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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 induces a mesenchymal–amoeboid switch in 3D invasive morphology. (A) Western blot (left) showing that H1299 cells were stably depleted of LKB1 using a targeting shRNA lentivirus. Empty pLKO.1 vector was used as a control. Spheroids from H1299 pLKO.1 and shLKB1 cells were embedded in a collagen type I matrix and imaged at 24 h postembedding (right). Bottom, zoomed images. Amoeboid and mesenchymal cell morphologies were quantified as a percentage back to the total number of cells invaded in each spheroid. Three spheroids. Scale, 50 μm. Arrows, mesenchymal cells; arrowheads, amoeboid cells. (B) Schematic illustration of how mesenchymal and amoeboid cells were quantified. Any cell whose length was greater than or equal to twice its width was considered a mesenchymal cell. Right, examples of each cell morphology. Arrow, mesenchymal cell; arrowhead, amoeboid cell. (C) Experiment from A repeated in H1792 NSCLC cells. Bottom, Western blot confirming LKB1 knockdown. (D) H157 cells (LKB1-) were transiently transfected with either empty pcDNA3-GFP or pEGFP-C1 LKB1 WT vector. Spheroids were analyzed at 24 h for invasive phenotypes. Western blot confirms expression of GFP-LKB1 (right). Bottom, bar graph showing the percentage of mesenchymal cells in empty GFP control and LKB1-transfected H157 cells. (E–G) H1299 pLKO.1 and shLKB1 spheroids were embedded in a collagen matrix and imaged using live-cell confocal. (E) Bar graph showing the percentage of amoeboid cells calculated over time in H1299 pLKO.1 and shLKB1 cells. Eight spheroids. (F) Cell tracks were plotted from a single point of origin in H1299 pLKO.1 and shLKB1 invasive amoeboid and mesenchymal cells. (G) Bar graph showing meandering index and velocity of H1299 shLKB1 and pLKO.1 subtypes; 11 cells. *p ≤ 0.05, **p ≤ 0.01, and ***p < 0.001.
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Figure 1: LKB1 induces a mesenchymal–amoeboid switch in 3D invasive morphology. (A) Western blot (left) showing that H1299 cells were stably depleted of LKB1 using a targeting shRNA lentivirus. Empty pLKO.1 vector was used as a control. Spheroids from H1299 pLKO.1 and shLKB1 cells were embedded in a collagen type I matrix and imaged at 24 h postembedding (right). Bottom, zoomed images. Amoeboid and mesenchymal cell morphologies were quantified as a percentage back to the total number of cells invaded in each spheroid. Three spheroids. Scale, 50 μm. Arrows, mesenchymal cells; arrowheads, amoeboid cells. (B) Schematic illustration of how mesenchymal and amoeboid cells were quantified. Any cell whose length was greater than or equal to twice its width was considered a mesenchymal cell. Right, examples of each cell morphology. Arrow, mesenchymal cell; arrowhead, amoeboid cell. (C) Experiment from A repeated in H1792 NSCLC cells. Bottom, Western blot confirming LKB1 knockdown. (D) H157 cells (LKB1-) were transiently transfected with either empty pcDNA3-GFP or pEGFP-C1 LKB1 WT vector. Spheroids were analyzed at 24 h for invasive phenotypes. Western blot confirms expression of GFP-LKB1 (right). Bottom, bar graph showing the percentage of mesenchymal cells in empty GFP control and LKB1-transfected H157 cells. (E–G) H1299 pLKO.1 and shLKB1 spheroids were embedded in a collagen matrix and imaged using live-cell confocal. (E) Bar graph showing the percentage of amoeboid cells calculated over time in H1299 pLKO.1 and shLKB1 cells. Eight spheroids. (F) Cell tracks were plotted from a single point of origin in H1299 pLKO.1 and shLKB1 invasive amoeboid and mesenchymal cells. (G) Bar graph showing meandering index and velocity of H1299 shLKB1 and pLKO.1 subtypes; 11 cells. *p ≤ 0.05, **p ≤ 0.01, and ***p < 0.001.

Mentions: To probe the role of LKB1 in regulating 3D invasion, we stably depleted LKB1 in H1299 non–small cell lung cancer cells (LKB1 wild type) and compared them with isogenic parental pLKO.1 vector control cells (Figure 1A). Stable knockdown of LKB1 resulted in cells switching from a mesenchymal morphology to an amoeboid morphology (Figure 1A). In the pLKO.1 cells, 47% of the invasive cells showed an amoeboid morphology, as compared with 73% in the shLKB1 cells. Mesenchymal cells were defined as cells that had a length greater than twice their width, as previously described (Nogawa and Mizuno, 1981; Ladhani et al., 2011; Figure 1B). A similar transition was observed in H1792 cells (LKB1 wild-type NSCLC), with stable LKB1 depletion (Figure 1C). Similarly, a second LKB1-targeted short hairpin RNA (shRNA) in H1299 cells, as well as transient knockdown of LKB1 in H1299s and H1792s using small interfering RNA (siRNA), had similar effects, resulting in amoeboid-like morphology during invasion (Supplemental Figure S1). To confirm that this was an LKB1-dependent effect on cell invasion, we performed the reverse experiment and used H157 non–small cell lung cancer cells (LKB1-) expressing either green fluorescent protein (GFP)–tagged full-length (wild type [WT]) LKB1 or empty GFP control vector to analyze invasive morphology. Reexpression of WT LKB1 caused invaded cells to switch from an amoeboid-like morphology to an elongated, mesenchymal-like morphology (Figure 1D). Of the invasive cells expressing LKB1, 61% of the cells were mesenchymal in morphology, as compared with 27% in the LKB1- parental cells.


