Sunday, April 20, 2014

An Introduction to NOTCH1's Dual Carcinogous Properties

            In Aaron Madden and I’s cancer project, we explore the mutated NOTCH1 gene and how it can act either as a tumor suppressor gene or an oncogene, which we will ultimately use to observe how genes like these with seemingly contradictory functions alter how cancer should be perceived. The gene was initially noted as acting as an oncogene, however, recent research has been suggesting that it can also function as a tumor suppressor gene.  In our project, we will focus on the NOTCH1 gene’s effects in T-Cell acute lymphoblastic leukemia and head and neck squamous cell carcinoma.  NOTCH1's involvement in cancer was first observed in T-Cell acute lymphoblastic leukemia as an oncogene, but recent research has suggested that it can also acting as a tumor suppressor gene in some types of cancer. In this blog post, I will evaluate a study that suggests that the mutated NOTCH1 has tumor suppressor properties in head and neck squamous cell carcinoma and will briefly explain the potential of NOTCH1 to act both as a tumor suppressor gene and as an oncogene.

      In general, NOTCH1 codes for a transmembrane receptor protein called Notch1 that plays a prominent role in cell-fate determination, which allows it to influence cell proliferation, apoptosis, and cell-differentiation. When a Notch1 receptor attaches to a ligand attached to a neighboring cell, the protein is left exposed to two proteolytic cleavages from y-secretase, which release the cytoplasmic end of the protein, Notch-IC, into the cytoplasm. Notch-IC then travels to the nucleus to act as a transcription factor that promotes numerous genes that code for proteins that effect the cell cycle. Notch1 receptors are expressed significantly during tissue development due to its ability to regulate the differentiation fate of cells. Because of its role in cell proliferation, cell death, and cell growth suppression, the mutated gene protein product can act as a oncoprotein or a tumor suppressor. Whether the Notch1 protein acts like an oncoprotein or a tumor suppressor is contingent to “the cellular context and the crosstalk with other signal-transduction pathways”(Radtke and Raj 757). In other words, the Notch1 receptor's actions are dependent on the cellular environment and the type
 cell it is. Because of this, it is possible that it can act as an oncogene in some cells and as a tumor suppressing gene in other cells.

                                                                                               (Tables from reference 1)

 In the article,
Exome Sequencing of 
Head and Neck Squamous Cell Carcinoma Reveals Inactivating Mutations in NOTCH1, the authors used whole-exome sequencing to sequence 18,000 protein-encoding genes in 32 patients who had head and neck squamous cell carcinoma. DNA of the tumors and non cancerous cells on each patient were purified and sequenced, which was used to compare the genome of the tumor to the genome of a normal cell. DNA was sequenced by massive parallel sequencing initially, which 
found 911 total somatic mutations in the tumors. In order to verify the accuracy of the sequencing, the DNA was evaluated again by Sanger sequencing, which confirmed 609 mutations. If a mutated gene was observed in more than 2 different patients, it would be selected for further analysis. In this analysis, the genomes of tumor cells and normal cells in 88 more patients were sequenced. This analysis found that 15% of patients displayed loss of function mutations in NOTCH1.
 In total, there were 28 NOTCH1 mutations in 21 out of 120 patients (32 patients from the first analysis and 88 from the second analysis). 34% of the mutations reduced the size of notch1 through nonsense and deletion mutations (Table 1). The rest of the patients had missense mutations of NOTCH1 (Table 2). In order to complement the data found through sequencing, the authors utilized Affymetrix SNP6.0 microarrays to observe the copy number of the genes most mutated in head and neck squamous cell carcinoma tumors. The authors analyzed the number of NOTCH1 gene copies of 3 tumors with NOTCH1 mutations, which displayed that 2 of the 3 genomes suffered from a loss of one copy of the gene. Because the data suggests that NOTCH1 either suffers from a loss of function mutation or a complete deletion of the gene when it is mutated in head and neck squamous cell carcinoma, the authors concluded that it acts as a tumor suppressor gene in this type of cancer. The results also showed that NOTCH1 mutations are the second most frequent mutation to appear in this type of cancer. 

