Regulation of γ-Secretase/Notch Signaling in Cancer Cells

Notch signaling has emerged as an appealing pathway for cancer therapy due to its central role in controlling cell differentiation, growth, and survival in a broad spectrum of tumors. A recent report showing that more than 50 percent of all human T cell acute lymphoblastic leukemias have activating Notch-1 mutations (25) further highlights the critical role of Notch in carcinogenesis. Moreover, Notch signaling has been linked with other malignancies and cancer stem cells (26). γ-Secretase inhibitors (GSIs) that are widely used to probe Notch signaling in cancer cells have advanced to clinical studies. However, little is known about the regulation and function of γ-secretase in cancer cells. Recently, we have demonstrated that γ-secretase complexes vary in different types of tumor cells (21). Clearly, the intricacies of γ-secretase in tumor cells remain to be investigated. It is noteworthy to point out that although Notch is a key substrate of γ-secretase, it also cleaves other substrates that could play a role in tumor development. Therefore, an outcome due to down- and up regulation of γ-secretase can not always be attributed to Notch signaling. Therefore, characterization of the γ-secretase complex and elucidation of its regulatory mechanism will provide critical insight into the function of γ-secretase in cancer cells and offer a comprehensive understanding of this target for effective therapies.

γ-Secretase/Notch signaling in lymphomas

Notch signaling is an essential regulator of hematopoiesis and lymphopoiesis and is necessary for T cell commitment and development (27). Interestingly, it is Notch-1 signaling that controls T cell development (28), whereas Notch-2 regulates splenic marginal zone B cell function (29). Despite an abundance of evidence for the oncogenic potential of Notch, its role in various B cell malignancies remains in debate. Our preliminary studies have indicated that GSIs suppress the proliferation of lymphoma cells and that the γ-secretase complex in a B cell tumor line appears to be different than γ-secretase in other cell types, which likely control their activity and specificity, as well as sensitivity to GSIs. We focus on characterization of the γ-secretase complex in B cell lymphoma lines and examination of their specificity for APP and Notch-1 cleavage as well as sensitivity to different classes of GSIs. We elucidate the role of Notch in B cell lymphomas using dominant negative mastermind-like 1 (DNMAML1) that interferes with multiple Notch family members (30) and biological reagents, such as neutralizing Notch antibodies. We plan to evaluate the ability of GSI-34 to reduce tumor burden in an in vivo model of Burkitt’s lymphoma (31).

γ-Secretase/Notch signaling in breast cancer

Increasing evidence suggests that aberrant Notch signaling plays an important role in breast cancer development. The first indication that Notch signaling contributes to mammary tumorigenesis came from studies in the mouse mammary tumor virus (MMTV) (32) (33), in which both loci of Notch-1 and Notch-4 were found as frequent insertion sites of MMTV, resulting in a truncated and constitutively active Notch protein. Moreover, these active forms of Notch have been shown to contribute to tumorigenesis in animal models (33) (34). Elevated levels of Notch-1 and its ligand Jagged-1 have been associated with poor survival in breast cancer patients (35) (36). Numb, a negative regulator of the Notch pathway is frequently silenced in 50 percent of human breast tumors (37). Active Notch signaling has been found to play a critical role in breast cancer stem cells or cancer-initiating cells (38). Lee et al. (39) (40) found that Notch-1 and survivin co-segregated as a functional gene signature in basal breast cancer. In addition, increased Notch signaling has been suggested as a survival mechanism for drug resistance (41). Collectively, Notch signaling plays an important role in breast cancer development, as well as in drug resistance. We have found that hypoxia significantly induces γ-secretase activity in breast cancer cells and HIF1α is involved in modulating γ-secretase activity. Importantly, inhibition of γ-secretase considerably reduces migration and invasion of 4T1 and MDA-MB231 cells under hypoxia. γ-Secretase inhibitor treatment reduces the 4T1 primary tumor load and blocks 4T1 metastasis to the lung. We are delineating the connection of the HIF-1α pathway and γ-secretase using pharmacological and genetic approaches and elucidating the molecular basis of γ-secretase regulation under hypoxia. We are also examining the role of Notch signaling in breast tumor migration and invasion and the effect of γ-secretase inhibition on tumor development in cellular and animal models. Characterization of γ-secretase and understanding its regulatory mechanism under hypoxia in breast cancer cells is crucial to developing effective target-based therapies.

