Proto-oncogene, Pim-3 with serine/threonine kinase activity, is aberrantly
expressed in human colon cancer cells and can prevent Bad-mediated
Popivanova, Boryana Konstantinova; Li, Ying-Yi; Zheng, Huachuan;
Omura, Kenji; Fujii, Chifumi; Tsuneyama, Koichi; Mukaida, Naofumi
CitationCancer Science, 98(3): 321-328
A proto-oncogene, Pim-3 with serine/threonine kinase activity, is aberrantly expressed
in human colon cancer cells and can prevent Bad-mediated apoptosis
Boryana Konstantinova Popivanova,1, 2 Ying-Yi Li,1, 2 Huachuan Zheng,4 Kenji Omura,3
Chifumi Fujii,1 Koichi Tsuneyama,4 and Naofumi Mukaida1, 2
1Division of Molecular Bioregulation, Cancer Research Institute,
Laboratory, 3Department of General and Cardiothoracic Surgery, School of Medicine,
Kanazawa University and 4Department of Pathology and 21st Century Center of Excellence
Program, Toyama University Faculty of Medicine
Correspondence should be addressed to Naofumi Mukaida, MD, PhD, Division of
Molecular Bioregulation, Cancer Research Institute, Kanazawa University, 13-1 Takara-
machi, Kanazawa 920-0934, Japan.
Tel, +81-76-265-2767; Fax, +81-76-234-4520; E-mail, email@example.com-
Short Running title: Pim-3 expression in human colon cancer
We previously observed that Pim-3 with serine/threonine kinase activity, was aberrantly
expressed in malignant lesions of endoderm-derived organs, liver and pancreas. Because
Pim-3 protein was not detected in normal colon mucosal tissues, we evaluated Pim-3
expression in malignant lesions of human colon, another endoderm-derived organ. Pim-3
was detected immunohistochemically in well-differentiated (43/68 cases) and moderately-
differentiated (23/41 cases) but not poorly- differentiated colon adenocarcinomas (0/5
cases). Moreover, Pim-3 proteins were detected in adenoma (35/40 cases) and normal
mucosa (26/111 cases), which are adjacent to adenocarcinoma. Pim-3 was constitutively
expressed in SW480 cells and the transfection with Pim-3 short hairpin RNA promoted
apoptosis. In the same cell line, a pro-apoptotic molecule, Bad, was phosphorylated at Ser112
and Ser136, sites of phosphorylation that are representative of its inactive form. Ser112 but not
Ser136 phosphorylation in this cell line was abrogated by Pim-3 knockdown. Furthermore, in
human colon cancer tissues, Pim-3 co-localized with Bad in all cases (9/9) and with
phospho-Ser112Bad in most cases (6/9). These observations suggest that Pim-3 can
inactivate Bad by phosphorylating its Ser112 in human colon cancer cells and thus may
prevent apoptosis and promote progression of human colon cancer.
Pim-3 was originally identified as depolarization-induced gene, KID-1, in PC12 cells,
a rat pheochromocytoma cell line(1). Because KID-1 shows a high sequence similarity with
the proto-oncogene Pim family that expresses serine/threonine kinase activity, it was
renamed as Pim-3(2). Accumulating evidence indicates the potential involvement of other
Pim family members, Pim-1 and Pim-2, in various types of carcinogenesis(3-6). Indeed,
Deneen and colleagues demonstrated that Pim-3 gene transcription was enhanced in
EWS/ETS-induced malignant transformation of NIH 3T3 cells(7). Moreover, we revealed
that Pim-3 was selectively expressed in malignant lesions but not normal tissues of
endoderm-derived organs, liver(8) and pancreas(9). These observations suggest the potential
involvement of Pim-3 in carcinogenesis, particularly in endoderm-derived organs.
Apoptosis is an important mechanism during normal crypt formation in another
endoderm-derived organ, colon(10). The molecular mechanism of colon cancer progression
is known to involve several key events and mutation of a number of crucial genes(11). A
partial suppression of apoptosis occurs as a result of the inactivation of both alleles of the
APC gene and the suppression of apoptosis allows APC-deficient cells to develop
adenomatous polyps(12). Further suppression of apoptosis occurs as these cells develop
additional genetic mutations and phenotypic changes(13). Thus, the dysregulation of
apoptosis is crucially involved in colon carcinogenesis.
Accumulating evidence indicates that Pim kinases can phosphorylate substrates that
regulate apoptosis, and that activated Pim kinases might contribute to the pathogenesis of a
wide variety of malignancies(14). Similarly as observed on other members of Pim family(15,
16), we demonstrated that aberrantly-expressed Pim-3 can inactivate a pro-apoptotic BH3-
only Bcl-2-like molecule, Bad, by phosphorylating its serine residue and eventually prevent
apoptosis in human pancreatic cancer cell lines(9). These observations suggest that Pim-3
may contribute to carcinogenesis by negatively modulating apoptosis.
