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Background: Progesterone receptors (PR) are potent modifiers of endocrine responses. In aberrant signalling cancer contexts, phosphorylation events dramatically alter steroid hormone receptor action., Methods: The transcriptomes of primary tumours and metastases in mice harbouring ER+ breast cancer patient-derived xenografts (PDXs) were analysed following single-cell RNAseq. In vitro assays were employed to delineate mechanisms of endocrine resistance and stemness., Results: A 16-gene phospho-Ser294 PR (p-PR) signature predicted poor outcome in ER+ breast cancer. Relative to primary PDX tumours, metastatic lesions expressed abundant p-PR and exhibited an activated PR gene programme with elevated expression of PGR and IRS-1. Breast cancer models of activated PR lost the expression of IGF1R and acquired insulin hypersensitivity with tamoxifen insensitivity. Activated p-PR+ breast cancer cells formed increased tumourspheres with enlarged ALDH+ and CD24-/CD44 populations. E2 induced PR/IRS-1 interaction and exchange of IGF1Rβ for IRS-1 in p-PR-containing transcriptional complexes. Inhibition of IRS-1 or IR and inducible IRS-1 knockdown reduced tumourspheres. Endocrine-resistant models of luminal B breast cancer induced p-PR in 3D cultures and required PR and IRS-1 for tumoursphere formation., Conclusions: Phospho-PR-B cooperates with IRS-1 to promote outgrowth of endocrine-resistant and stem-like breast cancer cells. Targeting phospho-PR/IRS-1 crosstalk may block the emergence of endocrine resistance.

Steroid hormone receptors (SRs) are classically defined as ligand-activated transcription factors that function as master regulators of gene programs important for a wide range of processes governing adult physiology, development, and cell or tissue homeostasis. A second function of SRs includes the ability to activate cytoplasmic signaling pathways. Estrogen (ER), androgen (AR), and progesterone (PR) receptors bind directly to membrane-associated signaling molecules including mitogenic protein kinases (i.e. c-SRC and AKT), G-proteins, and ion channels to mediate context-dependent actions via rapid activation of downstream signaling pathways. In addition to making direct contact with diverse signaling molecules, SRs are further fully integrated with signaling pathways by virtue of their N-terminal phosphorylation sites that act as regulatory hot-spots capable of sensing the signaling milieu. In particular, ER, AR, PR, and closely related glucocorticoid receptors (GR) share the property of accepting (i.e. sensing) ligand-independent phosphorylation events by proline-directed kinases in the MAPK and CDK families. These signaling inputs act as a 'second ligand' that dramatically impacts cell fate. In the face of drugs that reliably target SR ligand-binding domains to block uncontrolled cancer growth, ligand-independent post-translational modifications guide changes in cell fate that confer increased survival, EMT, migration/invasion, stemness properties, and therapy resistance of non-proliferating SR+ cancer cell subpopulations. The focus of this review is on MAPK pathways in the regulation of SR+ cancer cell fate. MAPK-dependent phosphorylation of PR (Ser294) and GR (Ser134) will primarily be discussed in light of the need to target changes in breast cancer cell fate as part of modernized combination therapies.

Background: Altered signaling pathways typify breast cancer and serve as direct inputs to steroid hormone receptor sensors. We previously reported that phospho-Ser134-GR (pS134-GR) species are elevated in triple-negative breast cancer (TNBC) and cooperate with hypoxia-inducible factors, providing a novel avenue for activation of GR in response to local or cellular stress., Methods: We probed GR regulation by factors (cytokines, growth factors) that are rich within the tumor microenvironment (TME). TNBC cells harboring endogenous wild-type (wt) or S134A-GR species were created by CRISPR/Cas knock-in and subjected to transwell migration, invasion, soft-agar colony formation, and tumorsphere assays. RNA-seq was employed to identify pS134-GR target genes that are regulated both basally (intrinsic) or by TGFβ1 in the absence of exogenously added GR ligands. Regulation of selected basal and TGFβ1-induced pS134-GR target genes was validated by qRT-PCR and chromatin immunoprecipitation assays. Bioinformatics tools were used to probe public data sets for expression of pS134-GR 24-gene signatures., Results: In the absence of GR ligands, GR is transcriptionally activated via p38-dependent phosphorylation of Ser134 as a mechanism of homeostatic stress-sensing and regulated upon exposure of TNBC cells to TME-derived agents. The ligand-independent pS134-GR transcriptome encompasses TGFβ1 and MAPK signaling gene sets associated with TNBC cell survival and migration/invasion. Accordingly, pS134-GR was essential for TNBC cell anchorage-independent growth in soft-agar, migration, invasion, and tumorsphere formation, an in vitro readout of cancer stemness properties. Both pS134-GR and expression of the MAPK-scaffolding molecule 14-3-3ζ were essential for a functionally intact p38 MAPK signaling pathway downstream of MAP3K5/ASK1, indicative of a feedforward signaling loop wherein self-perpetuated GR phosphorylation enables cancer cell autonomy. A 24-gene pS134-GR-dependent signature induced by TGFβ1 predicts shortened overall survival in breast cancer patients., Conclusions: Phospho-S134-GR is a critical downstream effector of p38 MAPK signaling and TNBC migration/invasion, survival, and stemness properties. Our studies define a ligand-independent role for GR as a homeostatic "sensor" of intrinsic stimuli as well as extrinsic factors rich within the TME (TGFβ1) that enable potent activation of the p38 MAPK stress-sensing pathway and nominate pS134-GR as a therapeutic target in aggressive TNBC.

Progesterone receptors (PRs) are key modifiers of estrogen receptor (ER) target genes and drivers of luminal breast cancer progression. Total PR expression, rather than isoform-specific PR expression, is measured in breast tumors as an indicator of functional ER. We identified phenotypic differences between PR-A and PR-B in luminal breast cancer models with a focus on tumorsphere biology. Our findings indicated that PR-A is a dominant driver of cancer stem cell (CSC) expansion in T47D models, and PR-B is a potent driver of anchorage-independent proliferation. PR-A+ tumorspheres were enriched for aldehyde dehydrogenase (ALDH) activity, CD44+/CD24-, and CD49f+/CD24- cell populations relative to PR-B+ tumorspheres. Progestin promoted heightened expression of known CSC-associated target genes in PR-A+ but not PR-B+ cells cultured as tumorspheres. We report robust phosphorylation of PR-A relative to PR-B Ser294 and found that this residue is required for PR-A-induced expression of CSC-associated genes and CSC behavior. Cells expressing PR-A S294A exhibited impaired CSC phenotypes but heightened anchorage-independent cell proliferation. The PR target gene and coactivator, FOXO1, promoted PR phosphorylation and tumorsphere formation. The FOXO1 inhibitor (AS1842856) alone or combined with onapristone (PR antagonist), blunted phosphorylated PR, and tumorsphere formation in PR-A+ and PR-B+ T47D, MCF7, and BT474 models. Our data revealed unique isoform-specific functions of phosphorylated PRs as modulators of distinct and opposing pathways relevant to mechanisms of late recurrence. A clear understanding of PR isoforms, phosphorylation events, and the role of cofactors could lead to novel biomarkers of advanced tumor behavior and reveal new approaches to pharmacologically target CSCs in luminal breast cancer.

