downstaging in rectal cancer, such as the
circumferential extent of the tumor, distance from the anal verge,
and serum levels of carcinoembryonic antigen (CEA) (8–10).
However, the validation of these predictors remains
insufficient.
The phosphorylation of histone H2AX (into γ-H2AX) is
induced by DNA double-strand breaks (DSB). As tumor cells are
usually deficient in DNA damage response (DDR) pathways, it has
been suggested that constitutive expression of histone γ-H2AX might
indicate the disruption of DDR pathways and genomic instability
(11). γ-H2AX expression gradually,
but significantly, increases during tumor progression in human CRC
(12). In vitro, increasing
levels of γ-H2AX, after irradiation, have been correlated with
radiosensitivity in 18 human cell lines (13). Additionally, high γ-H2AX expression
is associated with poor prognosis in CRC patients (14). Therefore, we hypothesized that the
expression level of γ-H2AX is a predictor of radiosensitivity in
rectal cancer. In this study, we sought to clarify the relationship
between γ-H2AX expression and radiosensitivity in CRC, using in
vivo and in vitro experiments.
Materials and methods
Data set analysis
The Oncomine™ Research Platform (Thermo Fisher
Scientific, Inc.) was used in this study. The mRNA expression
levels of H2AX were investigated in each cohort study. The detailed
methodology of these studies is available in the references
(15–22).
Cell culture
Human CRC cell lines WiDr and DLD-1 were purchased
from JCRB (Japanese Collection of Research Bio Resources) Cell
Bank. Both lines were authenticated by short tandem repeat (STR)
sequence profiling by JCRB. STR examination showed that the WiDr
was identical to HT-29 (23). All
cells were cultured in RPMI-1640 (HyClone; GE Healthcare Life
Sciences) supplemented with 10% (v/v) heat-inactivated fetal bovine
serum at 37°C, in a humidified atmosphere containing 5%
CO2.
Patient samples
Six pairs of CRC tissues and adjacent normal mucosa
tissues, and eleven pairs of endoscopic biopsy samples from CRC
tissues and adjacent normal mucosa, were obtained from patients who
had undergone surgical resection of their tumor between 2013 and
2015 at Osaka Medical College Hospital (Takatsuki, Osaka, Japan).
Collection and investigation of the samples were approved by the
research Ethics Committee of Osaka Medical College (approval no.
1280, 2 September 2013) in accordance with the Declaration of
Helsinki. Before treatment, each patient provided written, informed
consent regarding the use of their tissues in our research. All
tissue sample pairs were collected from the same patient. Detailed
clinical information is shown in Tables
I and II. Pathological staging
of the cancers was performed according to postoperative
pathological reports, using guidelines for the treatment of
colorectal cancer from the Japanese Society for Cancer of the Colon
and Rectum 2010 (24). Each ‘grade
of effect’, induced by PCRT, was evaluated histologically by our
hospital's pathologist, using surgically resected specimens. The
criteria for the assessment of response to PCRT are defined as
follows: Grade 0 (no effect): No tumor cell necrosis or
degeneration was observed. Grade 1 (mild effect): Tumor cell
necrosis or degeneration is present in less than one third of the
entire lesion (minimal effect) or in more than one third but less
than two thirds of the entire lesion (mild effect). Grade 2
(moderate effect): Although prominent tumor cell necrosis,
degeneration, lytic change, and/or disappearance is present in more
than two thirds of the entire lesion, viable tumor cells remain.
Grade 3 (marked effect): Necrosis and/or lytic change is present
throughout the entire lesion, accompanied by replacement of
fibrosis, and viable tumor cells were not observed. Assessment was
performed on as many pathological specimens as possible, including
those prepared from the section of the whole tumor at the point of
maximum diameter (25).
 | Table I.Clinical and pathological features of
patients with CRC without any preoperative therapy. |
Table I.
Clinical and pathological features of
patients with CRC without any preoperative therapy.
| Case | Age | Sex | Location | Type | Tumor diameter
(mm) | Pathology | Tumora | Nodea |
Metastasisa | Stagea |
|---|
| 1 | 62 | M | A | 1 | 23×18 | tub1 | 2 | 0 | 0 | I |
| 2 | 62 | M | S | 2 | 64×38 | tub2, tub1 | 3 | 0 | 0 | IIA |
| 3 | 58 | M | R | 2 | 53×50 |
tub1=pap>tub2 | 3 | 1a | 1 | IV |
| 4 | 67 | M | T | 3 | 56×54 | tub2>tub1 | 3 | 1a | 1 | IV |
| 5 | 68 | F | R | 2 | 54×44 | tub2,
tub1>por2 | 3 | 1a | 0 | IIIB |
| 6 | 58 | M | R | 0 | 15×12 | tub1, tub2 | 1 | 0 | 0 | I |
| Table II.Clinical and pathological features of
patients with rectal cancer receiving preoperative
chemoradiotherapy. |
Table II.
