Increased K-Ras Protein and Activity in Mouse and Human Lung Epithelial Cells at Confluence Cell Growth and Differentiation Vol. 13, 441-448, September.

Increased K-Ras Protein Increased K-Ras Protein and Activity in Mouse and and Activity in Mouse and

Human Lung Epithelial Human Lung Epithelial Cells at ConfluenceCells at Confluence

Cell Growth and Differentiation Vol. 13, 441-448, Cell Growth and Differentiation Vol. 13, 441-448, September 2002September 2002

Authors: Wafa Dammouni, Gayatri Ramakrishna, Authors: Wafa Dammouni, Gayatri Ramakrishna, Gunamani Sithanandan, George T. Smith, Laura W. Gunamani Sithanandan, George T. Smith, Laura W.

Fornwalk, Akira Masuda, Takashi Takahashi, and Lucy Fornwalk, Akira Masuda, Takashi Takahashi, and Lucy M. AndersonM. Anderson

Presented by: Linda Bean and Jayna BretzmanPresented by: Linda Bean and Jayna Bretzman

Lung Cancer Lung Cancer SymptomsSymptoms Cough that is persistent or Cough that is persistent or

becomes intensebecomes intense Changes in color, volume, or Changes in color, volume, or

blood in spitblood in spit WheezingWheezing FatigueFatigue Loss of AppetiteLoss of Appetite

Lung Cancer StatisticsLung Cancer Statistics

173,770 173,770 Americans will be Americans will be diagnosed…diagnosed…160,440 will die160,440 will die

Total cost spent Total cost spent on treating Lung on treating Lung Cancer in the Cancer in the United States is United States is $5 billion per year$5 billion per year

Healthy Healthy Lung/Cancerous LungLung/Cancerous Lung

Classic Ras ParadigmClassic Ras Paradigm

Studies based largely on H-ras p21 in Studies based largely on H-ras p21 in fibroblasts suggest a mechanism of fibroblasts suggest a mechanism of where mutant Ras proteins are where mutant Ras proteins are responsible for cancer responsible for cancer

This mechanism involved oncogenic This mechanism involved oncogenic mutations in the Ras protein which mutations in the Ras protein which prevents down regulation of Ras by prevents down regulation of Ras by GAP, therefore Ras remains active and GAP, therefore Ras remains active and signals for proliferation and survival signals for proliferation and survival through Raf/MAPK(Erk) and P13K/PKB through Raf/MAPK(Erk) and P13K/PKB

Researcher’s QuestionResearcher’s Question

Recently, in both rat and mouse,Recently, in both rat and mouse,

K-ras p21 showed increasing K-ras p21 showed increasing expression in the development of expression in the development of fetal lung followed by a large fetal lung followed by a large increase as growth slowed and increase as growth slowed and the cells began to differentiate, the cells began to differentiate, and finally reaching maximal and finally reaching maximal levels in adult lung levels in adult lung

Question cont.Question cont.

Previous study finding is not Previous study finding is not consistent with proliferation of consistent with proliferation of lung type II cells but instead K-ras lung type II cells but instead K-ras p21 seemed more associated with p21 seemed more associated with growth arrest and differentiationgrowth arrest and differentiation

So…What is the signaling function So…What is the signaling function of K-ras p21 in lung epithelial of K-ras p21 in lung epithelial cells????cells????

Results from this studyResults from this study

In this study of peripheral lung In this study of peripheral lung epithelial cells in culture, the epithelial cells in culture, the researchers measured total K-ras researchers measured total K-ras p21 and the amount of activated p21 and the amount of activated GTP Ras.GTP Ras.

Their results confirmed K-ras p21 Their results confirmed K-ras p21 expression and activity are highest expression and activity are highest in growth-arrested, confluent cells.in growth-arrested, confluent cells.

Possible PathwayPossible Pathway

/Sos1

K-ras p21Raf

Mek

Erk

Akt

K-ras p21

Figure 1Figure 1

malignant

80% E9 cells

subconfluent

Figure 1: Ras activation Figure 1: Ras activation assay in subconfluent E9 assay in subconfluent E9 and E10 cell linesand E10 cell lines Ras activation assay determined by Ras activation assay determined by

Western immunoblotWestern immunoblot Based on the theory that only activated Based on the theory that only activated

GTP-bound ras p21 binds to the RBD GTP-bound ras p21 binds to the RBD (Ras binding domain) of raf-1 (MAPKKK)(Ras binding domain) of raf-1 (MAPKKK)

Part 1A: used 80% confluent E9 cells Part 1A: used 80% confluent E9 cells (malignant mouse lung cells), (malignant mouse lung cells), measured total K-Ras p21 and measured total K-Ras p21 and Activated K-Ras GTP, an average of Activated K-Ras GTP, an average of 62% is recovered as K-Ras GTP62% is recovered as K-Ras GTP

Figure 1 cont.Figure 1 cont.