Steroid hormone receptors (SRs) have a multitude of functions in human biology and disease progression. The SR family of related ligand-activated transcription factors includes androgen, estrogen, glucocorticoid, mineralocorticoid, and progesterone receptors. Antiestrogen or estrogen receptor (ER)-targeted therapies to block ER action remain the primary treatment of luminal breast cancers. Although this strategy is successful, ∼40% of patients eventually relapse due to endocrine resistance. The majority of hormone-independent tumors retain some level of SR expression, but sidestep hormone ablation treatments. SRs are known to crosstalk extensively with kinase signaling pathways, and this interplay has been shown to bypass ER-targeted therapies in part by providing alternative proliferation and survival signals that enable hormone independence. Modified receptors adopt alternate conformations that resist antagonism or promote agonism. SR-regulated transcription and SR-binding events have been classically studied as single receptor events using single hormones. However, it is becoming increasingly evident that individual steroids and SRs rarely act alone. Emerging evidence shows that coexpressed SRs crosstalk with each other in hormone-driven cancers, such as breast and prostate. Crosstalk between related SRs allows them to modulate signaling and transcriptional responses to noncognate ligands. This flexibility can lead to altered genomic binding and subsequent changes in SR target gene expression. This review will discuss recent mechanistic advances in elucidating SR crosstalk and the implications for treating hormone-driven cancers. Understanding this crosstalk (i.e., both opposing and collaborative) is a critical step toward expanding and modernizing endocrine therapies and will ultimately improve patient outcomes.

The metastatic cascade is a complex process that requires cancer cells to survive despite conditions of high physiologic stress. Previously, cooperation between the glucocorticoid receptor (GR) and hypoxia-inducible factors (HIF) was reported as a point of convergence for host and cellular stress signaling. These studies indicated p38 MAPK-dependent phosphorylation of GR on Ser134 and subsequent p-GR/HIF-dependent induction of breast tumor kinase (PTK6/Brk), as a mediator of aggressive cancer phenotypes. Herein, p-Ser134 GR was quantified in human primary breast tumors ( n = 281) and the levels of p-GR were increased in triple-negative breast cancer (TNBC) relative to luminal breast cancer. Brk was robustly induced following exposure of TNBC model systems to chemotherapeutic agents (Taxol or 5-fluorouracil) and growth in suspension [ultra-low attachment (ULA)]. Notably, both Taxol and ULA resulted in upregulation of the Aryl hydrocarbon receptor (AhR), a known mediator of cancer prosurvival phenotypes. Mechanistically, AhR and GR copurified and following chemotherapy and ULA, these factors assembled at the Brk promoter and induced Brk expression in an HIF-dependent manner. Furthermore, Brk expression was upregulated in Taxol-resistant breast cancer (MCF-7) models. Ultimately, Brk was critical for TNBC cell proliferation and survival during Taxol treatment and in the context of ULA as well as for basal cancer cell migration, acquired biological phenotypes that enable cancer cells to successfully complete the metastatic cascade. These studies nominate AhR as a p-GR binding partner and reveal ways to target epigenetic events such as adaptive and stress-induced acquisition of cancer skill sets required for metastatic cancer spread. Implication: Breast cancer cells enlist intracellular stress response pathways that evade chemotherapy by increasing cancer cell survival and promoting migratory phenotypes. Mol Cancer Res; 16(11); 1761-72. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Background: Development of distant metastases involves a complex multistep biological process termed the invasion-metastasis cascade, which includes dissemination of cancer cells from the primary tumor to secondary organs. NOTCH developmental signaling plays a critical role in promoting epithelial-to-mesenchymal transition, tumor stemness, and metastasis. Although all four NOTCH receptors show oncogenic properties, the unique role of each of these receptors in the sequential stepwise events that typify the invasion-metastasis cascade remains elusive., Methods: We have established metastatic xenografts expressing high endogenous levels of NOTCH3 using estrogen receptor alpha-positive (ERα + ) MCF-7 breast cancer cells with constitutive active Raf-1/mitogen-associated protein kinase (MAPK) signaling (vMCF-7 Raf-1 ) and MDA-MB-231 triple-negative breast cancer (TNBC) cells. The critical role of NOTCH3 in inducing an invasive phenotype and poor outcome was corroborated in unique TNBC cells resulting from a patient-derived brain metastasis (TNBC-M25) and in publicly available claudin-low breast tumor specimens collected from participants in the Molecular Taxonomy of Breast Cancer International Consortium database., Results: In this study, we identified an association between NOTCH3 expression and development of metastases in ERα + and TNBC models. ERα + breast tumor xenografts with a constitutive active Raf-1/MAPK signaling developed spontaneous lung metastases through the clonal expansion of cancer cells expressing a NOTCH3 reprogramming network. Abrogation of NOTCH3 expression significantly reduced the self-renewal and invasive capacity of ex vivo breast cancer cells, restoring a luminal CD44 low /CD24 high /ERα high phenotype. Forced expression of the mitotic Aurora kinase A (AURKA), which promotes breast cancer metastases, failed to restore the invasive capacity of NOTCH3-null cells, demonstrating that NOTCH3 expression is required for an invasive phenotype. Likewise, pharmacologic inhibition of NOTCH signaling also impaired TNBC cell seeding and metastatic growth. Significantly, the role of aberrant NOTCH3 expression in promoting tumor self-renewal, invasiveness, and poor outcome was corroborated in unique TNBC cells from a patient-derived brain metastasis and in publicly available claudin-low breast tumor specimens., Conclusions: These findings demonstrate the key role of NOTCH3 oncogenic signaling in the genesis of breast cancer metastasis and provide a compelling preclinical rationale for the design of novel therapeutic strategies that will selectively target NOTCH3 to halt metastatic seeding and to improve the clinical outcomes of patients with breast cancer.

Competing Interests: CONFLICTS OF INTEREST The authors declare no potential conflicts of interest.

