VDX-111 targets proliferative pathways in canine cancer cell lines.

VDX-111 (also identified as AMPI-109) is a vitamin D derivative which has shown anticancer activity. To further assess the function of this compound against multiple cancer types, we examined the efficacy of VDX-111 against a panel of 30 well characterized canine cancer cell lines. Across a variety of cancer types, VDX-111 induced widely variable growth inhibition, cell death, and migration inhibition, at concentrations ranging from 10 nM to 1 µM. Growth inhibition sensitivity did not correlate strongly with tumor cell histotype; however, it was significantly correlated with the expression of genes in multiple cell signaling pathways, including the MAPK and PI3K-AKT pathways. We confirmed inhibition of these signaling pathways as likely participants in the effects of VDX-111. These results suggest that a subset of canine tumors may be sensitive to treatment with VDX-111, and suggests possible predictive markers of drug sensitivity and pharmacodynamic biomarkers of drug exposure that could be employed in future clinical trials.

VDX-111, a novel small molecule, induces necroptosis to inhibit ovarian cancer progression.

Epithelial ovarian cancers that are nonhomologous recombination deficient, as well as those that are recurrent and in a platinum-resistant state, have limited therapeutic options. The objectives of this study were to characterize the mechanism of action and investigate the therapeutic potential of a small molecule, VDX-111, against ovarian cancer. We examined the ability of VDX-111 to inhibit the growth of a panel of ovarian cancer cell lines, focusing on BRCA wild-type lines. We found that VDX-111 causes a dose-dependent loss of cell viability across ovarian cancer cell lines. Reverse phase protein array (RPPA) analysis was used to identify changes in cell signaling in response to VDX-111 treatment. An RPPA analysis performed on cells treated with VDX-111 detected changes in cell signaling related to autophagy and necroptosis. Immunoblots of OVCAR3 and SNU8 cells confirmed a dose-dependent increase in LC3A/B and RIPK1. Incucyte live cell imaging was used to measure cell proliferation and death in response to VDX-111 alone and with inhibitors of apoptosis, necroptosis, and autophagy. Annexin/PI assays suggested predominantly nonapoptotic cell death, while real-time kinetic imaging of cell growth indicated the necroptosis inhibitor, necrostatin-1, attenuates VDX-111-induced loss of cell viability, suggesting a necroptosis-dependent mechanism. Furthermore, VDX-111 inhibited tumor growth in patient-derived xenograft and syngeneic murine models. In conclusion, the cytotoxic effects of VDX-111 seen in vitro and in vivo appear to occur in a necroptosis-dependent manner and may promote an antitumor immune response.

Expression Differences in BCL2 Family Members between Uveal and Cutaneous Melanomas Account for Varying Sensitivity to BH3 Mimetics.

Uveal melanoma (UM) is a subtype of melanoma. Although they share a melanocytic origin with cutaneous melanoma (CM), patients with UM have few treatment options. BCL2 homologous 3 mimetics are small-molecule drugs that mimic proapoptotic BCL2 family members. We compared BCL2 family member expression between UM and CM using immunoblot and The Cancer Genome Atlas transcriptomic analysis. UM has a unique signature of low BFL1 and high PUMA proteins compared with CM and 30 other cancer types, making them an attractive candidate for BCL2 homologous 3 protein mimetics. We tested the efficacy of a BCL2 inhibitor and MCL1 inhibitor (MCL1i) in UM, with viability assays, live-cell imaging, sphere assays, and mouse xenograft models. UM had a higher sensitivity to MCL1i than CM. Overexpression of BFL1 or knockdown of PUMA made the UM more resistant to MCL1i. In contrast, MAPK/extracellular signal‒regulated kinase inhibitor treatment in CM made them more sensitive to MCL1i. However, MCL1i-alone treatment was not very effective to reduce the UM initiating cells; to overcome this, we employed a combination of MCL1i with BCL2 inhibitor that synergistically inhibited UM initiating cell's capacity to expand. Overall, we identify a distinct expression profile of BCL2 family members for UM that makes them susceptible to BCL2 homologous 3 mimetics.

A Novel Regimen for Treating Melanoma: MCL1 Inhibitors and Azacitidine.

