Mode of action analysis for fluxapyroxad-induced rat liver tumour formation: evidence for activation of the constitutive androstane receptor and assessment of human relevance.

The fungicide fluxapyroxad (BAS 700 F) has been shown to significantly increase the incidence of liver tumours in male Wistar rats at dietary levels of 1500 and 3000 ppm and in female rats at a dietary level of 3000 ppm via a non-genotoxic mechanism. In order to elucidate the mode of action (MOA) for fluxapyroxad-induced rat liver tumour formation a series of in vivo and in vitro investigative studies were undertaken. The treatment of male and female Wistar rats with diets containing 0 (control), 50, 250, 1500 and 3000 ppm fluxapyroxad for 1, 3, 7 and 14 days resulted in a dose-dependent increases in relative weight at 1500 and 3000 ppm from day 3 onwards in both sexes, with an increase in relative liver weight being also observed in male rats given 250 ppm fluxapyroxad for 14 days. Examination of liver sections revealed a centrilobular hepatocyte hypertrophy in some fluxapyroxad treated male and female rats. Hepatocyte replicative DNA synthesis (RDS) was significantly increased in male rats given 1500 and 3000 ppm fluxapyroxad for 3 and 7 days and in female rats given 50-3000 ppm fluxapyroxad for 7 days and 250-3000 ppm fluxapyroxad for 3 and 14 days; the maximal increases in RDS in both sexes being observed after 7 days treatment. The treatment of male and female Wistar rats with 250-3000 ppm fluxapyroxad for 14 days resulted in significant increases in hepatic microsomal total cytochrome P450 (CYP) content and CYP2B subfamily-dependent enzyme activities. Male Wistar rat hepatocytes were treated with control medium and medium containing 1-100 µM fluxapyroxad or 500 µM sodium phenobarbital (NaPB) for 4 days. Treatment with fluxapyroxad and NaPB increased CYP2B and CYP3A enzyme activities and mRNA levels but had little effect on markers of CYP1A and CYP4A subfamily enzymes and of the peroxisomal fatty acid ß-oxidation cycle. Hepatocyte RDS was significantly increased by treatment with fluxapyroxad, NaPB and 25 ng/ml epidermal growth factor (EGF). The treatment of hepatocytes from two male human donors with 1-100 µM fluxapyroxad or 500 µM NaPB for 4 days resulted in some increases in CYP2B and CYP3A enzyme activities and CYP mRNA levels but had no effect on hepatocyte RDS, whereas treatment with EGF resulted in significant increase in RDS in both human hepatocyte preparations. Hepatocytes from male Sprague-Dawley wild type (WT) and constitutive androstane receptor (CAR) knockout (CAR KO) rats were treated with control medium and medium containing 1-16 µM fluxapyroxad or 500 µM NaPB for 4 days. While both fluxapyroxad and NaPB increased CYP2B enzyme activities and mRNA levels in WT hepatocytes, only minor effects were observed in CAR KO rat hepatocytes. Treatment with both fluxapyroxad and NaPB only increased RDS in WT and not in CAR KO rat hepatocytes, whereas treatment with EGF increased RDS in both WT and CAR KO rat hepatocytes. In conclusion, a series of in vivo and in vitro investigative studies have demonstrated that fluxapyroxad is a CAR activator in rat liver, with similar properties to the prototypical CAR activator phenobarbital. A robust MOA for fluxapyroxad-induced rat liver tumour formation has been established. Based on the lack of effect of fluxapyroxad on RDS in human hepatocytes, it is considered that the MOA for fluxapyroxad-induced liver tumour formation is qualitatively not plausible for humans.

UDP-6-glucose dehydrogenase in hormonally responsive breast cancers.