Our previous Northern blotting analysis failed to detect Pim-3 mRNA in normal
human endoderm-derived organs including liver, pancreas, and colon(8). Given that Pim-3
can prevent apoptosis, we assumed that Pim-3 might be aberrantly expressed in malignant
lesions of the colon and might contribute to colon carcinogenesis. To address this
hypothesis, we examined Pim-3 expression in human colon cancer tissues and found that
Pim-3 protein was detected in a substantial portion of human colon cancer samples,
particularly at the less advanced stages of cancer development. Moreover, the ablation of
endogenous Pim-3 in the colon cancer cell line reduced the amount of phosphorylated Bad
and eventually promoted apoptosis. Furthermore, we demonstrated that Pim-3 was co-
localized with Bad in cancer cells in human colon cancer tissues.
Materials and Methods
The following antibodies were used: mouse anti-Bad and rabbit anti-actin antibodies, Santa
Cruz Biotechnology (Santa Cruz, CA); rabbit anti-heat shock protein (Hsp)60 antibodies,
StressGen Biotechnologies Corp., (Victoria, Canada); Alexa Fluor® 488 donkey anti-rabbit
IgG, and Alexa Fluor® 594 donkey anti-mouse IgG, Molecular Probes Inc.; rabbit anti-
phospho-Ser112Bad, anti-phospho-Ser136Bad, and anti-phospho-Ser155Bad antibodies, Cell
Signaling Technology (Beverly, MA); ImmunoPure® peroxidase-conjugated goat anti-
mouse and goat anti-rabbit antibodies, Pierce Biotechnology, Inc. (Rockford, IL).
A well-differentiated colon cancer-derived cell line, SW480(17) was maintained in
RPMI1640 medium (Sigma Chemical CO.); while a moderately-differentiated colon cancer-
derived cell line, HT29(18), and a poorly-differentiated cell line, HCT116(19) were
maintained in McCoy’s 5A Modified Medium (Invitrogen Corporation, Carlsbad, CA). The
media were supplemented with 10 % heat-inactivated fetal bovine serum (FBS, Atlanta
Biologicals, Norcross, GA) and the cells were incubated in a humidified incubator at 37ºC
Colon tissue samples
Colon tissue specimens were obtained with an informed consent from individuals as shown in
Table 2. As controls, colon tissues were also obtained from 4 patients with diverticulitis but
without any malignant lesions. These samples were collected and paraffin-embedded at the
Toyama University Hospital with approval from the Human Subjects Research Ethical
Committee of Toyama University, Faculty of Medicine. An additional human colon tissues
was obtained from a patient upon surgery for colon cancer with his informed consent and with
approval from the Human Subjects Research Ethical Committe of Kanazawa University
Hospital. We separated the tumor and normal tissue, which was at least 2 cm apart from the
edge of tumor foci and was judged histologically to be free from adenocarcinoma cells, and
stored them immediately at -80oC until protein extraction.
Preparation of polyclonal anti-Pim-3 antibodies
White Japanese rabbits were immunized with a synthetic, keyhole limpet hemocyanin-
conjugated Pim-3 peptide (CGPGGVDHLPVKILQPAKAD), that corresponds to the amino
acid residues between 13 and 32 in human Pim-3. This region is well conserved in human
and murine Pim-3, but shows a high diversity with other Pim family members, Pim-1 and
Pim-2(8). An equal volume of the antigen (2 mg/ml) and an adjuvant, Titer Max®Gold
(CytRx®Corp., Atlanta, GA), were mixed and emulsified by sonication. Rabbits were
immunized 11 times (2 week interval), by subcutaneous injection of the antigen mixture
into their back. IgG fractions were purified from serum utilizing a Protein G SepharoseTM 4
Fast Flow column (Amersham Biosciences AB, Uppsala, Sweden) according to the
manufacturer’s instructions. The resultant IgG fractions were further purified using
HiTrapTM NHS-activated HP colum (Amersham Biosciences AB) conjugated with the
peptide used for the immunization. The concentration of the affinity-purified antibody was
determined by measuring the absorbance at 280 nm.
