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.

Targeting LIF/LIFR signaling in cancer.

Leukemia inhibitory factor (LIF), and its receptor (LIFR), are commonly over-expressed in many solid cancers and recent studies have implicated LIF/LIFR axis as a promising clinical target for cancer therapy. LIF/LIFR activate oncogenic signaling pathways including JAK/STAT3 as immediate effectors and MAPK, AKT, mTOR further downstream. LIF/LIFR signaling plays a key role in tumor growth, progression, metastasis, stemness and therapy resistance. Many solid cancers show overexpression of LIF and autocrine stimulation of the LIF/LIFR axis; these are associated with a poorer relapse-free survival. LIF/LIFR signaling also plays a role in modulating multiple immune cell types present in tumor micro environment (TME). Recently, two targeted agents that target LIF (humanized anti-LIF antibody, MSC-1) and LIFR inhibitor (EC359) were under development. Both agents showed effectivity in preclinical models and clinical trials using MSC-1 antibody are in progress. This article reviews the significance of LIF/LIFR pathways and inhibitors that disrupt this process for the treatment of cancer.

The Pleiotropic role, functions and targeted therapies of LIF/LIFR axis in cancer: Old spectacles with new insights.

The dysregulation of leukemia inhibitory factor (LIF) and its cognate receptor (LIFR) has been associated with multiple cancer initiation, progression, and metastasis. LIF plays a significant tumor-promoting role in cancer, while LIFR functions as a tumor promoter and suppressor. Epithelial and stromal cells secrete LIF via autocrine and paracrine signaling mechanism(s) that bind with LIFR and subsequently with co-receptor glycoprotein 130 (gp130) to activate JAK/STAT1/3, PI3K/AKT, mTORC1/p70s6K, Hippo/YAP, and MAPK signaling pathways. Clinically, activating the LIF/LIFR axis is associated with poor survival and anti-cancer therapy resistance. This review article provides an overview of the structure and ligands of LIFR, LIF/LIFR signaling in developmental biology, stem cells, cancer stem cells, genetics and epigenetics of LIFR, LIFR regulation by long non-coding RNAs and miRNAs, and LIF/LIFR signaling in cancers. Finally, neutralizing antibodies and small molecule inhibitors preferentially blocking LIF interaction with LIFR and antagonists against LIFR under pre-clinical and early-phase pre-clinical trials were discussed.

Growth Inhibitory Efficacy of Chinese Herbs in a Cellular Model for Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is characterized by the absence of estrogen receptor-α progesterone receptor and human epidermal growth factor receptor-2. Treatment for this breast cancer subtype is restricted to multidrug chemotherapy and survival pathway-based molecularly targeted therapy. The long-term treatment options are associated with systemic toxicity, spontaneous and/or acquired tumor resistance and the emergence a of drug-resistant stem cell population. These limitations lead to advanced stage metastatic cancer. Current emphasis is on research directions that identify efficacious, naturally occurring agents representing an unmet need for testable therapeutic alternatives for therapy resistant breast cancer. Chinese herbs are widely used in traditional Chinese medicine in women for estrogen related health issues and also for integrative support for cancer treatment. This review discusses published evidence on a TNBC model for growth inhibitory effects of several mechanistically distinct nontoxic Chinese herbs, most of them nutritional in nature, and identifies susceptible pathways and potential molecular targets for their efficacy. Documented anti-proliferative and pro-apoptotic effects of these herbs are associated with downregulation of RB, RAS, PI3K, and AKT signaling, modulation of Bcl-2/BAX protein expressions and increased caspase activity. This review provides a proof of concept for Chinese herbs as testable alternatives for prevention/therapy of TNBC.

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.

Growth inhibitory efficacy and anti-aromatase activity of Tabebuia avellanedae in a model for post-menopausal Luminal A breast cancer.