Fig 1B: amounts of total K-ras p21 were Fig 1B: amounts of total K-ras p21 were 50% less in the nontransformed E10 cells 50% less in the nontransformed E10 cells compared with the malignant E9 cellscompared with the malignant E9 cells

In malignant E9 cells, the K-ras p21 In malignant E9 cells, the K-ras p21 protein traveled faster than the K-ras protein traveled faster than the K-ras protein in the E10 cellsprotein in the E10 cells

Fig 1C: amounts of active K-ras p21 (GTP Fig 1C: amounts of active K-ras p21 (GTP bound) was 30 fold greater in the E9 cellsbound) was 30 fold greater in the E9 cells

Figure 2Figure 2

malignant

nontransformed

K-Ras

Active

K-Ras p21

Figure 2: Figure 2: Total and K-ras Total and K-ras p21-GTP in E10 and E9 cells p21-GTP in E10 and E9 cells during culture growth and during culture growth and confluenceconfluence Fig 2A: the amounts of total K-ras p21 Fig 2A: the amounts of total K-ras p21

increased progressively, by about 5 increased progressively, by about 5 fold, through 50% confluence through fold, through 50% confluence through two days post confluencetwo days post confluence

E9 cells total K-ras increased 2 foldE9 cells total K-ras increased 2 fold Fig 2B: measured active K-ras in E10 Fig 2B: measured active K-ras in E10

cells increased 20 fold where as the cells increased 20 fold where as the amount of active K-ras increased 7 fold amount of active K-ras increased 7 fold in the E9 cellsin the E9 cells

Figure 3Figure 3

E10 cell line

Reference point for

normalization

Increase

Increase

Increase

Figure 3: Quantification of Figure 3: Quantification of increases in total and active K-increases in total and active K-ras p21 at post confluent ras p21 at post confluent growth arrestgrowth arrest Fig 3A: In the E10 cell line at 100% Fig 3A: In the E10 cell line at 100%

confluence there was a 206-fold confluence there was a 206-fold increase of active K-Ras when compared increase of active K-Ras when compared to the active K-Ras at 50% confluence to the active K-Ras at 50% confluence (Erk1 and Erk2 were used as reference (Erk1 and Erk2 were used as reference in amount of protein Fig. 3B)in amount of protein Fig. 3B)

There is a little variation in experiments There is a little variation in experiments due to the subjective call of when the due to the subjective call of when the cells are at 50% confluencecells are at 50% confluence

Figure 3 cont.Figure 3 cont.

Fig 3C: At 100% confluence there was an Fig 3C: At 100% confluence there was an increase on average by a factor of 196 in increase on average by a factor of 196 in active K-ras when compared to the 50% active K-ras when compared to the 50% confluence; total K-ras increased by a factor confluence; total K-ras increased by a factor of 4.1 in post-confluent cells (100%); of 4.1 in post-confluent cells (100%);

Fig 3C: In 50% confluent cells 0.03% of total Fig 3C: In 50% confluent cells 0.03% of total K-ras p21 was in the GTP bound state; In K-ras p21 was in the GTP bound state; In 100% confluent cells 1.4% of total K-ras was 100% confluent cells 1.4% of total K-ras was in the active statein the active state

In summary, total K-Ras p21 and active K-ras In summary, total K-Ras p21 and active K-ras p21 both increased in post-confluence p21 both increased in post-confluence growth arrestgrowth arrest

Figure 4Figure 4

Figure 4: Testing Figure 4: Testing other cell lines other cell lines including humanincluding human Used C10 mouse and HPL1D human cellsUsed C10 mouse and HPL1D human cells Fig 4A/B: In mouse cells there was an Fig 4A/B: In mouse cells there was an

increase of 3.8 fold for total K-Ras p21 and a increase of 3.8 fold for total K-Ras p21 and a 9.3 fold increase for K-Ras GTP between 50 9.3 fold increase for K-Ras GTP between 50 and 100% confluenceand 100% confluence

Fig 4D: In human HPL1D cells, total K-ras Fig 4D: In human HPL1D cells, total K-ras p21 increased 3.7 fold and K-ras GTP 8.7 fold p21 increased 3.7 fold and K-ras GTP 8.7 fold in 100% versus 50% confluent cellsin 100% versus 50% confluent cells

In summary, an increase in both total K-Ras In summary, an increase in both total K-Ras and active K-Ras with growth arrest at and active K-Ras with growth arrest at confluency was a general characteristic of confluency was a general characteristic of mouse and human peripheral epithelial cellsmouse and human peripheral epithelial cells

Figure 5Figure 5

60% subconfluent E10

Figure 5: Serum Figure 5: Serum Activation of the ERK 1/2 Activation of the ERK 1/2 and Akt in 50% confluent and Akt in 50% confluent E10 cellsE10 cells The cells were serum starved for 48 hours The cells were serum starved for 48 hours

then replaced with the complete medium then replaced with the complete medium which restarted the cell cyclewhich restarted the cell cycle

Fig 5A/B: Neither total K-Ras p21 nor K-Fig 5A/B: Neither total K-Ras p21 nor K-Ras GTP increased after serum simulation Ras GTP increased after serum simulation at this timeat this time

Fig 5D: shows an increase in the Fig 5D: shows an increase in the activated Erk1/2 starting at 10 mins but activated Erk1/2 starting at 10 mins but this increase was not due to the this increase was not due to the activation of K-Ras p21activation of K-Ras p21

