Time-Series Analysis of Tumorigenesis in a Murine Skin Carcinogenesis Model

www.nature.com/scientificreports OPEN Received: 17 January 2018 Accepted: 17 August 2018 Published: xx xx xxxx Time-Series Analysis of Tumorigenesis in a Murine Skin Carcinogenesis Model Yoshimasa Aoto1, Kazuhiro Okumura2, Tsuyoshi Hachiya3, Sumitaka Hase1, Yuichi Wakabayashi2, Fuyuki Ishikawa4 & Yasubumi Sakakibara1 Recent years have witnessed substantial progress in understanding tumor heterogeneity and the process of tumor progression; however, the entire process of the transition of tumors from a benign to metastatic state remains poorly understood. In the present study, we performed a prospective cancer genome-sequencing analysis by employing an experimental carcinogenesis mouse model of squamous cell carcinoma to systematically understand the evolutionary process of tumors. We surgically collected a part of a lesion of each tumor and followed the progression of these tumors in vivo over time. Comparative time-series analysis of the genomes of tumors with different fates, i.e., those that eventually metastasized and regressed, suggested that these tumors acquired and inherited different mutations. These findings suggest that despite the occurrence of an intra-tumor selection event for malignant alteration during the transformation from early- to late-stage papilloma, the fate determination of tumors might be determined at an even earlier stage. Cancer is a result of genomic disorders represented by DNA mutations that typically lead to loss of DNA repair function and gain of abnormal proliferation function. Numerous reports on the process of malignant alterations suggest that benign tumors progress in a stepwise fashion while acquiring driver and passenger mutations, which eventually invade surrounding tissues to finally migrate to distant tissues1–6. The consortium projects represented by The Cancer Genome Atlas (TCGA) have catalogued the main cancer driver mutations and identified diverse driver genes from an identical cancer type as well as from more than 60 primary sites5. Since these driver genes promote cancer progression by conferring cells with abnormal biological functions such as limitless proliferation and neo-angiogenesis3,4, they are regarded as candidate therapeutic targets. However, the high variation of driver genes within an identical cancer type reflects not only the inter-tumor heterogeneity but also the difficulty of cancer therapy3,7. Moreover, recent studies focusing on the intra-tumor environment have suggested a polyclonal structure of tumors due to genomic instability6–8. Although tumors initially form from a single cell type, as each tumor cell randomly acquires somatic mutations and then proliferates, the polyclonal cell population is formed based on the different genetic backgrounds among tumor cells6–8. This genetic diversity of tumor cells generates the physiological diversity and differences in therapeutic sensitivity among tumor cells. Accordingly, the polyclonal structure of tumors is considered to be the most critical cause of treatment resistance and the recurrence of cancer6–8. More recently, the polyclonal structure of tumors has been addressed under the field termed “intra-tumor heterogeneity”, which has emerged as an essential aspect required for disclosing the entire landscape of tumor progression and delineating the specific causes of resistance to cancer treatment. To best understand the full spectrum of intra-tumor heterogeneity, evolutionary analysis has been performed using multi-region samples, which are obtained from multiple sites of a single malignant tumor, and/or the primary and metastatic tumors from the same individual; accordingly, several models have been proposed to explain the process of tumor progression and the origin of tumors9–12. An adaptive (Darwinian) tumor progression model was suggested by which only certain sub-groups (i.e., sub-clones) that could gain advantageous traits to survive would remain in the tumor 1 Department of Biosciences and Informatics, Keio University, 3-13-1 Hiyoshi, Kohoku-ku, Yokohama, 223-8522, Japan. 2Department of Carcinogenesis Research, Division of Experimental Animal Research, Chiba Cancer Center Research Institute, 666-2 Nitonacho, Chuo Ward, Chiba, Chiba, 260-8717, Japan. 3Iwate Medical Megabank Organization, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba-cho, Shiwa-gun, Iwate, 028-3694, Japan. 4 Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan. Correspondence and requests for materials should be addressed to Y.S. (email: yasu@bio. keio.ac.jp) Scientific REPOrTS | (2018) 8:12994 | DOI:10.1038/s41598-018-31349-x 1 www.nature.com/scientificreports/ environment. In the process of stepwise cancer progression, tumors have to overcome several barriers such as the lack of nutrients, immune response from the surrounding tissues, and lack of growth space, among others4. These barriers impose a type of selection pressure for tumor cells so that only those that are best adapted to the given tumor environment will survive to proliferate, and will thus acquire various mutations in the process to lead to intra-tumor heterogeneity that contributes to the diversity in treatment sensitivity and sustainable progression of tumors6–8,11. In contrast, the neutral evolution theory of tumor progression proposes that tumor cells are derived from an initial malignant cell such as a cancer stem cell that neutrally expands with random mutations, thereby resulting in intra-tumor heterogeneity11,13. Moreover, several hypotheses have been proposed to explain the origin of tumors, including a founder cancer stem cell that already possesses multiple driver mutations and then rapidly grows and forms a tumor via the acquisition of new driver (trigger) mutations, or development of an initial driver mutation that causes an undetectable tumor, which gradually grows in size owing to the acquisition of new driver mutations12. Despite these advances in the general understanding of inter-/intra-tumor heterogeneity and the process of tumor progression, the entire evolutionary process of tumors, from a benign to metastatic state, is still poorly understood. Gaining a detailed understanding the process of tumor evolution over time is expected to improve the confidence of early diagnostics and prognostic predictions. However, to date, the majority of cancer studies use specimens that have already transitioned to malignancy, and research based on tracking the transitions from an early benign tumor to a metastatic tumor is relatively limited. Given this background, we have begun to address this issue with a prospective cancer study with the goal of systematically understanding the evolutionary process of tumors. Toward this end, we have employed an experimental carcinogenesis mouse model, which promotes the formation of squamous cell carcinoma (SCC) on the back skin of the mice. We performed a classical two-stage carcinogen (...truncated)