Carcinoma associated fibroblasts (CAFs) promote breast cancer motility by suppressing mammalian Diaphanous-related formin-2 (mDia2)

Abstract

The tumor microenvironment (TME) promotes tumor cell invasion and metastasis. An important step in the shift to a pro-cancerous microenvironment is the transformation of normal stromal fibroblasts to carcinoma-associated fibroblasts (CAFs). CAFs are present in a majority of solid tumors and can directly promote tumor cell motility via cytokine, chemokine and growth factor secretion into the TME. The exact effects that the TME has upon cytoskeletal regulation in motile tumor cells remain enigmatic. The conserved formin family of cytoskeleton regulating proteins plays an essential role in the assembly and/or bundling of unbranched actin filaments. Mammalian Diaphanous-related formin 2 (mDia2/DIAPH3/Drf3/Dia) assembles a dynamic F-actin cytoskeleton that underlies tumor cell migration and invasion. We therefore sought to understand whether CAF-derived chemokines impact breast tumor cell motility through modification of the formin-assembled F-actin cytoskeleton. In MDA-MB-231 cells, conditioned media (CM) from WS19T CAFs, a human breast tumor-adjacent CAF line, significantly and robustly increased wound closure and invasion relative to normal human mammary fibroblast (HMF)-CM. WS19T-CM also promoted proteasome-mediated mDia2 degradation in MDA-MB-231 cells relative to control HMF-CM and WS21T CAF-CM, a breast CAF cell line that failed to promote robust MDA-MB-231 migration. Cytokine array analysis of CM identified up-regulated secreted factors in WS19T relative to control WS21T CM. We identified CXCL12 as a CM factor influencing loss of mDia2 protein while increasing MDA-MB-231 cell migration. Our data suggest a mechanism whereby CAFs promote tumor cell migration and invasion through CXCL12 secretion to regulate the mDia2-directed cytoskeleton in breast tumor cells.

Fig 1. WS19T carcinoma associated fibroblasts (CAFs) and WS19T-CM significantly increase MDA-MB-231 breast adenocarcinoma cell motility.

A-E. MDA-MB-231 breast tumor cells, WS19T CAFs, and MDA-MB-231 cells + WS19T CAFs co-culture (1:1 ratio) were plated and grown to confluency. Wound closures were also assessed over 16h in the presence of DMEM growth media, NIH 3T3-CM, HMF-CM, WS19T-CM, or WS21T-CM. For B, *p<0.001; **p<0.03 relative to DMEM control. For E, *p<0.008 relative to DMEM control. Each experiment was performed in triplicate and repeated thrice. Scale bars = 1000μm.

Fig 2. WS19T-CM reduces MDA-MB-231 cell proliferation.

A. MTT analysis of MDA-MB-231 cells + DMEM or WS19T-CM for 16-72h. *p<0.0001 relative to DMEM at the corresponding time point. B. 50,000 MDA-MB-231 cells were initially plated into DMEM or WS19T-CM and grown through 72h, manually counting at the indicated time points. *p<0.0001 relative to DMEM control at the corresponding time point. Three independent experiments each consisting of nine replicates per condition.

Fig 3. WS19T-CM increases MDA-MB-231 tumor cell invasion.

A. Representative images from MDA-MB-231 spheroids embedded in 2 mg/ml Type 1 collagen and incubated with DMEM growth media or WS19T-CM through 72h. Scale bars = 1000 μm. B. MDA-MB-231 spheroids were embedded in collagen and incubated with serum-free media (SFM), human mammary fibroblast-CM (HMF), DMEM + 10% FBS (DMEM), or WS19T-CM (CM). The area of each spheroid was measured at the time of embedding (T0) and at the indicated time points. Data are expressed as change in area relative to T0. Each condition was performed in triplicate and repeated thrice. *p<0.0001; **p<0.03; ***p<0.003.

Fig 4. WS19T-CM reduces mDia2 protein expression.

A. MDA-MB-231 cells were incubated in monolayers with WS19T-CM for 8-72h and western blotted for the indicated proteins. B. Densitometry was performed on A using Image J and mDia2 expression was normalized to GAPDH and compared to MDA-MB-231 cells in DMEM. *p<0.001 relative to DMEM (0h). C. MDA-MB-231 cells spheroids were cultured in WS19T-CM for 48-72h and cell lysates were blotted for the indicated proteins. Mono = MDA-MB-231 monolayer lysate. The DMEM condition were cells held in DMEM for the duration of the experiment. The D/D condition are cells that were plated in DMEM and underwent a media change at the same time point as the CM condition but was changed back into DMEM. D. Densitometry was performed on C using Image J with mDia2 expression normalized to GAPDH and compared to MDA-MB-231 spheroids in DMEM. *p<0.004 relative to DMEM. Each experiment was repeated thrice. E-G. MDA-MB-231 cells were incubated in monolayers with HMF-CM, 3T3-CM, or WS21T-CM for 8-72h and western blotted for the indicated proteins.

