Jessica Finlay-Schultz

Progestin suppression of miR-29 potentiates dedifferentiation of breast cancer cells via KLF4

The female hormone progesterone (P4) promotes the expansion of stem-like cancer cells in estrogen receptor (ER)- and progesterone receptor (PR)-positive breast tumors. The expanded tumor cells lose expression of ER and PR, express the tumor-initiating marker CD44, the progenitor marker cytokeratin 5 (CK5) and are more resistant to standard endocrine and chemotherapies. The mechanisms underlying this hormone-stimulated reprogramming have remained largely unknown. In the present study, we investigated the role of microRNAs in progestin-mediated expansion of this dedifferentiated tumor cell population. We demonstrate that P4 rapidly downregulates miR-29 family members, particularly in the CD44+ cell population. Downregulation of miR-29 members potentiates the expansion of CK5+ and CD44+ cells in response to progestins, and results in increased stem-like properties in vitro and in vivo. We demonstrate that miR-29 directly targets Krüppel-like factor 4 (KLF4), a transcription factor required for the reprogramming of differentiated cells to pluripotent stem cells, and for the maintenance of breast cancer stem cells. These results reveal a novel mechanism, whereby progestins increase the stem cell-like population in hormone-responsive breast cancers, by decreasing miR-29 to augment PR-mediated upregulation of KLF4. Elucidating the mechanisms whereby hormones mediate the expansion of stem-like cells furthers our understanding of the progression of hormone-responsive breast cancers.

Progesterone downregulation of miR-141 contributes to expansion of stem-like breast cancer cells through maintenance of progesterone receptor and Stat5a

Progesterone (P4) has emerged as an important hormone-regulating mammary stem cell (MaSC) populations. In breast cancer, P4 and synthetic analogs increase the number of stem-like cells within luminal estrogen receptor (ER)- and progesterone receptor (PR)-positive breast cancers. These cells gain expression of de-differentiated cell markers CD44 and cytokeratin 5 (CK5), lose luminal markers ER and PR, and are more therapy resistant. We previously described that P4 downregulation of microRNA (miR)-29a contributes to the expansion of CD44high and CK5+ cells. Here we investigated P4 downregulation of miR-141, a member of the miR-200 family of tumor suppressors, in facilitating an increase in stem-like breast cancer cells. miR-141 was the sole member of the miR-200 family P4-downregulated at the mature miRNA level in luminal breast cancer cell lines. Stable inhibition of miR-141 alone increased the CD44high population, and potentiated P4-mediated increases in both CD44high and CK5+ cells. Loss of miR-141 enhanced both mammosphere formation and tumor initiation. miR-141 directly targeted both PR and signal transducer and activator of transcription 5A (Stat5a), transcription factors important for MaSC expansion. miR-141 depletion increased PR protein levels, even in cell lines where PR expression is estrogen dependent. Stat5a suppression via small interfering RNA or a small-molecule inhibitor reduced the P4-dependent increase in CK5+ and CD44high cells. These data support a mechanism by which P4-triggered loss of miR-141 facilitates breast cancer cell de-differentiation through deregulation of PR and Stat5a, two transcription factors important for controlling mammary cell fate.

Fibroblast Subtypes Regulate Responsiveness of Luminal Breast Cancer to Estrogen

Purpose: Antiendocrine therapy remains the most effective treatment for estrogen receptor–positive (ER+) breast cancer, but development of resistance is a major clinical complication. Effective targeting of mechanisms that control the loss of ER dependency in breast cancer remains elusive. We analyzed breast cancer–associated fibroblasts (CAF), the largest component of the tumor microenvironment, as a factor contributing to ER expression levels and antiendocrine resistance. Experimental Design: Tissues from patients with ER+ breast cancer were analyzed for the presence of CD146-positive (CD146pos) and CD146-negative (CD146neg) fibroblasts. ER-dependent proliferation and tamoxifen sensitivity were evaluated in ER+ tumor cells cocultured with CD146pos or CD146neg fibroblasts. RNA sequencing was used to develop a high-confidence gene signature that predicts for disease recurrence in tamoxifen-treated patients with ER+ breast cancer. Results: We demonstrate that ER+ breast cancers contain two CAF subtypes defined by CD146 expression. CD146neg CAFs suppress ER expression in ER+ breast cancer cells, decrease tumor cell sensitivity to estrogen, and increase tumor cell resistance to tamoxifen therapy. Conversely, the presence of CD146pos CAFs maintains ER expression in ER+ breast cancer cells and sustains estrogen-dependent proliferation and sensitivity to tamoxifen. Conditioned media from CD146pos CAFs with tamoxifen-resistant breast cancer cells are sufficient to restore tamoxifen sensitivity. Gene expression profiles of patient breast tumors with predominantly CD146neg CAFs correlate with inferior clinical response to tamoxifen and worse patient outcomes. Conclusions: Our data suggest that CAF composition contributes to treatment response and patient outcomes in ER+ breast cancer and should be considered a target for drug development. Clin Cancer Res; 23(7); 1710–21. ©2016 AACR.

