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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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Average traction stress and contractility decreases when MYOF is depleted from MDA-MB-231 cells.Myoferlin-depleted cells exert (A) less average traction stress (mean ± SEM) on the substrate than the MDA-231LTVC cells. (B) The MDA-231MYOF-KD cells were more rounded cells with a more consistent traction stress over the cell area, whereas the MDA-231LTVC cells were more fibroblastic morphologically with a larger range of traction stress. Statistical significance (two-tailed Student’s t-test) was between the two cell types shown, where *p<0.05.
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pone-0086110-g006: Average traction stress and contractility decreases when MYOF is depleted from MDA-MB-231 cells.Myoferlin-depleted cells exert (A) less average traction stress (mean ± SEM) on the substrate than the MDA-231LTVC cells. (B) The MDA-231MYOF-KD cells were more rounded cells with a more consistent traction stress over the cell area, whereas the MDA-231LTVC cells were more fibroblastic morphologically with a larger range of traction stress. Statistical significance (two-tailed Student’s t-test) was between the two cell types shown, where *p<0.05.

Mentions: Finally, we used TFM to investigate how depletion of MYOF alters the traction stress generated by MDA-MB-231 tumor cells. For these studies, cells were plated at sparse density to reduce error when analyzing the fluorescent microbead displacements used to calculate the traction stress in the substrate. TFM measures the traction stresses exerted by the cell on the underlying substrate as shown in Figure 6. These raw data were quantified by calculating the average traction stress exerted by individual cells according to Eqn (6). The average traction stress exerted by MDA-231MYOF-KD cells was significantly lower (Figure 6A, p<0.05) than the traction stress exerted by MDA-231LTVC cells. The more rounded MDA-231MYOF-KD cells exert less traction on the substrate than MDA-231LTVC. We also noted that the MDA-231LTVC cells included a larger range of traction stresses, whereas the MDA-231MYOF-KD cells maintained a more consistent traction stress over the cell area (Figure 6B). Since contractile force generation by internal actin-myosin interactions is related to the amount of traction stress exerted by the cell, our data indicate that depletion of MYOF resulted in a significant reduction in the cell’s contractility or contractile force generation.


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)

Average traction stress and contractility decreases when MYOF is depleted from MDA-MB-231 cells.Myoferlin-depleted cells exert (A) less average traction stress (mean ± SEM) on the substrate than the MDA-231LTVC cells. (B) The MDA-231MYOF-KD cells were more rounded cells with a more consistent traction stress over the cell area, whereas the MDA-231LTVC cells were more fibroblastic morphologically with a larger range of traction stress. Statistical significance (two-tailed Student’s t-test) was between the two cell types shown, where *p<0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3935829&req=5

pone-0086110-g006: Average traction stress and contractility decreases when MYOF is depleted from MDA-MB-231 cells.Myoferlin-depleted cells exert (A) less average traction stress (mean ± SEM) on the substrate than the MDA-231LTVC cells. (B) The MDA-231MYOF-KD cells were more rounded cells with a more consistent traction stress over the cell area, whereas the MDA-231LTVC cells were more fibroblastic morphologically with a larger range of traction stress. Statistical significance (two-tailed Student’s t-test) was between the two cell types shown, where *p<0.05.
Mentions: Finally, we used TFM to investigate how depletion of MYOF alters the traction stress generated by MDA-MB-231 tumor cells. For these studies, cells were plated at sparse density to reduce error when analyzing the fluorescent microbead displacements used to calculate the traction stress in the substrate. TFM measures the traction stresses exerted by the cell on the underlying substrate as shown in Figure 6. These raw data were quantified by calculating the average traction stress exerted by individual cells according to Eqn (6). The average traction stress exerted by MDA-231MYOF-KD cells was significantly lower (Figure 6A, p<0.05) than the traction stress exerted by MDA-231LTVC cells. The more rounded MDA-231MYOF-KD cells exert less traction on the substrate than MDA-231LTVC. We also noted that the MDA-231LTVC cells included a larger range of traction stresses, whereas the MDA-231MYOF-KD cells maintained a more consistent traction stress over the cell area (Figure 6B). Since contractile force generation by internal actin-myosin interactions is related to the amount of traction stress exerted by the cell, our data indicate that depletion of MYOF resulted in a significant reduction in the cell’s contractility or contractile force generation.

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