Proline, glutamic acid, leucine-rich protein 1 (PELP1) is overexpressed in approximately 80% of invasive breast tumors. PELP1 dynamically shuttles between the nucleus and cytoplasm, but is primarily nuclear in normal breast tissue. However, altered localization of PELP1 to the cytoplasm is an oncogenic event that promotes breast cancer initiation and progression. Herein, interacting partners unique to cytoplasmic PELP1 and the mechanisms by which these interactions promote oncogenic PELP1 signaling were sought. AIB1 (amplified in breast cancer 1; also known as SRC-3 or NCOA3) was identified as a novel binding partner of cytoplasmic PELP1 in both estrogen receptor-positive (ER + ) and ER-negative cell lines. Cytoplasmic PELP1 expression elevated basal phosphorylation levels (i.e., activation) of AIB1 at Thr24, enhanced ALDH + tumorsphere formation, and upregulated specific target genes independently of hormone stimulation. Direct manipulation of AIB1 levels using shRNA abrogated cytoplasmic PELP1-induced tumorsphere formation and downregulated cytoplasmic PELP1-specific target genes. SI-2, an AIB1 inhibitor, limited the PELP1/AIB1 interaction and decreased cytoplasmic PELP1-induced tumorsphere formation. Similar results were observed in a murine-derived MMTV-AIB1 tumor cell line. Furthermore, in vivo syngeneic tumor studies revealed that PELP1 knockdown resulted in increased survival of tumor-bearing mice as compared with mice injected with control cells. Implications: These data demonstrate that cytoplasmic PELP1/AIB1-containing complexes function to promote advanced cancer phenotypes, including outgrowth of stem-like cells, associated with estrogen-independent breast cancer progression. Mol Cancer Res; 16(4); 707-19. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Estrogen is the major mitogenic stimulus of mammary gland development during puberty wherein ER signaling acts to induce abundant PR expression. PR signaling, in contrast, is the primary driver of mammary epithelial cell proliferation in adulthood. The high circulating levels of progesterone during pregnancy signal through PR, inducing expression of the prolactin receptor (PRLR). Cooperation between PR and prolactin (PRL) signaling, via regulation of downstream components in the PRL signaling pathway including JAKs and STATs, facilitates the alveolar morphogenesis observed during pregnancy. Indeed, these pathways are fully integrated via activation of shared signaling pathways (i.e. JAKs, MAPKs) as well as by the convergence of PRs and STATs at target genes relevant to both mammary gland biology and breast cancer progression (i.e. proliferation, stem cell outgrowth, tissue cell type heterogeneity). Thus, rather than a single mediator such as ER, transcription factor cascades (ER>PR>STATs) are responsible for rapid proliferative and developmental programming in the normal mammary gland. It is not surprising that these same mediators typify uncontrolled proliferation in a majority of breast cancers, where ER and PR are most often co-expressed and may cooperate to drive malignant tumor progression. This review will primarily focus on the integration of PR and PRL signaling in breast cancer models and the importance of this cross-talk in cancer progression in the context of mammographic density. Components of these PR/PRL signaling pathways could offer alternative drug targets and logical complements to anti-ER or anti-estrogen-based endocrine therapies., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Breast cancer is the second largest cause of cancer death among U.S. women and the leading cause of cancer death among women worldwide. Genome-wide association studies (GWAS) have identified several genetic variants associated with susceptibility to breast cancer, but these still explain less than half of the estimated genetic contribution to the disease. Combinations of variants (i.e. genetic interactions) may play an important role in breast cancer susceptibility. However, due to a lack of statistical power, the current tests for genetic interactions from GWAS data mainly leverage prior knowledge to focus on small sets of genes or SNPs that are known to have an association with breast cancer. Thus, many genetic interactions, particularly among novel variants, remain understudied. Reverse-genetic interaction screens in model organisms have shown that genetic interactions frequently cluster into highly structured motifs, where members of the same pathway share similar patterns of genetic interactions. Based on this key observation, we recently developed a method called BridGE to search for such structured motifs in genetic networks derived from GWAS studies and identify pathway-level genetic interactions in human populations. We applied BridGE to six independent breast cancer cohorts and identified significant pathway-level interactions in five cohorts. Joint analysis across all five cohorts revealed a high confidence consensus set of genetic interactions with support in multiple cohorts. The discovered interactions implicated the glutathione conjugation, vitamin D receptor, purine metabolism, mitotic prometaphase, and steroid hormone biosynthesis pathways as major modifiers of breast cancer risk. Notably, while many of the pathways identified by BridGE show clear relevance to breast cancer, variants in these pathways had not been previously discovered by traditional single variant association tests, or single pathway enrichment analysis that does not consider SNP-SNP interactions.

Background: Estrogen and progesterone are potent breast mitogens. In addition to steroid hormones, multiple signaling pathways input to estrogen receptor (ER) and progesterone receptor (PR) actions via posttranslational events. Protein kinases commonly activated in breast cancers phosphorylate steroid hormone receptors (SRs) and profoundly impact their activities., Methods: To better understand the role of modified PRs in breast cancer, we measured total and phospho-Ser294 PRs in 209 human breast tumors represented on 2754 individual tissue spots within a tissue microarray and assayed the regulation of this site in human tumor explants cultured ex vivo. To complement this analysis, we assayed PR target gene regulation in T47D luminal breast cancer models following treatment with progestin (promegestone; R5020) and antiprogestins (mifepristone, onapristone, or aglepristone) in conditions under which the receptor is regulated by Lys388 SUMOylation (K388 intact) or is SUMO-deficient (via K388R mutation to mimic persistent Ser294 phosphorylation). Selected phospho-PR-driven target genes were validated by qRT-PCR and following RUNX2 shRNA knockdown in breast cancer cell lines. Primary and secondary mammosphere assays were performed to implicate phospho-Ser294 PRs, epidermal growth factor signaling, and RUNX2 in breast cancer stem cell biology., Results: Phospho-Ser294 PR species were abundant in a majority (54%) of luminal breast tumors, and PR promoter selectivity was exquisitely sensitive to posttranslational modifications. Phospho-PR expression and target gene programs were significantly associated with invasive lobular carcinoma (ILC). Consistent with our finding that activated phospho-PRs undergo rapid ligand-dependent turnover, unique phospho-PR gene signatures were most prevalent in breast tumors clinically designated as PR-low to PR-null (luminal B) and included gene sets associated with cancer stem cell biology (HER2, PAX2, AHR, AR, RUNX). Validation studies demonstrated a requirement for RUNX2 in the regulation of selected phospho-PR target genes (SLC37A2). In vitro mammosphere formation assays support a role for phospho-Ser294-PRs via growth factor (EGF) signaling as well as RUNX2 as potent drivers of breast cancer stem cell fate., Conclusions: We conclude that PR Ser294 phosphorylation is a common event in breast cancer progression that is required to maintain breast cancer stem cell fate, in part via cooperation with growth factor-initiated signaling pathways and key phospho-PR target genes including SLC37A2 and RUNX2. Clinical measurement of phosphorylated PRs should be considered a useful marker of breast tumor stem cell potential. Alternatively, unique phospho-PR target gene sets may provide useful tools with which to identify patients likely to respond to selective PR modulators that block PR Ser294 phosphorylation as part of rational combination (i.e., with antiestrogens) endocrine therapies designed to durably block breast cancer recurrence.

The vascular endothelial growth factor (VEGF) isoform VEGF165 stimulates vascular growth and hyperpermeability. Whereas blood vessel growth is essential to sustain organ health, chronic hyperpermeability causes damaging tissue edema. By combining in vivo and tissue culture models, we show here that VEGF165-induced vascular leakage requires both VEGFR2 and NRP1, including the VEGF164-binding site of NRP1 and the NRP1 cytoplasmic domain (NCD), but not the known NCD interactor GIPC1. In the VEGF165-bound receptor complex, the NCD promotes ABL kinase activation, which in turn is required to activate VEGFR2-recruited SRC family kinases (SFKs). These results elucidate the receptor complex and signaling hierarchy of downstream kinases that transduce the permeability response to VEGF165. In a mouse model with choroidal neovascularisation akin to age-related macular degeneration, NCD loss attenuated vessel leakage without affecting neovascularisation. These findings raise the possibility that targeting NRP1 or its NCD interactors may be a useful therapeutic strategy in neovascular disease to reduce VEGF165-induced edema without compromising vessel growth., (© 2017 Fantin et al.)

Background: Approximately 18-20% of all human breast cancers have overexpressed human epidermal growth factor receptor 2 (HER2). Standard clinical practice is to treat only overexpressed HER2 (HER2+) cancers with targeted anti-HER2 therapies. However, recent analyses of clinical trial data have found evidence that HER2-targeted therapies may benefit a sub-group of breast cancer patients with non-overexpressed HER2. This suggests that measurement of other biological factors associated with HER2 cancer, such as HER2 signaling pathway activity, should be considered as an alternative means of identifying patients eligible for HER2 therapies., Methods: A new biosensor-based test (CELx TM HSF) that measures HER2 signaling activity in live cells is demonstrated using a set of 19 human HER2+ and HER2- breast cancer reference cell lines and primary cell samples derived from two fresh patient tumor specimens. Pathway signaling is elucidated by use of highly specific agonists and antagonists. The test method relies upon well-established phenotypic, adhesion-related, impedance changes detected by the biosensor., Results: The analytical sensitivity and analyte specificity of this method was demonstrated using ligands with high affinity and specificity for HER1 and HER3. The HER2-driven signaling quantified ranged 50-fold between the lowest and highest cell lines. The HER2+ cell lines were almost equally divided into high and low signaling test result groups, suggesting that little correlation exists between HER2 protein expression and HER2 signaling level. Unexpectedly, the highest HER2-driven signaling level recorded was with a HER2- cell line., Conclusions: Measurement of HER2 signaling activity in the tumor cells of breast cancer patients is a feasible approach to explore as a biomarker to identify HER2-driven cancers not currently diagnosable with genomic techniques. The wide range of HER2-driven signaling levels measured suggests it may be possible to make a distinction between normal and abnormal levels of activity. Analytical validation studies and clinical trials treating HER2- patients with abnormal HER2-driven signaling would be required to evaluate the analytical and clinical validity of using this functional biomarker as a diagnostic test to select patients for treatment with HER2 targeted therapy. In clinical practice, this method would require patient specimens be delivered to and tested in a central lab.

