Increased K-Ras Protein and Activity in Mouse and Human Lung Epithelial Cells at Confluence

1. Increased K-Ras Protein and Activity in Mouse and Human Lung Epithelial Cells at Confluence Cell Growth and Differentiation Vol. 13, 441-448, September 2002 Authors: Wafa Dammouni, Gayatri Ramakrishna, Gunamani Sithanandan, George T. Smith, Laura W. Fornwalk, Akira Masuda, Takashi Takahashi, and Lucy M. Anderson Presented by: Linda Bean and Jayna Bretzman

2. Lung Cancer Symptoms • Cough that is persistent or becomes intense • Changes in color, volume, or blood in spit • Wheezing • Fatigue • Loss of Appetite

3. Lung Cancer Statistics • 173,770 Americans will be diagnosed…160,440 will die • Total cost spent on treating Lung Cancer in the United States is $5 billion per year

4. Healthy Lung/Cancerous Lung

5. Classic Ras Paradigm • Studies based largely on H-ras p21 in fibroblasts suggest a mechanism of where mutant Ras proteins are responsible for cancer • This mechanism involved oncogenic mutations in the Ras protein which prevents down regulation of Ras by GAP, therefore Ras remains active and signals for proliferation and survival through Raf/MAPK(Erk) and P13K/PKB

6. Researcher’s Question • Recently, in both rat and mouse, K-ras p21 showed increasing expression in the development of fetal lung followed by a large increase as growth slowed and the cells began to differentiate, and finally reaching maximal levels in adult lung

7. Question cont. • Previous study finding is not consistent with proliferation of lung type II cells but instead K-ras p21 seemed more associated with growth arrest and differentiation • So…What is the signaling function of K-ras p21 in lung epithelial cells????

8. Results from this study • In this study of peripheral lung epithelial cells in culture, the researchers measured total K-ras p21 and the amount of activated GTP Ras. • Their results confirmed K-ras p21 expression and activity are highest in growth-arrested, confluent cells.

9. Possible Pathway /Sos1 K-ras p21 K-ras p21 Raf Mek Erk Akt

10. Figure 1 80% E9 cells subconfluent malignant

11. Figure 1: Ras activation assay in subconfluent E9 and E10 cell lines • Ras activation assay determined by Western immunoblot • Based on the theory that only activated GTP-bound ras p21 binds to the RBD (Ras binding domain) of raf-1 (MAPKKK) • Part 1A: used 80% confluent E9 cells (malignant mouse lung cells), measured total K-Ras p21 and Activated K-Ras GTP, an average of 62% is recovered as K-Ras GTP

12. Figure 1 cont. • Fig 1B: amounts of total K-ras p21 were 50% less in the nontransformed E10 cells compared with the malignant E9 cells • In malignant E9 cells, the K-ras p21 protein traveled faster than the K-ras protein in the E10 cells • Fig 1C: amounts of active K-ras p21 (GTP bound) was 30 fold greater in the E9 cells

13. Figure 2 nontransformed malignant K-Ras Active K-Ras p21

14. Figure 2: Total and K-ras p21-GTP in E10 and E9 cells during culture growth and confluence • Fig 2A: the amounts of total K-ras p21 increased progressively, by about 5 fold, through 50% confluence through two days post confluence • E9 cells total K-ras increased 2 fold • Fig 2B: measured active K-ras in E10 cells increased 20 fold where as the amount of active K-ras increased 7 fold in the E9 cells

15. Figure 3 E10 cell line Increase Reference point for normalization Increase Increase

16. Figure 3: Quantification of increases in total and active K-ras p21 at post confluent growth arrest • Fig 3A: In the E10 cell line at 100% confluence there was a 206-fold increase of active K-Ras when compared to the active K-Ras at 50% confluence (Erk1 and Erk2 were used as reference in amount of protein Fig. 3B) • There is a little variation in experiments due to the subjective call of when the cells are at 50% confluence

