Functional analysis of S6K1 regulation of apoptosis control Lindsay M. Webb, Catherine A. Gallo, David R. Plas Department of Cancer Cell Biology, University of Cincinnati Abstract Increased glucose-dependence characterizes the altered metabolism of tumor cells. S6K1 is a protein kinase that promotes glycolysis and glucose-dependent survival in leukemia cells. In cells expressing activated S6K1 or the vector control, we tested cell survival by treatment with an S6K1 inhibitor. S6K1 was required for survival, but activated S6K1 was not sufficient for survival. The data support the potential efficacy of S6K1 inhibition in cancer chemotherapy. Introduction PTEN deficiency activates protein kinase, S6K1, which activates glycolysis and glucose-dependent survival. ΔnΔc S6K1 is a constitutively active S6K1 protein kinase. Therefore, cells containing the ΔnΔc S6K1 construct should maintain a higher viability under apoptotic conditions. AD80, an S6K1 inhibitor, should promote apoptosis. Results Figure 1: Cloning of ΔnΔc S6K1 into MIT vector. A. B Map of ΔnΔc S6K1 construct C. Digest verification Figure 2: Transfection and transduction of ΔnΔc S6K1 into three FL5.12 cell lines A B C Figure 4: Role of S6K1 in survival control A. B. Figure 4: S6K1 is necessary, but not sufficient for survival. A. Structure of AD80, an S6K1 inhibitor. B. Cells were starved of IL3, a growth factor, to test survival control. PTEN deficient cells survive better than control or FL5.12s in vehicle treated cells. ΔnΔc S6K1 did not improve survival in any cell type. The S6K1 inhibitor, AD80, prevented the survival of PTEN deficient cells. Conclusion Activated S6K1 is not sufficient for survival. S6K1 inhibition shows that S6K1 is required for survival. Insufficient: Required: Acknowledgements University Honors Program SURF Program Plas Research Group, University of Cincinnati Shokat Research Group, University of California, San Francisco Presentation: Introducing the topic I am interested in looking at apoptosis control in cancer. PTEN is a tumor-suppressor in a pathway that controls cell proliferation and cell survival. PTEN deficiency results in increased cell survival Previous data in my lab has shown that a protein kinase S6K1, downstream of PTEN, is also very important in cell survival. AD80, an S6K1 inhibitor, should promote apoptosis, testing whether S6K1 is necessary for survival or not. I am also testing to see if delta N delta C S6K1, an activated form of S6K1, is sufficient to increase survival. Results First, I successfully cloned dNdC S6K1 into the retroviral vector, MIT. In Figure 1A I have a model of S6K1. The dNdC construct contains amino acids 55 to 423. In B, there is a map of dNdC S6K1 construct in MIT. This construct contains a myc tag. Also, the MIT retroviral vector contains an IRES and Thy1-1. In C, I verified that the construct was correctly inserted into the vector through restriction enzyme digest. Using NcoI, I got the expected 6kb and 1.2kb fragments. In Figure 2, I show that I transfected and transduced cells using dNdC and the vector control. A shows the three different cell lines that I transduced. FL5.12 cells are the parental cells, PTEN- cells are PTEN deficient and Vec cells are the vector control of PTEN deficient cells. I picked to work with these cells because FL5.12 cells themselves are not cancer cells; they need a growth factor called Il3 to survive. By knocking out PTEN in FL5s, you are able to study the specific effect of PTEN deficiency, rather than in a cancer genome where there are many mutations. B shows my transfection/transduction model. dNdC S6K1 plasmid and the vector control were transfected into 293T cells, which then produced virus that I was able to collect This virus was used to transduce the three different cell lines with dNdC and vector control. C shows my sort data on the Aria. In order to see which cells were transduced, I stained for Thy1-1, which is present in MIT. When cells were first transduced, they were about 20-30% Thy1-1 positive. However, after they were sorted, cells were greater than 95% Thy1-1 positive. I realize that I am missing a negative control that would show non transduced cells. I have the data, but I just did not include in on this poster. In Figure 3, I have a validation of dNdC S6K1 expression. In A, The myc tag indicates expression of the dNdC construction in all cell types. The PTEN blot shows that PTEN- cells are PTEN deficient. In B, I tested the substrate of S6K1. S6 is a small subunit ribosomal protein that is a substrate of S6K1. Phosphorylation of S6 occurs the most in PTEN deficient cells but there is little difference in dNdC cells. (This is a disappointment but I have evidence that dNdC conveys some rapamycin resistance.) In Figure 4, I show the role of S6K1 in survival control. In A, There is the structure of the S6K1 inhibitor , AD80. This structure has a purine ring structure, similar to adenine. Therefore, AD80 would be a competitive inhibitor of ATP. In B, I did an experiment to test survival control in which cells were starved of the growth factor Il3 and treated with vehicle control or AD80. As expected, PTEN deficient cells survive better than other cell types in vehicle treatments. Also, cell viability is low in AD80 treated cells. dNdC did not improve the survival rates, as there appears to be no difference between the vector control and dNdC cells treated with vehicle or AD80. Conclusions dNdC S6K1, or activated S6K1, was not sufficient for cell survival. However, AD80 decreased cell survival, showing the S6K1 is required for survival. Questions to be prepared for: What are the next steps in this project? As I have mentioned before, the dNdC construct has been shown to convey some rapamycin resistance. The next step would be to do an experiment in which I would starve cells of Il3 and treat them with rapamycin and AD80 to compare the effects of both drugs. I would also blot for pT389 of S6K1 and pS6 to show the differences. Fds;alskjdf sort 97% 27% Figure 2: Immortalized, hematopoietic FL5.12 cell lines were transduced and sorted. A. Parental, Control, PTEN deficient cell lines. B. Model of retroviral production and target cell transduction. C. Transduced cells were stained for Thy1-1 (a transduction marker) then sorted to >95% Thy1-1+. Figure 3: ΔnΔc S6K1 Validation A Testing Expression B Testing Substrates Vec PTEN- FL5.12 Vec PTEN- FL5.12 ΔnΔc - + - + - + Myc tag PTEN ΔnΔc - + - + - + S6 pS6 NcoI uncut ladder Figure 3: ΔnΔc S6K1 construct was validated by Western blot. A. Myc tag indicates the expression of the ΔnΔc construct in Vector, PTEN-, and FL5.12. B. S6 is increasingly phosphorylated in PTEN-deficient cells. There is little significant difference of ΔnΔc S6K1 on phosphorylation of S6 in all three cell types. Figure 1: Cloned ΔnΔc S6K1 into retroviral vector, MIT. A. Model of S6K1. ΔnΔc S6K1 has previously been shown to be an activated kinase. B. Map of ΔnΔc S6K1 MIT. B. Verification digest by NcoI produced 6.0 kb and 1.2 kb fragments.