A COL11A1-Correlated Pan-Cancer Gene Signature Of
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ARTICLE IN PRESS Cancer Letters ■■ (2016) ■■–■■ Contents lists available at ScienceDirect Cancer Letters journal homepage: www.elsevier.com/locate/canlet 1 Q2 Original Articles 2 3 A COL11A1-correlated pan-cancer gene signature of activated 4 fibroblasts for the prioritization of therapeutic targets 5 6 Q1 Dongyu Jia a, Zhenqiu Liu b, Nan Deng b, Tuan Zea Tan c, Ruby Yun-Ju Huang c, 7 Barbie Taylor-Harding a, Dong-Joo Cheon d, Kate Lawrenson a, Wolf R. Wiedemeyer a, 8 Ann E. Walts e, Beth Y. Karlan a,f, Sandra Orsulic a,f,* 9 a Women’s Cancer Program, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA 10 b Biostatistics and Bioinformatics Research Center, Cedars-Sinai Medical Center, Los Angeles, CA, USA 11 c Cancer Science Institute of Singapore, Center for Translational Medicine, National University of Singapore, Singapore 12 d Center for Cell Biology and Cancer Research, Albany Medical College, Albany, NY, USA 13 e Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA 14 f Department of Obstetrics and Gynecology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA 15 16 17 ARTICLE INFO ABSTRACT 18 2019 Article history: Although cancer-associated fibroblasts (CAFs) are viewed as a promising therapeutic target, the design 2221 Received 10 July 2016 of rational therapy has been hampered by two key obstacles. First, attempts to ablate CAFs have re- 2423 Received in revised form 30 August 2016 sulted in significant toxicity because currently used biomarkers cannot effectively distinguish activated 25 Accepted 1 September 2016 26 CAFs from non-cancer associated fibroblasts and mesenchymal progenitor cells. Second, it is unclear whether 2827 CAFs in different organs have different molecular and functional properties that necessitate organ- 3029 Keywords: specific therapeutic designs. Our analyses uncovered COL11A1 as a highly specific biomarker of activated 31 Cancer-associated fibroblasts 32 CAFs. Using COL11A1 as a ‘seed’, we identified co-expressed genes in 13 types of primary carcinoma in 33 Myofibroblasts 3534 Pan-cancer The Cancer Genome Atlas. We demonstrated that a molecular signature of activated CAFs is conserved 3736 Therapeutic targets in epithelial cancers regardless of organ site and transforming events within cancer cells, suggesting that 3938 Tumor microenvironment targeting fibroblast activation should be effective in multiple cancers. We prioritized several potential 40 pan-cancer therapeutic targets that are likely to have high specificity for activated CAFs and minimal tox- 41 icity in normal tissues. 42 © 2016 The Author(s). Published by Elsevier Ireland Ltd. This is an open access article under the CC BY- 43 NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 44 45 Introduction nisms by which activated CAFs contribute to such diverse aspects 66 46 of cancer progression are unclear, it is thought that fibroblasts 67 47 Under normal physiological conditions, collagen-rich fibro- together with increased collagen deposition and altered extracel- 68 48 blasts maintain tissue architecture and serve as a barrier to epithelial lular matrix (ECM) remodeling serve as a rich depot of cancer- 69 49 cell migration. However, cancer cells have the ability to convert promoting growth factors, cytokines, and chemokines [1,2]. 70 50 the surrounding fibroblasts into activated CAFs, which secrete spe- Additionally, altered levels of enzymes responsible for collagen 71 51 cific collagens, growth factors, and enzymes that promote cancer cross-link formation, such as lysyl oxidase (LOX) [4], increase tissue 72 52 growth, angiogenesis, invasion, and metastasis [1–3]. At the same stiffness and modify mechanotransduction resulting in the reorga- 73 53 time, these CAFs suppress anticancer immunity, confer drug resis- nization of loose connective tissue into tense linear tracks of fibers 74 54 tance and/or limit the access of chemotherapies, anti-angiogenic that serve as highways to promote chemotaxis of cancer cells 75 55 therapies, and immunotherapies [1–3]. Although the exact mecha- [5,6]. 76 56 Recognizing the crucial role of CAFs in most aspects of cancer 77 progression, it has been proposed that rational anticancer therapy 78 57 58 Abbreviations: αSMA, α-smooth muscle actin; CAFs, cancer-associated fibro- design should not only target the cancer cells but also the CAFs [7,8]. 