Introduction

Cholangiocarcinoma (CCA) comprises a heterogeneous group of malignant tumors with dismal prognosis. Alterations in post-translational modifications (PTMs), including SUMOylation, result in abnormal protein dynamics, cell disturbances and disease. Here, we investigate the role of SUMOylation in CCA development and progression.

Aims & Methods

Levels and function of SUMOylation, together with response to S-adenosylmethionine (SAMe) and ML792 (SUMOylation inhibitors) or CRISPR/Cas9 against UBE2I were evaluated in vitro, in vivo and/or in patients with CCA. The impact of SUMOylation in CCA cells on tumor-stroma crosstalk was assessed performing co-culture experiments with CCA-derived cancer-associated fibroblasts (CAFs), human endothelial cells and monocytes. Proteomic analyses were carried out by mass spectrometry.

Results

The SUMOylation machinery was found overexpressed and overactivated in human CCA cells and tumors, correlating with poor prognosis. Most SUMOylated proteins found upregulated in CCA cells, after SUMO1-immunoprecipitation and further proteomics, participate in cell proliferation, survival or cell homeostasis. Genetic (CRISPR/Cas9-UBE2I) and pharmacological (SAMe and ML792) inhibition of SUMOylation reduced CCA cell proliferation and impeded colony formation in vitro. Moreover, both SAMe and ML792 induced apoptotic cell death in CCA cells in vitro. SUMOylation depletion (SAMe, ML792 or CRISPR/Cas9-UBE2I) halted tumorigenesis in subcutaneous models of CCA in vivo. Furthermore, SUMOylation deficiency in CCA cells reduced cancer-associated fibroblast and endothelial cell proliferation and impaired macrophage polarization towards an anti-inflammatory M2-like phenotype.

Conclusion

Aberrant protein SUMOylation contributes to cholangiocarcinogenesis by promoting cell survival and proliferation. Moreover, SUMOylation impacts the CCA-stroma crosstalk. Impaired SUMOylation halts CCA growth and, thus, may represent a potential new therapeutic strategy for patients with CCA.

Introduction

Cholangiocarcinoma (CCA) includes a diverse range of biliary malignant tumours characterized by dismal prognosis. Tumours are commonly diagnosed when patients present locally-advanced or metastatic disease, limiting the access to potentially curative surgery. The genetic heterogeneity of CCAs has been reported, indicating that the most common gain-of-function mutations in K-RAS and IDH1, and FGFR2-translocations only occur in 16%, 10% and 15% of patients, respectively. These activating mutations are found in a complex genomic landscape where large number of genes are mutated at low frequencies. The role of these heterogeneously mutated genes in CCA progression is unknown and whether these accessory mutations modify the effect of more common mutations to promote tumour metastasis remains to be studied. Here, we investigate the genetics behind CCA dissemination, describing how cell intrinsic changes impact on cancer cell behaviour and shape the tumour microenvironment making it more permissive to metastasis.

Aims & Methods

Analysing whole genome/exome sequencing data from 277 patients diagnosed with intrahepatic CCA we identified a patient-led list of CCA mutations. We then developed an in vivo screen where we expressed gain of function K-Ras^G12Dwith a CRISPR/Cas9 gRNA library targeting genes mutated in the patient data. Immunohistochemistry analysis was performed to characterize the primary and metastatic tumours and their microenvironments. Using CRISPR/Cas9-mediated gene silencing, we interrogated whether these low frequency mutations promote epithelial-to-mesenchymal transition, migration and tumour dissemination in human CCA cells.

