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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

Loss of LKB1 results in increased collagen remodeling during invasion. (A) H1299 pLKO.1 and shLKB1 spheroids and the collagen matrix were imaged using SHG microscopy. Spheroids were dyed using CellTracker Red in order to visualize cells during invasion. Images were obtained at 0, 6, and 21 h postembedding. Scale, 50 μm. (B) Images from A were quantified using collagen alignment analysis. A single z-stack image (i) is used in CT-FIRE software to extract collagen fibers (green; ii). The software automatically determines various fiber lengths in the image, represented as different line colors (iii). A yellow line represents the manually selected tumor boundary. (C) i) Example histogram generated via CT-FIRE analysis of collagen alignment coefficients. ii) Surface and iii) contour plots of local alignment show topography of alignment patterns. (D) Alignment analysis was performed as described in B and C for H1299 pLKO.1 (blue) and shLKB1 (red) spheroids at 6 and 21 h, with the 0-h baseline alignment subtracted to remove any initial bias.
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Figure 7: Loss of LKB1 results in increased collagen remodeling during invasion. (A) H1299 pLKO.1 and shLKB1 spheroids and the collagen matrix were imaged using SHG microscopy. Spheroids were dyed using CellTracker Red in order to visualize cells during invasion. Images were obtained at 0, 6, and 21 h postembedding. Scale, 50 μm. (B) Images from A were quantified using collagen alignment analysis. A single z-stack image (i) is used in CT-FIRE software to extract collagen fibers (green; ii). The software automatically determines various fiber lengths in the image, represented as different line colors (iii). A yellow line represents the manually selected tumor boundary. (C) i) Example histogram generated via CT-FIRE analysis of collagen alignment coefficients. ii) Surface and iii) contour plots of local alignment show topography of alignment patterns. (D) Alignment analysis was performed as described in B and C for H1299 pLKO.1 (blue) and shLKB1 (red) spheroids at 6 and 21 h, with the 0-h baseline alignment subtracted to remove any initial bias.

Mentions: Because we found that LKB1 loss results in a unique amoeboid cell population, we wanted to determine whether this provides an invasive advantage while navigating the microenvironment. To do this, we performed multiphoton imaging on H1299 pLKO.1 and shLKB1 spheroids to visualize collagen remodeling and its relationship to cell type and invasive potential. LKB1 loss resulted in an increase in collagen alignment at 6 and 21 h (Figure 7A). We used a novel local alignment coefficient to quantify the heterogeneous alignment patterns. We used curvelet transform fiber extraction (CT-FIRE) software to extract collagen fibers (Figure 7B, i and ii). All fibers were quantized with a 5-pixel length. Then, for every pixel, we measured the local alignment coefficient parameter for every pixel by selecting all fiber segments within a circular neighborhood of 20 pixels (Figure 7Biii) to generate the alignment field (Supplemental Figure S7 explains the optimization of the local alignment coefficient calculation). Using this parameter, we generated histograms of local alignment coefficients, surface plots, and contour plots to quantify alignment (Figure 7C). Using this quantification of the local alignment coefficient and comparing to the 0-h baseline value, we found that at 6 h, shLKB1 cells show an increase in collagen alignment, which is further accentuated at 21 h; on the other hand, pLKO.1 control cells show in a decrease in the number of aligned fibers over time (Figure 7D), suggesting that LKB1 might actually negatively regulate remodeling during invasion. Thus LKB1-depleted cells are more efficient at realigning collagen fibers as they invade. Of interest, this realignment of the collagen matrix seems to occur via a matrix metalloproteinase (MMP)–independent mechanism. When treated with the pan-MMP inhibitor GM6001, shLKB1 showed no significant change in invasion compared with vehicle control (Supplemental Figure S8). These data suggest that LKB1 loss promotes collagen remodeling in an MMP-independent manner.


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)

Loss of LKB1 results in increased collagen remodeling during invasion. (A) H1299 pLKO.1 and shLKB1 spheroids and the collagen matrix were imaged using SHG microscopy. Spheroids were dyed using CellTracker Red in order to visualize cells during invasion. Images were obtained at 0, 6, and 21 h postembedding. Scale, 50 μm. (B) Images from A were quantified using collagen alignment analysis. A single z-stack image (i) is used in CT-FIRE software to extract collagen fibers (green; ii). The software automatically determines various fiber lengths in the image, represented as different line colors (iii). A yellow line represents the manually selected tumor boundary. (C) i) Example histogram generated via CT-FIRE analysis of collagen alignment coefficients. ii) Surface and iii) contour plots of local alignment show topography of alignment patterns. (D) Alignment analysis was performed as described in B and C for H1299 pLKO.1 (blue) and shLKB1 (red) spheroids at 6 and 21 h, with the 0-h baseline alignment subtracted to remove any initial bias.
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Figure 7: Loss of LKB1 results in increased collagen remodeling during invasion. (A) H1299 pLKO.1 and shLKB1 spheroids and the collagen matrix were imaged using SHG microscopy. Spheroids were dyed using CellTracker Red in order to visualize cells during invasion. Images were obtained at 0, 6, and 21 h postembedding. Scale, 50 μm. (B) Images from A were quantified using collagen alignment analysis. A single z-stack image (i) is used in CT-FIRE software to extract collagen fibers (green; ii). The software automatically determines various fiber lengths in the image, represented as different line colors (iii). A yellow line represents the manually selected tumor boundary. (C) i) Example histogram generated via CT-FIRE analysis of collagen alignment coefficients. ii) Surface and iii) contour plots of local alignment show topography of alignment patterns. (D) Alignment analysis was performed as described in B and C for H1299 pLKO.1 (blue) and shLKB1 (red) spheroids at 6 and 21 h, with the 0-h baseline alignment subtracted to remove any initial bias.
Mentions: Because we found that LKB1 loss results in a unique amoeboid cell population, we wanted to determine whether this provides an invasive advantage while navigating the microenvironment. To do this, we performed multiphoton imaging on H1299 pLKO.1 and shLKB1 spheroids to visualize collagen remodeling and its relationship to cell type and invasive potential. LKB1 loss resulted in an increase in collagen alignment at 6 and 21 h (Figure 7A). We used a novel local alignment coefficient to quantify the heterogeneous alignment patterns. We used curvelet transform fiber extraction (CT-FIRE) software to extract collagen fibers (Figure 7B, i and ii). All fibers were quantized with a 5-pixel length. Then, for every pixel, we measured the local alignment coefficient parameter for every pixel by selecting all fiber segments within a circular neighborhood of 20 pixels (Figure 7Biii) to generate the alignment field (Supplemental Figure S7 explains the optimization of the local alignment coefficient calculation). Using this parameter, we generated histograms of local alignment coefficients, surface plots, and contour plots to quantify alignment (Figure 7C). Using this quantification of the local alignment coefficient and comparing to the 0-h baseline value, we found that at 6 h, shLKB1 cells show an increase in collagen alignment, which is further accentuated at 21 h; on the other hand, pLKO.1 control cells show in a decrease in the number of aligned fibers over time (Figure 7D), suggesting that LKB1 might actually negatively regulate remodeling during invasion. Thus LKB1-depleted cells are more efficient at realigning collagen fibers as they invade. Of interest, this realignment of the collagen matrix seems to occur via a matrix metalloproteinase (MMP)–independent mechanism. When treated with the pan-MMP inhibitor GM6001, shLKB1 showed no significant change in invasion compared with vehicle control (Supplemental Figure S8). These data suggest that LKB1 loss promotes collagen remodeling in an MMP-independent manner.

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