Cancer Signalling Pathway, Epidermal Growth Factor Receptor - Coursework Example

Introduction In the past few decades humans have made spectacular advances in medical sciences however even to this day finding a perfect cure to cancer remains a big challenge. Cancer oriented research have tried to unravel the various pathways of the disease, the molecules involved and the genetics and epigenetics of the problem in order to completely understand cancer and design appropriate therapies for treatment. One such therapeutic option that had been devised was the administration of monoclonal antibodies such as cetuximab and panitumumab targeted particularly against Epidermal Growth Factor Receptor (EGFR) in order to treat cancers such as metastatic colorectal cancer or lung cancer (Jean and Shah, 2008). In the cancer signalling pathway, EGFR, which belongs to the ErbB family of proteins, is activated upon binding with other receptors such as the peptide growth receptors (Normanno et al, 2006). Studies have shown that in patients with cancer the EGFR associated with peptide growth receptors is over expressed. Therefore, monoclonal antibodies which had been developed as inhibitors of EGFR along with conventional chemotherapy helped in prolongation of life in patients afflicted with cancer. However, complexity arose when certain patients with genetic alterations such as mutation in K-ras or PTEN instability showed resistance to the administration of the monoclonal antibodies which inhibited the success of the anti-cancer therapies and added to patient mortality. Several researchers focussed their studies on strategies to eliminate the resistance developed by the patients which called for an in-depth understanding of the resistance pathways. This paper, focuses particularly on two such studies- Acquired resistance to cetuximab is mediated by increased PTEN instability and leads cross-resistance to gefitinib in HCC827 NSCLC cells by Kim et al (2010) and MEK1/2 Inhibitors AS703026 and AZD6244 May Be Potential Therapies for KRAS Mutated Colorectal Cancer That Is Resistant to EGFR Monoclonal Antibody Therapy by Yoon et al (2011). Comparing and Contrasting Both of the papers have devised novel way to understand the development of resistance against treatment. In terms of theory both the papers looked into different aspects of the problem in terms of activation of EGFR. Yoon et al focused to understand the KRAS mutation induced resistance in colorectal cancer and come up with a plausible method to solve the problem. Colorectal cancer is a common form of cancer and has shown elevated expression of EGRF in the cells. However, the molecular therapy involving administration of monoclonal antibodies have shown their efficacy in only a small percentage of patients while those patients with metastatic form of colorectal cancer bearing KRAS mutation of the cells develop resistance towards the antibody therapy by reducing G-protein downstream of EGFR and the associated response to the therapeutic action of the monoclonal antibodies . Therefore it had been deduced that that the non-mutated form of KRAS is required for the substantial efficacy of the administered antibodies (Amado et al, 2008). Thus the main aim of the authors was too investigate the status of the mutation of KRAS present in the patient and also inhibit such mutations and also investigate the efficacy of MEK1/2 inhibitors as AS703026 and AZD6244 as therapies to be used in patients with KRAS mutations. . In comparison to Yoon et al’s method of approaching the problem, Kim et al have focused on the role played by EGRF upon the activation of MAPK and Akt pathways which are known to be responsible for abnormal cell proliferations and onset of cancer such as non-small cell lung cancer (NSCLC) and the importance of mutated form of EGFR. The first line of treatment in most severe forms of NSCLC is treatment with cetuximab however upon failure of the first line of treatment a second treatment option is available and involves the use of gefitinib. However, several studies have shown that patients who show positive response towards treatment with gefitinib express mutated forms of EGFR gene (Kobayashi et al, 2005). It has thus been established that the sensitive mutation in EGFR leads to elevation in the signaling of the receptor and results in an enhanced susceptibility towards gefitinib therapy. The main of the authors were to understand the main reason behind cetuximab resistance and investigate the phenomena of cross-resistance between cetuximab and gefitinib as a potential problem in the clinical settings. Given the experimental designs for both the papers the methodology adopted were also different. Yoon et al used human colorectal cancer cell lines which had either wild type or the mutated form of KRAS which had been derived from DLD-1 human colorectal cell. The procured cell lines were then cultured in the laboratory settings at temperature of 37 degrees in a medium containing fetal bovine serum, penicillin and streptomycin. Mutational analysis of KRAS mutations was conducted using a kit. The cells were then placed and treated with either AS703026 or AZD6244 both with and