Timeframe: 2017 – 2019
Goal: Evaluate the role of Hedgehog and YAP signaling in fibrolamellar carcinoma
Principal Investigators: Cynthia Guy, MD and Anna Mae Diehl, MD
Fibrolamellar carcinoma has a unique histological appearance consisting of large tumor cells surrounded by thick fibrous bands, the stroma. Molecular crosstalk between tumor and stroma is important in tumor maintenance and progression. Decoding the important signals in these interactions will reveal new potential therapeutic targets.
This effort aimed to study the role of Hedgehog (Hh) signaling in tumor-stromal interactions. Hh signaling is important in normal liver development and regeneration as well as tumor-stromal interactions in other cancers. Active Hh signaling also activates a protein called Yap, which results in stroma accumulation and primitive stem cells, both of which are seen in FLC. The study examined the role of Hh and Yap signaling in tumor-stroma interactions and their effect on tumor growth and progression.
Key Findings: This study generated a FLC xenograft model in mice by injecting human FLC cells into immunodeficient mice. After confirming that the tumor exhibited typical features of the primary human FLC, the tumor was disaggregated and the resulting cell suspension was examined for functional characteristics of stem-like cells (e.g., anchorage independent growth capacity) and subjected to single cell RNA sequencing (SC RNA seq) to characterize the various communities of cells in the tumor.
In the study, data from SC RNA seq showed a decrease in miR 375 (which was reported by the Sethupathy lab as the most downregulated microRNA in human FLC) and increased levels of YAP1. This was significant because miR375 inhibits YAP1 and PKA is known to suppress miR375. Overexpressing miR 375 in a FLC cell line reduced YAP1 activity and suppressed FLC growth and migratory capacity in culture. Additionally, treatment with a Yap1 inhibitor inhibited Yap target gene expression in the xenograft and changed the histologic features of the cancer. It reduced the stroma and caused the cancer cells to appear more like mature hepatocytes.
Further analysis of SC RNA- Seq data showed transcriptionally distinct cluster of cells, with one of them showing a significant enrichment with mesenchymal genes, including multiple types of collagen and other matrix molecules as well as expression of Yap and Igfsbp3 (insulin like growth factor-2 RNA binding protein-3). Igf2bp3 is a known marker of various tumor initiating stem-like cells (TISCs), including TISCS for hepatocellular carcinoma and cholangiocarcinoma.
These results suggest that the malignant human FLC cells promoted accumulation of nonmalignant mouse cells with TISC-like characteristics, including expression of autocrine/paracrine reprogramming factors (e.g., Yap). The concept that nonmalignant mouse cells in the removed tumor might have been “reprogrammed” was supported by analysis of other mouse cell clusters which showed expression of markers associated with malignant FLC cells.
Results of this investigation were included in an article published by the American Journal of Pathology in January 2020. The full text of that article can be read here: https://ajp.amjpathol.org/article/S0002-9440(19)30770-9/fulltext
Implications: This work provided insight into the pathogenesis of FLC, specifically the induction of epithelial cells to transition towards a more fibrogenic, mesenchymal, and stem-like state, while limiting mesenchymal cells from acquiring less fibrogenic and more epithelial characteristics. Understanding these pathways would be beneficial to better understanding the drivers of metastasis in FLC.