             I am satisfied with the methods that the authors used for determining the presence of mutations in head and neck squamous cell carcinoma tumor genomes, however, I am skeptical of the validity of the results of this study because of the sample sizes used throughout their experiment. The authors did a an excellent job of confirming which genes have a significant role in head and neck squamous cell carcinoma by using both massive parallel sequencing and Sanger sequencing techniques, however, they lacked an adequate sample size (32 patients at the time) to make these assumptions. Also, when the authors sequenced 88 more patients and emphasized observing the genes that were mutated most frequently in the tumors, the results were still obscured by the lack of patients (120 patients in total at the time), but by using both massive parallel sequencing and Sanger sequencing techniques, the results had more credibility. In short, I believe that using both massive parallel sequencing and Sanger sequencing to verify the amount of mutations gives robust results, but the inadequate sample sizes hinders the conclusions that can be drawn from the data. Also, when the authors were testing for the amount of NOTCH1 genes in the genome tumors, they solely used 3 patients, which makes the results of this portion of the experiment lack adequate support to draw conclusions from. I can infer that the small sample sizes for this experiment was contingent to the time and money that are necessary to be invested into whole-exome sequencing and gene number copy analysis. As time progresses, hopefully such techniques will become more inexpensive and available to more research facilities and hospitals. Overall, I cannot fully entrust this study, but its results definitely suggest that NOTCH1 acts like a tumor suppressor gene in head and neck squamous cell carcinoma.

     Due to its versatility, NOTCH1 mutations can create conditions that are conducive to sustaining cell proliferation, resist programed death, and evading tumor suppressors. The gene’s role in cancer was initially recognized as solely an oncogene, but new studies, such as the one I highlighted earlier, are beginning to find tumors where a loss of function of the gene is observed. Besides its dual function in cancer, there are only a small amount of tumors that have NOTCH1 mutations, which make its identification, “difficult to distinguish from passenger mutations” (1156)1. The properties of NOTCH1 illustrate that there are potentially a plethora of genes hidden in our genome that could have both oncogene and tumor suppressor gene attributes. NOTCH1 and similar genes are important to study because they display why cancer treatment should be subjective to the type of cancer it is targeting. If one dismissed NOTCH1 as an oncogene, treatment that targets against it would allow the cancer to thrive more efficiently in cancers where it acts as a tumor suppressor gene. As displayed by the study I elaborated on, research on cancer derived from genomic sequencing is robust, but due the amount of time and money necessary to conduct this research makes it difficult to use abundantly. Once genomic sequencing and analysis become less expensive, researchers will be able to find more genes like NOTCH1 and ultimately develop better treatments to counteract these anomalous genes. Overall, the reason why we find the NOTCH1 gene and its inconsistent influence on cancer to be intriguing is because it truly reflects how complex cancer is and how difficult it truly is to treat appropriately.

 1. Agrawal, N., M. J. Frederick, C. R. Pickering, C. Bettegowda, K. Chang, R. J. Li, C. Fakhry, T.-X. Xie, J. Zhang, J. Wang, N. Zhang, A. K. El-Naggar, S. A. Jasser, J. N. Weinstein, L. Trevino, J. A. Drummond, D. M. Muzny, Y. Wu, L. D. Wood, R. H. Hruban, W. H. Westra, W. M. Koch, J. A. Califano, R. A. Gibbs, D. Sidransky, B. Vogelstein, V. E. Velculescu, N. Papadopoulos, D. A. Wheeler, K. W. Kinzler, and J. N. Myers. "Exome Sequencing of Head and Neck Squamous Cell Carcinoma Reveals Inactivating Mutations in NOTCH1." Science333.6046 (2011): 1154-157. Web.
 2. Radtke, Freddy, and Kenneth Raj. "The Role of Notch in Tumorigenesis: Oncogene or Tumour Suppressor?" Nature Reviews Cancer 3.10 (2003): 756-67. Web. 20 Apr. 2014.

3. Brakenhoff, R. H. "Another NOTCH for Cancer." Science 333.6046 (2011): 1102-103. Web. 20 Apr. 2014.