Figure 6Figure 6

Figure 6: Activation by Figure 6: Activation by serum of Erk 1/2 and AKt serum of Erk 1/2 and AKt in E10, C10, and HPL1D in E10, C10, and HPL1D cellscells Fig 6 A/B/C/D/F: after serum stimulation, E10, Fig 6 A/B/C/D/F: after serum stimulation, E10,

C10, and HPL1D cells had a rapid marked C10, and HPL1D cells had a rapid marked increase in all phosphorylated (activated) increase in all phosphorylated (activated) Erk1, Erk2, and Akt (implicated as a Erk1, Erk2, and Akt (implicated as a downstream target of p21 in some systems)downstream target of p21 in some systems)

HPL1D cells appeared to have constitutive Akt HPL1D cells appeared to have constitutive Akt activation even under serum-starved conditionactivation even under serum-starved condition

These results were the same for both These results were the same for both subconfluent and confluent growthsubconfluent and confluent growth

In summary, Erk1/2 and Akt were quite In summary, Erk1/2 and Akt were quite dissimiliar to K-ras p21 with regard to dissimiliar to K-ras p21 with regard to expression and activation patternsexpression and activation patterns

Figure 7Figure 7

Figure 7: Increase of Figure 7: Increase of Grb2/Sos1 complex at Grb2/Sos1 complex at confluenceconfluence Since K-ras p21 activation is associated with Since K-ras p21 activation is associated with

the upstream Grb2/Sos1 complex, these the upstream Grb2/Sos1 complex, these levels were assessed at 50% and 100% levels were assessed at 50% and 100% confluence in E10, C10, and HPL1D cell linesconfluence in E10, C10, and HPL1D cell lines

Fig 7A/C: Large increase in the Grb2/Sos1 Fig 7A/C: Large increase in the Grb2/Sos1 complex at 100% confluence can be complex at 100% confluence can be correlated to the activated K-ras at 100%correlated to the activated K-ras at 100%

Fig 7B: The C10 cells were more variable Fig 7B: The C10 cells were more variable and fell short of statistical significance and fell short of statistical significance

DiscussionDiscussion

This study confirmed K-ras p21 This study confirmed K-ras p21 expression and activation in lung expression and activation in lung peripheral epithelial cells are higher peripheral epithelial cells are higher in growth-arrested cells than in those in growth-arrested cells than in those that are rapidly proliferating that are rapidly proliferating (growing) (growing)

K-ras activation at confluence was K-ras activation at confluence was paralleled by an increase in Grb2-paralleled by an increase in Grb2-Sos1 complex (Fig. 7)Sos1 complex (Fig. 7)

DiscussionDiscussion

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

DiscussionDiscussion

These results lead to many more These results lead to many more questions??? questions???

1. Is the K-ras p21 1. Is the K-ras p21 responsible for growth arrest responsible for growth arrest

or is it a result of the or is it a result of the beginning of beginning of cell differentiation?cell differentiation?

2. What pathway carries the signal 2. What pathway carries the signal to to the nucleus since activated Erks the nucleus since activated Erks and Akt and Akt do not appear to be involved?do not appear to be involved?

DiscussionDiscussion

The pathway(s) possibly used by the The pathway(s) possibly used by the increased K-ras p21 activity to bring increased K-ras p21 activity to bring about growth arrest and/or about growth arrest and/or differentiation are unknown at differentiation are unknown at presentpresent

Lack of any correlation with Lack of any correlation with activation of the P13K/Akt and activation of the P13K/Akt and raf/Erk1/2 pathways make these raf/Erk1/2 pathways make these unlikely downstream targetsunlikely downstream targets

DiscussionDiscussion

In summary, the control of the In summary, the control of the increase in total and active K-ras p21 increase in total and active K-ras p21 during growth arrest in lung type II during growth arrest in lung type II cells, and the consequences of this cells, and the consequences of this increase in the maintenance of growth increase in the maintenance of growth arrest and differentiation, are topics arrest and differentiation, are topics for further researchfor further research

This further research can reveal critical This further research can reveal critical targets for intervention in lung cancer targets for intervention in lung cancer initiation, development, and malignant initiation, development, and malignant progressionprogression

DiscussionDiscussion

If activated K-ras p21 is related to growth If activated K-ras p21 is related to growth arrest and differentiation, how do mutations arrest and differentiation, how do mutations in it, leading to continuous activation, in it, leading to continuous activation, contribute to tumorigenesis?contribute to tumorigenesis?

One possibility, is that mutant oncogenic K-One possibility, is that mutant oncogenic K-ras p21 interacts abnormally with ras p21 interacts abnormally with downstream pathways not utilized by normal downstream pathways not utilized by normal K-ras p21 such as jun and jun kinase.K-ras p21 such as jun and jun kinase.

Another is that the mutation prevents Another is that the mutation prevents interaction with a downstream effector that interaction with a downstream effector that leads to growth arrest/differentiation, e.g. leads to growth arrest/differentiation, e.g. GAP.GAP.