Heat shock protein 27 (Hsp27) is a chaperone protein, and its expression is increased in response to various stress stimuli including anticancer chemotherapy, which allows the cells to survive and causes drug resistance. We previously identified lead compounds that bound to Hsp27 and tubulin via proteomic approaches. Systematic ligand based optimization in the current study significantly increased the cell growth inhibition and apoptosis inducing activities of the compounds. Compared to the lead compounds, one of the new derivatives exhibited much better potency to inhibit tubulin polymerization but a decreased activity to inhibit Hsp27 chaperone function, suggesting that the structural modification dissected the dual targeting effects of the compound. The most potent compounds 20 and 22 exhibited strong cell proliferation inhibitory activities at subnanomolar concentration against 60 human cancer cell lines conducted by Developmental Therapeutic Program at the National Cancer Institute and represented promising candidates for anticancer drug development.

Peroxisome proliferator–activated receptor-γ (PPARγ) is a ligand-activated nuclear receptor which controls lipid and glucose metabolism. It is also the master regulator of adipogenesis. In adipocytes, ligand-dependent PPARγ activation is associated with proteasomal degradation; therefore, regulation of PPARγ degradation may modulate its transcriptional activity. Here, we show that neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4), an E3 ubiquitin ligase, interacts with the hinge and ligand binding domains of PPARγ and is a bona fide E3 ligase for PPARγ. NEDD4 increases PPARγ stability through the inhibition of its proteasomal degradation. Knockdown of NEDD4 in 3T3-L1 adipocytes reduces PPARγ protein levels and suppresses adipocyte conversion. PPARγ correlates positively with NEDD4 in obese adipose tissue. Together, these findings support NEDD4 as a novel regulator of adipogenesis by modulating the stability of PPARγ.

It has been demonstrated that two-dimensional (2D) monolayer cancer cell proliferation assay for anti-cancer drug screening is a very artificial model and cannot represent the characteristics of three-dimensional (3D) solid tumors. The multi-cellular in vitro 3D tumor spheroid model is of intermediate complexity, and can provide a bridge to the gap between the complex in vivo tumors and simple in vitro monolayer cell cultures. In this study, a simple and cost-effective cancer 3D spheroid assay suitable for small molecule anti-cancer compound screening was developed, standardized and validated on H292 non-small lung cancer cell line. A pilot screening with this assay was performed utilizing a compound library consisting of 41 anti-cancer agents. The traditional 2D monolayer cell proliferation assay was also performed with the same cell line and compounds. A correlational study based on the IC(50) values from the 2D and 3D assays was conducted. There is low correlation with the two sets of biological data, suggesting the two screening methods provide different information regarding the potency of the tested drug candidates.

High frequency of KRAS and TP53 mutations is a unique genetic feature of pancreatic ductal adenocarcinoma (PDAC). TP53 mutation not only renders PDAC resistance to chemotherapies but also drives PDAC invasiveness. Therapies targeting activating mutant KRAS are not available and the outcomes of current PDAC treatment are extremely poor. Here, we report that MMRi62, initially identified as an MDM2-MDM4–targeting small molecule with p53-independent pro-apoptotic activity, shows anti-PDAC activity in vitro and in vivo. We show that MMRi62 inhibits proliferation, clonogenic, and spheroid growth of PDAC cells by induction of cell death. MMRi62-induced cell death in PDAC is characteristic of ferroptosis that is associated with increased autophagy, increased reactive oxygen species, and lysosomal degradation of NCOA4 and ferritin heavy chain (FTH1). In addition to induced degradation of FTH1, MMRi62 also induces proteasomal degradation of mutant p53. Interestingly, MMRi62-induced ferroptosis occurs in PDAC cell lines harboring either KRAS and TP53 double mutations or single TP53 mutation. In orthotopic xenograft PDAC mouse models, MMRi62 was capable of inhibiting tumor growth in mice associated with downregulation of NCOA4 and mutant p53 in vivo. Strikingly, MMRi62 completely abrogated metastasis of orthotopic tumors to distant organs, which is consistent with MMRi62's ability to inhibit cell migration and invasion in vitro. These findings identified MMRi62 as a novel ferroptosis inducer capable of suppressing PDAC growth and overcoming metastasis.

Tubulin and heat shock protein 27 (Hsp27) are well-characterized molecular targets for anti-cancer drug development. We previously identified lead compounds that inhibited both Hsp27 and tubulin. These compounds exhibited extensive anti-cancer activities against the proliferation of various human cancer cell lines. In the current study, a systematic ligand based structural optimization led to new analogs that significantly inhibited the growth of a panel of breast cancer cell lines. Furthermore, the most potent compounds were examined with tubulin polymerization assay and Hsp27 chaperone activity assay. The compounds showed potent tubulin polymerization inhibition but no Hsp27 inhibitory effect. The structural optimization dissected the dual activity and improved the selectivity of the compounds for tubulin. The results revealed several structural moieties of the lead compounds that are critical for Hsp27 inhibition. The modification of these structural fragments eliminated Hsp27 inhibition, but did not harm tubulin-targeting effects of the compounds. This result further defined the structure-activity relationship between the tubulin and Hsp27 effects of these compounds.