KSR1- and KSR2-mediated Signaling Pathways Regulating Tumorigenesis and Metabolism
The laboratory's goal is to understand the signal transduction mechanisms that regulate energy expenditure and response to energy stress that are critical for the development of normal tissue and tumor cell survival. To that end, the lab's research focus resides primarily on the role of molecular scaffolds Kinase Suppressor of Ras 1 (KSR1) and KSR2 in these processes.
KSR1 was first identified in genetic screens of Drosophila and C. elegans (Kornfeld et al., 1995; Sundaram & Han, 1995; Therrien et al., 1995). These genetic screens identified mutated alleles that reversed the abnormal phenotype caused by activated Ras in both species. In Drosophila, loss-of-function ksr alleles reversed the rough-eye phenotype of activated Ras but not activated Raf. In C. elegans, mutated ksr alleles were found to reverse the abnormal multivulval phenotype caused by activated let-60 Ras, the ras homolog in C. elegans. On the other hand, mutant ksr alleles had no effect on the phenotype of activated Raf in either Drosophila or C. elegans. These genetic studies led to the preliminary suggestion that KSR interacted in the MAP kinase pathway above or parallel to Raf signaling in worms and flies. KSR1-/- mice are fertile with subtle developmental defects but are resistant to tumorigenesis by oncogenic ras. Studies with mammalian KSR1 defined the function of the protein as a molecular scaffold for the Raf/MEK/ERK MAP kinase cascade required for cell transformation by oncogenic ras (Kortum & Lewis, 2004; Nguyen et al., 2002; Roy et al., 2002; Therrien et al., 1996).
A related gene, KSR2, was identified in C. elegans, mouse, and humans (Channavajhala et al., 2003; Costanzo-Garvey et al., 2009; Ohmachi et al., 2002). KSR2 in C. elegans is required for Ras-mediated signaling during germline meiotic progression and functions redundantly with KSR1 in excretion, vulva development, and spicule formation. Mammalian KSR2 mediates calcium-induced Ras-to-ERK1/2 signaling and it is regulated by the phosphatase Calcineurin (Dougherty et al., 2009). In contrast to KSR1-/- mice, adult KSR2-/- mice are obese and insulin resistant. In vivo, ex vivo, and in vitro experiments reveal that KSR2 interacts with and affects the activation of the primary cellular energy sensor, AMP kinase. Thus, deletion of KSR2 appears to disrupt cellular mechanisms sensing energy stress (Costanzo-Garvey et al., 2009; Fernandez et al., 2012).
Lewis Lab Contributions (for details, see publications section)
The molecular scaffold KSR1 modulates ERK signaling to dictate cell fate
Our work reveals that the KSR proteins function to regulate tumorigenesis and metabolism in normal and tumor cells. KSR1 regulates the duration and intensity of ERK1/2 activation and it is required for H-RasV12-induced transformation, H-RasV12-induced senescence, and proper activation of the adipogenic differentiation program (Kortum et al., 2005; Kortum et al., 2006; Kortum & Lewis, 2004).
KSR1 phosphorylation regulates protein stability and function
Our earlier work identified ten phosphorylation sites on the N-terminus end of KSR1 (Volle et al., 1999). Further characterization of these phosphorylation sites revealed that mutation of Threonine-274 and Serine-392 redistributed KSR1 from the cytoplasm to the nucleus (Brennan et al., 2002), increased KSR1 protein stability, promoted ERK activity, cell proliferation and transformation (Razidlo et al., 2004).
KSR1 regulates cell cycle response to DNA damage
KSR1 mediates ERK activation induced by DNA damage. KSR1 is required for re-initiation of the cell cycle and escape from G2/M arrest following exposure to the DNA interstrand cross-linking agent mitomycin C (Razidlo et al., 2009).
KSR1 regulates metabolic capacity in cells transformed by oncogenic Ras
We have shown the KSR1-dependent, but ERK-independent, regulation of PGC-1α and ERRα to mediate H-RasV12-induced transformation and maximize the overall capacity of oxidative phosphorylation and aerobic glycolysis (Fisher et al., 2011).
KSR1 regulates glucose homeostasis
KSR1-/- mice do not exhibit any developmental defects, however, they have hypertrophic adipocytes (Kortum et al., 2005). In this study we determined KSR1-/- mice are modestly glucose intolerant and exhibit elevated serum insulin levels, indicating KSR1 plays a role in glucose homeostasis (Klutho et al., 2011).
KSR2 disruption causes obesity in mice
KSR2-/- mice are normal size when born, grow poorly as neonates. Those KSR2-/- mice that survive to weaning are more active than wild type littermates yet become obese and insulin resistant as adults. Despite obesity, and the co-incident elevation of circulating leptin, KSR2-/- mice respond to acute leptin with reduced food consumption. In the DBA1Lac/J strain, KSR2 deletion causes obesity despite the fact that these mice at slightly less (as measured per mouse or per gram body weight) than wild type littermates. This, effectively genetic, pair-feeding experiment demonstrates that KSR2-/- mice have reduced energy expenditure but are energy efficient. Underlying this phenotype is a defect in the activation of AMP kinase in white adipose tissue and a decreased core temperature, likely due to defective metabolism of lipids in intrascapular brown adipose tissue (Costanzo-Garvey et al., 2009).
