Iron Metabolism Markers in Colorectal Cancer Development and Progression

1. Iron metabolism markersRole inColorectal cancer Development and Progression Presented by Nesreen Fayyad Al-Khofash MSc in molecular medicine and translational research College of medicine University of Sharjah, UAE Supervised by:Dr. Wael Abdel-Rahman Hassan & Dr. Mawieh Hamad

2. Table of index: • Introduction • Aims and significance of the study • Materials • Methods • Results • Take home messages • Further directions

3. Iron in Cancer • Pathways involved in iron acquisition, trafficking, storage and regulation are all defected, suggesting that iron metabolism is important for tumor cell survival. Initiation and progression of different forms of cancer ROS The very attribute that makes iron useful enzymatically also enables iron to participate in potentially deleterious free radical-generating reactions

4. AIMS • Identify iron regulators that participate in CRC development and progression. • Find out if iron overloud could be a driving factor. Significance of the study: • Provides an insight into the expression levels of iron regulators in CRC. • Defines the correlation between iron regulators them selves and between and clinical and pathological characteristics of tumors. • Provides bases for functional analysis.

5. Methods:

6. Results:

7. A-IHC analysis on CRC samples: • Ferroportin, TfR1 and catalase respectively showed elevated expression. • Ferroportin showing the highest frequency; 97% of the total number of samples showed positive expression. • 51% of tumors showed ferritin expression. • TfR2 was moderately expressed in 32% of CRC samples.

8. Ferroportin staining via IHC: Tumor: Strong. Normal mucosa: positive Tumor:“Negative” Normal mucosa:negative Tumor:Strong in islands. Normal mucosa is positive

9. B-Statistical analysis on CRC samples: r = 0.267  r = 0.438  Significant correlation p<0.05 r = 0.287 r = 0.460

10. C-Western blot analysis: • TfR1 was found to be highly over expressed in the cell line of primary CRC with Wild type P53 the rest of the cell lines had mutated P53 and showed significant lower levels of TfR1expression levels. The colon epithelial cell line with wild type P53 showed the lowest level of TfR1expression. High TfR1 in primary tumor with WT P53 Mutant P53 A follow up experiment to determine iron content in cells will need to be performed to confirm the suspected TfR1 role in supporting ROS stress that is suspected to drive cancer development and progression.

11. Elevated TfR1 and TfR2 along with slight increase in Ferritin, suggest that increased iron uptake and reduced iron storage may contribute to iron overload.

12. Hepcidin Ferroportin We will attempt to verify this finding, however, as such, a question remains as to why theses tumors preferentially upregulate Ferroportin • Hepcidin (Hep) was found to have an inversely related expression with Ferroportin in all CRC cell lines regardless of their stage or pathological characteristics with no constant ratio. • All CRC cell lines had high hepcidin content that was dramatically higher than Ferroportin content in any of the investigated CRC cell lines.

13. HO-1 Several lines of evidence have supported the implication of through modulating expression of angiogenic factors.

14. Ferroportin Silencing • Successful silencingof FPN. ____________ • Hepcidin:3-folds of increase. • Ferritin:slightly increasing

15. Take home messages: • All 5 iron regulators showed elevated expression in CRC compared to epithelial cells, making them good markers for CRC • Hepcidin showed dramatic increase in expression making it a good marker for CRC and particularly this might be applied in early stage detection. • Elevated TfR1 and TfR2 along with down regulated Ferritin, suggest that increased iron uptake and reduced iron storage may contribute to iron overload. Our findings suggest that colorectal cancer associates with disrupted iron metabolism and it shows significant heterogeneity in the expression profile of key iron regulatory proteins.

16. Further Directions: • Verification of data using different antibodies and larger tumor series; tumors of different lineages such as breast cancer will be examined. • Statistical Correlation between Hepcidin, HO-1 and iron regulators. • Iron staining assay • Oxidative stress analysis • Functional analyses ,post translational modifications.

17. Acknowledgement • I would like to thank His Highness Sheikh Doctor Sultan Bin Mohammad Al-Qassimi for his unlimited support and for offering MSc scholarship in Molecular medicine and translational research. • Prof. Qutaiba • Dr. Wael Abdel-Rahman & Dr. Mawieh Hamad • Dr. Samir Awad-Allah • Also, I am grateful to Mrs. Vidhya Anish, Mrs. Jasmin Shafarin, Mrs. IsraSati, Dr. KhuloudBajbouj and Dr. Asha Caroline for assistance. • I acknowledge the financial support of the student Project support fund, the Cancer Research Group, SIMR, Terry Fox and Al Jalila foundation grants to Dr Wael. • My thanks are extended to the University of Sharjah and Sharjah Institute of Medical Research for the financial support as well as the wonderful facility. • I would like to dedicate my work to my supporting family.