Immunohistochemical analysis of human colon cancer tissues
Paraffin-embedded human colon adenocarcinoma tissues were deparaffinized in xylene,
rehydrated through graded concentrations of ethanol and washed with PBS. Endogenous
peroxidase activity was blocked with 3% H2O2 (DakoCytomation, Carpinteria, CA) for 5
minutes. For antigen retrieval, sections were heated in 10 mM sodium citrate buffer
(pH6.0), followed by blocking with Non-Specific Staining Blocking reagent
(DakoCytomation). The slides were incubated with rabbit polyclonal anti-Pim-3 IgG (3
μg/ml) or with normal rabbit IgG overnight at 4ºC. The slides were further incubated with
horseradish peroxidase (HRP)-labelled anti-rabbit polymer EnVision+ System
(DakoCytomation) at room temperature for 30 minutes. Immune complexes were visualized
with Peroxidase Substrate DAB kit (Vector Laboratories, Inc., Burlingame, CA),
counterstained with hematoxylin, dehydrated and coverslipped. Positive cells exhibit either
a supranuclear or cytoplasmic staining pattern. A researcher without a prior knowledge of
the clinical samples evaluated the proportion of Pim-3-positive cells in colon carcinoma
tissues in a semi-quantitative manner, without assessing the intensities, as follows; -, absent
positive cells; +, positive cells in less than 5 % of total; +, positive cells with 5 to 25 %; ++,
positive cells with 25 to 50 %; +++, positive cells with greater than 50 %.
Cells were seeded on Lab-Teck® chamber slides (Nalge Nunc International Corp.,
Naperville, IL) and incubated at 37ºC with 5% CO2. Forty-eight hours later, they were fixed
with 4% paraformaldehyde and permeabilized with 0,1% Triton X-100/PBS for 10 minutes.
Immunostaining was performed by incubating the slides with rabbit polyclonal anti-Pim-3
(3 μg/ml), or with the combination of mouse anti-Bad (1:30) and rabbit anti-Hsp60
antibodies (1:200), followed by incubation with Alexa Fluor® 488 donkey anti-rabbit and/or
Alexa Fluor® 594 donkey anti-mouse (1:100) fluorescent antibodies for 1 hour at room
temperature. As a negative control, slides were incubated with normal donkey serum instead
of the primary antibodies. In some experiments, after washing with PBS, cells were
incubated with propidium iodide (Annexin V-FITC kit, Bender MedSystems, GmbH,
Vienna, Austria, 1μg/ml) for 1 min. Images were captured on a Carl Zeiss LSM510
confocal microscope (Carl Zeiss Microimage Inc., Thornwood, NY).
Immunoprecipitation and Western blotting
Human colon tissues were minced into small pieces and homogenized in RIPA Lysis buffer
(Santa Cruz Biotechnology, Inc.,CA), supplemented with protease inhibitors. The lysates
were sonicated and centrifuged at 14,000 rpm at 4˚C for 15 min. The resultant cell lysates
were used for Western blotting with anti-Pim-3 IgG as previously described.(9) Cell lysates
from colon cancer cell lines were subjected to immunoprecipitation with anti-Pim-3 IgG
followed by Western blotting analysis with anti-Pim-3 IgG as previously described.(9) Cell
lysates immunoprecipitated with control rabbit IgG were used as a negative control, while
those from HEK293 cells transfected with human Pim-3 cDNA were used as a positive
control. In some experiments, mouse anti-Bad antibody (1:100) was used instead of anti-Pim-
3 antibody in Western blot analyses following immunoprecipitation with anti-Pim-3 antibody.
Transfection with short hairpin RNA (shRNA)
SW480, HT29 and HCT116 cells were transfected with shRNA for Pim-3 (5’-
GCACGUGGUGAAGGAGCGG-3’) and Scramble shRNA (5’-
GCGCGCUUUGUAGGAUUCG-3’), inserted into the pSilencerTM 3.1-H1 Neo expression
vector (Ambion, Austin, TX), using LipofectamineTM 2000 Reagent (Invitrogen) according
to the manufacturer’s instructions. In our preliminary experiments, Pim-1 and Pim-2 mRNA
was faintly detected in SW480, HT29 and HCT116 cells. However, we further confirmed
that the transfection of Pim-3 shRNA reduced the mRNA expression of Pim-3, but not Pim-
1 and Pim-2 (data not shown).
Cell cycle and cell apoptosis analysis
At the indicated time intervals following the transfection with either Pim-3 or Scramble
shRNA, the cells were harvested and fixed with 70% ethanol at -20oC. The fixed cells were
incubated with 50 μg/ml propidium iodide (Molecular Probes, Inc.) and 1 μg/ml RNase A
for 30 min at room temperature. DNA content was then analyzed on a FACS Calibur system
(Becton Dickinson, Bedford, MA). The distribution of cells in each cell-cycle phase was
determined by using cell ModFitLT Software (Becton Dickinson). In some experiments,
phosphatidylserine exposure level was determined by staining the cells with human
Annexin V-FITC Kit (Bender MedSystem GmbH, Vienna, Austria), according to the
manufacturer’s instructions. At least 20,000 stained cells were analyzed on a FACS Calibur
system for each determination.
