Inhibitors of ERp44, PDIA1, and AGR2 induce disulfide-mediated oligomerization of Death Receptors 4 and 5 and cancer cell death

Abstract

Breast cancer mortality remains unacceptably high, indicating a need for safer and more effective therapeutic agents. Disulfide bond Disrupting Agents (DDAs) were previously identified as a novel class of anticancer compounds that selectively kill cancers that overexpress the Epidermal Growth Factor Receptor (EGFR) or its family member HER2. DDAs kill EGFR+ and HER2+ cancer cells via the parallel downregulation of EGFR, HER2, and HER3 and activation/oligomerization of Death Receptors 4 and 5 (DR4/5). However, the mechanisms by which DDAs mediate these effects are unknown. Affinity purification analyses employing biotinylated-DDAs reveal that the Protein Disulfide Isomerase (PDI) family members AGR2, AGR3, PDIA1, and ERp44 are DDA target proteins. Further analyses demonstrate that shRNA-mediated knockdown of AGR2 and ERp44, or expression of ERp44 mutants, enhance basal and DDA-induced DR5 oligomerization. DDA treatment of breast cancer cells disrupts PDIA1 and ERp44 mixed disulfide bonds with their client proteins. Together, these results reveal DDAs as the first small molecule, active site inhibitors of AGR2 and ERp44, demonstrate a role for AGR2 and ERp44 in regulating the activity, stability, and localization of DR4 and DR5, and nominate ERp44 as a new molecular target for anticancer therapeutics.

Repurposing Tranexamic Acid as an Anticancer Agent

“…TA treatment reduced Lys acetylation, but TA did not override the increased acetylation induced by SAHA co-treatment ( Figure 3E ). In contrast, treatment with the DDA dMtcyDTDO ( Law et al, 2021 ) decreased baseline Lys acetylation and blocked the SAHA-driven increase in acetylation. The p300 protein is a major cellular histone acetyltransferase (HAT).…”

“…Our group identified a class of compounds with anticancer activity termed DDAs that interfere with disulfide bond formation, induce endoplasmic reticulum (ER) stress, and inhibit protein synthesis ( Ferreira et al, 2015 ; Ferreira et al, 2017 ; Wang et al, 2019a ; Wang et al, 2019b ). Results posted in a preprint suggest that DDAs act by inhibiting a subset of Protein Disulfide Isomerases ( Law et al, 2021 ). Therefore, we examined if DDAs induce overlapping responses with TA and therefore may be useful in combination regimens against cancer.…”

“…Results posted in a preprint suggest that DDAs may suppress tumor growth by inhibiting a subset of Protein Disulfide Isomerases (PDIs) ( Law et al, 2021 ). Interestingly, DDAs exhibit some activities that overlap with those of TA, including decreased MYC expression, sensitivity enhancement by MYC overexpression, lowering of STAT3 tyrosine phosphorylation and S6K1 phosphorylation on Thr 389 and Ser 411 , inhibition of protein synthesis, and reduced histone acetylation.…”

Roles of Protein Disulfide Isomerase in Breast Cancer

“…Most PDI inhibitors are known to or likely to bind to catalytic sites of the a or a' domain of PDI: they include PACMA31 [114], P1 [115], 16F16 [116], AS15 [117], CCF642 [118], S-CW3554 [119], Origamicin [120], (±)-dMtcyDTDO [121], Ga-1 [122], 35G8 [123], Copper (II) complex 1 [124], and SK053 [125]. PACMA31 belongs to the class of propynoic acid carbamoyl methyl amides (PACMAs), and it covalently binds to the Cys400 of the active site of PDI, and it shows cytotoxicity on a broad range of human cancer cells [81].…”

“…CCF642, S-CW3554, and Origamicin are also irreversible PDI inhibitors, showing cytotoxicity in multiple myeloma cells (CCF642 and S-CW3554) [118,119] and in neuroblastoma cells (Origamicin) [120]. Disulfide bond disrupting agents (DDAs) are a new class of irreversible PDI inhibitors, and dimethoxy-tcyDTDO and (±)-dMtcyDTDO are confirmed to bind to the active site cysteines of AGR2/3 and ERp44 [121]. It seems to interact with PDIA1, but its binding site on PDIA1 has not yet been determined.…”