Suppression of Spry4 enhances cancer stem cell properties of human MDA-MB-231 breast carcinoma cells
© Jing et al. 2016
Received: 6 November 2015
Accepted: 25 February 2016
Published: 11 March 2016
Cancer stem cells contribute to tumor initiation, heterogeneity, and recurrence, and are critical targets in cancer therapy. Sprouty4 (Spry4) is a potent inhibitor of signal transduction pathways elicited by receptor tyrosine kinases, and has roles in regulating cell proliferation, migration and differentiation. Spry4 has been implicated as a tumor suppressor and in modulating embryonic stem cells.
The purpose of this research was to test the novel idea that Spry4 regulates cancer stem cell properties in breast cancer.
Loss-of function of Spry4 in human MDA-MB-231 cell was used to test our hypothesis. Spry4 knockdown or control cell lines were generated using lentiviral delivery of human Spry4 or non-targeting control shRNAs, and then selected with 2 μg/ml puromycin. Cell growth and migratory abilities were determined using growth curve and cell cycle flow cytometry analyses and scratch assays, respectively. Xenograft tumor model was used to determine the tumorigenic activity and metastasis in vivo. Cancer stem cell related markers were evaluated using immunoblotting assays and fluorescence-activated cell sorting. Cancer stem cell phenotype was evaluated using in vitro mammosphere formation and drug sensitivity tests, and in vivo limiting dilution tumor formation assay.
Two out of three tested human Spry4 shRNAs significantly suppressed the expression of endogenous Spry4 in MDA-MB-231 cells. Suppressing Spry4 expression increased MDA-MB-231 cell proliferation and migration. Suppressing Spry4 increased β3-integrin expression, and CD133+CD44+ subpopulation. Suppressing Spry4 increased mammosphere formation, while decreasing the sensitivity of MDA-MB-231 cells to Paclitaxel treatment. Finally, suppressing Spry4 increased the potency of MDA-MB-231 cell tumor initiation, a feature attributed to cancer stem cells.
Our findings provide novel evidence that endogenous Spry4 may have tumor suppressive activity in breast cancer by suppressing cancer stem cell properties in addition to negative effects on tumor cell proliferation and migration.
KeywordsSprouty4 (Spry4) Cancer stem cells Beta3- integrin (CD61) CD133 Receptor tyrosine kinases (RTK)
Breast cancer is the most common cancer among women, and despite tremendous advances in diagnosis and treatment at an early stage, it is still the second leading cause of cancer related deaths among women in the United States . Recurrence and metastasis of the primary tumor are thought to be key contributors to the incurable nature of metastatic breast cancer. Accumulating evidence suggests that tumor recurrence, metastasis and poor clinical outcome of cancer patients is strongly influenced by a small subset of stem-like cells, also called cancer stem cells (CSCs) [2–4]. CSCs are tumor initiating cells that evade the effects of systemic therapies. They have the capacity to self-renew and differentiate into bulk tumor cells, and demonstrate resistance to standard chemotherapy [3, 4]. Despite the recognition that CSCs are a critical target for tumor eradication, the molecular regulators of CSC phenotype remain poorly understood.
Receptor tyrosine kinases (RTK) play central roles in multiple biological processes including proliferation, survival, differentiation and migration , and are often associated with normal and CSC identity mainly through activating Ras/ERK and PI3K/Akt signaling pathways [6–8]. Spry4 is a feedback regulator that is induced by RTK/MAPK kinase and restrains RTK signaling output. Spry4 displays tumor suppressor activity by inhibiting tumor cell migration and proliferation in human cancers including lung , prostate  and breast cancers . This study tests the novel idea that Spry4 regulates properties of the CSC in breast carcinoma. We used lentiviral delivery of human Spry4 shRNAs to suppress endogenous Spry4 expression in human MDA-MB-231 cells, and found this efficiently altered the phenotype of the CSC subpopulation, leading to a more malignant and drug-resistant phenotype. Our studies suggest that the endogenous activity of Spry4 targets CSCs to promote the tumor suppressive phenotype.