Although treatment options for melanoma patients have expanded in recent years with the approval of immunotherapy and targeted therapy, there is still an unmet need for new treatment options for patients that are ineligible for, or resistant to these therapies. BH3 mimetics, drugs that mimic the activity of pro-apoptotic BCL2 family proteins, have recently achieved remarkable success in the clinical setting. The combination of BH3 mimetic ABT-199 (venetoclax) plus azacitidine has shown substantial benefit in treating acute myelogenous leukemia. We evaluated the efficacy of various combinations of BH3 mimetic + azacitidine in fourteen human melanoma cell lines from cutaneous, mucosal, acral and uveal subtypes. Using a combination of cell viability assay, BCL2 family knockdown cell lines, live cell imaging, and sphere formation assay, we found that combining inhibition of MCL1, an anti-apoptotic BCL2 protein, with azacitidine had substantial pro-apoptotic effects in multiple melanoma cell lines. Specifically, this combination reduced cell viability, proliferation, sphere formation, and induced apoptosis. In addition, this combination is highly effective at reducing cell viability in rare mucosal and uveal subtypes. Overall, our data suggest this combination as a promising therapeutic option for some patients with melanoma and should be further explored in clinical trials.

MCL1 inhibitors S63845/MIK665 plus Navitoclax synergistically kill difficult-to-treat melanoma cells.

Current treatment for patients with metastatic melanoma include molecular-targeted therapies and immune checkpoint inhibitors. However, a subset of melanomas are difficult-to-treat. These melanomas include those without the genetic markers for targeted therapy, non-responsive to immunotherapy, and those who have relapsed or exhausted their therapeutic options. Therefore, it is necessary to understand and explore other biological processes that may provide new therapeutic approaches. One of most appealing is targeting the apoptotic/anti-apoptotic system that is effective against leukemia. We used genetic knockdown and pharmacologic approaches of BH3 mimetics to target anti-apoptotic BCL2 family members and identified MCL1 and BCLXL as crucial pro-survival members in melanoma. We then examined the effects of combining BH3 mimetics to target MCL1 and BCLXL in vitro and in vivo. These include clinical-trial-ready compounds such as ABT-263 (Navitoclax) and S63845/S64315 (MIK655). We used cell lines derived from patients with difficult-to-treat melanomas. In vitro, the combined inhibition of MCL1 and BCLXL resulted in significantly effective cell killing compared to single-agent treatment (p < 0.05) in multiple assays, including sphere assays. The combination-induced cell death was independent of BIM, and NOXA. Recapitulated in our mouse xenograft model, the combination inhibited tumor growth, reduced sphere-forming capacity (p < 0.01 and 0.05, respectively), and had tolerable toxicity (p > 0.40). Taken together, this study suggests that dual targeting of MCL1 and BCLXL should be considered as a treatment option for difficult-to-treat melanoma patients.

Role of GDF15 in methylseleninic acid-mediated inhibition of cell proliferation and induction of apoptosis in prostate cancer cells.

The growth inhibitory efficacy of methylseleninic acid (MSA) in prostate cancer cells has been documented extensively. However, our understanding of the immediate targets that are key to the growth inhibitory effects of MSA remains limited. Here, using multiple preclinical prostate cancer models, we demonstrated in vitro and in vivo that GDF15 is a most highly induced, immediate target of MSA. We further showed that knockdown of GDF15 mitigates MSA inhibition of cell proliferation and induction of apoptosis. Analysis of gene expression data from over 1000 primary and 200 metastatic prostate cancer samples revealed that GDF15 expression is decreased in metastatic prostate cancers compared to primary tumors and that lower GDF15 levels in primary tumors are associated with higher Gleason scores and shorter survival of the patients. Additionally, pathways that are negatively correlated with GDF15 levels in clinical samples are also negatively correlated with MSA treatment in cultured cells. Since most, if not all, of these pathways have been implicated in prostate cancer progression, suppressing their activities by inducing GDF15 is consistent with the anticancer effects of MSA in prostate cancer. Overall, this study provides support for GDF15 as an immediate target of MSA in prostate cancer cells.

Reversal of Triple-Negative Breast Cancer EMT by miR-200c Decreases Tryptophan Catabolism and a Program of Immunosuppression.

