Thursday, May 29, 2014

Notch1 Helps or Hinders Tumor Formation in Conjunction with HPV?

In HPV positive cervix cancer, one cannot develop cancer solely from having HPV; additional mutations, such as NOTCH1 mutations, are necessary to instigate tumorigenesis. Different studies have yielded contradictory results of whether Notch1 acts as either a tumor suppressor protein or an oncoprotein in HPV positive cervical cancer. In this article1, the authors observed that when Notch1 is expressed excessively, it acts as a tumor suppressor in HPV positive cervical cancer; however, when it is expressed in moderate amounts in conjugation with constitutively active PI3K, it presented oncogenic properties.

Figure 1. Effects of overexpression of Notch1-IC on cell proliferation
 in HeLa, C4-1, and C-33 cell lines evaluated by thymidine 
incorporation assay analysis. Cells were either transfected with
 a vector containing a gene coding for Notch1-IC attached to
 a CMV promoter (displayed as CMV Notch1 IC) or were 
devoid of a vector (shown as control). Assays were run in triplicate.
Experiment 2 is a replication of experiment 1.
In order to observe how overexpression of Notch1 influences cell proliferation, the authors transfected HeLa and C4-I cell lines, which both are derived from HPV 18 infected cervical cancer lesions, and a C-33 cell line, which is made up of HPV-negative keratinocytes, with a vector containing a gene coding for Notch1-IC (active form of Notch1). The vectors contain a cytomegalovirus early promoter, which promotes the overexpression of Notch1-IC. In order to evaluate cell proliferation, thymidine uptake, which increases when cells proliferate, was evaluated by thymidine incorporation assaysThe thymidine uptake of cell lines that were transfected with Notch1-IC DNA were compared to the same cell lines without Notch1-IC DNA transfection (control). It appears that the C4-1 cells yield the most variable and unreliable data out of the 3 cell lines, which is apparent because of the difference in the data between experiment one and two, which have the same variables, and the big error bars on the control data (figure 1). The data suggests that Notch1-IC expression reduced cell proliferation in all the cells sampled, and this reduction was more significant in cells with HPV DNA, which indicates that Notch1’s ability to prevent cell proliferation is more prominent in cervical cancer cells that are infected with HPV than healthy keratinocytes (figure 1). These results support that Notch1 can act as a tumor repressor protein in HPV positive cervical cancer when expressed excessively.

Figure 2. Anchorage-independent growth in soft agar of primary human 
foreskin keratinocytes that infected with HPV 16 in various conditions
 (without Notch1-IC or abnormal PI3k, moderate presence of 
Notch1-IC and/or presence of constitutively activated PI3K) after
 2 weeks of incubation. Labels correspond to DNA integrated into the
 keratinocytes genomes by retroviral transduction. FSKE6/7 represents 
HPV 16 DNA (HPV infection). Notch1 IC DNA represents 
Notch1-IC DNA (Notch1-IC moderate presence). 
P110αCAAX DNA represents p110α DNA (constitutively active PI3k). 
In order to see if moderate levels of Notch1 expression contribute to tumorigenesis, the authors virally transduced Notch1-IC DNA, which allows Notch1-IC to be expressed moderately. They also observed how overactivated PI3K influenced tumorigenesis by virally transducing a gene into that codes for a constitutively active p110α domain of PI3K into cells. Primary human foreskin keratinocytes (FSK) infected with HPV 16 were exposed to retroviruses that contained the Notch1-IC gene, p110α gene, both of these genes, or none of these genes (control). The cells were incubated onto soft agar and the authors observed anchorage-independent growthwhich indicates growth of transformed tumor cells, and the results are shown in figure 2. I can infer from these pictures that overactivated PI3K in HPV positive cervix cells induces the creation of aggressive, fast growing tumors, while the moderate expression of Notch1 increases the likelihood that cells undergo abnormal growth, however, this growth is slower than overactivated PI3K induced growth (figure 2). These pictures reflect that HPV infection, moderate expression of Notch1, and overactivated PI3K alone do not significantly contribute to tumorigenesis,but the synergy of these factors express robust oncogenic characteristics.

This is a fairly robust study, however, the authors derived many of their conclusions from the soft agar analysis in figure 2, which slightly obscures their conclusions because evaluating tumor growth by observing colony growth is very subjective. One cannot derive quantitative data, which hinders their ability to support their claims with statistical evidence, from these pictures except for colony count, which the authors did not include. Robust conclusions are made from the soft agar assay, however, because healthy cells simply cannot grow efficiently independent of a physical attachment to a substrate. I believe the thymidine uptake assay analysis in figure 1 obtained fairly robust data because the assays were run in triplicate and the non-control assays were run in triplicate twice. There seemed to be a significant difference between the control and experiment data, however, the authors did not provide the statistical evidence (p values) to support this difference. Also, the conclusions are further obscured by solely using constitutively active Notch1 (Notch1-IC). Overall, the data did support their conclusions, however, due to their omission of critical data and use of Notch1-IC, I am skeptical. Because other research suggests contradictory functions of Notch1’s influence on tumorigenisis in HPV positive cervix cancer, these authors offer a plausible explanation for this contradiction— Notch1 could act as a tumor suppressor protein or an oncoprotein depending on its amount of expression—but more research, especially in vivo, must be conducted to support these claims.

1. Lathion, Stéphanie, Janina Schaper, and Peter Beard. "Notch1 Can Contribute to Viral-Induced Transformation of Primary Human Keratinocytes." Cancer Research 63: 8687-694. Web. 27 May 2014.
2. Min Jie Alvin Tan, Elizabeth A. White, Mathew E. Sowa, J. Wade Harper, Jon C. Aster, and Peter M. Howley. "Cutaneous β-human papillomavirus E6 Proteins Bind Mastermind-like Coactivators and Repress Notch Signaling" PNAS 109.23:1473–E1480. Web. 27 May 2014.