MDA-MB-231 breast cancer cell from ATCC were cultured in α-MEM containing 10 % FBS supplemented with 1 % non-essential amino acids (invitrogen) and penicillin/streptomycin/amphotericin B. To generate stable Spry4 knockdown cells, low passage MDA-MB-231 cells (passage 10–15) were transduced with human Spry4 shRNA lentiviruses or non-targeting control lentiviruses (Open Biosystems), and selected in medium containing 2 μg/ml puromycin.
Cells were lysed in HNTG buffer [20 mM HEPES pH7.4, 150 mM NaCl, 10 % glycerol, 1 % Triton X-100, 1.5 mM MgCl2, 1.0 mM EGTA and proteinase inhibitor cocktail (Roche)]. Cell lysates were subjected to SDS-PAGE separation. Immunoblotting was performed with antibodies against Spry4, EGFR, ERK, β1-integrin, β3-integrin and Src (Santa Cruz), phosphor-ERK, phosphor-Akt, Akt, pSrc (Cell Signaling), and tubulin (Sigma).
Cell growth curve analysis and anchorage-independent colony forming assay
Spry4 knockdown (S4kd) or non-targeting (NT) control stable cell lines were trypsinized and counted. For growth curve analysis, 5 × 103 S4kd or NT stable cells were plated in each well of 12 well plates in triplicate, cultured in growth media, and counted by Coulter counter (Beckman Coulter, Inc.). For anchorage-independent colony formation, 1 × 105 S4kd or NT stable cells were mixed with medium containing 0.4 % agar and were spread on top of a bottom agar layer (0.8 % agar in growth medium). Cells were grown for 2 weeks, and colonies were counted and photographed. The diameter of the colonies was measured using Image J software (NIH).
Mammosphere assays were performed as described by Dontu  with modifications. Briefly, 5000 of NT or S4kd cells were suspended in serum-free αMEM containing 20 ng/ml FGF2, 20 ng/ml EGF (R&D Systems) and 1xB27 serum free supplement (invitrogen) and cultured on ultra-low attachment 6-well plates. Mammospheres were monitored daily by phase-contrast microscopy to ensure that they were derived from a single cell. The number of mammospheres was counted at day 10, and their size was measured using ImageJ (NIH).
Fluorescence-activated cell sorting (FACS) analysis
NT and S4kd cells were trypsinized, stained with fluor- conjugated antibodies: anti-CD61-APC, anti-CD29-PE, anti-CD133-PE, anti-CD24-FITC, anti-CD49f-FITC and anti-CD44-APC, and analyzed on FACSCalibur (BD Biosciences). The data were analyzed using FlowJo software.
NT and S4kd cells were plated in 6 well plates in triplicate at subconfluence and cultured for 24 h. Confluent cells were treated with 2 μg/ml mitomycin C for 2 h prior to cell denudation using a 1 ml pipette tips. Cells were washed with growth medium and continually cultured in growth medium containing 1 μg/ml mitomycin C for 48 h. The progress of migration was photographed in eight regions at 0, 24 and 48 h. Denuded areas were measured and quantified with Image J.
Animal xenograft analysis
Six to eight-week old NOD/SCID female mice (Jackson Laboratory) were used for xenograft tumor studies according to previous report. NT or S4kd MDA-MB-231 stable cells were harvested in the exponential growth phase using EDTA solution and washed twice with ice cold PBS, and resuspended in PBS at the dose of 1 × 106 per 200 ul. 200 ul of cells were injected into the left inguinal mammary fat pad, five mice were used per cell line. Tumor length and width was measured with a caliper weekly, and tumor volume calculated using the formula W2L/2 (L = length, W = width) . Nine weeks later when tumors were approximately 10–15 mm at their largest diameter, tumors and lungs were removed and snap frozen or fixed in 10 % formalin for further analysis. For in vivo limiting dilution assay (LDA), mice were injected with 1 × 103, 1 × 104 or 1 × 105 cells, and monitored daily. Tumor formation was verified at end-stage after 4 month after tumor cell injection. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of Maine Medical Center, and conducted in compliance with regulatory guidelines involving the use of vertebrate animals in biomedical research.
