Hormone-sensitive prostate cancer typically progresses to castration resistant prostate cancer (CRPC) after the androgen deprivation therapy. We investigated the impact of microRNAs (miRs) in the transition of prostate cancer to CRPC. MiR-221/-222 was highly expressed in bone metastatic CRPC tumor specimens. We previously demonstrated that transient overexpression of miR-221/-222 in LNCaP promoted the development of the CRPC phenotype. In current study, we show that stably overexpressing miR-221 confers androgen independent (AI) cell growth in LNCaP by rescuing LNCaP cells from growth arrest at G1 phase due to the lack of androgen. Overexpressing of miR-221 in LNCaP reduced the transcription of a subgroup of androgen-responsive genes without affecting the androgen receptor (AR) or AR-androgen integrity. By performing systematic biochemical and bioinformatical analyses, we identified two miR-221 targets, HECTD2 and RAB1A, which could mediate the development of CRPC phenotype in multiple prostate cancer cell lines. Downregulation of HECTD2 significantly affected the androgen-induced and AR-mediated transcription, and downregulation of HECTD2 or RAB1A enhances AI cell growth. As a result of the elevated expression of miR-221, expression of many cell cycle genes was altered and pathways promoting epithelial to mesenchymal transition/tumor metastasis were activated. We hypothesize that a major biological consequence of upregulation of miR-221 is reprogramming of AR signaling, which in turn may mediate the transition to the CRPC phenotype.
Ischemia-reperfusion injury is a common feature of ischemic stroke, which occurs when blood supply is restored after a period of ischemia. Reperfusion can be achieved either by thrombolysis using thrombolytic reagents such as tissue plasminogen activator (tPA), or through mechanical removal of thrombi. Spontaneous reperfusion also occurs after ischemic stroke. However, despite the beneficial effect of restored oxygen supply by reperfusion, it also causes deleterious effect compared with permanent ischemia. With the recent advancement in endovascular therapy including thrombectomy and thrombus disruption, reperfusion-injury has become an increasingly critical challenge in stroke treatment. It is therefore of extreme importance to understand the mechanisms of ischemia-reperfusion injury in the brain in order to develop effective therapeutics. Accumulating experimental evidence have suggested that the mechanisms of ischemia-reperfusion injury include oxidative stress, leukocyte infiltration, platelet adhesion and aggregation, complement activation, mitochondrial mediated mechanisms, and blood-brain-barrier (BBB) disruption, which altogether ultimately lead to edema or hemorrhagic transformation (HT) in the brain. Potential therapeutic strategies against ischemia-reperfusion injury may be developed targeting these mechanisms. In this review, we briefly discuss the pathophysiology and cellular and molecular mechanisms of cerebral ischemia-reperfusion injury, and potential therapeutic strategies.
ER+ breast cancers depend on ER signaling throughout disease progression, including after acquired resistance to existing endocrine agents, providing a rationale for further optimization and development of ER-targeting agents. Fulvestrant is unique amongst currently approved ER ligand therapeutics due to its classification as a full ER antagonist, which is thought to be achieved through degradation of ERα protein. However, the full clinical potential of fulvestrant is believed to be limited by poor physiochemical properties and exposure limitations due to its administration by intramuscular injection. Strategies to generate orally bioavailable molecules that retain fulvestrant's full antagonist profile but with considerably improved drug-like properties are thus being widely employed to identify next generation ER therapeutics. However, we find that therapeutic candidates that have recently emerged from prospective optimization of ER degradation, including GDC-0810 and GDC-0927, are not mechanistically equivalent. GDC-0810, GDC-0927, and fulvestrant display unique profiles