Plasma Membrane Channel TRPM4 Mediates Immunogenic Therapy-Induced Necrosis.

Several emerging therapies kill cancer cells primarily by inducing necrosis. As necrosis activates immune cells, potentially, uncovering the molecular drivers of anticancer therapy-induced necrosis could reveal approaches for enhancing immunotherapy efficacy. To identify necrosis-associated genes, we performed a genome-wide CRISPR-Cas9 screen with negative selection against necrosis-inducing preclinical agents BHPI and conducted follow-on experiments with ErSO. The screen identified transient receptor potential melastatin member 4 (TRPM4), a calcium-activated, ATP-inhibited, sodium-selective plasma membrane channel. Cancer cells selected for resistance to BHPI and ErSO exhibited robust TRPM4 downregulation, and TRPM4 reexpression restored sensitivity to ErSO. Notably, TRPM4 knockout (TKO) abolished ErSO-induced regression of breast tumors in mice. Supporting a broad role for TRPM4 in necrosis, knockout of TRPM4 reversed cell death induced by four additional diverse necrosis-inducing cancer therapies. ErSO induced anticipatory unfolded protein response (a-UPR) hyperactivation, long-term necrotic cell death, and release of damage-associated molecular patterns that activated macrophages and increased monocyte migration, all of which was abolished by TKO. Furthermore, loss of TRPM4 suppressed the ErSO-induced increase in cell volume and depletion of ATP. These data suggest that ErSO triggers initial activation of the a-UPR but that it is TRPM4-mediated sodium influx and cell swelling, resulting in osmotic stress, which sustains and propagates lethal a-UPR hyperactivation. Thus, TRPM4 plays a pivotal role in sustaining lethal a-UPR hyperactivation that mediates the anticancer activity of diverse necrosis-inducing therapies. SIGNIFICANCE: A genome-wide CRISPR screen reveals a pivotal role for TRPM4 in cell death and immune activation following treatment with diverse necrosis-inducing anticancer therapies, which could facilitate development of necrosis-based cancer immunotherapies.

A small-molecule activator of the unfolded protein response eradicates human breast tumors in mice.

Metastatic estrogen receptor α (ERα)-positive breast cancer is presently incurable. Seeking to target these drug-resistant cancers, we report the discovery of a compound, called ErSO, that activates the anticipatory unfolded protein response (a-UPR) and induces rapid and selective necrosis of ERα-positive breast cancer cell lines in vitro. We then tested ErSO in vivo in several preclinical orthotopic and metastasis mouse models carrying different xenografts of human breast cancer lines or patient-derived breast tumors. In multiple orthotopic models, ErSO treatment given either orally or intraperitoneally for 14 to 21 days induced tumor regression without recurrence. In a cell line tail vein metastasis model, ErSO was also effective at inducing regression of most lung, bone, and liver metastases. ErSO treatment induced almost complete regression of brain metastases in mice carrying intracranial human breast cancer cell line xenografts. Tumors that did not undergo complete regression and regrew remained sensitive to retreatment with ErSO. ErSO was well tolerated in mice, rats, and dogs at doses above those needed for therapeutic responses and had little or no effect on normal ERα-expressing murine tissues. ErSO mediated its anticancer effects through activation of the a-UPR, suggesting that activation of a tumor protective pathway could induce tumor regression.

Combined Targeting of Estrogen Receptor Alpha and Exportin 1 in Metastatic Breast Cancers.

The majority of breast cancer specific deaths in women with estrogen receptor positive (ER+) tumors occur due to metastases that are resistant to therapy. There is a critical need for novel therapeutic approaches to achieve tumor regression and/or maintain therapy responsiveness in metastatic ER+ tumors. The objective of this study was to elucidate the role of metabolic pathways that undermine therapy efficacy in ER+ breast cancers. Our previous studies identified Exportin 1 (XPO1), a nuclear export protein, as an important player in endocrine resistance progression and showed that combining selinexor (SEL), an FDA-approved XPO1 antagonist, synergized with endocrine agents and provided sustained tumor regression. In the current study, using a combination of transcriptomics, metabolomics and metabolic flux experiments, we identified certain mitochondrial pathways to be upregulated during endocrine resistance. When endocrine resistant cells were treated with single agents in media conditions that mimic a nutrient deprived tumor microenvironment, their glutamine dependence for continuation of mitochondrial respiration increased. The effect of glutamine was dependent on conversion of the glutamine to glutamate, and generation of NAD+. PGC1α, a key regulator of metabolism, was the main driver of the rewired metabolic phenotype. Remodeling metabolic pathways to regenerate new vulnerabilities in endocrine resistant breast tumors is novel, and our findings reveal a critical role that ERα-XPO1 crosstalk plays in reducing cancer recurrences. Combining SEL with current therapies used in clinical management of ER+ metastatic breast cancer shows promise for treating and keeping these cancers responsive to therapies in already metastasized patients.

