Pharmacological inhibition of the LIF/LIFR autocrine loop reveals vulnerability of ovarian cancer cells to ferroptosis.

Of all gynecologic cancers, epithelial-ovarian cancer (OCa) stands out with the highest mortality rates. Despite all efforts, 90% of individuals who receive standard surgical and cytotoxic therapy experience disease recurrence. The precise mechanism by which leukemia inhibitory factor (LIF) and its receptor (LIFR) contribute to the progression of OCa remains unknown. Analysis of cancer databases revealed that elevated expression of LIF or LIFR was associated with poor progression-free survival of OCa patients and a predictor of poor response to chemotherapy. Using multiple primary and established OCa cell lines or tissues that represent five subtypes of epithelial-OCa, we demonstrated that LIF/LIFR autocrine signaling is active in OCa. Moreover, treatment with LIFR inhibitor, EC359 significantly reduced OCa cell viability and cell survival with an IC50 ranging from 5-50 nM. Furthermore, EC359 diminished the stemness of OCa cells. Mechanistic studies using RNA-seq and rescue experiments unveiled that EC359 primarily induced ferroptosis by suppressing the glutathione antioxidant defense system. Using multiple in vitro, ex vivo and in vivo models including cell-based xenografts, patient-derived explants, organoids, and xenograft tumors, we demonstrated that EC359 dramatically reduced the growth and progression of OCa. Additionally, EC359 therapy considerably improved tumor immunogenicity by robust CD45+ leukocyte tumor infiltration and polarizing tumor-associated macrophages (TAMs) toward M1 phenotype while showing no impact on normal T-, B-, and other immune cells. Collectively, our findings indicate that the LIF/LIFR autocrine loop plays an essential role in OCa progression and that EC359 could be a promising therapeutic agent for OCa.

The LIFR Inhibitor EC359 Effectively Targets Type II Endometrial Cancer by Blocking LIF/LIFR Oncogenic Signaling.

Endometrial cancer (ECa) is the most common female gynecologic cancer. When comparing the two histological subtypes of endometrial cancer, Type II tumors are biologically more aggressive and have a worse prognosis than Type I tumors. Current treatments for Type II tumors are ineffective, and new targeted therapies are urgently needed. LIFR and its ligand, LIF, have been shown to play a critical role in the progression of multiple solid cancers and therapy resistance. The role of LIF/LIFR in the progression of Type II ECa, on the other hand, is unknown. We investigated the role of LIF/LIFR signaling in Type II ECa and tested the efficacy of EC359, a novel small-molecule LIFR inhibitor, against Type II ECa. The analysis of tumor databases has uncovered a correlation between diminished survival rates and increased expression of leukemia inhibitory factor (LIF), suggesting a potential connection between altered LIF expression and unfavorable overall survival in Type II ECa. The results obtained from cell viability and colony formation assays demonstrated a significant decrease in the growth of Type II ECa LIFR knockdown cells in comparison to vector control cells. Furthermore, in both primary and established Type II ECa cells, pharmacological inhibition of the LIF/LIFR axis with EC359 markedly decreased cell viability, long-term cell survival, and invasion, and promoted apoptosis. Additionally, EC359 treatment reduced the activation of pathways driven by LIF/LIFR, such as AKT, mTOR, and STAT3. Tumor progression was markedly inhibited by EC359 treatment in two different patient-derived xenograft models in vivo and patient-derived organoids ex vivo. Collectively, these results suggest LIFR inhibitor EC359 as a possible new small-molecule therapeutics for the management of Type II ECa.

Inhibition of LIFR Blocks Adiposity-Driven Endometrioid Endometrial Cancer Growth.