Determination of Bad proteins after Pim-3 shRNA transfection.
At the indicated time
intervals following shRNA transfection, whole cell lysates were prepared by using
CelLyticTM-M mammalian Cell Lysis/Extraction Reagent containing complete protein
inhibitor cocktail as previously described(9). Aliquots (50 μg) of the obtained supernatants
were separated on a 15 % SDS-PAGE and transferred onto an Immobilon-P Transfer
membrane. After being saturated with 3% BSA, the membrane was incubated with rabbit
anti-Pim-3, mouse anti-Bad, rabbit anti-phopho-Ser112Bad, anti-phospho-Ser136Bad, or anti-
phopho-Ser155Bad antibodies followed by the incubation with ImmunoPure® peroxidase-
conjugated anti-mouse or anti-rabbit IgG. The blotted membrane was then treated with the
Super Signal West Dura Extended Duration Substrate and signals were detected by LAS-
3000 mini CCD camera.
The data on human colon carcinoma tissues were analyzed statistically by using Spearman’s
correlation analysis to analyze the rank data. p<0.05 was accepted as statistically significant.
In other experiments, data were expressed as mean ± SD and analyzed by using one-way
ANOVA, followed by Fisher’s Protected Least Significant Difference test. Differences were
considered significant, when p<0.05.
Enhanced Pim-3 protein expression in human colon cancer tissues. Pim-3-positive cells
were not detected in colon tissues that lacked any malignant lesions (data not shown), or
normal colon epithelium distant to adenocarcinoma (Fig. 1A-a). In contrast, a substantial
number of Pim-3-positive cells were detected in normal epithelium adjacent to
adenocarcinoma tissues (Fig. 1A-b) or adenoma tissues (Fig 1A-c), as well as
adenocarcinoma (Fig. 1A-d and e). The positive reaction was not observed when control
IgG was used as the primary antibody instead of anti-Pim-3 antibodies (Fig 1B), indicating
the specificity of the reaction. Pim-3 protein was detected mainly in the cytoplasm of
adenoma and adenocarcinoma cells (Fig. 1A-c to e). Moreover, Western blotting analysis
detected Pim-3 protein in tumor but not normal colon tissue derived from a patient with
well-differentiated colon carcinoma (Fig. 1C), further indicating the specificity of the used
antibody. Although some adenocarcinoma tissues consisted mostly of Pim-3-positive cells
(Fig. 1A-d), positive rates were the highest in adenoma (Table 1). Poorly-differentiated
adenocarcinoma (n=5) did not show any positive staining (Fig. 1A-f), while well- and
moderately-differentiated adenocarcinoma exhibited similar Pim-3 staining (Fig. 1A-c and d,
and Table 2). Moreover, tumor sizes and invasive depth did not have any significant effects
on the extent of Pim-3 staining (Table 2). In contrast, the presence of lymph node or liver
metastatic foci resulted in decreased Pim-3 expression, compared with adenocarcinoma
without a distant metastasis (Table 2). When sixty samples from a different group, were
simultaneously analyzed for Pim-3 expression in both primary foci and metastatic lesions,
Pim-3 expression in primary foci did not show any significant correlation with that in
metastatic foci (Table 3). These observations suggest that Pim-3 expression was enhanced
more frequently at the early phase rather than the later stages of colon carcinogenesis when
distant metastasis occurs.
Constitutive Pim-3 expression in human colon cancer cell lines. Pim-3 expression in
colon cancer tissues prompted us to examine Pim-3 expression in colon cancer-derived cell
lines, SW480, HT29, and HCT116, which are derived from well-, moderately-, and poorly-
differentiated cancer cells. RT-PCR analysis detected Pim-3 mRNA in these cell lines (data
not shown). Immunoblotting analysis demonstrated that SW480 and HT29 contained
similar but greater amounts of Pim-3 than HCT116 (Fig. 2 A and B). An
immunofluorescence analysis further demonstrated a lower Pim-3 expression in HCT119,
compared with SW480 and HT29 (Fig. 2C), consistent with a lower incidence of Pim-3
expression in human poorly-differentiated colon cancer (Table 2). Because Pim-3 can
inactivate a pro-apoptotic BH3-only protein, Bad by phosphorylating Ser112
examined the phosphorylation states of Bad in these cell lines. Bad was constitutively
phosphorylated at Ser112, and to a lesser degree, Ser136 and Ser155 in these cell lines.
Furthermore, the amounts of total Bad and phospho-Ser112Bad but not other phosphorylated
forms of Bad were higher in SW480 and HT29 than HCT116 cells (Fig. 2A and B).
