Weiße et al. Molecular Cancer (2021) 20:88 https://doi.org/10.1186/s12943-021-01384-w RESEARCH Open Access Identification of lymphocyte cell-specific protein-tyrosine kinase (LCK) as a driver for invasion and migration of oral cancer by tumor heterogeneity exploitation Jonas Weiße1, Julia Rosemann1†, Lisa Müller2†, Matthias Kappler3, Alexander W. Eckert4, Markus Glaß5, Danny Misiak5, Stefan Hüttelmaier5, Wolfgang G. Ballhausen6, Mechthild Hatzfeld2, Monika Haemmerle7 and Tony Gutschner1* Abstract Background: Cancer metastases are the main cause of lethality. The five-year survival rate for patients diagnosed with advanced stage oral cancer is 30%. Hence, the identification of novel therapeutic targets is an urgent need. However, tumors are comprised of a heterogeneous collection of cells with distinct genetic and molecular profiles that can differentially promote metastasis making therapy development a challenging task. Here, we leveraged intratumoral heterogeneity in order to identify drivers of cancer cell motility that might be druggable targets for anti-metastasis therapy. Methods: We used 2D migration and 3D matrigel-based invasion assays to characterize the invasive heterogeneity among and within four human oral cancer cell lines in vitro. Subsequently, we applied mRNA-sequencing to map the transcriptomes of poorly and strongly invasive subclones as well as primary tumors and matched metastasis. Results: We identified SAS cells as a highly invasive oral cancer cell line. Clonal analysis of SAS yielded a panel of 20 subclones with different invasive capacities. Integrative gene expression analysis identified the Lymphocyte cell- specific protein-tyrosine kinase (LCK) as a druggable target gene associated with cancer cell invasion and metastasis. Inhibition of LCK using A-770041 or dasatinib blocked invasion of highly aggressive SAS cells. Interestingly, reduction of LCK activity increased the formation of adherens junctions and induced cell differentiation. Conclusion: Analysis of invasive heterogeneity led to the discovery of LCK as an important regulator of motility in oral cancer cells. Hence, small molecule mediated inhibition of LCK could be a promising anti-metastasis therapy option for oral cancer patients. Keywords: Clonal heterogeneity, Dasatinib, EMT, HNSCC, Invasion, ITH, Metastasis, OSCC, Src * Correspondence: [email protected] †Julia Rosemann and Lisa Müller contributed equally to this work. 1Junior Research Group ‘RNA biology and pathogenesis’, Faculty of Medicine, Martin Luther University Halle-Wittenberg, 06120 Halle, Germany Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Weiße et al. Molecular Cancer (2021) 20:88 Page 2 of 23 Background environments [15–17]. An important source of plasticity Oral cancers of the lip, tongue and oral cavity were esti- of malignancy is epithelial-to-mesenchymal transition mated to account for ~ 355,000 newly diagnosed neopla- (EMT), an epigenetically controlled program that en- sia and over 177,000 cancer deaths in 2018 [1, 2]. Oral ables transitions between different phenotypic states that squamous cell carcinomas (OSCC), a subgroup of head confer motility and enhance survival [5]. EMT is associ- and neck squamous cell carcinoma (HNSCC), account ated with a loss of cell adhesiveness and polarity as well for more than 90% of all oral cancers. Distinct pheno- as cytoskeletal and signaling changes thereby enhancing types are differentiated in the oral mucosa depending on the ability of cancer cells to migrate and invade. The their environment. Whereas the gingival epithelium con- underlying regulatory mechanisms have been extensively stitutes of a stratified squamous keratinized epithelium, studied and a critical role for EMT along the metastatic the oral sulcular epithelium appears to be stratified and cascade has been described in several cancer entities in- non-keratinized [3]. OSCC can originate from altered cluding oral cancers [18, 19]. However, some controver- stem cells or through dedifferentiation of early-stage dif- sies regarding the relevance of EMT exist and alternative ferentiated cells [4, 5]. Well-known risk factors of oral mechanisms of migration should be considered, also in cancer are tobacco and