I have utilised publicly available data to identify misexpressed genes involved in membrane trafficking in colorectal cancer patients from the TCGA datasets. I have also utilised DESeq2 on public RNA-seq and single cell RNA-seq datasets in which Apc is perturbed in the intestine of mice.
I screened 121 lines covering 63 genes, giving an average of two lines per gene, often including both knockdown and overexpression of targeted candidates. From this screen Rab35 emerged as a promising candidate. Rab35 belongs to the small family of GTPases and has previously been implicated in cytokinesis and fast recycling of proteins. Rab35 was of interest because it was identified as having a suppressive effect on proliferation when its YFP-tagged wild type (Rab35WT) or constitutively active (Rab35CA) form was expressed. Conversely, Rab35 enhanced proliferation when its function was perturbed with RNAi silencing (Rab35 RNAi) or a dominant negative YFP-tagged mutant (Rab35 DN).
To gain insight into how Rab35 regulates Wnt activity when Apc is depleted, we monitored the Wnt reporter Fz3 using a fluorescently tagged reporter line. Under homeostatic conditions, Wnt activity is highest at compartment boundaries. Remarkably, when Apc is silenced using the esgTS driver, Wnt signalling was predominantly activated in the anterior R2 portion of the intestine. To understand how Rab35 modulates the Wnt pathway, we performed epistasis experiments with Apc RNAi, which demonstrated that Rab35 is able to regionally enhance or suppress Wnt activity by co-expressing Apc RNAi with either Rab35DN or Rab35CA, respectively. This is of particular interest since the regionalisation of the intestine has not been properly understood but has important implication for proximal and distal CRC.
We next wanted to understand the Rab35-dependent downstream mechanisms that modulate Wnt signalling and progenitor proliferation. We investigated this by focusing on the Rho GTPase, Cdc42, since its activity has been reported to be regulated by Rab35 during neurite outgrowth in vitro. To understand the role of cdc42 for Wnt signalling and progenitor proliferation, we performed genetic interaction experiments between Cdc42 and Apc. Interestingly, co-expression of Cdc42WT with ApcRNAi led to significantly higher Wnt activity and progenitor proliferation when compared to Apc RNAi alone. Conversely, we could revert Apc RNAi-induced Wnt activation and progenitor proliferation back to wild type levels when Cdc42 was silenced with RNAi or by co-expressing a dominant negative Cdc42 mutant (Cdc42DN). Taken together, we show that Cdc42 is necessary and sufficient to modulate Wnt activity in the absence of Apc.
I next asked which signalling cascades modulate the Wnt pathway and progenitor proliferation. Previous reports indicate that Cdc42 regulates JNK signalling in vitro, but the role of this interaction in the intestine has not been studied before in vivo. To understand the convergence between JNK and Wnt signalling, we investigated their epistatic relationship in the intestine. Interestingly, when Wnt signalling was activated by Apc RNAi, co-expression of Bsk DN or puc RNAi either suppressed Wnt activity and progenitor proliferation or enhanced these two phenotypes, respectively
To determine whether JNK signalling has a conserved role in vertebrates, we utilised mouse colon organoids with a truncating mutation in Apc (Apc trunc) generated with CRISPR technology. We seeded WT and Apc trunc organoids, treated them with either DMSO or the JNK1 inhibitor DB07268 and extracted protein or measured proliferation rate. First, we observed that Apc trunc organoids displayed elevated levels of β-catenin resulting from its stabilisation upon activation of the Wnt pathway. Second, using a phospho-SAPK/JNK (Thr183/Tyr185) antibody, we find that Apc trunc organoids are in a JNK active state which could be suppressed by using DB07268. Third, treatment with DB07268 preferentially reduced the proliferation of rate of Apc trunc organoids and had a mild non-significant effect on WT organoids. These findings demonstrate that JNK signalling is a conserved modulator of mouse colon organoid proliferation when Apc is mutated.
