The SOX9 transcription factor ensures proper tissue development and homeostasis and has been implicated in promoting tumor progression. However, the role of SOX9 as a driver of lung adenocarcinoma (LUAD), or any cancer, remains unclear. Using CRISPR/Cas9 and Cre-LoxP gene knockout approaches in the KrasG12D-driven mouse LUAD model, we found that loss of Sox9 significantly reduces lung tumor development, burden and progression, contributing to significantly longer overall survival. SOX9 consistently drove organoid growth in vitro, but SOX9-promoted tumor growth was significantly attenuated in immunocompromised mice compared to syngeneic mice. We demonstrate that SOX9 suppresses immune cell infiltration and functionally suppresses tumor associated CD8+ T, natural killer and dendritic cells. These data were validated by flow cytometry, gene expression, RT-qPCR, and immunohistochemistry analyses in KrasG12D-driven murine LUAD, then confirmed by interrogating bulk and single-cell gene expression repertoires and immunohistochemistry in human LUAD. Notably, SOX9 significantly elevates collagen-related gene expression and substantially increases collagen fibers. We propose that SOX9 increases tumor stiffness and inhibits tumor-infiltrating dendritic cells, thereby suppressing CD8+ T cell and NK cell infiltration and activity. Thus, SOX9 drives KrasG12D-driven lung tumor progression and inhibits anti-tumor immunity at least partly by modulating the tumor microenvironment.
This is a preview of subscription content, access via your institution
Subscribe to this journal
Receive 50 print issues and online access
$259.00 per year
only $5.18 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
The datasets generated during this study can be accessed here: https://doi.org/10.6084/m9.figshare.20810164.
Jo A, Denduluri S, Zhang B, Wang Z, Yin L, Yan Z, et al. The versatile functions of Sox9 in development, stem cells, and human diseases. Genes Dis. 2014;1:149–61.
Lefebvre V, Angelozzi M, Haseeb A. SOX9 in cartilage development and disease. Curr Opin Cell Biol. 2019;61:39–47.
Haseeb A, Kc R, Angelozzi M, de Charleroy C, Rux D, Tower RJ, et al. SOX9 keeps growth plates and articular cartilage healthy by inhibiting chondrocyte dedifferentiation/osteoblastic redifferentiation. Proc Natl Acad Sci USA. 2021;118:e2019152118.
Rockich BE, Hrycaj SM, Shih HP, Nagy MS, Ferguson MA, Kopp JL, et al. Sox9 plays multiple roles in the lung epithelium during branching morphogenesis. Proc Natl Acad Sci USA. 2013;110:E4456–4464.
Akiyama H, Chaboissier MC, Martin JF, Schedl A, de Crombrugghe B. The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes Dev. 2002;16:2813–28.
Akiyama H, Kim JE, Nakashima K, Balmes G, Iwai N, Deng JM, et al. Osteo-chondroprogenitor cells are derived from Sox9 expressing precursors. Proc Natl Acad Sci USA. 2005;102:14665–70.
Guven A, Kalebic N, Long KR, Florio M, Vaid S, Brandl H, et al. Extracellular matrix-inducing Sox9 promotes both basal progenitor proliferation and gliogenesis in developing neocortex. Elife. 2020;9:e49808.
Seymour PA, Freude KK, Tran MN, Mayes EE, Jensen J, Kist R, et al. SOX9 is required for maintenance of the pancreatic progenitor cell pool. Proc Natl Acad Sci USA. 2007;104:1865–70.
Foster JW, Dominguez-Steglich MA, Guioli S, Kwok C, Weller PA, Stevanovic M, et al. Campomelic dysplasia and autosomal sex reversal caused by mutations in an SRY-related gene. Nature. 1994;372:525–30.
Wagner T, Wirth J, Meyer J, Zabel B, Held M, Zimmer J, et al. Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9. Cell. 1994;79:1111–20.
Barrionuevo F, Bagheri-Fam S, Klattig J, Kist R, Taketo MM, Englert C, et al. Homozygous inactivation of Sox9 causes complete XY sex reversal in mice. Biol Reprod. 2006;74:195–201.
Ruan H, Hu S, Zhang H, Du G, Li X, Li X, et al. Upregulated SOX9 expression indicates worse prognosis in solid tumors: a systematic review and meta-analysis. Oncotarget. 2017;8:113163–73.
Aguilar-Medina M, Avendano-Felix M, Lizarraga-Verdugo E, Bermudez M, Romero-Quintana JG, Ramos-Payan R, et al. SOX9 stem-cell factor: clinical and functional relevance in cancer. J Oncol. 2019;2019:6754040.
Panda M, Tripathi SK, Biswal BK. SOX9: An emerging driving factor from cancer progression to drug resistance. Biochim Biophys Acta Rev Cancer. 2021;1875:188517.