Src couples estrogen receptor to the anticipatory unfolded protein response and regulates cancer cell fate under stress.

Accumulation of unfolded protein, or other stresses, activates the classical reactive unfolded protein response (UPR). In the recently characterized anticipatory UPR, receptor-bound estrogen, progesterone and other mitogenic hormones rapidly elicit phosphorylation of phospholipase C γ (PLCγ), activating the anticipatory UPR. How estrogen and progesterone activating their receptors couples to PLCγ phosphorylation and anticipatory UPR activation was unknown. We show that the oncogene c-Src is a rate-limiting regulator whose tyrosine kinase activity links estrogen and progesterone activating their receptors to anticipatory UPR activation. Supporting Src coupling estrogen and progesterone to anticipatory UPR activation, we identified extranuclear complexes of estrogen receptor α (ERα):Src:PLCγ and progesterone receptor:Src:PLCγ. Moreover, Src inhibition protected cancer cells against cell death. To probe Src's role, we used the preclinical ERα biomodulator, BHPI, which kills cancer cells by inducing lethal anticipatory UPR hyperactivation. Notably, Src inhibition blocked BHPI-mediated anticipatory UPR activation and the resulting rapid increase in intracellular calcium. After unbiased long-term selection for BHPI-resistant human breast cancer cells, 4/11 BHPI-resistant T47D clones, and nearly all MCF-7 clones, exhibited reduced levels of normally growth-stimulating Src. Notably, Src overexpression by virus transduction restored sensitivity to BHPI. Furthermore, in wild type cells, several-fold knockdown of Src, but not of ERα, strongly blocked BHPI-mediated UPR activation and subsequent HMGB1 release and necrotic cell death. Thus, Src plays a previously undescribed pivotal role in activation of the tumor-protective anticipatory UPR, thereby increasing the resilience of breast cancer cells. This is a new role for Src and the anticipatory UPR in breast cancer.

Protein kinase C-α is upregulated by IMP1 in melanoma and is linked to poor survival.

The oncofetal mRNA-binding protein, IMP1 or insulin-like growth factor-2 mRNA-binding protein 1 (IGF2BP1), promotes the overexpression of several oncogenic proteins by binding to and stabilizing their mRNAs. IMP1 is frequently overexpressed in melanoma and is associated with a poor prognosis, but the full spectrum of IMP1 target transcripts remains unknown. Here, we report the identification of protein kinase C-α (PKCα), as a novel molecular target of IMP1. Overexpression of IMP1 resulted in increased levels of PKCα, while RNAi knockdown of IMP1 resulted in decreased PKCα mRNA stability, PKCα protein levels, and MAPK/ERK activation. In addition to IMP1 acting as a positive regulator of PKCα mRNA, we also report the identification of miR-340 as a negative regulator of PKCα mRNA. In melanoma cancer cells, inhibition of miR-340 led to increased PKCα protein levels. PKCα plays important roles in numerous signaling pathways including the MAPK/ERK signaling pathway. PKCα activates RAF1, which in turn activates MEK1, and activates downstream transcriptional targets of MAPK through activation of JNK signaling. Together, these pathways provide a way to activate MAPK signaling downstream of BRAF and MEK1 inhibitors, which are commonly used to treat melanoma. Analysis of 117 melanoma tumors samples showed that overexpression of PKCα is associated with poorer overall survival. In patients harboring BRAF or NRAS mutations, PKCα overexpression is associated with an 11-fold increased risk of death. Thus, PKCα mRNA is a novel target of IMP1, which is commonly overexpressed in melanoma and is linked to poorer overall survival.