79 59 blasts; CSPG4, chondroitin sulfate proteoglycan 4; ECM, extracellular matrix; LOX, Unlike cancer cells, CAFs are genetically stable [9], which reduce the 80 60 lysyl oxidase; EMT, epithelial–mesenchymal transition; FAP, fibroblast activation risk of therapy-induced clonal selection, resistance, and cancer re- 81 61 protein; PALLD, palladin; PDGFRα, platelet-derived growth factor receptor α; PDPN, currence. Furthermore, targeting CAFs could potentially affect 82 62 podoplanin; TGFβ, transforming growth factor β; TNC, tenascin-C; PRECOG, PREdiction multiple biochemical pathways to prevent cancer progression 83 63 of Clinical Outcomes from Genomic Profiles. 64 * Corresponding author. Fax: +1 310 423 9537. and recurrence. CAF-targeting therapeutic approaches in different 84 65 E-mail address: [email protected] (S. Orsulic). experimental mouse cancer models have been shown to improve 85 http://dx.doi.org/10.1016/j.canlet.2016.09.001 0304-3835/© 2016 The Author(s). Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/). Please cite this article in press as: Dongyu Jia, et al., A COL11A1-correlated pan-cancer gene signature of activated fibroblasts for the prioritization of therapeutic targets, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.09.001 ARTICLE IN PRESS 2 D. Jia et al. / Cancer Letters ■■ (2016) ■■–■■ 86 tumoral immune response, intratumoral drug delivery, and thera- Materials and methods 152 87 peutic efficacy [10–15]. These studies confirm the key role of CAFs Human tissues 153 88 in cancer progression and demonstrate their effectiveness as a ther- 89 apeutic target. However, targeting CAFs in some cancer models Studies involving human tissue samples were approved by the Cedars-Sinai In- 154 90 actually promoted cancer progression. For example, depletion of stitutional Review Board (IRB 15425). The samples included a tissue microarray from 155 91 αSMA+ stroma in a mouse pancreatic cancer model resulted in in- 42 patients with matched primary, metastatic, and recurrent ovarian cancer. 156 92 creased cancer aggressiveness, enhanced hypoxia and epithelial– In situ hybridization 157 93 mesenchymal transition (EMT), suppressed anticancer immunity, 94 and reduced survival [16]. The RNA hybridization kit (RNAscope 2.0 FFPE Assay) and probes for COL11A1, 158 95 The contradictory results in different cancer models could be ex- the bacterial gene dapB (negative control), and the housekeeping gene HPRT (pos- 159 96 plained by different roles of CAFs in different cancer types, i.e. CAFs itive control), were from Advanced Cell Diagnostics, Inc. Formalin-fixed, paraffin- 160 97 could be promoting breast cancer and inhibiting pancreatic cancer. embedded tissue section slides were processed by the Cedars-Sinai Biobank and 161 Translational Research Core following the protocol provided with the RNAscope In 162 98 Alternatively, in all cancer types CAFs prevent cancer progression Situ Hybridization kit from Advanced Cell Diagnostics, Inc. The slides were coun- 163 99 until they receive activating signals from cancer cells and convert terstained with Mayer’s hematoxylin. 164 100 into ‘activated CAFs’, which in turn confer invasive and metastatic 101 abilities upon cancer cells [7]. Therapies that target all CAFs are coun- Immunohistochemistry 165 102 terproductive and likely to result in the death of normal fibroblasts Immunohistochemical detection of αSMA was performed on formalin-fixed, 166 103 and significant toxicity. Preferential targeting of activated CAFs has paraffin-embedded tissue sections using the protocol provided with the pre- 167 104 been challenging because activated CAFs are poorly understood at diluted asm-1 clone antibody from Leica Microsystems. Staining was done by the 168 105 the molecular level. During activation, CAFs exhibit phenotypic Cedars-Sinai Pathology Service on the Ventana Benchmark Ultra automated slide 169 106 changes that partially overlap with myofibroblastic changes during stainer. The staining was visualized using the Ventana OptiView DAB Detection System. 170 The slides were counterstained with Mayer’s hematoxylin. 171 107 wound healing, inflammation, and fibrosis, including secretion of 108 specific ECM components, cytokines and growth factors [1,17]. In vitro co-culture experiments 172 109 Several markers have been used to distinguish activated from non- 110 activated CAFs: α-smooth muscle actin (αSMA, encoded by gene The ovarian cancer cell lines OVCAR3-GFP, KURAMOCHI-GFP and OVSAHO- 173 111 ACTA2) [3], fibroblast activation protein (FAP) [12], podoplanin GFP were maintained in RPMI-1640 (Corning) supplemented with 10% fetal bovine 174 serum (FBS) and 1x penicillin/streptomycin (Corning). Cell line authenticity was con- 175 112 (PDPN) [18], palladin (PALLD) [19,20], tenascin-C (TNC) [21], platelet- firmed by Laragen using the short tandem repeat (STR) method. The immortalized 176 113 derived growth factor receptor α (PDGFRα) [22,23], and chondroitin normal ovarian fibroblasts INOF-tdTomato cell lines [34]