Mutations in foregut SOX2+ cells induce efficient proliferation via CXCR2 pathway
Identification of the precise molecular pathways involved in oncogene-induced transformation may help us gain a better understanding of tumor initiation and promotion. Here, we demonstrate that SOX2+ foregut epithelial cells are prone to oncogenic transformation upon mutagenic insults, such as KrasG12D and p53 deletion. GFP-based lineage-tracing experiments indicate that SOX2+ cells are the cells-of-origin of esophagus and stomach hyperplasia. Our observations indicate distinct roles for oncogenic KRAS mutation and P53 deletion. p53 homozygous deletion is required for the acquisition of an invasive potential, and KrasG12D expression, but not p53 deletion, suffices for tumor formation. Global gene expression analysis reveals secreting factors upregulated in the hyperplasia induced by oncogenic KRAS and highlights a crucial role for the CXCR2 pathway in driving hyperplasia. Collectively, the array of genetic models presented here demonstrate that stratified epithelial cells are susceptible to oncogenic insults, which may lead to a better understanding of tumor initiation and aid in the design of new cancer therapeutics.
KeywordsSox2 tumor CXCR2 stratified epithelia
Cancer arises from a progressive accumulation of genetic mutations in proto-oncogenes and tumor suppressor genes (Visvader and Lindeman, 2012; Blanpain and Simons, 2013). For example, the oncogene Kras and the tumor suppressor gene p53 are frequently mutated in a wide range of human cancers (Serrano et al. 1997; Kuilman et al., 2010) and are known to induce tumor initiation in a variety of mouse models (Jackson et al., 2001; Singh et al., 2010).
Abnormal proliferative signals of oncogenic insults including oncogenic KRAS are known to activate a senescent phenotype in cells, presumably designed to prevent the growth of oncogene-transformed cells and to preserve the tumor in a non-aggressive state (Collado and Serrano, 2006). Senescent cells, in turn, secrete large amounts of cytokines and chemokines in a phenomenon known as Senescence-Associated Secretory Phenotype (SASP). Among SASP-related factors, CXC chemokines that bind to CXC chemokine receptor 2 (CXCR2) have been shown to reinforce senescence, which results in growth arrest, further preventing tumor progression (Acosta et al., 2008). However, SASP components can also dangerously stimulate a malignant phenotype and have tumor-promoting responses. Some of the factors secreted by senescent cells such as GROα, CXCL-12 or IL-8 lead to activate proliferation in the surrounding epithelial cells (Krtolica et al., 2001; Coppé et al., 2008). Therefore, the effect of SASP on cell behavior is context-dependent.
Not only is the specific genetic mutation a determining factor for tumor initiation but the cell type from which the tumor originates is also important. Cellular populations that seem to have particularly high tumorigenic potential include adult stem cells (ASCs) and progenitor cells (PCs), which normally play crucial roles in tissue homeostasis and repair (Huels and Sansom, 2015; Sanchez-Danes et al., 2016; Zhu et al., 2016). These cells might be ideal candidates to serve as the cells-of-origin for cancers and as such ASCs/PCs have been intensively studied. However, it still remains to be fully understood which cell population is prone to oncogenic transformation and what kind of oncogenic insults induce tumor initiation from certain ASCs/PCs.
Here, we sought to identify proliferative ASCs/PCs that are the most susceptible to oncogenic mutations. By initially focusing on oncogenic Kras, together with the loss of p53, we found that foregut basal cells that express SOX2 efficiently proliferated to hyperplasia in response to oncogenic mutations. We also revealed distinct roles of oncogenic KRAS and P53 deletion in driving hyperplasia. Furthermore, oncogenic Kras elevated expression of SASP-related chemokines, which contributed to the oncogenic proliferation through a CXCR2-dependent signaling pathway. Taken together, these results suggest that SOX2+ epithelial basal cells in the esophagus and stomach are highly susceptible to oncogenic stimuli. Our findings may help elucidate early events in tumor formation and the cells-of-origin of tumors, which could in turn provide insights towards a better understanding of neoplasia.