Survival for metastatic breast cancer is low and thus, continued efforts to treat and prevent metastatic progression are critical. Estrogen is shown to promote aggressive phenotypes in multiple cancer models irrespective of estrogen receptor (ER) status. Similarly, UDP-Glucose 6-dehydrogenase (UGDH) a ubiquitously expressed enzyme involved in extracellular matrix precursors, as well as hormone processing increases migratory and invasive properties in cancer models. While the role of UGDH in cellular migration is defined, how it intersects with and impacts hormone signaling pathways associated with tumor progression in metastatic breast cancer has not been explored. Here we demonstrate that UGDH knockdown blunts estrogen-induced tumorigenic phenotypes (migration and colony formation) in ER+ and ER- breast cancer in vitro. Knockdown of UGDH also inhibits extravasation of ER- breast cancer ex vivo, primary tumor growth and animal survival in vivo in both ER+ and ER- breast cancer. We also use single cell RNA-sequencing to demonstrate that our findings translate to a human breast cancer clinical specimen. Our findings support the role of estrogen and UGDH in breast cancer progression provide a foundation for future studies to evaluate the role of UGDH in therapeutic resistance to improve outcomes and survival for breast cancer patients.

Single-cell RNA sequencing comparison of the human metastatic prostate spine tumor microenvironment.

Spinal column tumors can be difficult to process for single-cell omic studies, given the heterogeneity in tissue. Here, we present a protocol for operating room-to-benchtop single-cell processing of clinical specimens from a prostate cancer patient. We describe steps for sample homogenization, red blood cell lysis, cryopreservation, and single-cell sequencing analysis. This protocol can be used to identify prognostic markers and therapeutic targets for patients with osseous spine metastases and better inform eligibility for clinical trials.

Targeting CaMKK2 Inhibits Actin Cytoskeletal Assembly to Suppress Cancer Metastasis.

Triple-negative breast cancers (TNBC) tend to become invasive and metastatic at early stages in their development. Despite some treatment successes in early-stage localized TNBC, the rate of distant recurrence remains high, and long-term survival outcomes remain poor. In a search for new therapeutic targets for this disease, we observed that elevated expression of the serine/threonine kinase calcium/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) is highly correlated with tumor invasiveness. In validation studies, genetic disruption of CaMKK2 expression or inhibition of its activity with small molecule inhibitors disrupted spontaneous metastatic outgrowth from primary tumors in murine xenograft models of TNBC. High-grade serous ovarian cancer (HGSOC), a high-risk, poor prognosis ovarian cancer subtype, shares many features with TNBC, and CaMKK2 inhibition effectively blocked metastatic progression in a validated xenograft model of this disease. Mechanistically, CaMKK2 increased the expression of the phosphodiesterase PDE1A, which hydrolyzed cyclic guanosine monophosphate (cGMP) to decrease the cGMP-dependent activity of protein kinase G1 (PKG1). Inhibition of PKG1 resulted in decreased phosphorylation of vasodilator-stimulated phosphoprotein (VASP), which in its hypophosphorylated state binds to and regulates F-actin assembly to facilitate cell movement. Together, these findings establish a targetable CaMKK2-PDE1A-PKG1-VASP signaling pathway that controls cancer cell motility and metastasis by impacting the actin cytoskeleton. Furthermore, it identifies CaMKK2 as a potential therapeutic target that can be exploited to restrict tumor invasiveness in patients diagnosed with early-stage TNBC or localized HGSOC. SIGNIFICANCE: CaMKK2 regulates actin cytoskeletal dynamics to promote tumor invasiveness and can be inhibited to suppress metastasis of breast and ovarian cancer, indicating CaMKK2 inhibition as a therapeutic strategy to arrest disease progression.

Ca 2+ /Calmodulin Dependent Protein Kinase Kinase-2 (CaMKK2) promotes Protein Kinase G (PKG)-dependent actin cytoskeletal assembly to increase tumor metastasis.