Estrogen-independent Myc overexpression confers endocrine therapy resistance on breast cancer cells expressing ERαY537S and ERαD538G mutations.

Approximately 30% of metastatic breast cancers harbor estrogen receptor α (ERα) mutations associated with resistance to endocrine therapy and reduced survival. Consistent with their constitutive proliferation, T47D and MCF7 cells in which wild-type ERα is replaced by the most common mutations, ERαY537S and ERαD538G, exhibit partially estrogen-independent gene expression. A novel invasion/dissociation/rebinding assay demonstrated that the mutant cells have a higher tendency to dissociate from invasion sites and rebind to a second site. Compared to ERαD538G breast tumors, ERαY537S tumors exhibited a dramatic increase in lung metastasis. Transcriptome analysis showed that the ERαY537S and ERαD538G mutations each elicit a unique gene expression profile. Gene set enrichment analysis showed Myc target pathways are highly induced in mutant cells. Moreover, chromatin immunoprecipitation showed constitutive, fulvestrant-resistant, recruitment of ERα mutants to the Myc enhancer region, resulting in estrogen-independent Myc overexpression in mutant cells and tumors. Knockdown and virus transduction showed Myc is necessary and sufficient for ligand-independent proliferation of the mutant cells but had no effect on metastasis-related phenotypes. Thus, Myc plays a key role in aggressive proliferation-related phenotypes exhibited by breast cancer cells expressing ERα mutations.

Structurally Novel Antiestrogens Elicit Differential Responses from Constitutively Active Mutant Estrogen Receptors in Breast Cancer Cells and Tumors.

Many estrogen receptor α (ERα)-positive breast cancers develop resistance to endocrine therapy via mutation of ERs whose constitutive activation is associated with shorter patient survival. Because there is now a clinical need for new antiestrogens (AE) against these mutant ERs, we describe here our development and characterization of three chemically novel AEs that effectively suppress proliferation of breast cancer cells and tumors. Our AEs are effective against wild-type and Y537S and D538G ERs, the two most commonly occurring constitutively active ERs. The three new AEs suppressed proliferation and estrogen target gene expression in WT and mutant ER-containing cells and were more effective in D538G than in Y537S cells and tumors. Compared with WT ER, mutants exhibited approximately 10- to 20-fold lower binding affinity for AE and a reduced ability to be blocked in coactivator interaction, likely contributing to their relative resistance to inhibition by AE. Comparisons between mutant ER-containing MCF7 and T47D cells revealed that AE responses were compound, cell-type, and ERα-mutant dependent. These new ligands have favorable pharmacokinetic properties and effectively suppressed growth of WT and mutant ER-expressing tumor xenografts in NOD/SCID-γ mice after oral or subcutaneous administration; D538G tumors were more potently inhibited by AE than Y537S tumors. These studies highlight the differential responsiveness of the mutant ERs to different AEs and make clear the value of having a toolkit of AEs for treatment of endocrine therapy-resistant tumors driven by different constitutively active ERs. Cancer Res; 77(20); 5602-13. ©2017 AACR.

A Novel IMP1 Inhibitor, BTYNB, Targets c-Myc and Inhibits Melanoma and Ovarian Cancer Cell Proliferation.