Endometrial cancer (EC) is the fourth most common cancer in women, and half of the endometrioid EC (EEC) cases are attributable to obesity. However, the underlying mechanism(s) of obesity-driven EEC remain(s) unclear. In this study, we examined whether LIF signaling plays a role in the obesity-driven progression of EEC. RNA-seq analysis of EEC cells stimulated by adipose conditioned medium (ADP-CM) showed upregulation of LIF/LIFR-mediated signaling pathways including JAK/STAT and interleukin pathways. Immunohistochemistry analysis of normal and EEC tissues collected from obese patients revealed that LIF expression is upregulated in EEC tissues compared to the normal endometrium. Treatment of both primary and established EEC cells with ADP-CM increased the expression of LIF and its receptor LIFR and enhanced proliferation of EEC cells. Treatment of EEC cells with the LIFR inhibitor EC359 abolished ADP-CM induced colony formation andcell viability and decreased growth of EEC organoids. Mechanistic studies using Western blotting, RT-qPCR and reporter assays confirmed that ADP-CM activated LIF/LIFR downstream signaling, which can be effectively attenuated by the addition of EC359. In xenograft assays, co-implantation of adipocytes significantly enhanced EEC xenograft tumor growth. Further, treatment with EC359 significantly attenuated adipocyte-induced EEC progression in vivo. Collectively, our data support the premise that LIF/LIFR signaling plays an important role in obesity-driven EEC progression and the LIFR inhibitor EC359 has the potential to suppress adipocyte-driven tumor progression.

A First-in-Class Inhibitor of ER Coregulator PELP1 Targets ER+ Breast Cancer.

Most patients with estrogen receptor alpha-positive (ER+) breast cancers initially respond to treatment but eventually develop therapy resistance with disease progression. Overexpression of oncogenic ER coregulators, including proline, glutamic acid, and leucine-rich protein 1 (PELP1), are implicated in breast cancer progression. The lack of small molecules that inhibits PELP1 represents a major knowledge gap. Here, using a yeast-two-hybrid screen, we identified novel peptide inhibitors of PELP1 (PIP). Biochemical assays demonstrated that one of these peptides, PIP1, directly interacted with PELP1 to block PELP1 oncogenic functions. Computational modeling of PIP1 revealed key residues contributing to its activity and facilitated the development of a small-molecule inhibitor of PELP1, SMIP34, and further analyses confirmed that SMIP34 directly bound to PELP1. In breast cancer cells, SMIP34 reduced cell growth in a dose-dependent manner. SMIP34 inhibited proliferation of not only wild-type (WT) but also mutant (MT) ER+ and therapy-resistant breast cancer cells, in part by inducing PELP1 degradation via the proteasome pathway. RNA sequencing analyses showed that SMIP34 treatment altered the expression of genes associated with estrogen response, cell cycle, and apoptosis pathways. In cell line-derived and patient-derived xenografts of both WT and MT ER+ breast cancer models, SMIP34 reduced proliferation and significantly suppressed tumor progression. Collectively, these results demonstrate SMIP34 as a first-in-class inhibitor of oncogenic PELP1 signaling in advanced breast cancer. SIGNIFICANCE: Development of a novel inhibitor of oncogenic PELP1 provides potential therapeutic avenues for treating therapy-resistant, advanced ER+ breast cancer.

LIFR inhibition enhances the therapeutic efficacy of HDAC inhibitors in triple negative breast cancer.

Histone deacetylase inhibitors (HDACi) are identified as novel therapeutic agents, however, recent clinical studies suggested that they are marginally effective in treating triple negative breast cancer (TNBC). Here, we show that first-in-class Leukemia Inhibitory Factor Receptor (LIFRα) inhibitor EC359 could enhance the therapeutic efficacy of HDACi against TNBC. We observed that both targeted knockdown of LIFR with CRISPR or treatment with EC359 enhanced the potency of four different HDACi in reducing cell viability, cell survival, and enhanced apoptosis compared to monotherapy in TNBC cells. RNA-seq studies demonstrated oncogenic/survival signaling pathways activated by HDACi were attenuated by the EC359 + HDACi therapy. Importantly, combination therapy potently inhibited the growth of TNBC patient derived explants, cell derived xenografts and patient-derived xenografts in vivo. Collectively, our results suggest that targeted inhibition of LIFR can enhance the therapeutic efficacy of HDACi in TNBC.

