TGF-β Type I Receptor Signaling in Melanoma Liver Metastases Increases Metastatic Outgrowth

SIMPLE SUMMARY: Melanoma patients with liver metastasis have a poor prognosis, despite progress in therapeutic strategies. The cytokine Transforming Growth Factor β (TGF-β) plays a role in melanoma cells and acts on cells in the liver. We hypothesized that this cytokine influences the metastatic outgrowth of melanoma in liver. To investigate this, we generated a model to turn on and off TGF-β signaling in the B16F10 melanoma cells. In vitro, TGF-β activation repressed B16F10 melanoma cell growth and migration, while in vivo, sustained TGF-β activation increased outgrowth in liver. In the tumor microenvironment, TGF-β activation led to changes in immune cells. Analysis of newly secreted proteins revealed that B16F10 cells in which the TGF-β pathway is overly active secrete more matrix remodeling proteins. Such proteins that surround cells could directly or indirectly lead to changes in immune cell compartments during liver metastasis. Our results contribute to the understanding of TGF-β in liver metastasis of melanoma cells. ABSTRACT: Despite advances in treatment for metastatic melanoma patients, patients with liver metastasis have an unfavorable prognosis. A better understanding of the development of liver metastasis is needed. The multifunctional cytokine Transforming Growth Factor β (TGF-β) plays various roles in melanoma tumors and metastasis, affecting both tumor cells and cells from the surrounding tumor microenvironment. To study the role of TGF-β in melanoma liver metastasis, we created a model to activate or repress the TGF-β receptor pathway in vitro and in vivo in an inducible manner. For this, we engineered B16F10 melanoma cells to have inducible ectopic expression of a constitutively active (ca) or kinase-inactive (ki) TGF-β receptor I, also termed activin receptor-like kinase (ALK5). In vitro, stimulation with TGF-β signaling and ectopic caALK5 expression reduced B16F10 cell proliferation and migration. Contrasting results were found in vivo; sustained caALK5 expression in B16F10 cells in vivo increased the metastatic outgrowth in liver. Blocking microenvironmental TGF-β did not affect metastatic liver outgrowth of both control and caALK5 expressing B16F10 cells. Upon characterizing the tumor microenvironment of control and caALk5 expressing B16F10 tumors, we observed reduced (cytotoxic) T cell presence and infiltration, as well as an increase in bone marrow-derived macrophages in caALK5 expressing B16F10 tumors. This suggests that caALK5 expression in B16F10 cells induces changes in the tumor microenvironment. A comparison of newly synthesized secreted proteins upon caALK5 expression by B16F10 cells revealed increased secretion of matrix remodeling proteins. Our results show that TGF-β receptor activation in B16F10 melanoma cells can increase metastatic outgrowth in liver in vivo, possibly through remodeling of the tumor microenvironment leading to altered infiltration of immune cells. These results provide insights in the role of TGF-β signaling in B16F10 liver metastasis and could have implications regarding the use of TGF-β inhibitors for the treatment of melanoma patients with liver metastasis.