We have previously demonstrated that endoxifen is the most important tamoxifen metabolite responsible for eliciting the anti-estrogenic effects of this drug in breast cancer cells expressing estrogen receptor-alpha (ERα). However, the relevance of ERβ in mediating endoxifen action has yet to be explored. Here, we characterize the molecular actions of endoxifen in breast cancer cells expressing ERβ and examine its effectiveness as an anti-estrogenic agent in these cell lines.

TGF-β Inducible Early Gene-1 (TIEG1) is a Krüppel-like transcription factor (KLF10) that was originally cloned from human osteoblasts as an early response gene to TGF-β treatment. As reported previously, TIEG1(-/-) mice have decreased cortical bone thickness and vertebral bone volume and have increased spacing between the trabeculae in the femoral head relative to wildtype controls. Here, we have investigated the role of TIEG1 in osteoclasts to further determine their potential role in mediating this phenotype. We have found that TIEG1(-/-) osteoclast precursors differentiated more slowly compared to wildtype precursors in vitro and high RANKL doses are able to overcome this defect. We also discovered that TIEG1(-/-) precursors exhibit defective RANKL-induced phosphorylation and accumulation of NFATc1 and the NFATc1 target gene DC-STAMP. Higher RANKL concentrations reversed defective NFATc1 signaling and restored differentiation. After differentiation, wildtype osteoclasts underwent apoptosis more quickly than TIEG1(-/-) osteoclasts. We observed increased AKT and MEK/ERK signaling pathway activation in TIEG1(-/-) osteoclasts, consistent with the roles of these kinases in promoting osteoclast survival. Adenoviral delivery of TIEG1 (AdTIEG1) to TIEG1(-/-) cells reversed the RANKL-induced NFATc1 signaling defect in TIEG1(-/-) precursors and eliminated the differentiation and apoptosis defects. Suppression of TIEG1 with siRNA in wildtype cells reduced differentiation and NFATc1 activation. Together, these data provide evidence that TIEG1 controls osteoclast differentiation by reducing NFATc1 pathway activation and reduces osteoclast survival by suppressing AKT and MEK/ERK signaling.

Mammalian target of rapamycin (mTOR) represents a key downstream intermediate for a myriad of oncogenic receptor tyrosine kinases. In the case of the insulin-like growth factor (IGF) pathway, the mTOR complex (mTORC1) mediates IGF-1 receptor (IGF-1R)-induced estrogen receptor alpha (ERα) phosphorylation/activation and leads to increased proliferation and growth in breast cancer cells. As a result, the prevalence of mTOR inhibitors combined with hormonal therapy has increased in recent years. Conversely, activated mTORC1 provides negative feedback regulation of IGF signaling via insulin receptor substrate (IRS)-1/2 serine phosphorylation and subsequent proteasomal degradation. Thus, the IGF pathway may provide escape (e.g. de novo or acquired resistance) from mTORC1 inhibitors. It is therefore plausible that combined inhibition of mTORC1 and IGF-1R for select subsets of ER-positive breast cancer patients presents as a viable therapeutic option.

Although 70-80% of newly diagnosed ovarian cancer patients respond to first-line therapy, almost all relapse and five-year survival remains below 50%. One strategy to increase five-year survival is prolonging time to relapse by improving first-line therapy response. However, no biomarker today can accurately predict individual response to therapy. In this study, we present analytical and prospective clinical validation of a new test that utilizes primary patient tissue in 3D cell culture to make patient-specific response predictions prior to initiation of treatment in the clinic. Test results were generated within seven days of tissue receipt from newly diagnosed ovarian cancer patients obtained at standard surgical debulking or laparoscopic biopsy. Patients were followed for clinical response to chemotherapy. In a study population of 44, the 32 test-predicted Responders had a clinical response rate of 100% across both adjuvant and neoadjuvant treated populations with an overall prediction accuracy of 89% (39 of 44, p < 0.0001). The test also functioned as a prognostic readout with test-predicted Responders having a significantly increased progression-free survival compared to test-predicted Non-Responders, p = 0.01. This correlative accuracy establishes the test's potential to benefit ovarian cancer patients through accurate prediction of patient-specific response before treatment.

