Paper written after the summer of the Rutgers research experience.

1. The expression of ITPK in normal colon and colorectal cancer cells
Juan E. Maldonado Weng1
, Ishwarya Murali2
, Fernando Vidal-Vanaclocha3
and Lawrence Gaspers2
Universidad de Puerto Rico1
, Cayey, Puerto Rico; Rutgers Graduate School of Biomedical Sciences2
, Newark,
New Jersey; Universidad San Pablo CEU3
, Madrid, Spain
Abstract
The liver is a major target tissue for metastases from a variety of tumors including colorectal cancers. Despite the
high prevalence of tumor formation in the liver, the mechanisms required for implantation and colonization of
tumor cells in the liver have not been fully delineated. Increases in cytosolic Ca2+
([Ca2+
]i) has been implicated in
many aspects of tumorigenesis including cell proliferation, adhesion and migration. The pathways generating these
Ca2+
responses and the downstream effector molecules have been extensively studied. In contrast, the role of
molecules terminating the Ca2+
signals have received less attention in carcinogenesis and may represent new
therapeutic targets to treat cancer or prognostic indicators of metastatic potential. Our preliminary studies suggests
that the over expression of inositol 1,4,5-trisphosphate kinase isoform C (ITPKC) inhibits the binding of human colon
cancer cells to liver sinusoidal endothelial cells. ITPKs are a family enzymes of that catalyzes the phosphorylation of
inositol 1,4,5-trisphosphate (InsP3) to inositol 1,3,4,5- tetrakisphosphate (InsP4). This reaction terminates InsP3-
dependent Ca2+
release from the endoplasmic reticulum and generates another potential signaling molecule, InsP4,
which has been implicated in the regulation of Ca2+
influx.
The preliminary data suggest that ITPKC is an anti-adhesive protein, and is expected to suppress the formation of
metastasis. On the other hand, previous studies have implicated ITPK isoform A as a pro-metastatic gene. ITPKA is
overexpressed in metastases derived from lung and mammillary cancers, whereas protein and mRNA levels of ITPKA
are significantly decreased in oral squamous cell carcinomas. These data suggest that ITPKA may play either a pro-
metastatic or an anti-metastatic role in cancer pathology that depends upon the cell-specific context. The goal of
this summer research project was to determine the mRNA and protein levels of all three ITPK isoforms in
immortalized human colonic epithelial cells (HCEC) derived from normal adult biopsies versus the levels found
human colorectal cancer cells. We will test that hypothesis that ITPKC is down regulated in colorectal carcinomas
verses the normal colon cells.
Introduction
In 2012, there were over 1.4 million cases of
colorectal cancer across the world (American
Cancer Society, 2008). Colorectal cancer affects
the majority of the countries like the United States,
China, and most of Europe. Because of this serious
threat, there is a non-stop search for treatments
and cures. Colorectal cancer is a tumor formed
from within the inner lining of the colon. It starts
as a non-benign polyp that eventually develops
into an invasive tumor. Most symptoms of the
disease go unnoticed by the victims until it is too
late. By the time of diagnosis, more tumors would
be found in other parts of the body. One of the
major targets of colorectal cancer metastases is the
liver. A patient that is diagnosed in time has an
overall five year survivability of sixty per cent
(American Cancer Society, 2008), but once the
cancer invades the liver, a patient’s survival rate
lowers to five per cent (What are the survival rates
for colorectal cancer by stage?, 2014).
The mechanism in which a colorectal tumor
invades the liver is the key to find a therapeutic
alternative for victims of this disease. Not much is
known about this mechanism of invasion. It has
been shown that contact with the liver endothelial
does increase levels of calcium ions within the cells.
This increase of Ca2+
is interpreted to be the result