without cetuximab. This was done for 72 hours. The MTT reagent was then added and MTT assay was conducted following standard protocol.MTT assay is conducted to test cell sensitivity and account for cell viability (Van Meerloo et al, 2011). Similar study using mice as a model organism was also used after obtaining approval from ethical committee. The mice were used for tumor xenograft study to test the efficacy of the MEK inhibitors where focus was also based on colorectal cell line. In comparison to Yoon et al’s methodology, cetuximab-resistant cell lines were generated by Kim et al, using HCC827 NSCLC cells. The cetuximab-resistant cell line was (HCC827-CR) which was prepared by the researchers themselves. Phospho RTK-array was used as a tool to detect receptor phosphorylation of RTKs (Morishita et al, 2010). Protein detection was done and EGFR was measured using flow cytometry. Transfection was followed by analysis of the PTEN using RT-PCR (reverse transcriptase Polymerase Chain Reaction). Therefore comparing both the methodologies that have been adopted and followed by the authors of the two papers we can clearly see that both the methodologies have employed various molecular techniques. Yoon et al have utilized techniques such as DNA extraction, MTT assay, Brd-U incorporation, Immunoblotting and immunohistochemistry while KIM et al employed methods such as transfection, RT-PCR, Western Blotting and Flow Cytometry. The main difference in the methodology lied in the fact Yoon et al, conducted the study on both human cancer cell lines as well as on a model organism i.e. in-vivo test was also conducted while the study done by Kim et al, made use of cell lines only. However, compared to the cell lines bought from an external laboratory by Yoon et al, Kim et al constructed their own cetuximab-resistant cell lines for investigation. In accordance with the study methodologies the results extracted by both the studies were different. Yoon et al, studies both the wild-type and mutated KRAS type cell lines and found that on treatment with high levels of cetuximab both the cell lines proliferated to an extent. The proliferation was measured using MTT assay. In accordance to the hypothesis proposed by the researchers it was seen that the mutated cells that had KRAS mutation showed resistance to the anti-cancer therapy while excessive cellular growth was clearly inhibited upon treatment with cetuximab on the wild type.MTT analysis revealed that the cetuximab successfully inhibited cancerous growth by 30% in the wild type (See Appendix 1, Fig-1). In vivo test on mice xenograft established a similar trend where mice injected with b-MUT tumors i.e. the mutated form when treated with cetuximab failed to show any kind of reduction in the volume of the cells afflicted with cancer. These results fulfilled the first aim of the experiment i.e. to establish the efficacy of cetuximab on KRAS mutated forms of cell. The methodology also investigated the efficacy of MEK inhibitors- as AS703026 and AZD6244  since MEK ½ inhibition action is one of the most important cancer therapeutic approaches (Zhao and Adjei, 2014). It was observed that in the wild type cell when treated with cetuximab showed inhibition of ERK pathway by reducing the load of RAS but this did not happen when cetuximab was administered to the mutated forms. ERK pathway is known to be hyper activated in cancer patients (Samatar and Poulikakos, 2014). However when these mutated forms were treated with the MEK inhibitors it was seen that ERK pathway was inhibited and thus no cellular proliferation was detected. The efficacy of the MEK inhibitors was measured using BrdU and MTT assays. Again mouse model was used to confirm the in-vivo action of the MEK inhibitors in those cells which are cetuximab resistant induced by KRAS mutation. Immunohistochemical analysis revealed and confirmed that the inhibitors worked in cetuximab resistant cells and inhibited cancerous growth. Kim et al conducted the experiment according to their selected methodology and confirmed that HCC827 NSCLC cell lines were devoid of any mutations. The researchers hypothesized about the role played by hyperactivity of AKt pathway induced cetuximab resistance and that inhibition of this hyperactivity would help in restoring the sensitivity towards the anti-cancer therapy. Immunoblotting was employed to measure the Akt and EKT pathways and the hypothesis was supported by the immunoblot results. Since it was confirmed that Akt pathway was involved the researchers also focused on PTEN since reduction in PTEN resulted in hyperactivity of the Akt pathway and resulted in cellular proliferation (Carnero et al, 2008) (See appendix 1, Figure 2). Molecular studies helped in establishing the fact that PTEN instability was the underlying cause of the Akt activation especially in HCC827-CR cells. In addition to this, the researchers had also designed to understand the mechanism of de-novo resistance of cells to the second line of treatment with gefitinib. MTT assay was employed to measure cell viability of cells treated with gefitinib and which were resistant to cetuximab. It was confirmed that HCC827-CR cells had de-novo