Eric Zluhan, Eric Miller and Scott Blystone!

The Formin FMNL1 is a Key Component for Metastasis!of Breast Cancer Cells MDA-MB-231!

Metastasis!

Methods!

Results!Conclusions!

Breast cancer is one of the most common types of cancer and a leading cause of death in women. The metastatic spread of cancer cells that break apart from primary tumors is the most dangerous characteristic of the disease and causes about ninety percent of breast cancer related deaths. The tumor cells can migrate to sites such as the brain, bone or lungs, and develop new deadly tumors. ! ! The actin cytoskeleton is crucial for cancer cells’ ability to metastasize. Actin filaments are organized in both adhesion stress fibers and proteolytic structures, termed invadopodia, both of which which are key for migration and invasion.!

The formin family of proteins have been shown to be key for actin assembly, facilitating the polymerization of G-actin to F-actin (see Figure 2). One of these formins, FMNL1, has been shown to be crucial in macrophage migration. Reduction of FMNL1 decreases macrophage construction of podosomes and, therefore, its migratory capabilities. ! ! FMNL1 has been shown to be present in the prototypical breast cancer cells, MDA-MB-231. We hypothesize that 1.) FMNL1 is crucial for formation of invadopodia structures in MDA-MB-231 cells due its role in assembling actin and therefore 2.) knockdown of FMNL1 will reduce invasive capabilities in MDA-MB-231 cells.!

Department of Cell & Developmental Biology!SUNY Upstate Medical University Syracuse, NY 13210!

Figure 1. Metastasis. !(1.) Cancer cell from primary tumor migrates through tissue via proteolytic invadopodia containing actin (pink). !(2.) Tumor cell trafficking to new tissue site. (3.) Cancer cell invades and establishes itself and forms a new tumor at secondary site. !

Figure 2. Active FMNL1. !Formins act as a dimeric protein unit. The NH2-terminus contains a Rho GTPase binding domain. When a Rho GTPase binds, the DID and DAD domain dissociate and the formin becomes active. The FH1 domain recruits profilin-actin complexes to the growing barbed end where the FH2 domain elongates the actin filament through a processive capping movement.!

Ø Cultured a spontaneous transformed cell line of MDA-MB-231 breast cancer cells for experiments!

Ø Administered siRNA molecules targeting FMNL1 transcripts for RNA interference!

Ø Western-blotted knockdown and control cells for levels of FMNL1 protein and transaldolase (control) !

Ø Invasion assay – Cells were placed on BD BioCoatTM MatrigelTM Invasion Chambers which acts as an in vitro basement membrane!

Ø Counted invasive cells in membrane under light microscope!

*

0

50

100

150

200

250

No Treatment 400nM Neg. Ctrl. siRNA 400nM FMNL1 siRNA

# of

Cel

ls

MDA-MB-231 Invasion Assay

Future Directions!

Figure 5. Invasion Assay Results. Numbers of invasive cells were counted under light microscope. There were fewer invasive cells observed in the membrane when they were treated with FMNL1 siRNA – about a 33% reduction compared to no treatment and negative control (scramble) siRNA. !

Ø  More Complex Invasion Assays – these will allow a more detailed analysis of how cell movement is affected!

Ø  Reduce other formins and examine the effects on cell movement!

Ø  Rescue experiment – reduce endogenous FMNL1, rescue the levels by introducing exogenous FMNL1!

Ø  In vivo studies – Our lab has developed a line of FMNL1 floxed allele mice. Crossing the floxed allele mouse with a p53 dependent spontaneous tumor forming mouse with breast epithelial Cre recombinase - followed by evaluation of tumor behavior - would be a good in vivo model and definitive test to determine metastatic behavior of FMNL1 deficient animal tumors.!

Ø  RNA interference using siRNA for FMNL1 is effective at reducing FMNL1 protein levels in MDA-MB-231 cells.!

Ø  FMNL1 is key for MDA-MB-231 cell movement. Cells with depleted levels of FMNL1 have reduced invasive capabilities as shown by the amount of cells that moved through the membrane in the invasion assay.!

Ø  There are other factors that contribute to the movement of MDA-MB-231 because while the number of cells that were able to move through the membrane are reduced, there was still a significant portion of them that successfully moved through membrane.!

Figure 3. Immunofluorescence microscopy image of MDA-MB-231 cell. Rhodamine phalloidin (red) staining of actin and FITC (green) staining of FMNL1. !

! ! Punctate actin co-localizes ! ! in regions with punctate ! ! FMNL1, suggesting !!! ! invadopodia structures are ! ! present in these areas.!

Figure 4. Western Blot of FMNL1 siRNA treated cells and control cells. Cells that were treated with FMNL1 siRNA displayed reduced levels of FMNL1 protein compared with cells treated with negative control (scramble) siRNA. 96 hour treatments with siRNA had lower levels of FMNL1 than 48 hour treatments. It was important that FMNL1 was reduced substantially at 96 hours because the invasion assay could be carried out before FMNL1 levels were restored in the cells. !

Abstract!Cancer cells utilize the actin cytoskeleton to carry out migration and invasion in metastasis. The recently identified formin family of actin assembly proteins could become a therapeutic target to reduce or prevent metastasis. One formin, FMNL1, is present in the breast cancer cells MDA-MB-231 and is implicated in the formation of actin in the invadopodias that the cells utilize for migration and invasion. We hypothesized that the reduction of FMNL1 in MDA-MB-231 (MDA) cells will reduce their metastatic capabilities. To test this, we reduced FMNL1 via RNA interference. MDA cells were treated with FMNL1 siRNA and applied to a matrigel invasion assay. Western blots were performed in parallel to confirm that FMNL1 protein levels were reduced substantially and long enough for the invasion assay to take place. FMNL1 siRNA treated cells exhibited reduced invasive capabilities. The results supported our hypothesis that FMNL1 is significant in metastasis due to its ability to assemble actin filaments in key migratory and invasive cytoskeletal structures. !!

Formins!

Adapted from Chesarone et. al 2010

Actin

FMNL1

Merge