Wang HY, Lian P, Zheng PS. SOX9, a potential tumor suppressor in cervical cancer, transactivates p21WAF1/CIP1 and suppresses cervical tumor growth. Oncotarget. 2015;6:20711–22.
Passeron T, Valencia JC, Namiki T, Vieira WD, Passeron H, Miyamura Y, et al. Upregulation of SOX9 inhibits the growth of human and mouse melanomas and restores their sensitivity to retinoic acid. J Clin Invest. 2009;119:954–63.
Zhou H, Qin Y, Ji S, Ling J, Fu J, Zhuang Z, et al. SOX9 activity is induced by oncogenic Kras to affect MDC1 and MCMs expression in pancreatic cancer. Oncogene. 2018;37:912–23.
Capaccione KM, Hong X, Morgan KM, Liu W, Bishop JM, Liu L, et al. Sox9 mediates Notch1-induced mesenchymal features in lung adenocarcinoma. Oncotarget. 2014;5:3636–50.
Ling S, Chang X, Schultz L, Lee TK, Chaux A, Marchionni L, et al. An EGFR-ERK-SOX9 signaling cascade links urothelial development and regeneration to cancer. Cancer Res. 2011;71:3812–21.
Song S, Ajani JA, Honjo S, Maru DM, Chen Q, Scott AW, et al. Hippo coactivator YAP1 upregulates SOX9 and endows esophageal cancer cells with stem-like properties. Cancer Res. 2014;74:4170–82.
Schmidlin CJ, Zeng T, Liu P, Wei Y, Dodson M, Chapman E, et al. Chronic arsenic exposure enhances metastatic potential via NRF2-mediated upregulation of SOX9. Toxicol Appl Pharmacol. 2020;402:115138.
Zhang S, Che D, Yang F, Chi C, Meng H, Shen J, et al. Tumor-associated macrophages promote tumor metastasis via the TGF-beta/SOX9 axis in non-small cell lung cancer. Oncotarget. 2017;8:99801–15.
Hong X, Liu W, Song R, Shah JJ, Feng X, Tsang CK, et al. SOX9 is targeted for proteasomal degradation by the E3 ligase FBW7 in response to DNA damage. Nucleic Acids Res. 2016;44:8855–69.
Shao N, Huang H, Idris M, Peng X, Xu F, Dong S, et al. KEAP1 Mutations Drive Tumorigenesis by Suppressing SOX9 Ubiquitination and Degradation. Adv Sci (Weinh). 2020;7:2001018.
Luanpitpong S, Li J, Manke A, Brundage K, Ellis E, McLaughlin SL, et al. SLUG is required for SOX9 stabilization and functions to promote cancer stem cells and metastasis in human lung carcinoma. Oncogene. 2016;35:2824–33.
Suryo Rahmanto A, Savov V, Brunner A, Bolin S, Weishaupt H, Malyukova A, et al. FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma. EMBO J. 2016;35:2192–212.
Huang JQ, Wei FK, Xu XL, Ye SX, Song JW, Ding PK, et al. SOX9 drives the epithelial-mesenchymal transition in non-small-cell lung cancer through the Wnt/beta-catenin pathway. J Transl Med. 2019;17:143.
Tripathi SK, Biswal BK. SOX9 promotes epidermal growth factor receptor-tyrosine kinase inhibitor resistance via targeting beta-catenin and epithelial to mesenchymal transition in lung cancer. Life Sci. 2021;277:119608.
Tripathi SK, Sahoo RK, Biswal BK. SOX9 as an emerging target for anticancer drugs and a prognostic biomarker for cancer drug resistance. Drug Discov Today. 2022;27:2541–50.
Cancer Genome Atlas Research N. Comprehensive molecular profiling of lung adenocarcinoma. Nature. 2014;511:543–50.
Jiang SS, Fang WT, Hou YH, Huang SF, Yen BL, Chang JL, et al. Upregulation of SOX9 in lung adenocarcinoma and its involvement in the regulation of cell growth and tumorigenicity. Clin Cancer Res. 2010;16:4363–73.
Kopp JL, von Figura G, Mayes E, Liu FF, Dubois CL, Morris JPt, et al. Identification of Sox9-dependent acinar-to-ductal reprogramming as the principal mechanism for initiation of pancreatic ductal adenocarcinoma. Cancer Cell. 2012;22:737–50.
Grimm D, Bauer J, Wise P, Kruger M, Simonsen U, Wehland M, et al. The role of SOX family members in solid tumours and metastasis. Semin Cancer Biol. 2020;67:122–53.
Zhou CH, Ye LP, Ye SX, Li Y, Zhang XY, Xu XY, et al. Clinical significance of SOX9 in human non-small cell lung cancer progression and overall patient survival. J Exp Clin Cancer Res. 2012;31:18.