LIF/LIFR oncogenic signaling is a novel therapeutic target in endometrial cancer.

Endometrial cancer (EC) is the fourth most common cancer in women. Advanced-stage EC has limited treatment options with a poor prognosis. There is an unmet need for the identification of actionable drivers for the development of targeted therapies in EC. Leukemia inhibitory factor receptor (LIFR) and its ligand LIF play a major role in cancer progression, metastasis, stemness, and therapy resistance. However, little is known about the functional significance of the LIF/LIFR axis in EC progression. In this study using endometrial tumor tissue arrays, we identified that expression of LIF, LIFR is upregulated in EC. Knockout of LIFR using CRISPR/Cas9 in two different EC cells resulted in a significant reduction of their cell viability and cell survival. In vivo studies demonstrated that LIFR-KO significantly reduced EC xenograft tumor growth. Treatment of established and primary patient-derived EC cells with a novel LIFR inhibitor, EC359 resulted in the reduction of cell viability with an IC50 in the range of 20-100 nM and induction of apoptosis. Further, treatment with EC359 reduced the spheroid formation of EC cancer stem cells and reduced the levels of cancer stem cell markers SOX2, OCT4, NANOG, and Axin2. Mechanistic studies demonstrated that EC359 treatment attenuated the activation of LIF-LIFR driven pathways, including STAT3 and AKT/mTOR signaling in EC cells. Importantly, EC359 treatment resulted in a significant reduction of the growth of EC patient-derived explants ex vivo, EC cell line-derived xenografts, and patient-derived xenografts in vivo. Collectively, our work revealed the oncogenic potential of the LIF/LIFR axis in EC and support the utility of LIFR inhibitor, EC359, as a novel targeted therapy for EC via the inhibition of LIF/LIFR oncogenic signaling.

EC313-a tissue selective SPRM reduces the growth and proliferation of uterine fibroids in a human uterine fibroid tissue xenograft model.

Uterine fibroids (UFs) are associated with irregular or excessive uterine bleeding, pelvic pain or pressure, or infertility. Ovarian steroid hormones support the growth and maintenance of UFs. Ulipristal acetate (UPA) a selective progesterone receptor (PR) modulator (SPRM) reduce the size of UFs, inhibit ovulation and lead to amenorrhea. Recent liver toxicity concerns with UPA, diminished enthusiasm for its use and reinstate the critical need for a safe, efficacious SPRM to treat UFs. In the current study, we evaluated the efficacy of new SPRM, EC313, for the treatment for UFs using a NOD-SCID mouse model. EC313 treatment resulted in a dose-dependent reduction in the fibroid xenograft weight (p < 0.01). Estradiol (E2) induced proliferation was blocked significantly in EC313-treated xenograft fibroids (p < 0.0001). Uterine weight was reduced by EC313 treatment compared to UPA treatment. ER and PR were reduced in EC313-treated groups compared to controls (p < 0.001) and UPA treatments (p < 0.01). UF specific desmin and collagen were markedly reduced with EC313 treatment. The partial PR agonism and no signs of unopposed estrogenicity makes EC313 a candidate for the long-term treatment for UFs. Docking studies have provided a structure based explanation for the SPRM activity of EC313.

EC359: A First-in-Class Small-Molecule Inhibitor for Targeting Oncogenic LIFR Signaling in Triple-Negative Breast Cancer.