The results were presented as mean ± SD and analyzed with Student’s t test. P < 0.05 was denoted as statistically significant.
Suppression of Spry4 in MDA-MB-231 cells promotes cell proliferation and migration in vitro
Suppression of Spry4 potentiates MDA-MB-231 cell in vitro anchorage-independent growth, and in vivo tumor growth and lung metastasis
To test whether the in vitro features of Spry4 knockdown cells are maintained in vivo, we performed orthotopic xenograft analysis to test if knockdown of Spry4 affects the tumor formation by injecting 1 × 106 NT or S4kd#1 cells into the mammary fat pads of immunodeficient NOD/SCID mice. Tumor growth was monitored and measured weekly. All injected mice developed palpable tumors within 2 weeks. However, S4kd tumors grew to a greater final size compared to control tumors (Fig. 2c, d). Furthermore, mice with S4kd tumors had an increased rate of spontaneous lung metastases compared to mice bearing NT tumors. This was quantified by counting representative metastatic lung foci from H&E stained histological sections (Fig. 2e, f), and by using RT-qPCR to identify levels of human HPRT mRNA in the mouse lungs (Fig. 2g). Thus, the increased malignant phenotype due to loss of Spry4 was maintained in vivo in primary tumors as well as secondary, metastatic tumors.
Suppression of Spry4 increases β3-intergin expression of MDA-MB-231 cells
Suppression of Spry4 increases the CD133+ subpopulation and enhances tumorigenic potential of MDA-MB-231 cells
Suppressing Spry4 expression decreases the sensitivity of MDA-MB-231 cells to Paclitaxel treatment
Drug resistance is a feature attributed to CSCs, and is a serious obstacle to cancer therapy [3, 4]. Since suppression of Spry4 enhances the CSC phenotype, we tested cell sensitivity to Paclitaxel, a common therapy for breast cancer treatment. In clonogenic assays, S4kd cells formed more and larger colonies following a single high dosage of Paclitaxel treatment compared to NT cells (Fig. 4h, i). Measurement of cell viability using the MTT assay also showed that suppressing Spry4 decreased the sensitivity of MDA-MB-231 to Paclitaxel treatment in a range from 0.001–5 μM and increased cell survival after 24 h of treatment. Paclitaxel had higher killing potential against NT than S4kd cells (Fig. 4j). These results suggest that endogenous Spry4 in human breast cancer MDA-MB-231 cells contributes to drug sensitivity.
CSCs play critical roles in cancer progression and metastasis. Spry4 has been shown to function as tumor suppressor [9–11]. The objective of this study was to test whether the suppressive role of Spry4 in tumorigenesis involves modulation of CSCs. Using the MDA-MB-231 model, we demonstrate that suppressing endogenous Spry4 increased cell growth and migration in vitro, xenograft tumor growth and metastasis in vivo, and these effects were accompanied by an increase in β3-integrin expression. We demonstrate that Spry4 knockdown MDA-MB-231 cells led to enhancement of CSC features, including increased CD133+CD44+ subpopulation and mammosphere formation, decreased sensitivity to Paclitaxel treatment in vitro, and increased capacity for xenograft tumor initiation in vivo. Thus, our results for the first time demonstrate a role of Spry4 in modulating CSC phenotype in the MDA-MB-231 breast cancer cell model.