The oncofetal mRNA-binding protein, IMP1 or insulin-like growth factor-2 mRNA-binding protein 2 (IGF2BP1), binds to and stabilizes c-Myc, ß-TrCP1, and other oncogenic mRNAs, leading to increased expression of the proteins encoded by its target mRNAs. IMP1 is frequently overexpressed in cancer and is strongly correlated with a poor prognosis and reduced survival in melanoma, ovarian, breast, colon, and lung cancer. While IMP1 is an attractive anticancer drug target, there are no small molecule inhibitors of IMP1. A fluorescence anisotropy-based assay was used to screen 160,000 small molecules for their ability to inhibit IMP1 binding to fluorescein-labeled c-Myc mRNA. The small molecule, BTYNB, was identified as a potent and selective inhibitor of IMP1 binding to c-Myc mRNA. In cells, BTYNB downregulates several mRNA transcripts regulated by IMP1. BTYNB destabilizes c-Myc mRNA, resulting in downregulation of c-Myc mRNA and protein. BTYNB downregulates ß-TrCP1 mRNA and reduces activation of nuclear transcriptional factors-kappa B (NF-κB). The oncogenic translation regulator, eEF2, emerged as a new IMP1 target mRNA, enabling BTYNB to inhibit tumor cell protein synthesis. BTYNB potently inhibited proliferation of IMP1-containing ovarian cancer and melanoma cells with no effect in IMP1-negative cells. Overexpression of IMP1 reversed BTYNB inhibition of cell proliferation. BTYNB completely blocked anchorage-independent growth of melanoma and ovarian cancer cells in colony formation assays. With its ability to target c-Myc and to inhibit proliferation of difficult-to-target melanomas and ovarian cancer cells, and with its unique mode of action, BTYNB is a promising small molecule for further therapeutic evaluation and mechanistic studies.

Antiestrogen Resistant Cell Lines Expressing Estrogen Receptor α Mutations Upregulate the Unfolded Protein Response and are Killed by BHPI.

Outgrowth of metastases expressing ERα mutations Y537S and D538G is common after endocrine therapy for estrogen receptor α (ERα) positive breast cancer. The effect of replacing wild type ERα in breast cancer cells with these mutations was unclear. We used the CRISPR-Cas9 genome editing system and homology directed repair to isolate and characterize 14 T47D cell lines in which ERαY537S or ERαD538G replace one or both wild-type ERα genes. In 2-dimensional, and in quantitative anchorage-independent 3-dimensional cell culture, ERαY537S and ERαD538G cells exhibited estrogen-independent growth. A progestin further increased their already substantial proliferation in micromolar 4-hydroxytamoxifen and fulvestrant/ICI 182,780 (ICI). Our recently described ERα biomodulator, BHPI, which hyperactivates the unfolded protein response (UPR), completely blocked proliferation. In ERαY537S and ERαD538G cells, estrogen-ERα target genes were constitutively active and partially antiestrogen resistant. The UPR marker sp-XBP1 was constitutively activated in ERαY537S cells and further induced by progesterone in both cell lines. UPR-regulated genes associated with tamoxifen resistance, including the oncogenic chaperone BiP/GRP78, were upregulated. ICI displayed a greater than 2 fold reduction in its ability to induce ERαY537S and ERαD538G degradation. Progestins, UPR activation and perhaps reduced ICI-stimulated ERα degradation likely contribute to antiestrogen resistance seen in ERαY537S and ERαD538G cells.

Interplay between steroid hormone activation of the unfolded protein response and nuclear receptor action.

To identify new pathways of estrogen action and novel estrogen receptor α (ERα) biomodulators, we performed high throughput screening and used follow on assays and bioinformatics to identify small molecule ERα inhibitors with a novel mode of action. These studies led to identification of rapid extranuclear activation of the endoplasmic reticulum stress sensor, the unfolded protein response (UPR), as a new pathway of estrogen-ERα action. Moreover, increasing evidence indicates that the mechanism underlying anticipatory activation of the UPR is shared among steroid and peptide hormones and is conserved from insects to humans. It is likely that this newly unveiled extranuclear pathway is used by diverse mitogenic hormones to prepare cells for the increased protein folding load that will occur during subsequent cell proliferation. Demonstrating biological relevance, elevated expression of a UPR gene signature in ERα positive breast cancer is a powerful new prognostic marker tightly correlated with subsequent resistance to tamoxifen, tumor recurrence and poor survival. In addition, overexpression of epidermal growth factor receptor and HER2/neu is positively correlated with increased UPR activation in breast cancer. This review describes recent research that demonstrates the importance of anticipatory UPR activation in therapy resistant tumors and discusses a promising small molecule biomodulator that inhibits tumor growth by tuning this UPR signaling pathway.

Estrogen receptor α inhibitor activates the unfolded protein response, blocks protein synthesis, and induces tumor regression.