Sanchez-Rivera FJ, Papagiannakopoulos T, Romero R, Tammela T, Bauer MR, Bhutkar A, et al. Rapid modelling of cooperating genetic events in cancer through somatic genome editing. Nature. 2014;516:428–31.
Jackson EL, Olive KP, Tuveson DA, Bronson R, Crowley D, Brown M, et al. The differential effects of mutant p53 alleles on advanced murine lung cancer. Cancer Res. 2005;65:10280–8.
Ambrogio C, Gomez-Lopez G, Falcone M, Vidal A, Nadal E, Crosetto N, et al. Combined inhibition of DDR1 and Notch signaling is a therapeutic strategy for KRAS-driven lung adenocarcinoma. Nat Med. 2016;22:270–7.
Shultz LD, Lyons BL, Burzenski LM, Gott B, Chen X, Chaleff S, et al. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells. J Immunol. 2005;174:6477–89.
Mikucki ME, Fisher DT, Matsuzaki J, Skitzki JJ, Gaulin NB, Muhitch JB, et al. Non-redundant requirement for CXCR3 signalling during tumoricidal T-cell trafficking across tumour vascular checkpoints. Nat Commun. 2015;6:7458.
Spranger S, Dai D, Horton B, Gajewski TF. Tumor-Residing Batf3 Dendritic Cells Are Required for Effector T Cell Trafficking and Adoptive T Cell Therapy. Cancer Cell. 2017;31:711–23 e714.
Harlin H, Meng Y, Peterson AC, Zha Y, Tretiakova M, Slingluff C, et al. Chemokine expression in melanoma metastases associated with CD8+ T-cell recruitment. Cancer Res. 2009;69:3077–85.
Muthuswamy R, McGray AR, Battaglia S, He W, Miliotto A, Eppolito C, et al. CXCR6 by increasing retention of memory CD8(+) T cells in the ovarian tumor microenvironment promotes immunosurveillance and control of ovarian cancer. J Immunother Cancer. 2021;9:e003329.
Waskow C, Liu K, Darrasse-Jeze G, Guermonprez P, Ginhoux F, Merad M, et al. The receptor tyrosine kinase Flt3 is required for dendritic cell development in peripheral lymphoid tissues. Nat Immunol. 2008;9:676–83.
Wang Y, Qi Z, Zhou M, Yang W, Hu R, Li G, et al. Stanniocalcin1 promotes cell proliferation, chemoresistance and metastasis in hypoxic gastric cancer cells via Bcl2. Oncol Rep. 2019;41:1998–2008.
Chakraborty A, Brooks H, Zhang P, Smith W, McReynolds MR, Hoying JB, et al. Stanniocalcin-1 regulates endothelial gene expression and modulates transendothelial migration of leukocytes. Am J Physiol Renal Physiol. 2007;292:F895–904.
Lin H, Kryczek I, Li S, Green MD, Ali A, Hamasha R, et al. Stanniocalcin 1 is a phagocytosis checkpoint driving tumor immune resistance. Cancer Cell. 2021;39:480–93 e486.
Takizawa N, Hironaka T, Mae K, Ueno T, Horii Y, Nagasaka A, et al. GPRC5B promotes collagen production in myofibroblasts. Biochem Biophys Res Commun. 2021;561:180–6.
Ricard-Blum S. The collagen family. Cold Spring Harb Perspect Biol. 2011;3:a004978.
Xiao Q, Jiang Y, Liu Q, Yue J, Liu C, Zhao X, et al. Minor type IV collagen alpha5 chain promotes cancer progression through discoidin domain receptor-1. PLoS Genet. 2015;11:e1005249.
Skinner SJ, Somervell CE, Buch S, Post M. Transferrin gene expression and transferrin immunolocalization in developing foetal rat lung. J Cell Sci. 1991;99:651–6. Pt 3.
Thorsson V, Gibbs DL, Brown SD, Wolf D, Bortone DS, Ou Yang TH, et al. The immune landscape of cancer. Immunity. 2018;48:812–830 e814.
Kim N, Kim HK, Lee K, Hong Y, Cho JH, Choi JW, et al. Single-cell RNA sequencing demonstrates the molecular and cellular reprogramming of metastatic lung adenocarcinoma. Nat Commun. 2020;11:2285.
Borgenvik A, Holmberg KO, Bolin S, Zhao M, Savov V, Rosén G, et al. Dormant SOX9-Positive Cells Facilitate MYC-Driven Recurrence of Medulloblastoma. Cancer Research. 2022;82:4586–603.
Scharf GM, Kilian K, Cordero J, Wang Y, Grund A, Hofmann M, et al. Inactivation of Sox9 in fibroblasts reduces cardiac fibrosis and inflammation. JCI Insight. 2019;4:e126721.