Tryptophan-2,3-dioxygenase (TDO2), a rate-limiting enzyme in the tryptophan catabolism pathway, is induced in triple-negative breast cancer (TNBC) by inflammatory signals and anchorage-independent conditions. TNBCs express extremely low levels of the miR-200 family compared with estrogen receptor-positive (ER+) breast cancer. In normal epithelial cells and ER+ breast cancers and cell lines, high levels of the family member miR-200c serve to target and repress genes involved in epithelial-to-mesenchymal transition (EMT). To identify mechanism(s) that permit TNBC to express TDO2 and other proteins not expressed in the more well-differentiated ER+ breast cancers, miRNA-200c was restored in TNBC cell lines. The data demonstrate that miR-200c targeted TDO2 directly resulting in reduced production of the immunosuppressive metabolite kynurenine. Furthermore, in addition to reversing a classic EMT signature, miR-200c repressed many genes encoding immunosuppressive factors including CD274/CD273, HMOX-1, and GDF15. Restoration of miR-200c revealed a mechanism, whereby TNBC hijacks a gene expression program reminiscent of that used by trophoblasts to suppress the maternal immune system to ensure fetal tolerance during pregnancy. IMPLICATIONS: Knowledge of the regulation of tumor-derived immunosuppressive factors will facilitate development of novel therapeutic strategies that complement current immunotherapy to reduce mortality for patients with TNBC.

PRL-3 engages the focal adhesion pathway in triple-negative breast cancer cells to alter actin structure and substrate adhesion properties critical for cell migration and invasion.

Triple-negative breast cancers (TNBCs) are among the most aggressive cancers characterized by a high propensity to invade, metastasize and relapse. We previously reported that the TNBC-specific inhibitor, AMPI-109, significantly impairs the ability of TNBC cells to migrate and invade by reducing levels of the metastasis-promoting phosphatase, PRL-3. Here, we examined the mechanisms by which AMPI-109 and loss of PRL-3 impede cell migration and invasion. AMPI-109 treatment or knock down of PRL-3 expression were associated with deactivation of Src and ERK signaling and concomitant downregulation of RhoA and Rac1/2/3 GTPase protein levels. These cellular changes led to rearranged filamentous actin networks necessary for cell migration and invasion. Conversely, overexpression of PRL-3 promoted TNBC cell invasion by upregulating matrix metalloproteinase 10, which resulted in increased TNBC cell adherence to, and degradation of, the major basement membrane component laminin. Our data demonstrate that PRL-3 engages the focal adhesion pathway in TNBC cells as a key mechanism for promoting TNBC cell migration and invasion. Collectively, these data suggest that blocking PRL-3 activity may be an effective method for reducing the metastatic potential of TNBC cells.

Genome-wide functional genetic screen with the anticancer agent AMPI-109 identifies PRL-3 as an oncogenic driver in triple-negative breast cancers.

Triple-negative breast cancers (TNBC) are among the most aggressive and heterogeneous cancers with a high propensity to invade, metastasize and relapse. Here, we demonstrate that the anticancer compound, AMPI-109, is selectively efficacious in inhibiting proliferation and inducing apoptosis of multiple TNBC subtype cell lines as assessed by activation of pro-apoptotic caspases-3 and 7, PARP cleavage and nucleosomal DNA fragmentation. AMPI-109 had little to no effect on growth in the majority of non-TNBC cell lines examined. We therefore utilized AMPI-109 in a genome-wide shRNA screen in the TNBC cell line, BT-20, to investigate the utility of AMPI-109 as a tool in helping to identify molecular alterations unique to TNBC. Our screen identified the oncogenic phosphatase, PRL-3, as a potentially important driver of TNBC growth, migration and invasion. Through stable lentiviral knock downs and transfection with catalytically impaired PRL-3 in TNBC cells, loss of PRL-3 expression, or functionality, led to substantial growth inhibition. Moreover, AMPI-109 treatment, downregulation of PRL-3 expression or impairment of PRL-3 activity reduced TNBC cell migration and invasion. Histological evaluation of human breast cancers revealed PRL-3 was significantly, though not exclusively, associated with the TNBC subtype and correlated positively with regional and distant metastases, as well as 1 and 3 year relapse free survival. Collectively, our study is proof-of-concept that AMPI-109, a selectively active agent against TNBC cell lines, can be used as a molecular tool to uncover unique drivers of disease progression, such as PRL-3, which we show promotes oncogenic phenotypes in TNBC cells.