Leukemia inhibitory factor receptor (LIFR) and its ligand LIF play a critical role in cancer progression, metastasis, stem cell maintenance, and therapy resistance. Here, we describe a rationally designed first-in-class inhibitor of LIFR, EC359, which directly interacts with LIFR to effectively block LIF/LIFR interactions. EC359 treatment exhibits antiproliferative effects, reduces invasiveness and stemness, and promotes apoptosis in triple-negative breast cancer (TNBC) cell lines. The activity of EC359 is dependent on LIF and LIFR expression, and treatment with EC359 attenuated the activation of LIF/LIFR-driven pathways, including STAT3, mTOR, and AKT. Concomitantly, EC359 was also effective in blocking signaling by other LIFR ligands (CTF1, CNTF, and OSM) that interact at LIF/LIFR interface. EC359 significantly reduced tumor progression in TNBC xenografts and patient-derived xenografts (PDX), and reduced proliferation in patient-derived primary TNBC explants. EC359 exhibits distinct pharmacologic advantages, including oral bioavailability, and in vivo stability. Collectively, these data support EC359 as a novel targeted therapeutic that inhibits LIFR oncogenic signaling.See related commentary by Shi et al., p. 1337.

Utilizing cell line-derived organoids to evaluate the efficacy of a novel LIFR-inhibitor, EC359 in targeting pancreatic tumor stroma.

Survival of pancreatic cancer (PC) patient is poor due to lack of effective treatment modalities, which is partly due to the presence of dense desmoplasia that impedes the delivery of chemotherapeutics. Therefore, PC stroma-targeting therapies are expected to improve the efficacy of chemotherapeutics. However, in vitro evaluation of stromal-targeted therapies requires a culture system which includes components of both tumor stroma and parenchyma. We aim to generate a cell line-derived 3D organoids to test the efficacy of stromal-targeted, LIFR-inhibitor EC359. Murine PC (FC1245) and stellate (ImPaSC) cells were cultured to generate organoids that recapitulated the histological organization of PC with the formation of ducts by epithelial cells surrounded by activated fibroblasts, as indicated by CK19 and α-SMA staining, respectively. Analysis by qRT-PCR demonstrated a significant downregulation of markers of activated stroma, POSTN, FN1, MMP9, and SPARC (p<0.0001), when treated with gemcitabine in combination with EC359. Concurrently, collagen proteins including COL1A1, COL1A2, COL3A1, and COL5A1 were significantly downregulated (p <0.0001) after treatment with gemcitabine in combination with EC359. Overall, our study demonstrates the utility of cell lines-derived 3D organoids to evaluate the efficacy of stroma-targeted therapies as well as the potential of EC359 to target activated stroma in PC.

Esters of levonorgestrel and etonogestrel intended as single, subcutaneous-injection, long-lasting contraceptives.

An effort with the goal of discovering single-dose, long-lasting (>6 months) injectable contraceptives began using levonorgestrel (LNG)-17-ß esters linked to a sulfonamide function purposed as human carbonic anhydrase II (hCA 2) ligands. One single analog from this first series showed noticeably superior anti-ovulatory activity in murine models, and a subsequent structure-activity relationship (SAR, the relationship between a compound's molecular structure and its biological activity) study based on this compound identified a LNG-phenoxyacetic acid ester analog exhibiting longer anti-ovulatory properties using the murine model at 2 and 4 mg dose than medroxyprogesterone acetate (MPA). The same ester function linked to etonogestrel (ENG) furnished a compound which inhibited ovulation at 2 mg for 60 days, the longest duration of all compounds tested at these doses. By comparison, MPA at the same dose inhibited ovulation for 32 days.

Progesterone receptor isoforms, agonists and antagonists differentially reprogram estrogen signaling.