Recurrent estrogen receptor α (ERα)-positive breast and ovarian cancers are often therapy resistant. Using screening and functional validation, we identified BHPI, a potent noncompetitive small molecule ERα biomodulator that selectively blocks proliferation of drug-resistant ERα-positive breast and ovarian cancer cells. In a mouse xenograft model of breast cancer, BHPI induced rapid and substantial tumor regression. Whereas BHPI potently inhibits nuclear estrogen-ERα-regulated gene expression, BHPI is effective because it elicits sustained ERα-dependent activation of the endoplasmic reticulum (EnR) stress sensor, the unfolded protein response (UPR), and persistent inhibition of protein synthesis. BHPI distorts a newly described action of estrogen-ERα: mild and transient UPR activation. In contrast, BHPI elicits massive and sustained UPR activation, converting the UPR from protective to toxic. In ERα(+) cancer cells, BHPI rapidly hyperactivates plasma membrane PLCγ, generating inositol 1,4,5-triphosphate (IP3), which opens EnR IP3R calcium channels, rapidly depleting EnR Ca(2+) stores. This leads to activation of all three arms of the UPR. Activation of the PERK arm stimulates phosphorylation of eukaryotic initiation factor 2α (eIF2α), resulting in rapid inhibition of protein synthesis. The cell attempts to restore EnR Ca(2+) levels, but the open EnR IP3R calcium channel leads to an ATP-depleting futile cycle, resulting in activation of the energy sensor AMP-activated protein kinase and phosphorylation of eukaryotic elongation factor 2 (eEF2). eEF2 phosphorylation inhibits protein synthesis at a second site. BHPI's novel mode of action, high potency, and effectiveness in therapy-resistant tumor cells make it an exceptional candidate for further mechanistic and therapeutic exploration.

High-throughput fluorescence anisotropy screen for inhibitors of the oncogenic mRNA binding protein, IMP-1.

Cancer cell proliferation is regulated by oncogenes, such as c-Myc. An alternative approach to directly targeting individual oncogenes is to target IMP-1, an oncofetal protein that binds to and stabilizes messenger RNAs (mRNAs), leading to elevated expression of c-Myc and other oncogenes. Expression of IMP-1 is tightly correlated with a poor prognosis and reduced survival in ovarian, lung, and colon cancer. Small-molecule inhibitors of IMP-1 have not been reported. We established a fluorescence anisotropy/polarization microplate assay (FAMA) for analyzing binding of IMP-1 to a fluorescein-labeled 93 nucleotide c-Myc mRNA target (flMyc), developed the assay as a highly robust (Z' factor = 0.60) FAMA-based high-throughput screen for inhibitors of binding of IMP-1 to flMyc, and carried out a successful pilot screen of 17,600 small molecules. Our studies support rapidly filtering out toxic nonspecific inhibitors using an early cell-based assay in control cells lacking the target protein. The physiologic importance of verified hits from the in vitro high-throughput screen was demonstrated by identification of the first small-molecule IMP-1 inhibitor, a lead compound that selectively inhibits proliferation of IMP-1-positive cancer cells with very little or no effect on proliferation of IMP-1-negative cells.

Evaluation of a luciferase-based reporter assay as a screen for inhibitors of estrogen-ERα-induced proliferation of breast cancer cells.

Estrogens, acting through estrogen receptor α (ERα), stimulate breast cancer proliferation, making ERα an attractive drug target. Since 384-well format screens for inhibitors of proliferation can be challenging for some cells, inhibition of luciferase-based reporters is often used as a surrogate end point. To identify novel small-molecule inhibitors of 17ß-estradiol (E(2))-ERα-stimulated cell proliferation, we established a cell-based screen for inhibitors of E(2)-ERα induction of an estrogen response element (ERE)(3)-luciferase reporter. Seventy-five "hits" were evaluated in tiered follow-up assays to identify where hits failed to progress and evaluate their effectiveness as inhibitors of E(2)-ERα-induced proliferation of breast cancer cells. Only 8 of 75 hits from the luciferase screen inhibited estrogen-induced proliferation of ERα-positive MCF-7 and T47D cells but not control ERα-negative MDA-MB-231 cells. Although 12% of compounds inhibited E(2)-ERα-stimulated proliferation in only one of the ERα-positive cell lines, 40% of compounds were toxic and inhibited growth of all the cell lines, and ~37% exhibited little or no ability to inhibit E(2)-ERα-stimulated cell proliferation. Representative compounds were evaluated in more detail, and a lead ERα inhibitor was identified.