Triple-negative breast cancers (TNBCs) tend to become highly invasive early during cancer development. Despite some successes in the initial treatment of patients diagnosed with early-stage localized TNBC, the rate of metastatic recurrence remains high with poor long-term survival outcomes. Here we show that elevated expression of the serine/threonine-kinase, Calcium/Calmodulin (CaM)-dependent protein kinase kinase-2 (CaMKK2), is highly correlated with tumor invasiveness. We determined that genetic disruption of CaMKK2 expression, or inhibition of its activity, disrupted spontaneous metastatic outgrowth from primary tumors in murine xenograft models of TNBC. High-grade serous ovarian cancer (HGSOC), a high-risk, poor-prognosis ovarian cancer subtype, shares many genetic features with TNBC, and importantly, CaMKK2 inhibition effectively blocked metastatic progression in a validated xenograft model of this disease. Probing the mechanistic links between CaMKK2 and metastasis we defined the elements of a new signaling pathway that impacts actin cytoskeletal dynamics in a manner which increases cell migration/invasion and metastasis. Notably, CaMKK2 increases the expression of the phosphodiesterase PDE1A which decreases the cGMP-dependent activity of protein kinase G1 (PKG1). This inhibition of PKG1 results in decreased phosphorylation of Vasodilator-Stimulated Phosphoprotein (VASP), which in its hypophosphorylated state binds to and regulates F-actin assembly to facilitate contraction/cell movement. Together, these data establish a targetable CaMKK2-PDE1A-PKG1-VASP signaling pathway that controls cancer cell motility and metastasis. Further, it credentials CaMKK2 as a therapeutic target that can be exploited in the discovery of agents for use in the neoadjuvant/adjuvant setting to restrict tumor invasiveness in patients diagnosed with early-stage TNBC or localized HGSOC.

Inhibition of estrogen signaling in myeloid cells increases tumor immunity in melanoma.

Immune checkpoint blockade (ICB) therapies have significantly prolonged patient survival across multiple tumor types, particularly in melanoma. Interestingly, sex-specific differences in response to ICB have been observed, with males receiving a greater benefit from ICB than females, although the mechanism or mechanisms underlying this difference are unknown. Mining published transcriptomic data sets, we determined that the response to ICBs is influenced by the functionality of intratumoral macrophages. This puts into context our observation that estrogens (E2) working through the estrogen receptor α (ERα) stimulated melanoma growth in murine models by skewing macrophage polarization toward an immune-suppressive state that promoted CD8+ T cell dysfunction and exhaustion and ICB resistance. This activity was not evident in mice harboring macrophage-specific depletion of ERα, confirming a direct role for estrogen signaling within myeloid cells in establishing an immunosuppressed state. Inhibition of ERα using fulvestrant, a selective estrogen receptor downregulator (SERD), decreased tumor growth, stimulated adaptive immunity, and increased the antitumor efficacy of ICBs. Further, a gene signature that determines ER activity in macrophages predicted survival in patients with melanoma treated with ICB. These results highlight the importance of E2/ER signaling as a regulator of intratumoral macrophage polarization, an activity that can be therapeutically targeted to reverse immune suppression and increase ICB efficacy.

Current and emerging estrogen receptor-targeted therapies for the treatment of breast cancer.

Nearly 80% of all breast cancers are estrogen receptor positive (ER+) and require the activity of this transcription factor for tumor growth and survival. Thus, endocrine therapies, which target the estrogen signaling axis, have and will continue to be the cornerstone of therapy for patients diagnosed with ER+ disease. Several inhibitors of ER activity exist, including aromatase inhibitors (AIs), selective estrogen receptor modulators (SERMs), selective estrogen receptor degraders/down-regulators (SERDs), and ER proteolysis-targeting chimeras (ER PROTACs); drugs which differ in the mechanism(s) by which they inhibit this signaling pathway. Notwithstanding their significant impact on the management of this disease, resistance to existing endocrine therapies remains a major impediment to durable clinical responses. Although the mechanisms of resistance are complex and varied, dependence on ER is typically retained after progression on SERMs and AIs, suggesting that ER remains a bona fide therapeutic target. The discovery and development of orally bioavailable drugs that eliminate ER expression (SERDs and ER PROTACs) will likely aid in treating this growing patient population. All of the existing endocrine therapies were developed with the intent of inhibiting the cancer cell intrinsic actions of ER and/or with the objective of achieving extreme estrogen deprivation and most achieve that goal. A longstanding question that remains to be addressed, however, is how actions of existing interventions extrinsic to the cancer cells influence tumor biology. We believe that these issues need to be addressed in the development of strategies to develop the next generation of ER-modulators optimized for positive activities in both cancer cells and other cells within the tumor microenvironment (TME).

