Co-evolution of AR gene copy number and structural complexity in endocrine therapy resistant prostate cancer.

Androgen receptor (AR) inhibition is standard of care for advanced prostate cancer (PC). However, efficacy is limited by progression to castration-resistant PC (CRPC), usually due to AR re-activation via mechanisms that include AR amplification and structural rearrangement. These two classes of AR alterations often co-occur in CRPC tumors, but it is unclear whether this reflects intercellular or intracellular heterogeneity of AR. Resolving this is important for developing new therapies and predictive biomarkers. Here, we analyzed 41 CRPC tumors and 6 patient-derived xenografts (PDXs) using linked-read DNA-sequencing, and identified 7 tumors that developed complex, multiply-rearranged AR gene structures in conjunction with very high AR copy number. Analysis of PDX models by optical genome mapping and fluorescence in situ hybridization showed that AR residing on extrachromosomal DNA (ecDNA) was an underlying mechanism, and was associated with elevated levels and diversity of AR expression. This study identifies co-evolution of AR gene copy number and structural complexity via ecDNA as a mechanism associated with endocrine therapy resistance.

Receptor for hyaluronan-mediated motility (RHAMM) defines an invasive niche associated with tumor progression and predicts poor outcomes in breast cancer patients.

Breast cancer invasion and metastasis result from a complex interplay between tumor cells and the tumor microenvironment (TME). Key oncogenic changes in the TME include aberrant synthesis, processing, and signaling of hyaluronan (HA). Hyaluronan-mediated motility receptor (RHAMM, CD168; HMMR) is an HA receptor enabling tumor cells to sense and respond to this aberrant TME during breast cancer progression. Previous studies have associated RHAMM expression with breast tumor progression; however, cause and effect mechanisms are incompletely established. Focused gene expression analysis of an internal breast cancer patient cohort confirmed that increased RHAMM expression correlates with aggressive clinicopathological features. To probe mechanisms, we developed a novel 27-gene RHAMM-related signature (RRS) by intersecting differentially expressed genes in lymph node (LN)-positive patient cases with the transcriptome of a RHAMM-dependent model of cell transformation, which we validated in an independent cohort. We demonstrate that the RRS predicts for poor survival and is enriched for cell cycle and TME-interaction pathways. Further analyses using CRISPR/Cas9-generated RHAMM-/- breast cancer cells provided direct evidence that RHAMM promotes invasion in vitro and in vivo. Immunohistochemistry studies highlighted heterogeneous RHAMM protein expression, and spatial transcriptomics associated the RRS with RHAMM-high microanatomic foci. We conclude that RHAMM upregulation leads to the formation of 'invasive niches', which are enriched in RRS-related pathways that drive invasion and could be targeted to limit invasive progression and improve patient outcomes. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Nuclear corepressors NCOR1/NCOR2 regulate B cell development, maintain genomic integrity and prevent transformation.

The nuclear corepressors NCOR1 and NCOR2 interact with transcription factors involved in B cell development and potentially link these factors to alterations in chromatin structure and gene expression. Herein, we demonstrate that Ncor1/2 deletion limits B cell differentiation via impaired recombination, attenuates pre-BCR signaling and enhances STAT5-dependent transcription. Furthermore, NCOR1/2-deficient B cells exhibited derepression of EZH2-repressed gene modules, including the p53 pathway. These alterations resulted in aberrant Rag1 and Rag2 expression and accessibility. Whole-genome sequencing of Ncor1/2 DKO B cells identified increased number of structural variants with cryptic recombination signal sequences. Finally, deletion of Ncor1 alleles in mice facilitated leukemic transformation, whereas human leukemias with less NCOR1 correlated with worse survival. NCOR1/2 mutations in human leukemia correlated with increased RAG expression and number of structural variants. These studies illuminate how the corepressors NCOR1/2 regulate B cell differentiation and provide insights into how NCOR1/2 mutations may promote B cell transformation.

Combining nilotinib and PD-L1 blockade reverses CD4+ T-cell dysfunction and prevents relapse in acute B-cell leukemia.

