Uveal melanoma cells use ameboid and mesenchymal mechanisms of cell motility crossing the endothelium

Abstract

Uveal melanomas (UM) are malignant cancers arising from the pigmented layers of the eye. UM cells spread through the bloodstream, and circulating UM cells are detectable in patients before metastases appear. Extravasation of UM cells is necessary for formation of metastases, and transendothelial migration (TEM) is a key step in extravasation. UM cells execute TEM via a stepwise process involving the actin-based processes of ameboid blebbing and mesenchymal lamellipodial protrusion. UM cancers are driven by oncogenic mutations that activate Gαq/11, and this activates TRIO, a guanine nucleotide exchange factor (GEF) for RhoA and Rac1. We found that pharmacologic inhibition of Gαq/11 in UM cells reduced TEM. Inhibition of the RhoA pathway blocked amoeboid motility but led to enhanced TEM; in contrast, inhibition of the Rac1 pathway decreased mesenchymal motility and reduced TEM. Inhibition of Arp2/3 complex allowed cells to transmigrate without intercalation, a direct mechanism similar to the one often displayed by immune cells. BAP1-deficient (+/-) UM subclones displayed motility behaviors and increased levels of TEM, similar to the effects of RhoA inhibitors. We conclude that RhoA and Rac1 signaling pathways, downstream of oncogenic Gαq/11, combine with pathways regulated by BAP1 to control the motility and transmigration of UM cells. Movie S1: Representative DIC movies of UM cells added to endothelial monolayers after treatment with vehicle control (DMSO) or FR900359. 20 sec intervals between frames; 2 hrs total elapsed time. Download Original Video (.8 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e20-04-0241/20210104/media/mc-e20-04-0241-s05.,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518515abb28bda92fbabe7b929571bd415190bf44d1790b7324fb90f184f91fab10e395327a1bef9725d0c99a358bbbe69c082a95f9d49957eb2932b2be9a6f328f5ec3ab3a0b97d9f37f310d42fd5299f545a21c350266480cca58f95ee1f0fec14c99b7f17a77c808f3513fe04ec6c3580c678ac782d0cac8b7ac9eb8c781d014b001be1c9e054975e297452d045b8de1551764e95e0 Movie S2: Representative DIC movies of UM cells added to endothelial monolayers after treatment with ROCK inhibitor (Y27632) or myosin inhibitor (Blebbistatin). 20 sec intervals between frames; 2 hrs total elapsed time. Download Original Video (.8 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e20-04-0241/20210104/media/mc-e20-04-0241-s06.,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518515abb28bda92fbabe7b929571bd415190bf44d1790b7324fb90f184f91fab10e395327a1bef9725d0c99a358bbbe69c082a95f9d49957eb2932b2be9a6f328f5ec3ab3a0b97d9f37f310d42fd5299f545a21c350266480cca58f95ee1f0fec14c99b7f17a77c808f3513fe04ef6c3580c678ac7833f728f707bf2375ce9a6836701d3190abe0fd1f6828e7efa9e6ec1982a146466e Movie S3: Representative DIC movies of UM cells added to endothelial monolayers after treatment with Rac inhibitor (NSC23766) or PAK inhibitor (IPA3). 20 sec intervals between frames; 2 hrs total elapsed time. Download Original Video (.8 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e20-04-0241/20210104/media/mc-e20-04-0241-s07.,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518515abb28bda92fbabe7b929571bd415190bf44d1790b7324fb90f184f91fab10e395327a1bef9725d0c99a358bbbe69c082a95f9d49957eb2932b2be9a6f328f5ec3ab3a0b97d9f37f310d42fd5299f545a21c350266480cca58f95ee1f0fec14c99b7f17a77c808f3513fe04ee6c3580c678ac78c1c705a83ea80db8d9058ce647259482a40ede122f512bf0e89abfb6d168fb25dd Movie S4: Representative DIC movies of UM cells added to endothelial monolayers after treatment with Arp2/3 inhibitor (CK666). Left panel shows incomplete intercalation; right panel shows direct transmigration without intercalation. 20 sec intervals between frames; 2 hrs total elapsed time. Download Original Video (.8 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e20-04-0241/20210104/media/mc-e20-04-0241-s08.,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518515abb28bda92fbabe7b929571bd415190bf44d1790b7324fb90f184f91fab10e395327a1bef9725d0c99a358bbbe69c082a95f9d49957eb2932b2be9a6f328f5ec3ab3a0b97d9f37f310d42fd5299f545a21c350266480cca58f95ee1f0fec14c99b7f17a77c808f3513fe04e16c3580c678ac78e67fa6e271c5ab5b4c7fe6d59a40cc5027959d6074417a7c857a84e1b65267b0c8 Movie S5: Representative DIC movies of BAP1+/- 92.1 UM subclones, 2D3 and 1D7, added to endothelial monolayers. 20 sec intervals between frames; 2 hrs total elapsed time. Download Original Video (.8 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e20-04-0241/20210104/media/mc-e20-04-0241-s09.,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518515abb28bda92fbabe7b929571bd415190bf44d1790b7324fb90f184f91fab10e395327a1bef9725d0c99a358bbbe69c082a95f9d49957eb2932b2be9a6f328f5ec3ab3a0b97d9f37f310d42fd5299f545a21c350266480cca58f95ee1f0fec14c99b7f17a77c808f3513fe04e06c3580c678ac7820b0a56dd68b1ff9dedb5ddacd606171f246c3c3917cf3a9ae9f7a23ef33cab0a4