Research and Advanced Education
Development and characterization of a cellular model of acquired resistance to cetuximab in colorectal cancer KRAS - and BRAF-wt human cell lines
In a degree where the clinical aspect of medicine predominates, the GAPIC (Office for the Support of Scientific and Technological Research) offers students an opportunity to become familiar with experimental research and develop their scientific reasoning. Given that we were both keen to participate in a lab research project and deepen our knowledge and practice of basic science, we decided to get in touch with Professor Teresa Pacheco, of the Clinical Oncology Research Unit of the IMM, whom we already knew as our biochemistry lecturer. Following a brief discussion, Professor Teresa Pacheco accepted to be our supervisor and this is how we succeeded to be involved in a recent project of the Unit. The part of it we are responsible for will be developed during the 2012/2013 academic year and is called“Development and characterization of a cellular model of acquired resistance to cetuximab in colorectal cancer KRAS - and BRAF-wt human cell lines”.
Rationale for this project
Colorectal cancer is the third most common cancer worldwide¹, and about 40-50% of diagnoses are made when the disease is already at metastatic stage. At this stage, patients show a very poor prognosis, with an estimated average overall survival rate of 18-21 months¹.
The pathogenesis of colorectal cancer involves the deregulation of cell signalling pathways4, of which those initiated by the Epidermal Growth Factor Receptor (EGFR), involved in the survival, proliferation and invasiveness of tumour cells, stand out ¹.
In a new approach to developing therapies directed at specific targets, the monoclonal antibodies cetuximab and panitumumab, which are inhibitors of the EGFR activity and are used especially in cases of metastatic colorectal cancer, were produced. However, their efficacy has been demonstrated in only 10-20% of cases ¹, which suggests the presence of cellular changes that resist, at primary or secondary level, this treatment.
One of the mechanisms of primary resistance to anti-EGFR therapy involves the overactivation of downstream participants in the signalling pathways initiated by EGFR, the KRAS protein being the most important in this context. Patients with mutations in the corresponding gene present constitutively active forms of the KRAS protein, which cancels off the benefit of the aforesaid biological therapy4. In addition to primary resistance mechanisms, it appears that tumours with no KRAS mutation and initially sensitive to anti-EGFR therapy often acquire secondary resistance to it ¹.
Besides the aforementioned cellular changes, other changes involved in tumour resistance to anti-EGFR therapy have been described, including variations in the activity of BRAF, PI3K and PTEN proteins, although their clinical correlation is not as strong as that observed with KRAS¹.
However, there are still a percentage of tumours which, while having none of the four mutations described above, still do not respond to anti-EGFR therapy, and this has an influence on treatment possibilities. This fact suggests the presence of other signalling paths involved in the physiopathology of cancer which remain undiscovered. One of these still unidentified resistance mechanisms may lie in the PI3K/AKT/mTOR pathway. The mammalian Target of Rapamycin (mTOR) is a proto-oncogene that encodes a kinase² located downstream in the PI3K/AKT3 signalling pathway, which is initiated by EGFR activation. Its action on the cell is related to the organization of the actin cytoskeleton, cell proliferation, survival and metabolism5.
Indeed, the scientific literature published to date reveals that mTOR hyperactivation is a very frequent event in human cancers5. In addition, a study conducted with cetuximab resistant colorectal cancer cells showed that the administration of one anti-EGFR drugs concurrently with everolimus, an mTOR inhibitor, allowed restoring sensitivity to biological therapy, with the two drugs acting in synergy². Accordingly, this is something that requires further research, considering the mTOR overactivation as a cetuximab resistant mechanism in metastatic colorectal cancer.
What are the objectives?
In order to investigate the aforementioned hypothesis, the aim of this work is to develop a cellular model of cetuximab acquired resistance from human cellular lines derived from KRAS- and BRAF-wild type colorectal cancer and do their molecular characterization by comparing the selected lines (cetuximab resistant) with the corresponding parental lines. This analysis will be done using immunoblotting techniques and PCR, which we intend to improve and integrate in our technical skills. This work will be done until November 2013, and results will be disclosed in December at Research Day.
Currently, this project is still at optimization stage, and the laboratory work is due to start soon. As the several planned stages progress, we hope to gather theoretical and practical knowledge essential to the research component, develop the necessary mental skills to be able to address several scientific issues and gain new insights into the obstacles arising every day. When we conclude this stage of our academic life, we also hope to have contributed, to a large or small extent, to the resolution of this increasingly emerging pathology that threatens so many lives worldwide.
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References
1. Bardeli, A., Siena, S. (2010). Molecular Mechanisms of Resistance to Cetuximab and Panitumumab in Colorectal Cancer. Journal of Clinical Oncology, 28(7): 1254-1261
2. Bianco, R., Garofalo, S., Rosa, R., Damiano, V., et al. (2008). Inhibition of mTOR pathway by everolimus cooperates with EGFR inhibitors in human tumors sensitive and resistant to anti-EGFR drugs. British Journal of Cancer, 98: 923-930
3. Hsieh, A.C., Liu, Y., Edlind, M.P., Ingolia, N.T., Janes, M.R., et al. (2012). The translational landscape of mTOR signaling steers cancer initiation and metastasis. Nature, 485: 55-60
4. Pritchard, C.C., Grady, W.M. (2011). Colorectal Cancer Molecular Biology Moves Into Clinical Practice. Gut, 60(1): 116-129
5. Zaytseva, Y.Y., Valentino, J.D., Gulhati, P., Evers, B.M. (2012). mTOR inhibitors in cancer therapy. Cancer Letters, 319: 1-7
Sara Félix Dâmaso
year 4 student of the IMM
sara-damaso@campus.ul.pt
Maria Madalena da Palma Rosário
year 4 student of the IMM
m.rosario@campus.ul.pt
Rationale for this project
Colorectal cancer is the third most common cancer worldwide¹, and about 40-50% of diagnoses are made when the disease is already at metastatic stage. At this stage, patients show a very poor prognosis, with an estimated average overall survival rate of 18-21 months¹.
