Cell state plasticity is carefully regulated in adult epithelia to prevent cancer. The aberrant expansion of the normally restricted capability for cell state plasticity in neoplasia is poorly defined. Using genetically engineered and carcinogen-induced mouse models of intestinal neoplasia, we observed that impaired differentiation is a conserved event preceding cancer development. Single-cell RNA sequencing (scRNA-seq) of premalignant lesions from mouse models and a patient with hereditary polyposis revealed that cancer initiates by adopting an aberrant transcriptional state characterized by regenerative activity, marked by Ly6a (Sca-1), and reactivation of fetal intestinal genes, including Tacstd2 (Trop2). Genetic inactivation of Sox9 prevented adenoma formation, obstructed the emergence of regenerative and fetal programs, and restored multilineage differentiation by scRNA-seq. Expanded chromatin accessibility at regeneration and fetal genes upon Apc inactivation was reduced by concomitant Sox9 suppression. These studies indicate that aberrant cell state plasticity mediated by unabated regenerative activity and developmental reprogramming precedes cancer development.

Cell state (phenotypic) plasticity is a carefully regulated feature of adult epithelial cells that enables adaptive responses to injury, inflammation, and other forms of stress. Aberrant expansion of the normally restricted capability for cell state plasticity to escape terminal differentiation is a critical aspect of neoplasia. The nongenetic factors and specific programs that mediate aberrant cell state plasticity and impaired differentiation require deeper characterization to understand this elusive aspect of cancer pathogenesis. Using genetically engineered and carcinogen-induced murine models of intestinal neoplasia, we demonstrate that impaired differentiation is a conserved event preceding cancer development. Single cell RNA-sequencing (scRNA-seq) of neoplastic intestinal lesions from both mouse models and a patient with familial adenomatous polyposis revealed that cancer initiates by adopting an aberrant transcriptional state characterized by nonoverlapping expression of a regenerative pathway, marked by Ly6a (Sca-1), and a fetal intestinal program, positive for Tacstd2 (Trop2). Genetic inactivation of Sox9 prevented adenoma formation in ApcKO mice, obstructed emergence of aberrant regenerative and fetal intestinal programs, and restored multi-lineage differentiation by scRNA-seq. Expanded chromatin accessibility at regeneration and fetal genes upon Apc inactivation was reduced by concomitant Sox9 suppression. These studies indicate that cancer initiation requires aberrant cell state plasticity mediated by unabated regenerative activity and developmental reprogramming.

Cell state plasticity – the ability of cells to acquire new states via differentiation programs - is an important feature of embryogenesis, allowing for tissue specification during development, and adult homeostasis, enabling adaptive responses to physiological cues and pathological states. Epigenetic governance and gene regulation endow epithelia with restricted cell state plasticity that enable tissue homeostasis while avoiding properties that may facilitate neoplasia. There is emerging evidence that aberrant expansion of the normally restricted capability for cell state plasticity to escape terminal differentiation is a key aspect of cancer initiation. The nongenetic factors and specific programs that mediate aberrant cell state plasticity require deeper characterization to understand this elusive aspect of cancer pathogenesis. Previously challenging to study, cell state plasticity can now be investigated using single cell technology, offering unprecedented molecular resolution. We applied state-of-the-art methods on primary human tissue and derivative organoids as well as different mouse models to (a) characterize aberrant transcriptional cell states enabling cancer initiation and (b) define a functional mediator of expanded cell state plasticity. Using genetically engineered and carcinogen-induced murine models of intestinal neoplasia, we demonstrate that inappropriate Sox9 expression and loss of post-mitotic villus differentiation are early events preceding cancer development. By applying single cell RNA-sequencing (scRNA-seq) to flow-sorted epithelial cells from the Lgr5Cre; Apcf/f; R26tdT genetic model, we observed that preneoplastic cells express specific intestinal stem cell genes but are otherwise transcriptionally rewired, evading resemblance to normal intestinal cell types; we therefore refer to these cells as aberrant stem cell-like (AbSC). We further analyzed the AbSC transcriptional state, searching for features that explain its distinguished gene expression profile, and found that it is characterized by impaired differentiation, enhanced regenerative capacity, and reactivation of fetal genes. The evaluation of preneoplastic colonic lesions from the carcinogen-induced mouse model and a patient with familial adenomatous polyposis (FAP) by scRNA-seq confirmed these findings. Notably, while chromatin accessibility increased at regenerative genes, new accessibility was observed at fetal intestinal genes by ATAC-seq. Genetic inactivation of Sox9 prevented adenoma formation in ApcKO mice, obstructed emergence of the aberrant transcriptional state, including genes reserved for fetal intestinal development, and restored multi-lineage differentiation by scRNA-seq. SOX9 knockdown in human adenoma organoids compromised expression of fetal genes and induced differentiation. These studies indicate that cancer initiation requires aberrant phenotypic plasticity mediated by unabated regenerative activity and developmental reprogramming. Citation Format: Pratyusha Bala, Jonathan Rennhack, Clare Morris, Daulet Aitymbayev, Sydney M. Moyer, Gina N. Duronio, William Hahn, Nilay S. Sethi. Aberrant cell state plasticity mediates colorectal cancer initiation [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr PR012.

Background and aims: DNA repair deficiency is a common feature of cancer. Homologous recombination (HR) and nucleotide excision repair (NER) are the two most frequently disabled DNA repair pathways in solid tumors. HR deficient breast, ovarian, pancreatic and prostate cancers respond well to platinum chemotherapy and PARP inhibitors. However, the frequency of DNA repair pathway deficiency in gastric and esophageal adenocarcinoma (GEA) still lacks diagnostic and functional validation. Furthermore, whether DNA repair deficient GEA have enhanced responsiveness to platinum chemotherapy and sensitivity to PARP inhibitors is not well characterized. Methods: Using whole exome and genome sequencing data, we measured various HR deficiency-associated mutational signatures in patient specimen of gastric, esophageal and colorectal cancer specimens and gastric cancer cell lines. Gold-standard immunofluorescence assays were used to confirm HR and NER deficiency in cancer cell lines. The relationship between PARP inhibitor treatment and tumor response was evaluated in patients with gastric cancer. Drug sensitivity was determined using standard in vitro cell culture assays. Single-cell RNA-sequencing was performed to evaluate gastric cancer response to commonly used chemotherapeutics. Results: We found that a significant subset of GEA, but very few colorectal tumors, show evidence of HR deficiency by mutational signature analysis (HRD score). Gastric cancer cell lines with high HRD mutational signature scores demonstrated functional HR deficiency by RAD51 assay and increased sensitivity to platinum and PARP inhibitors. There was a positive association between HRD scores and tumor response in patients with gastric cancer treated with a PARP inhibitor on a clinical trial. A gastric cancer cell line with strong sensitivity to cisplatin showed HR proficiency but exhibited NER deficiency by DDB2 proteo-probe assay. Single-cell RNA-sequencing revealed that, in addition to inducing general apoptosis, cisplatin treatment triggered ferroptosis in a NER-deficient gastric cancer, which may explain the outlier sensitivity. Conclusion: A subset of upper gastrointestinal tumors have genomic features of HR and NER deficiency and therefore may be more likely to benefit from platinum chemotherapy and PARP inhibition.