Results

The in vivo screening showed that K-Ras alone was insufficient to initiate tumour formation. In combination with our gRNA library, however K-Ras-driven tumours developed within 8 weeks. Exome sequencing of these tumours identified CRISPR-induced mutations in 53 genes which facilitated mutant K-Ras-induced tumour growth. We found a range of chromatin modifiers, including Ncor1, that upon deletion, together with Tp53 loss and K-Ras^G12Doverexpression, resulted in metastatic CCA. Although animal survival and tumour burden were not significantly different compared to those harbouring K-Ras^G12D;Tp53^-/- tumours, K-RasG12D;Tp53^-/-;Ncor1^-/- mice presented tumours in various sites other than the liver. Importantly, histological analysis of the primary tumours with metastatic potential revealed phenotypic changes in cancer cells as well as differences in the stroma. Furthermore, deletion of NCOR1 in human CCA cell lines increased their migratory capacity.

Conclusion

Low-frequency mutations in chromatin modifying genes, such as Ncor1, cooperate with common driver mutations to promote tumour progression and dissemination by altering cancer cell properties and modifying the tumour microenvironment.

References

1. Banales JM, et al., Nature Reviews Gastroenterology & Hepatology 2020
2. Kendre G, Murugesan K , et al., Journal of Hepatology 2023
3. Younger NT, Wilson ML, et al., Cancer Research 2022

Introduction

Cholangiocarcinoma (CCA) comprises a heterogeneous group of malignant tumours with dismal prognosis. During the last years, different studies have highlighted the key role of the immune system in the development of intrahepatic CCA (iCCA) and several combinational therapies targeting the tumour microenvironment (TME) have shown promising results for anti-cancer therapy. In this regard, the macrophage receptor with collagenous structure (MARCO) is a class A scavenger receptor found on particular subsets of macrophages that has been described to play a determining role in macrophage polarization and consequently in adaptive immune responses in many solid tumours. However, its role in iCCA is still unknown. This study aims to unravel the role of MARCO in iCCA development and progression.

Aims & Methods

The cell-type specific MARCO expression was examined in iCCA human tumors by using publicly available single-cell RNA sequencing data from different studies and MARCO-expressing tumor-associated macrophages (TAMs) were phenotypically characterized. MARCO mRNA expression was analyzed in human control and iCCA liver tissue samples and associated to different immune cell types and immune-functionality scores employing state-of-the-art technologies as ConsensusTME, Tumour Immune Dysfunction and Exclusion (TIDE) tool and Tumour Immunophenotype Profiling (TIP). To study the role of MARCO in murine cholangiocarcinogenesis, wild type (WT) and Marco^-/- mice were subjected to 3 different iCCA murine models and flow cytometry analysis of the TME was carried out to characterize different lymphocytic and myeloid populations.

Results

Single-cell RNA sequencing data indicate that MARCO is expressed in a specific subtype of TAMs in patients with iCCA. Besides, high MARCO expression levels in the liver samples of patients with iCCA are linked with worse clinical outcome. In line with this, MARCO expression in human iCCA tumours is associated to cell types involved in tumour progression such as M2 macrophages, and related with T cell dysfunction. Regarding the potential role of MARCO in murine models of iCCA, Marco^-/- mice show a trend to be protected from iCCA development, the mechanisms behind this effect being likely associated to a reduction of the innate immune cells such as CD9⁺Ly6C^-F4/80⁺ resident macrophages and type-2 innate lymphoid cells (ILC2). Noteworthy, in a context of a syngeneic orthotopic experimental model, Marco^-/- mice exhibit a reduced presence of immune checkpoint molecules in innate and adaptive immune cells, including a lower percentage of PD-L1⁺Ly6C^-F4/80⁺ resident macrophages and, PD-1⁺ and CTLA-4⁺ cytotoxic CD8⁺ T cells in comparison to WT mice. In addition, MARCO deficiency significantly improves the overall survival of mice subjected to the orthotopic injection of syngeneic CCA cells into the liver, due to a reduction of the tumour metastasis in the lungs.

Conclusion

High MARCO expression is associated to a worse outcome in patients with iCCA and is associated to an immunosuppressive TME. Importantly, Marco^-/- mice display a reduced presence of immunosuppressive cell populations and present an increased overall survival. Therefore, MARCO arises as a novel therapeutic target for iCCA.