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LKB1 kinase-dependent and -independent defects disrupt polarity and adhesion signaling to drive collagen remodeling during invasion.

Konen J, Wilkinson S, Lee B, Fu H, Zhou W, Jiang Y, Marcus AI - Mol. Biol. Cell (2016)

Bottom Line: The majority of LKB1 mutations are truncations that disrupt its kinase activity and remove its C-terminal domain (CTD).Instead, cell polarity is overseen by the kinase-independent function of its CTD and more specifically its farnesylation.This occurs through a mesenchymal-amoeboid morphological switch that signals through the Rho-GTPase RhoA.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Medical Oncology, Winship Cancer Institute of Emory University, Atlanta, GA 30322 Graduate Program in Cancer Biology, Emory University, Atlanta, GA 30322.

No MeSH data available.


Related in: MedlinePlus

LKB1-depleted amoeboid cells depend on pFAK during invasion. (A) Western analysis of pFAKY397 expression in H1299 pLKO.1 and shLKB1 cells (top). A similar experiment was performed in H1792 LKB1 siRNA treated cells (bottom). Bar graph shows densitometry of the phospho to total FAK ratio in the H1792 LKB1 knockdown cells compared with control siRNA. (B) H1299 pLKO.1 and shLKB1 spheroids were analyzed for expression of pFAKY397 using IF imaging. Scale, 20 μm. (C) Quantification of pFAKY397 site intensity (left) and total number of pFAKY397 sites per cell (right). (D) Activity of downstream FAK signaling was analyzed using IF imaging of pPAXY118 in H1299 pLKO.1 and shLKB1 spheroids. Scale, 20 μm. (E) Quantification of pPAXY118 site intensity (left) and total number of pPAXY118 sites per cell (right). (F) Zoomed images of pFAK immunofluorescence imaging (described in B). Mesenchymal and amoeboid cell types in H1299 pLKO.1 and shLKB1. Scale, 10 μm. (G–I) H1299 pLKO.1 and shLKB1 spheroids were exposed to either 1 μM PF-573228 FAK inhibitor or vehicle control and imaged over time. (G) Still images of the live-cell imaging experiment were taken every 8 h. Bottom, quantification of total cells invaded, showing significantly decreased cells invaded in shLKB1 cells treated with FAK inhibitor. Scale, 100 μm. (H) Cell tracks from the FAK inhibitor experiment showed that LKB1-depleted amoeboid cells have a significantly decreased velocity during invasion compared with vehicle control; 8–15 cells. (I) The percentage of amoeboid cells in the live cell FAK inhibitor experiment was assessed at 0, 8, 12, and 16 h; five or six spheroids. *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001.
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Figure 6: LKB1-depleted amoeboid cells depend on pFAK during invasion. (A) Western analysis of pFAKY397 expression in H1299 pLKO.1 and shLKB1 cells (top). A similar experiment was performed in H1792 LKB1 siRNA treated cells (bottom). Bar graph shows densitometry of the phospho to total FAK ratio in the H1792 LKB1 knockdown cells compared with control siRNA. (B) H1299 pLKO.1 and shLKB1 spheroids were analyzed for expression of pFAKY397 using IF imaging. Scale, 20 μm. (C) Quantification of pFAKY397 site intensity (left) and total number of pFAKY397 sites per cell (right). (D) Activity of downstream FAK signaling was analyzed using IF imaging of pPAXY118 in H1299 pLKO.1 and shLKB1 spheroids. Scale, 20 μm. (E) Quantification of pPAXY118 site intensity (left) and total number of pPAXY118 sites per cell (right). (F) Zoomed images of pFAK immunofluorescence imaging (described in B). Mesenchymal and amoeboid cell types in H1299 pLKO.1 and shLKB1. Scale, 10 μm. (G–I) H1299 pLKO.1 and shLKB1 spheroids were exposed to either 1 μM PF-573228 FAK inhibitor or vehicle control and imaged over time. (G) Still images of the live-cell imaging experiment were taken every 8 h. Bottom, quantification of total cells invaded, showing significantly decreased cells invaded in shLKB1 cells treated with FAK inhibitor. Scale, 100 μm. (H) Cell tracks from the FAK inhibitor experiment showed that LKB1-depleted amoeboid cells have a significantly decreased velocity during invasion compared with vehicle control; 8–15 cells. (I) The percentage of amoeboid cells in the live cell FAK inhibitor experiment was assessed at 0, 8, 12, and 16 h; five or six spheroids. *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001.

Mentions: We next wanted to determine the effect of LKB1 loss specifically on the amoeboid population because these cells showed a significant increase in velocity during invasion, even more so than amoeboid cells in the wild-type population (Figure 1F). We first assessed whether FAK was hyperactive in LKB1-depleted cells. Western blot confirmed an increase in pFAKY397 in H1299 and H1792 LKB1-depleted cells compared with controls (Figure 6A); however, this provided information only on the whole population. Therefore we then analyzed single invasive cells for their pFAK status based on morphology in H1299 pLKO.1 and shLKB1 spheroids via immunofluorescence. We confirmed that LKB1 loss resulted in pFAKY397 hyperactivation compared with pLKO.1 control cells (Figure 6B). The mean intensity of each individual pFAK site increased significantly from 748 in pLKO.1 cells to 1751 in shLKB1 cells (Figure 6C, left), and the total number of pFAK sites per cell showed a significant increase from 3 to 72 sites/cell with LKB1 depletion (Figure 6C, right). This increase in pFAKY397 also resulted in increased downstream adhesion signaling, since both the mean phospho-paxillin (pPaxY118) site intensity and total number of pPaxY118 sites per cell significantly increased (Figure 6, D and E).