Major roadblocks to developing effective progesterone receptor (PR)-targeted therapies in breast cancer include the lack of highly-specific PR modulators, a poor understanding of the pro- or anti-tumorigenic networks for PR isoforms and ligands, and an incomplete understanding of the cross talk between PR and estrogen receptor (ER) signaling. Through genomic analyses of xenografts treated with various clinically-relevant ER and PR-targeting drugs, we describe how the activation or inhibition of PR differentially reprograms estrogen signaling, resulting in the segregation of transcriptomes into separate PR agonist and antagonist-mediated groups. These findings address an ongoing controversy regarding the clinical utility of PR agonists and antagonists, alone or in combination with tamoxifen, for breast cancer management. Additionally, the two PR isoforms PRA and PRB, bind distinct but overlapping genomic sites and interact with different sets of co-regulators to differentially modulate estrogen signaling to be either pro- or anti-tumorigenic. Of the two isoforms, PRA inhibited gene expression and ER chromatin binding significantly more than PRB. Differential gene expression was observed in PRA and PRB-rich patient tumors and PRA-rich gene signatures had poorer survival outcomes. In support of antiprogestin responsiveness of PRA-rich tumors, gene signatures associated with PR antagonists, but not PR agonists, predicted better survival outcomes. The better patient survival associated with PR antagonists versus PR agonists treatments was further reflected in the higher in vivo anti-tumor activity of therapies that combine tamoxifen with PR antagonists and modulators. This study suggests that distinguishing common effects observed due to concomitant interaction of another receptor with its ligand (agonist or antagonist), from unique isoform and ligand-specific effects will inform the development of biomarkers for patient selection and translation of PR-targeted therapies to the clinic.

Effects of Combination of Estradiol with Selective Progesterone Receptor Modulators (SPRMs) on Human Breast Cancer Cells In Vitro and In Vivo.

Use of estrogen or estrogen/progestin combination was an approved regimen for menopausal hormonal therapy (MHT). However, more recent patient-centered studies revealed an increase in the incidence of breast cancer in women receiving menopausal hormone therapy with estrogen plus progestin rather than estrogen alone. Tissue selective estrogen complex (TSEC) has been proposed to eliminate the progesterone component of MHT with supporting evidences. Based on our previous studies it is evident that SPRMs have a safer profile on endometrium in preventing unopposed estrogenicity. We hypothesized that a combination of estradiol (E2) with selective progesterone receptor modulator (SPRM) to exert a safer profile on endometrium will also reduce mammary gland proliferation and could be used to prevent breast cancer when used in MHT. In order to test our hypothesis, we compared the estradiol alone or in combination with our novel SPRMs, EC312 and EC313. The compounds were effectively controlled E2 mediated cell proliferation and induced apoptosis in T47D breast cancer cells. The observed effects were found comparable that of BZD in vitro. The effects of SPRMs were confirmed by receptor binding studies as well as gene and protein expression studies. Proliferation markers were found downregulated with EC312/313 treatment in vitro and reduced E2 induced mammary gland proliferation, evidenced as reduced ductal branching and terminal end bud growth in vivo. These data supporting our hypothesis that E2+EC312/EC313 blocked the estrogen action may provide basic rationale to further test the clinical efficacy of SPRMs to prevent breast cancer incidence in postmenopausal women undergoing MHT.

A new approach towards the synthesis of drospirenone and steroidal spirolactones.

A general methodology for the synthesis of different steroidal 17-spirolactones is described. This method uses lithium acetylide of ethyl propiolate as the three carbon synthon and the method was successfully applied for the process development of drospirenone.

Synthesis and biological evaluation of 11' imidazolyl antiprogestins and mesoprogestins.

