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-231MYOF-KD) leads to an epithelial morphology compared to the mesenchymal morphology observed in control (MDA- 231LTVC) and wild-type cells. Knockdown of MYOF led to significant reductions in cell migration velocity and MDA- 231MYOF-KD cells migrated directionally and collectively, while MDA-231LTVC cells exhibited single cell migration. Decreased migration velocity and collective migration were accompanied by significant changes in cell mechanics. MDA-231MYOF-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- 231MYOF-KD cells, tumors were smaller and demonstrated lower tumor burden. Moreover, MDA- 231MYOF-KD tumors were highly circularized and did not invade locally into the adventia in contrast to MDA- 231LTVC-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.
The human amnion is a major intrauterine source of prostaglandin (PG) E(2), a potent mediator of uterine contractions and cervical ripening. During parturition, inflammatory cytokines promote PGE(2) production through increased prostaglandin-endoperoxide synthase-2 (PTGS2, also known as cyclooxygenase-2) expression. This is mediated, in part, through activation of the transcription factor nuclear factor kappa B (NFkappaB). Prostaglandin E synthase (PTGES, also known as microsomal PGE synthase-1) acts downstream of PTGS2 and is inducibly expressed in most systems. We hypothesized that NFkappaB might regulate cytokine-induced PTGES expression in amnion cells. With amnion mesenchymal cells, we found that proinflammatory cytokines coordinately upregulated PTGS2 and PTGES mRNA expression. In parallel, increased expression of the PTGS2 and PTGES proteins was observed. In comparison, the expression of two other PGE synthases (PTGES2 and PTGES3) was unmodified. PTGES induction was blocked both in the presence of pharmacological NFkappaB inhibitors and following adenovirus-mediated overexpression of a dominant-negative NFkappaB pathway protein. In cells transiently transfected with a luciferase reporter bearing a portion (-597/+33) of the human PTGES gene promoter, interleukin-1beta (IL1B) produced a moderate increase in luciferase activity; this effect was abrogated in the presence of an indirect NFkappaB inhibitor (MG-132). Finally, a kappaB-like regulatory element was identified that, when mutated, markedly attenuated IL1B-responsive PTGES promoter activity. In conclusion, our results support a role for NFkappaB in cytokine-induced PTGES expression in amnion mesenchymal cells in vitro. By coordinately regulating PTGS2 and PTGES, NFkappaB may contribute to an inducible PGE(2) biosynthesis pathway during human parturition.
The PAT family of lipid storage droplet proteins comprised five members, each of which has become an established regulator of cellular neutral lipid metabolism. Perilipin 5 (also known as lsdp-5, MLDP, PAT-1, and OXPAT), the most recently discovered member of the family, has been shown to localize to two distinct intracellular pools: the lipid storage droplet (LD), and a poorly characterized cytosolic fraction. We have characterized the denser of these intracellular pools and find that a population of perilipin 5 not associated with large LDs resides in complexes with a discrete density (~ 1.15 g/ml) and size (~ 575 kDa). Using immunofluorescence, western blotting of isolated sucrose density fractions, native gradient gel electrophoresis, and co-immunoprecipitation, we have shown that these small (~ 15 nm), perilipin 5-encoated structures do not contain the PAT protein perilipin 2 (ADRP), but do contain perilipin 3 and several other as of yet uncharacterized proteins. The size and density of these particles as well as their susceptibility to degradation by lipases suggest that like larger LDs, they have a neutral lipid rich core. When treated with oleic acid to promote neutral lipid deposition, cells ectopically expressing perilipin 5 experienced a reorganization of LDs in the cell, resulting in fewer, larger droplets at the expense of smaller ones. Collectively, these data demonstrate that a portion of cytosolic perilipin 5 resides in high density lipid droplet complexes that participate in cellular neutral lipid accumulation.
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