Cancer-associated exportin-6 upregulation inhibits the transcriptionally repressive and anticancer effects of nuclear profilin-1

Abstract

SUMMARY Aberrant expression of nuclear transporters and deregulated subcellular localization of their cargo proteins are emerging as drivers and therapeutic targets of cancer. Here, we present evidence that the nuclear exporter exportin-6 and its cargo profilin-1 constitute a functionally important and frequently deregulated axis in cancer. Exportin-6 upregulation occurs in numerous cancer types and is associated with poor patient survival. Reducing exportin-6 level in breast cancer cells triggers antitumor effects by accumulating nuclear profilin-1. Mechanistically, nuclear profilin-1 interacts with eleven-nineteen-leukemia protein (ENL) within the super elongation complex (SEC) and inhibits the ability of the SEC to drive transcription of numerous pro-cancer genes including MYC . XPO6 and MYC are positively correlated across diverse cancer types including breast cancer. Therapeutically, exportin-6 loss sensitizes breast cancer cells to the bromodomain and extra-terminal (BET) inhibitor JQ1. Thus, exportin-6 upregulation is a previously unrecognized cancer driver event by spatially inhibiting nuclear profilin-1 as a tumor suppressor.

Ser71 Phosphorylation Inhibits Actin-Binding of Profilin-1 and Its Apoptosis-Sensitizing Activity

“…Our prior study suggested that some of these anticancer activities may stem from nuclear Pfn1 (Diamond et al, 2015). Our more recent work supported this theory and demonstrated that nuclear Pfn1 functions as a transcriptional repressor by binding and inhibiting the Super Elongation Complex (SEC), a positive regulator of transcriptional elongation of many pro-cancer genes (Zhu et al, 2021). Furthermore, we provided evidence that Pfn1 undergoes spatial deregulation in a broad range of cancer due to overexpression of its nuclear exporter exportin-6.…”

“…Untagged, Myc-tagged, and HA-tagged Pfn1 in pcDNA3, His-tagged Pfn1 in pRK172, untagged Pfn1 in pLenti-CMV/TO-Neo-DEST, and YFP-Pfn1 with and without NES or NLS tag in pFLRu-FH vector have been described previously ( Shao et al, 2008b ; Diamond et al, 2015 ; Zhu et al, 2021 ). Point mutations (S71A and S71D) within Pfn1 were introduced by site-directed mutagenesis using QuikChange.…”

“…For drug treatment in 2D cultures, MDA-MB-231 or BT-549 stable cells were seeded at 1,000 cells per well in 96-well plates or 500 cells per well in 24-well plates, and treated on the next day with vehicle or paclitaxel in quadruplicate wells. Viable cells were quantified 5–7 days later by the Alamar Blue assay ( Diamond et al, 2015 ; Zhu et al, 2021 ). Briefly, they were incubated with 100ul (for 96-well) or 500ul (for 24-well) growth media containing 44 μM resazurin for 2–4 h at 37°C, and the fluorescence intensity of resorufin (converted product) in the media was measured at 540λ Ex/590λ Em on a fluorescence plate reader (Tecan Infinite M200).…”

“…Five-week old female NOD/SCID and NU/NU mice were purchased from Charles River and kept under standard institutional care. Experimental details for orthotopically inoculating MDA-MB-231 stable cells were previously described ( Diamond et al, 2015 ; Zhu et al, 2021 ). For paclitaxel dosing, when tumors in the NU/NU mice reached an average of ∼70 mm 3 , they were randomly divided and treated with 0.9% sodium chloride or paclitaxel (10 mg/kg) by weekly intraperitoneal injection for two consecutive weeks.…”

“…We have found in prior and recent studies that PLP-binding is important for the tumor-inhibitory function of Pfn1 ( Diamond et al, 2015 ), at least partially due to the direct interaction of nuclear Pfn1 with ENL, a PLP-containing protein, in the SEC complex ( Zhu et al, 2021 ). We and others found that PLP-binding of Pfn1 can be abolished by Ser 137 phosphorylation in its C-terminus ( Shao et al, 2008b ; Diamond et al, 2015 ).…”