The results of clinical trials evaluating the efficacy of HER2 inhibitors in patients with breast cancer indicate that the correlation between HER2 receptor levels and patient outcomes is as low as 50%. The relatively weak correlation between HER2 status and response to HER2-targeting drugs suggests that measurement of HER2 signaling activity, rather than absolute HER2 levels, may more accurately diagnose HER2-driven breast cancer. A new diagnostic test, the CELx HER2 Signaling Profile (CELx HSP) test, is demonstrated to measure real-time HER2 signaling function in live primary cells. In the present study, epithelial cells extracted fresh from breast cancer patient tumors classified as HER2 negative (HER2-, n = 34 of which 33 were estrogen receptor positive) and healthy subjects (n = 16) were evaluated along with reference breast cancer cell lines (n = 19). Live cell response to specific HER2 agonists (NRG1b and EGF) and antagonist (pertuzumab) was measured. Of the HER2- breast tumor cell samples tested, 7 of 34 patients (20.5%; 95% CI = 10%-37%) had HER2 signaling activity that was characterized as abnormally high. Amongst the tumor samples there was no correlation between HER2 protein status (by cell cytometry) and HER2 signaling activity (hyperactive or normal) (Regression analysis P = 0.144, R2 = 0.068). One conclusion is that measurement of HER2 signaling activity can identify a subset of breast cancers with normal HER2 receptor levels with abnormally high levels of HER2 signaling. This result constitutes a new subtype of breast cancer that should be considered for treatment with HER2 pathway inhibitors.

Progesterone Receptors (PRs) are critical effectors of estrogen receptor (ER) signaling required for mammary gland development and reproductive proficiency. In breast and reproductive tract malignancies, PR expression is a clinical prognostic marker of ER action. While estrogens primarily regulate PR expression, other factors likely contribute to a dynamic range of receptor expression across diverse tissues. In this study, we identified estrogen-independent but progestin (R5020)-dependent regulation of ER target genes including PGR in ER+/PR+ cancer cell lines. R5020 (10nM-10μM range) induced dose-dependent PR mRNA and protein expression in the absence of estrogen but required both PR and ERα. Antagonists of either PR (RU486, onapristone) or ERα (ICI 182,780) attenuated R5020 induction of TFF1, CTSD, and PGR. Chromatin immunoprecipitation (ChIP) assays performed on ER+/PR+ cells demonstrated that both ERα and PR were recruited to the same ERE/Sp1 site-containing region of the PGR proximal promoter in response to high dose progestin (10μM). Recruitment of ERα and PR to chromatin and subsequent PR mRNA induction were dependent upon rapid activation of MAPK/ERK and AKT; inhibition of these kinase pathways via U0126 or LY294002 blocked these events. Overall, we have identified a novel mechanism of ERα activation initiated by rapid PR-dependent kinase pathway activation and associated with phosphorylation of ERα Ser118 for estrogen-independent but progestin-dependent ER/PR cross talk. These studies may provide insight into mechanisms of persistent ER-target gene expression during periods of hormone (i.e. estrogen) ablation and suggest caution following prolonged treatment with aromatase or CYP17 inhibitors (i.e. contexts when progesterone levels may be abnormally elevated)., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background and Objective: The introduction of optical coherence tomography angiography (OCTA) provides new insights into the retinal vasculature. The aim of this study was to explore the value of OCTA in imaging retinal hemangioblastomas and monitoring laser treatment in patients with von Hippel-Lindau (VHL) disease., Patients and Methods: Ten eyes of 10 patients with VHL disease were included in this retrospective case series. All patients underwent complete ophthalmological work-up including OCTA for retinal and optic nerve head hemangioblastoma., Results: Two patients showed retinal scars and no recurrence of hemangioblastoma in OCTA. Three patients revealed recurrent hemangioblastomas. Two patients demonstrated a new hemangioblastoma. Three patients showed hemangioblastomas of the optic nerve head. Successful laser photocoagulation could be monitored with OCTA., Conclusions: Screening for retinal hemangioblastomas with OCTA alone is not possible. OCTA may help to distinguish hemangioblastomas and other lesions in VHL disease, especially after treatment and allows the differentiation from harmless non-vascular lesions in questionable cases. [Ophthalmic Surg Lasers Imaging Retina. 2016;47:935-946.]., (Copyright 2016, SLACK Incorporated.)

The (1)H nuclear magnetic resonance spectrum of hexamethylbenzene orientationally ordered in the nematic liquid crystal ZLI-1132 is analysed using covariance matrix adaptation evolution strategy. The spectrum contains over 350 000 lines with many overlapping transitions, from which four independent direct dipolar couplings are obtained. The rotations of the six methyl groups appear to be correlated due to mutual steric hindrance. Adjacent methyl groups show counter-rotating or geared motion. Hexamethylbenzene thus behaves as a molecular hexagonal gear.

Background: The progesterone receptor (PR) is expressed by ∼70% of early breast tumours and is implicated in the progression of breast cancer. In cancerous tissues PR may be activated in the absence of a ligand, or when ligand concentrations are very low, resulting in aberrantly activated PR (APR). The presence of APR may indicate that patients with breast cancer are more likely to respond to antiprogestins. The aims of this study were to describe and classify the histological subnuclear morphology of active and inactive PR in archival breast cancer samples., Methods: Archived tumour specimens from 801 women with invasive breast cancer were collected. Tissue samples (n=789) were analysed for PR isoforms A and B (PRA and PRB), Ki67 and estrogen receptors (ERα) status, using immunohistochemistry. Medical records were used to determine human epidermal growth factor 2 (HER2) status, tumour stage and grade., Results: A total of 79% of tumours stained positive for either PRA or PRB, and of these 25% of PRA-positive and 23% of PRB-positive tumours had PR present in the activated form. APRA was associated with higher tumour grade (p=0.001). APRB was associated with a higher tumour grade (p=0.046) and a trend for a more advanced stage. Patients with PR-positive tumours treated with antiestrogens had better disease-free survival (DFS) than those with PR-negative tumours (p<0.0001). Cumulative progression rate and DFS were similar irrespective of APR status. Both APRA and APRB were independent of HER2, ERα and Ki67 expression., Conclusions: APR had a binary mode of expression in the breast cancer specimens tested, allowing separation into two tumour subsets. APR is an independent target at the cellular and tumour level and may therefore be a suitable predictive marker for antiprogestins, such as onapristone. Using the described technique, a companion diagnostic is under development to identify APR in solid tumours., Competing Interests: JBon is an investigator of an ARNO Therapeutics study and has received honoraria payments, research credits and trip fees for presentations at various congresses from Invivis Pharmaceuticals Inc. PJ has received travel fees for presentations at various congresses from Invivis Pharmaceuticals Inc. EMG holds a leadership position at Invivis Pharmaceuticals Inc. AAZ is the CEO of ARNO Therapeutics Inc. JO has received honoraria payments from ARNO Therapeutics Inc.