Interest in preclinical drug development for ovarian cancer has stimulated development of patient-derived xenograft (PDX) or tumorgraft models. However, the unintended formation of human lymphoma in severe combined immunodeficiency (SCID) mice from Epstein-Barr virus (EBV)-infected human lymphocytes can be problematic. In this study, we have characterized ovarian cancer PDXs which developed human lymphomas and explore methods to suppress lymphoproliferative growth. Fresh human ovarian tumors from 568 patients were transplanted intraperitoneally in SCID mice. A subset of PDX models demonstrated atypical patterns of dissemination with mediastinal masses, hepatosplenomegaly, and CD45-positive lymphoblastic atypia without ovarian tumor engraftment. Expression of human CD20 but not CD3 supported a B-cell lineage, and EBV genomes were detected in all lymphoproliferative tumors. Immunophenotyping confirmed monoclonal gene rearrangements consistent with B-cell lymphoma, and global gene expression patterns correlated well with other human lymphomas. The ability of rituximab, an anti-CD20 antibody, to suppress human lymphoproliferation from a patient's ovarian tumor in SCID mice and prevent growth of an established lymphoma led to a practice change with a goal to reduce the incidence of lymphomas. A single dose of rituximab during the primary tumor heterotransplantation process reduced the incidence of CD45-positive cells in subsequent PDX lines from 86.3% (n = 117 without rituximab) to 5.6% (n = 160 with rituximab), and the lymphoma rate declined from 11.1% to 1.88%. Taken together, investigators utilizing PDX models for research should routinely monitor for lymphoproliferative tumors and consider implementing methods to suppress their growth.

Progressive scarring is ubiquitous postoperatively and in an array of chronic systemic diseases. Recent studies indicate that such scarring has a high female propensity; females are also almost exclusively affected by endometriosis, a common sex steroid-dependent fibrotic disease. Endometriosis-related fibrosis is regulated epigenetically through transcription factor Krüppel-like factor 11 (KLF11). In response to surgical induction of endometriosis, Klf11-/- female mice develop significant fibrosis in contrast to wild-type mice. We therefore hypothesized that female fibrotic predilection was mediated by differential sex steroid regulation of KLF11/collagen 1a1 signaling and investigated the fibrotic response in wild-type and Klf11-/- male and female animals using a sterile peritonitis model. Fibrosis selectively developed in Klf11-/- females. Fibrosis in these animals was almost completely abrogated by ovariectomy. Ovariectomized animals were selectively supplemented with estradiol, medroxyprogesterone acetate (MPA), or dihydrotestosterone; fibrosis was only observed in mice exposed to MPA. Fibrosis therefore selectively developed in Klf11-/- female mice in response to physiological or pharmacological progesterone. The fibrotic response in these animals was also mitigated in response to antiprogestin therapy. Profibrotic gene expression was activated in a primary human peritoneal cell line in response to KLF11 short hairpin RNA and MPA but not estradiol. KLF11/collagen 1a1 signaling previously shown to be linked to fibrosis was thus selectively dysregulated in MPA-treated cells. Our in vivo and in vitro findings in an animal model and human cells, respectively, suggest that progressive fibrotic scarring is a sexually dimorphic response irrespective of etiology; moreover, it is responsive to novel, individualized therapeutic intervention.

Metabolic dysfunction underlies several chronic diseases, many of which are exacerbated by obesity. Dietary interventions can reverse metabolic declines and slow aging, although compliance issues remain paramount. 17α-estradiol treatment improves metabolic parameters and slows aging in male mice. The mechanisms by which 17α-estradiol elicits these benefits remain unresolved. Herein, we show that 17α-estradiol elicits similar genomic binding and transcriptional activation through estrogen receptor α (ERα) to that of 17β-estradiol. In addition, we show that the ablation of ERα completely attenuates the beneficial metabolic effects of 17α-E2 in male mice. Our findings suggest that 17α-E2 may act through the liver and hypothalamus to improve metabolic parameters in male mice. Lastly, we also determined that 17α-E2 improves metabolic parameters in male rats, thereby proving that the beneficial effects of 17α-E2 are not limited to mice. Collectively, these studies suggest ERα may be a drug target for mitigating chronic diseases in male mammals.