Clinical and pathological features of
patients with rectal cancer receiving preoperative
chemoradiotherapy.
| Case | Age | Sex | Type | Pathology | Tumora | Nodea |
Metastasisa | Stagea |
|---|
| A, Grade 1a +
1b |
| 1 | 41 | M | 2 | tub1, tub2 | 3 | 1 | 0 | IIIB |
| 2 | 77 | M | 3 | tub1, tub2 | 3 | 1 | 0 | IIIB |
| 3 | 62 | F | 3 | tub2 | 3 | 0 | 0 | IIA |
| B, Grade 2 |
| 1 | 72 | M | 3 | tub1, tub2 | 3 | 1 | 0 | IIIB |
| 2 | 67 | F | 2 | tub1 | 3 | 1 | 0 | IIIB |
| 3 | 74 | M | 3 | tub2 | 3 | 0 | 0 | IIA |
| 4 | 62 | M | 2 | tub2>tub1 | 3 | 0 | 0 | IIA |
| C, Grade 3 |
| 1 | 57 | M | 2 | tub2>por | 3 | 2a | 0 | IIIB |
| 2 | 65 | M | 2 | tub1 | 3 | 0 | 0 | IIA |
| 3 | 67 | M | 2 | tub2 | 3 | 1 | 0 | IIIB |
| 4 | 71 | M | 2 | tub1, pap | 3 | 0 | 0 | IIA |
Irradiation time course
experiments
CRC cells were seeded in 6-well plates at a
concentration of 0.4×104 cells per well (10-30%
confluence) the day before irradiation. After irradiation at 10 Gy,
cells were incubated for 24, 48, 72 and 96 h, and the effects were
assessed.
Gene silencing in irradiation
experiments
siRNA (siR) for H2AX was purchased from Santa Cruz
Biotechnology. Silencer negative control siRNA (Invitrogen; Thermo
Fisher Scientific, Inc.) was used as a control for nonspecific
effects. Cells were transfected with 10 nM siRNAs using
Lipofectamine™ RNAiMAX (Invitrogen; Thermo Fisher Scientific, Inc.)
according to the manufacturer's protocol. After 24 h of
transfection, the cells were irradiated with 10 Gy and subsequently
collected after 72 h.
Cell viability
MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT)
solution was purchased from Sigma-Aldrich; Merck KGaA. The detailed
protocol is described in a previous report (26). Absorbance at 540 nm was measured
using an SH-1000 Lab microplate reader (Corona Electric Co.,
Ltd.).
Immunohistochemistry (IHC)
Detailed protocol information is described in our
previous reports (26,27). Anti-phospho-histone H2A.X (Ser139)
antibody (EMD Millipore) was used. Images were taken with a BZ-x700
microscope (Keyence Co.).
Western blot analysis
Detailed protocol information is described in our
previous reports (26–28). The primary antibodies used were as
follows: anti-phospho-histone H2A.X (Merck Millipore; 1:1,000) and
anti-β-actin (Cell Signaling Technology, Inc.; 1:1,000).
HRP-conjugated horse anti-mouse and anti-rabbit IgGs (Cell
Signaling Technology; 1:1,000) were used as secondary antibodies.
Immunoblots were detected and visualized using Fusion-FX7 (Vilber
Lourmat).
Statistical analysis
Each experiment was performed in triplicate. The
data are presented as the mean ± SE. All statistical analyses were
performed using JMP® 12 (SAS Institute Inc.).
Statistical differences between the mean values of multiple groups
were determined using analysis of variance followed by Student's
t-test or one-way analysis of variance (ANOVA). Tukey-Kramer test
was performed post hoc following one-way ANOVA. P-values <0.05
indicated statistical significance.
Results
Significant upregulation of H2AX mRNA
in CRC tissues
The expression of γ-H2AX is increased in advanced
CRC and is associated with poor prognosis (12,14).
First, the mRNA levels of H2AX were investigated using datasets
from cohort studies. Our dataset analysis showed that the mRNA
levels of H2AX were significantly upregulated in CRC tissues
compared to those in normal tissues, except for one cohort study
(Fig. 1A). The expression levels of
H2AX in CRC patients with distant metastasis (M1 Primary),
including liver metastasis (Liver Met), was higher than that in
patients without metastasis (M0 Primary; Fig. 1B). Interestingly, H2AX mRNA levels in
the group positive for microsatellite instability (MSI) were higher
than those in the MSI-negative group (Fig. 1C). These results suggest that
upregulation of H2AX mRNA is associated with the development of
CRC, in a similar way to the expression of γ-H2AX.