2. of signaling pathways, which have been extensively
studied. It is also implicated that the constant
increase of Ca2+
also leads to the further
proliferation and adhesion of the cancerous cells.
Not many studies have analyzed the molecules
with the function of terminating these calcium
signals.
The mechanism in which the levels of
cytosolic calcium increase is the Phospholipase C
(PLC)/ Inositol 1,4,5-trisphosphate (InsP3) pathway.
The PLC/InsP3 pathway is activated by the binding
of a growth hormone or ligand to its counter
receptor on the colorectal cancer cell surface. PLC
catalyzes the hydrolysis of a lipid resulting in
diacylglycerol and InsP3. The InsP3 reacts with an
InsP3 receptor (insP3R) on the endoplasmic
reticulum that releases all the Ca2+
inside. This
depletion of calcium within the endoplasmic
reticulum causes the store-operated Ca2+
entry
(SOCE) channel to open causing more Ca2+
to enter
the cytoplasm. The molecule that has the function
of terminating the calcium entry signal is Inositol
1,4,5-triphosphate kinase (ITPK). ITPK is an enzyme
that ends the calcium increase by phosphorylating
the InsP3 into Inositol 1,3,4,5-tetrakisphosphate
(InsP4) which ends the binding between InsP3 and
the InsP3R (Xia & Yang, 2005).
ITPK is a molecule with different isoforms
which includes isoform A, B and C. This molecule is
involved in inositol signaling pathway, calcium
signal transduction, brain development, gene
transcription and many other important
mechanisms. This molecule is found in three
isoforms: A, B and C. Each isoform is different in
their molecular mass, intracellular distribution and
tissue expression (Xia & Yang, 2005).
Dr. Vidal-Vanaclocha and his lab were able
to find the molecule Inositol 1,4,5-triphosphate
kinase isoform C (ITPK C) as an overexpressed
protein from their non-adherent colorectal cancer
sample utilizing a random homozygous gene
perturbation technique (Functional Genetics, Inc.).
The preliminary data suggests that ITPKC is an anti-
adhesive gene. Moreover, analysis of microarray
data sets submitted to NCBI ‘s GEO website
indicate that ITPKC may be down regulated in
breast and colon cancers. These results imply that
colorectal cancer cells have a down regulation of
ITPKC. Colorectal cancer was caused by the
constant increase of calcium entering the cell. If
one of the functions of ITPKC is to terminate the
Ca2+ entry signals, then colorectal cancer cells
must have a down regulated expression of ITPKC in
comparison to expression levels of normal colon
cells.
The goal is to measure the gene expression
and protein abundance of ITPK isoforms in
colorectal cancer and normal colon tissues. This
will help test the hypothesis that ITPKC is down
regulated in colorectal carcinomas versus the
normal colon cells.

3. Methods
This research project required the
measurement of gene expression and protein
abundance in colorectal cancer cells and normal
colon cells. The candidate colon cancer cells were:
HCT116 which is highly metastatic, HT29 and
SW620 which are metastatic, and SW480 which is
lowly metastatic. The normal colon cells utilized
were Immortalized human colon epithelial cells
(HCEC) obtained from normal adult biopsies from
Dr. J. W. Shay (UT South western Medical Center).
These samples were kept in HCEC supplemented X-
media in the presence of 5% oxygen. HCEC cells
were harvested during logarithmic growth or
growth arrested to induce cell differentiation.
Total RNA was extracted utilizing TRIzol
reagent. This procedure was guided by the
protocols from ambion©. The isolated RNA was
tested for quality with gel electrophoresis in a 1.5%
agarose. This qualitative test was to ensure
minimal contamination and intact RNA. A
quantitative RNA absorbance assay measured the
amount of nanograms per microliter before loading
onto the gels to ensure that RNA was loaded
equally between samples. Whole cell lysates
were prepared with SDS and used for Western blot
analysis.
Results
Total RNA was extracted from normal colon
cells or colorectal cancer cell lines. Analysis of the
initial RNA samples indicated that the RNA was
degraded and heavily contaminated with genomic
DNA. Therefore, these experiments were repeated
and freshly isolated RNA. The images in Figure 1
show the total RNA extracted from the indicated
cell lines and separated by electrophoresis on a
1.5% agarose gel. The data show that the RNA was
intact in our second preparation. RNA quality is
indicated by the strong bands for 28S, 18S and 5S
RNA. The RNA samples will be treated with DNase I
Figure 1