Enhanced apoptosis of human colon cancer cell lines resulting from the ablation of
endogenous Pim-3 protein.
In order to clarify the role of endogenous Pim-3 in cell
survival, we ablated Pim-3 protein by the transfection into SW480 of Pim-3 shRNA.
Transfection of Pim-3 shRNA but not Scramble shRNA into SW480 cells markedly
diminished Pim-3 protein by 48 hr posttransfection (Fig. 3A). The transfection of Pim-3
shRNA resulted in a higher ratio of sub-G1 cell populations with reduced G1 and G2/M
populations, compared to the cells transfected with Scramble shRNA (Fig. 3Bi to Biii).
Moreover, Pim-3 shRNA transfectants contained a markedly higher ratio of apoptotic cells
as evidenced by enhanced phosphatidylserine externalization (Fig. 3Biv to Bvi). Similar
phenomena were observed when HT29 cells were transfected with Pim-3 shRNA (data not
shown). We next examined the effects of Pim-3 ablation on Bad phosphorylation. The
ablation of Pim-3 reduced the amount of phospho-Ser112Bad but not the amounts of total
Bad and phospho-Ser136Bad (Fig. 3A). Several lines of evidence indicate that Bad was
translocated from cytoplasm to mitochondria upon its dephosphorylation.(20) Hence, we
explored the intracellular localization of Bad upon dephosphorylation of Ser112 but
Ser136Bad arising from Pim-3 ablation. We employed anti-Hsp60 antibodies to detect
mitochondria, because Hsp60 is exclusively present in mitochondria.(21) Bad was not co-
loclaized with Hsp60 in untreated cells but Pim-3 ablation induced the co-localization of
Bad with Hsp60 (Fig. 3C), suggesting its mitochondrial localization. Similar phenomena
were observed on another colon cancer cell line, HT29 (data not shown). Thus, these
observations suggest that Pim-3, aberrantly expressed in human colon cancer cells, can
inactivate the potent pro-apoptotic factor, Bad, and thereby prevent its translocation to
mitochondria, by phosphorylating Bad Ser112.
Co-localization of Pim-3 with Bad protein in primary human colon cancer. In order to
address whether there was evidence to suggest that Pim-3 might phosphorylate Bad in
primary human colon cancer cells, we performed a double-color immunofluorescence
analysis on human colon cancer tissues. Pim-3 protein was detected in tumor cells in 9 out
of 10 human colon cancer samples that we examined. In all these Pim-3-positive samples,
Bad protein was co-localized with Pim-3 proteins in both the horizontal (xy axis) and
orthogonal planes (xz and yz axes) (Fig. 4A). Pim-3 proteins did not co-localize with
GAPDH, a protein that is abundantly present in the cytoplasm (data not shown). Moreover,
an immunoprecipitation with anti-Pim-3 antibodies co-precipitated Bad in a human colon
cancer tissue (Fig. 4B). These observations would further indicate the co-localization of
Pim-3 with Bad in human colon cancer cells. Finally, in 6 out of 9 Pim-3-positive samples,
Pim-3 protein co-localized with phospho-Ser112Bad in both the horizontal (xy axis) and
orthogonal planes (xz and yz axes) (Fig. 4C). Thus, Pim-3 may phosphorylate Bad at Ser112
even in primary colon cancer, thereby regulating apoptosis of the cancer cells.
Orderly apoptosis of colon epithelial cells is essential for normal colon crypt
formation(10). A partial deregulation of apoptosis occurs due to the inactivation of both
alleles of the APC gene, the first key event in colon carcinogenesis(12). Further suppression
of apoptosis occurs as these cells develop additional genetic mutations and phenotypic
changes(13). However, it remains elusive on the molecular mechanisms underlying apoptosis
deregulation in colon carcinogenesis. Accumulating evidence suggests that the regulation of
apoptosis of colon cancer cells involves several kinases including Erk1/2, p38 mitogen-
activated kinase, and Akt(22-26). Pim kinases can phosphorylate a similar range of substrates
as Akt and thus can provide survival signals to various types of tumor cells(14). Our
observation that Pim-3 expression was enhanced in the malignant lesions of endoderm-
derived organs, the liver(8) and pancreas(9), prompted us to evaluate Pim-3 expression in
colon carcinoma tissue. Indeed, Pim-3 was expressed in a substantial portion of primary
colon cancer tissues. Moreover, we have determined that aberrantly expressed Pim-3 may
counteract apoptosis by phosphorylating a pro-apoptotic molecule, Bad.
