Bindu Santhamma

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

IntroductionProteasome 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.MethodsWe 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.ResultsWe 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.ConclusionsWe 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.

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.

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.

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.

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.

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

Graphical abstract Figure. No Caption available. HighlightsCarbodiimide‐mediated esterification of levonogestrel and etonogestrel.First examples of sulfonamide‐derived norgestrel analogs.Two compounds identified as effective anti‐ovulation agents in the murine model. Abstract An effort with the goal of discovering single‐dose, long‐lasting (>6 months) injectable contraceptives began using levonorgestrel (LNG)‐17‐&bgr; 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.

Abstract 5792: Development of cell line derived organoids to evaluate stroma targeted therapies in pancreatic cancer

Background: Most solid tumors have extensive stroma that not only facilitates the tumor progression but also impedes the delivery of the chemotherapeutic agents. Due to lack of any in-vitro system, presently it is difficult to evaluate any stroma-targeted therapies. Therefore, we developed an organoid system using labeled pancreatic cancer and stellate cell lines. Methods: Murine (FC 1295 and imPSCc-2) cell lines cultured in different combinations were grown as an organoid system using matrigel. The organoids, starting day four were treated with either gemcitabine or EC359, a novel mifepristone derived steroidal cytotoxic agent that targets tumor stroma, or both in combination. qRT-PCR analysis of activated stroma signature genes was performed on the mRNA isolated from different treatment groups. HE 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5792. doi:10.1158/1538-7445.AM2017-5792

Abstract LB-208: First-in-class steroidal leukemia inhibitory factor (LIF) inhibitor in targeted cancer therapy

The leukemia inhibitory factor (LIF), an IL-6 family member, is a pleotropic cytokine has been demonstrated as an oncogene and involved in multiple cellular mechanisms during the initiation and progression of various human malignancies. Overexpression of LIF was significantly correlated with advanced tumor stage, larger tumor size and worse relapse free survival rate. In the present study EC330, a novel steroidal LIF inhibitor showed cytotoxicity in various cancer cell lines and NCI-60 cell line panel at low nano-molar range. EC330 blocked formation of colonies in soft agar and inhibited angiogenesis (tube formation) in human umbilical vein endothelial cells. EC330 binds to LIF/LIFR complex by insilico docking studies and reduced the STAT3 phosphorylation in a dose dependent manner through blocking LIF-JAK-STAT signaling. EC330 showed marked specificity in MCF-7 cells overexpressing LIF verses MCF-7 cells. The compound further showed cytoskeletal disruption and targeting cancer associated fibroblasts (CAFs). EC330 treatment (0.5 and 2.5mg/kg) dose dependently reduced tumor burden in ovarian (IGROV-1) and triple negative breast cancer (MDA-MB-231) cell xenografted mouse models. These findings establish EC330 as a novel LIF inhibitor having targeted therapeutic perspectives for patients with aggressive primary tumors. LIF targeting may result in the blockade of cancer fibroblast associated pro invasive tumor microenvironment in regular as well as therapy resistant tumors. Citation Format: Hareesh B. Nair, Bindu Santhamma, Surya Viswanadhapa, Klaus J. Nickisch. First-in-class steroidal leukemia inhibitory factor (LIF) inhibitor in targeted cancer therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-208.