6Characterization of tropomyosin receptor kinase signaling in intrahepatic cholangiocarcinoma
Georgios Koulios, Ting-Fang Lee, Kevin C Ray, Vincent Quoc-Huy Trinh, Frank Revetta, M Kay Washington, Kemal M Akat, Daniela Sia, Youngmin A LeeAbstract
Background
Tropomyosin receptor kinases (TRK) TRKA, TRKB and TRKC (encoded by NTRK1, NTRK2 and NTRK3, respectively), regulate survival, proliferation and differentiation of neurons through ligand-dependent activation of downstream signaling pathways including MAPK, PI3K and PKC. TRK-fusion positive cancers have ligand-independent activation of TRKs and have emerged as potent oncogenic drivers that can be very effectively targeted by FDA-approved TRK inhibitors. In intrahepatic cholangiocarcinoma (iCCA), however, NTRK fusions account only for a very small subset (<1%) and TRK-inhibitors have therefore been little explored in iCCA. The ligands of TRKs are neurotrophins, members of the nerve growth factor family, which include neurotrophin-3 (NTF3) and brain-derived neurotrophic factor (BDNF). NTF3 is the high-affinity ligand of TRKC but can bind to TRKA, TRKB and TRKC while BDNF is the high-affinity ligand of TRKB. As we recently demonstrated that hepatic stellate cells (HSCs), which are precursors of cancer-associated fibroblasts, release NTF3, promoting hepatocyte proliferation via paracrine TRKB signaling, we investigated if TRK signaling is activated in iCCA to assess the therapeutic potential of pharmacologic TRK inhibition.
Methods
Gene expression of NTF3, BDNF, NTRK1, NTRK2 and NTRK3 were assessed in publicly available iCCA datasets. Human iCCA specimens were analyzed by western blotting and immunohistochemistry for protein expression of TRK receptors and their ligands. NTF3- or BDNF-dependent cell proliferation was studied in human iCCA cell lines HuCCT1, Huh28 and liver fibroblast cell line LX2. Larotrectinib and taletrectinib, FDA-approved TRK inhibitors, were used for inhibition studies. iCCA patient-derived organoid lines were generated and used to study NTF3- or BDNF-dependent EdU uptake by immunofluorescence.
Results
Analysis of publicly available transcriptomic CCA datasets showed significant overexpression of NTRK1, NTRK2, BDNF in CCAs compared to normal livers. Detailed transcriptomic analysis in another dataset of 122 patients showed highest enrichment of NTRK1, NTRK2, NTRK3, BDNF or NTF3 in the ‘immune classical’ and ‘inflammatory stroma’ subtype which are characterized by more abundant stroma and inflammation representing approximately 36% (44/122) of patients in this dataset. Similarly, western blot analysis of paired iCCA and adjacent non-tumorous liver tissue demonstrated increased expression of TRKB, TRKC, and NTF3 in a subset of human iCCA samples. Co-immunofluorescence analysis with reelin, a CAF marker, demonstrated increased expression of BDNF and NTF3 in CAFs. Incubation of HuCCT1, Huh28 and LX2 cells with BDNF or NTF3 resulted in a significant increase in cellular proliferation, which was inhibited by co-incubation with larotrectinib or taletrectinib. Consistently, BDNF and NTF3 induced increased EdU uptake in iCCA PDOs which was significantly reduced following co-incubation with larotrectinib.
Conclusion
These findings demonstrated that TRKB, TRKC and NTF3 are overexpressed in human iCCA. Moreover, NTF3 and BDNF induced proliferation in iCCA and HSC cell lines, as well as iCCA PDOs, which was significantly decreased by co-incubation with TRK inhibitors larotrectinib or taletrectinib. Collectively, our data support TRK signaling as a growth signaling pathway in iCCA which merits further investigation of pan-TRK inhibitors as a potential therapeutic strategy for iCCA.