RTK signaling not only regulates normal embryonic stem cells, but also plays important roles in acquisition and maintenance of CSCs in many cancers including glioblastoma, breast, head and neck squamous cell carcinomas [6, 8, 25–27]. The MAPK/ERK and PI3K/Akt signaling pathways play important roles in maintaining the “stemness” of normal and CSCs. Spry family proteins function as RTK signaling modulators and regulate stem cell self-renewal, survival and differentiation [28–31]. Our findings suggest that Spry4 also regulates CSCs, and this effect may not be restrained to MDA-MB-231 cells because MAPK/ERK and PI3 K/Akt pathways are shared in different cell types. In fact, we performed a similar Spry4 knockdown analysis in HTB-126, another breast cancer cell line, and found a similar increase of CSC properties in those cells (Additional file 1: Figure S2). Further study is warranted to evaluate whether this function of Spry4 is broadly conserved in multiple cancer types and stages of progression.
The mechanism of Spry4 in regulating tumor cell migration remains unclear. Expression of integrins is correlated with disease progression and metastasis in various tumor types including lung, melanoma and breast [10, 15, 32–36]. In MDA-MB-231 cell overexpression of β3-integrin promotes cell migration and invasion in vitro, and xenograft tumor cell lung metastasis in vivo . Expression of β3-integrin has also been reported to promote spontaneous metastasis of breast tumors to bone [15, 38–40], and serves as a marker of CSCs in some murine  and human  breast tumors. The expression of integrins is also critical for mammary stem cell/progenitor behavior [42, 43] and breast carcinogenesis . Studies have shown that sustained activation of the Raf-MEK-ERK signaling pathway induced expression of β3-integrin is associated with transformed cell . PI3K/Akt signaling has also been shown to mediate IL-8 induced αvβ3 expression and motility in human chondrosarcoma cells . MDA-MB-231 cells harbor an activating mutation in Ras, suppressing Spry4 expression had mild but significant increase on pERK activation, and chemical inhibition of MEK/MAPK signaling did not eliminate the increase of β3-integrin due to suppressing Spry4. We also observe an increase of pAkt with loss of Spry4 expression in MDA-MB-231 cells, however chemical inhibition of PI3K/pAkt signaling by PI3 K inhibitor did not normalize the expression of β3-integrin in S4kd cells. We have shown that Spry4 regulates β3-integrin degradation in endothelial cell by inhibiting VEGFR mediated Src activation , however, suppressing Spry4 in MDA-MB-231 cells appears to have no effect on Src activation when cells are cultured in growth medium. Additional study of how Spry4 regulates β3-integrin expression, and further examination whether acquisition of β3-integrin is necessary for the enhanced CSC phenotype of Spry4 knockdown cells is importance for better understanding CSC biology.
HJ acquired the data, LL, CV, RF, and SCH analyzed and interpreted the data, and XY designed the experiments, analyzed the data and drafted the report, and all authors reviewed and revised it critically and approved the final version to be published. All authors read and approved the final manuscript.
We thank the MMCRI histology core Armie Mangoba, Katrina Abramo and Dr. Volkhard Lindner for histochemistry analysis. This study was supported by the Maine Cancer Foundation Accelerate Grant and a Maine Medical Center Research Program Grant to XY.
The authors declare that they have no competing interests.