Expressing oncogenic Kras and p53 deletion in SOX2+ cells induces hyperplasia in the esophagus and forestomach
Differential impacts of oncogenic KRAS and P53 deletion on gene expression signature
SASP-related factors are involved in oncogenic Kras-mediated cellular proliferation
p53 deletion results in an invasive phenotype
ASCs/PCs are found in many tissues and organs in the adult body and are important for tissue homeostasis and regeneration upon injury but, at the same time, these cells might be ideal candidates to be the cells-of-origin for cancers (Arnold et al., 2011). Here we found that SOX2+ foregut ASCs/PCs are prone to oncogenic transformation despite the presence of SOX2+ cells in other organs, such as the lungs. Our observations indicate distinct roles for oncogenic KRAS mutation and P53 deletion in tumor formation. Global gene expression analysis reveals that secreting factors contribute to the development of oncogenic KRAS-induced tumors and highlights a crucial role for the CXCR2 pathway in driving tumor formation.
SOX2 has been reported to play an important role not only in development and somatic reprogramming but also in cancer initiation/progression. For example, amplification of the SOX2 gene has been reported in human squamous cell carcinomas (SCC) of the lung and esophagus, small-cell lung cancer (SCLC) and glioblastoma (Bass et al., 2009; Annovazzi et al., 2011; Rudin et al., 2012). Overexpression of Sox2 leads to hyperplasia and tumor formation in several tissues (Lu et al., 2010; Liu et al., 2013; Mukhopadhyay et al., 2014). Furthermore, Sox2 expression marks the tumor-initiating cell population of skin squamous cell carcinomas once Sox2 expression is induced during tumorigenesis (Boumahdi et al., 2014). SOX2+ cells are also responsible for propagating medulloblastoma and targeting them prevented tumor growth (Vanner et al., 2014). Taken together, these results indicate the importance of the SOX2 molecule and SOX2+ cells in tumor development. However, tumor susceptibility of SOX2+ cells seems oncogene-specific. A previous report showed that the loss of APC in pyloric SOX2+ cells generated tumors (Sarkar et al., 2016). Similarly, targeted expression of oncogenic β-catenin in SOX2+ cells is reported to give rise to other tumor types in a non-cell-autonomous manner (e.g., pituitary tumors) (Andoniadou et al., 2013). However, we did not observe abnormal proliferation in the glandular region as well as in the pituitary in our system. These results suggest distinct oncogenic mutation susceptibilities in SOX2+ cells throughout different tissue niches.
We also found distinct roles for KRAS and P53 in oncogenic transformation of SOX2+ cells. Oncogenic Kras expression, but not p53 deletion, was sufficient to induce a hyperplasic phenotype; and p53 deletion accelerated tumorigenic proliferation in KrasG12D-induced hyperplasia. Similarly, others have found that the loss of p53 in stem cells of the colon results in tumor formation only when combined with DNA damage and chronic inflammation (Schwitalla et al., 2013; Davidson et al., 2015). Importantly, p53 homozygous deletion along with the Kras mutation led to an invasive phenotype and highly malignant tumors, highlighting the role of P53 in tumor invasion.
We identified SASP-related chemokines as responsible factors for oncogenic Kras-dependent proliferation in the forestomach and esophagus. It is thought that SASP may be induced in senescent cells to potentiate cell proliferation of surrounding pre-tumor cells and to functionally disrupt normal tissues (Krtolica et al., 2001; Coppe et al., 2008). Some of the SASP-related chemokines activate the CXCR2-dependent signaling pathway, known to trigger a secretory network that results in growth arrest, further preventing tumor progression (Acosta et al., 2008). In fact, a previous paper showed that CXCR2 is a blockade to drive oncogene-induced senescence in pancreatic tumors (Lesina et al., 2016). Inconsistent with these reports, we found that oncogenic KRAS increased the expression of SASP-related chemokines in foregut basal cells, which contributed to oncogenic proliferation. Given that epithelial cells in the esophagus and forestomach are highly proliferative, similar to pre-tumor cells, these cells might have unique characteristics, which allow them to proliferate in response to SASP-related chemokines. Interestingly, esophageal epithelial cells express some of the pluripotency factors (unpublished data), highlighting the uniqueness of these cells.