The transcription factor Krüppel-like factor 10 (Klf10), also known as Tieg1 for TGFβ (Inducible Early Gene-1) is known to control numerous genes in many cell types that are involved in various key biological processes (differentiation, proliferation, apoptosis, inflammation), including cell metabolism and human disease. In skeletal muscle, particularly in the soleus, deletion of the Klf10 gene (Klf10 KO) resulted in ultrastructure fiber disorganization and mitochondrial metabolism deficiencies, characterized by muscular hypertrophy. To determine the metabolic profile related to loss of Klf10 expression, we analyzed blood and soleus tissue using UHPLC-Mass Spectrometry. Metabolomics analyses on both serum and soleus revealed profound differences between wild-type (WT) and KO animals. Klf10 deficient mice exhibited alterations in metabolites associated with energetic metabolism. Additionally, chemical classes of aromatic and amino-acid compounds were disrupted, together with Krebs cycle intermediates, lipids and phospholipids. From variable importance in projection (VIP) analyses, the Warburg effect, citric acid cycle, gluconeogenesis and transfer of acetyl groups into mitochondria appeared to be possible pathways involved in the metabolic alterations observed in Klf10 KO mice. These studies have revealed essential roles for Klf10 in regulating multiple metabolic pathways whose alterations may underlie the observed skeletal muscle defects as well as other diseases.

Survey data from the Mayo Clinic Breast Disease Registry were used to assess fertility counseling and fertility preservation strategies in a modern cohort of young women with breast cancer. One hundred respondents were identified who were under age 50 at the time of breast cancer diagnosis and who expressed interest in future childbearing near the time of diagnosis and/or 1 year later. Ninety-three percent of the 81 respondents to the year one survey recalled fertility counseling prior to cancer treatment. Most who reported a high level of fertility concern declared that this concern had impacted their treatment decisions, often shortening their planned duration of endocrine therapy. Approximately half had taken steps to preserve future fertility, and a third had used a gonadotropin-releasing hormone agonist either alone or combined with another method (e.g., embryo or oocyte cryopreservation).

Cdc20 is an activator of the anaphase-promoting complex/cyclosome that initiates anaphase onset by ordering the destruction of cyclin B1 and securin in metaphase. To study the physiological significance of Cdc20 in higher eukaryotes, we generated hypomorphic mice that express small amounts of this essential cell cycle regulator. In this study, we show that these mice are healthy and not prone to cancer despite substantial aneuploidy. Cdc20 hypomorphism causes chromatin bridging and chromosome misalignment, revealing a requirement for Cdc20 in efficient sister chromosome separation and chromosome-microtubule attachment. We find that cyclin B1 is newly synthesized during mitosis via cytoplasmic polyadenylation element-binding protein-dependent translation, causing its rapid accumulation between prometaphase and metaphase of Cdc20 hypomorphic cells. Anaphase onset is significantly delayed in Cdc20 hypomorphic cells but not when translation is inhibited during mitosis. These data reveal that Cdc20 is particularly rate limiting for cyclin B1 destruction because of regulated de novo synthesis of this cyclin after prometaphase onset.

A genome-wide association study identified LMO1, which encodes an LIM-domain-only transcriptional cofactor, as a neuroblastoma susceptibility gene that functions as an oncogene in high-risk neuroblastoma. Here we show that dβh promoter-mediated expression of LMO1 in zebrafish synergizes with MYCN to increase the proliferation of hyperplastic sympathoadrenal precursor cells, leading to a reduced latency and increased penetrance of neuroblastomagenesis. The transgenic expression of LMO1 also promoted hematogenous dissemination and distant metastasis, which was linked to neuroblastoma cell invasion and migration, and elevated expression levels of genes affecting tumor cell-extracellular matrix interaction, including loxl3, itga2b, itga3, and itga5. Our results provide in vivo validation of LMO1 as an important oncogene that promotes neuroblastoma initiation, progression, and widespread metastatic dissemination.

