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

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%

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

Tumor cell adhesion to the Liver endothelial cells

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.

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

Role of ITPK in Calcium Signaling

PLC = Phospholipase CIP3 = Inositol triphosphateIP3R = InsP3 ReceptorITPK = IP3 3-KinaseIP4 = Inositol Tetrakisphosphate

PLC = Phospholipase CIP3 = Inositol triphosphateIP3R = InsP3 ReceptorITPK = IP3 3-KinaseIP4 = Inositol Tetrakisphosphate

Role of ITPK in Calcium Signaling

LSEC

Hypothesis

ITPKC levels are down regulated in colon carcinomas versus normal colon cells

TumorCell

PLC IP3Adhesion &

Migration

IP4

Ca2+

ITPKC

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).

Cell lines used in this study

SW620

HCT-116

HT-29

SW480

CaCo-2

HCEC 1ct

HCEC 2ct

HCEC 1ct

HCEC 2ct

Metastatic

Po

ten

tial

Growth arrested to induce differentiation

Non-Differentiated

Colorectal cancer cells

Human colon epithelial cells

Results Testing the Primers for ITPK A, B & C

Total RNA extracted from normal HCEC

Intact, little contamination, good ratio between 28s and 18s proves good RNA isolation

The Total RNA extracted from colon cancer samples

Both groups of samples were isolated well, but need to be decontaminated before continuing

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

The Protein levels of ITPK A isoforms in colon cancer

samples & normal colon tissue

SW620

HCT-116

HT-29

SW480

CaCo-2

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.

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

Acknowledgements

Lawrence Gaspers, PhD.

Fernando Vidal-Vanaclocha, M.D.

Guoqiang Wang, PhD

Ishwarya Murali

Questions?

Other Sources

ITPKC is down regulated in in Breast and Colon cancer

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 = DoxycyclineLSECs = Liver sinusoidal endothelial cells.

Total RNA extracted from normal HCEC

Intact, little contamination, good ratio between 28s and 18s proves good RNA isolation

The Total RNA extracted from colon cancer samples

Both groups of samples were isolated well, but need to be decontaminated before continuing

The Protein levels of ITPK A isoforms in colon cancer

samples & normal colon tissue

SW620

HCT-116

HT-29

SW480

CaCo-2