This document summarizes a student's summer research project investigating expression levels of inositol trisphosphate kinase (ITPK) isoforms in normal colon cells and colorectal cancer cells. Preliminary studies found ITPKC overexpression inhibits cancer cell binding to liver cells, suggesting it is anti-metastatic. The student aimed to measure ITPK mRNA and protein levels to test if ITPKC is downregulated in cancer versus normal cells. Initial results showed ITPKA protein levels varied between cancer cell lines, and an additional higher molecular weight protein was lower in cancer cells. RNA extraction was initially unsuccessful but a second attempt yielded intact RNA for further analysis.

1. FIGURE 4
The expression of ITPK in normal colon and colorectal cancer cells
Juan E. Maldonado Weng1, Ishwarya Murali2, Fernando Vidal-Vanaclocha,M.D.3 and Lawrence D. Gaspers,PhD2
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 of enzymes 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 (Fig. 1).
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 the
hypothesis that ITPKC is down regulated in colorectal carcinomas verses the normal
colon cells.
PRELIMINARY STUDIES
The laboratory of Dr. Vidal-Vanaclocha carried a genetic screen using a random
homozygous gene perturbation technique (RHGP, Functional Genetics, Inc.) to identify
candidate genes regulating the adhesion of colorectal colon cancer cells to liver
endothelial cells (Marquez et al., 2013). The screen identified increased expression of
inositol 1,4,5-trisphosphate kinase isoform C (ITPKC) in non-adherent cancer cells.
Moreover, analysis of microarray data sets submitted to NCBI ‘s GEO website indicate
that ITPKC may be down regulated in breast and colon cancers (Fig. 2). Taken together,
these data suggest that ITPKC is an anti-metastatic gene.
FIGURE 1
REFERENCES
Marquez, J., Kohli, M., Arteta, B., Chang, S., Li, W.B., Goldblatt, M., and Vidal-Vanaclocha, F.
(2013). Identification of hepatic microvascular adhesion-related genes of human colon cancer
cells using random homozygous gene perturbation. Int J Cancer 133, 2113-2122.
METHODS
Six colorectal cancer cell lines (CaCo-2, HCT-116, HT-29, SW1116, SW620 and SW480)
were obtained from ATCC or the NJMS Cancer Institute and certified mycoplasma free
prior to experimentation. Please note that the SW480 and SW620 cell lines were
derived from either the primary tumor or metastases from the same individual.
Immortalized human colon epithelial cells (HCEC) were obtained from Dr. J. W. Shay (UT
Southwestern Medical Center). The HCEC cell lines are from normal adult biopsies. All
cell lines were maintained in HCEC supplemented X-media in the presence of 5%
oxygen atmosphere. HCEC cells were harvested during logarithmic growth or growth
arrested to induce cell differentiation. Total RNA was extracted using TRIzol reagent.
Whole cell lysates were prepared with SDS and used for Western blot analysis.
RESULTS-PROTEIN
We carried out initial studies to validate the specificity of the isoform specific α-ITPK
antibodies. The results are shown in Figure 4. Cells were transiently transfected with
human IPTK cDNAs and protein levels were determined. Fig 4a shows the expression
of ITPKA protein in non-transfected (NT) and transfected cultures (ITPKA).
Immunodetection was inhibited by a blocking peptide (Fig. 4b) 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. 4c), but could not detect
endogenous levels of ITPKB (not shown). We are currently testing α-ITPKC antibodies
from different vendors.
The endogenous levels of ITPKA protein were determined in normal colon epithelial
cells and colorectal cancer cell lines. The results are shown in Figure 5. 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.
RESULTS-RNA
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 with
freshly isolated RNA. The images in Figure 3 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 to remove any genomic DNA contamination and then used to prepare cDNA.
The resulting cDNA was to 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).
SUMMARY
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 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.
FIGURE 5
FIGURE 2 FIGURE 3