KRAS Amplification and Acquired Resistance to Cetuximab

In my last blog post, I examined questionable factors of a paper whose authors conclude that KRAS mutations drive acquired resistance to an anti-EGFR therapy drug called cetuximab for colorectal cancers. My main concern is with the strength of the authors’ conclusion and if the data is sufficient enough to lead to a causal relationship. This paper presents two main overall sets of data. The first is in regards to the molecular mechanisms of secondary resistance to anti-EGFR therapies, while the second set attempts to determine clinically if KRAS mutations or amplifications are related to acquired cetuximab resistance. For this blog post, I will examine one part of the first data set.

           
The study was conducted with two colorectal cancer cellular models. One is called DiFi, the other Lim1215. Both lines were generated to be similar on a molecular level to the colorectal cancer patients who most likely would respond to cetuxtimab. DiFi cells overexpress EGFR due to the amplification of the EGFR gene. On the other hand, Lim1215 cells express normal levels of EGFR, while still being similarly sensitive to cetuximab as DiFi cells. From these two models, the researchers produced the cetuximab-resistant variants (DiFi-R, Lim1215-R), which are extremely sensitive to EGFR inhibition. One concern is that the methods section of the paper did not delve much into the specifics of how the cells were generated, so I am unsure how the cells function and how they are equally resistant to cetuximab. 

This blog post will focus on the DiFi cells and their resistant lines.

Graph (a) depicts the resistance of DiFi R1 and DiFi R2 to increasing concentrations of cetuximab over the course of a week. The significant difference between the wild type DiFi and the R lines shows that there is a resistance. The figure does not distinguish the difference between R1 and R2, but it is later clarified that DiFi R1 was generated by exposure to a constant dose of cetuximab for a year, while R2 was exposed to stepwise increasing cetuximab dosage over a year. In addition, graph (a) shows that the cell viability of R1 and R2 are extremely similar and almost equally resistant. The inclusion of these two different drug treatment protocols reduces the possibility that the method of treatment could affect resistance. Parts (b) and (c) display the amplification of the KRAS gene in DiFi R. Part (b) compares the exome gene copy number between the control DiFi and resistant DiFi R, clearly showing the difference and transition from EGFR expression to KRAS expression. Part (c) provides the reader with a visual representation confirming the amplification of KRAS in DiFi R. However, the study consisted of two R lines, so which one is actually shown?

Western blot analysis of the different cells in parts (d) and (e) gives us a better look at the protein levels. The parental DiFi line clearly has a lot of EGFR present and very little KRAS. DiFi R1 and R2 are similar in that both have low levels of EGFR but high levels of KRAS. However, a slight difference between R1 and R2 after exposed to cetuximab is that R2 has more pMEK and pERK, possibly as a result of method of treatment. Despite these minor differences, the resistant lines clearly depict the decrease of EGFR but increase of KRAS from the control, continuing to support the hypothesis.  Part (e) was obtained by infecting DiFi cells with a KRAS lentivirus, which is used for gene manipulation because it specializes in infecting non-diving cells. Unfortunately, the function of the lentivirus was not expanded upon neither in the article nor the methods section, but I assumed that it silenced the KRAS gene according to outside sources. The similar presence of KRAS in “KRAS over” (which I assumed to mean “overexpressed”), R1, and R2 connects the resistant lines to having an overexpression of KRAS as well. The details of this Western blot analysis are lacking, which could undermine its significance as a part of the study.

Graph (f) looks again at cell viability in relation to cetuximab concentration but this time in DiFi cells overexpressing KRAS, wildtype cells, and empty vectors. The significant difference between the cell viability of DiFi “KRAS over” and DiFi wildtype and DiFi empty show a resistance in DiFi “KRAS over”. However, at higher concentrations of cetuximab, the cell viability of DiFi “KRAS over” is very close to DiFi wildtype and DiFi empty. In addition, the cell viability of DiFi wildtype in graph (f) is extremely different from graph (a), where graph (a) seemed to show that cetuximab was more effective, while graph (f) showed more resistance in the wildtype. The authors use graph (f) to connect overexpression of KRAS to cetuximab resistance, but with these two possible points, the conclusion may have been weakened.

From this data, the authors conclude that in DiFi cells, KRAS amplification mediates the acquired resistance to cetuximab. The data strongly supports the correlation and relationship between the two variables, but without a molecular mechanism, a causation claim would be too strong. This data is a great indication and starting place for future research on the subject for the mechanism.    

My next post will look into the data of the other model, Lim1215. 

References:

 

Sandra Misale, Rona Yaeger, Sebastijan Hobor, Elisa Scala, Manickam Janakiraman, David Liska, Emanuele Valtorta, Roberta Schiavo, Michela Buscarino, Giulia Siravegna, Katia Bencardino, Andrea Cercek, Chin-Tung Chen, Silvio Veronese, Carlo Zanon, Andrea Sartore-Bianchi, Marcello Gambacorta, Margherita Gallicchio, Efsevia Vakiani, Valentina Boscaro, Enzo Medico, Martin Weiser, Salvatore Siena, Federica Di Nicolantonio, David Solit, and Alberto Bardelli. “Emergence of KRAS mutations and acquired resistance to anti EGFR therapy in colorectal cancer.” Nature (2012) 486:7404. Web May 3, 2014.