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Open Full Page Published OnlineFirst September 28, 2017; DOI: 10.1158/0008-5472.CAN-17-0569 Cancer Tumor and Stem Cell Biology Research Mechano-Signal Transduction in Mesenchymal Stem Cells Induces Prosaposin Secretion to Drive the Proliferation of Breast Cancer Cells Seiichiro Ishihara1, David R. Inman1, Wan-Ju Li2, Suzanne M. Ponik1, and Patricia J. Keely1,† Abstract In response to chemical stimuli from cancer cells, mesenchymal cancer cells expressed increased levels of a-smooth muscle actin, a stem cells (MSC) can differentiate into cancer-associated fibro- marker of CAF, compared with MSC cultured on a soft matrix. By blasts (CAF) and promote tumor progression. How mechanical contrast, MSC cultured on a stiff matrix secreted prosaposin that stimuli such as stiffness of the extracellular matrix (ECM) con- promoted proliferation and survival of mammary carcinoma cells tribute to MSC phenotype in cancer remains poorly understood. but inhibited metastasis. Our findings suggest that in addition to Here, we show that ECM stiffness leads to mechano-signal trans- chemical stimuli, increased stiffness of the ECM in the tumor duction in MSC, which promotes mammary tumor growth in part microenvironment induces differentiation of MSC to CAF, trig- through secretion of the signaling protein prosaposin. On a stiff gering enhanced proliferation and survival of mammary cancer matrix, MSC cultured with conditioned media from mammary cells. Cancer Res; 77(22); 6179–89. Ó2017 AACR. Introduction many types of tissues (6). In cancer, it is thought that MSCs are recruited to the tumor microenvironment and show both protu- Tumor tissue contains multiple components, including cancer mor and antitumor phenotypes (7, 8). Recently it is reported that, cells, stromal cells, immune cells, and extracellular matrices (1), in several cancers, MSCs differentiate to CAFs and promote tumor which can interact with each other via chemical and mechanical progression in response to chemical stimuli, such as soluble stimuli and create complex biochemical and biophysical proper- factors secreted from cancer cells (9–11). However, the contribu- ties of the tumor microenvironment. For example, chemical cues tion of mechanical stimuli including stiffness of ECMs, to differ- such as cytokines and growth factors secreted from stromal and entiation of MSCs in the tumor microenvironment has not been immune cells stimulate cancer cells to promote or suppress tumor shown clearly. progression (1). Mechanical stimuli are also critical for cancer Prosaposin (PSAP, Psap) is the precursor protein for saposin A- progression, as cancer cells response to mechanical cues, such as D, which are lysosomal activator proteins that facilitate the tissue stiffness, and as a result, acquire malignant phenotypes (2, hydrolysis of sphingolipids (12). In addition to the function in 3). Many previous studies reported that interaction between lysosome, PSAP is also secreted as full-length prosaposin that cancer cells and chemical or mechanical cues play a role in cancer regulates neurotrophic action in the nerve system (12). Recently, progression or suppression; however, the interaction between PSAP was reported to promote ERK (Mapk1) activation and breast non-cancerous cells and these cues in the tumor microenviron- cancer growth (13) and saposin C, active domain of PSAP, ment has not been clearly defined. enhances survival via Akt activation in prostate cancer cells In the tumor microenvironment, carcinoma-associated fibro- (14). In contrast, PSAP inhibits metastasis via stimulating the blasts (CAF) are thought to contribute to cancer progression via expression of thorombospondin-1 in fibroblasts present in tumor chemical stimuli and modification of the extracellular environ- (15). However, the source of PSAP and its role in the tumor ment (4, 5). The origin of CAFs remains controversial, and may microenvironment are not yet fully understood. include resident fibroblasts, adipocytes, and infiltrating mesen- In this study, we investigated whether MSCs respond to ECM chymal stem cells (MSC). MSCs are multipotent cells present in stiffness and regulate growth or progression of mammary carci- noma. We revealed that stiff ECM induces differentiation of MSCs to CAFs via mechano-signal transduction, including regulation of 1Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, Wisconsin. 2Departments of Orthopedics and Rehabilitation, and YAP (Yap1) and myosin light chain (MLC, Myl9) activation. Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin. Although there has been documentation that MSCs respond to matrix stiffness to control different normal cell fates (e.g., oste- Note: Supplementary data for this article are available at Cancer Research fi Online (http://cancerres.aacrjournals.org/). ogenic, neurogenic, and myogenic; ref. 16), this is the rst study to show that a stiff matrix regulates the procarcinoma phenotype of †Deceased. mesenchymal stem cells to promote cancer growth. We also CorrespondingAuthor: Seiichiro Ishihara, University of Wisconsin-Madison, 1111 identified that PSAP is secreted by MSCs in a stiff tumor micro- Highland Avenue Room 4528, Madison, WI 53705. Phone: 608-265-5094; Fax: environment and promotes proliferation and survival in mam- 608-262-7306; E-mail: sishihara@wisc.edu mary cancer cells via Akt activation. Conditioned media of mam- doi: 10.1158/0008-5472.CAN-17-0569 mary cancer cells induced growth and morphological changes in Ó2017 American Association for Cancer Research. MSCs, suggesting that bidirectional communication between www.aacrjournals.org 6179 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst September 28, 2017; DOI: 10.1158/0008-5472.CAN-17-0569 Ishihara et al. mammary cancer cells and MSCs in a stiff tumor microenviron- Supplementary Fig. S1. After 6 days, the cells were washed with ment is critical for mammary cancer progression. PBS three times and cultured for an additional 24 hours with DMEM (nonserum, 1% antibiotics and antimycotics). Then, the Materials and Methods media was collected and centrifuged (1,300 rpm, 3 minutes). The Cell culture supernatant was filtered through a 0.22-mm pore filter. The MSCs were established from a BALBc mouse (BALB/cByJ, concentration of conditioned media is adjusted to MSC cell female, 7-weeks-old, from The Jackson Laboratory) and a B6 number quantified in collagen gels by fluorescence staining of mouse (C57BL/6, female, 7-weeks-old, The Jackson Laboratory). nuclei or calcein AM (3.4 mmol/L) treatment. The conditioned As reported previously (17), we isolated femurs from mice and media were stored at À20 CorÀ80 C. flushed the bone marrow from both ends with 1 mL of MSC- media (DMEM, Gibco, 11885092) supplemented with 20% FBS Establishment of transgenic cells (GEMINI Bio-products, 100-106) and 1% antibiotics and anti- We constructed pBABE-puro-EGFP-hYAP vector for establish- mycotics (Sigma-Aldrich, A5955). Then, the bone marrow sus- ment of EGFP-hYAP–overexpressed MSCs. For the construc- pension was filtered with a 70-mm pore cell strainer and seeded tion, we cloned the EGFP-hYAP sequence from pEGFP-C3- onto a 75-mL cell culture flask or a 6-cm cell culture plastic dish hYAP vector (a gift from Marius Sudol, Cancer Research UK with MSC-media. After 24 hours, cells were washed with phos- London Research Institute, London, United Kingdom; Addgene phate-buffered saline (PBS) and cultured with MSC-media. Media plasmid # 17843; ref. 21) by PCR with Pfu ultra-polymerase were changed every 3 days until the cells reached 70% confluence. Agilent Technologies, 600670) and inserted it into multiclon- At this point, we treated the cells with 0.25% trypsin-EDTA for 2 ing site of pBABE-puro vector [a gift from Hartmut Land, Jay minutes to collect and propagate the floating cells in a new dish Morgenstern (Imperial Cancer Research Fund), and Bob Wein- with MSC-media as MSCs. The differentiation potential to adi- berg (Whitehead Institute for Biomedical Research, Cambridge, pocytes and osteocytes was confirmed by differentiation assay. For MA), Addgene plasmid # 1764; ref. 22] by using sequence- this assay, we cultured the cells with adipogenic media (Cyagen and ligation-independent cloning method (23) with the 0 Biosciences, GUXMX-90031) or osteogenic media (Cyagen Bio- primers (forward: 5 -tccttctctaggcgccggccggatctataagcagagctggtt- 0 0 sciences, GUXMX-90021) and stained with Oil Red O or Alizarin tagtgaaccgtca-3 ,reverse:5-actgacacacattccacagggtcgacct- 0 Red S to confirm adipocytes and osteocytes, respectively. 4T1 cells gagggctctataaccatgtaagaaagc-3 ). The construction was con- (ATCC, 2012) were cultured with RPMI-1640 media (Gibco, firmed by diagnostic digestion and sequencing. IRES-EGFP– 11875119) supplemented with 10% FBS and 1% antibiotics and overexpressed MSCs; (EGFP-MSCs, control for MSCs with antimycotics. E0771 cells (kind gift from the Dr. Friedl's labora- hYAP) and EGFP-hYAP–overexpressed MSCs (YAP-MSC) were tory, 2016, University of Wisconsin-Madison) were cultured with established by retrovirus transfection of pBABE-puro-IRES- RPMI1640 media supplemented with 10% FBS, 10 mmol/L EGFP (a gift from L. Miguel Martins, MRC Toxicology Unit, HEPES, and 1% antibiotics and antimycotics. The cells cultured Leicester, United Kingdom; Addgene plasmid # 14430) and for less than 30 passages were used. 4T1 cells were authenticated pBABE-puro-EGFP-hYAP, respectively. Empty pGIPZ-transgen- and tested for mycoplasma
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