Final Report Summary - COLONGEVA (Implications of copy number variants of the genome in the etiology and progression of colorectal cancer) Colorectal cancer (CRC) is one of the most devastating diseases in the Western world. Despite great scientific achievements, there is still the need to improve the diagnosis, prognosis and prediction to treatment response to determine the biological bases for personalized medicine. The main goal of this project is to decipher the role of genomic mutations and copy number variants (CNVs) in the prognosis and progression of CRC, to better understand which genes, transcripts and molecular pathways are affected in the colorectal tumorigenesis, as well as the individualized response to treatment.Specifically, the project objectives described in the original grant proposal are: 1- Discovery of copy number variants associated with an increased colorectal cancer susceptibility and progression: The ultimate goal of this translational effort is to identify genomic biomarkers to risk-stratify patients according to prognostic factors. Clinical management of stage II colon cancer (CC) patients represents a major therapeutic challenge since adjuvant chemotherapy is not systematically indicated. However, between 10 to 20% of them show recurrence within 5 years after surgery. Therefore, by SNP-arrays, we assessed copy number alterations of stage II CC samples with clinical follow-up of five years after surgery. Preliminary data show that tumors of patients with recurrence have a greater amount of CNVs. Moreover, our results suggest some candidate genomic regions to discern patients at risk of recurrence, which are currently under a validation phase.In addition, genomic copy number gains and losses as well as copy neutral LOH (also referred as uniparental disomies, UPDs) have been assessed in tumor samples and lymphocyte blood DNA of CRC patients and controls. Integrative analysis of SNP-arrays, array CGH and methylation status has led to the identification of unequivocal UPD profiles that unveil novel candidate tumor suppressor genes using an own sample set and a validation set from The Cancer Genome Atlas CRC data set (Torabi et al., Carcinogenesis, 2015). A follow-up study aims at deciphering UPD profiles for other gastrointestinal cancers, including esophageal, stomach and liver cancer. The presence of mosaic large structural variants and UPDs in lymphocyte blood DNA is indicative of the presence of germline structural alterations with a causative role in the tumorigenesis (Torabi et al., submitted).Finally, in order to study the mechanisms by which genomic instability, a hallmark of CRC, fuels intratumor heterogeneity, we generated diploid and tetraploid isogenic CRC cell lines, and found that tetraploid cells showed greater levels of replication stress causing DNA damage, lagging chromosomes and aberrant mitosis (Wangsa et al., FASEB J, in press).2- Genomic alterations and mutations in radiochemotherapy resistant CRC tumors:Genomic changes assessed in rectal primary tumors before and after being radiochemotherapeutically treated will define genetic markers that drive tumors to become resistant to radiochemotherapeutic agents. This ongoing project is interrogating the copy number changes of six genes involved in CRC at the single cell level to determine whether subclonal populations present in pre-treated locally advanced rectal cancers are responsible for generating resistance after radiochemoradiotherapy treatment. These results will also provide evidence as to whether levels of chromosomal instability might be of use as biomarkers for predicting treatment response.Furthermore, granted that multi-resistant metastatic colorectal tumors evade second and third line of treatments with targeted therapies, we have identified that the nuclear localization of the insulin growth factor 1 receptor (IGF1-R) is associated with progression-free survival and overall survival after treating CRC patients with first line of chemotherapeutic agents as well as targeted therapies (e.g. ganitumab). These results suggest that nuclear IGF1-R is a surrogate for resistance to treatment (Codony-Servat et al., Br J Cancer, 2017).3- Mechanisms of formation of genomic alterations at the sites of CNVs:The integration of the nuclear organization with the etiology of human diseases, including cancer, strengthens the relationship between structure, function and time (4D Nucleome). The main goal of this research line is to understand how the higher order of chromatin organization and the nuclear architecture influence the formation of CNV and their gene expression, which ultimately will result in cellular advantages for the tumor cell. Using chromosome conformation capture (Hi-C) to study the genome structure of CRC cells, unprecedented CNVs were identified at high resolution as well as their relationship between chromatin organization and gene expression (Seaman et al., Mol Cancer Res, 2017).Additionally, in order to investigate the association between gene expression levels and the radial positioning of genes within their corresponding chromosome territories, we performed three-dimensional fluorescence in situ hybridization (3D-FISH) experiments in two CRC cell lines. Assessing the radial position of genes over- and underexpressed we could suggest a non-random distribution of the interphase chromatin in which over-expressed genes are located more towards the periphery of their respective chromosome territories (Torabi et al., Chromosoma, 2017).• Potential impact:The execution of this project fits perfectly with the current challenges of the society, specially in health and wellness. We expect to develop tools to apply translational research based on -omic technologies to colon cancer, which represents one of the diseases with the highest prevalence in Spain and in the Western countries. The application of genomic and transcriptomic analysis will be instrumental in promoting the discovery of biomarkers associated with prognosis in patients with non-metastatic colon cancer and with treatment response to those patients who already developed metastasis. This might be used to implement new diagnostic techniques that allow an individualized treatment of patients and stratification of those that present a higher risk of relapse of this disease.Specifically, the results of this proposal will characterize the genetic and transcriptomic profile of tumor tissues from patients with stage II CC, with the final goal of identifying a panel of biomarkers that can help to predict the risk of recurrence, and therefore guide the use of adjuvant chemotherapy. High mortality rates in CRC are associated with late detection, underscoring the need for early-stage detection, risk assessment, and clinical intervention. There can be no question that improving the clinical assessment of non-metastatic cancer is the best strategy for reducing morbidity and mortality of CRC. However, currently available screening and risk stratification based on conventional histological diagnosis has its own flaws. Similarly, locally advanced rectal cancer patients usually receive neoadjuvant radiochemotherapy. Nevertheless, not all patients respond to the same extent, and while some tumors have regressed at the time of surgery, some other did not. Our hypothesis is that genetic biomarkers and intratumor heterogeneity might be responsible for these clinical outcomes. Next-generation sequencing (NGS) technologies have surpassed the limitations of the traditional Sanger sequencing and therefore are capable of massive parallel sequencing. Moreover, the recent availability and affordability of genome-wide NGS platforms provide a more comprehensive, systematic and prudent biomarker discovery tool. We are taking advantage of this technology to characterize the colorectal carcinogenesis at the genetic and transcriptomic level. Furthermore, addressing intratumor heterogeneity to assess molecular markers will result in a comprehensive understanding of the tumor biology and its association with clinical outcome.