Physical function declines in old age, portending disability, increased health expenditures, and mortality. Cellular senescence, leading to tissue dysfunction, may contribute to these consequences of aging, but whether senescence can directly drive age-related pathology and be therapeutically targeted is still unclear. Here we demonstrate that transplanting relatively small numbers of senescent cells into young mice is sufficient to cause persistent physical dysfunction, as well as to spread cellular senescence to host tissues. Transplanting even fewer senescent cells had the same effect in older recipients and was accompanied by reduced survival, indicating the potency of senescent cells in shortening health- and lifespan. The senolytic cocktail, dasatinib plus quercetin, which causes selective elimination of senescent cells, decreased the number of naturally occurring senescent cells and their secretion of frailty-related proinflammatory cytokines in explants of human adipose tissue. Moreover, intermittent oral administration of senolytics to both senescent cell-transplanted young mice and naturally aged mice alleviated physical dysfunction and increased post-treatment survival by 36% while reducing mortality hazard to 65%. Our study provides proof-of-concept evidence that senescent cells can cause physical dysfunction and decreased survival even in young mice, while senolytics can enhance remaining health- and lifespan in old mice.

Cancer progresses through distinct stages, and mouse models recapitulating traits of this progression are frequently used to explore genetic, morphological, and pharmacological aspects of tumor development. To complement genomic investigations of this process, we here quantify phosphoproteomic changes in skin cancer development using the SILAC mouse technology coupled to high-resolution mass spectrometry. We distill protein expression signatures from our data that distinguish between skin cancer stages. A distinct phosphoproteome of the two stages of cancer progression is identified that correlates with perturbed cell growth and implicates cell adhesion as a major driver of malignancy. Importantly, integrated analysis of phosphoproteomic data and prediction of kinase activity revealed PAK4-PKC/SRC network to be highly deregulated in SCC but not in papilloma. This detailed molecular picture, both at the proteome and phosphoproteome level, will prove useful for the study of mechanisms of tumor progression.

The anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase functions with the E2 ubiquitin-conjugating enzyme UbcH10 in the orderly progression through mitosis by marking key mitotic regulators for destruction by the 26-S proteasome. UbcH10 is overexpressed in many human cancer types and is associated with tumor progression. However, whether UbcH10 overexpression causes tumor formation is unknown. To address this central question and to define the molecular and cellular consequences of UbcH10 overexpression, we generated a series of transgenic mice in which UbcH10 was overexpressed in graded fashion. In this study, we show that UbcH10 overexpression leads to precocious degradation of cyclin B by the APC/C, supernumerary centrioles, lagging chromosomes, and aneuploidy. Importantly, we find that UbcH10 transgenic mice are prone to carcinogen-induced lung tumors and a broad spectrum of spontaneous tumors. Our results identify UbcH10 as a prominent protooncogene that causes whole chromosome instability and tumor formation over a wide gradient of overexpression levels.

Mutations in the VHL gene are associated with highly vascular tumors of kidney, brain, retina, and adrenal gland. The inability of the mutant VHL protein to destabilize HIF-1 plays a crucial role in malignant angiogenesis. VHL is also associated with ECM assembly but the molecular mechanisms of this activity remain unclear. We used expression arrays and cell lines with different VHL status to identify ECM-associated genes controlled by VHL. One of them, adhesion-associated TGFBI, was repressed by VHL and overexpressed in renal, gastrointestinal, brain, and other tumors. Analyzing the mechanism of TGFBI up-regulation in clear cell carcinoma, we identified a novel VHL target, a Kruppel-like transcriptional factor 10 (KLF10). The TGFBI promoter, which we isolated and studied in Luc-reporter assay, was induced by KLF10 but not hypoxia. These data provide the molecular basis for the observed VHL effect on TGFBI and stimulate further research into the KLF10 and TGFBI roles in cancer.

We describe a phase II clinical trial of the combination of ribociclib and letrozole for treatment of relapsed oestrogen receptor (ER)-positive ovarian cancer (OC) and endometrial cancer (EC). The primary endpoint was the proportion of patients alive, progression-free survival (PFS), and still on treatment at 12 weeks (PFS12), with 45% or greater considered positive.