Dissecting Steroid Receptor Function by Analytical Ultracentrifugation.

Steroid receptors comprise a family of ligand-activated transcription factors. The members include the androgen receptor (AR), estrogen receptor (ER), glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and progesterone receptor (PR). Each receptor controls distinct sets of genes associated with development, metabolism, and homeostasis. Although a qualitative understanding of how individual receptors mediate gene expression has come into focus, quantitative insight remains less clear. As a step toward delineating the physical mechanisms by which individual receptors activate their target genes, we have carried out a systematic dissection of receptor interaction energetics with their multisite regulatory elements. Analytical ultracentrifugation (AUC) has proved indispensable in these studies, in part by revealing the energetics of receptor self-association and its thermodynamic coupling to DNA binding. Here, we discuss these findings in the context of understanding specificity of receptor-mediated gene control. We first highlight the role of sedimentation velocity and sedimentation equilibrium in addressing receptor assembly state, and present a comparative analysis across the receptor family. We then use these results for understanding how receptors assemble at multisite regulatory elements, and hypothesize how these findings might play a role in receptor-specific gene regulation. Finally, we examine receptor behavior in a cellular context, with a view toward linking our in vitro studies with in vivo function.

Glucocorticoid Receptor-DNA Dissociation Kinetics Measured in Vitro Reveal Exchange on the Second Time Scale.

The glucocorticoid receptor (GR) is a member of the steroid receptor family of ligand-activated transcription factors. Recent live cell imaging studies have revealed that interactions of GR with chromatin are highly dynamic, with average receptor residence times of only seconds. These findings were surprising because early kinetic studies found that GR-DNA interactions in vitro were much slower, having calculated residence times of minutes to hours. However, these latter analyses were conducted at a time when it was possible to work with only either partially purified holoreceptor or its purified but isolated DNA binding domain. Noting these limitations, we reexamined GR-DNA dissociation kinetics using a highly purified holoreceptor shown to be amenable to rigorous study. We first observe that GR-DNA interactions in vitro are not slow as previously thought but converge with in vivo behavior, having residence times of only seconds to tens of seconds. This rapid exchange is seen at six individual response elements and the multisite MMTV promoter used in live cell imaging. Second, GR dissociation rates are identical for all response elements. Thus, previously observed differences in receptor affinity toward these sequences are not due to differences in off rate but in on rate. Finally, dissociation kinetics are biphasic in character. A minimal kinetic model consistent with the data is that in which DNA-bound GR interconverts between states on a second time scale, with dissociation occurring via a multistep process. We speculate that receptor interconversion in this time frame can be recognized by the coregulatory proteins that interact with GR, leading to unique transcriptional responses.

Reduced expression of GDF-15 is associated with atrophic inflammatory lesions of the prostate.

BACKGROUND: Accumulating evidence suggests that chronic prostatic inflammation may lead to prostate cancer development. Growth differentiation factor-15 (GDF-15) is highly expressed in the prostate and has been associated with inflammation and tumorigenesis. METHODS: To examine the relationship between GDF-15 and prostatic inflammation, GDF-15 expression was measured by immunohistochemical (IHC) staining in human prostatectomy specimens containing inflammation. The relationship between GDF-15 and specific inflammatory cells was determined using non-biased computer image analysis. To provide insight into a potential suppressive role for GDF-15 in inflammation, activation of inflammatory mediator nuclear factor of kappa B (NFκB) was measured in PC3 cells. RESULTS: GDF-15 expression in luminal epithelial cells was decreased with increasing inflammation severity, suggesting an inverse association between GDF-15 and inflammation. Quantification of IHC staining by image analysis for GDF-15 and inflammatory cell markers revealed an inverse correlation between GDF-15 and CD3+, CD4+, CD8+, CD68+, and inos+ leukocytes. GDF-15 suppressed NFκB activity in luciferase reporter assays. Expression of the NFκB target, interleukin 8 (IL-8), was downregulated by GDF-15. CONCLUSIONS: The inverse relationship between GDF-15 and inflammation demonstrates a novel expression pattern for GDF-15 in the human prostate and suppression of NFκB activity may shed light on a potential mechanism for this inverse correlation.

