Polyclonal interactions in intestinal tumorigenesis

Abstract

The adenoma-carcinoma sequence posits that colorectal cancers result from the stepwise accumulation of mutations in a number of cancer driver genes, with mutations in the tumour suppressor gene \textit{APC} thought to be necessary and sufficient for transformation. However, large-scale sequencing efforts have uncovered significant intratumoural genetic heterogeneity which is inconsistent with such a linear model of tumour progression. Alternative models of tumour evolution have been put forward but they all assume an initial clonal expansion originating from a single transformed cell. This contrasts with reports of polyclonality in a number of neoplastic lesions. In the first part of this thesis, I combine multicolour lineage tracing with a mouse model of intestinal tumorigenesis to show that a significant proportion of tumours have a polyclonal origin following the loss of one allele of \textit{Apc} and subsequent mutagenesis. These polyclonal tumours cannot be explained by the random collision of independent tumours but instead, appear to be the result of recruitment of a poorly- or partially-transformed clone. Next, using a highly multiplexed targeted amplicon sequencing approach, the nature of the mutagen-induced variants in Apc is characterised in monoclonal and polyclonal tumours. By microdissecting polyclonal tumours based on their lineage marker status, I show that mutations landing in the N-terminus of Apc are depleted in monoclonal tumours and are instead selected for in minor clones. This suggests that certain Apc mutations are unable to lead to transformation via the monoclonal route but instead have to be rescued through clonal interactions. In the final part of this thesis, I go on to show that clones within polyclonal tumours are complementary in their Kras and Myc pathway activation levels, which suggests that the well-established phenomenon of oncogenic cooperation between these two pathways can sometimes be non-cell autonomous. These transcriptomic differences are reflected in an observed variation in cell phenotypes, with pronounced differences in cancer stem cell identities with an Lgr5 and Anxa1 dichotomy. Importantly, this intratumoural heterogeneity in cancer stem cell identity corresponds to lineage marker territories, indicating that founder cell multiplicity can contribute to intratumoural heterogeneity that persists during the life of a tumour. In summary, this work adds to a growing literature that proposes an alternative to the predominant monoclonal theory of tumour formation.