DETERMINATION OF K-RAS MUTATION IN COLORECTAL AND LUNG CANCER
E.Andreas1,2,3, Ali Mujahid1,3 , Attia Youssef1, Seidi Armin1
1 Department of Medical Biotechnology, IMC UAS Krems (3500) International Campus Piaristengasse Austria. 2 Department of Genetics,University of Cambridge (CB2 3EH ),United Kingdom
Keywords: K-RAS, Mutation, Tumorigenesis, EGFR, Metastasis, Monoclonal Antibody
Abstract

A mutated K-RAS gene is the originator of several characters of colorectal cancer among them tumor initiation, growth, survival, metastasis formation and even immune response. Thus; the aim of our experiments is to establish if the tumor samples express the K-RAS mutation before starting the monoclonal antibody-based therapy. Otherwise it makes no sense to block the receptor binding site if the signaling cascade is independent of the binding of the ligand to the receptor. Colorectal cancer, a cancer resulting from uncontrolled cell growth in the colon or rectum is the fourth most common cause of cancer death. The stage of its development affects the method of treatment of this disease. A prominent method to treat this type of cancer is the blockage of the EGFR by monoclonal antibodies. This kind of approach however seems only to be efficient as long as there is no mutation of K-Ras, a GTPase thought to play an important role at an early stage of the development of colorectal cancer. In case of a mutation in the RAS protein, the Ras GTPase is activated to such an extent that the subsequent over activation of downstream signaling pathways leads to tumor genesis.

Article Information

Identifiers and Pagination:
Year:2012
Volume:4
First Page:244
Last Page:248
Publisher Id:JAppPharm (2012 ). 4. 244-248
Article History:
Received:July 15, 2012
Accepted:September 21, 2012
Collection year:2012
First Published:October 1, 2012

INTRODUCTION

Colon cancer is one of the most common cancers besides breast and lung cancer. In the treatment of metastatic colorectal cancer one of the most significant therapeutic advances in cancer treatment has occurred. It will become more important in the future to look on an individual tumor’s genetic profile rather than treating a specific tumor type (Astin M & Griffin, T, 2011). One particularly interesting biomarker is the epidermal growth factor receptor (EGFR), which’s treatment, is very efficacious. However, patients with a tumor positive K-Ras mutation show resistance to EGFR blocking. It has been reported that approximately 30 to 50% of colon cancer patients have K-Ras mutations. This mutation in the Ras-oncogene lead to over activation of downstream signaling which in turn leads to cell transformation and tumorigenesis. Activated K-Ras not only promotes tumor initiation but also tumor growth, survival, progression, local invasion, metastasis formation, angiogenesis, and even immune response (Curr Med Chem 2006). In 2000, a study in the US showed that 32% of the assessed K-Ras mutations in colon cancer tumors showed mutations in either codon 12 or 13. Codon 12 mutations were associated with advanced stage of colon cancer and a more aggressive tumor. Patients with the specific mutation glycine to valine had a significant poorer prognosis for survival. Also the codon 13 mutation showed a 40% increase in short-term mortality. It is therefore of indispensable importance to check for mutations of the K-Ras gene to see if an EGFR treatment will be successful or not. To analyze possible mutations, ARMS-PCR can be used which is described in the result section (Clin Ther, 2008).

 

METHODOLOGY

Immunohistochemistry staining (IHC) was the second method of choice to observe cell aggregation, invasiveness, hyper-proliferation and other cancer hallmarks. An anti-E-cadherin-antibody was used to stain E-cadherin which is mainly found on the cell membrane of the intestinal villi. As E-cadherin is thus used as a marker for the cell membrane, disordered or aggregated tumors can be seen easily through fluorescence microscopy. E-cadherin expression is often down-regulated in highly invasive, poorly differentiated tumors. The loss of expression of E-cadherin appears to be an important step in tumorigenic progression. The IHC is used in this protocol to see the tumor progression and invasiveness of the colon cancer patients (Yamanaka Y, Friess H; Anticancer Res 1993).