Endometriosis is a highly prevalent, chronic, heterogeneous, fibro-inflammatory disease that remains recalcitrant to conventional therapy. We previously showed that loss of KLF11, a transcription factor implicated in uterine disease, results in progression of endometriosis. Despite extensive homology, co-expression, and human disease association, loss of the paralog Klf10 causes a unique inflammatory, cystic endometriosis phenotype in contrast to fibrotic progression seen with loss of Klf11. We identify here for the first time a novel role for KLF10 in endometriosis. In an animal endometriosis model, unlike wild-type controls, Klf10(-/-) animals developed cystic lesions with massive immune infiltrate and minimal peri-lesional fibrosis. The Klf10(-/-) disease progression phenotype also contrasted with prolific fibrosis and minimal immune cell infiltration seen in Klf11(-/-) animals. We further found that lesion genotype rather than that of the host determined each unique disease progression phenotype. Mechanistically, KLF10 regulated CD40/CD154-mediated immune pathways. Both inflammatory as well as fibrotic phenotypes are the commonest clinical manifestations in chronic fibro-inflammatory diseases such as endometriosis. The complementary, paralogous Klf10 and Klf11 models therefore offer novel insights into the mechanisms of inflammation and fibrosis in a disease-relevant context. Our data suggests that divergence in underlying gene dysregulation critically determines disease-phenotype predominance rather than the conventional paradigm of inflammation being precedent to fibrotic scarring. Heterogeneity in clinical progression and treatment response are thus likely from disparate gene regulation profiles. Characterization of disease phenotype-associated gene dysregulation offers novel approaches for developing targeted, individualized therapy for recurrent and recalcitrant chronic disease.

Triple Negative Breast Cancer (TNBC) accounts for 15-20% of all breast cancer cases, yet is responsible for a disproportionately high percentage of breast cancer mortalities. Thus, there is an urgent need to identify novel biomarkers and therapeutic targets based on the molecular events driving TNBC pathobiology. Estrogen receptor beta (ERβ) is known to elicit anti-cancer effects in TNBC, however its mechanisms of action remain elusive. Here, we report the expression profiles of ERβ and its association with clinicopathological features and patient outcomes in the largest cohort of TNBC to date. In this cohort, ERβ was expressed in approximately 18% of TNBCs, and expression of ERβ was associated with favorable clinicopathological features, but correlated with different overall survival outcomes according to menopausal status. Mechanistically, ERβ formed a co-repressor complex involving enhancer of zeste homologue 2/polycomb repressive complex 2 (EZH2/PRC2) that functioned to suppress oncogenic NFκB/RELA (p65) activity. Importantly, p65 was shown to be required for formation of this complex and for ERβ-mediated suppression of TNBC. Our findings indicate that ERβ+ tumors exhibit different characteristics compared to ERβ- tumors and demonstrate that ERβ functions as a molecular switch for EZH2, repurposing it for tumor suppressive activities and repression of oncogenic p65 signaling.

CUB-domain containing protein 1 (CDCP1) is a cancer associated cell surface protein that amplifies pro-tumorigenic signalling by other receptors including EGFR and HER2. Its potential as a cancer target is supported by studies showing that anti-CDCP1 antibodies inhibit cell migration and survival in vitro, and tumor growth and metastasis in vivo. Here we characterize two anti-CDCP1 antibodies, focusing on immuno-conjugates of one of these as a tool to detect and inhibit ovarian cancer. Methods: A panel of ovarian cancer cell lines was examined for cell surface expression of CDCP1 and loss of expression induced by anti-CDCP1 antibodies 10D7 and 41-2 using flow cytometry and Western blot analysis. Surface plasmon resonance analysis and examination of truncation mutants was used to analyse the binding properties of the antibodies for CDCP1. Live-cell spinning-disk confocal microscopy of GFP-tagged CDCP1 was used to track internalization and intracellular trafficking of CDCP1/antibody complexes. In vivo, zirconium 89-labelled 10D7 was detected by positron-emission tomography imaging, of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. The efficacy of cytotoxin-conjugated 10D7 was examined against ovarian cancer cells in vitro and in vivo. Results: Our data indicate that each antibody binds with high affinity to the extracellular domain of CDCP1 causing rapid internalization of the receptor/antibody complex and degradation of CDCP1 via processes mediated by the kinase Src. Highlighting the potential clinical utility of CDCP1, positron-emission tomography imaging, using zirconium 89-labelled 10D7, was able to detect subcutaneous and intraperitoneal xenograft ovarian cancers in mice, including small (diameter <3 mm) tumor deposits of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. Furthermore, cytotoxin-conjugated 10D7 was effective at inhibiting growth of CDCP1-expressing ovarian cancer cells in vitro and in vivo. Conclusions: These data demonstrate that CDCP1 internalizing antibodies have potential for killing and detection of CDCP1 expressing ovarian cancer cells.

To determine relevant provider opinions on uterus transplantation (UTx).