Another member of the Pim family, Pim-1, becomes constitutively active without
any alteration in conformation, due to the absence of any regulatory domains(27). Pim-3
shows a high sequence identity with Pim-1, even in the kinase domain and lacks any
regulatory domains(8). Thus, when aberrantly expressed in colon cancer cells, Pim-3 can be
active without any further modification. Recently, the human Pim-3 gene has been assigned
to chromosome 22q13. A comparative genomic hybridization analysis demonstrated that the
gain at 22q13.3 represented the minimal chromosomal changes found specifically in colon
cancer tissues with microsatellite instability(28). However, we failed to detect Pim-3 gene
amplification in several human colon cancer cell lines including SW480 and HT29 (our
unpublished data). Thus, it is not likely that constitutive enhanced expression of Pim-3 in
colon cancer cells is a result of Pim-3 gene amplification.
Several lines of evidence suggest that a transcription factor, Stat3, is constitutively
activated in colon cancer(29-31) and that both Pim-1 and Pim-2 can be a transcriptional target
of Stat3(32, 33). Moreover, we also observed that the transfection of a dominant negative form
of Stat3 reduced Pim-3 expression in SW480 cells (our unpublished data), suggesting that
Stat3 can regulate Pim-3 gene transcription. Of interest is the fact that Pim-1 can
synergistically induce Stat3-mediated cell cycle progression and anti-apoptotic process(31).
Aberrantly expressed Pim-3 may also synergistically augment the Stat3-induced malignant
phenotype of colon cancer as Pim-3 has a remarkable sequence identity with Pim-1. If this
is indeed the case, blocking Pim-3 activity may reduce Stat3 activity and thus inhibit colon
Akt shows a similar substrate specificity as the Pim kinases including Pim-3 and is
presumed to have an essential role in the regulation of apoptosis(14). Activation of Akt can
contribute to the pathogenesis of a wide variety of malignancies. Indeed, Roy and
colleagues demonstrated that activated Akt was detected in a substantial portion of colon
adenoma (57% positive) and adenocarcinoma (57% positive) but not in normal colonic
epithelium and hyperplastic polyps(25). Based on these observations, these authors claimed
that Akt overexpression may be an early event during colon carcinogenesis. Pim-3 exhibited
a similar staining pattern as Akt. Pim-3 was also detected in normal colon mucosal tissues
that are adjacent to adenocarcinoma but do not exhibit any morphological abnormalities.
Thus, Pim-3 overexpression might be an earlier event than that of Akt during the
development of the malignant phenotype. Moreover, the incidence of Pim-3 expression was
higher in adenoma than adenocarcinoma. We previously observed that precancerous lesions
in liver showed a higher incidence of Pim-3 expression than hepatocellular carcinoma
lesions.(8) Thus, it is tempting to speculate that aberrant Pim-3 expression might generally
have a crucial role in the early phase but not later phase of carcinogenesis.
Genetic analysis has revealed that somatic missense mutations in the gene of Bad, a
pro-apoptotic BH3-only protein, occur in a small proportion of colon adenocarcinoma
samples(34). It was postulated that somatic mutation of Bad may contribute to colon
carcinogenesis due to decreased apoptosis-inducing activities of a mutant Bad, when
compared with wild-type Bad. The pro-apoptotic activity of Bad is regulated by its
phoshporylation at serine residues. Unphosphorylated Bad binds and eventually inactivates
anti-apoptotic family members, primarily Bcl-XL but also Bcl-2(35-37). Bad can be
inactivated by the phosphorylation at either Ser112 or Ser136. Phosphorylated Bad loses its
capacity to bind to anti-apoptotic molecules such as Bcl-XL and Bcl-2 and its movement
from the surface of mitochondria to the cytosol requires the 14-3-3 protein. Upon liberation
from Bad, Bcl-XL and Bcl-2 can maintain mitochondrial membrane potential and
subsequently prevent apoptosis(38, 39). In this report, we demonstrated that Bad is
constitutively phosphorylated at Ser112 and to a lesser degree, Ser136, in colon cancer cell
lines. Moreover, the ablation of Pim-3 reduced phosphorylation of Ser112, similarly
observed on human pancreatic cancer cell lines (9). Furthermore, our in vitro kinase assay
revealed that Pim-3 phosphorylates Bad Ser112 (our unpublished data), which represents an
inactive form of Bad(15, 16). Considering that Bad mutants with lower pro-apoptotic activities
were detected in colon carcinomas(34), Pim-3-mediated phosphorylation and subsequent
inactivation of Bad protein may also contribute to colon carcinogenesis by preventing the
The Akt consensus phosphorylation site contains arginine at the -5 and -3 in relation
to the phosphorylation site(40), similar to that required for Pim-1 activity(32). Although both
Bad Ser136 and Ser112 conform to this motif, Akt preferentially phosphorylates Bad Ser136 (38).