 

RESULTS AND DISCUSSION


Table 1: Ct values of the cDNA

cDNA

H1975

SW480

FFPE

NTC

WT

27,37

27,42

25,05

28,5

MUT1

27,2

33,87

33,9

33,46

MUT3

35,94

34,34

33,29

34,18

 

WT/MUT1

WT

WT/??

 

Graph 1: Comparison of the cDNA expression levels of Wt, Mut1 and Mut3

 




Genomic DNA

H1975: WT

SW480: WT and Mut1 almost equal: possible case of Heterozygosity WT/MUT1.Booth allele are present on the genomic DNA level.

FFPE: It is possible that there is another type of mutation

 

 

 

 

Table 2: Ct values of the gDNA

gDNA

 H1975

SW480

FFPE

NTC

WT

20,3

20,77

33,12

28,5

MUT1

33,7

19,64

33,53

33,46

MUT3

33,2

40,81

32,8

34,18

 

WT

WT/MUT1

 

 

 

Immunohistochemistry

The immunohistochemistry assay was not successful in our case. The assay for the wild type didn’t work. A possible cause is that the cDNA synthesis did not work. This affects all the other results. But supposing that the assay worked, we can say that the most mutation observed in the samples is MUT1 and it is observed in a heterozygotic state (Mut1/Wt) (Nat Clin Pract Oncol, Clin Ther, 2008)

IHC: It is also very important to determine if the E-cadherin have been down regulated (Cadherin switch) to be able to make a prognosis. Most of the cases an E cadherin down regulation is sign of an invasive behavior of the tumor. In this case the surgical treatment is not a good alternative and the chemotherapy seems to be more appropriate (Astsaturov I, Cohen RB, Harari P; Expert Rev Anticancer Ther 2006).

 

The results of the ICH assay were not satisfactory. Many causes can lead to that.

Example of possible cause:

*Dehydration steps may be not long enough (time)

*Incubation time with the antibodies

*Non-reactive tissue can lead to lack of staining

* The antibody itself can be deteriorated or not adequately used and conserved (repeated thawing freezing cycles)

 



CONCLUSION

The determination of K- Ras mutation in the colorectal cancer cells is a pre-requisite before starting any monoclonal antibody-based therapy (Klein S and Levitzki A, 2009). Many mutations can affect the signaling of K- Ras and thus the samples should be tested for all of the known mutations but at least the most frequent ones.

 

ACKNOWLEDGEMENTS

We  thank Dr. Elisabeth Hofmann for technical assistance  and Dr. Eger helped for qPCR, ARM-PCR, as well as statistical assistance .The isolated cancerous tissue used for this study was kindly given by miss Tina IMC Krems, Krems  Austria.

This study work was supported by Grant gratefully given by Department of Medical Biotechnology, IMC University of applied sciences Krems, Austria.

 

REFERENCES

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2.      Barr, F.A.et al: Polo-like Kinases and the orchestration of cell division .Nat. Rev. Mol. Cell Biol.2004, 5:429-440.

3.      Marumoto, T.et al: Aurora-A—a guardian of poles. Nat. Rev. Cancer 2005, 5:42-50.

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Welcome to the research group of Prof. Dr. Cornelia M. Keck in Marburg. Cornelia M. Keck is a pharmacist and obtained her PhD in 2006 from the Freie Universität (FU) in Berlin. In 2009 she was appointed as Adjunct Professor for Pharmaceutical and Nutritional Nanotechnology at the University Putra Malaysia (UPM) and in 2011 she obtained her Venia legendi (Habilitation) at the Freie Universität Berlin and was appointed as a Professor for Pharmacology and Pharmaceutics at the University of Applied Sciences Kaiserslautern. Since 2016 she is Professor of Pharmaceutics and Biopharmaceutics at the Philipps-Universität Marburg. Her field of research is the development and characterization of innovative nanocarriers for improved delivery of poorly soluble actives for healthcare and cosmetics. Prof. Keck is executive board member of the German Association of Nanotechnology (Deutscher Verband Nanotechnologie), Vize-chairman of the unit „Dermocosmetics“ at the German Society of Dermopharmacy, active member in many pharmaceutical societies and member of the BfR Committee for Cosmetics at the Federal Institute for Risk Assessment (BfR).

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