Friday, May 30, 2014

Tumor Suppressor Functions and Mutations of NOTCH1 in HNSCC

Figure 1. Duality of Notch functions in keratinocyte commitment to differentiation and cell survival. Notch signaling impacts on, and is regulated by a complex signaling network with an important role in skin homeostasis and tumor development. Like Notch, many other components of this network exert a duality of functions, impinging on keratinocyte growth/differentiation control as well as cell survival. This has potentially important implications for cancer therapy. An attractive approach could be the use of selective inhibitors, which can suppress the Notch prosurvival function while leaving intact or enhancing its ability to induce differentia- tion. Alternatively, Notch inhibitors, or inducers, could be used as part of a combination therapy in conjunction with other compounds with growth inhibitory and/or pro-apoptotic functions. 

In the review article, Notch tumor suppressor function, figure 1 is presented, showing a few of the pathways of how Notch can act as a tumor suppressor in keratinocyte tumors.3 Since the figure is somewhat complicated, the first thing to notice is both the upper and lower parts of the figure. The upper part diagrams Notch’s tumor suppressor role through inhibitions of stem cell renewal and cell differentiation signaling pathways and the lower part portrays Notch’s tumor suppressor role by either inhibition or activation of apoptotic pathways. A few of the most important pathways to notice are the Notch1, p63, p53, p21, and Wnts pathway along with the FoxO3a and NFkB/Akt pathway. In the Notch1, p63, p53, p21, and Wnts pathway, Wnts triggers the activation of products such as Cyclin D1, which is a main component that causes a cell to enter the cell cycle and go from G1phase into S phase. Therefore the whole cycle of Notch1, p63, p53, and p21 has a big role in controlling cell’s to enter the cell cycle. On the other hand, FoxO3a is a pro-apoptotic gene and NFkB/Akt are pro-interferon genes. Notch inhibits FoxO3a and activates NFkB, which essentially creates this balance between stopping cell death and promoting the immune system to kill those cells that are infected with viruses. This balance is important for regulating cell’s such that they don't enter a hyperplastic or neoplastic state while still maintaining appropriate levels of cell proliferation. Essentially, combined with information from previous blogs, mutations in Notch can cause it to transform into an oncogene or a tumor suppressor, depending on how and where the mutations take place.

Figure 2. NOTCH gene mutations identified in head and neck cancer. A. schematic diagram of the domain structure of NOTCH1. (domain structures of NOTCH2 and NOTCH3 are similar). All nonsense mutations occur upstream of the TAD domain, which is required for transactivation of target genes. Each arrowhead represents a single point mutation in an individual tumor, of the class indicated to the left. 

Figure 3. Schematic depiction of mutations in NOTCH1. (A) Previously observed NOTCH1 mutations in hematopoietic malignancies. EGF, epidermal growth factor; LNR, Lin12-Notch repeats; TMD, trans- membrane domain; RAM, recombination signal-binding protein 1 for J-k (RBPjk) association module; NLS, nuclear localization signal; PEST, proline, glutamic acid, serine/threonine-rich motifs. Red bars represent previously described mutation hotspots (amino acids 1575 to 1630 and 2250 to 2550). (B) Previously observed NOTCH1 mutations in solid tumors. Colored arrow (missense mutation) and “X” (truncating mutation) depict mutations found in different tumor types: pink, breast cancer; black, glioma; blue, lung cancer; green, pancreatic adenocarcinoma; red, esophageal squamous cell carcinoma; purple, tongue squamous cell carcinoma. (C) Mutations in NOTCH1 in HNSCC observed in this study. Black arrow indicates missense mutation and red “X” indicates truncating mutation (13, 22–24). 

