Makepeace, C. M. | bioRxiv Science

bioRxiv2024

Examination of INPP5A in uveal melanoma uncovers novel calcium transients

Uveal melanoma is a highly aggressive intraocular cancer that metastasizes in about half of patients whereupon it is inexorably fatal. Uveal melanomas (UM) are distinct from other melanomas because they are driven by constitutively activating mutation in the heterotrimeric G protein alpha subunits Gq (GNAQ) and G11 (GNA11). This results in constitutive production of inositol trisphosphate (IP3) by phospholipase C-beta downstream of Gq/11. In normal cells, increased IP3 causes calcium release from the endoplasmic reticulum, which would be cytotoxic if maintained chronically, but UM cells are able to survive constitutive IP3 production. INPP5A, which dephosphorylates and thus inactivates IP3, is highly upregulated in UM cells compared to other melanomas and another study has shown that INPP5A is necessary for UM cell survival. To understand the mechanism of calcium regulation in response to IP3, we collected single-cell calcium measurements and found that UM cells driven by constitutively active Gq/11 produce spontaneous calcium transients. These calcium oscillations are not seen in any other melanoma cell lines unless induced by an agonist, but they are present in patient UM tumor samples. Moreover, these calcium oscillations are lost in UM cells treated with the Gq/11 inhibitor FR900359, demonstrating their dependence on constitutive Gq/11 activity. We found that the INPP5A inhibitor YU144369 causes significant changes in calcium oscillations in UM cells, demonstrating a role for INPP5A in this system. INPP5A is tethered to membranes by C-terminal prenylation and palmitoylation, suggesting that localization may play a role in INPP5A regulation of IP3 levels. GFP-tagged INPP5A was localized to plasma membrane, nuclear envelop, endoplasmic reticulum, and lysosomes. Mutation of the palmitoylation site significantly reduced localization to the plasma membrane, while mutation of the prenylation site resulted in purely nucleoplasmic localization of INPP5A. These results demonstrate a role of palmitoylation in the regulation of INPP5A localization and mobilization in UM cells.

cancer biology↗

bioRxiv2021

Targeting primary and metastatic uveal melanoma with a G protein inhibitor

Uveal melanoma (UM) is the most common intraocular tumor in adults. Nearly half of UM patients develop metastatic disease and often succumb within months because effective therapy is lacking. A novel therapeutic approach has been suggested by the discovery that UM cell lines driven by mutant constitutively active Gq or G11 can be targeted by FR900359 (FR) or YM-254890, which are bioavailable, selective inhibitors of the Gq/11/14 subfamily of heterotrimeric G proteins. Here, we have addressed the therapeutic potential of FR for UM. We found that FR inhibited all oncogenic Gq/11 mutants reported in UM. FR arrested growth of all Gq/11-driven UM cell lines tested, but induced apoptosis only in a few. Similarly, FR inhibited growth of, but did not efficiently kill, UM tumor cells from biopsies of primary or metastatic tumors. FR evoked melanocytic redifferentiation of UM tumor cells with low (class 1), but not high (class 2), metastatic potential. FR administered systemically below its LD50 strongly inhibited growth of PDX-derived class 1 and class 2 UM tumors in mouse xenograft models, and reduced blood pressure transiently. FR did not regress xenografted UM tumors, or significantly affect heart rate, liver function, hematopoiesis, or behavior. These results indicated the existence of a therapeutic window in which FR can be explored for treating UM, and potentially other diseases caused by constitutively active Gq/11.

cancer biology↗