Patients with acute lymphoblastic leukemia have experienced significantly improved outcomes due to the advent of chimeric antigen receptor (CAR) T cells and bispecific T-cell engagers, although a proportion of patients still relapse despite these advances. T-cell exhaustion has been recently suggested to be an important driver of relapse in these patients. Indeed, phenotypic exhaustion of CD4+ T cells is predictive of relapse and poor overall survival in B-cell acute lymphoblastic leukemia (B-ALL). Thus, therapies that counter T-cell exhaustion, such as immune checkpoint blockade, may improve leukemia immunosurveillance and prevent relapse. Here, we used a murine model of Ph+ B-ALL as well as human bone marrow biopsy samples to assess the fundamental nature of CD4+ T-cell exhaustion and the preclinical therapeutic potential for combining anti-PD-L1 based checkpoint blockade with tyrosine kinase inhibitors targeting the BCR-ABL oncoprotein. Single-cell RNA-sequence analysis revealed that B-ALL induces a unique subset of CD4+ T cells with both cytotoxic and helper functions. Combination treatment with the tyrosine kinase inhibitor nilotinib and anti-PD-L1 dramatically improves long-term survival of leukemic mice. Depletion of CD4+ T cells prior to therapy completely abrogates the survival benefit, implicating CD4+ T cells as key drivers of the protective anti-leukemia immune response. Indeed, treatment with anti-PD-L1 leads to clonal expansion of leukemia-specific CD4+ T cells with the aforementioned helper/cytotoxic phenotype as well as reduced expression of exhaustion markers. These findings support efforts to use PD1/PD-L1 checkpoint blockade in clinical trials and highlight the importance of CD4+ T-cell dysfunction in limiting the endogenous anti-leukemia response.

Single-cell analysis identifies dynamic gene expression networks that govern B cell development and transformation.

Integration of external signals and B-lymphoid transcription factor activities organise B cell lineage commitment through alternating cycles of proliferation and differentiation, producing a diverse repertoire of mature B cells. We use single-cell transcriptomics/proteomics to identify differentially expressed gene networks across B cell development and correlate these networks with subtypes of B cell leukemia. Here we show unique transcriptional signatures that refine the pre-B cell expansion stages into pre-BCR-dependent and pre-BCR-independent proliferative phases. These changes correlate with reciprocal changes in expression of the transcription factor EBF1 and the RNA binding protein YBX3, that are defining features of the pre-BCR-dependent stage. Using pseudotime analysis, we further characterize the expression kinetics of different biological modalities across B cell development, including transcription factors, cytokines, chemokines, and their associated receptors. Our findings demonstrate the underlying heterogeneity of developing B cells and characterise developmental nodes linked to B cell transformation.

Characterizing Macrophage Diversity in Metastasis-Bearing Lungs Reveals a Lipid-Associated Macrophage Subset.

While macrophages are among the most abundant immune cell type found within primary and metastatic mammary tumors, how their complexity and heterogeneity change with metastatic progression remains unknown. Here, macrophages were isolated from the lungs of mice bearing orthotopic mammary tumors for single-cell RNA sequencing (scRNA-seq). Seven distinct macrophage clusters were identified, including populations exhibiting enhanced differential expression of genes related to antigen presentation (H2-Aa, Cd74), cell cycle (Stmn1, Cdk1), and interferon signaling (Isg15, Ifitm3). Interestingly, one cluster demonstrated a profile concordant with lipid-associated macrophages (Lgals3, Trem2). Compared with nontumor-bearing controls, the number of these cells per gram of tissue was significantly increased in lungs from tumor-bearing mice, with the vast majority costaining positively with the alveolar macrophage marker Siglec-F. Enrichment of genes implicated in pathways related to lipid metabolism as well extracellular matrix remodeling and immunosuppression was observed. In addition, these cells displayed reduced capacity for phagocytosis. Collectively, these findings highlight the diversity of macrophages present within metastatic lesions and characterize a lipid-associated macrophage subset previously unidentified in lung metastases. SIGNIFICANCE: scRNA-seq of macrophages isolated from lung metastases reveals extensive macrophage heterogeneity and identifies a novel subpopulation enriched for genes involved in lipid metabolism, extracellular matrix remodeling, and immunosuppression.

AR gene rearrangement analysis in liquid biopsies reveals heterogeneity in lethal prostate cancer.

