Periodic Reporting for period 1 - TWSCC (Trafficking of Wnt Signaling Components in Cancer)
Periodo di rendicontazione: 2021-09-01 al 2023-08-31
Colorectal cancer (CRC) is a leading cause of cancer related deaths throughout the world. Inactivating mutations to the tumour suppressor gene Adenomatous polyposis coli (Apc) are considered to be an early event that initiates this disease in approx. 80% of cases. Apc is a component of the Wnt signalling pathway and has several roles including inhibiting β-catenin, which functions as a transcriptional coactivator in the nucleus and is a component of the adherens junction. The loss of Apc function constitutively activates Wnt signalling to drive proliferation and provide cells with a competitive advantage over their surrounding wild type cells, which initiates tumour formation. Over the past three decades we have gained a good understanding of the mutations that are involved in the progression of this disease. However, we currently lack insight into the membrane trafficking mechanisms that are involved in modulating dysregulated Wnt signalling.
Current efforts at developing effective therapies for CRC have had limited success. Therefore, there is an urgent need to develop novel therapeutics that can extend patient life, especially during late stage CRC. Discovering such therapies will have a significant positive impact on society since CRC is currently a major health burden, costing the EU approx. €19.1 billion per year.
The overall objective of this proposal is to directly address what roles membrane trafficking events have in modulating dysregulated Wnt signalling. We aim to 1) identify novel trafficking regulators that are involved in Wnt signalling when Apc is mutated, 2) discover the mechanisms that are involved in modulating Wnt signalling, 3) perform translation experiments to understand the conservation of mechanisms involved.
Current efforts at developing effective therapies for CRC have had limited success. Therefore, there is an urgent need to develop novel therapeutics that can extend patient life, especially during late stage CRC. Discovering such therapies will have a significant positive impact on society since CRC is currently a major health burden, costing the EU approx. €19.1 billion per year.
The overall objective of this proposal is to directly address what roles membrane trafficking events have in modulating dysregulated Wnt signalling. We aim to 1) identify novel trafficking regulators that are involved in Wnt signalling when Apc is mutated, 2) discover the mechanisms that are involved in modulating Wnt signalling, 3) perform translation experiments to understand the conservation of mechanisms involved.
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.
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.
I have used cutting edge methods including high resolution and super-resolution microscopy as well as single cell RNA-sequencing with elaborate genetics to understand how membrane trafficking events are involved in Wnt signalling and intestinal stem cell proliferation. This led to translation experiments in mouse colon organoids where we showed that inhibitors of the JNK pathways preferentially reduce cancer organoid growth, thus demonstrating the potential for this to function as a therapeutic target.
CRC costs the EU approx. €19.1 billion per year. Previous studies have identified socioeconomic status, gender and education level as important factors in determining risk to CRC. The socioeconomic and educational factors are of particular interest since they can be avoidable through appropriate public health policies. In most cases, CRC is treatable if diagnosed at an early stage and can be preventable when risk patients are placed under surveillance, including undergoing regular colonoscopies. By understanding how tumour location impacts CRC prognosis, we can raise awareness and design better biopsies strategies developed to obtain more tissues from disease regions and increase coverage, aiding in early diagnosis. Additionally, insight into the genetic mechanisms that confer regional susceptibility to CRC can help identify risk associated genes and better guide patient treatment.
CRC costs the EU approx. €19.1 billion per year. Previous studies have identified socioeconomic status, gender and education level as important factors in determining risk to CRC. The socioeconomic and educational factors are of particular interest since they can be avoidable through appropriate public health policies. In most cases, CRC is treatable if diagnosed at an early stage and can be preventable when risk patients are placed under surveillance, including undergoing regular colonoscopies. By understanding how tumour location impacts CRC prognosis, we can raise awareness and design better biopsies strategies developed to obtain more tissues from disease regions and increase coverage, aiding in early diagnosis. Additionally, insight into the genetic mechanisms that confer regional susceptibility to CRC can help identify risk associated genes and better guide patient treatment.