LKB1 kinase-dependent and -independent defects disrupt polarity and adhesion signaling to drive collagen remodeling during invasion.

Konen J, Wilkinson S, Lee B, Fu H, Zhou W, Jiang Y, Marcus AI - Mol. Biol. Cell (2016)

LKB1-depleted amoeboid cells depend on pFAK during invasion. (A) Western analysis of pFAKY397 expression in H1299 pLKO.1 and shLKB1 cells (top). A similar experiment was performed in H1792 LKB1 siRNA treated cells (bottom). Bar graph shows densitometry of the phospho to total FAK ratio in the H1792 LKB1 knockdown cells compared with control siRNA. (B) H1299 pLKO.1 and shLKB1 spheroids were analyzed for expression of pFAKY397 using IF imaging. Scale, 20 μm. (C) Quantification of pFAKY397 site intensity (left) and total number of pFAKY397 sites per cell (right). (D) Activity of downstream FAK signaling was analyzed using IF imaging of pPAXY118 in H1299 pLKO.1 and shLKB1 spheroids. Scale, 20 μm. (E) Quantification of pPAXY118 site intensity (left) and total number of pPAXY118 sites per cell (right). (F) Zoomed images of pFAK immunofluorescence imaging (described in B). Mesenchymal and amoeboid cell types in H1299 pLKO.1 and shLKB1. Scale, 10 μm. (G–I) H1299 pLKO.1 and shLKB1 spheroids were exposed to either 1 μM PF-573228 FAK inhibitor or vehicle control and imaged over time. (G) Still images of the live-cell imaging experiment were taken every 8 h. Bottom, quantification of total cells invaded, showing significantly decreased cells invaded in shLKB1 cells treated with FAK inhibitor. Scale, 100 μm. (H) Cell tracks from the FAK inhibitor experiment showed that LKB1-depleted amoeboid cells have a significantly decreased velocity during invasion compared with vehicle control; 8–15 cells. (I) The percentage of amoeboid cells in the live cell FAK inhibitor experiment was assessed at 0, 8, 12, and 16 h; five or six spheroids. *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001.
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Figure 6: LKB1-depleted amoeboid cells depend on pFAK during invasion. (A) Western analysis of pFAKY397 expression in H1299 pLKO.1 and shLKB1 cells (top). A similar experiment was performed in H1792 LKB1 siRNA treated cells (bottom). Bar graph shows densitometry of the phospho to total FAK ratio in the H1792 LKB1 knockdown cells compared with control siRNA. (B) H1299 pLKO.1 and shLKB1 spheroids were analyzed for expression of pFAKY397 using IF imaging. Scale, 20 μm. (C) Quantification of pFAKY397 site intensity (left) and total number of pFAKY397 sites per cell (right). (D) Activity of downstream FAK signaling was analyzed using IF imaging of pPAXY118 in H1299 pLKO.1 and shLKB1 spheroids. Scale, 20 μm. (E) Quantification of pPAXY118 site intensity (left) and total number of pPAXY118 sites per cell (right). (F) Zoomed images of pFAK immunofluorescence imaging (described in B). Mesenchymal and amoeboid cell types in H1299 pLKO.1 and shLKB1. Scale, 10 μm. (G–I) H1299 pLKO.1 and shLKB1 spheroids were exposed to either 1 μM PF-573228 FAK inhibitor or vehicle control and imaged over time. (G) Still images of the live-cell imaging experiment were taken every 8 h. Bottom, quantification of total cells invaded, showing significantly decreased cells invaded in shLKB1 cells treated with FAK inhibitor. Scale, 100 μm. (H) Cell tracks from the FAK inhibitor experiment showed that LKB1-depleted amoeboid cells have a significantly decreased velocity during invasion compared with vehicle control; 8–15 cells. (I) The percentage of amoeboid cells in the live cell FAK inhibitor experiment was assessed at 0, 8, 12, and 16 h; five or six spheroids. *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001.
Mentions: We next wanted to determine the effect of LKB1 loss specifically on the amoeboid population because these cells showed a significant increase in velocity during invasion, even more so than amoeboid cells in the wild-type population (Figure 1F). We first assessed whether FAK was hyperactive in LKB1-depleted cells. Western blot confirmed an increase in pFAKY397 in H1299 and H1792 LKB1-depleted cells compared with controls (Figure 6A); however, this provided information only on the whole population. Therefore we then analyzed single invasive cells for their pFAK status based on morphology in H1299 pLKO.1 and shLKB1 spheroids via immunofluorescence. We confirmed that LKB1 loss resulted in pFAKY397 hyperactivation compared with pLKO.1 control cells (Figure 6B). The mean intensity of each individual pFAK site increased significantly from 748 in pLKO.1 cells to 1751 in shLKB1 cells (Figure 6C, left), and the total number of pFAK sites per cell showed a significant increase from 3 to 72 sites/cell with LKB1 depletion (Figure 6C, right). This increase in pFAKY397 also resulted in increased downstream adhesion signaling, since both the mean phospho-paxillin (pPaxY118) site intensity and total number of pPaxY118 sites per cell significantly increased (Figure 6, D and E).

Bottom Line: The majority of LKB1 mutations are truncations that disrupt its kinase activity and remove its C-terminal domain (CTD).Instead, cell polarity is overseen by the kinase-independent function of its CTD and more specifically its farnesylation.This occurs through a mesenchymal-amoeboid morphological switch that signals through the Rho-GTPase RhoA.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Medical Oncology, Winship Cancer Institute of Emory University, Atlanta, GA 30322 Graduate Program in Cancer Biology, Emory University, Atlanta, GA 30322.

No MeSH data available.


Related in: MedlinePlus