Antiprogestins with a 4' para imidazolylphenyl moiety were synthesized and their biochemical interactions with the progesterone and glucocorticoid receptor were investigated. Depending on the substitution pattern at the 17 position partial progesterone receptor (PR)-agonistic derivatives like compounds EC339 and EC336 or pure antagonists like compound EC317 were obtained. EC317 was investigated in vivo and found to be significantly more potent than RU 486 in cycling and pregnant guinea pigs. For testing the biological action progesterone receptor modulators (PRM), guinea pigs appears as a specific model when compare to pregnant human uterus. This model correlates to human conditions such as softening and widening of the cervix, the elevation of the uterine responsiveness to prostaglandins and oxytocin, and finally to induction of labor. The use of non-pregnant guinea pigs permitted the simultaneous assessment of PR-agonistic and PR-antagonistic properties and their physiological interactions with uterine and vaginal environment. These can histologically be presumed from the presence of estrogen or progesterone dominance in the genital tract tissues. The ovarian histology indicated the effects on ovulation. Corpora lutea in guinea pigs further reflects inhibitory effects of the progesterone-dependent uterine prostaglandin secretion. PRMs are initially synthesized as analogues of RU 486. They represent a heterogeneous group of compounds with different ratios of PR-agonistic and-antagonistic properties. PR-agonistic properties may be essential for uterine anti-proliferative effects. In various clinical studies these were also attributed to RU 486 or Ulipristal [1,2]. Adjusted PR-agonistic PRMs (EC312, EC313) [3] may be more effective in achieving a mitotically resting endometrium and superior uterine tumor inhibition. For the use in termination of pregnancy, progesterone-inhibitory effects are essentially needed. Even minor PR-agonistic properties compromise the therapeutic goals. Pure PR-antagonists, as EC317, clearly exceeded the gold standard RU 486 with respect to labor inducing effects. Mechanistically it is surprising that both types of compound may be potent inhibitors of ovulation.

Effects of a novel proteasome inhibitor BU-32 on multiple myeloma cells.

Proteasome inhibition is associated with substantial antitumor effects in preclinical models of multiple myeloma (MM) as well as in patients. However, results of recent clinical trials to evaluate the effect of the proteasome inhibitor Bortezomib (Velcade(®), also called PS-341) in MM patients have shown limited activity when used as a single agent. This underscores the need to find new efficacious and less toxic proteasome inhibitors. Recently, carfilzomib was approved for the treatment of refractory/relapsed MM and several new agents have been introduced into the clinic, including marizomib and MLN9708, and trials investigating these second-generation proteasome inhibitors have demonstrated promising results. We have recently synthesized a novel proteasome inhibitor, BU-32, and tested its growth inhibitory effects in different human MM cells including RPMI8226, MM.1S, MM.1R, and U266. In this study, we evaluate the efficacy of the novel proteasome inhibitor BU-32 (NSC D750499) using an in vitro MM model. BU-32 exhibits strong cytotoxicity in a panel of MM cell lines--RPMI8226, MM1S, MM1R, and U266. In addition, we demonstrate by proteasome inhibition assay that BU-32 potently inhibits the chymotryptic- and caspase-like activities of the 26S proteasome. We further show from Annexin V-FITC binding studies that BU-32, like Bortezomib, induces apoptosis in a panel of MM cell lines but the effect is more pronounced with BU-32-treated cells. Invasion assay with the MM.1S cell line indicates that BU-32 inhibits the invasiveness of myeloma cells. Results from our studies using real-time PCR array analyses show that BU-32 effectively downregulates an array of angiogenesis and inflammatory markers. Our results suggest that BU-32 might be a potential chemotherapeutic agent with promising antitumor activity for the treatment of MM.

Design, synthesis, and biological evaluation of bone-targeted proteasome inhibitors for multiple myeloma.

Multiple myeloma (MM) is an incurable neoplasm characterized by devastating and progressive bone destruction. Standard chemotherapeutic agents have not been effective at significantly prolonging the survival of MM patients and these agents are typically associated with often severe, dose-limiting side effects. There is great need for methods to target the delivery of novel, effective cytotoxic agents specifically to bone, where myeloma cells reside. We have synthesized and evaluated the effects of the bone-targeted proteasome inhibitors PS-341-BP-1, PS-341-BP-2 and MG-262-BP on cell proliferation using the mouse 5TGM1 and human RPMI 8226 cell lines in vitro. The compounds exhibit strong cytotoxicity on MM cell lines and reduce the number of viable cells in a dose dependent manner.

Synthesis of substituted estradiols by the selective aromatization of A-ring of steroidal 19-nor-Δ-4-3-ketones with phenylselenyl halides/hydrogen peroxide.