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- Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin. 2014;64(1):9–29.View ArticlePubMedGoogle Scholar
- Liu H, Patel MR, Prescher JA, Patsialou A, Qian D, Lin J, Wen S, Chang YF, Bachmann MH, Shimono Y, et al. Cancer stem cells from human breast tumors are involved in spontaneous metastases in orthotopic mouse models. Proc Natl Acad Sci USA. 2010;107(42):18115–20.View ArticlePubMedPubMed CentralGoogle Scholar
- Smalley M, Piggott L, Clarkson R. Breast cancer stem cells: obstacles to therapy. Cancer Lett. 2013;338(1):57–62.View ArticlePubMedGoogle Scholar
- Geng SQ, Alexandrou AT, Li JJ. Breast cancer stem cells: multiple capacities in tumor metastasis. Cancer Lett. 2014;349(1):1–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell. 2000;103(2):211–25.View ArticlePubMedGoogle Scholar
- Gotoh N. Control of stemness by fibroblast growth factor signaling in stem cells and cancer stem cells. Curr Stem Cell Res Ther. 2009;4(1):9–15.View ArticlePubMedGoogle Scholar
- Feng Y, Dai X, Li X, Wang H, Liu J, Zhang J, Du Y, Xia L. EGF signalling pathway regulates colon cancer stem cell proliferation and apoptosis. Cell Prolif. 2012;45(5):413–9.View ArticlePubMedGoogle Scholar
- Dvorak P, Dvorakova D, Hampl A. Fibroblast growth factor signaling in embryonic and cancer stem cells. FEBS Lett. 2006;580(12):2869–74.View ArticlePubMedGoogle Scholar
- Tennis MA, Van Scoyk MM, Freeman SV, Vandervest KM, Nemenoff RA, Winn RA. Sprouty-4 inhibits transformed cell growth, migration and invasion, and epithelial-mesenchymal transition, and is regulated by Wnt7A through PPARgamma in non-small cell lung cancer. Mol Cancer Res. 2010;8(6):833–43.View ArticlePubMedPubMed CentralGoogle Scholar
- Wang J, Thompson B, Ren C, Ittmann M, Kwabi-Addo B. Sprouty4, a suppressor of tumor cell motility, is down regulated by DNA methylation in human prostate cancer. Prostate. 2006;66(6):613–24.View ArticlePubMedGoogle Scholar
- Vanas V, Muhlbacher E, Kral R, Sutterluty-Fall H. Sprouty4 interferes with cell proliferation and migration of breast cancer-derived cell lines. Tumour Biol. 2014;35(5):4447–56.View ArticlePubMedGoogle Scholar
- Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, Kawamura MJ, Wicha MS. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev. 2003;17(10):1253–70.View ArticlePubMedPubMed CentralGoogle Scholar
- Price JE. Xenograft models in immunodeficient animals: I. Nude mice: spontaneous and experimental metastasis models. Methods Mol Med. 2001;58:205–13.PubMedGoogle Scholar
- Liu H, Radisky DC, Yang D, Xu R, Radisky ES, Bissell MJ, Bishop JM. MYC suppresses cancer metastasis by direct transcriptional silencing of alphav and beta3 integrin subunits. Nat Cell Biol. 2012;14(6):567–74.View ArticlePubMedPubMed CentralGoogle Scholar
- Felding-Habermann B. Integrin adhesion receptors in tumor metastasis. Clin Exp Metastasis. 2003;20(3):203–13.View ArticlePubMedGoogle Scholar
- Gong Y, Yang X, He Q, Gower L, Prudovsky I, Vary CP, Brooks PC, Friesel RE. Sprouty4 regulates endothelial cell migration via modulating integrin beta3 stability through c-Src. Angiogenesis. 2013;16(4):861–75.View ArticlePubMedPubMed CentralGoogle Scholar
- Sasaki A, Taketomi T, Kato R, Saeki K, Nonami A, Sasaki M, Kuriyama M, Saito N, Shibuya M, Yoshimura A. Mammalian Sprouty4 suppresses Ras-independent ERK activation by binding to Raf1. Nat Cell Biol. 2003;5(5):427–32.View ArticlePubMedGoogle Scholar