The protein expression of γ-H2AX was
upregulated in CRC specimens compared to adjacent normal
tissues
We examined the protein expression of γ-H2AX in
advanced CRC tissue without preoperative therapy (Table I). IHC showed that almost all nuclei
in cancer cells were stained, indicating strong expression of
γ-H2AX compared to that in adjacent normal tissues (Fig. 2A). The same tendency was observed
during western blot analysis (Fig. 2B
and C). These findings support the results of a previous study
(12), indicating that the
expression levels of γ-H2AX were upregulated in advanced CRC
tissue.
The suppression of γ-H2AX facilitated
the inhibition of cell viability induced by irradiation in CRC
cells
To assess the protective effect of γ-H2AX against
irradiation, changes in the expression levels of γ-H2AX were
measured after irradiation treatment, in two CRC cell lines. As
shown in Fig. 3A and B, the
expression level of γ-H2AX increased in correlation with
irradiation-induced inhibition of cell viability, in a
time-dependent manner. Subsequently, we examined the effects of
combination treatment (irradiation and knockdown of H2AX by siRNA)
in these cell lines. As expected, additional inhibition of growth
was observed with the combination therapy, compared to that with
irradiation or siR-H2AX alone, in both cell lines (Fig. 3C and D). These results imply that the
expression of γ-H2AX is associated with radiosensitivity in CRC
cells.
 | Figure 3.Association between irradiation and
γ-H2AX expression in CRC cells. (A) Cell viabilities at 24, 48, 72
and 96 h after 10 Gy irradiation in two CRC cell lines. (B) The
protein expression of γ-H2AX in irradiation-treated CRC cells. The
experimental conditions were the same as in (A). (C) Cell viability
after irradiation and siR-H2AX combination treatment in CRC cells.
The effects were assessed 72 h after irradiation. C, control; Si,
siR-H2AX; Ra, irradiation. (D) The protein expression levels of
γ-H2AX after single or combination treatment. The experimental
conditions were the same as in (C). ***P<0.001 as indicated.
H2AX, H2A histone family member X; CRC, colorectal cancer; siR or
Si, small interfering RNA; C, control; Ra, irradiation. |
Sensitivity to preoperative
chemoradiotherapy was enhanced in the low γ-H2AX-expression group
of patients with advanced rectal cancer
Finally, we investigated the role of γ-H2AX in
vivo. We examined the response to PCRT in biopsy samples, based
on preoperative inspection (Table
II). As shown in Fig. 4A and B,
the expression levels of γ-H2AX in grade 2 or grade 3 tissues were
significantly lower than those of the grade 1 group. These results
suggest that low expression of γ-H2AX enhances radiosensitivity in
CRC cells (Fig. 4C).
 | Figure 4.Association between preoperative
chemoradiation therapy and γ-H2AX expression in clinical rectal
cancer specimens. (A) Representative protein expression of γ-H2AX
in each grade of preoperative chemoradiation therapy. (B)
Quantification western blot analysis results in 11 patients with
advanced rectal cancer. (C) The effectiveness of radiotherapy
depends on γ-H2AX expression in CRC. CRC cell sensitivity to
radiotherapy with low expressions of γ-H2AX is high. Namely, the
expression of γ-H2AX is associated with the potential for
radiotherapy resistance. *P<0.05 as indicated. H2AX, H2A histone
family member X; CRC, colorectal cancer; grade 1a, minimal effect;
grade 1b, mild effect; grade 2, moderate effect; grade 3, marked
effect in rectal cancer patients; N, normal, T, tumor. |
Discussion
In this study, we found that the mRNA levels of H2AX
and the expression of γ-H2AX in CRC tissue were both higher than in
normal tissues. γ-H2AX has also been reported as a diagnostic and
prognostic marker in other types of cancer, such as cancer of the
breast, bladder, and ovary (29–31).
These reports support our results, and suggest that H2AX,
especially in its phosphorylated form, may be a gene which is
universally associated with cancer. Importantly, the expression
levels of H2AX seemed to be related to MSI. Recently, MSI has been
recognized as one of the key mechanisms of carcinogenesis due to
lack of mismatch repair (MMR), and has been associated with immune
checkpoint blockade therapy using pembrolizumab (32). DSB immediately phosphorylates H2AX to
form γ-H2AX (33), and γ-H2AX is a
known sensitive marker for DSB (11). Hence, the results met our
expectations, and the elucidation of the detailed association
between the roles of H2AX (non-phosphorylated form) and the
acquisition of MSI remains an important issue.
In this study, we also evaluated the potential of
γ-H2AX to predict the effectiveness of preoperative radiotherapy.