Small molecule inhibitors as probes for estrogen and androgen receptor action.

Because activated estrogen (ER) and androgen (AR) receptors stimulate cell proliferation in breast and prostate cancer, inhibiting their actions represents a major therapeutic goal. Most efforts to modulate ER and AR activity have focused on inhibiting the synthesis of estrogens or androgens or on the identification of small molecules that act by competing with agonist hormones for binding in the ligand-binding pocket of the receptor. An alternative approach is to implement screens for small molecule inhibitors that target other sites in the pathway of steroid receptor action. Many of these second-site inhibitors directly target ER or AR; others have still unknown sites of action. Small molecule inhibitors that target second sites represent new leads with clinical potential; they serve as novel modulators of receptor action; and they can reveal new and as yet unidentified interactions and pathways that modulate ER and AR action.

A noncompetitive small molecule inhibitor of estrogen-regulated gene expression and breast cancer cell growth that enhances proteasome-dependent degradation of estrogen receptor {alpha}.

The mechanisms responsible for 17ß-estradiol (E(2))-stimulated breast cancer growth and development of resistance to tamoxifen and other estrogen receptor α (ERα) antagonists are not fully understood. We describe a new tool for dissecting ERα action in breast cancer, p-fluoro-4-(1,2,3,6,-tetrahydro-1,3-dimethyl-2-oxo-6-thionpurin-8-ylthio) (TPSF), a potent small-molecule inhibitor of estrogen receptor α that does not compete with estrogen for binding to ERα. TPSF noncompetitively inhibits estrogen-dependent ERα-mediated gene expression with little inhibition of transcriptional activity by NF-κB or the androgen or glucocorticoid receptor. TPSF inhibits E(2)-ERα-mediated induction of the proteinase inhibitor 9 gene, which is activated by ERα binding to estrogen response element DNA, and the cyclin D1 gene, which is induced by tethering ERα to other DNA-bound proteins. TPSF inhibits anchorage-dependent and anchorage-independent E(2)-ERα-stimulated growth of MCF-7 cells but does not inhibit growth of ER-negative MDA-MB-231 breast cancer cells. TPSF also inhibits ERα-dependent growth in three cellular models for tamoxifen resistance; that is, 4-hydroxytamoxifen-stimulated MCF7ERαHA cells that overexpress ERα, fully tamoxifen-resistant BT474 cells that have amplified HER-2 and AIB1, and partially tamoxifen-resistant ZR-75 cells. TPSF reduces ERα protein levels in MCF-7 cells and several other cell lines without altering ERα mRNA levels. The proteasome inhibitor MG132 abolished down-regulation of ERα by TPSF. Thus, TPSF affects receptor levels at least in part due to its ability to enhance proteasome-dependent degradation of ERα. TPSF represents a novel class of ER inhibitor with significant clinical potential.

A new small molecule inhibitor of estrogen receptor alpha binding to estrogen response elements blocks estrogen-dependent growth of cancer cells.

Estrogen receptor alpha (ERalpha) plays an important role in several human cancers. Most current ERalpha antagonists bind in the receptor ligand binding pocket and compete for binding with estrogenic ligands. Instead of the traditional approach of targeting estrogen binding to ER, we describe a strategy using a high throughput fluorescence anisotropy microplate assay to identify small molecule inhibitors of ERalpha binding to consensus estrogen response element (cERE) DNA. We identified small molecule inhibitors of ERalpha binding to the fluorescein-labeled (fl)cERE and evaluated their specificity, potency, and efficacy. One small molecule, theophylline, 8-[(benzylthio)methyl]-(7CI,8CI) (TPBM), inhibited ERalpha binding to the flcERE (IC(50) approximately 3 microm) and inhibited ERalpha-mediated transcription of a stably transfected ERE-containing reporter gene. Inhibition by TPBM was ER-specific, because progesterone and glucocorticoid receptor transcriptional activity were not significantly inhibited. In tamoxifen-resistant breast cancer cells that overexpress ERalpha, TPBM inhibited 17beta-estradiol (E(2))-ERalpha (IC(50) 9 microm) and 4-hydroxytamoxifen-ERalpha-mediated gene expression. Chromatin immunoprecipitation showed TPBM reduced E(2).ERalpha recruitment to an endogenous estrogen-responsive gene. TPBM inhibited E(2)-dependent growth of ERalpha-positive cancer cells (IC(50) of 5 microm). TPBM is not toxic to cells and does not affect estrogen-independent cell growth. TPBM acts outside of the ER ligand binding pocket, does not act by chelating the zinc in ER zinc fingers, and differs from known ERalpha inhibitors. Using a simple high throughput screen for inhibitors of ERalpha binding to the cERE, a small molecule inhibitor has been identified that selectively inhibits ERalpha-mediated gene expression and estrogen-dependent growth of cancer cells.

