A Soft Microenvironment Protects from Failure of Midbody Abscission and Multinucleation Downstream of the EMT-Promoting Transcription Factor Snail Allison K
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Published OnlineFirst February 26, 2018; DOI: 10.1158/0008-5472.CAN-17-2899 Cancer Molecular Cell Biology Research A Soft Microenvironment Protects from Failure of Midbody Abscission and Multinucleation Downstream of the EMT-Promoting Transcription Factor Snail Allison K. Simi1, Alisya¸ A. Anlas¸1, Melody Stallings-Mann2, Sherry Zhang1, Tiffaney Hsia1, Magdalena Cichon2, Derek C. Radisky2, and Celeste M. Nelson1,3 Abstract Multinucleation is found in more than one third of tumors as well as multinucleation in a human patient sample of andislinkedtoincreasedtoleranceformutation,resistanceto metaplastic carcinoma of the breast, a rare classification char- chemotherapy, and invasive potential. The integrity of the acterized by deposition of collagen fibers and active EMT. In genome depends on proper execution of the cell cycle, which contrast, a soft microenvironment protected mammary epithe- can be altered through mechanotransduction pathways as the lial cells from becoming multinucleated by preventing Snail- tumor microenvironment stiffens during tumorigenesis. Here, induced upregulation of septin-6. Our data suggest that tissue we show that signaling downstream of matrix metalloprotei- stiffening during tumorigenesis synergizes with oncogenic sig- nase-3 (MMP3) or TGFb, known inducers of epithelial–mes- naling to promote genomic abnormalities that drive cancer enchymal transition (EMT), also promotes multinucleation in progression. stiff microenvironments through Snail-dependent expression Significance: These findings reveal tissue stiffening during of the filament-forming protein septin-6, resulting in midbody tumorigenesis synergizes with oncogenic signaling to promote persistence, abscission failure, and multinucleation. Consis- genomic abnormalities that drive cancer progression. Cancer Res; tently, we observed elevated expression of Snail and septin-6 78(9); 2277–89. Ó2018 AACR. Introduction doubling (WGD) and that WGD typically precedes other somatic copy-number alterations (4). A comparison of in situ All cancers are genomically unstable and the most common breast carcinomas revealed that multinucleation is more com- type of genomic instability is chromosomal instability, or an mon in pleomorphic lobular carcinoma than in the less inva- increased rate of chromosome missegregation. Chromosomal sive classical lobular or ductal carcinomas, suggesting that instability is characterized by aneuploidy, which is defined as multinucleation may be associated with invasive potential an abnormal number of chromosomes and is found in 85% of (5). Furthermore, induction of tetraploidy in human cells solid cancers, including 85% of breast tumors (1). Even in the promotes increased tolerance for mutation, resistance to che- absence of other defects, induction of aneuploidy is sufficient motherapeutic drugs, and transformation in culture (6). to promote tumorigenesis in vivo (2). Failure of mitosis that In addition to being genomically unstable, tumors are gen- results in multinucleation can lead to aneuploidy—multinu- erally stiffer than normal tissue. Enhanced deposition and cleated cells that continue to divide will commonly produce crosslinking increases the density, and consequently the stiff- aneuploid progeny of varying chromosomal arrangements (3), ness, of the extracellular matrix(ECM)intumors,andstiffness and there is mounting evidence that multinucleation is both is a common diagnostic parameter. Mechanosensors mediate common in cancer and indicative of tumor prognosis: An the balance between extracellular forces and intracellular cyto- analysis of eleven types of cancer from The Cancer Genome skeletal tension and in doing so translate physical changes in Atlas found that 37% percent of tumors exhibit whole-genome the microenvironment to chemical signals inside the cell. Signaling triggered by stiffening of the ECM can induce changes in phenotype and gene expression that result in deleterious 1Department of Chemical and Biological Engineering, Princeton University, consequences, including cancer progression. For example, the Princeton, New Jersey. 