Steroid Hormones, Steroid Receptors, and Breast Cancer Stem Cells

The ovarian hormones progesterone and estrogen play important roles in breast cancer etiology, proliferation, and treatment. Androgens may also contribute to breast cancer risk and progression. In recent years, significant advances have been made in defining the roles of these steroid hormones in stem cell homeostasis in the breast. Stem cells are potential origins of breast cancer and may dictate tumor phenotype. At least a portion of breast cancers are proposed to be driven by cancer stem cells (CSCs), cells that mimic the self-renewing and repopulating properties of normal stem cells, and can confer drug resistance. Progesterone has been identified as the critical hormone regulating normal murine mammary stem cell (MaSC) populations and normal human breast stem cells. Synthetic progestins increase human breast cancer risk; one theory speculates that this occurs through increased stem cells. Progesterone treatment also increases breast CSCs in established breast cancer cell lines. This is mediated in part through progesterone regulation of transcription factors, signal transduction pathways, and microRNAs. There is also emerging evidence that estrogens and androgens can regulate breast CSC numbers. The evolving concept that a breast CSC phenotype is dynamic and can be influenced by cell signaling and external cues emphasizes that steroid hormones could be crucial players in controlling CSC number and function. Here we review recent studies on steroid hormone regulation of breast CSCs, and discuss mechanisms by which this occurs.

Transcriptional repression of the α7 nicotinic acetylcholine receptor subunit gene (CHRNA7) by activating protein-2α (AP-2α).

Background: Transcription of the α7 nicotinic receptor gene (CHRNA7) is decreased in schizophrenia. Results: Activating protein-2α (AP-2α) is a potent repressor of CHRNA7 transcription. Conclusion: AP-2α may contribute to the decreased expression of the CHRNA7 gene in schizophrenia. Significance: This is the first report of a transcriptional repressor in the human CHRNA7 gene. The CHRNA7 gene, which encodes the α7 nicotinic acetylcholine receptor (α7*nAChR), has been implicated as a candidate gene in schizophrenia. Expression of the α7*nAChR mRNA and protein are reduced in multiple regions of post-mortem brain from patients diagnosed with schizophrenia. Transcriptional regulation may therefore be an important mechanism for the regulation of this gene. A 230-bp proximal promoter fragment, necessary for transcription in cultured neuroblastoma cells, was used to study a putative AP-2α binding site. Mutation of the site indicates that AP-2α plays a negative role in regulating CHRNA7 transcription. This was confirmed through knockdown and overexpression of AP-2α. Electrophoretic mobility shift assays (EMSAs) identified positive DNA-protein interaction at this same site, and supershift assays indicate that the complex includes AP-2α. The interaction was confirmed in cells using chromatin immunoprecipitation (ChIP). DNA methylation was discovered as an anomalous mechanism for CHRNA7 regulation in one cell line. These studies suggest a role for AP-2α regulation of CHRNA7 mRNA expression in multiple tissues during development.

Breast cancer suppression by progesterone receptors is mediated by their modulation of estrogen receptors and RNA polymerase III

Greater than 50% of estrogen receptor (ER)-positive breast cancers coexpress the progesterone receptor (PR), which can directly and globally modify ER action to attenuate tumor growth. However, whether this attenuation is mediated only through PR-ER interaction remains unknown. To address this question, we assessed tumor growth in ER/PR-positive patient-derived xenograft models of breast cancer, where both natural and synthetic progestins were found to antagonize the mitogenic effects of estrogens. Probing the genome-wide mechanisms by which this occurs, we documented that chronic progestin treatment blunted ER-mediated gene expression up to 2-fold at the level of mRNA transcripts. Unexpectedly, <25% of all ER DNA binding events were affected by the same treatment. The PR cistrome displayed a bimodal distribution. In one group, >50% of PR binding sites were co-occupied by ER, with a propensity for both receptors to coordinately gain or lose binding in the presence of progesterone. In the second group, PR but not ER was associated with a large fraction of RNA polymerase III-transcribed tRNA genes, independent of hormone treatment. Notably, we discovered that PR physically associated with the Pol III holoenzyme. Select pre-tRNAs and mature tRNAs with PR and POLR3A colocalized at their promoters were relatively decreased in estrogen + progestin-treated tumors. Our results illuminate how PR may indirectly impede ER action by reducing the bioavailability of translational molecules needed for tumor growth. Cancer Res; 77(18); 4934-46. ©2017 AACR.