in terms of ER degradation, transcriptional phenotypes and anti-proliferative potential across a panel of ER+ breast cancer cell lines. In HCI-011 (ER.WT) and HCI-013 (ER.Y537S) ER+ patient-derived breast cancer xenograft (PDX) models, GDC-0927 achieves more robust transcriptional suppression of ER than GDC-0810, and also and greater efficacy. Although displaying a more desirable mechanistic profile than GDC-0810, GDC-0927 has more rapid clearance and poor oral bioavailability, leading to a high pill burden and potential exposure limitation. Here, we describe for the first time GDC-9545, in which the distinct liabilities of GDC-0810, GDC-0927 and fulvestrant are addressed. GDC-9545 is a non-steroidal ER ligand that is highly potent in competing with estradiol for binding and in driving an antagonist conformation within the ER ligand binding domain. Like fulvestrant, and displaying some improvements over GDC-0927, GDC-9545 consistently induces ER turnover and drives deep transcriptional suppression of ER, resulting in robust in vitro anti-proliferative activity. GDC-9545 exhibits reduced metabolism and increased oral bioavailability relative to GDC-0927, resulting in an overall improved oral exposure in multiple species. As a result of both its mechanistic pharmacology and improved oral exposure, GDC-9545 can achieve the same degree of anti-tumor activity as GDC-0927 but at 100-fold lower doses in the HCI-013 PDX model. The in vivo efficacy of GDC-9545 in this model is greater than GDC-0810 and fulvestrant at clinically relevant exposures. The highly potent in vivo efficacy of GDC-9545 likely arises due to the particular combination of high binding potency, full suppression of ER signaling, and an improved DMPK profile when compared to GDC-0927 and fulvestrant. GDC-9545 is currently being evaluated in Phase 1 clinical trials (ClinicalTrials.gov Identifier: NCT03332797). Citation Format: Metcalfe C, Ingalla E, Blake RA, Chang J, Daemen A, De Bruyn T, Giltnane JM, Guan J, Hafner M, Hartman S, Kategaya L, Kleinheinz T, Liang J, Mody V, Nannini M, Oeh J, Ubhayakar S, Wertz I, Young A, Zbieg J, Zhou W, Sampath D, Friedman LS, Wang X. GDC-9545: A novel ER antagonist and clinical candidate that combines desirable mechanistic and pre-clinical DMPK attributes [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-07.
Neurovascular disease often involves multi-organ system injury. For example, patent foramen ovale (PFO) related ischemic strokes involve not just the brain, but also the heart, the lung, and the peripheral vascular circulation. For higher-risk but high-reward systemic therapy (e.g., thrombolytics, therapeutic hypothermia, PFO closure) to be implemented safely, very careful patient selection and close monitoring of disease progression and therapeutic efficacy are imperative. For example, more than a decade after the approval of therapeutic hypothermic and intravenous thrombolysis treatments, they both remain extremely underutilized, in part due to lack of clinical tools for patient selection or to follow therapeutic efficacy. Therefore, in order to understand the complexity of the global effects of clinical neurovascular diseases and their therapies, a systemic approach may offer a unique perspective and provide tools with clinical utility. Clinical proteomic approaches may be promising to monitor systemic changes in complex multi-organ diseases – especially where the disease process can be “sampled” in clinically accessible fluid such as blood, urine and CSF. Here, we describe a “pharmaco-proteomic” approach to three major challenges in translational neurovascular research directly at bedside – in order to better stratify risk, widen therapeutic windows, explore novel targets to be validated at the bench – 1) thrombolytic treatment for ischemic stroke, 2) therapeutic hypothermia for post cardiac arrest syndrome, and 3) treatment for patent foramen ovale (PFO) related paradoxical embolic stroke. In the future, this clinical proteomics approach may help to improve patient selection, ensure more precise clinical phenotyping for clinical trials, and individualize patient treatment.