The array of genetic tumor models generated, combined with the lineage tracing experiments and global expression analyses described here, may open new paths for a better understanding of neoplasia. They may also help the future design of therapeutics targeting the initial stages of tumor formation and progression as well as facilitate the identification of novel parameters for earlier tumor diagnosis.
Materials and Methods
Mcm2CreER/WT (Pruitt et al., 2007), Sox2CreER/WT (Arnold et al., 2011), Lgr5CreER/WT (Barker et al., 2007), KrasLSL-G12D/WT (Jackson et al., 2001), p53Flox/Flox (Jonkers et al., 2001), ROSALSL-PIK3CA(H1047R)/LSL-PIK3CA(H1047R) (Adams et al., 2011), ROSALSL-Luc/LSL-Luc (Safran et al., 2003), and ROSALSL-GFP/LSL-GFP (Mao et al., 2001) have been previously described. We used both male and female mice for this study but the same gender was used for each experiment unless otherwise stated.
To activate CRE in the mice carrying CreER, TAM, dissolved in corn oil, was given orally (50 mg/mL) or intraperitoneally (20 mg/mL) to 6- to 10-week-old animals for 5 consecutive days, unless otherwise stated.
Tissue preparation and IHC
For IHC, tissues were harvested, fixed in 10% neutralized Formalin for 2 days and then stored in 70% ethanol until further processing. H&E staining, PAS staining and IHC on paraffin-sections were performed following standard protocols. The following antibodies were used for IHC: anti-GFP (Abcam, 6673, 1:200; Clontech, JL-8, 1:100); Ki67 (Cell signaling, 12202, 1:200); Proton-pump (MBL, D032-3H, 1:100); Gastrin (Santa Cruz, sc-783, 1:200); anti-p63 (Santa Cruz, sc-56188, 1:200); anti-CK13 (Abcam, 92551, 1:1000); anti-Loricrin (Abcam, 24722, 1:1000); anti-CXCL7 (Bioss Inc., A-21235, 1:200); anti-CXCR2 (Abcam, 14935, 1:200).
Mice were examined at 3 or 4 weeks post TAM administration by BLI performed using an IVIS Kinetic 2200 from Caliper Life sciences. Mice were i.p. injected with 150 mg/kg D-Luciferin (BIOSYNTH), anesthetized with isoflurane and dorsal images were then captured 10 min post luciferin injection.
Isolated tissues were homogenized with a polytron in TRIzol. The extracted RNA was purified using the RNeasy Micro Kit (Qiagen) from the homogenates. RNA quality was assessed and all samples had a minimum RNA integrity number (RIN) of 7.8. RNA library preps were prepared using the Illumina TruSeq Stranded Total RNA Sample Prep kit with Ribo-zero Gold (cat. no. RS-122-2301). Briefly, RNA was depleted of ribosomal RNA and mitochondrial RNA, then fragmented and reverse transcribed. cDNA was end-repaired, adenylated, ligated with sequencing primers and PCR amplified. Libraries were pooled and sequenced on the HiSeq 2500 using v4 sequencing reagents at single-end 50 base-pair (bp) to a depth of 15–20 million reads per experiment. Reads were mapped to the mouse genome (NCBI37/mm9) using STAR (PMID: 23104886). Gene expression levels and Gene Ontology enrichment were calculated using HOMER (PMID: 20513432) and clustering was performed using Cluster 3.0 and Java TreeView. Differential expression was defined using a false discovery rate (FDR) cut-off of 5% and a fold change of at least 2 using edgeR (PMID: 19910308). RNA-Seq data have been deposited in the Gene Expression Omnibus under accession code GSE66457.