resistance to both cetuximab and gefitinib however on acquirement of mutation i.e. upon inhibition of the activity of Akt pathway; gefitinib could be used for treatment. Discussion and Conclusion A close analysis of both the experimental designs gives us a different perspective on the resistance of the cells to monoclonal antibodies. Both the studies have successfully presented a good approach towards solving the problem. There exist few glitches in the adopted methodology of either group of researchers. In Yoon et al’s paper, we see utilization of MEK ½ which are under clinical trials and even though the choice was very good from the experimental point of view the fact that the inhibitors are still under trial to be used in humans is not as application oriented as desired (Kirkwood et al, 2012). In comparison the approach adopted by Kim et al, i.e. elevation of PTEN stability and reduced activation of the Akt pathway to restore sensitivity to the gefitinib therapy is a much more application oriented approach. However, from a theoretical point of view, Yoon et al’s made good use of molecular techniques and took care to involve both in-vitro and in-vivo experiments to establish their hypothesis whereas Kim et al did not consider to use any kind of model organism. I believe cellular studies are always a good way to approach and solve problems however given the fact that cancer is a challenging concept and needs to be handled with great caution and use of model organisms is one of the best ways to study disease mechanisms (Aitman et al, 2011). Both the research methodology have utilized MTT assay to measure cell viability. MTT assay is an excellent choice and is most often used to determine anticancer activity. Therefore the assay was the right choice in the experimental designs. However even though both he studies have tried to solve the problem of cetuximab resistance of the cancer cells none of the experiments have successfully confirmed the underlying cause of the resistance. In all probability this means that the acquired resistance of the cells may involve a complex interplay between various pathways and protein products. The most important result of both the experiments is that both have highlighted important facts about he acquired resistance and have successfully investigated the solutions to the problem well. Thus, in spite of being vastly different in their approaches, Yoon et al and Kim et al have presented very accurate approaches towards acquiring the answer to the research question. APPENDIX I Figure 1: Sensitivity of Wild type and mutated KRAS upon treatment with Cetuximab at 24, 48 and 72 hour intervals (Yoon et al, 2011). Figure 2:PTEN-P13-AKT-TOR pathway- How PTEN inactivation activated AKT pathway resulting in cancer. (Song et al, 2012). Reference Aitman,T.J. et al .(2011). The future of model organisms in human disease research. Nature Reviews Genetics, 12, p.575- 582. Amado,R.G. et al.(2008). Wild-Type KRAS Is Required for Panitumumab Efficacy in Patients With Metastatic Colorectal Cancer.American Association of Clinical oncology, 26 (31), p.5139. Carnero,A . et al. (2008). The PTEN/PI3K/AKT signalling pathway in cancer, therapeutic implications. Current Cancer Drug targets, 8 (3), p.187-198. Jean,G.W. and Shah, S.R.(2008). Epidermal growth factor receptor monoclonal antibodies for the treatment of metastatic colorectal cancer. Pharmacotherapy, 28 (6), p.742-754. Kim,S.M. et al.(2010). Acquired resistance to cetuximab is mediated by increased PTEN instability and leads cross-resistance to gefitinib in HCC827 NSCLC cells.Cancer Letters, 296 (2), 150-159. Kirkwood,J.M. et al. (2012). Phase II, open-label, randomized trial of the MEK1/2 inhibitor selumetinib as monotherapy versus temozolomide in patients with advanced melanoma. Clinical cancer Research, 18 (2), p.555-567. Kobayashi,S. et al.(2005). EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. The New England Journal of Medicine, 352 (8), p.786-792. Morishita,A. et al. (2010). The use of protein array to identify targetable receptor tyrosine kinases for treatment of human colon cancer. International Journal of Oncology, 37 (4), p.829-835. Normanno, N. et al.(2006). Epidermal growth factor receptor (EGFR) signaling in cancer. Gene, 366 (1), p.2-16. Samatar, A.A. and Poulikakos,P.I. (2014). Targeting RAS–ERK signaling in cancer: promises and challenges. Nature Reviews, 13, p.928-942. VanMeerloo,J et al. (2011). Cell sensitivity assays: the MTT assay. Methods in Molecular Biology, 731, p.237-245. Yoon,J. et al.(2011). MEK1/2 Inhibitors AS703026 and AZD6244 May Be Potential Therapies for KRAS Mutated Colorectal Cancer That Is Resistant to EGFR Monoclonal Antibody Therapy. Cancer Research, 71, p.445-453. Zhao,Y and Adjei, A.A. (2014). The clinical development of MEK inhibitors. Nature, 11, p.385-400. Appendix Song, M.S. et al (2012). The functions and regulation of the PTEN tumour suppressor.Nature Reviews, 13, p.283- 296. Yoon,J. et al.(2011). MEK1/2 Inhibitors AS703026 and AZD6244 May Be Potential Therapies for KRAS Mutated Colorectal Cancer That Is Resistant to EGFR Monoclonal Antibody Therapy. Cancer Research, 71, p.445-453. Read More