Castration-resistant prostate cancer (CRPC) is driven by AR gene aberrations that arise during androgen receptor (AR)-targeted therapy. AR amplification and mutations have been profiled in circulating tumor cells (CTCs), but whether AR gene rearrangements can be assessed in CTCs is unknown. In this study, we leveraged CRPC cell lines with defined AR gene rearrangements to develop and validate a CTC DNA analysis approach that utilized whole genome amplification and targeted DNA-sequencing of AR and other genes important in CRPC. We tested the utility of this approach by analyzing matched CTC DNA and plasma cell-free DNA (cfDNA) from a case series of ten CRPC patients. One of ten CTC samples and two of ten cfDNA samples were positive for AR gene rearrangements. All AR gene rearrangements were discordant between matched liquid biopsy samples. One patient harbored separate AR gene rearrangements in CTC DNA and cfDNA, but concordant AR amplification and AR T878A mutation. This patient also displayed concordant loss of TP53 and PTEN, but the loss of RB1 in cfDNA only. The overall frequency of discordant alterations in these genes between matched CTC DNA and cfDNA was high. This study establishes the technical feasibility of analyzing structural rearrangements, mutations, and copy number variants in AR and other CRPC genes using two different sources of DNA from a single blood sample. Paired CTC DNA and cfDNA analysis may have utility for capturing the heterogeneity of genetic alterations in CRPC patients.

Erratum: Witschen, P.M., et al. Tumor Cell Associated Hyaluronan-CD44 Signaling Promotes Pro-Tumor Inflammation in Breast Cancer. Cancers 2020, 12, 1325.

The authors wish to make the following corrections to this paper [...].

Tumor Cell Associated Hyaluronan-CD44 Signaling Promotes Pro-Tumor Inflammation in Breast Cancer.

Cancer has been conceptualized as a chronic wound with a predominance of tumor promoting inflammation. Given the accumulating evidence that the microenvironment supports tumor growth, we investigated hyaluronan (HA)-CD44 interactions within breast cancer cells, to determine whether this axis directly impacts the formation of an inflammatory microenvironment. Our results demonstrate that breast cancer cells synthesize and fragment HA and express CD44 on the cell surface. Using RNA sequencing approaches, we found that loss of CD44 in breast cancer cells altered the expression of cytokine-related genes. Specifically, we found that production of the chemokine CCL2 by breast cancer cells was significantly decreased after depletion of either CD44 or HA. In vivo, we found that CD44 deletion in breast cancer cells resulted in a delay in tumor formation and localized progression. This finding was accompanied by a decrease in infiltrating CD206+ macrophages, which are typically associated with tumor promoting functions. Importantly, our laboratory results were supported by human breast cancer patient data, where increased HAS2 expression was significantly associated with a tumor promoting inflammatory gene signature. Because high levels of HA deposition within many tumor types yields a poorer prognosis, our results emphasize that HA-CD44 interactions potentially have broad implications across multiple cancers.

JAK/STAT inhibition in macrophages promotes therapeutic resistance by inducing expression of protumorigenic factors.

Tumor-associated macrophages contribute to tumor progression and therapeutic resistance in breast cancer. Within the tumor microenvironment, tumor-derived factors activate pathways that modulate macrophage function. Using in vitro and in vivo models, we find that tumor-derived factors induce activation of the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway in macrophages. We also demonstrate that loss of STAT3 in myeloid cells leads to enhanced mammary tumorigenesis. Further studies show that macrophages contribute to resistance of mammary tumors to the JAK/STAT inhibitor ruxolitinib in vivo and that ruxolitinib-treated macrophages produce soluble factors that promote resistance of tumor cells to JAK inhibition in vitro. Finally, we demonstrate that STAT3 deletion and JAK/STAT inhibition in macrophages increases expression of the protumorigenic factor cyclooxygenase-2 (COX-2), and that COX-2 inhibition enhances responsiveness of tumors to ruxolitinib. These findings define a mechanism through which macrophages promote therapeutic resistance and highlight the importance of understanding the impact of targeted therapies on the tumor microenvironment.

CDK11 Loss Induces Cell Cycle Dysfunction and Death of BRAF and NRAS Melanoma Cells.

Cyclin dependent kinase 11 (CDK11) is a protein kinase that regulates RNA transcription, pre-mRNA splicing, mitosis, and cell death. Targeting of CDK11 expression levels is effective in the experimental treatment of breast and other cancers, but these data are lacking in melanoma. To understand CDK11 function in melanoma, we evaluated protein and RNA levels of CDK11, Cyclin L1 and Cyclin L2 in benign melanocytes and BRAF- as well as NRAS-mutant melanoma cell lines. We investigated the effectiveness of reducing expression of this survival kinase using RNA interference on viability, clonal survival, and tumorsphere formation in melanoma cell lines. We examined the impact of CDK11 loss in BRAF-mutant melanoma on more than 700 genes important in cancer signaling pathways. Follow-up analysis evaluated how CDK11 loss alters cell cycle function in BRAF- and NRAS-mutant melanoma cells. We present data on CDK11, CCNL1 and CCNL2 mRNA expression in melanoma patients, including prognosis for survival. In sum, we found that CDK11 is necessary for melanoma cell survival, and a major impact of CDK11 loss in melanoma is to cause disruption of the cell cycle distribution with accumulation of G1- and loss of G2/M-phase cancer cells.