BrdU labeling was performed using BrdU In-Situ Detection Kit (BD Biosciences, 550803) according to the manufacturer’s instructions. Briefly, the mice were i.p. injected with 1 mg of BrdU and the tissues were collected from the injected mice at 24 hr post injection, followed by paraffin embedding and sectioning. After being deparaffinized and antigen-retrieved, the section was stained using biotinylated anti-BrdU and Streptavidin HRP together with DAB substrate and BrdU+ cells were counted for quantification.
Mouse primary esophageal cells were derived as previously described (Kalabis et al., 2008). Briefly, the esophagi were isolated, opened longitudinally, washed in PBS followed by Dispase (1 U/mL) for 15–20 min at 37 °C. The opened esophagi were minced with forceps and incubated with TrypLE for 10 min at 37 °C. After inactivation of TrypLE with FBS, the cell suspension was filtered through 100-μm and 40-μm cell strainers. The obtained cells were centrifuged and re-suspended in keratinocyte serum-free medium (Life Technologies), followed by plating on matrigel-coated plates. Human primary esophageal epithelial cells were obtained from Cell Biologics. Het-1A cell line was obtained from ATCC. OE21 cell line was obtained from sigma. The cells were cultured according to manufacturer’s instructions.
Single cell suspension of the esophagus and the forestomach was obtained as mentioned above. Lung cell isolation was performed as previously described (Gereke et al., 2012). Briefly, lungs were perfused with PBS and the salivary glands were removed to expose the trachea, followed by instillation with 1 U/mL dispase and 1% low-melting agarose. After gel solidification with ice, the lungs were isolated and washed with PBS, and incubated with dispase at room temperature for 45 min. The lungs were minced and filtered through 100-μm and 40-μm cell strainers to obtain a single cell suspension. The single cell suspension was subjected to FACS analysis.
The cells of interest were cultured in 0.5% soft agarose layered on harder agarose in 60-mm dishes. After 14 days, the colonies were counted.
We thank Dr. Manching Ku for next-generation sequencing, Drs. Kimberly McIntyre and Varki Nissi for histology. We also thank May Schwarz and Peter Schwarz for administrative help, Marie N. Krause, Ilir Dubova, Keiichiro Suzuki, Masakazu Kurita, April Goebl, Emi Aizawa-Suzuki, Na Young Kim, and Rupa Devi Soligalla for experimental help, Elena Vicario-Orri, Toshiro Hara and Eiji Yoshihara for critical advice, and David O’Keefe and Michael Nunn for help with manuscript preparation. This work was supported by the National Key Research and Development Program of China (2015CB964800), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16010100), the National Natural Science Foundation of China (81625009, 81330008, 91749202, 81861168034), Program of Beijing Municipal Science and Technology Commission (Z151100003915072), Advanced Innovation Center for Human Brain Protection (117212, 3500-1192012), and Beijing Municipal Commission of Health and Family Planning PXM2018_026283_000002). Work in the laboratory of J.C.I.B was supported by a Cancer Center Support Grant, the G. Harold and Leila Y, Mathers Charitable Foundation, The Leona M. and Harry B. Helmsley Charitable Trust (2012-PG-MED002), The Moxie Foundation, Fundacion Dr. Pedro Guillen and Universidad Católica San Antonio de Murcia (UCAM). T.H. was supported by a Pioneer Fund Postdoctoral Scholar Award, Nomis Fellowship, and Uehara Memorial Foundation research fellowship.