Steroid hormone receptors (SRs) are heavily posttranslationally modified by the reversible addition of a variety of molecular moieties, including phosphorylation, acetylation, methylation, SUMOylation, and ubiquitination. These rapid and dynamic modifications may be combinatorial and interact (i.e. may be sequential, complement, or oppose each other), creating a vast array of uniquely modified receptor subspecies that allow for diverse receptor behaviors that enable highly sensitive and context-dependent hormone action. For example, in response to hormone or growth factor membrane-initiated signaling events, posttranslational modifications (PTMs) to SRs alter protein-protein interactions that govern the complex process of promoter or gene-set selection coupled to transcriptional repression or activation. Unique phosphorylation events allow SRs to associate or disassociate with specific cofactors that may include pioneer factors and other tethering partners, which specify the resulting transcriptome and ultimately change cell fate. The impact of PTMs on SR action is particularly profound in the context of breast tumorigenesis, in which frequent alterations in growth factor-initiated signaling pathways occur early and act as drivers of breast cancer progression toward endocrine resistance. In this article, with primary focus on breast cancer relevance, we review the mechanisms by which PTMs, including reversible phosphorylation events, regulate the closely related SRs, glucocorticoid receptor and progesterone receptor, allowing for precise biological responses to ever-changing hormonal stimuli., (© 2016 Society for Endocrinology.)

Cancer cells use stress response pathways to sustain their pathogenic behavior. In breast cancer, stress response-associated phenotypes are mediated by the breast tumor kinase, Brk (PTK6), via the hypoxia-inducible factors HIF-1α and HIF-2α. Given that glucocorticoid receptor (GR) is highly expressed in triple-negative breast cancer (TNBC), we investigated cross-talk between stress hormone-driven GR signaling and HIF-regulated physiologic stress. Primary TNBC tumor explants or cell lines treated with the GR ligand dexamethasone exhibited robust induction of Brk mRNA and protein that was HIF1/2-dependent. HIF and GR coassembled on the BRK promoter in response to either hypoxia or dexamethasone, indicating that Brk is a direct GR/HIF target. Notably, HIF-2α, not HIF-1α, expression was induced by GR signaling, and the important steroid receptor coactivator PELP1 was also found to be induced in a HIF-dependent manner. Mechanistic investigations showed how PELP1 interacted with GR to activate Brk expression and demonstrated that physiologic cell stress, including hypoxia, promoted phosphorylation of GR serine 134, initiating a feed-forward signaling loop that contributed significantly to Brk upregulation. Collectively, our findings linked cellular stress (HIF) and stress hormone (cortisol) signaling in TNBC, identifying the phospho-GR/HIF/PELP1 complex as a potential therapeutic target to limit Brk-driven progression and metastasis in TNBC patients., (©2016 American Association for Cancer Research.)

Unlabelled: Progesterone promotes differentiation coupled to proliferation and prosurvival in the breast, but inhibits estrogen-driven growth in the reproductive tract and ovaries. Herein, it is demonstrated, using progesterone receptor (PR) isoform-specific ovarian cancer model systems, that PR-A and PR-B promote distinct gene expression profiles that differ from PR-driven genes in breast cancer cells. In ovarian cancer models, PR-A primarily regulates genes independently of progestin, while PR-B is the dominant ligand-dependent isoform. Notably, FOXO1 and the PR/FOXO1 target gene p21 (CDKN1A) are repressed by PR-A, but induced by PR-B. In the presence of progestin, PR-B, but not PR-A, robustly induced cellular senescence via FOXO1-dependent induction of p21 and p15 (CDKN2B). Chromatin immunoprecipitation (ChIP) assays performed on PR isoform-specific cells demonstrated that while each isoform is recruited to the same PRE-containing region of the p21 promoter in response to progestin, only PR-B elicits active chromatin marks. Overexpression of constitutively active FOXO1 in PR-A-expressing cells conferred robust ligand-dependent upregulation of the PR-B target genes GZMA, IGFBP1, and p21, and induced cellular senescence. In the presence of endogenous active FOXO1, PR-A was phosphorylated on Ser294 and transactivated PR-B at PR-B target genes; these events were blocked by the FOXO1 inhibitor (AS1842856). PR isoform-specific regulation of the FOXO1/p21 axis recapitulated in human primary ovarian tumor explants treated with progestin; loss of progestin sensitivity correlated with high AKT activity., Implications: This study indicates FOXO1 as a critical component for progesterone signaling to promote cellular senescence and reveals a novel mechanism for transcription factor control of hormone sensitivity., (©2015 American Association for Cancer Research.)

Objectives/hypothesis: Tumor volume has been postulated to be an important prognostic factor for oncological outcome after radiotherapy or chemoradiotherapy. This postulate was retrospectively investigated in a consecutively treated cohort of T3-T4 larynx cancer patients., Study Design: Retrospective cohort study., Methods: For 166 patients with T3-T4 larynx cancer (1999-2008), pretreatment computed tomography and magnetic resonance imaging scans were available for tumor volume delineation. Patients were treated with radiotherapy, chemoradiotherapy, or total laryngectomy with postoperative radiotherapy. Both a dedicated head and neck radiologist and the first author determined all tumor volumes. Statistical analysis was by Kaplan-Meier plots and Cox proportional hazard models., Results: Patients with T3 larynx cancer had significantly smaller tumor volumes than patients with T4 larynx cancer (median = 8.1 cm(3) and 15.8 cm(3), respectively; P < .0001). In the group treated with total laryngectomy and postoperative radiotherapy, no association was found between tumor volume and local or locoregional control or overall survival. In the group treated with radiotherapy, a nonsignificant trend was observed between local control and tumor volume. In the chemoradiotherapy group, however, a significant impact of tumor volume was found on local control (hazard ratio = 1.07; 95% confidence interval = 1.01-1.13; P = .028)., Conclusions: Tumor volume was not significantly associated with local control, locoregional control, or overall survival in the surgically treated group. In the group treated with radiotherapy, there was no statistically significant association, but a trend was observed between local control and tumor volume. Only in patients treated with concurrent chemoradiotherapy was a significant impact of tumor volume on local control found., Level of Evidence: 4., (© 2015 The American Laryngological, Rhinological and Otological Society, Inc.)

Objective: Ocular neovascularization (ONV) is a pathological feature of sight-threatening human diseases, such as diabetic retinopathy and age-related macular degeneration. Macrophage depletion in mouse models of ONV reduces the formation of pathological blood vessels, and myeloid cells are widely considered an important source of the vascular endothelial growth factor A (VEGF). However, the importance of VEGF or its upstream regulators hypoxia-inducible factor-1α (HIF1α) and hypoxia-inducible factor-2α (HIF2α) as myeloid-derived regulators of ONV remains to be determined., Approach and Results: We used 2 mouse models of ONV, choroidal neovascularization and oxygen-induced retinopathy, to show that Vegfa is highly expressed by several cell types, but not myeloid cells during ONV. Moreover, myeloid-specific VEGF ablation did not reduce total ocular VEGF during choroidal neovascularization or oxygen-induced retinopathy. In agreement, the conditional inactivation of Vegfa, Hif1a, or Epas1 in recruited and resident myeloid cells that accumulated at sites of neovascularization did not significantly reduce choroidal neovascularization or oxygen-induced retinopathy., Conclusions: The finding that myeloid cells are not a significant local source of VEGF in these rodent models of ONV suggests that myeloid function in neovascular eye disease differs from skin wound healing and other neovascular pathologies., (© 2015 The Authors.)