Analysis of RNA-protein interactions by a microplate-based fluorescence anisotropy assay.

Quantitative studies of RNA-protein interactions are quite cumbersome using traditional methods. We developed a rapid microplate-based fluorescence anisotropy (FA)/fluorescence polarization assay that works well, even with RNA probes >90 nucleotides long. We analyzed binding of RNA targets by vigilin/DDP1/SCP160p and by c-myc coding region instability determinant (CRD) binding protein, CRD-BP. Vigilin is essential for cell viability and functions in heterochromatin formation and mRNA decay. The CRD-BP stabilizes c-myc mRNA. Vigilin bound to a vitellogenin mRNA 3'-UTR probe with a two to three-fold lower affinity than to a Drosophila dodecasatellite ssDNA binding site and bound to the c-myc CRD with a two- to three-fold lower affinity than to the vitellogenin mRNA 3'-UTR. Competition between vigilin and CRD-BP for binding to the CRD may therefore play a role in regulating c-myc mRNA degradation. We analyzed suitability of the microplate-based FA assay for high-throughput screening for small-molecule regulators of RNA-protein interactions. The assay exhibits high reproducibility and precision and works well in 384-well plates and in 5 microl to 20 microl samples. To demonstrate the potential of this assay for screening libraries of small molecules to identify novel regulators of RNA-protein interactions, we identified neomycin and H33342 as inhibitors of binding of vigilin to the vitellogenin mRNA 3'-UTR.

Structure and sequence of the human fast skeletal troponin T (TNNT3) gene: insight into the evolution of the gene and the origin of the developmentally regulated isoforms.

We describe the cloning, sequencing and structure of the human fast skeletal troponin T (TNNT3) gene located on chromosome 11p15.5. The single-copy gene encodes 19 exons and 18 introns. Eleven of these exons, 1-3, 9-15 and 18, are constitutively spliced, whereas exons 4-8 are alternatively spliced. The gene contains an additional subset of developmentally regulated and alternatively spliced exons, including a foetal exon located between exon 8 and 9 and exon 16 or alpha (adult) and 17 or beta (foetal and neonatal). Exon phasing suggests that the majority of the alternatively spliced exons located at the 5' end of the gene may have evolved as a result of exon shuffling, because they are of the same phase class. In contrast, the 3' exons encoding an evolutionarily conserved heptad repeat domain, shared by both TnT and troponin I (TnI), may be remnants of an ancient ancestral gene. The sequence of the 5' flanking region shows that the putative promoter contains motifs including binding sites for MyoD, MEF-2 and several transcription factors which may play a role in transcriptional regulation and tissue-specific expression of TnT. The coding region of TNNT3 exhibits strong similarity to the corresponding rat sequence. However, unlike the rat TnT gene, TNNT3 possesses two repeat regions of CCA and TC. The exclusive presence of these repetitive elements in the human gene indicates divergence in the evolutionary dynamics of mammalian TnT genes. Homologous muscle-specific splicing enhancer motifs are present in the introns upstream and downstream of the foetal exon, and may play a role in the developmental pattern of alternative splicing of the gene. The genomic correlates of TNNT3 are relevant to our understanding of the evolution and regulation of expression of the gene, as well as the structure and function of the protein isoforms. The nucleotide sequence of TNNT3 has been submitted to EMBL/GenBank under Accession No. AF026276.