Development of Notch-signal modulators

Recently, we have developed small molecules that selectively inhibit γ-secretase for cleavage of Notch-1 over APP and their action of mechanism is under investigation. In addition, we have performed cell panning using Notch-1, -2 or -3 overexpressing cells to screen Notch binders from two phage peptide libraries (the CX10C library contains 1.9 x 109 cysteine-constrained random sequences that are 10-amino acids long and the NNS20 library includes 2.2 x 109 linear random sequences of 20-mer linear peptides). We have also developed a high content cell-based assay that can screen Notch-1 (N1), Notch-2 (N2) and Notch-3 (N3) modulators. We plan to screen chemical libraries to discover molecules that modulate either an individual pathway or multiple pathways. Identification of small molecules or peptides that are capable of selectively targeting a single Notch pathway will offer significant progress in the development of molecular probes and drug candidates.

  1. Selkoe, D. J., and Wolfe, M. S. (2007) Cell 131, 215-221

  2. Brown, M. S., Ye, J., Rawson, R. B., and Goldstein, J. L. (2000) Cell 100, 391-398

  3. Erez, E., Fass, D., and Bibi, E. (2009) Nature 459, 371-378

  4. Li, Y. M., Lai, M. T., Xu, M., Huang, Q., DiMuzio-Mower, J., Sardana, M. K., Shi, X. P., Yin, K. C., Shafer, J. A., and Gardell, S. J. (2000) Proc Natl Acad Sci U S A 97, 6138-6143

  5. Francis, R., McGrath, G., Zhang, J., Ruddy, D. A., Sym, M., Apfeld, J., Nicoll, M., Maxwell, M., Hai, B., Ellis, M. C., Parks, A. L., Xu, W., Li, J., Gurney, M., Myers, R. L., Himes, C. S., Hiebsch, R., Ruble, C., Nye, J. S., and Curtis, D. (2002) Dev Cell 3, 85-97

  6. Goutte, C., Tsunozaki, M., Hale, V. A., and Priess, J. R. (2002) Proc Natl Acad Sci U S A 99, 775-779

  7. Li, Y. M., Xu, M., Lai, M. T., Huang, Q., Castro, J. L., DiMuzio-Mower, J., Harrison, T., Lellis, C., Nadin, A., Neduvelil, J. G., Register, R. B., Sardana, M. K., Shearman, M. S., Smith, A. L., Shi, X. P., Yin, K. C., Shafer, J. A., and Gardell, S. J. (2000) Nature 405, 689-694

  8. Ahn, K., Shelton, C. C., Tian, Y., Zhang, X., Gilchrist, M. L., Sisodia, S. S., and Li, Y.-M. (2010) Proc Natl Acad Sci U S A 107, 21435-21440

  9. Takasugi, N., Tomita, T., Hayashi, I., Tsuruoka, M., Niimura, M., Takahashi, Y., Thinakaran, G., and Iwatsubo, T. (2003) Nature 422, 438-441

  10. He, G., Luo, W., Li, P., Remmers, C., Netzer, W. J., Hendrick, J., Bettayeb, K., Flajolet, M., Gorelick, F., Wennogle, L. P., and Greengard, P. (2010) Nature 467, 95-98

  11. Kopan, R., and Ilagan, M. X. (2004) Nat Rev Mol Cell Biol 5, 499-504

  12. Sherrington, R., Rogaev, E. I., Liang, Y., Rogaeva, E. A., Levesque, G., Ikeda, M., Chi, H., Lin, C., Li, G., Holman, K., and et al. (1995) Nature 375, 754-760

  13. Lessard, C. B., Wagner, S. L., and Koo, E. H. (2010) Proceedings of the National Academy of Sciences 107, 21236-21237

  14. Zheng, H., and Koo, E. H. (2006) Mol Neurodegener 1, 5

  15. Placanica, L., Tarassishin, L., Yang, G., Peethumnongsin, E., Kim, S. H., Zheng, H., Sisodia, S. S., and Li, Y. M. (2009) J Biol Chem 284, 2967-2977

