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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

KRAS inhibition dephosphorylates Crk-II on serine 41. Western blots demonstrating loss of Crk-II phosphorylation on serine 41 in Rh2 NSCLC cells following 24 hour exposure of cells to combination of (F) farnesyltransferase inhibitor (BMS-225975 at 2 μM) and (G) geranylgeranyltransferase inhibitor (P61A6 at 2 μM).
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Fig6: KRAS inhibition dephosphorylates Crk-II on serine 41. Western blots demonstrating loss of Crk-II phosphorylation on serine 41 in Rh2 NSCLC cells following 24 hour exposure of cells to combination of (F) farnesyltransferase inhibitor (BMS-225975 at 2 μM) and (G) geranylgeranyltransferase inhibitor (P61A6 at 2 μM).

Mentions: We previously showed that PAK1 phosphorylates Crk-II on serine 41 [5] as PAK1 silencing dephosphorylated Crk-II on this residue and the amino acid sequence of Crk-II at the vicinity of Serine 41 was identical to the PAK1 phosphorylation sequence site on Raf and also this sequence showed a high degree of homology to the PAK1 phosphorylation consensus sequence in other PAK1 effectors (i.e., MEK, Snail and LIMK-1) [5,14]. Considering PAK1 is shown to be functioning downstream of KRAS, we sought to examine whether KRAS signal is transduced through Crk as well. In order to answer this question, we inhibited KRAS by a combination of farnesyltransferase inhibition (FTI) and geranylgeranyltransferase inhibition (GGTI) in NSCLC cells and examined Crk phosphorylation. Recently, we developed novel small molecule geranylgeranyltransferase type I inhibitors (GGTIs) [15-18] through a high-throughput screen. One of the identified GGTIs (i.e., P61A6) has inhibited both proliferation and cell cycle progression in a variety of human cancer cell lines including NSCLC cells [19]. Interestingly, exposure of Rh2 cells to FTI (BMS-225975) and GGTI (P61A6) combination for 24 hours completely dephosphorylated Crk-II on Serine 41 as demonstrated in a western blot assay (Figure 6). On the other hand, exposure of each inhibitor individually had little or no effect on Crk-II phosphorylation. This finding demonstrates that adaptor protein/proto-oncogene Crk is functioning downstream of KRAS. Considering (i) PAK1 is shown to be operative downstream of KRAS [1-4] and (ii) PAK1 phosphorylates Crk on serine 41 [5], it is most likely that the link from KRAS to Crk is transduced through PAK1.Figure 6


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)

KRAS inhibition dephosphorylates Crk-II on serine 41. Western blots demonstrating loss of Crk-II phosphorylation on serine 41 in Rh2 NSCLC cells following 24 hour exposure of cells to combination of (F) farnesyltransferase inhibitor (BMS-225975 at 2 μM) and (G) geranylgeranyltransferase inhibitor (P61A6 at 2 μM).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: KRAS inhibition dephosphorylates Crk-II on serine 41. Western blots demonstrating loss of Crk-II phosphorylation on serine 41 in Rh2 NSCLC cells following 24 hour exposure of cells to combination of (F) farnesyltransferase inhibitor (BMS-225975 at 2 μM) and (G) geranylgeranyltransferase inhibitor (P61A6 at 2 μM).
Mentions: We previously showed that PAK1 phosphorylates Crk-II on serine 41 [5] as PAK1 silencing dephosphorylated Crk-II on this residue and the amino acid sequence of Crk-II at the vicinity of Serine 41 was identical to the PAK1 phosphorylation sequence site on Raf and also this sequence showed a high degree of homology to the PAK1 phosphorylation consensus sequence in other PAK1 effectors (i.e., MEK, Snail and LIMK-1) [5,14]. Considering PAK1 is shown to be functioning downstream of KRAS, we sought to examine whether KRAS signal is transduced through Crk as well. In order to answer this question, we inhibited KRAS by a combination of farnesyltransferase inhibition (FTI) and geranylgeranyltransferase inhibition (GGTI) in NSCLC cells and examined Crk phosphorylation. Recently, we developed novel small molecule geranylgeranyltransferase type I inhibitors (GGTIs) [15-18] through a high-throughput screen. One of the identified GGTIs (i.e., P61A6) has inhibited both proliferation and cell cycle progression in a variety of human cancer cell lines including NSCLC cells [19]. Interestingly, exposure of Rh2 cells to FTI (BMS-225975) and GGTI (P61A6) combination for 24 hours completely dephosphorylated Crk-II on Serine 41 as demonstrated in a western blot assay (Figure 6). On the other hand, exposure of each inhibitor individually had little or no effect on Crk-II phosphorylation. This finding demonstrates that adaptor protein/proto-oncogene Crk is functioning downstream of KRAS. Considering (i) PAK1 is shown to be operative downstream of KRAS [1-4] and (ii) PAK1 phosphorylates Crk on serine 41 [5], it is most likely that the link from KRAS to Crk is transduced through PAK1.Figure 6

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