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Significance of KRAS/PAK1/Crk pathway in non-small cell lung cancer oncogenesis.

Mortazavi F, Lu J, Phan R, Lewis M, Trinidad K, Aljilani A, Pezeshkpour G, Tamanoi F - BMC Cancer (2015)

Bottom Line: Furthermore, KRAS mutant tumors expressed higher p-PAK1(Thr423) compared to KRAS wild type.KRAS prenylation inhibition by (FTI + GGTI) completely dephosphorylated proto-oncogene c-Crk on Serine 41 while Crk phosphorylation did not change by individual prenylation inhibitors or diluent.Combination of PAK1 inhibition and partial inhibition of all other KRAS effectors by (FTI + GGTI) dramatically altered morphology, motility and proliferation of H157 and A549 cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology, West Los Angeles VA, Los Angeles, CA, USA. fredmortazavi@ucla.edu.

ABSTRACT

Background: Key effector(s) of mutated KRAS in lung cancer progression and metastasis are unknown. Here we investigated the role of PAK1/Crk axis in transduction of the oncogenic KRAS signal in non-small cell lung cancer (NSCLC).

Methods: We used NSCLC clinical specimens to examine the correlation among KRAS mutations (codon 12, 13 and 61); PAK1/Crk axis activation [p-PAK1(Thr423), p-Crk(Ser41)]; and adhesion molecules expression by immunohistochemistry. For assessing the role of proto-oncogene c-Crk as a KRAS effector, we inhibited KRAS in NSCLC cells by a combination of farnesyltransferase inhibitor (FTI) and geranylgeranyltransferase inhibitor (GGTI) and measured p-Crk-II(Ser41) by western blotting. Finally, we disrupted the signaling network downstream of KRAS by blocking KRAS/PAK1/Crk axis with PAK1 inhibitors (i.e., IPA-3, FRAX597 or FRAX1036) along with partial inhibition of all other KRAS effectors by prenylation inhibitors (FTI + GGTI) and examined the motility, morphology and proliferation of the NSCLC cells.

Results: Immunohistochemical analysis demonstrated an inverse correlation between PAK1/Crk phosphorylation and E-cadherin/p120-catenin expression. Furthermore, KRAS mutant tumors expressed higher p-PAK1(Thr423) compared to KRAS wild type. KRAS prenylation inhibition by (FTI + GGTI) completely dephosphorylated proto-oncogene c-Crk on Serine 41 while Crk phosphorylation did not change by individual prenylation inhibitors or diluent. Combination of PAK1 inhibition and partial inhibition of all other KRAS effectors by (FTI + GGTI) dramatically altered morphology, motility and proliferation of H157 and A549 cells.

Conclusions: Our data provide evidence that proto-oncogene c-Crk is operative downstream of KRAS in NSCLC. Previously we demonstrated that Crk receives oncogenic signals from PAK1. These data in conjunction with the work of others that have specified the role of PAK1 in transduction of KRAS signal bring forward the importance of KRAS/PAK1/Crk axis as a prominent pathway in the oncogenesis of KRAS mutant lung cancer.

No MeSH data available.


Related in: MedlinePlus

PAK1 activation and Crk phosphorylation are positively correlated while establish a negative correlation with E-cadherin in NSCLC. Dot plots demonstrating the correlation between A-E-cadherin and p-PAK1(Thr423); B-E-cadherin and p-Crk-II(Ser41); C-p-PAK1(Thr423) and p-Crk-II(Ser41) expression by immunohistochemistry in 17 NSCLC clinical specimens. The average intensity of protein expression across the slide is quantified in a scale from 0-3+. The correlation between the expressions of each two marker was examined by utilizing Spearman Rank Correlation statistical test.
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Fig2: PAK1 activation and Crk phosphorylation are positively correlated while establish a negative correlation with E-cadherin in NSCLC. Dot plots demonstrating the correlation between A-E-cadherin and p-PAK1(Thr423); B-E-cadherin and p-Crk-II(Ser41); C-p-PAK1(Thr423) and p-Crk-II(Ser41) expression by immunohistochemistry in 17 NSCLC clinical specimens. The average intensity of protein expression across the slide is quantified in a scale from 0-3+. The correlation between the expressions of each two marker was examined by utilizing Spearman Rank Correlation statistical test.

Mentions: We observed a concordance between expression of p-PAK1(Thr423) and p-Crk-II(Ser41) (Figure 1). Interestingly, tumors expressing phosphorylated PAK1/Crk had very low level of p120-catenin and E-cadherin (Figure 1). Of note, we did not observe an association between total PAK1 and Crk-II expression with that of p120-catenin/E-cadherin (data not shown). In order to examine the statistical correlation between the intensity of PAK1/Crk phosphorylation with E-cadherin expression, we quantified the phosphorylation and expression of these proteins in a scale of 1-3 as described in the Methods section. Subsequently, the correlation between the intensity of p-PAK1(Thr423), p-Crk-II(Ser41) and E-cadherin was examined in a Spearman Rank Correlation analysis (Figure 2). E-cadherin expression in the examined samples showed a statistically significant negative correlation with the expression of p-PAK1(Thr423) and p-Crk-II(Ser41) (p < 0.0072 and p < 0.047 respectively) while p-PAK1(Thr423) and p-Crk-II(Ser41) established a positive correlation with each other (p < 0.0097). These findings highly suggest that PAK1 activation by upstream stimuli results in Crk mediated suppression of p120-catenin and E-cadherin in lung cancer.Figure 1


Significance of KRAS/PAK1/Crk pathway in non-small cell lung cancer oncogenesis.