2Department of Cancer Biology, Mayo Clinic Cancer 3 ability of matrix metalloproteinase-3 (MMP3; stromelysin-1) Center, Jacksonville, Florida. Department of Molecular Biology, Princeton to induce epithelial–mesenchymal transition (EMT) in mam- University, Princeton, New Jersey. mary epithelial cells depends on the subcellular localization Note: Supplementary data for this article are available at Cancer Research of the Rac1 splice variant Rac1b, which is modulated by Online (http://cancerres.aacrjournals.org/). the stiffness of the surrounding microenvironment. MMP3 is Corresponding Author: Celeste M. Nelson, Princeton University, 303 Hoyt a secreted protease commonly upregulated in cancer that Laboratory, William Street, Princeton, NJ 08544. Phone: 609-258-8851; Fax: induces expression of Rac1b. Stiff substrata, with compliances 609-258-1247; E-mail: [email protected] characteristic of breast tumors, cause activation and clustering doi: 10.1158/0008-5472.CAN-17-2899 of integrins, which promotes localization of Rac1b to the Ó2018 American Association for Cancer Research. plasma membrane, activation of NADPH oxidase, production www.aacrjournals.org 2277 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst February 26, 2018; DOI: 10.1158/0008-5472.CAN-17-2899 Simi et al. of reactive oxygen species (ROS), and elevated expression of 25 mmol/L; N-acetyl-cysteine (NAC, Sigma Aldrich), 10 mmol/L; the key EMT effector and transcription factor, Snail. Soft sub- recombinant TGFb1 (R&D Systems), 10 ng/mL. Cells were strata, with compliances characteristic of normal mammary exposed to H2O2 or NAC for 72 hours and to TGFb1 for 48 tissue, prevent Rac1b membrane localization and protect hours. To increase proliferation of SCp2 cells on soft substrata, against EMT (7). 100 ng/mL of EGF was added to the culture medium for 48 hours. Through their primary role in remodeling the ECM, MMPs induce many changes in the surrounding cells, EMT serving as just Synthetic substrata one example. Notably, MMPs were previously linked to genomic Polyacrylamide (PA) substrata were generated as described instability in culture and in vivo. MMP3 in particular was found to previously (7, 14). Briefly, PA gels were polymerized directly on increase the resistance of mouse mammary epithelial cells to N- 31-mm-diameter glass coverslips as follows: 12.5% (vol/vol) (phosphonacetyl)-L-aspartate (PALA) through amplification of acrylamide was mixed with bis-acrylamide in water at either the CAD locus downstream of ROS, in addition to causing other 0.5% (vol/vol) or 17.5% (vol/vol). Polymerization was initiated genomic amplifications and deletions similar to those observed in by adding 10% ammonium persulfate (Bio-Rad) at a 1:200 transgenic mice that ectopically express MMP3 in the mammary dilution and N,N,N0,N0-tetramethylethylenediamine (Sigma gland (8, 9). Similarly, TGFb is a potent inducer of EMT that has Aldrich) at a 1:2,000 dilution. Thirty-six mL of the mixture was also been implicated in genomic instability, including promotion sandwiched between coverslips for 40 minutes at room temper- of multinucleation in MCF10A mammary epithelial cells (10). ature. PA gels were stored in PBS at 4C and characterized as The ability of TGFb to induce EMT in mammary epithelial cells is previously described (7). likewise regulated by substratum stiffness (11). The surfaces of the PA gels were then functionalized with 200 Although the mechanical properties of the microenvironment mg/mL fibronectin (BD Biosciences) as described previously using have previously been shown to regulate the cell cycle (12, 13), and the heterobifunctional crosslinker Sulfo-SANPAH (Thermo Sci- proper regulation of the cell cycle is intrinsically connected to the entific; ref. 14). Before plating cells, gels were washed three times stability of the genome, a link between abnormal mechanical with PBS and incubated with culture medium for 30 minutes at properties in the extracellular microenvironment and induction 37C. To achieve a final density of approximately 15,000 cells/ of genomic instability has not been established. We show here cm2, we initially seeded 8 Â 105,4Â 105, or 1.5 Â 105 cells onto that matrix stiffness regulates multinucleation in mammary epi- soft or stiff gels or into empty wells of a 6-well plate. thelial cells. By evaluating how cells respond to EMT inducers when cultured on engineered two-dimensional (2D) polyacryl- Transfections and viral transductions amide substrata of varying stiffness, we found that multinuclea- SCp2 cells were transfected or transduced in medium contain- tion is increased on stiff substrata through failure of midbody ing tetracycline, with the exception of the MMP3 Æ NAC and abscission as a consequence of the expression of septin-6, a novel MMP3 Æ shSnail experiments, which were performed under target of Snail. A soft microenvironment appears to protect the tetracycline withdrawal. pMK782 Septin6-GFP was obtained stability of the genome in epithelial cells by preventing septin-6 from AddGene (plasmid # 38296). pEYFP-C1 was obtained from overexpression. Taken together, our data provide evidence that BD Biosciences (catalog #6005-1). Plasmids were transfected tissue stiffening during tumorigenesis synergizes with EMT-asso- using FuGENE HD Transfection Reagent (Promega). Briefly, ciated pathways to promote genomic abnormalities that drive