Aim: Preoperative detection of extranodal spread (ENS) in head and neck cancer can have important consequences for patient management. The aim of this study was to determine whether 18-fluorodeoxyglucose positron emission tomography ([18F]FDG PET) or a combination with Magnetic Resonance Imaging (MRI) could more accurately predict ENS, especially with the near availability of fully integrated [18F]FDG PET/MRI scanners., Methods: In retrospective cohort design a total of twelve patients, with 18 lymphnode metastases were studied with [18F]FDG PET and MRI. Presence of ENS was scored on MRI, and [18F]FDG PET images using a SUV max cut-off point of 12. Histopathology results were used as reference standard. Sensitivity, specificity and accuracy were calculated., Results: The sensitivity, specificity and accuracy of [18F]FDG PET for ENS reached 70%,100% and 83%, respectively. The mean SUVmax of ENS positive lymphnodes was 13.6 versus 8.7 for lymphnode metastases without ENS (P=0.03). The sensitivity, specificity and accuracy of MRI for ENS were 70%, 100% and 83%, respectively. When the [18F]FDG PET and MRI findings were combined sensitivity, specificity and accuracy were 80%, 100% and 89%, respectively. Thus, accuracy increased from 83% to 89%., Conclusion: When there is no ENS or doubt of ENS on MRI, [18F]FDG PET seems to have additional value since it improves sensitivity and resolves uncertainty in case of high FDG uptake. This benefit needs to be confirmed prospectively in a larger cohort.

The NMR spectrum of n-hexane orientationally ordered in the nematic liquid crystal ZLI-1132 is analysed using covariance matrix adaptation evolution strategy (CMA-ES). The spectrum contains over 150 000 transitions, with many sharp features appearing above a broad, underlying background signal that results from the plethora of overlapping transitions from the n-hexane as well as from the liquid crystal. The CMA-ES requires initial search ranges for NMR spectral parameters, notably the direct dipolar couplings. Several sets of such ranges were utilized, including three from MD simulations and others from the modified chord model that is specifically designed to predict hydrocarbon-chain dipolar couplings. In the end, only inaccurate dipolar couplings from an earlier study utilizing proton-proton double quantum 2D-NMR techniques on partially deuterated n-hexane provided the necessary estimates. The precise set of dipolar couplings obtained can now be used to investigate conformational averaging of n-hexane in a nematic environment.

Progesterone and progesterone receptors (PRs) are essential for the development and cyclical regulation of hormone-responsive tissues including the breast and reproductive tract. Altered functions of PR isoforms contribute to the pathogenesis of tumors that arise in these tissues. In the breast, progesterone acts in concert with estrogen to promote proliferative and pro-survival gene programs. In sharp contrast, progesterone inhibits estrogen-driven growth in the uterus and protects the ovary from neoplastic transformation. Progesterone-dependent actions and associated biology in diverse tissues and tumors are mediated by two PR isoforms, PR-A and PR-B. These isoforms are subject to altered transcriptional activity or expression levels, differential crosstalk with growth factor signaling pathways, and distinct post-translational modifications and cofactor-binding partners. Herein, we summarize and discuss the recent literature focused on progesterone and PR isoform-specific actions in breast, uterine, and ovarian cancers. Understanding the complexity of context-dependent PR actions in these tissues is critical to developing new models that will allow us to advance our knowledge base with the goal of revealing novel and efficacious therapeutic regimens for these hormone-responsive diseases., (© 2015 Society for Endocrinology.)

To characterize the molecular mechanism and map the response element used by progesterone (P) to upregulate tissue factor (TF) in breast cancer cells. TF expression and mRNA levels were analyzed in breast cancer ZR-75 and T47D cells, using Western blot and real-time PCR, respectively. Mapping of the TF promoter was performed using luciferase vectors. Progesterone receptor (PR) and specificity protein 1 (Sp1) binding to the TF promoter were analyzed by chromatin immuno precipitation assay. Specific or selective inhibitors were used for the MEK1/2 and the c-Src pathways (UO126 and PP2, respectively). TF mRNA increase peaks at 18 h following P treatment in ZR-75 and T47D cells. P upregulation occurs via a transcriptional mechanism that depends on PR and MEK1/2 activation, PR and Sp1 transcription factors bind to a region in the TF promoter that contains three Sp1 sites. TF mRNA upregulation requires an intact PR proline-rich site (mPRO), but it is independent from c-Src. TF upregulation by P is mediated by Sp1 sites in the TF promoter region. Transcriptional upregulation in breast cancer cells occurs via a new mechanism that requires MEK1/2 activation and the mPRO site but independent of c-Src activity. PR Phosphorylation at serine 294 and 345 is not essential.

Progesterone and estrogen are important drivers of breast cancer proliferation. Herein, we probed estrogen receptor-α (ER) and progesterone receptor (PR) cross-talk in breast cancer models. Stable expression of PR-B in PR-low/ER+ MCF7 cells increased cellular sensitivity to estradiol and insulin-like growth factor 1 (IGF1), as measured in growth assays performed in the absence of exogenous progestin; similar results were obtained in PR-null/ER+ T47D cells stably expressing PR-B. Genome-wide microarray analyses revealed that unliganded PR-B induced robust expression of a subset of estradiol-responsive ER target genes, including cathepsin-D (CTSD). Estradiol-treated MCF7 cells stably expressing PR-B exhibited enhanced ER Ser167 phosphorylation and recruitment of ER, PR and the proline-, glutamate- and leucine-rich protein 1 (PELP1) to an estrogen response element in the CTSD distal promoter; this complex co-immunoprecipitated with IGF1 receptor (IGFR1) in whole-cell lysates. Importantly, ER/PR/PELP1 complexes were also detected in human breast cancer samples. Inhibition of IGF1R or phosphoinositide 3-kinase blocked PR-B-dependent CTSD mRNA upregulation in response to estradiol. Similarly, inhibition of IGF1R or PR significantly reduced ER recruitment to the CTSD promoter. Stable knockdown of endogenous PR or onapristone treatment of multiple unmodified breast cancer cell lines blocked estradiol-mediated CTSD induction, inhibited growth in soft agar and partially restored tamoxifen sensitivity of resistant cells. Further, combination treatment of breast cancer cells with both onapristone and IGF1R tyrosine kinase inhibitor AEW541 was more effective than either agent alone. In summary, unliganded PR-B enhanced proliferative responses to estradiol and IGF1 via scaffolding of ER-α/PELP1/IGF1R-containing complexes. Our data provide a strong rationale for targeting PR in combination with ER and IGF1R in patients with luminal breast cancer.

The proton NMR spectra of n-pentane orientationally ordered in two nematic liquid-crystal solvents are studied over a wide temperature range and analysed using covariance matrix adaptation evolutionary strategy. Since alkanes possess small electrostatic moments, their anisotropic intermolecular interactions are dominated by short-range size-and-shape effects. As we assumed for n-butane, the anisotropic energy parameters of each n-pentane conformer are taken to be proportional to those of ethane and propane, independent of temperature. The observed temperature dependence of the n-pentane dipolar couplings allows a model-free separation between conformer degrees of order and conformer probabilities, which cannot be achieved at a single temperature. In this way for n-pentane 13 anisotropic energy parameters (two for trans trans, tt, five for trans gauche, tg, and three for each of gauche+ gauche+, pp, and gauche+ gauche-, pm), the isotropic trans-gauche energy difference Etg and its temperature coefficient Etg(') are obtained. The value obtained for the extra energy associated with the proximity of the two methyl groups in the gauche+ gauche- conformers (the pentane effect) is sensitive to minute details of other assumptions and is thus fixed in the calculations. Conformer populations are affected by the environment. In particular, anisotropic interactions increase the trans probability in the ordered phase.