GREB1 regulates PI3K/Akt signaling to control hormone-sensitive breast cancer proliferation.

Over 70% of breast cancers express the estrogen receptor (ER) and depend on ER activity for survival and proliferation. While hormone therapies that target receptor activity are initially effective, patients invariably develop resistance which is often associated with activation of the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway. While the mechanism by which estrogen regulates proliferation is not fully understood, one gene target of ER, growth regulation by estrogen in breast cancer 1 (GREB1), is required for hormone-dependent proliferation. However, the molecular function by which GREB1 regulates proliferation is unknown. Herein, we validate that knockdown of GREB1 results in growth arrest and that exogenous GREB1 expression initiates senescence, suggesting that an optimal level of GREB1 expression is necessary for proliferation of breast cancer cells. Under both of these conditions, GREB1 is able to regulate signaling through the PI3K/Akt/mTOR pathway. GREB1 acts intrinsically through PI3K to regulate phosphatidylinositol (3,4,5)-triphosphate levels and Akt activity. Critically, growth suppression of estrogen-dependent breast cancer cells by GREB1 knockdown is rescued by expression of constitutively activated Akt. Together, these data identify a novel molecular function by which GREB1 regulates breast cancer proliferation through Akt activation and provides a mechanistic link between estrogen signaling and the PI3K pathway.

Comparative studies on the effects of sodium phenobarbital and two other constitutive androstane receptor (CAR) activators on induction of cytochrome P450 enzymes and replicative DNA synthesis in cultured hepatocytes from wild type and CAR knockout rats.

Nongenotoxic chemicals can produce liver tumours in rats and mice by a mitogenic mode of action involving activation of the constitutive androstane receptor (CAR). The aim of this study was to evaluate the usefulness of cultured hepatocytes from normal (wild type; WT) and CAR knockout (KO) rats to screen compounds as potential activators of rat CAR and to validate this test system. Cultured hepatocytes from male Sprague-Dawley WT and CAR KO rats were treated with either 100 and 1000 µM sodium phenobarbital (NaPB), 3-100 µM fluquinconazole (FQZ), or 3-300 µM 3-(difluoromethyl)-1-methyl-N-(3´,4´,6-trifluoro[1,1´-biphenyl]-2-yl)-1H-pyrazole-4-carboxamide (TI1) for 96 h. Induction of cytochrome P450 (CYP) enzymes was monitored by measurement of 7-pentoxyresorufin O-depentylase (PROD), 7-benzyloxyresorufin O-debenzylase (BROD) and 7-benzyloxyquinoline O-debenzylase (BQ) activities. Hepatocytes undergoing replicative DNA synthesis (RDS) were labelled by adding 10 µM 5-bromo-2´-deoxyuridine to the culture medium for determination of the hepatocyte labelling index. The treatment of WT, but not of CAR KO, rat hepatocytes with NaPB, FQZ and TI1 increased hepatocyte RDS and induced CYP2B-dependent PROD activity. In contrast, all three compounds increased CYP2B/3A-dependent BROD and CYP3A-dependent BQ activities in both WT and CAR KO rat hepatocytes. Hepatocyte RDS was increased in both WT and CAR KO rat hepatocytes by treatment with 25 ng/ml epidermal growth factor as a positive control. Overall, these results demonstrate that the effects of three CAR activators on RDS and CYP2B enzyme induction are abolished in cultured CAR KO rat hepatocytes. As demonstrated by this validation study, the CAR KO hepatocyte model is a useful in vitro mechanistic tool for the rapid screening of chemicals as potential activators of rat CAR.