KSR2 regulates cell transformation via AMP kinase
KSR2 mediates cell proliferation, transformation and metabolism. KSR2 overexpression promotes cell proliferation and cooperates with oncogenic H-Ras to promote cell transformation in KSR1-/- MEFs. KSR2 knockdown in tumor cells diminished the ability of cell to metabolize nutrients and maximize aerobic glycolysis and oxidative phosphorylation. The effects of KSR2 on cell transformation were mediated through AMPK activation and were not dependent on the function of KSR2 as a scaffold of Raf/MEK/ERK (Fernandez et al., 2012).
Using genomics, proteomics, in vitro, and in vivo models, we are identifying downstream effectors of KSR1 and KSR2 that are regulated dependently or independently of the Raf/MEK/ERK kinase cascade.
KSR1 is the focus of two research efforts. The first effort is directed at identifying mechanisms through which KSR1 promotes the expression of transcriptional regulators critical for the expansion of glycolytic and oxidative metabolism in human tumors bearing Ras oncogenes.
The complementary effort is a coordinated gene expression-based genome-wide RNAi and small molecule screen using KSR1 as a reference standard. In this research we are using the fact that KSR1 is an essential contributor to Ras-driven tumor development and tumor maintenance but is dispensable for normal development. Effectors of Ras-induced and KSR1-mediated signaling or "KSR1-like" gene products should be similarly selective for tumor cell survival in comparison to that of normal cells from the same histotype. Thus, our screen is intended to identify genes whose disruption will kill tumors cells but not normal cells, revealing potential targets with a high therapeutic index. The small molecule screen mimics the approach of the RNAi screen and is intended to identify molecules that kill tumor cells, but not normal cells.
Another major effort in the lab is to understand the physiological role of KSR2 in regulating energy expenditure. Current efforts are directed toward a biochemical, endocrine, and phenotypic dissection of KSR2-/‑ mice during postnatal development. Preliminary data support a working hypothesis that KSR2 is a critical component in metabolic programming early in postnatal development. One direction in this project is to identify the endocrine pathways regulated by KSR2 during postnatal growth. A complementary approach is to identify the critical tissue(s) expressing KSR2 essential to normal metabolic programming in neonates and subsequent metabolic homeostasis in adult mice. We have recently generated floxed alleles of KSR2 to facilitate our effort.
Channavajhala, P.L., Wu, L., Cuozzo, J.W., Hall, J.P., Liu, W., Lin, L.L. & Zhang, Y. (2003). J Biol Chem, 278, 47089-97.
Costanzo-Garvey, D.L., Pfluger, P.T., Dougherty, M.K., Stock, J.L., Boehm, M., Chaika, O., Fernandez, M.R., Fisher, K., Kortum, R.L., Hong, E.G., Jun, J.Y., Ko, H.J., Schreiner, A., Volle, D.J., Treece, T., Swift, A.L., Winer, M., Chen, D., Wu, M., Leon, L.R., Shaw, A.S., McNeish, J., Kim, J.K., Morrison, D.K., Tschop, M.H. & Lewis, R.E. (2009). Cell Metab, 10, 366-78.
Dougherty, M.K., Ritt, D.A., Zhou, M., Specht, S.I., Monson, D.M., Veenstra, T.D. & Morrison, D.K. (2009). Mol Cell, 34, 652-62.
Kornfeld, K., Hom, D.B. & Horvitz, H.R. (1995). Cell, 83, 903-13.
Nguyen, A., Burack, W.R., Stock, J.L., Kortum, R., Chaika, O.V., Afkarian, M., Muller, W.J., Murphy, K.M., Morrison, D.K., Lewis, R.E., McNeish, J. & Shaw, A.S. (2002). Mol Cell Biol, 22, 3035-45.
Ohmachi, M., Rocheleau, C.E., Church, D., Lambie, E., Schedl, T. & Sundaram, M.V. (2002). Curr Biol, 12, 427-33.
Roy, F., Laberge, G., Douziech, M., Ferland-McCollough, D. & Therrien, M. (2002). Genes Dev, 16, 427-38.
Sundaram, M. & Han, M. (1995). Cell, 83, 889-901.
Therrien, M., Chang, H.C., Solomon, N.M., Karim, F.D., Wassarman, D.A. & Rubin, G.M. (1995). Cell, 83, 879-88.
Therrien, M., Michaud, N.R., Rubin, G.M. & Morrison, D.K. (1996). Genes Dev, 10, 2684-95.