A porcine enterovirus G associated with enteric disease contains a novel papain-like cysteine protease.

Identification of unknown pathogens in pigs displaying enteric illness is difficult due to the large diversity of bacterial and viral species found within faecal samples. Current methods often require bacterial or viral isolation, or testing only a limited number of known species using quantitative PCR analysis. Herein, faeces from two 25-day-old piglets with diarrhoea from Texas, USA, were analysed by metagenomic next-generation sequencing to rapidly identify possible pathogens. Our analysis included a bioinformatics pipeline of rapid short-read classification and de novo genome assembly which resulted in the identification of a porcine enterovirus G (EV-G), a complete genome with substantial nucleotide differences (>30 %) among current sequences, and a novel non-structural protein similar in sequence to the Torovirus papain-like cysteine protease (PLpro). This discovery led to the identification and circulation of an EV-G with a novel PLpro in the USA that has not been previously reported.

Posttranslationally modified progesterone receptors direct ligand-specific expression of breast cancer stem cell-associated gene programs.

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.

Cytoplasmic Localization of Proline, Glutamic Acid, Leucine-rich Protein 1 (PELP1) Induces Breast Epithelial Cell Migration through Up-regulation of Inhibitor of κB Kinase ϵ and Inflammatory Cross-talk with Macrophages.

Cytoplasmic localization of proline, glutamic acid, leucine-rich protein 1 (PELP1) is observed in ∼40% of women with invasive breast cancer. In mouse models, PELP1 overexpression in the mammary gland leads to premalignant lesions and eventually mammary tumors. In preliminary clinical studies, cytoplasmic localization of PELP1 was seen in 36% of women at high risk of developing breast cancer. Here, we investigated whether cytoplasmic PELP1 signaling promotes breast cancer initiation in models of immortalized human mammary epithelial cells (HMECs). Global gene expression analysis was performed on HMEC lines expressing vector control, PELP1-wt, or mutant PELP1 in which the nuclear localization sequence was altered, resulting in cytoplasmic localization of PELP1 (PELP1-cyto). Global gene expression analysis identified that PELP1-cyto expression in HMECs induced NF-κB signaling pathways. Western blotting analysis of PELP1-cyto HMECs showed up-regulation of inhibitor of κB kinase ϵ (IKKϵ) and increased phosphorylation of the NF-κB subunit RelB. To determine whether secreted factors produced by PELP1-cyto HMECs promote macrophage activation, THP-1 macrophages were treated with HMEC-conditioned medium (CM). PELP1-cyto CM induced changes in THP-1 gene expression as compared with control cell CM. Double conditioned medium (DCM) from the activated THP-1 cells was then applied to HMECs to determine whether paracrine signaling from PELP1-cyto-activated macrophages could in turn promote migration of HMECs. PELP1-cyto DCM induced robust HMEC migration, which was reduced in DCM from PELP1-cyto HMECs expressing IKKϵ shRNA. Our findings suggest that cytoplasmic localization of PELP1 up-regulates pro-tumorigenic IKKϵ and secreted inflammatory signals, which through paracrine macrophage activation regulates the migratory phenotype associated with breast cancer initiation.

Breast Tumor Kinase (Brk/PTK6) Is Induced by HIF, Glucocorticoid Receptor, and PELP1-Mediated Stress Signaling in Triple-Negative Breast Cancer.

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.

Active FOXO1 Is a Key Determinant of Isoform-Specific Progesterone Receptor Transactivation and Senescence Programming.

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.

Progesterone action in breast, uterine, and ovarian cancers.

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.

Progesterone receptor-cyclin D1 complexes induce cell cycle-dependent transcriptional programs in breast cancer cells.

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.

Tracking progesterone receptor-mediated actions in breast cancer.

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.

A Common Docking Domain in Progesterone Receptor-B links DUSP6 and CK2 signaling to proliferative transcriptional programs in breast cancer cells.

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.