Steroid receptor-DNA interactions: toward a quantitative connection between energetics and transcriptional regulation.

Steroid receptors comprise an evolutionarily conserved family of transcription factors. Although the qualitative aspects by which individual receptors regulate transcription are well understood, a quantitative perspective is less clear. This is primarily because receptor function is considerably more complex than that of classical regulatory factors such as phage or bacterial repressors. Here we discuss recent advances in placing receptor-specific transcriptional regulation on a more quantitative footing, specifically focusing on the role of macromolecular interaction energetics. We first highlight limitations and challenges associated with traditional approaches for assessing the role of energetics (more specifically, binding affinity) with functional outcomes such as transcriptional activation. We next demonstrate how rigorous in vitro measurements and straightforward interaction models quantitatively relate energetics to transcriptional activity within the cell, and follow by discussing why such an approach is unexpectedly effective in explaining complex functional behavior. Finally, we examine the implications of these findings for considering the unique gene regulatory properties of the individual receptors.

Endocrine disrupting activities of the flavonoid nutraceuticals luteolin and quercetin.

Dietary plant flavonoids have been proposed to contribute to cancer prevention, neuroprotection, and cardiovascular health through their anti-oxidant, anti-inflammatory, pro-apoptotic, and antiproliferative activities. As a consequence, flavonoid supplements are aggressively marketed by the nutraceutical industry for many purposes, including pediatric applications, despite inadequate understanding of their value and drawbacks. We show that two flavonoids, luteolin and quercetin, are promiscuous endocrine disruptors. These flavonoids display progesterone antagonist activity beneficial in a breast cancer model but deleterious in an endometrial cancer model. Concurrently, luteolin possesses potent estrogen agonist activity while quercetin is considerably less effective. These results highlight the promise and peril of flavonoid nutraceuticals and suggest caution in supplementation beyond levels attained in a healthy, plant-rich diet.

Growth differentiation factor-15 (GDF-15) suppresses in vitro angiogenesis through a novel interaction with connective tissue growth factor (CCN2).

Growth differentiation factor-15 (GDF-15) and the CCN family member, connective tissue growth factor (CCN2), are associated with cardiac disease, inflammation, and cancer. The precise role and signaling mechanism for these factors in normal and diseased tissues remains elusive. Here we demonstrate an interaction between GDF-15 and CCN2 using yeast two-hybrid assays and have mapped the domain of interaction to the von Willebrand factor type C domain of CCN2. Biochemical pull down assays using secreted GDF-15 and His-tagged CCN2 produced in PC-3 prostate cancer cells confirmed a direct interaction between these proteins. To investigate the functional consequences of this interaction, in vitro angiogenesis assays were performed. We demonstrate that GDF-15 blocks CCN2-mediated tube formation in human umbilical vein endothelial (HUVEC) cells. To examine the molecular mechanism whereby GDF-15 inhibits CCN2-mediated angiogenesis, activation of αV ß3 integrins and focal adhesion kinase (FAK) was examined. CCN2-mediated FAK activation was inhibited by GDF-15 and was accompanied by a decrease in αV ß3 integrin clustering in HUVEC cells. These results demonstrate, for the first time, a novel signaling pathway for GDF-15 through interaction with the matricellular signaling molecule CCN2. Furthermore, antagonism of CCN2 mediated angiogenesis by GDF-15 may provide insight into the functional role of GDF-15 in disease states.

Analysis of a glucocorticoid-estrogen receptor chimera reveals that dimerization energetics are under ionic control.

Steroid receptors assemble at DNA response elements as dimers, resulting in coactivator recruitment and transcriptional activation. Our work has focused on dissecting the energetics associated with these events and quantitatively correlating the results with function. A recent finding is that different receptors dimerize with large differences in energetics. For example, estrogen receptor-α (ER-α) dimerizes with a ΔG=-12.0 kcal/mol under conditions in which the glucocorticoid receptor (GR) dimerizes with a ΔG≤-5.1 kcal/mol. To determine the molecular forces responsible for such differences, we created a GR/ER chimera, replacing the hormone-binding domain (HBD) of GR with that of ER-α. Cellular and biophysical analyses demonstrate that the chimera is functionally active. However, GR/ER dimerization energetics are intermediate between the parent proteins and coupled to a strong ionic linkage. Since the ER-α HBD is the primary contributor to dimerization, we suggest that GR residues constrain an ion-regulated HBD assembly reaction.