Background: Electronic health records (EHRs) promise to enable broad-ranging discovery with power exceeding that of conventional research cohort studies. However, research using EHR datasets may be subject to selection bias, which can be compounded by missing data, limiting the generalizability of derived insights. Methods: Mass General Brigham (MGB) is a large New England-based healthcare network comprising seven tertiary care and community hospitals with associated outpatient practices. Within an MGB-based EHR warehouse of >3.5 million individuals with at least one ambulatory care visit, we approximated a community-based cohort study by selectively sampling individuals longitudinally attending primary care practices between 2001-2018 (n=520,868), which we named the Community Care Cohort Project (C3PO). We also utilized pre-trained deep natural language processing (NLP) models to recover vital signs (i.e., height, weight, and blood pressure) from unstructured notes in the EHR. We assessed the validity of C3PO by deploying established risk models including the Pooled Cohort Equations (PCE) and the Cohorts for Aging and Genomic Epidemiology Atrial Fibrillation (CHARGE-AF) score, and compared model performance in C3PO to that observed within typical EHR Convenience Samples which included all individuals from the same parent EHR with sufficient data to calculate each score but without a requirement for longitudinal primary care. All analyses were facilitated by the JEDI Extractive Data Infrastructure pipeline which we designed to efficiently aggregate EHR data within a unified framework conducive to regular updates. Results: C3PO includes 520,868 individuals (mean age 48 years, 61% women, median follow-up 7.2 years, median primary care visits per individual 13). Estimated using reports, C3PO contains over 2.9 million electrocardiograms, 450,000 echocardiograms, 12,000 cardiac magnetic resonance images, and 75 million narrative notes. Using tabular data alone, 286,009 individuals (54.9%) had all vital signs available at baseline, which increased to 358,411 (68.8%) after NLP recovery (31% reduction in missingness). Among individuals with both NLP and tabular data available, NLP-extracted and tabular vital signs obtained on the same day were highly correlated (e.g., Pearson r range 0.95-0.99, p<0.01 for all). Both the PCE models (c-index range 0.724-0.770) and CHARGE-AF (c-index 0.782, 95% 0.777-0.787) demonstrated good discrimination. As compared to the Convenience Samples, AF and MI/stroke incidence rates in C3PO were lower and calibration error was smaller for both PCE (integrated calibration index range 0.012-0.030 vs. 0.028-0.046) and CHARGE-AF (0.028 vs. 0.036). Conclusions: Intentional sampling of individuals receiving regular ambulatory care and use of NLP to recover missing data have the potential to reduce bias in EHR research and maximize generalizability of insights.
#5046 Introduction: Basal-like breast cancer is associated with high grade, poor prognosis, and younger patient age. Because tumors are estrogen receptor (ER) and HER2 negative, effective targeted treatment for basal breast cancer remains elusive. The lack of efficacy of currently used chemotherapies may reflect their inability to eradicate cancer stem cells (CSC) within a basal breast tumor.
 Chondroitin sulfate proteoglycan 4 (CSPG4), a molecule initially identified in melanoma cells, is a cell surface antigen involved in migration and invasion of tumor cells. The rat homolog of the human CSPG4, named NG2, is expressed by progenitor cells in several types of tissues. Furthermore, NG2 expression is typically maximal when progenitor cells are mitotic and motile. As one important characteristic of cancer stem cells is their ability to migrate and metastasize, and both CSPG4 and NG2 play a role in tumor cell migration, we tested whether CSPG4 is expressed on cells with the CSC phenotype in ER negative/HER2 negative (basal-like) breast cancer, and can thus serve as a target for immunotherapy of breast CSC.
 Methods and Results: The mRNA levels of CSPG4 in ER- and Her2- breast cancer were compared to ER-/Her2+, ER+/Her2-, and ER+/Her2+ breast cancer using a publicly available, clinically annotated breast cancer data set. The gene expression level of CSPG4 was elevated in the ER- and Her2- subgroup when compared to ER-/Her2+ (Mann Whitney, p=0.03), ER+/Her2- (Mann Whitney, p=0.01), and ER+/Her2+ (Mann Whitney, p=0.06). These results suggest that CSPG4 expression is best correlated with the basal phenotype of breast cancer. Flow cytometric analysis showed that CSPG4 is expressed on CD44+, CD24-/lo cells in the human basal breast cancer cell lines, i.e., MDA-MD-231, MDA-MB-435, HS578T and SUM149, but is not detectable on the luminal breast cancer cell lines, i.e., MCF-7, SK-BR-3 and T-47D. Furthermore CSPG4 is expressed on the cell surface of putative breast CSC identified as lineage-negative (CD2,3,10,16,18,31,45,64,140b), CD44+, CD24-/lo cells in pleural effusions of 12 patients with breast cancer. Further, inoculation of flow cytometric sorted CD44+CD24-/low CSPG4+ cells from the MDA-MB-231-luciferease expressing cell line, to the mammary fat pads of immunodeficient mice, led to the development of tumors detected by whole-body bioluminescent reporter imaging 3 weeks after the injection. In contrast, inoculation of CD44+CD24-/low CSPG4- cells, sorted from the same cell line, did not generate tumors. More importantly, CSPG4-specific mAb significantly inhibited post-surgery tumor recurrence and lung metastases in SCID mice transplanted with the human basal breast cancer cell line MDA-MB-435, which has the putative breast CSC phenotype (CD44+, CD24-/lo) in more than 99% of cells.