Comparison of the hepatic and thyroid gland effects of sodium phenobarbital and pregnenolone-16α-carbonitrile in wild-type and constitutive androstane receptor (CAR)/pregnane X receptor (PXR) knockout rats.

The hepatic and thyroid gland effects of the constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) and the pregnane X receptor (PXR) activator pregnenolone-16α-carbonitrile (PCN) were examined in male Sprague-Dawley wild-type (WT) and knockout (KO) rats lacking both hepatic CAR and PXR receptors (CAR KO/PXR KO rats). The treatment of WT rats for 7 d with 500 ppm NaPB in the diet and 100 mg/kg/d PCN by gavage resulted in increased relative liver weight, hepatocyte hypertrophy, increased hepatocyte replicative DNA synthesis (RDS) and induction of cytochrome P450 CYP2B and CYP3A subfamily enzymes. NaPB and PCN also induced thyroid gland follicular cell RDS and hepatic microsomal UDP-glucuronosyltransferase activity towards thyroxine as substrate. These effects were not observed in the liver and thyroid gland of CAR KO/PXR KO rats. Male C57BL/6 J (WT) and CAR KO/PXR KO mice were given 1000 ppm NaPB in the diet for 7 d. In WT, but not in CAR KO/PXR KO, mice NaPB treatment resulted in liver hypertrophy and induction of hepatocyte RDS and Cyp2b enzymes. These results suggest that the CAR KO/PXR KO rat and mouse models are useful experimental models for mode of action studies with rodent CAR activators.

GREB1 isoforms regulate proliferation independent of ERα co-regulator activities in breast cancer.

Activation of the transcription factor estrogen receptor α (ERα) and the subsequent regulation of estrogen-responsive genes play a crucial role in the development and progression of the majority of breast cancers. One gene target of ERα, growth regulation by estrogen in breast cancer 1 (GREB1), is associated with proliferation and regulation of ERα activity in estrogen-responsive breast cancer cells. The GREB1 gene encodes three distinct isoforms: GREB1a, GREB1b and GREB1c, whose molecular functions are largely unknown. Here, we investigate the role of these isoforms in regulation of ERα activity and proliferation. Interaction between GREB1 and ERα was mapped to the amino terminus shared by all GREB1 variants. Analysis of isoform-specific regulation of ERα activity suggests none of the GREB1 isoforms possess potent co-regulator activity. Exogenous expression of GREB1a resulted in elevated expression of some ER-target genes, independent of ERα activity. Despite this slight specificity of GREB1a for gene regulation, exogenous expression of either GREB1a or GREB1b resulted in decreased proliferation in both ER-positive and ER-negative breast carcinoma cell lines, demonstrating an ER-independent function of GREB1. Interestingly, we show an increase in the expression of GREB1b and GREB1c mRNA in malignant breast tissue compared to normal patient samples, suggesting a selective preference for these isoforms during malignant transformation. Together, these data suggest GREB1a has an isoform-specific function as a transcriptional regulator while all isoforms share an ER-independent activity that regulates proliferation.

Comparison of the hepatic and thyroid gland effects of sodium phenobarbital in wild type and constitutive androstane receptor (CAR) knockout rats and pregnenolone-16α-carbonitrile in wild type and pregnane X receptor (PXR) knockout rats.