Glucocorticoid receptor-DNA interactions: binding energetics are the primary determinant of sequence-specific transcriptional activity.

The glucocorticoid receptor (GR) is a member of the steroid receptor family of ligand-activated transcription factors. A long-standing question has focused on how GR and other receptors precisely control gene expression. One difficulty in addressing this is that GR function is influenced by multiple factors including ligand and coactivator levels, chromatin state, and allosteric coupling. Moreover, the receptor recognizes an array of DNA sequences that generate a range of transcriptional activities. Such complexity suggests that any single parameter-DNA binding affinity, for example-is unlikely to be a dominant contributor to function. Indeed, a number of studies have suggested that for GR and other receptors, binding affinity toward different DNA sequences is poorly correlated with transcriptional activity. As a step toward determining the factors most predictive of GR function, we rigorously examined the relationship between in vitro GR-DNA binding energetics and in vivo transcriptional activity. We first demonstrate that previous approaches for assessing affinity-function relationships are problematic due to issues of data transformation and linearization. Thus, the conclusion that binding energetics and transcriptional activity are poorly correlated is premature. Using more appropriate analyses, we find that energetics and activity are in fact highly correlated. Furthermore, this correlation can be quantitatively accounted for using simple binding models. Finally, we show that the strong relationship between energetics and transcriptional activity is recapitulated in multiple promoter contexts, cell lines, and chromatin environments. Thus, despite the complexity of GR function, DNA binding energetics are the primary determinant of sequence-specific transcriptional activity.

Effect of dietary vitamin D and calcium on the growth of androgen-insensitive human prostate tumor in a murine model.

Vitamin D deficiency has been associated with increased risk of prostate cancer (PC) in epidemiologic and prospective studies. An association has also been made between high dietary calcium and increased PC risk. In this study, we evaluated the effect of dietary vitamin D and calcium on the growth of human androgen-insensitive prostate tumor in an athymic mouse model. We observed highest tumor growth in the normal calcium - vitamin D-deficient group, while tumor growth between the normal calcium - vitamin D-sufficient, high calcium - vitamin D-sufficient and high calcium - vitamin D-deficient diet-groups did not significantly differ but was significantly lower than that in the normal calcium - vitamin D-deficient group. Our results suggest an important role of dietary vitamin D as a preventive agent in androgen-insensitive PC.

From steroid receptors to cytokines: the thermodynamics of self-associating systems.

Since 1987, the Gibbs Conference on Biothermodynamics has maintained a focus on understanding the quantitative aspects of gene regulatory systems. These studies coupled rigorous techniques with exact theory to dissect the linked reactions associated with bacterial and lower eukaryotic gene regulation. However, only in the last ten years has it become possible to apply this approach to clinically relevant, human gene regulatory systems. Here we summarize our work on the thermodynamics of human steroid receptors and their interactions with multi-site promoter sequences, highlighting results not available from more traditional biochemical and structural approaches. Noting that the Gibbs Conference has also served as a vehicle to promote the broader use of thermodynamics in understanding biology, we then discuss collaborative work on the hydrodynamics of a cytokine implicated in tumor suppression, prostate derived factor (PDF).

1α,25-Dihydroxyvitamin D3-3ß-bromoacetate, a potential cancer therapeutic agent: synthesis and molecular mechanism of action.

Synthesis of 1α,25-dihydroxyvitamin D(3)-3ß-bromoacetate (1,25(OH)(2)D(3)-3-BE), a potential anti-cancer agent is presented. We also report that mechanism of action of 1,25(OH)(2)D(3)-3-BE may involve reduction of its catabolism, as evidenced by the reduced and delayed expression of 1α,25-dihydroxyvitamin D(3)-24-hydroxylase (CYP24) gene in cellular assays.