Promoter methylation and tissue-specific transcription of the α7 nicotinic receptor gene, CHRNA7.

The α7 nicotinic acetylcholine receptor is known to regulate a wide variety of developmental and secretory functions in neural and non-neural tissues. The mechanisms that regulate its transcription in these varied tissues are not well understood. Epigenetic processes may play a role in the tissue-specific regulation of mRNA expression from the α7 nicotinic receptor subunit gene, CHRNA7. Promoter methylation was correlated with CHRNA7 mRNA expression in various tissue types and the role of DNA methylation in regulating transcription from the gene was tested by using DNA methyltransferase (DNMT1) inhibitors and methyl donors. CHRNA7 mRNA expression was silenced in SH-EP1 cells and bisulfite sequencing PCR revealed the CHRNA7 proximal promoter was hypermethylated. The proximal promoter was hypomethylated in the cell lines HeLa, SH-SY5Y, and SK-N-BE which express varying levels of CHRNA7 mRNA. Expression of CHRNA7 mRNA was present in SH-EP1 cells after treatment with the methylation inhibitor, 5-aza-2-deoxycytidine (5-Aza-CdR), and increased in SH-EP1 and HeLa cells using another methylation inhibitor, zebularine (ZEB). Transcription from the CHRNA7 promoter in HeLa cells was increased when the methyl donor methionine (MET) was absent from the media. Using methylation-sensitive restriction enzyme analysis (MSRE), there was a strong inverse correlation between CHRNA7 mRNA levels and promoter DNA methylation across several human tissue types. The results support a role for DNA methylation of the proximal promoter in regulation of CHRNA7 transcription.

Abstract P3-05-03: A novel progesterone receptor (PR)-RNA polymerase III association represses estrogen-dependent growth in breast tumor patient-derived xenografts

Background: Progesterone (P) is an important hormone for development and normal function of the breast; however, its role in established breast cancers is less clear. P has been implicated in regulating tumor cell growth, signaling, differentiation state, and stem/progenitor properties in breast cancer cells. Progesterone receptors (PRs) are considered positive prognostic indicators yet potential targets for treatment, although the dilemma of positive or negative targeting persists. P can either inhibit or stimulate breast cancer cell growth in the absence of estrogens (E), and usually blocks E mediated growth. Recent studies using breast cancer cell lines have deciphered a mechanism by which P suppresses E dependent growth through modulation of the estrogen receptor (ER) cistrome through a physical ER/PR association. However, the ER/PR association is weak to undetectable in our ER+PR+ breast cancer patient-derived xenografts (PDX). We therefore hypothesize that PR can regulate E-dependent breast cancer growth independent of direct interference of ER transcription. The purpose of this study was to use unbiased genomics and proteomics of breast cancer PDX to uncover additional mechanisms of P repression of breast cancer growth. Methods: These studies used two luminal ER+PR+ PDX (UCD4 and UCD65) that contain high levels of ER and PR (>90%), and where P inhibits E-dependent growth. Tumors were grown in vivo in female NSG mice under continuous placebo, E (17b-estradiol), E plus P, or E plus the synthetic progestin medroxyprogesterone acetate (MPA) for 8-10 weeks. RNAseq, chromatin immunoprecipitation sequencing (ChIPseq), and rapid immunoprecipitation followed by mass spectrometry of endogenous proteins (RIME) were performed to analyze differential transcriptional, cistromic, and protein-protein interactions in E compared to E plus P or MPA treated tumors. Co-immunoprecipitation (co-IP) and ChIP were used to verify results. Results: Both P and MPA potently inhibited E-dependent growth of both tumor lines. Gene expression studies found that both hormones reversed transcription of over one third of the estrogen/ER transcriptome in both tumors. RIME for PR uncovered significant interactions between PR and multiple RNA polymerase III subunits (POLR3). Co-IP using POLR3A and POLR3B confirmed PR associates with the POLR3 holoenzyme. Furthermore, ChIPseq revealed that PR binds to one third of POLR3 regulated tRNA genes. PR also associated with Maf1 in one tumor, a known POLR3 suppressor. Conclusions: Here we describe a novel association of PR with POLR3 in two luminal breast cancer PDX, an interaction not described in breast cancer cell lines. Our data suggest this is a negative regulatory interaction that may occur through recruiting a POLR3 repressor. These data implicate multifaceted P control of E dependent tumor growth; in addition to antagonizing E regulated genes at the transcription level, P can regulate translation though depletion of amino acid carrying tRNAs, thus slowing protein synthesis. Identifying which tumors utilize this growth-suppressive mechanism may pinpoint appropriate candidates for progestin therapy and/or provide a prognostic tool for predicting tumor progression. Citation Format: Finlay-Schultz J, Gillen AE, Brechbuhl HM, Ivie J, Bentley DL, Kabos P, Sartorius CA. A novel progesterone receptor (PR)-RNA polymerase III association represses estrogen-dependent growth in breast tumor patient-derived xenografts [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-05-03.