A number of chemicals produce liver and thyroid gland tumours in rodents by nongenotoxic modes of action (MOAs). In this study the hepatic and thyroid gland effects of the constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) were examined in male Sprague-Dawley wild type (WT) rats and in CAR knockout (CAR KO) rats and the effects of the pregnane X receptor (PXR) activator pregnenolone-16α-carbonitrile (PCN) were examined in WT and PXR knockout (PXR KO) rats. Rats were either fed diets containing 0 (control) or 500 ppm NaPB or were dosed with 0 (control) or 100 mg/kg/day PCN orally for 7 days. The treatment of WT rats with NaPB and PCN for 7 days resulted in increased relative liver weight, increased hepatocyte replicative DNA synthesis (RDS) and the induction of cytochrome P450 CYP2B and CYP3A subfamily enzyme, mRNA and protein levels. In marked contrast, the treatment of CAR KO rats with NaPB and PXR KO rats with PCN did not result in any increases in liver weight and induction of CYP2B and CYP3A enzymes. The treatment of CAR KO rats with NaPB had no effect on hepatocyte RDS, while PCN produced only a small increase in hepatocyte RDS in PXR KO rats. Treatment with NaPB had no effect on thyroid gland weight in WT and CAR KO rats, whereas treatment with PCN resulted in an increase in relative thyroid gland weight in WT, but not in PXR KO, rats. Thyroid gland follicular cell RDS was increased by the treatment of WT rats with NaPB and PCN, with NaPB also producing a small increase in thyroid gland follicular cell RDS in CAR KO rats. Overall, the present study with CAR KO rats demonstrates that a functional CAR is required for NaPB-mediated increases in liver weight, stimulation of hepatocyte RDS and induction of hepatic CYP enzymes. The studies with PXR KO rats demonstrate that a functional PXR is required for PCN-mediated increases in liver weight and induction of hepatic CYP enzymes; with induction of hepatocyte RDS also being largely mediated through PXR. The hepatic effects of NaPB in CAR KO rats and of PCN in PXR KO rats are in agreement with those observed in other recent literature studies. These results suggest that CAR KO and PXR KO rats are useful experimental models for liver MOA studies with rodent CAR and PXR activators and may also be useful for thyroid gland MOA studies.

Comparison of the effects of sodium phenobarbital in wild type and humanized constitutive androstane receptor (CAR)/pregnane X receptor (PXR) mice and in cultured mouse, rat and human hepatocytes.

Phenobarbital (PB), a constitutive androstane receptor (CAR) activator, produces liver tumours in rodents by a mitogenic mode of action involving CAR activation. In this study, the hepatic effects of sodium phenobarbital (NaPB) were compared in male C57BL/6J wild type (WT) mice and in humanized mice, where both the mouse CAR and pregnane X receptor (PXR) have been replaced by their human counterparts (hCAR/hPXR mice). Investigations were also performed in cultured male C57BL/6J and CD-1 mouse, male Sprague-Dawley rat and male and female human hepatocytes. The treatment of WT and hCAR/hPXR mice with 186-984 ppm NaPB in the diet for 7 days resulted in increased relative liver weight, hypertrophy and induction of cytochrome P450 (CYP) enzyme activities. Treatment with NaPB also produced dose-dependent increases in hepatocyte replicative DNA synthesis (RDS), with the effect being more marked in WT than in hCAR/hPXR mice. While the treatment of cultured C57BL/6J and CD-1 mouse, Sprague-Dawley rat and human hepatocytes with 100 and/or 1000 µM NaPB for 4 days induced CYP enzyme activities, increased RDS was only observed in mouse and rat hepatocytes. However, as a positive control, epidermal growth factor increased RDS in hepatocytes from all three species. In summary, although human hepatocytes are refractory to the mitogenic effects of NaPB, treatment with NaPB induced RDS in vivo in hCAR/hPXR mice, which is presumably due to the human CAR and PXR receptors operating in a mouse hepatocyte regulatory environment. As the response of the hCAR/hPXR mouse to the CAR activator NaPB differs markedly from that of human hepatocytes, the hCAR/hPXR mouse is thus not a suitable animal model for studies on the hepatic effects of nongenotoxic rodent CAR activators.