Mortazavi F, Lu J, Phan R, Lewis M, Trinidad K, Aljilani A, Pezeshkpour G, Tamanoi F - BMC Cancer (2015)

PAK1 activation and Crk phosphorylation are positively correlated while establish a negative correlation with E-cadherin in NSCLC. Dot plots demonstrating the correlation between A-E-cadherin and p-PAK1(Thr423); B-E-cadherin and p-Crk-II(Ser41); C-p-PAK1(Thr423) and p-Crk-II(Ser41) expression by immunohistochemistry in 17 NSCLC clinical specimens. The average intensity of protein expression across the slide is quantified in a scale from 0-3+. The correlation between the expressions of each two marker was examined by utilizing Spearman Rank Correlation statistical test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4477307&req=5

Fig2: PAK1 activation and Crk phosphorylation are positively correlated while establish a negative correlation with E-cadherin in NSCLC. Dot plots demonstrating the correlation between A-E-cadherin and p-PAK1(Thr423); B-E-cadherin and p-Crk-II(Ser41); C-p-PAK1(Thr423) and p-Crk-II(Ser41) expression by immunohistochemistry in 17 NSCLC clinical specimens. The average intensity of protein expression across the slide is quantified in a scale from 0-3+. The correlation between the expressions of each two marker was examined by utilizing Spearman Rank Correlation statistical test.
Mentions: We observed a concordance between expression of p-PAK1(Thr423) and p-Crk-II(Ser41) (Figure 1). Interestingly, tumors expressing phosphorylated PAK1/Crk had very low level of p120-catenin and E-cadherin (Figure 1). Of note, we did not observe an association between total PAK1 and Crk-II expression with that of p120-catenin/E-cadherin (data not shown). In order to examine the statistical correlation between the intensity of PAK1/Crk phosphorylation with E-cadherin expression, we quantified the phosphorylation and expression of these proteins in a scale of 1-3 as described in the Methods section. Subsequently, the correlation between the intensity of p-PAK1(Thr423), p-Crk-II(Ser41) and E-cadherin was examined in a Spearman Rank Correlation analysis (Figure 2). E-cadherin expression in the examined samples showed a statistically significant negative correlation with the expression of p-PAK1(Thr423) and p-Crk-II(Ser41) (p < 0.0072 and p < 0.047 respectively) while p-PAK1(Thr423) and p-Crk-II(Ser41) established a positive correlation with each other (p < 0.0097). These findings highly suggest that PAK1 activation by upstream stimuli results in Crk mediated suppression of p120-catenin and E-cadherin in lung cancer.Figure 1

Bottom Line: Furthermore, KRAS mutant tumors expressed higher p-PAK1(Thr423) compared to KRAS wild type.KRAS prenylation inhibition by (FTI + GGTI) completely dephosphorylated proto-oncogene c-Crk on Serine 41 while Crk phosphorylation did not change by individual prenylation inhibitors or diluent.Combination of PAK1 inhibition and partial inhibition of all other KRAS effectors by (FTI + GGTI) dramatically altered morphology, motility and proliferation of H157 and A549 cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology, West Los Angeles VA, Los Angeles, CA, USA. fredmortazavi@ucla.edu.

ABSTRACT

Background: Key effector(s) of mutated KRAS in lung cancer progression and metastasis are unknown. Here we investigated the role of PAK1/Crk axis in transduction of the oncogenic KRAS signal in non-small cell lung cancer (NSCLC).

Methods: We used NSCLC clinical specimens to examine the correlation among KRAS mutations (codon 12, 13 and 61); PAK1/Crk axis activation [p-PAK1(Thr423), p-Crk(Ser41)]; and adhesion molecules expression by immunohistochemistry. For assessing the role of proto-oncogene c-Crk as a KRAS effector, we inhibited KRAS in NSCLC cells by a combination of farnesyltransferase inhibitor (FTI) and geranylgeranyltransferase inhibitor (GGTI) and measured p-Crk-II(Ser41) by western blotting. Finally, we disrupted the signaling network downstream of KRAS by blocking KRAS/PAK1/Crk axis with PAK1 inhibitors (i.e., IPA-3, FRAX597 or FRAX1036) along with partial inhibition of all other KRAS effectors by prenylation inhibitors (FTI + GGTI) and examined the motility, morphology and proliferation of the NSCLC cells.

Results: Immunohistochemical analysis demonstrated an inverse correlation between PAK1/Crk phosphorylation and E-cadherin/p120-catenin expression. Furthermore, KRAS mutant tumors expressed higher p-PAK1(Thr423) compared to KRAS wild type. KRAS prenylation inhibition by (FTI + GGTI) completely dephosphorylated proto-oncogene c-Crk on Serine 41 while Crk phosphorylation did not change by individual prenylation inhibitors or diluent. Combination of PAK1 inhibition and partial inhibition of all other KRAS effectors by (FTI + GGTI) dramatically altered morphology, motility and proliferation of H157 and A549 cells.

Conclusions: Our data provide evidence that proto-oncogene c-Crk is operative downstream of KRAS in NSCLC. Previously we demonstrated that Crk receives oncogenic signals from PAK1. These data in conjunction with the work of others that have specified the role of PAK1 in transduction of KRAS signal bring forward the importance of KRAS/PAK1/Crk axis as a prominent pathway in the oncogenesis of KRAS mutant lung cancer.

No MeSH data available.


Related in: MedlinePlus