Cytokeratin 5-Positive Cells Represent a Therapy Resistant subpopulation in Epithelial Ovarian Cancer.

Objective Cytokeratin 5 (CK5) is an epithelial cell marker implicated in stem and progenitor cell activity in glandular reproductive tissues and endocrine and chemotherapy resistance in estrogen receptor (ER)+ breast cancer. The goal of this study was to determine the prevalence of CK5 expression in ovarian cancer and the response of CK5+ cell populations to cisplatin therapy. Materials and Methods Cytokeratin 5 expression was evaluated in 2 ovarian tissue microarrays, representing 137 neoplasms, and 6 ovarian cancer cell lines. Cell lines were treated with IC50 (half-maximal inhibitory concentration) cisplatin, and the prevalence of CK5+ cells pretreatment and posttreatment was determined. Proliferation of CK5+ versus CK5− cell populations was determined using 5-bromo-2′-deoxyuridine incorporation. Chemotherapy-induced apoptosis in CK5+ versus CK5− cells was measured using immunohistochemical staining for cleaved caspase-3. Results Cytokeratin 5 was expressed in 39.3% (42 of 107) of epithelial ovarian cancers with a range of 1% to 80% positive cells. Serous and endometrioid histologic subtypes had the highest percentage of CK5+ specimens. Cytokeratin 5 expression correlated with ER positivity (38 of 42 CK5+ tumors were also ER+). Cytokeratin 5 was expressed in 5 of 6 overall and 4 of 4 ER+ epithelial ovarian cancer cell lines ranging from 2.4% to 52.7% positive cells. Cytokeratin 5+ compared with CK5− cells were slower proliferating. The prevalence of CK5+ cells increased after 48-hour cisplatin treatment in 4 of 5 cell lines tested. Cytokeratin 5+ ovarian cancer cells compared with CK5− ovarian cancer cells were more resistant to cisplatin-induced apoptosis. Conclusions Cytokeratin 5 is expressed in a significant proportion of epithelial ovarian cancers and represents a slower proliferating chemoresistant subpopulation that may warrant cotargeting in combination therapy.

Recovery and analysis of transcriptome subsets from pooled single-cell RNA-seq libraries

Single-cell RNA sequencing (scRNA-seq) methods generate sparse gene expression profiles for thousands of single cells in a single experiment. The information in these profiles is sufficient to classify cell types by distinct expression patterns but the high complexity of scRNA-seq libraries often prevents full characterization of transcriptomes from individual cells. To extract more focused gene expression information from scRNA-seq libraries, we developed a strategy to physically recover the DNA molecules comprising transcriptome subsets, enabling deeper interrogation of the isolated molecules by another round of DNA sequencing. We applied the method in cell-centric and gene-centric modes to isolate cDNA fragments from scRNA-seq libraries. First, we resampled the transcriptomes of rare, single megakaryocytes from a complex mixture of lymphocytes and analyzed them in a second round of DNA sequencing, yielding up to 20-fold greater sequencing depth per cell and increasing the number of genes detected per cell from a median of 1,313 to 2,002. We similarly isolated mRNAs from targeted T cells to improve the reconstruction of their VDJ-rearranged immune receptor mRNAs. Second, we isolated CD3D mRNA fragments expressed across cells in a scRNA-seq library prepared from a clonal T cell line, increasing the number of cells with detected CD3D expression from 59.7% to 100%. Transcriptome resampling is a general approach to recover targeted gene expression information from single-cell RNA sequencing libraries that enhances the utility of these costly experiments, and may be applicable to the targeted recovery of molecules from other single-cell assays.