Presentation based on Summer internship in Rutgers 2014

1. The expression of ITPK in
normal colon and colorectal
cancer cells
Juan Enrique Maldonado Weng
Lawrence Gaspers, Ph.D.
Rutgers New Jersey Medical School
Department of Pharmacology & Physiology

2. Colorectal Cancer
• Usually begins as a non-cancerous tumor on the
inner lining of the colon or rectum
• In the United States, the overall five-year survival
rate is 65%

3. http://www.sirtex.com/us/patients/about-
cancer/colorectal-cancer-in-the-liver/
• Nearly 1.4 million cases of
colorectal cancer in 2012
• Approximately 60 to 70
percent of people with
colorectal cancer will
eventually develop a liver
tumor, which drops survival
rates to 6%
Colon cancer often
metastasize to the liver

4. Tumor cell adhesion to the Liver
endothelial cells

5. Authors proposed that ITPKC
could be anti-metastatic gene.
A previous functional genetic
screen identified increased
expression of ITPKC in non-
adherent colorectal cancer
cells.
Inositol 1,4,5-trisphosphate 3’ kinase
Marquez, J…. and Vidal-Vanaclocha, F. (2013). Int J Cancer.

6. Inositol 1,4,5-trisphosphate 3’ kinase
• Part of the phosphoinositide-dependent calcium signaling pathway. It is involved
in regulating calcium homeostasis, gene transcription and development.
• There are three isoforms in humans
• Each isoform has a different molecular mass, intracellular localization and
unique tissue distribution

7. Role of ITPK in Calcium Signaling
PLC = Phospholipase C
IP3 = Inositol
triphosphate
IP3R = InsP3 Receptor
ITPK = IP3 3-Kinase
IP4 = Inositol
Tetrakisphosphate

8. PLC = Phospholipase C
IP3 = Inositol
triphosphate
IP3R = InsP3 Receptor
ITPK = IP3 3-Kinase
IP4 = Inositol
Tetrakisphosphate
Role of ITPK in Calcium Signaling

9. LSEC
Hypothesis
ITPKC levels are down regulated in colon
carcinomas versus normal colon cells
Tumor
Cell
PLC IP3
Adhesion &
Migration
IP4
Ca2+
ITPKC

10. Aims
• Determine the mRNA levels and protein abundance of all
three ITPK isoforms in:
• Coloretcal cancer cell lines vs immortalized normal
human colon epithelial cells (HCEC).

11. Cell lines used in this study
 SW620
 HCT-116
 HT-29
 SW480
 CaCo-2
 HCEC 1ct
 HCEC 2ct
 HCEC 1ct
 HCEC 2ct
Metastatic
Potential
Growth arrested
to induce
differentiation
Non-Differentiated
Colorectal
cancer cells
Human colon
epithelial cells

12. Results
 Testing the Primers for ITPK A, B & C

13.  Total RNA extracted from normal HCEC
Intact, little contamination, good ratio between
28s and 18s proves good RNA isolation

14.  The Total RNA extracted from colon cancer samples
Both groups of samples were isolated well, but
need to be decontaminated before continuing

15.  Testing the antibodies for ITPK A, B & C
ITPKA antibodies
bind to
ITPKA protein
ITPKB antibodies
bind to
ITPKB protein only when
overexpressed
Obtaining decent ITPKC
antibodies is currently in
progress

16.  The Protein levels of ITPK A isoforms in colon cancer
samples & normal colon tissue
SW620
HCT-116
HT-29
SW480
CaCo-2

17. Conclusion
 We were able to extract intact total RNA with little
genomic DNA contamination.
 We determined the protein levels of ITPKA in
normal colon and colorectal cancer cells.
 Initial results indicate a wide range of ITPKA
expression within colon cancer cell lines.

18. Future Works
• Determine the RNA expression in CRC cells and normal colon cells:
• Decontaminate the samples from genomic DNA
• Perform first strand synthesis to obtain cDNA
• Measure expression qRT-PCR
• Determine the protein abundance in CRC and normal colon cells:
• Find and characterize ITPKB and ITPKC antibodies
• Repeat ITPKA measurement with replicate samples

19. Acknowledgements
Lawrence Gaspers, PhD.
Fernando Vidal-Vanaclocha, M.D.
Guoqiang Wang, PhD
Ishwarya Murali

20. Questions?

21. Other Sources
ITPKC is down regulated in in
Breast and Colon cancer

24. Cell Adhesion Assay
Down regulation of pro-adhesive
genes or upregulation of anti-
adhesive genes.
Upregulation of pro-adhesive
genes or downregulation of anti-
adhesive genes.
RSL-1 = Doxycycline
LSECs = Liver sinusoidal endothelial cells.

25.  Total RNA extracted from normal HCEC
Intact, little contamination, good ratio between
28s and 18s proves good RNA isolation

26.  The Total RNA extracted from colon cancer samples
Both groups of samples were isolated well, but
need to be decontaminated before continuing

27.  The Protein levels of ITPK A isoforms in colon cancer
samples & normal colon tissue
SW620
HCT-116
HT-29
SW480
CaCo-2