Aromatase inhibitors (AIs) represent a treatment option for post-menopausal estrogen receptor-positive (ER+) breast cancer as monotherapy, or in combination with cyclin-dependent kinase 4/6 or mTOR inhibitors. Long-term treatment with these agents leads to dose-limiting toxicity and drug resistance. Natural substances provide testable alternatives to current therapy. Tabebuia avellanedae (TA) tree is indigenous to the Amazon rainforest. The inner bark of TA represents a medicinal dietary supplement known as Taheebo. Non-fractionated aqueous extract from TA is an effective growth inhibitor in the Luminal A and triple negative breast cancer models. The quinone derivative naphthofurandione (NFD) is a major bioactive agent in TA. The present study examined the efficacy of finely ground powder from the inner bark of TA, available under the name of Taheebo-NFD-Marugoto (TNM). The ER+ MCF-7 cells stably transfected with the aromatase gene MCF-7AROM represented a model for aromatase-expressing post-menopausal breast cancer. Anchorage-independent colony formation, cell cycle progression, pro-apoptotic caspase 3/7 activity, apoptosis-specific gene expression, aromatase activity and select estradiol (E2) target gene expression represented the mechanistic end points. Treatment of MCF-7AROM cells with TNM induced a dose-dependent reduction in E2-promoted anchorage-independent colony number. Mechanistic assays on TNM-treated MCF-7AROM cells demonstrated that TNM at a concentration of 10 µg (NFD content: 2 ng), induced S-phase arrest, increased pro-apoptotic caspase 3/7 activity, increased pro-apoptotic BAX and decreased anti-apoptotic BCL-2 gene expression, and inhibited aromatase activity. Additionally, TNM treatment downregulated ESR-1 (gene for ER-α), aromatase and progesterone gene expression and reduced mRNA levels of E2 target genes pS2, GRB2 and cyclin D1. Inhibition of aromatase activity, based on the NFD content of TNM was superior to the clinical AIs Letrozole and Exemestane. These data demonstrated the potential efficacy of TNM as a nutritional alternative for current therapy of aromatase positive, post-menopausal breast cancer.

Growth inhibitory efficacy of Cornus officinalis in a cell culture model for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) lacks the expressions of estrogen receptor-α, progesterone receptor and human epidermal growth factor receptor-2. The treatment options for TNBC include anthracyclin/taxol based conventional chemotherapy and small molecular inhibitor based targeted therapy. However, the therapeutic efficacy is limited by systemic toxicity and acquired tumor resistance; identification of less toxic testable alternatives is urgently required. Non-toxic nutritional herbs are commonly used in traditional Chinese herbal medicine for general health management and may additionally represent a testable therapeutic alternative for TNBC. The present study examined the growth inhibitory efficacy of the nutritional herb Cornus officinalis (CO) in MDA-MB-231 cells, which represent a cell culture model for TNBC, and identified potential mechanistic leads. In MDA-MB-231 cells, CO induced dose-dependent cytostatic growth arrest [inhibitory concentration (IC)50, 0.1% and IC90, 0.5%], and inhibited anchorage independent colony formation. Mechanistically, CO inhibited G1 to S phase transition leading to G1 arrest and decreased the expression of cyclin D1 and phosphorylated-retinoblastoma proteins. CO additionally altered apoptosis specific BCL-2 associated X protein/B-cell lymphoma-2 expression and upregulated pro-apoptotic caspase-3/7 activity. Collectively, these data provided mechanistic evidence for the efficacy of CO, and validated a mechanism-based approach to prioritize efficacious nutritional herbs as testable alternatives for secondary prevention/treatment of TNBC.

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.

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.

A prodrug design for improved oral absorption and reduced hepatic interaction.

A series of estradiol-17-ß esters of N-(p-sulfomylbenzamide)-amino acids were prepared and evaluated for systemic and hepatic estrogenic activity after oral administration in ovariectomized rats. The alkyl substitution at nitrogen of amino acids such as proline or N-methyl-alanine produced compounds that exhibit potent oral activity. The proline analog (EC508) was further evaluated along with 17ß-estradiol (E2) and ethinyl-estradiol (EE) and compared their effects on the uterus, angiotensin and HDL-cholesterol after oral administration to ovariectomized female rats. Orally administered EC508 produced systemic estrogenic activity 10 times greater than EE and a 100 times higher activity than E2 with no influence on levels of angiotensin and HDL-cholesterol, whereas EE and E2 reduced the HDL-cholesterol and increased the angiotensine plasma levels. EC508 might offer significant advantages in indications like fertility control and HRT based on its high oral bioavailability and lack of hepatic estrogenicity.

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.

An efficient model of human endometriosis by induced unopposed estrogenicity in baboons.