4. to remove any genomic DNA contamination and
then used to prepare cDNA. The resulting cDNA
was be used in QT-PCR assays to determine
expression levels of the three ITPK isoforms. The
expression will be normalized to the housekeeping
genes hypoxanthine phosphoribosyltransferase
(HPRT1), TATA-binding protein (TBP) and
transferrin receptor p90 subunit (TFRC).
We carried out initial studies to validate the
specificity of the isoform specific α-ITPK antibodies.
The results are shown in Figure 2. Cells were
transiently transfected with human IPTK cDNAs and
protein levels were determined. Fig 2a shows the
expression of ITPKA protein in non-transfected (NT)
and transfected cultures (ITPKA).
Immunodetection was inhibited by a blocking
peptide (Fig. 2b) indicating that the antibody
specifically recognizes ITPKA. The α-ITPKA
antibody did not detect the endogenous protein
under these conditions due to low levels of
expression. The α-ITPKB antibody recognizes the
overexpressed protein (Fig. 2c), but could not
detect endogenous levels of ITPKB (not shown).
We are currently testing α-ITPKC antibodies from
different vendors.
Figure 2

5. The endogenous levels of ITPKA protein were
determined in normal colon epithelial cells and
colorectal cancer cell lines. The results are shown
in Figure 3. ITPKA expression was highest in the
SW480 and SW620 and lowest in CaCo-2 cell lines.
ITPKA protein levels did not change significantly
upon differentiation and were similar to the levels
detected in some colorectal cancers. Interestingly,
the α-ITPKA antibody detected an additional higher
molecular weight protein band around 110 Kd,
which was significantly lower in the cancer cell
lines. The identity of this protein is currently
unknown.
Discussion
There are many victims of this disease.
Finding a less risky alternative treatment for
colorectal cancer will be a great advancement in
the field of medicine. Many cancer treatments
utilized currently are too harmful for patients. A
less destructive treatment will benefit victims of
colorectal cancer and especially beneficial for
patients who have metastasized liver cancer. ITPK
enzymes might be manipulated to prevent the
tumor from metastasizing and invading other parts
of the body. If by overexpressing the enzymes in
colorectal tumors, the constant increase of Ca2+
will be inhibited. The adhesion and metastasis will
also cease because of the overexpression of ITPK.
There is a possibility for a new drug target to arise
from this research project.
Conclusion
The goal for this summer research project
was to investigate the expression of ITPK isoforms
in normal colon cells and colorectal cancer cells.
This project gave me hands-on experience and
many laboratory techniques including tissue
culture, RNA isolation, RNA quantification and
quality, gel electrophoresis, RT-PCR, protein
determination and Western blot techniques. This
research project also gave me practical experience
in the difficulties of carrying out scientific research.
For example, the RNA extracted in the first attempt
was degraded and contaminated. We learned from
our mistakes and in the second attempt we
Figure 3

6. isolated intact RNA with little genomic DNA
contamination. We were able to measure protein
levels of ITPKA in normal colon and colorectal
cancer cells. Initial results indicate a wide range of
ITPK expression; however, these results need to be
confirmed with additional replicate samples. In
summary, there were many hurdles in my summer
research project, but all the lessons and skills
obtained are invaluable.
Future works
In the near future, the RNA expression and
protein abundance for the normal HCEC and
colorectal cancer cells will be determined.
Determining RNA expression will require the
decontamination of the total RNA isolated from all
the cell lines to eliminate genomic contamination.
Afterwards, a first strand synthesis will be
performed to obtain cDNA to then perform qRT-
PCR. A qRT-PCR will provide the measurements of
the RNA expression of ITPK. The next step would be
to determine protein abundance of ITPK in the cell
lines. This step requires the characterization of
ITPKB and ITPKC antibodies. The measurements of
ITPKA will be repeated to verify what has been
observed in the results above. With these results,
we will be able to confirm or reject our hypothesis.
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