ASCs, adult stem cells; BLI, bioluminescence imaging; Bp, Base-pair; Cs, progenitor cells; CXCR2, CXC chemokine receptor 2; ESCC, esophageal squamous cell carcinoma; FDR, false discovery rate; HDF, human dermis skin fibroblast; hpEEC, human primary esophageal epithelial cell; IHC, immunohistochemistry; Krt17, Keratin 17; Luc, luciferase; mpEEC, mouse primary esophageal epithelial cell; PAS, periodic acid-Schiff; RNA-Seq, RNA-sequencing; RIN, RNA integrity number; SASP, senescence-associated secretory phenotype; SCC, squamous cell carcinomas; SCLC, small-cell lung cancer; TAM, tamoxifen
Compliance with ethics guidelines
Tomoaki Hishida, Eric Vazquez-Ferrer, Yuriko Hishida-Nozaki, Ignacio Sancho-Martinez, Yuta Takahashi, Fumiyuki Hatanaka, Jun Wu, Alejandro Ocampo, Pradeep Reddy, Min-Zu Wu, Laurie Gerken, Reuben J. Shaw, Concepcion Rodriguez-Esteban, Christopher Benner, Hiroshi Nakagawa, Pedro Guillen Garcia, Estrella Nuñez Delicado, Antoni Castells, Josep M. Campistol, Guang-Hui Liu and Juan Carlos Izpisua Belmonte declare that they have no conflict of interest.
All animal experiments were approved by the Committee on Animal Care at the Salk Institute. All institutional and national guidelines for the care and use of laboratory animals were followed. This article does not contain any studies with human subjects performed by any of the authors.
- Andoniadou CL, Matsushima D, Mousavy Gharavy SN, Signore M, Mackintosh AI, Schaeffer M, Gaston-Massuet C, Mollard P, Jacques TS, Le Tissier P et al (2013) Sox2(+) stem/progenitor cells in the adult mouse pituitary support organ homeostasis and have tumor-inducing potential. Cell Stem Cell 13:433–445CrossRefGoogle Scholar
- Annovazzi L, Mellai M, Caldera V, Valente G, Schiffer D (2011) SOX2 expression and amplification in gliomas and glioma cell lines. Cancer Genom Proteom 8:139–147Google Scholar
- Feng Y, Bommer GT, Zhao J, Green M, Sands E, Zhai Y, Brown K, Burberry A, Cho KR, Fearon ER (2011) Mutant KRAS promotes hyperplasia and alters differentiation in the colon epithelium but does not expand the presumptive stem cell pool. Gastroenterology 141(1003–1013):e1001–1010Google Scholar
- Gereke M, Autengruber A, Grobe L, Jeron A, Bruder D, Stegemann-Koniszewski S (2012) Flow cytometric isolation of primary murine type II alveolar epithelial cells for functional and molecular studies. J Vis Exp.Google Scholar
- Kalabis J, Oyama K, Okawa T, Nakagawa H, Michaylira CZ, Stairs DB, Figueiredo JL, Mahmood U, Diehl JA, Herlyn M et al (2008) A subpopulation of mouse esophageal basal cells has properties of stem cells with the capacity for self-renewal and lineage specification. J Clin Invest 118:3860–3869PubMedPubMedCentralGoogle Scholar
- Schwitalla S, Ziegler PK, Horst D, Becker V, Kerle I, Begus-Nahrmann Y, Lechel A, Rudolph KL, Langer R, Slotta-Huspenina J et al (2013) Loss of p53 in enterocytes generates an inflammatory microenvironment enabling invasion and lymph node metastasis of carcinogen-induced colorectal tumors. Cancer Cell 23(1):93–106CrossRefGoogle Scholar
- White JR, Lee JM, Young PR, Hertzberg RP, Jurewicz AJ, Chaikin MA, Widdowson K, Foley JJ, Martin LD, Griswold DE et al (1998) Identification of a potent, selective non-peptide CXCR45 antagonist that inhibits interleukin-8-induced neutrophil migration. J Biol Chem 273:10095–10098CrossRefGoogle Scholar
- Zhu L, Finkelstein D, Gao C, Shi L, Wang Y, Lopez-Terrada D, Wang K, Utley S, Pounds S, Neale G et al (2016) Multi-organ Mapping of Cancer Risk. Cell 166(1132–1146):e1137Google Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.