The endogenous expression levels of γ-H2AX in WiDr cells without
any therapy was higher than that in DLD-1 cells, and the effect of
γ-H2AX suppression on cell viability in WiDr cells was stronger
than that in DLD-1 cells after irradiation. Taken together, these
findings imply that the expression level of γ-H2AX is important for
radiosensitivity, and CRC cells with an elevated expression of
γ-H2AX possess a certain tolerance to irradiation. Various
predictive factors for radiotherapy have been previously reported,
such as p53, ki67, Bax, Bcl-2, cyclooxygenase-2 and CD133 (34–37).
However, a bona fide predictive marker for radiotherapy has yet to
be established, and further research is required to identify
reliable candidates.
In addition, the underlying mechanism which
associates γ-H2AX with cell death, after irradiation, is unknown.
Several molecular mechanisms have been reported, relating to genes
associated with cell death and γ-H2AX. It has been shown that the
inhibition of caspase-4 activation interferes with γ-H2AX in CRC
cells (38). The relationship with
poly(ADP-ribose) polymerase (PARP), which is activated by DNA
damage similar to H2AX, is extremely important because
PARP-inhibitors are, clinically, expected to become novel
anticancer drugs (39). In addition,
epigenetic regulation should be considered. For example,
microRNA-138 regulates DNA damage by targeting H2AX (40), and H2AX phosphorylation regulates
apoptosis in lung cancer cells via the microRNA-3196/PUMA pathway
(41).
The present study had some limitations. First, the
number of cases used in the investigation of sensitivity to
preoperative chemoradiotherapy was small (only eleven cases). To
enhance the reliability of our findings, larger studies and
confirmatory studies are needed. Second, we also have to consider
the influence of chemotherapy on γ-H2AX expression. In many cases,
radiotherapy is not performed alone, as a preoperative therapy for
CRC. Fluorouracil and oxaliplatin, which are generally used in
preoperative chemoradiotherapy, induce DNA damage. In this study,
we attempted to focus on the association between H2AX and
radiosensitivity, and avoided an extremely complicated experimental
system. Third, regarding the experiments using CRC cells, the
association of γ-H2AX with radiosensitivity was examined in a
limited environment. Although a comparative investigation in a
xenografted mouse model, using CRC cells with either high- or low-
γ-H2AX expression, might support our findings, we selected human
specimens because of concerns that the immune response would be
insufficient in an animal model. Fourth, our findings of an
association between H2AX and MSI were preliminary results.
Considering recent advances in immune checkpoint blockade therapy,
it is time to clarify the detailed mechanisms of this
association.
In conclusion, the expression levels of H2AX and
γ-H2AX were upregulated in CRC cells. Moreover, this upregulation
may be associated with MSI. In radiotherapy, sensitivity was
enhanced by the suppression of γ-H2AX, and γ-H2AX showed potential
as a novel predictive marker of the effectiveness of preoperative
radiotherapy in CRC patients.
Acknowledgements
The authors would like to thank Ms. Akiko Miyamoto
at the Laboratory of General and Gastroenterological Surgery, as
well as Mr. Rintaro Oide and Mr. Teruo Ueno at the Osaka Medical
College Research and Development Center for their technical
support. The authors would also like to acknowledge Professor Asako
Nakamura (Department of Biological Sciences, College of Science,
Ibaraki University) for advice.
Funding
The current study was supported by the Japan Society
for the Promotion of Science KAKENHI (grant no. 17K10680) and by
Osaka Medical College (OMC) Internal Research.
Availability of data and materials
All data generated or analyzed during this study are
included in this published article.
Authors' contributions
SK, KTas and KTani conceived and designed the
current study. SK performed in vivo experiments, NK
performed in vitro experiments and KK analyzed datasets. TT,
SK, NK, KTas, KTani, YInom, YIm, RT, YInou, MK, KK, KU, MY, JO,
KTana and SWL interpreted and analyzed the data. SK, KTani, MY,
KTana and JO provided materials and funding. SK, KTas, KTani wrote
and revised the manuscript. KU supervised the current study. All
authors read and approved the final manuscript.
Ethics approval and consent to
participate
The present study was approved by the research
Ethics Committee of Osaka Medical College (approval no. 1280; 2nd
September 2013) and was conducted in accordance with the Helsinki
Declaration. Before treatment, each patient provided written
informed consent regarding the use of their tissues in this
research.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing
interests.
Glossary
Abbreviations
Abbreviations:
|
CRC
|
colorectal cancer
|
|
DDR
|
DNA damage response
|
|
DMSO
|
dimethyl sulfoxide
|
|
DSB
|
double-strand breaks
|
|
IHC
|
immunohistochemistry
|
|
PARP
|
poly (ADP-ribose) polymerase
|
|
PCRT
|
preoperative chemoradiotherapy
|
|
siRNA
|
small interfering RNA
|
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