17. Figure 3 cont. • Fig 3C: At 100% confluence there was an increase on average by a factor of 196 in active K-ras when compared to the 50% confluence; total K-ras increased by a factor of 4.1 in post-confluent cells (100%); • Fig 3C: In 50% confluent cells 0.03% of total K-ras p21 was in the GTP bound state; In 100% confluent cells 1.4% of total K-ras was in the active state • In summary, total K-Ras p21 and active K-ras p21 both increased in post-confluence growth arrest

18. Figure 4

19. Figure 4: Testing other cell lines including human • Used C10 mouse and HPL1D human cells • Fig 4A/B: In mouse cells there was an increase of 3.8 fold for total K-Ras p21 and a 9.3 fold increase for K-Ras GTP between 50 and 100% confluence • Fig 4D: In human HPL1D cells, total K-ras p21 increased 3.7 fold and K-ras GTP 8.7 fold in 100% versus 50% confluent cells • In summary, an increase in both total K-Ras and active K-Ras with growth arrest at confluency was a general characteristic of mouse and human peripheral epithelial cells

20. Figure 5 60% subconfluent E10

21. Figure 5: Serum Activation of the ERK 1/2 and Akt in 50% confluent E10 cells • The cells were serum starved for 48 hours then replaced with the complete medium which restarted the cell cycle • Fig 5A/B: Neither total K-Ras p21 nor K-Ras GTP increased after serum simulation at this time • Fig 5D: shows an increase in the activated Erk1/2 starting at 10 mins but this increase was not due to the activation of K-Ras p21

22. Figure 6

23. Figure 6: Activation by serum of Erk 1/2 and AKt in E10, C10, and HPL1D cells • Fig 6 A/B/C/D/F: after serum stimulation, E10, C10, and HPL1D cells had a rapid marked increase in all phosphorylated (activated) Erk1, Erk2, and Akt (implicated as a downstream target of p21 in some systems) • HPL1D cells appeared to have constitutive Akt activation even under serum-starved condition • These results were the same for both subconfluent and confluent growth • In summary, Erk1/2 and Akt were quite dissimiliar to K-ras p21 with regard to expression and activation patterns

24. Figure 7

25. Figure 7: Increase of Grb2/Sos1 complex at confluence • Since K-ras p21 activation is associated with the upstream Grb2/Sos1 complex, these levels were assessed at 50% and 100% confluence in E10, C10, and HPL1D cell lines • Fig 7A/C: Large increase in the Grb2/Sos1 complex at 100% confluence can be correlated to the activated K-ras at 100% • Fig 7B: The C10 cells were more variable and fell short of statistical significance

26. Discussion • This study confirmed K-ras p21 expression and activation in lung peripheral epithelial cells are higher in growth-arrested cells than in those that are rapidly proliferating (growing) • K-ras activation at confluence was paralleled by an increase in Grb2-Sos1 complex (Fig. 7)

27. Discussion • Serum did not activate K-ras 21 (Fig. 5) however after serum stimulation, Erks and Akt were both activated but there was no increase in the amount of phosphorylated Erks and Akt at confluence (Fig. 6)

28. Discussion • These results lead to many more questions??? 1. Is the K-ras p21 responsible for growth arrest or is it a result of the beginning of cell differentiation? 2. What pathway carries the signal to the nucleus since activated Erks and Akt do not appear to be involved?

29. Discussion • The pathway(s) possibly used by the increased K-ras p21 activity to bring about growth arrest and/or differentiation are unknown at present • Lack of any correlation with activation of the P13K/Akt and raf/Erk1/2 pathways make these unlikely downstream targets

30. Discussion • In summary, the control of the increase in total and active K-ras p21 during growth arrest in lung type II cells, and the consequences of this increase in the maintenance of growth arrest and differentiation, are topics for further research • This further research can reveal critical targets for intervention in lung cancer initiation, development, and malignant progression

31. Discussion • If activated K-ras p21 is related to growth arrest and differentiation, how do mutations in it, leading to continuous activation, contribute to tumorigenesis? • One possibility, is that mutant oncogenic K-ras p21 interacts abnormally with downstream pathways not utilized by normal K-ras p21 such as jun and jun kinase. • Another is that the mutation prevents interaction with a downstream effector that leads to growth arrest/differentiation, e.g. GAP.