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Loss of myoferlin redirects breast cancer cell motility towards collective migration.

Volakis LI, Li R, Ackerman WE, Mihai C, Bechel M, Summerfield TL, Ahn CS, Powell HM, Zielinski R, Rosol TJ, Ghadiali SN, Kniss DA - PLoS ONE (2014)

Bottom Line: Knockdown of MYOF led to significant reductions in cell migration velocity and MDA-231(MYOF-KD) cells migrated directionally and collectively, while MDA-231(LTVC) cells exhibited single cell migration.Moreover, MDA-231(MYOF-KD) tumors were highly circularized and did not invade locally into the adventia in contrast to MDA-231(LTVC)-injected animals.Thus MYOF loss is associated with a change in tumor formation in xenografts and leads to smaller, less invasive tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, The Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT
Cell migration plays a central role in the invasion and metastasis of tumors. As cells leave the primary tumor, they undergo an epithelial to mesenchymal transition (EMT) and migrate as single cells. Epithelial tumor cells may also migrate in a highly directional manner as a collective group in some settings. We previously discovered that myoferlin (MYOF) is overexpressed in breast cancer cells and depletion of MYOF results in a mesenchymal to epithelial transition (MET) and reduced invasion through extracellular matrix (ECM). However, the biomechanical mechanisms governing cell motility during MYOF depletion are poorly understood. We first demonstrated that lentivirus-driven shRNA-induced MYOF loss in MDA-MB-231 breast cancer cells (MDA-231(MYOF-KD)) leads to an epithelial morphology compared to the mesenchymal morphology observed in control (MDA-231(LTVC)) and wild-type cells. Knockdown of MYOF led to significant reductions in cell migration velocity and MDA-231(MYOF-KD) cells migrated directionally and collectively, while MDA-231(LTVC) cells exhibited single cell migration. Decreased migration velocity and collective migration were accompanied by significant changes in cell mechanics. MDA-231(MYOF-KD) cells exhibited a 2-fold decrease in cell stiffness, a 2-fold increase in cell-substrate adhesion and a 1.5-fold decrease in traction force generation. In vivo studies demonstrated that when immunocompromised mice were implanted with MDA-231(MYOF-KD) cells, tumors were smaller and demonstrated lower tumor burden. Moreover, MDA-231(MYOF-KD) tumors were highly circularized and did not invade locally into the adventia in contrast to MDA-231(LTVC)-injected animals. Thus MYOF loss is associated with a change in tumor formation in xenografts and leads to smaller, less invasive tumors. These data indicate that MYOF, a previously unrecognized protein in cancer, is involved in MDA-MB-231 cell migration and contributes to biomechanical alterations. Our results indicate that changes in biomechanical properties following loss of this protein may be an effective way to alter the invasive capacity of cancer cells.

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MYOF depletion in MDA-MB-231 cells alters overall morphology and the appearance of leading edge protrusions.(A–C) SEM images of (A) wild-type (MDA-231WT), (B) MDA-231LTVC, and (C) MDA-231MYOF-KD cells when seeded at subconfluent concentrations (∼30%). Note the mesenchymal-like morphology of the MDA-231WT and MDA-231LTVC cells (arrows) compared to the more epithelial morphology of MDA-231MYOF-KD cells (arrowheads). (D–G) Morphometric analyses of all MDA-MB-231 cell lines. Data included measurements on: (D) lamellipodia number per cell, (E) lamellipodia surface area per cell, (F) filopodia length, and (G) cell surface area. Bars represent mean ± SEM. Statistical significance was between the starred bar and the other two cell types shown (Kruskal Wallis with Dunn’s multiple comparison test), where *p<0.05, **p<0.01, and ***p<0.001. Scale bars = 10 µm.
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pone-0086110-g001: MYOF depletion in MDA-MB-231 cells alters overall morphology and the appearance of leading edge protrusions.(A–C) SEM images of (A) wild-type (MDA-231WT), (B) MDA-231LTVC, and (C) MDA-231MYOF-KD cells when seeded at subconfluent concentrations (∼30%). Note the mesenchymal-like morphology of the MDA-231WT and MDA-231LTVC cells (arrows) compared to the more epithelial morphology of MDA-231MYOF-KD cells (arrowheads). (D–G) Morphometric analyses of all MDA-MB-231 cell lines. Data included measurements on: (D) lamellipodia number per cell, (E) lamellipodia surface area per cell, (F) filopodia length, and (G) cell surface area. Bars represent mean ± SEM. Statistical significance was between the starred bar and the other two cell types shown (Kruskal Wallis with Dunn’s multiple comparison test), where *p<0.05, **p<0.01, and ***p<0.001. Scale bars = 10 µm.

Mentions: Using scanning electron microscopy, we evaluated the morphology (Figure 1A, B, and C) of MDA-MB-231 human breast tumor cells before (MDA-231WT) and after (MDA-231MYOF-KD) stable lentivirus-mediated transduction of shRNAs targeting MYOF as well as in a control line expressing a non-human, non-targeting shRNA (MDA-231LTVC). The parental MDA-231WT and MDA-231LTVC cell lines exhibited a mesenchymal phenotype with an average of 1.5–1.8 lamellipodia per cell and a lamellipodia surface area of ∼ 50 µm2 per cell (Figure 1A, B, D, and E). In contrast, when MYOF was diminished by >90%, cells reverted to an epithelial shape that was polygonal and typically contained a single broad lamellipodium with longer filopodia (Figure 1C and F). In addition, the average surface area of MYOF-depleted MDA-MB-231 cells was more than two-fold greater than either wild-type or MDA-231LTVC cells (Figure 1G).


Loss of myoferlin redirects breast cancer cell motility towards collective migration.