The molecular chaperone Hsp90 is important for the functional maturation of many client proteins, and inhibitors are in clinical trials for multiple indications in cancer. Hsp90 inhibition activates the heat shock response and can improve viability in a cell model of the P23H misfolding mutation in rhodopsin that causes autosomal dominant retinitis pigmentosa (adRP). Here, we show that a single low dose of the Hsp90 inhibitor HSP990 enhanced visual function and delayed photoreceptor degeneration in a P23H transgenic rat model. This was associated with the induction of heat shock protein expression and reduced rhodopsin aggregation. We then investigated the effect of Hsp90 inhibition on a different type of rod opsin mutant, R135L, which is hyperphosphorylated, binds arrestin and disrupts vesicular traffic. Hsp90 inhibition with 17-AAG reduced the intracellular accumulation of R135L and abolished arrestin binding in cells. Hsf-1(-/-) cells revealed that the effect of 17-AAG on P23H aggregation was dependent on HSF-1, whereas the effect on R135L was HSF-1 independent. Instead, the effect on R135L was mediated by a requirement of Hsp90 for rhodopsin kinase (GRK1) maturation and function. Importantly, Hsp90 inhibition restored R135L rod opsin localization to wild-type (WT) phenotype in vivo in rat retina. Prolonged Hsp90 inhibition with HSP990 in vivo led to a posttranslational reduction in GRK1 and phosphodiesterase (PDE6) protein levels, identifying them as Hsp90 clients. These data suggest that Hsp90 represents a potential therapeutic target for different types of rhodopsin adRP through distinct mechanisms, but also indicate that sustained Hsp90 inhibition might adversely affect visual function.

Ovarian steroid hormones contribute to breast cancer initiation and progression primarily through the actions of their nuclear transcription factors, the estrogen receptor alpha (ERα) and progesterone receptors (PRs). These receptors are important drivers of the luminal A and B subtypes of breast cancer, where estrogen-blocking drugs have been effective endocrine therapies for patients with these tumors. However, many patients do not respond, or become resistant to treatment. When endocrine therapies fail, the luminal subtypes of breast cancer are more difficult to treat because these subtypes are among the most heterogeneous in terms of mutation diversity and gene expression profiles. Recent evidence suggests that progestin and PR actions may be important drivers of luminal breast cancers. Clinical trial data has demonstrated that hormone replacement therapy with progestins drives invasive breast cancer and results in greater mortality. PR transcriptional activity is dependent upon cross-talk with growth factor signaling pathways that alter PR phosphorylation, acetylation, or SUMOylation as mechanisms for regulating PR target gene selection required for increased cell proliferation and survival. Site-specific PR phosphorylation is the primary driver of gene-selective PR transcriptional activity. However, PR phosphorylation and heightened transcriptional activity is coupled to rapid PR protein degradation; the range of active PR detected in tumors is likely to be dynamic. Thus, PR target gene signatures may provide a more accurate means of tracking PR's contribution to tumor progression rather than standard clinical protein-based (IHC) assays. Further development of antiprogestin therapies should be considered alongside antiestrogens and aromatase inhibitors., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The progesterone receptor (PR) and its coactivators are direct targets of activated cyclin-dependent kinases (CDKs) in response to peptide growth factors, progesterone, and deregulation of cell cycle inhibitors. Herein, using the T47D breast cancer model, we probed mechanisms of cell cycle-dependent PR action. In the absence of exogenous progestin, the PR is specifically phosphorylated during the G2/M phase. Accordingly, numerous PR target genes are cell cycle regulated, including HSPB8, a heat-shock protein whose high expression is associated with tamoxifen resistance. Progestin-induced HSPB8 expression required cyclin D1 and was insensitive to antiestrogens but blocked by antiprogestins or inhibition of specificity factor 1 (SP1). HSPB8 expression increased with or without ligand when cells were G2/M synchronized or contained high levels of cyclin D1. Knockdown of PRs abrogated ligand-independent HSPB8 expression in synchronized cells. Notably, PRs and cyclin D1 copurified in whole-cell lysates of transiently transfected COS-1 cells and in PR-positive T47D breast cancer cells expressing endogenous cyclin D1. PRs, cyclin D1, and SP1 were recruited to the HSPB8 promoter in progestin-treated T47D breast cancer cells. Mutation of PR Ser345 to Ala (S345A) or inhibition of CDK2 activity using roscovitine disrupted PR/cyclin D1 interactions with DNA and blocked HSPB8 mRNA expression. Interaction of phosphorylated PRs with SP1 and cyclin D1 provides a mechanism for targeting transcriptionally active PRs to selected gene promoters relevant to breast cancer progression. Understanding the functional linkage between PRs and cell cycle regulatory proteins will provide keys to targeting novel PR/cyclin D1 cross talk in both hormone-responsive disease and HSPB8-high refractory disease with high HSPB8 expression.

The ovarian steroid hormone, progesterone, and its nuclear receptor, the progesterone receptor, are implicated in the progression of breast cancer. Clinical trial data on the effects of hormone replacement therapy underscore the importance of understanding how progestins influence breast cancer growth. The progesterone receptor regulation of distinct target genes is mediated by complex interactions between the progesterone receptor and other regulatory factors that determine the context-dependent transcriptional action of the progesterone receptor. These interactions often lead to post-translational modifications to the progesterone receptor that can dramatically alter receptor function, both in the normal mammary gland and in breast cancer. This review highlights the molecular components that regulate progesterone receptor transcriptional action and describes how a better understanding of the complex interactions between the progesterone receptor and other regulatory factors may be critical to enhancing the clinical efficacy of anti-progestins for use in the treatment of breast cancer.

Proline, glutamic acid, and leucine rich protein 1 (PELP1) is a large multi-domain protein that has been shown to modulate an increasing number of pathways and biological processes. The first reports describing the cloning and characterization of PELP1 showed that it was an estrogen receptor coactivator. PELP1 has now been shown to be a coregulator for a growing number of transcription factors. Furthermore, recent reports have shown that PELP1 is a member of chromatin remodeling complexes. In addition to PELP1 nuclear functions, it has been shown to have cytoplasmic signaling functions as well. In the cytoplasm PELP1 acts as a scaffold molecule and mediates rapid signaling from growth factor and hormone receptors. PELP1 signaling ultimately plays a role in cancer biology by increasing proliferation and metastasis, among other cellular processes. Here we will review (1) the cloning and characterization of PELP1 expression, (2) interacting proteins, (3) PELP1 signaling, and (4) PELP1-mediated biology., (Copyright © 2013 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Progesterone receptors (PR) are transcription factors relevant to breast cancer biology. Herein, we describe an N-terminal common docking (CD) domain in PR-B, a motif first described in mitogen-activated protein kinases. Binding studies revealed PR-B interacts with dual-specificity phosphatase 6 (DUSP6) via the CD domain. Mutation of the PR-B CD domain (mCD) attenuated cell cycle progression and expression of PR-B target genes (including STAT5A and Wnt1); mCD PR-B failed to undergo phosphorylation on Ser81, a ck2-dependent site required for expression of these genes. PR-B Ser81 phosphorylation was dependent on binding with DUSP6 and required for recruitment of a transcriptional complex consisting of PR-B, DUSP6 and ck2 to an enhancer region upstream of the Wnt1 promoter. STAT5 was present at this site in the absence or presence of progestin. Furthermore, phospho-Ser81 PR-B was recruited to the STAT5A gene upon progestin treatment, suggestive of a feed-forward mechanism. Inhibition of JAK/STAT-signaling blocked progestin-induced STAT5A and Wnt1 expression. Our studies show that DUSP6 serves as a scaffold for ck2-dependent PR-B Ser81 phosphorylation and subsequent PR-B-specific gene selection in coordination with STAT5. Coregulation of select target genes by PR-B and STAT5 is likely a global mechanism required for growth promoting programs relevant to mammary stem cell biology and cancer.

