Figure 1. Low molecular weight cyclin E promotes tumorigenesis. In normal cells, cyclin E/CDK2 is tightly regulated and triggers the onset of S phase. In tumors, cyclin E undergoes proteolytic processing generating low molecular weight species that exhibit increased kinase activity and resistance to inhibition by cyclin kinase inhibitors p21/p27. The expression of LMW-E promotes aberrant acinar morphogenesis, centrosome amplification, and tumors associated with activation of the bRaf/ERK/mTOR pathway. LMW-E, low molecular weight cyclin E. doi:10.1371/journal.pgen.1002623.g001 length cyclin E in tumorigenesis cannot be excluded. In addition, Spruck et al. reported that the level of LMW-E corre- lates with full-length cyclin E, suggesting that LMW-E reflects the total cyclin E protein in primary breast tumors, cell lines, and even normal breast tissue [21]. To examine the effect of LMW-E in the absence of over-expression, and in the absence of full-length cyclin E, it will be important to use a knock-in model in which expression of LMW-E is driven from the endogenous cyclin E promoter. 2) What is the relationship between LMW-E and the b-Raf-ERK1/2-mTOR signaling pathway? The authors demonstrated that the b-Raf-ERK1/2-mTOR signaling pathway is activated in tumors expressing high levels of LMW-E. The b-Raf-ERK1/ References 1. Hwang HC, Clurman BE (2005) Cyclin E in normal and neoplastic cell cycles. Oncogene 24: 2776–2786. 2. Keyomarsi K, Tucker SL, Buchholz TA, Callister M, Ding Y, et al. (2002) Cyclin E and survival in patients with breast cancer. N Engl J Med 347: 1566–1575. 3. Porter PL, Malone KE, Heagerty PJ, Alexander GM, Gatti LA, et al. (1997) Expression of cell-cycle regulators p27Kip1 and cyclin E, alone and in combination, correlate with survival in young breast cancer patients. Nat Med 3: 222–225. 4. Loeb KR, Kostner H, Firpo E, Norwood T, Tsuchiya KD, et al. (2005) A mouse model for cyclin E-dependent genetic instability and tumor- igenesis. Cancer Cell 8: 35–47. 5. Minella AC, Swanger J, Bryant E, Welcker M, Hwang H, et al. (2002) p53 and p21 form an 2-mTOR pathway may be a downstream signaling pathway deregulated by LMW- E, or it could be a parallel survival pathway selected in LMW-E–expressing tumors. In particular, the fact that only combinational therapy targeting both cy- clin E–associated kinase activity and the b- Raf-ERK1/2-mTOR pathway generates an anti-tumor effect argues against a direct cause–effect relationship and is suggestive of a parallel pathway. 3) Is LMW-E expression required for tumor growth and does down-regulation of LMW-E alter tumor growth, invasion, or metastasis? The authors have generated an inducible model that should facilitate these studies. 4) Is the tumor-promoting activity of LMW-E due to enhanced deregulated kinase activity or to alternative target inducible barrier that protects cells against cyclin E-cdk2 deregulation. Curr Biol 12: 1817–1827. 6. Harwell RM, Porter DC, Danes C, Keyomarsi K (2000) Processing of cyclin E differs between normal and tumor breast cells. Cancer Res 60: 481–489. 7. Porter DC, Zhang N, Danes C, McGahren MJ, Harwell RM, et al. (2001) Tumor-specific pro- teolytic processing of cyclin E generates hyperac- tive lower-molecular-weight forms. Mol Cell Biol 21: 6254–6269. 8. Harwell RM, Mull BB, Porter DC, Keyomarsi K (2004) Activation of cyclin-dependent kinase 2 by full length and low molecular weight forms of cyclin E in breast cancer cells. J Biol Chem 279: 12695–12705. 9. Wingate H, Puskas A, Duong M, Bui T, Richardson D, et al. (2009) Low molecular weight specificity? The LMW-E construct used in these studies has an N-terminal deletion (40 amino acids) that eliminates the proposed nuclear localization signal (NLS) and potentially affects the intracel- lular localization and substrate specificity [22,23]. 5) How is LMW-E generated in tumors, and is it tumor-type specific? It has been proposed and demonstrated by Caruso et al. that many tumors have elevated protease activity and decreased levels of protease inhibitors such as elafin [24] that may contribute to the generation of LMW-E. Further characterization of the proteolytic pathways that target cyclin E in tumors may provide alternative therapeutic targets. cyclin E is specific in breast cancer and is associated with mechanisms of tumor progression. Cell Cycle 8: 1062–1068. 10. Akli S, Zheng PJ, Multani AS, Wingate HF, Pathak S, et al. (2004) Tumor-specific low molecular weight forms of cyclin E induce genomic instability and resistance to p21, p27, and antiestrogens in breast cancer. Cancer Res 64: 3198–3208. 11. Wingate H, Zhang N, McGarhen MJ, Bedrosian I, Harper JW, et al. (2005) The tumor-specific hyperactive forms of cyclin E are resistant to inhibition by p21 and p27. J Biol Chem 280: 15148–15157. 12. Akli S, Van Pelt CS, Bui T, Multani AS, Chang S, et al. (2007) Overexpression of the low molecular weight cyclin E in transgenic mice induces metastatic mammary carcinomas through the PLoS Genetics | www.plosgenetics.org 2 March 2012 | Volume 8 | Issue 3 | e1002623