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