Above are two figures that depict the mutations found in an array of HNSCC cases and previously discovered T-ALL cases. Each of these figures is created from their own individual study. The study for figure 2, taken from The Mutational Landscape of Head and Neck Squamous Cell Carcinoma, looked at 92 different HNSCC tumor/normal cell pairs, which about 12 had NOTCH1 mutations.4 Likewise, this is the same for figure 3 in diagram A of NOTCH1, taken from Exome Sequencing of Head and Neck Squamous Cell Carcinoma. This study included the exome sequencing of 32 different HNSCC tumors, 21 of with experienced NOTCH1 mutations.1 As seen in figure 2, T-ALL mutations happen mostly in the HD region (pink).  Similarly, the results from figure 3 revealed inactivating mutations in NOTCH1 within this same region. What is important to keep in mind is that this region and the TM region contain the cleavage sites that release the intra-cellular domain of NOTCH1 (ICN).2 Essentially, these mutations disrupt these sites in a way such that these cleavage sites are no longer protected and can constantly be cleaved or even to the point where the extracellular and cytoplasmic sides completely disassociate from each other. Either way, there is constantly and excess amount of ICN within T-ALL cells, which in turn triggers cells to turn cancerous. The mutations in the PEST region are also believed to be contributors to T-ALL but is unknown as to why because the gene activator region lies within the ANK repeats and TAD regions of this domain.
In HNSCC, however, the mutations are much more sporadic and located in regions that have much different physiological roles. In figure 2, the majority of the mutations in NOTCH1 occur in the extracellular domain within the EGF-like repeats. This region is the ligand-binding region. Therefore these mutations cause a loss of function of NOTCH1 mostly by the inability of ligand-binding. There are some cases too, with nonsense, splice, and deletion mutations where there is either a no presence of ICN or no cleavage site. Similarly in figure 3 under diagram C, the sporadic and high concentration of mutations in the EFG-like region suggests the same idea. However, in this study, more mutations were found, mainly in the Ankyrin repeats. The Ankyrin repeats domain is a domain that is essential for proper ICN function, since this is the region that acts as the gene activator. Therefore, these mutations also create a loss of function in NOTCH1, which gives it its roll in as a tumor suppressor in HNSCCs.
When looking at these mechanisms, one might conclude that creating therapies that could either activate NOTCH1 in HNSCC or deactivate NOTCH1 in T-ALL could be valid forms of therapy. However, due to the complexity and duality of the NOTCH1 signaling pathway, doing either of these has reciprocal problems that could be equal or even worse than the original carcinoma itself. For example, in T-ALL, a gamma secretase inhibitor was invented in order to stop the constant production of ICN. However, this reduction in ICN production led to the formation of skin cancer.3 Studies like these have not been done on HNSCC’s yet because no therapy has been made for NOTCH1 in HNSCC. However, the complex duality of NOTCH1 makes all reasonable therapies much less possible than they might sound.

Work Cited:

Agrawal, N., Y. Wu, V. E. Velculescu, N. Papadopoulos, D. A. Wheeler, K. W. Kinzler, D. M. Muzny, J. A. Drummond, L. Trevino, M. J. Frederick, C. R. Pickering, C. Bettegowda, L. D. Wood, 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, R. H. Hruban, J. N. Myers, W. H. Westra, W. M. Koch, J. A. Califano, R. A. Gibbs, D. Sidransky, and B. Vogelstein. "Exome Sequencing of Head and Neck Squamous Cell Carcinoma Reveals Inactivating Mutations in NOTCH1." Science 333.6046 (2011): 1154-1157. Science. Web. 10 Apr. 2014.

Aster, Jon C, Stephen C Blacklow, and Warren S Pear. "Notch signalling in T-cell lymphoblastic leukaemia/lymphoma and other haematological malignancies." The Journal of Pathology 223.2 (2011): 263-274. NCBI. Web. 15 May 2014.

Dotto, G P. "Notch tumor suppressor function." Oncogene 27.38 (2008): 5115-5123. Oncogene. Web. 10 May 2014.

Stransky, N., M. Parkin, W. Winckler, K. Ardlie, S. B. Gabriel, M. Meyerson, E. S. Lander, G. Getz, T. R. Golub, L. A. Garraway, R. C. Onofrio, C. Guiducci, M. Romkes, G. Saksena, A. M. Egloff, A. D. Tward, A. D. Kostic, K. Cibulskis, A. Sivachenko, G. V. Kryukov, M. S. Lawrence, C. Sougnez, A. Mckenna, J. L. Weissfeld, E. Shefler, A. H. Ramos, P. Stojanov, S. L. Carter, D. Voet, M. L. Cortes, D. Auclair, M. F. Berger, J. R. Grandis, R. R. Seethala, L. Wang, C. Rangel-Escareno, J. C. Fernandez-Lopez, A. Hidalgo-Miranda, and J. Melendez-Zajgla. "The Mutational Landscape of Head and Neck Squamous Cell Carcinoma." Science 333.6046 (2011): 1157-1160. NCBI. Web. 12 May 2014.