alcohol abuse, consumption of conjunction with EMT, to capture the full spectrum of areca nut products as well as infection with human pap- cell states, which is a prerequisite to develop effective illoma virus (HPV) [6–8]. Several genetic and epigenetic anti-metastasis strategies [20, 21]. Another important as- alterations that lead to genomic instability and loss of pect that needs to be considered is tumor heterogeneity. tumor suppressor genes (TP53, CDKN2A, RB1, RBL1/2) Heterogeneity exists on multiple levels: in between dif- as well as activation of oncogenic signaling pathways in- ferent patients of the same tumor entity (inter-tumoral cluding epithelial growth factor receptor (EGFR), heterogeneity), between metastasis of the same patient phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian (inter-metastatic heterogeneity) or among the cells of target of rapamycin (mTOR), mitogen-activated protein one tumor (intra-tumoral heterogeneity, ITH). ITH is a kinase (MAPK), and Janus kinase/signal transducers and key factor for drug resistance and metastasis formation activators of transcription (JAK/STAT) have been associ- [22]. In HNSCC and especially OSCC patients, a high ated with oral cancer [9, 10]. Despite significant ad- ITH was described by several studies [23–25]. Clonal vances in the diagnosis and treatment of oral cancers, heterogeneity accelerates metastasis dissemination by in- the identification of novel therapeutic targets is an ur- creasing the probability for invasive subclones. Here, we gent need given the 5-year survival rate of ~ 30% for pa- reasoned that pre-existing ITH within oral cancer cell tients with advanced disease [11, 12]. Once patients populations could be exploited not only to gain a deeper develop distant metastases, the median time to death is understanding of the molecular mechanisms of metasta- only 3.3 months [13]. Thus, thorough investigations of sis, but also to identify important and potentially drug- the mechanisms involved in oral cancer metastasis are gable target genes. By isolating individual subclones of a needed to develop effective therapeutics to extend the highly invasive oral cancer cell line, we identified the life of patients. However, our current understanding of Lymphocyte cell-specific protein-tyrosine kinase (LCK) the underlying molecular processes is still very limited as an important regulator of migration and invasion. In- and druggable targets involved in the metastatic dissem- hibition of LCK using a clinically approved drug, i.e. ination of oral cancer cells are yet to be revealed. Im- dasatinib, induced cancer cell differentiation and im- portantly, the invasion-metastasis cascade is a multistep paired cell motility by enhancing the formation of adhe- process including several steps that have to be taken by rens junctions. Hence, LCK might be a promising target tumor cells in order to spread from the primary tumor that should be further evaluated in pre-clinical and clin- site to a distant location [14]. It starts with cells detach- ical studies in the future. ing from the primary tumor and locally breaching through the basement membrane in order to invade the Methods surrounding extracellular matrix and connective tissue. Cell culture, subclone generation, and drug treatments Upon intravasation into the blood or lymphatic vessels The human squamous cell carcinoma cell lines FaDu cancer cells travel to distant anatomical sites where they (hypopharynx), Cal33 (oral tongue), XF354 (oral cavity) extravasate from the vessels and invade into the stroma and SAS (oral tongue) were a kind gift of Prof. Daniel of the metastatic site. Here, the tumor cells form micro- Zips and have been cultured in RPMI-1640 medium metastases and eventually start their proliferative pro- supplemented with 10% fetal bolvine serum (FBS), 100 gram in order to colonize the tissue. Importantly, Units/ml penicillin, 100 μg/ml streptomycin (Thermo phenotypic plasticity markedly influences the metastatic Fisher Scientific, Waltham), 2 mM L-Glutamine and 1 progression, as it enables tumor cells to tolerate and mM sodium pyruvate (Sigma Life Science, St. Louis) at adapt to several different stress factors and changing 37 °C and 5% CO2. SAS