Fig 5. WS19T-mediated mDia2 loss is recoverable upon washout.

A. MDA-MB-231 cells were incubated in DMEM with no media change (DMEM), WS19T-CM (CM), or DMEM with a media refresh of DMEM (D/D) for 8h. Cells in WS19T-CM were then released into DMEM after the 8h incubation. Western blots were performed on cell lysates for the indicated proteins. B. Densitometry was performed on A using Image J with mDia2 expression normalized to GAPDH and compared to DMEM. *p<0.03 relative to DMEM. The experiment was performed in quadruplicate.

Fig 6. WS19T-CM does not decrease mDia2 mRNA levels.

A. MDA-MB-231 cells were treated for the indicated times with either DMEM growth media or WS19T CM, and qRT-PCR performed in triplicate. Data are expressed as expression fold change (2^-ΔΔCt) relative to A. cyclophilin B (PPIB) or B. GAPDH. No significant differences were observed. The experiment was performed 4 times.

Fig 7. mDia2 functional inhibition does not affect mDia2 protein expression.

A. MDA-MB-231 cells were grown to 60% confluency and treated with vehicle (DMSO) or 40μM SMIFH2 for 1-16h prior to lysate collection and Western blotting. B. Densitometry from A. where mDia2 was normalized to GAPDH and compared relative to DMEM. C. MDA-MB-231 cells were treated with 40μM SMIFH2 starting at T0 continuously for 16h during a wound closure experiment. Experiments were repeated thrice.

Fig 8. WS19T-CM mediated mDia2 loss is proteasome-dependent.

A. MDA-MB-231 cells were treated with 10μg/mL cycloheximide (CHX) or WS19T-CM for 1-24h. Cell lysates assessed by Western blotting. mDia2 expression was normalized to GAPDH of the respective 0h condition and compared to mDia2 expression at the respective 0h condition. B. Normalized mDia2 expression was graphed versus time. A fit line was generated and the resulting equation was used for extrapolating unknown values. All experiments were repeated thrice. C. MDA-MB-231 cells were grown to 60% confluency. Cells were incubated in DMEM, vehicle (water), WS19T-CM (CM), lactacystin (Lact or L), or WS19T-CM+ lactacystin (Lact+CM, L+CM) for 16h prior to cell lysate collection and Western blotting. D. Densitometry was performed on C, where mDia2 expression was normalized to GAPDH expression and compared relative to MDA-MB-231 cells in full DMEM. *p<0.01 compared to DMEM. E. MDA-MB-231 cells were treated as in C. at T0h continuously for 16h during a wound closure experiment.

Fig 9. WS19T-CM upregulates a panel of cancer-associated cytokines.

A. Conditioned media from HMF, WS21T CAFs, and WS19T CAFs were analyzed using membrane-based cytokine antibody arrays. Samples were assessed in duplicate. The experiment was repeated three times. B. Densitometry of WS19T-CM and WS21T-CM treated cytokine membranes were normalized to HMF-CM.

Fig 10. CXCL12 mediates MDA-MB-231 mDia2 downregulation while cell migration.

A. HMF, WS21T, and WS19T fibroblasts were plated and CM collected concurrently for each replicate. HMF-CM, WS19T-CM, and WS21T-CM from three independent collections were applied in triplicate to a CXCL12/SDF1α ELISA assay. The experiment was repeated three times. CXCL12 levels were averaged for each CM and compared to HMF-CM. *p<0.0008 HMF-CM relative to WS21T-CM, WS21T-CM relative to WS19T-CM, and HMF-CM relative to WS19T-CM. p<0.001. B. MDA-MB-231 cells were treated with 15, 25, and 100ng/ml CXCL12 and wound closure assays performed for 16h. The assay performed in triplicate and repeated three times. *p<0.001 C. MDA-MB-231 cells were treated with 100ng/ml CXCL12 for 8-72h and cell lysates were Western blotted. mDia2 expression was normalized to GAPDH and compared to the DMEM control. The experiment was repeated three times. *p<0.01 D. MDA-MB-231 cells were treated with WS19T-CM as indicated. Cells were pretreated (P) for 15m with AMD3100, and/or simultaneously and continuously (C) with AMD3100 and CM (CM) or DMEM (DM) for 16h. Cell lysates were Western blotted as indicated.