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 induces a mesenchymal–amoeboid switch in 3D invasive morphology. (A) Western blot (left) showing that H1299 cells were stably depleted of LKB1 using a targeting shRNA lentivirus. Empty pLKO.1 vector was used as a control. Spheroids from H1299 pLKO.1 and shLKB1 cells were embedded in a collagen type I matrix and imaged at 24 h postembedding (right). Bottom, zoomed images. Amoeboid and mesenchymal cell morphologies were quantified as a percentage back to the total number of cells invaded in each spheroid. Three spheroids. Scale, 50 μm. Arrows, mesenchymal cells; arrowheads, amoeboid cells. (B) Schematic illustration of how mesenchymal and amoeboid cells were quantified. Any cell whose length was greater than or equal to twice its width was considered a mesenchymal cell. Right, examples of each cell morphology. Arrow, mesenchymal cell; arrowhead, amoeboid cell. (C) Experiment from A repeated in H1792 NSCLC cells. Bottom, Western blot confirming LKB1 knockdown. (D) H157 cells (LKB1-) were transiently transfected with either empty pcDNA3-GFP or pEGFP-C1 LKB1 WT vector. Spheroids were analyzed at 24 h for invasive phenotypes. Western blot confirms expression of GFP-LKB1 (right). Bottom, bar graph showing the percentage of mesenchymal cells in empty GFP control and LKB1-transfected H157 cells. (E–G) H1299 pLKO.1 and shLKB1 spheroids were embedded in a collagen matrix and imaged using live-cell confocal. (E) Bar graph showing the percentage of amoeboid cells calculated over time in H1299 pLKO.1 and shLKB1 cells. Eight spheroids. (F) Cell tracks were plotted from a single point of origin in H1299 pLKO.1 and shLKB1 invasive amoeboid and mesenchymal cells. (G) Bar graph showing meandering index and velocity of H1299 shLKB1 and pLKO.1 subtypes; 11 cells. *p ≤ 0.05, **p ≤ 0.01, and ***p < 0.001.
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Figure 1: LKB1 induces a mesenchymal–amoeboid switch in 3D invasive morphology. (A) Western blot (left) showing that H1299 cells were stably depleted of LKB1 using a targeting shRNA lentivirus. Empty pLKO.1 vector was used as a control. Spheroids from H1299 pLKO.1 and shLKB1 cells were embedded in a collagen type I matrix and imaged at 24 h postembedding (right). Bottom, zoomed images. Amoeboid and mesenchymal cell morphologies were quantified as a percentage back to the total number of cells invaded in each spheroid. Three spheroids. Scale, 50 μm. Arrows, mesenchymal cells; arrowheads, amoeboid cells. (B) Schematic illustration of how mesenchymal and amoeboid cells were quantified. Any cell whose length was greater than or equal to twice its width was considered a mesenchymal cell. Right, examples of each cell morphology. Arrow, mesenchymal cell; arrowhead, amoeboid cell. (C) Experiment from A repeated in H1792 NSCLC cells. Bottom, Western blot confirming LKB1 knockdown. (D) H157 cells (LKB1-) were transiently transfected with either empty pcDNA3-GFP or pEGFP-C1 LKB1 WT vector. Spheroids were analyzed at 24 h for invasive phenotypes. Western blot confirms expression of GFP-LKB1 (right). Bottom, bar graph showing the percentage of mesenchymal cells in empty GFP control and LKB1-transfected H157 cells. (E–G) H1299 pLKO.1 and shLKB1 spheroids were embedded in a collagen matrix and imaged using live-cell confocal. (E) Bar graph showing the percentage of amoeboid cells calculated over time in H1299 pLKO.1 and shLKB1 cells. Eight spheroids. (F) Cell tracks were plotted from a single point of origin in H1299 pLKO.1 and shLKB1 invasive amoeboid and mesenchymal cells. (G) Bar graph showing meandering index and velocity of H1299 shLKB1 and pLKO.1 subtypes; 11 cells. *p ≤ 0.05, **p ≤ 0.01, and ***p < 0.001.
Mentions: To probe the role of LKB1 in regulating 3D invasion, we stably depleted LKB1 in H1299 non–small cell lung cancer cells (LKB1 wild type) and compared them with isogenic parental pLKO.1 vector control cells (Figure 1A). Stable knockdown of LKB1 resulted in cells switching from a mesenchymal morphology to an amoeboid morphology (Figure 1A). In the pLKO.1 cells, 47% of the invasive cells showed an amoeboid morphology, as compared with 73% in the shLKB1 cells. Mesenchymal cells were defined as cells that had a length greater than twice their width, as previously described (Nogawa and Mizuno, 1981; Ladhani et al., 2011; Figure 1B). A similar transition was observed in H1792 cells (LKB1 wild-type NSCLC), with stable LKB1 depletion (Figure 1C). Similarly, a second LKB1-targeted short hairpin RNA (shRNA) in H1299 cells, as well as transient knockdown of LKB1 in H1299s and H1792s using small interfering RNA (siRNA), had similar effects, resulting in amoeboid-like morphology during invasion (Supplemental Figure S1). To confirm that this was an LKB1-dependent effect on cell invasion, we performed the reverse experiment and used H157 non–small cell lung cancer cells (LKB1-) expressing either green fluorescent protein (GFP)–tagged full-length (wild type [WT]) LKB1 or empty GFP control vector to analyze invasive morphology. Reexpression of WT LKB1 caused invaded cells to switch from an amoeboid-like morphology to an elongated, mesenchymal-like morphology (Figure 1D). Of the invasive cells expressing LKB1, 61% of the cells were mesenchymal in morphology, as compared with 27% in the LKB1- parental cells.

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