Basal-type triple-negative breast cancers (TNBC) are aggressive and difficult to treat relative to luminal-type breast cancers. TNBC often express abundant Met receptors and are enriched for transcriptional targets regulated by hypoxia-inducible factor-1α (HIF-1α), which independently predict cancer relapse and increased risk of metastasis. Brk/PTK6 is a critical downstream effector of Met signaling and is required for hepatocyte growth factor (HGF)-induced cell migration. Herein, we examined the regulation of Brk by HIFs in TNBC in vitro and in vivo. Brk mRNA and protein levels are upregulated strongly in vitro by hypoxia, low glucose, and reactive oxygen species. In HIF-silenced cells, Brk expression relied upon both HIF-1α and HIF-2α, which we found to regulate BRK transcription directly. HIF-1α/2α silencing in MDA-MB-231 cells diminished xenograft growth and Brk reexpression reversed this effect. These findings were pursued in vivo by crossing WAP-Brk (FVB) transgenic mice into the MET(Mut) knockin (FVB) model. In this setting, Brk expression augmented MET(Mut)-induced mammary tumor formation and metastasis. Unexpectedly, tumors arising in either MET(Mut) or WAP-Brk × MET(Mut) mice expressed abundant levels of Sik, the mouse homolog of Brk, which conferred increased tumor formation and decreased survival. Taken together, our results identify HIF-1α/2α as novel regulators of Brk expression and suggest that Brk is a key mediator of hypoxia-induced breast cancer progression. Targeting Brk expression or activity may provide an effective means to block the progression of aggressive breast cancers., (©2013 AACR.)

Objectives: The aim of this study is to determine a set of MRI lymph node characteristics predictive for extranodal tumor spread (ENS) in head and neck cancer patients., Methods: In 39 patients, 60 lymph nodes with on MRI a minimal axial diameter of more than 1cm or an inhomogeneous enhancement were studied. Two radiologists evaluated all MR-images for findings potentially indicative of the presence of ENS. Sensitivity, specificity and odds ratios based on logistic regression were calculated., Results: On MR-imaging, 20 lymph nodes were staged positive for ENS. On histopathology, 30 nodes were positive for ENS. In total, 14 nodes (23%) were scored differently on MR-imaging and histopathology. The MR-finding "infiltration of adjacent planes" established a specificity of 100% (lower 90% confidence bound: 91%) and sensitivity of 50% (95% confidence interval [CI]: 28-72%)., Conclusion: The MRI finding "infiltration of adjacent planes" may be high enough (100% in our study) to be used for treatment planning., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Loss of nuclear progesterone receptors (PR) and low circulating progesterone levels are associated with increased ovarian cancer (OC) risk. However, PR are abundantly expressed in a significant percentage of serous and endometrioid ovarian tumors; patients with PR+ tumors typically experience longer progression-free survival relative to those with PR-null tumors. The molecular mechanisms of these protective effects are poorly understood. To study PR action in OC in the absence of added estrogen (i.e., needed to induce robust PR expression), we created ES-2 OC cells stably expressing vector control or GFP-tagged PR-B (GFP-PR). Progestin (R5020) stimulation of ES-2 cells stably expressing GFP-PR induced cellular senescence characterized by altered cellular morphology, prolonged survival, senescence-associated β-galactosidase activity, G1 cell cycle arrest and upregulation of the cell cycle inhibitor, p21, as well as the Forkhead-box transcription factor, FOXO1; these results repeated in unmodified ER+/PR+ PEO4 OC cells. PR-B and FOXO1 were detected within the same PRE-containing regions of the p21 upstream promoter. Knockdown of p21 resulted in molecular compensation via FOXO1-dependent upregulation of numerous FOXO1 target genes (p15, p16, p27) and an increased rate of senescence. Inhibition of FOXO1 (with AS1842856) or stable FOXO1 knockdown inhibited progestin-induced p21 expression and blocked progestin-induced senescence. Overall, these findings support a role for PR as a tumor suppressor in OC cells, which exhibits inhibitory effects by inducing FOXO1-dependent cellular senescence. Clinical "priming" of the PR-FOXO1-p21 signaling pathway using PR agonists may provide a useful strategy to induce irreversible cell cycle arrest and thereby sensitize OC cells to existing chemotherapies as part of combination "two-step" therapies.

Background: Tumor volume is a significant prognostic factor in the treatment of malignant head and neck tumors. Unfortunately, it is not routinely measured because of the workload involved., Methods: Twenty-one patients, between 2009 and 2010, were studied. Dynamic contrast-enhanced MRI (DCE-MRI) at 3.0T was performed. A workstation previously developed for semi-automated segmentation of breast cancers on DCE-MRI was used to segment the head and neck cancers. The Pearson correlation analysis was used to assess the agreement between volumetric measurements and the manually derived gross tumor volume (GTV)., Results: In 90.5% of the patients (19 of 21) correlation could be made between DCE-MRI and the manually derived GTV. The Pearson correlation coefficient between the automatically derived tumor volume at DCE-MRI and the manually derived GTVs was R(2) = 0.95 (p < .001)., Conclusion: Semi-automated tumor volumes on DCE-MRI were representative of those derived from the manually derived GTV (R(2) = 0.95; p < .001)., (Copyright © 2012 Wiley Periodicals, Inc.)

Purpose: Two noninvasive delivery strategies for VEGF/PDGF receptor tyrosine kinase inhibitors (RTKI) were explored that exploited uveal retention as a means for establishing an ocular drug depot: a single oral "loading" dose and topical administration., Methods: Melanin binding was confirmed by centrifugation and mass spectrometry. Ocular retention was examined in pigmented and albino rats. Ocular release kinetics were measured 3 to 28 days postdosing in pigmented rats. Microautoradiography was used to demonstrate retention of RTKI in the uveal tract. A uveal drug depot of pazopanib was created by a single oral dose prior to induction of laser choroidal neovascularization (CNV). Choroid/retinal pigmented epithelium (RPE) retention of a related RTKI with enhanced topical bioavailability, GW771806, was confirmed by bioanalytics, and its ability to regress CNV compared with pazopanib., Results: Pazopanib and GW771806 directly bound melanin and were retained within the uveal tract of pigmented rats for weeks following a single oral dose. Pazopanib was undetectable systemically following a single oral administration prior to CNV induction, and reduced CNV as well as twice daily dosing. Topical ocular delivery of GW771806 at 5 mg/mL led to high choroidal/RPE exposure and significantly regressed CNV lesions; 2 mg/mL prevented lesion progression., Conclusions: Uveal retention of drugs such as pazopanib can be used to create a sustained-release depot. Topical GW771806 regressed CNV. These data indicate that topical or infrequent oral loading dose treatment with VEGF/PDGF RTKI retained in the choroid/RPE might allow noninvasive treatments for ocular neovascular disease.

Objective. This study was conducted to assess the value of CT and MR imaging in the preoperative evaluation of ICA encasement. Methods. Based upon three patient groups this study was performed. Retrospective analysis of 260 neck dissection reports from 2001 to 2010 was performed to determine unexpected peroperative-diagnosed encasement. Two experienced head and neck radiologists reviewed 12 scans for encasement. Results. In four out of 260 (1.5%) patients undergoing neck dissection, preoperative imaging was false negative as there was peroperative encasement of the ICA. Of 380 patients undergoing preoperative imaging, the radiologist reported encasement of the ICA in 25 cases. In 342 cases no encasement was described, 125 of these underwent neck dissection, and 2 had encasement peroperatively. The interobserver variation kappa varied from 0.273 to 1 for the different characteristics studied. Conclusion. These retrospectively studied cohorts demonstrate that preoperative assessment of encasement of the ICA using MRI and/or CT was of value in evaluation of ICA encasement and therefore contributively in selecting operable patients (without ICA encasement), since in only 1.5% encasement was missed. However, observer variation affects the reliability of this feature.