In colon cancer cell lines, Ser112 and to a lesser extent, Ser136 are constitutively
phosphorylated and the ablation of Pim-3 reduced the phosphorylation of Ser112 but not
Ser136. Of interest is that the inhibition of Akt induced apoptosis of colon cancer cells,
without dephosphorylating Bad Ser112 and Ser136.(41) Our in vitro kinase assay revealed that
Pim-3 phosphorylates Bad Ser112 (our unpublished data) and we observed that the amount
of phosphor-Ser112 but not phosphor-Ser136 Bad was proportional to the amount of Pim-3 in
human colon cancer cell lines. Moreover, we previously observed that the transfection of
Pim-3 enhanced the phosphorylation of Ser112 but not Ser136 Bad in human pancreatic cell
lines.(9) Thus, it is probable that Pim-3 directly phosphorylates Bad Ser112 and inactivates
Bad, thereby preventing the apoptosis of colon cancer cells. Thus, the inhibition of Pim-3
may induce apoptosis of colon cancer cells in a distinct way from the inhibition of Akt.
Moreover, Pim-3 may offer an advantage as a molecular target, due to its selective
expression in malignant lesions of liver(8), pancreas(9), and colon, in contrast to the
expression of Akt in most normal organs.
We would like to express our sincere gratitude to Drs. Howard Young and Debbie
Hodge (NCI-Frederick, USA) for their critical comments on the manuscript. We thank Dr.
Osamu Hori (Kanazawa University Graduate School of Medical Sciences) for his technical
advice on a double-color immunofluorescence analysis.
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- ± + ++ +++
85 10 15
mucosa adjacent to
Adenoma adjacent to
111 1 0 23.4*
40 5 7 25 3 0 87.5**
131 60 9 48 10 4 54.2***
Table 1. Pim-3 expression in colorectal mucosa, adenoma, and adenocarcinoma. The
proportion of Pim-3-positive cells were evaluated in mucosa and adenoma, that are adjacent
to adenocarcinoma, as well as the adenocarcinoma area, as described in Materials and
Methods. *, compared with adenoma, p=0.001, Spearman rank correlation coefficient
(Rs)=0.596; **, compared with adenocarcinoma, p=0.008; Rs=0.202; *** compared with
mucosa, p=0.001, Rs=0.347
n Pim-3 expression
- ± + ++ +++
Sex 0.057 0.544
Female 46 17 5 185 1 63.0
Male 68 31 4 255 3 54.4
Age 0.073 0.439
<60 19 101 60 2 47.4
≥60 95 388 3710 2 60.0
Tumor size 0.135 0.154
<4cm 46 162 225 1 65.2
≥4cm 68 327 215 3 52.9
Invasive depth 0.046 0.626
Above submucosa 16 4 2 91 0 75.0
Muscularis propria 13 6 0 70 0 53.8
Below subserosa 85 387 279 4 55.3
Lymph node metastatic foci 0.195 0.038
- 52 17 4 227 2 67.3
+ 62 315 213 2 50.0
Liver metastatic foci 0.459 0.001
- 95 30 8 4310 4 68.4
+ 19 181 0 0 0 5.3
Differentiation 0.121 0.200
Well-differentiated 68 257 285 3 63.2
Moderately-differentiated 41 182 155 1 56.1
Poorly-differentiated 5 5 0 00 0 0
Table 2. Pim-3 positive samples were analyzed in correlation with clinicopathological
features of colorectal adenocarcinoma. Rs, Spearman rank correlation coefficient.
Primary foci n
- ± + ++ +++
23 4 7
1 3 0
10 7 3
0 1 1
0 0 0
34 15 11
Table 3. Pim-3 expression in primary foci was analyzed in correlation with metastasis. Rs,
Spearman rank correlation coefficient.
Legends to Figures
Figure 1. Aberrant expression of Pim-3 protein in human colon cancer tissues
Immunostaining with anti-Pim-3 antibodies was performed on human normal colon tissue
distant to adenocarcinoma (A-a), normal colon tissue adjacent to adenocarcinoma (A-b),
adenoma tissues (A-c and B), well-differentiated adenocarcinoma (A-d), moderately-
differentiated adenocarcinoma (A-e), and poorly-differentiated adenocarcinoma (A-f) were
immunostained with either anti-Pim-3 antibodies (A and B-a) or normal rabbit IgG (B-b) as
described in Materials and Methods. Representative results are shown here. Original
C. Western blotting analysis of Pim-3 protein in human colon cancer tissues. Cell lysates
from colon cancer and normal tissues (3 mg) were subjected to an immunoblotting with
anti-Pim-3 antibody as described in Materials and Methods. HEK293 cells transfected with
human Pim-3 cDNA were used as a positive control.