- Furthauer M, Reifers F, Brand M, Thisse B, Thisse C. Sprouty4 acts in vivo as a feedback-induced antagonist of FGF signaling in zebrafish. Development. 2001;128(12):2175–86.PubMedGoogle Scholar
- Yang X, Gong Y, Tang Y, Li H, He Q, Gower L, Liaw L, Friesel RE. Spry1 and Spry4 differentially regulate human aortic smooth muscle cell phenotype via Akt/FoxO/myocardin signaling. PLoS One. 2013;8(3):e58746.View ArticlePubMedPubMed CentralGoogle Scholar
- Vaillant F, Asselin-Labat ML, Shackleton M, Forrest NC, Lindeman GJ, Visvader JE. The mammary progenitor marker CD61/beta3 integrin identifies cancer stem cells in mouse models of mammary tumorigenesis. Cancer Res. 2008;68(19):7711–7.View ArticlePubMedGoogle Scholar
- Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA. 2003;100(7):3983–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M, Jacquemier J, Viens P, Kleer CG, Liu S, et al. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell. 2007;1(5):555–67.View ArticlePubMedPubMed CentralGoogle Scholar
- Wu Y, Wu PY. CD133 as a marker for cancer stem cells: progresses and concerns. Stem Cells Dev. 2009;18(8):1127–34.View ArticlePubMedGoogle Scholar
- Grosse-Gehling P, Fargeas CA, Dittfeld C, Garbe Y, Alison MR, Corbeil D, Kunz-Schughart LA. CD133 as a biomarker for putative cancer stem cells in solid tumours: limitations, problems and challenges. J Pathol. 2013;229(3):355–78.View ArticlePubMedGoogle Scholar
- Emlet DR, Gupta P, Holgado-Madruga M, Del Vecchio CA, Mitra SS, Han SY, Li G, Jensen KC, Vogel H, Xu LW, et al. Targeting a glioblastoma cancer stem-cell population defined by EGF receptor variant III. Cancer Res. 2014;74(4):1238–49.View ArticlePubMedGoogle Scholar
- Korkaya H, Paulson A, Iovino F, Wicha MS. HER2 regulates the mammary stem/progenitor cell population driving tumorigenesis and invasion. Oncogene. 2008;27(47):6120–30.View ArticlePubMedPubMed CentralGoogle Scholar
- Abhold EL, Kiang A, Rahimy E, Kuo SZ, Wang-Rodriguez J, Lopez JP, Blair KJ, Yu MA, Haas M, Brumund KT, et al. EGFR kinase promotes acquisition of stem cell-like properties: a potential therapeutic target in head and neck squamous cell carcinoma stem cells. PLoS One. 2012;7(2):e32459.View ArticlePubMedPubMed CentralGoogle Scholar
- Felfly H, Klein OD. Sprouty genes regulate proliferation and survival of human embryonic stem cells. Sci Rep. 2013;3:2277.View ArticlePubMedPubMed CentralGoogle Scholar
- Shea KL, Xiang W, LaPorta VS, Licht JD, Keller C, Basson MA, Brack AS. Sprouty1 regulates reversible quiescence of a self-renewing adult muscle stem cell pool during regeneration. Cell Stem Cell. 2010;6(2):117–29.View ArticlePubMedPubMed CentralGoogle Scholar
- Urs S, Venkatesh D, Tang Y, Henderson T, Yang X, Friesel RE, Rosen CJ, Liaw L. Sprouty1 is a critical regulatory switch of mesenchymal stem cell lineage allocation. FASEB J. 2010;24(9):3264–73.View ArticlePubMedPubMed CentralGoogle Scholar
- Jung JE, Moon SH, Kim DK, Choi C, Song J, Park KS. Sprouty1 regulates neural and endothelial differentiation of mouse embryonic stem cells. Stem Cells Dev. 2012;21(4):554–61.View ArticlePubMedGoogle Scholar
- Kren A, Baeriswyl V, Lehembre F, Wunderlin C, Strittmatter K, Antoniadis H, Fassler R, Cavallaro U, Christofori G. Increased tumor cell dissemination and cellular senescence in the absence of beta1-integrin function. EMBO J. 2007;26(12):2832–42.View ArticlePubMedPubMed CentralGoogle Scholar