  16. Shelton, C. C., Tian, Y., Frattini, M. G., and Li, Y. M. (2009) Mol Neurodegener 4, 22

  17. Yang, G., Yin, Y. I., Chun, J., Shelton, C. C., Ouerfelli, O., and Li, Y. M. (2009) Bioorg Med Chem Lett 19, 922-925

  18. Tian, Y., Bassit, B., Chau, D., and Li, Y. M. (2010) Nat Struct Mol Biol 17, 151-158

  19. Shelton, C. C., Zhu, L., Chau, D., Yang, L., Wang, R., Djaballah, H., Zheng, H., and Li, Y. M. (2009) Proc Natl Acad Sci U S A 106, 20228-20233

  20. Artavanis-Tsakonas, S., Matsuno, K., and Fortini, M. E. (1995) Science 268, 225-232

  21. Placanica, L., Chien, J. W., and Li, Y. M. (2010) Biochemistry 49, 2796-2804

  22. Placanica, L., Zhu, L., and Li, Y. M. (2009) PLoS ONE 4, e5088

  23. Tian, Y., Crump, C. J., and Li, Y. M. (2010) J Biol Chem 285, 32549-32556

  24. Pufall, M. A., and Graves, B. J. (2002) Annu Rev Cell Dev Biol 18, 421-462

  25. Weng, A. P., Ferrando, A. A., Lee, W., Morris, J. P. t., Silverman, L. B., Sanchez-Irizarry, C., Blacklow, S. C., Look, A. T., and Aster, J. C. (2004) Science 306, 269-271

  26. Yin, L., Velazquez, O. C., and Liu, Z. J. (2010) Biochem Pharmacol

  27. Maillard, I., Fang, T., and Pear, W. S. (2005) Annual Review of Immunology 23, 945-974

  28. Radtke, F., Wilson, A., Mancini, S. J., and MacDonald, H. R. (2004) Nat Immunol 5, 247-253

  29. Saito, T., Chiba, S., Ichikawa, M., Kunisato, A., Asai, T., Shimizu, K., Yamaguchi, T., Yamamoto, G., Seo, S., Kumano, K., Nakagami-Yamaguchi, E., Hamada, Y., Aizawa, S., and Hirai, H. (2003) Immunity 18, 675-685

  30. Maillard, I., Weng, A. P., Carpenter, A. C., Rodriguez, C. G., Sai, H., Xu, L., Allman, D., Aster, J. C., and Pear, W. S. (2004) Blood 104, 1696-1702

  31. Ghetie, M. A., Richardson, J., Tucker, T., Jones, D., Uhr, J. W., and Vitetta, E. S. (1990) Int J Cancer 45, 481-485

  32. Gallahan, D., and Callahan, R. (1987) J Virol 61, 66-74

  33. Dievart, A., Beaulieu, N., and Jolicoeur, P. (1999) Oncogene 18, 5973-5981

  34. Jhappan, C., Gallahan, D., Stahle, C., Chu, E., Smith, G. H., Merlino, G., and Callahan, R. (1992) Genes Dev 6, 345-355

  35. Reedijk, M., Odorcic, S., Chang, L., Zhang, H., Miller, N., McCready, D. R., Lockwood, G., and Egan, S. E. (2005) Cancer Res 65, 8530-8537

  36. Dickson, B. C., Mulligan, A. M., Zhang, H., Lockwood, G., O’Malley, F. P., Egan, S. E., and Reedijk, M. (2007) Mod Pathol 20, 685-693

  37. Pece, S., Serresi, M., Santolini, E., Capra, M., Hulleman, E., Galimberti, V., Zurrida, S., Maisonneuve, P., Viale, G., and Di Fiore, P. P. (2004) J Cell Biol 167, 215-221

  38. Dontu, G., Jackson, K. W., McNicholas, E., Kawamura, M. J., Abdallah, W. M., and Wicha, M. S. (2004) Breast Cancer Res 6, R605-615

  39. Lee, C. W., Simin, K., Liu, Q., Plescia, J., Guha, M., Khan, A., Hsieh, C. C., and Altieri, D. C. (2008) Breast Cancer Res 10, R97

  40. Lee, C. W., Raskett, C. M., Prudovsky, I., and Altieri, D. C. (2008) Cancer Res 68, 5273-5281

  41. Osipo, C., Patel, P., Rizzo, P., Clementz, A. G., Hao, L., Golde, T. E., and Miele, L. (2008) Oncogene.