The pathogenesis of colorectal cancer involves the deregulation of cell signalling pathways4, of which those initiated by the Epidermal Growth Factor Receptor (EGFR), involved in the survival, proliferation and invasiveness of tumour cells, stand out ¹.
In a new approach to developing therapies directed at specific targets, the monoclonal antibodies cetuximab and panitumumab, which are inhibitors of the EGFR activity and are used especially in cases of metastatic colorectal cancer, were produced. However, their efficacy has been demonstrated in only 10-20% of cases ¹, which suggests the presence of cellular changes that resist, at primary or secondary level, this treatment.
One of the mechanisms of primary resistance to anti-EGFR therapy involves the overactivation of downstream participants in the signalling pathways initiated by EGFR, the KRAS protein being the most important in this context. Patients with mutations in the corresponding gene present constitutively active forms of the KRAS protein, which cancels off the benefit of the aforesaid biological therapy4. In addition to primary resistance mechanisms, it appears that tumours with no KRAS mutation and initially sensitive to anti-EGFR therapy often acquire secondary resistance to it ¹.
Besides the aforementioned cellular changes, other changes involved in tumour resistance to anti-EGFR therapy have been described, including variations in the activity of BRAF, PI3K and PTEN proteins, although their clinical correlation is not as strong as that observed with KRAS¹.
However, there are still a percentage of tumours which, while having none of the four mutations described above, still do not respond to anti-EGFR therapy, and this has an influence on treatment possibilities. This fact suggests the presence of other signalling paths involved in the physiopathology of cancer which remain undiscovered. One of these still unidentified resistance mechanisms may lie in the PI3K/AKT/mTOR pathway. The mammalian Target of Rapamycin (mTOR) is a proto-oncogene that encodes a kinase² located downstream in the PI3K/AKT3 signalling pathway, which is initiated by EGFR activation. Its action on the cell is related to the organization of the actin cytoskeleton, cell proliferation, survival and metabolism5.
Indeed, the scientific literature published to date reveals that mTOR hyperactivation is a very frequent event in human cancers5. In addition, a study conducted with cetuximab resistant colorectal cancer cells showed that the administration of one anti-EGFR drugs concurrently with everolimus, an mTOR inhibitor, allowed restoring sensitivity to biological therapy, with the two drugs acting in synergy². Accordingly, this is something that requires further research, considering the mTOR overactivation as a cetuximab resistant mechanism in metastatic colorectal cancer.
What are the objectives?
In order to investigate the aforementioned hypothesis, the aim of this work is to develop a cellular model of cetuximab acquired resistance from human cellular lines derived from KRAS- and BRAF-wild type colorectal cancer and do their molecular characterization by comparing the selected lines (cetuximab resistant) with the corresponding parental lines. This analysis will be done using immunoblotting techniques and PCR, which we intend to improve and integrate in our technical skills. This work will be done until November 2013, and results will be disclosed in December at Research Day.
Currently, this project is still at optimization stage, and the laboratory work is due to start soon. As the several planned stages progress, we hope to gather theoretical and practical knowledge essential to the research component, develop the necessary mental skills to be able to address several scientific issues and gain new insights into the obstacles arising every day. When we conclude this stage of our academic life, we also hope to have contributed, to a large or small extent, to the resolution of this increasingly emerging pathology that threatens so many lives worldwide.
--------------
References
1. Bardeli, A., Siena, S. (2010). Molecular Mechanisms of Resistance to Cetuximab and Panitumumab in Colorectal Cancer. Journal of Clinical Oncology, 28(7): 1254-1261
2. Bianco, R., Garofalo, S., Rosa, R., Damiano, V., et al. (2008). Inhibition of mTOR pathway by everolimus cooperates with EGFR inhibitors in human tumors sensitive and resistant to anti-EGFR drugs. British Journal of Cancer, 98: 923-930
3. Hsieh, A.C., Liu, Y., Edlind, M.P., Ingolia, N.T., Janes, M.R., et al. (2012). The translational landscape of mTOR signaling steers cancer initiation and metastasis. Nature, 485: 55-60
4. Pritchard, C.C., Grady, W.M. (2011). Colorectal Cancer Molecular Biology Moves Into Clinical Practice. Gut, 60(1): 116-129
5. Zaytseva, Y.Y., Valentino, J.D., Gulhati, P., Evers, B.M. (2012). mTOR inhibitors in cancer therapy. Cancer Letters, 319: 1-7
Sara Félix Dâmaso
year 4 student of the IMM
sara-damaso@campus.ul.pt
Maria Madalena da Palma Rosário
year 4 student of the IMM
m.rosario@campus.ul.pt