A range of 6-, 7-, and 11-substituted estradiols were synthesized by the selective aromatization of the A-ring of 19-nor steroids using phenylselenyl halides followed by oxidation with hydrogen peroxide. Established methods utilizing copper(II) halides failed or have given poor yields with these substrates.

Extranuclear functions of ER impact invasive migration and metastasis by breast cancer cells.

The molecular basis of breast cancer progression to metastasis and the role of estrogen receptor (ER) signaling in this process remain poorly understood. Emerging evidence suggests that ER participates in extranuclear signaling in addition to genomic functions. Recent studies identified proline-, glutamic acid-, and leucine-rich protein-1 (PELP1) as one of the components of ER signalosome in the cytoplasm. PELP1 expression is deregulated in metastatic breast tumors. We examined the mechanism and significance of ER-PELP1-mediated extranuclear signals in the cytoskeletal remodeling and metastasis. Using estrogen dendrimer conjugate (EDC) that uniquely activate ER extranuclear signaling and by using model cells that stably express PELP1 short hairpin RNA (shRNA), we show that PELP1 is required for optimal activation of ER extranuclear actions. Using a yeast two-hybrid screen, we identified integrin-linked kinase 1 (ILK1) as a novel PELP1-binding protein. Activation of extranuclear signaling by EDC uniquely enhanced E2-mediated ruffles and filopodia-like structures. Using dominant-negative and dominant-active reagents, we found that estrogen-mediated extranuclear signaling promotes cytoskeleton reorganization through the ER-Src-PELP1-phosphoinositide 3-kinase-ILK1 pathway. Using in vitro Boyden chamber assays and in vivo xenograft assays, we found that ER extranuclear actions contribute to cell migration. Collectively, our results suggest that ER extranuclear actions play a role in cell motility/metastasis, establishing for the first time that endogenous PELP1 serves as a critical component of ER extranuclear actions leading to cell motility/invasion and that the ER-Src-PELP1-ILK1 pathway represents a novel therapeutic target for preventing the emergence of ER-positive metastasis.

BU-32: a novel proteasome inhibitor for breast cancer.

INTRODUCTION: Proteasome inhibition provides an attractive approach to cancer therapy and may have application in the treatment of breast cancer. However, results of recent clinical trials to evaluate the effect of the proteasome inhibitor Bortezomib (Velcade, also called PS-341) in metastatic breast cancer patients have shown limited activity when used as a single agent. This underscores the need to find new and more efficacious proteasome inhibitors. In this study, we evaluate the efficacy of the novel proteasome inhibitor BU-32 (NSC D750499-S) using in vitro and in vivo breast cancer models. METHODS: We have recently synthesized a novel proteasome inhibitor (BU-32) and tested its growth inhibitory effects in different breast cancer cells including MCF-7, MDA-MB-231, and SKBR3 by in vitro cytotoxicity and proteasomal inhibition assays. The apoptotic potential of BU32 was tested using flow cytometry and analyzing cell cycle regulatory proteins. In vivo tumor xenograft studies for solid tumor as well as tumor metastasis were conducted using MDA-MB-231-GFP cells. RESULTS: We report for the first time that BU-32 exhibits strong cytotoxicity in a panel of cell lines: MDA-MB-231 (IC50 = 5.8 nM), SKBR3 (IC50 = 5.7 nM) and MCF-7 cells (IC50 = 5.8 nM). It downregulates a wide array of angiogenic marker genes and upregulates apoptotic markers, including Bid and Bax. Incubation of MDA-MB-231 cells with BU-32 results in the accumulation of cell cycle inhibitor proteins p21 and p27 and stabilization of the tumor suppressor protein p53. Studies in in vivo solid tumor and metastasis models show significant effect with a 0.06 mg/kg dose of BU-32 and marked reduction in tumor burden in the skeleton. CONCLUSIONS: We have shown that BU-32 is effective in cultured breast cancer cells and in breast cancer xenografts. The results suggest its potential benefit in breast cancer treatment.