- McCabe NP, De S, Vasanji A, Brainard J, Byzova TV. Prostate cancer specific integrin alphavbeta3 modulates bone metastatic growth and tissue remodeling. Oncogene. 2007;26(42):6238–43.View ArticlePubMedPubMed CentralGoogle Scholar
- Watson-Hurst K, Becker D. The role of N-cadherin, MCAM and beta3 integrin in melanoma progression, proliferation, migration and invasion. Cancer Biol Ther. 2006;5(10):1375–82.View ArticlePubMedGoogle Scholar
- Taherian A, Li X, Liu Y, Haas TA. Differences in integrin expression and signaling within human breast cancer cells. BMC Cancer. 2011;11:293.View ArticlePubMedPubMed CentralGoogle Scholar
- Desgrosellier JS, Barnes LA, Shields DJ, Huang M, Lau SK, Prevost N, Tarin D, Shattil SJ, Cheresh DA. An integrin alpha(v)beta(3)-c-Src oncogenic unit promotes anchorage-independence and tumor progression. Nat Med. 2009;15(10):1163–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Pecheur I, Peyruchaud O, Serre CM, Guglielmi J, Voland C, Bourre F, Margue C, Cohen-Solal M, Buffet A, Kieffer N, et al. Integrin alpha(v)beta3 expression confers on tumor cells a greater propensity to metastasize to bone. FASEB J. 2002;16(10):1266–8.PubMedGoogle Scholar
- Eckhardt BL, Parker BS, van Laar RK, Restall CM, Natoli AL, Tavaria MD, Stanley KL, Sloan EK, Moseley JM, Anderson RL. Genomic analysis of a spontaneous model of breast cancer metastasis to bone reveals a role for the extracellular matrix. Molecular cancer research: MCR. 2005;3(1):1–13.PubMedGoogle Scholar
- Zhao Y, Bachelier R, Treilleux I, Pujuguet P, Peyruchaud O, Baron R, Clement-Lacroix P, Clezardin P. Tumor alphavbeta3 integrin is a therapeutic target for breast cancer bone metastases. Cancer Res. 2007;67(12):5821–30.View ArticlePubMedGoogle Scholar
- Carter RZ, Micocci KC, Natoli A, Redvers RP, Paquet-Fifield S, Martin AC, Denoyer D, Ling X, Kim SH, Tomasin R, et al. Tumour but not stromal expression of beta3 integrin is essential, and is required early, for spontaneous dissemination of bone-metastatic breast cancer. J Pathol. 2015;235(5):760–72.View ArticlePubMedGoogle Scholar
- Seguin L, Kato S, Franovic A, Camargo MF, Lesperance J, Elliott KC, Yebra M, Mielgo A, Lowy AM, Husain H, et al. An integrin beta(3)-KRAS-RalB complex drives tumour stemness and resistance to EGFR inhibition. Nat Cell Biol. 2014;16(5):457–68.View ArticlePubMedPubMed CentralGoogle Scholar
- Desgrosellier JS, Lesperance J, Seguin L, Gozo M, Kato S, Franovic A, Yebra M, Shattil SJ, Cheresh DA. Integrin alphavbeta3 drives slug activation and stemness in the pregnant and neoplastic mammary gland. Dev Cell. 2014;30(3):295–308.View ArticlePubMedPubMed CentralGoogle Scholar
- Pontier SM, Muller WJ. Integrins in mammary-stem-cell biology and breast-cancer progression–a role in cancer stem cells? J Cell Sci. 2009;122(Pt 2):207–14.View ArticlePubMedGoogle Scholar
- Desgrosellier JS, Cheresh DA. Integrins in cancer: biological implications and therapeutic opportunities. Nat Rev Cancer. 2010;10(1):9–22.View ArticlePubMedPubMed CentralGoogle Scholar
- Woods D, Cherwinski H, Venetsanakos E, Bhat A, Gysin S, Humbert M, Bray PF, Saylor VL, McMahon M. Induction of beta3-integrin gene expression by sustained activation of the Ras-regulated Raf-MEK-extracellular signal-regulated kinase signaling pathway. Mol Cell Biol. 2001;21(9):3192–205.View ArticlePubMedPubMed CentralGoogle Scholar
- Lee CY, Huang CY, Chen MY, Lin CY, Hsu HC, Tang CH. IL-8 increases integrin expression and cell motility in human chondrosarcoma cells. J Cell Biochem. 2011;112(9):2549–57.View ArticlePubMedGoogle Scholar