Endometriosis is a chronic estrogen-dependent disease that occurs in approximately 10% of reproductive age women. Baboons offer a clear benefit for studying the initiation and progression of endometriosis since baboon is very close to humans phylogenetically. Progestins are used in the treatment of endometriosis. The therapeutic window of progestins depends on the ratio of its affinity towards progesterone receptor agonism verses antagonism. The present study is to determine the role of pure antiprogestin in baboon endometriosis. We hypothesize that pure antiprogestin will induce unopposed estrogenicity and spontaneous endometriosis in baboons. The rate of endometrial invasion and attachment through modeled peritoneum in the presence and absence of progesterone and antiprogestin was evaluated in this study. A baboon model of endometriosis induced by unopposed estrogenicity using progesterone receptor antagonist (EC304) was used in this study. We observed EC304 has induced unopposed estrogenicity that deregulated proteins involved in attachment, invasion, cell growth, and steroid hormone receptors in this model. Our data suggest that depleting progesterone levels in the endometrium will increase estrogen hyper-responsiveness that leads to increased endometriotic lesion progression in the baboon (Papio anubis) model. This study reports a refined model of human endometriosis in baboons that could potentially be used to develop new diagnostic and therapeutic strategies for the benefit of women suffering from endometriosis.

Synthesis and antiprogestational properties of novel 17-fluorinated steroids.

Progesterone receptor (PR) plays a key role in reproductive functions, and compounds that inhibit progesterone action (antiprogestins) have potential use in the treatment of estrogen- and progesterone-dependent diseases, including uterine leiomyomas and breast cancer. In the present study, we chemically synthesized novel 17-fluorinated steroids and evaluated the cytotoxicity profiles of these compounds in T47D breast cancer cells compared to the activity of known antiprogestins, including ZK230 211, RU-486, CDB2914, CDB4124 and ORG33628. We analyzed in vitro receptor-binding assays and PR-transactivation assays to establish the antiprogestational activity of these molecules. The representative antiprogestin EC304 was found to inhibit in vitro tumorigenicity in a dose-dependent fashion in T47D cells by a colony formation assay at 1 and 10nM concentrations. The potent in vivo antiprogestational activity of EC304 was also demonstrated in an antinidation assay for the interruption of early pregnancy in rats. The strong antiprogestational activity and absence of antiglucocorticoid activity in EC compounds may demonstrate their utility in the treatment of leiomyoma, endometriosis and breast cancer.

Gossypin as a novel selective dual inhibitor of V-RAF murine sarcoma viral oncogene homolog B1 and cyclin-dependent kinase 4 for melanoma.

Mutation in the BRAF gene (BRAFV600E) exists in nearly 70% of human melanomas. Targeted therapy against BRAFV600E kinase using a recently identified RAF-selective inhibitor, PLX4032, has been successful in early clinical trials. However, in patients with the normal BRAF allele (wild-type), PLX4032 is protumorigenic. This conundrum identifies the unmet need for novel therapeutic agents to target BRAFV600E kinase that are not counterproductive. We have identified gossypin, a pentahydroxy flavone, as a potent antimelanoma agent. Gossypin inhibited human melanoma cell proliferation, in vitro, in melanoma cell lines that harbor both BRAFV600E kinase and cyclin-dependent kinase 4 (CDK4) as well as in cells with BRAF wild-type allele. Gossypin inhibited kinase activities of BRAFV600E and CDK4, in vitro, possibly through direct binding of gossypin with these kinases, as confirmed by molecular docking studies. For cells harboring the BRAFV600E, gossypin inhibited cell proliferation through abrogation of the MEK-ERK-cyclin D1 pathway and in cells with BRAF wild-type allele, through attenuation of the retinoblastoma-cyclin D1 pathway. Furthermore, gossypin significantly inhibited melanoma growth in an organotypic three-dimensional skin culture mimicking human skin. Gossypin (10 and 100 mg/kg) treatment for 10 days in human melanoma (A375) cell xenograft tumors harboring BRAFV600E significantly reduced tumor volume through induction of apoptosis and increased survival rate in mice, and the effect was significantly superior to that of PLX4032 (10 mg/kg) or roscovitine 10 mg/kg. In summary, this study identified gossypin as a novel agent with dual inhibitory effects for BRAFV600E kinase and CDK4 for treatment of melanoma.