Ashkenazi S, Ortenberg R, Besser M, Schachter J, Markel G. SOX9 indirectly regulates CEACAM1 expression and immune resistance in melanoma cells. Oncotarget. 2016;7:30166–77.
Heng TS, Painter MW. Immunological Genome Project C. The Immunological Genome Project: networks of gene expression in immune cells. Nat Immunol. 2008;9:1091–4.
Kuczek DE, Larsen AMH, Thorseth ML, Carretta M, Kalvisa A, Siersbaek MS, et al. Collagen density regulates the activity of tumor-infiltrating T cells. J Immunother Cancer. 2019;7:68.
Gordon-Weeks A, Yuzhalin AE. Cancer extracellular matrix proteins regulate tumour immunity. Cancers (Basel). 2020;12:3331.
Nishiyama A, Xin L, Sharov AA, Thomas M, Mowrer G, Meyers E, et al. Uncovering early response of gene regulatory networks in ESCs by systematic induction of transcription factors. Cell Stem Cell. 2009;5:420–33.
Kohli K, Pillarisetty VG, Kim TS. Key chemokines direct migration of immune cells in solid tumors. Cancer Gene Ther. 2022;29:10–21.
Schulz O, Hammerschmidt SI, Moschovakis GL, Forster R. Chemokines and chemokine receptors in lymphoid tissue dynamics. Annu Rev Immunol. 2016;34:203–42.
Dangaj D, Bruand M, Grimm AJ, Ronet C, Barras D, Duttagupta PA, et al. Cooperation between constitutive and inducible chemokines enables T cell engraftment and immune attack in solid tumors. Cancer Cell. 2019;35:885–900 e810.
Tokunaga R, Zhang W, Naseem M, Puccini A, Berger MD, Soni S, et al. CXCL9, CXCL10, CXCL11/CXCR3 axis for immune activation - A target for novel cancer therapy. Cancer Treat Rev. 2018;63:40–47.
Kim CW, Kim KD, Lee HK. The role of dendritic cells in tumor microenvironments and their uses as therapeutic targets. BMB Rep. 2021;54:31–43.
Weiss A. T cell antigen receptor signal transduction: a tale of tails and cytoplasmic protein-tyrosine kinases. Cell. 1993;73:209–12.
Elder ME, Lin D, Clever J, Chan AC, Hope TJ, Weiss A, et al. Human severe combined immunodeficiency due to a defect in ZAP-70, a T cell tyrosine kinase. Science. 1994;264:1596–9.
Palacios EH, Weiss A. Distinct roles for Syk and ZAP-70 during early thymocyte development. J Exp Med. 2007;204:1703–15.
Ritthipichai K, Haymaker CL, Martinez M, Aschenbrenner A, Yi X, Zhang M, et al. Multifaceted role of BTLA in the Control of CD8(+) T-cell fate after antigen encounter. Clin Cancer Res. 2017;23:6151–64.
Taouk G, Hussein O, Zekak M, Abouelghar A, Al-Sarraj Y, Abdelalim EM, et al. CD56 expression in breast cancer induces sensitivity to natural killer-mediated cytotoxicity by enhancing the formation of cytotoxic immunological synapse. Sci Rep. 2019;9:8756.
Laughney AM, Hu J, Campbell NR, Bakhoum SF, Setty M, Lavallee VP, et al. Regenerative lineages and immune-mediated pruning in lung cancer metastasis. Nat Med. 2020;26:259–69.
Dost AFM, Moye AL, Vedaie M, Tran LM, Fung E, Heinze D, et al. Organoids model transcriptional hallmarks of oncogenic KRAS activation in lung epithelial progenitor cells. Cell Stem Cell. 2020;27:663–678 e668.
We thank Jia Peng for suggestions and discussions. We thank the Wenwei Hu laboratory for help with tissue sectioning and imaging. We thank Gina Castellano and Samantha Grabler for maintaining NSG mice for subcutaneous transplantation. We thank Lucyann Franciosa and Shafiq Bhat from CINJ histology laboratory for IHC staining. We thank Joshua Vieth and Shashi Sharma from the CINJ Immune Monitoring & Advanced Genomics core facility for the flow cytometry assay. Services and results in support of this research project were generated by the Rutgers Cancer Institute of New Jersey Histopathology and Immune Monitoring Shared Resources, supported, in part, with funding from NCI P30CA072720-5919. This work was supported by NCI R01CA190578 (SRP), R01CA237347-01A1 (JYG) and ACS 134036-RSG-19-165-01-TBG (JYG).
The authors declare no competing interest.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Zhong, H., Lu, W., Tang, Y. et al. SOX9 drives KRAS-induced lung adenocarcinoma progression and suppresses anti-tumor immunity. Oncogene 42, 2183–2194 (2023). https://doi.org/10.1038/s41388-023-02715-5