Volakis LI, Li R, Ackerman WE, Mihai C, Bechel M, Summerfield TL, Ahn CS, Powell HM, Zielinski R, Rosol TJ, Ghadiali SN, Kniss DA - PLoS ONE (2014)

MYOF depletion in MDA-MB-231 cells alters overall morphology and the appearance of leading edge protrusions.(A–C) SEM images of (A) wild-type (MDA-231WT), (B) MDA-231LTVC, and (C) MDA-231MYOF-KD cells when seeded at subconfluent concentrations (∼30%). Note the mesenchymal-like morphology of the MDA-231WT and MDA-231LTVC cells (arrows) compared to the more epithelial morphology of MDA-231MYOF-KD cells (arrowheads). (D–G) Morphometric analyses of all MDA-MB-231 cell lines. Data included measurements on: (D) lamellipodia number per cell, (E) lamellipodia surface area per cell, (F) filopodia length, and (G) cell surface area. Bars represent mean ± SEM. Statistical significance was between the starred bar and the other two cell types shown (Kruskal Wallis with Dunn’s multiple comparison test), where *p<0.05, **p<0.01, and ***p<0.001. Scale bars = 10 µm.
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Related In: Results  -  Collection

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pone-0086110-g001: MYOF depletion in MDA-MB-231 cells alters overall morphology and the appearance of leading edge protrusions.(A–C) SEM images of (A) wild-type (MDA-231WT), (B) MDA-231LTVC, and (C) MDA-231MYOF-KD cells when seeded at subconfluent concentrations (∼30%). Note the mesenchymal-like morphology of the MDA-231WT and MDA-231LTVC cells (arrows) compared to the more epithelial morphology of MDA-231MYOF-KD cells (arrowheads). (D–G) Morphometric analyses of all MDA-MB-231 cell lines. Data included measurements on: (D) lamellipodia number per cell, (E) lamellipodia surface area per cell, (F) filopodia length, and (G) cell surface area. Bars represent mean ± SEM. Statistical significance was between the starred bar and the other two cell types shown (Kruskal Wallis with Dunn’s multiple comparison test), where *p<0.05, **p<0.01, and ***p<0.001. Scale bars = 10 µm.
Mentions: Using scanning electron microscopy, we evaluated the morphology (Figure 1A, B, and C) of MDA-MB-231 human breast tumor cells before (MDA-231WT) and after (MDA-231MYOF-KD) stable lentivirus-mediated transduction of shRNAs targeting MYOF as well as in a control line expressing a non-human, non-targeting shRNA (MDA-231LTVC). The parental MDA-231WT and MDA-231LTVC cell lines exhibited a mesenchymal phenotype with an average of 1.5–1.8 lamellipodia per cell and a lamellipodia surface area of ∼ 50 µm2 per cell (Figure 1A, B, D, and E). In contrast, when MYOF was diminished by >90%, cells reverted to an epithelial shape that was polygonal and typically contained a single broad lamellipodium with longer filopodia (Figure 1C and F). In addition, the average surface area of MYOF-depleted MDA-MB-231 cells was more than two-fold greater than either wild-type or MDA-231LTVC cells (Figure 1G).

Bottom Line: Knockdown of MYOF led to significant reductions in cell migration velocity and MDA-231(MYOF-KD) cells migrated directionally and collectively, while MDA-231(LTVC) cells exhibited single cell migration.Moreover, MDA-231(MYOF-KD) tumors were highly circularized and did not invade locally into the adventia in contrast to MDA-231(LTVC)-injected animals.Thus MYOF loss is associated with a change in tumor formation in xenografts and leads to smaller, less invasive tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, The Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT
Cell migration plays a central role in the invasion and metastasis of tumors. As cells leave the primary tumor, they undergo an epithelial to mesenchymal transition (EMT) and migrate as single cells. Epithelial tumor cells may also migrate in a highly directional manner as a collective group in some settings. We previously discovered that myoferlin (MYOF) is overexpressed in breast cancer cells and depletion of MYOF results in a mesenchymal to epithelial transition (MET) and reduced invasion through extracellular matrix (ECM). However, the biomechanical mechanisms governing cell motility during MYOF depletion are poorly understood. We first demonstrated that lentivirus-driven shRNA-induced MYOF loss in MDA-MB-231 breast cancer cells (MDA-231(MYOF-KD)) leads to an epithelial morphology compared to the mesenchymal morphology observed in control (MDA-231(LTVC)) and wild-type cells. Knockdown of MYOF led to significant reductions in cell migration velocity and MDA-231(MYOF-KD) cells migrated directionally and collectively, while MDA-231(LTVC) cells exhibited single cell migration. Decreased migration velocity and collective migration were accompanied by significant changes in cell mechanics. MDA-231(MYOF-KD) cells exhibited a 2-fold decrease in cell stiffness, a 2-fold increase in cell-substrate adhesion and a 1.5-fold decrease in traction force generation. In vivo studies demonstrated that when immunocompromised mice were implanted with MDA-231(MYOF-KD) cells, tumors were smaller and demonstrated lower tumor burden. Moreover, MDA-231(MYOF-KD) tumors were highly circularized and did not invade locally into the adventia in contrast to MDA-231(LTVC)-injected animals. Thus MYOF loss is associated with a change in tumor formation in xenografts and leads to smaller, less invasive tumors. These data indicate that MYOF, a previously unrecognized protein in cancer, is involved in MDA-MB-231 cell migration and contributes to biomechanical alterations. Our results indicate that changes in biomechanical properties following loss of this protein may be an effective way to alter the invasive capacity of cancer cells.

Show MeSH
Related in: MedlinePlus