 Conclusion: These results suggest that the CSPG4 is a potential new immunotherapeutic target for basal-like breast cancer and breast CSC. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5046.
ER+ breast cancers can depend on ER signaling throughout disease progression, including after acquired resistance to existing endocrine agents, providing a rationale for further optimization and development of ER-targeting agents. Fulvestrant is unique amongst currently approved ER ligand therapeutics due to classification as a full ER antagonist, which is thought to be achieved through degradation of ER protein. However, the full clinical potential of fulvestrant is believed to be limited by poor bioavailability, spurring attempts to generate ligands capable of driving ER degradation but with improved drug-like properties. Here, we evaluate three ER ligand clinical candidates that recently emerged from prospective optimization of ER degradation – GDC-0810, AZD9496 and GDC-0927 - and show that they display distinct mechanistic features. GDC-0810 and AZD9496 are more limited in their ER degradation capacity relative to GDC-0927 and fulvestrant, display evidence of weak transcriptional activation of ER in breast cancer cells (i.e. partial agonist activity), and do not achieve the same degree of in vitro anti-proliferative activity as GDC-0927 and fulvestrant. In the HCI-013 (ER.Y537S) and HCI-011 (ER.WT) ER+ patient-derived xenograft models, GDC-0927 drives greater transcriptional suppression of ER, and greater anti-tumor activity relative to GDC-0810. We found that despite their full antagonist phenotype, GDC-0927 and fulvestrant promote association of ER with DNA, including at canonical ERE motifs, prior to ER degradation. Interestingly however, integration of ER ChIP-Seq and ATAC-Seq data revealed that ER complexed with fulvestrant or GDC-0927 fails to increase chromatin accessibility at DNA binding sites, in contrast to partial agonists which result in increased chromatin accessibility at ER binding sites. Thus, although ER contacts DNA when engaged with fulvestrant and GDC-0927, it is functionally inert. To further explore mechanistic features that might account for the differential activity of full antagonists and partial agonists that occurs prior to ER degradation, we used cell-based florescence recovery after photobleaching (FRAP) to measure the kinetics of ER diffusion within the nucleus. We demonstrate that while ER is generally highly mobile, including after engagement with GDC-0810 and AZD9496, GDC-0927 and fulvestrant immobilize intra-nuclear ER. A site saturating mutagenesis screen revealed a series of novel ER mutations that prevent ER immobilization by fulvestrant and GDC-0927. This class of “always mobile” ER variants promotes an antagonist-to-agonist transcriptional switch for fulvestrant and GDC-0927, and simultaneously prevents ER degradation by these molecules, implying that ER immobilization is a key functional determinant of robust transcriptional suppression. We thus propose that ER degradation is not a driver of full ER antagonism, but rather a downstream consequence of ER immobilization, occurring after a suppressive phenotype has been established at chromatin. We additionally argue that evaluating the transcriptional output of candidate ER therapeutics, both pre-clinically and clinically, will be critical for the identification of ER ligands with best-in-class potential. Citation Format: Metcalfe C, Zhou W, Guan J, Daemen A, Hafner M, Blake RA, Ingalla E, Young A, Oeh J, De Bruyn T, Ubhayakar S, Chen I, Giltnane JM, Li J, Wang X, Sampath D, Hager JH, Friedman LS. Prospective optimization of estrogen receptor degradation yields ER ligands with variable capacities for ER transcriptional suppression [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr GS3-05.
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