Figure 2. Constitutive Pim-3 expression in human colon cancer cell lines, SW480,
HT29 and HCT116.
A. Immunoblotting analysis of Pim-3 and various forms of Bad proteins in human colon
cancer cell lines. Cell lysates were obtained from human colon cancer cells. Aliqouts (2 mg)
were subjected to an immunoprecipitation with anti-Pim-3 antibodies or control rabbit IgG,
followed by an immunoblotting with anti-Pim-3 antibodies as described in Materials and
Methods. Another aliquots (50 μg) were immunoblotted with antibodies for various forms
of Bad proteins and β-actin. Representative results from 3 independent experiments are
B. Quantification of Pim-3 and various forms of Bad proteins in colon cancer cells. The
band intensities of Pim-3 and various forms of Bad in Figure 2A were measured with NIH
Image Analysis Software Ver. 1.62 (NIH, Bethesda, MD) and were normalized to those of
β-actin. Asterisk indicates **, p<0.01 compared with SW480 cells.
C. Immunofluorescence analysis of Pim-3 protein expression in human colon cancer cell
lines. A double-color mmunofluorescence analysis was carried out on human colon cancer
cell lines, HCT116 (left row) and HT29 cells (middle row) and SW480 (right row) with the
combination of anti-Pim-3 antibodies and propidium iodine as described in Materials and
Methods. Green and red colorations indicate Pim-3 protein and nuclei, respectively.
Representative results from 3 independent experiments are shown here. Original
magnification; upper panel, x200; lower panel, x800. Scale bars, 20μm.
Figure 3. The effects of ablation of endogenous Pim-3 protein on Bad phosphorylation
A . Cell lysates were obtained from human colon cancer cells, SW480, that were transfected
with Pim-3 shRNA, Scramble shRNA, or no shRNA, and the resultant lysates were
subjected to immunoblotting as described in Materials and Methods. Representative results
from 3 independent experiments are shown here. Similar results are obtained using HT29
B. A human colon cancer cell line, SW480, was either transfected with Scramble shRNA (i
and iv) or Pim-3 shRNA (ii, iii, v and vi). Cells were harvested 48 hr after transfection and
subjected to staining with propidium iodide (PI) (i to iii) or combined staining with PI and
annexin V (iv to vi) as described in Materials and Methods. The proportion of cells in each
cell cycle phase was determined (i to iii) as described in Materials and Methods. The
number in each quadrant (iv and v) indicates the proportion of the cells present in the
quadrant. The intensities of annexin-V staining are shown in vi, by overlaying the data in iv
and v. Representative results from 3 independent experiments are shown here. Similar
results were obtained using HT29 cells.
C. Immunofluorescence analysis of Bad intracellular localization in human colon cancer
cells. SW480 cells, transfected with Pim-3 shRNA, Scramble shRNA or no shRNA, were
seeded onto chamber slides. Thirty-two hours after the transfection, a double-color
immunofluorescence analysis was performed as described in Materials and Methods. Green,
red, and yellow colorations represent Hsp60, Bad, and co-localization of Hsp60 and Bad,
respectively. Representative results from 3 independent experiments are shown here.
Original magnification, x 1,600. Scale bar, 10μm.
Figure 4. Association of Pim-3 with Bad in human colon cancer tissues
A. Human colon cancer samples were immunostained with either the combination of anti-
Pim-3 and anti-Bad antibodies as described in Materials and Methods. The fluorescent
images were digitally merged in the left and right two panels. The box in the left panel
indicates the site, where a picture with a higher magnification was taken. Representative
results from 10 individual samples are shown here. Original magnification; left panel, x100;
other panels, x400. Scale bars, 50 μm.
B. Cell lysates were obtained from a human colon cancer tissue, HeLa cells and HEK293
cells transfected with human Pim-3 cDNA. The cell lysates from a human colon cancer
tissues were subjected to immunoprecipitation with anti-Pim-3 or control rabbit IgG. HeLa
cells express Bad, but not Pim-3, and HEK293 cells express Pim-3, but not Bad. Hence,
Hela cells a were subjected to immunoprecipitation with anti-Bad. The resultant precipitated
were further subjected to immunoblotting with anti-Bad (upper panel) or anti-Pim-3 (lower
panel) antibodies as described in Materials and Methods.
C. Human colon cancer samples were immunostained with either the combination of anti-
Pim-3 and anti-phosho-Ser112Bad antibodies as described in Materials and Methods. The
fluorescent images were digitally merged in the left and right two panels. The box in the left
panel indicates the site, where a picture with a higher magnification was taken.
Representative results from 10 individual samples are shown here. Original magnification;
left panel, x100;other panels, x400. Scale bars, 50 μm.
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