Basal-like Cells Research Articles

Acetylated a-tubulin sense the change of extracellular matrix to regulate the invasion and metastasis of breast cancer

Objective To reveal acetylated a-tubulin acts as pressure sensors, sensing the changes in extracellular matrix to impact on the invasion and metastasis of breast cancer. Methods The acetylated alpha microtubule protein expressions were detected, and its relationship with invasion and metastasis in breast cancer clinical specimens and different molecular subtypes of breast cancer cell lines were analyzed. The expression of acetylated a-tubulin was interfered through chemical methods, and cell morphology and the change of the invasion and metastasis ability were detected under the condition of different matrix hardness. Results The expression of acetylated a-tubulin was highest in basal-like breast cancer tissue and cell lines, and was lowest expressed in the luminal B breast cancer tissue and the breast cancer cell lines. The expression of acetylated a-microtubule was positively correlated with the occurrence of breast cancer. Under the condition of soft substrate cultivation, cell polarization was declined, becoming the circular or oval shape. The acetylated a-tubulin caused reduction in cell polarity, accompanied with less invasion and metastasis ability. Conclusions The acetylated a-tubulin acts as pressure sensors, sensing the sclerosis of extracellular matrix in the process of tumorigenesis, promoting invasion and metastasis of breast cancer. Key words: Tubulin/ME; Extracellular matrix/ME; Breast neoplasms; Neoplasm invasiveness; Neoplasm metastasis

Abstract P3-07-08: Molecular subtype-specific expression of long noncoding RNA regulates proliferation of basal-like breast cancer cells

Abstract Background: Basal-like breast cancer (BLBC) disproportionally affects younger women and African American women, displaying aggressive clinical behavior with poor outcomes. BLBC is difficult to treat due to its lack of defined molecular targets, and with current treatment, patients often develop metastatic disease. A growing body of evidence points to long noncoding (lnc) RNAs as mediators of tumor progression, suggesting a new class of targets in cancer therapy. While dynamic changes in lncRNA expression are observed across many types of cancer, our understanding of their role in cancer biology is limited. The aim of this study is to identify lncRNAs differentially expressed in BLBC and to characterize the functional features of candidate lncRNA. Methods: Breast tumors and normal breast tissues were recruited from the U of Chicago Breast Cancer Tissue Bank under IRB approved protocols. Microarray profiling was done using Human LncRNA Array v3 (Arraystar) containing 30,586 lncRNAs and 26,109 mRNAs. Non-poly(A) RNAs were included with ribo-zero RNA-seq (Illumina HiSeq 4000). Molecular subtype of breast tumors was determined by PAM50 intrinsic classifier. In addition, CpG methylation of lncRNA promoter regions was analyzed with TCGA HumanMethylation450 Array data from 588 breast tissues. We performed in vitro studies using antisense oligonucleotide (ASO) knockdown and CRISPR-on overexpression in BLBC cell lines. Phenotypic consequences of perturbations of specific lncRNA expression were assessed using proliferation, apoptosis, cell cycle and homologous recombination assays. Results: Microarray profiling of breast tissues from 30 African American women revealed a unique lncRNA signature in basal-like tumors compared to non-basal like tumors. Hundreds of lncRNAs were specifically expressed in BLBC, with >100-fold differences in some lncRNAs compared to tumors from other subtypes or normal breast tissues. Through statistical and in silico analyses we selected lncRNAs displaying increased expression in basal-like tumors (vs non-basal tumors) and BLBC cell lines (vs non-basal cell lines) with a trend of higher expression in cells isolated from women of African ancestry for functional studies. RNA-seq of 50 additional breast tissues recapitulated the distinct lncRNA clustering within the basal-like subtype. TCGA analysis of CpG islands in several lncRNA promoter regions revealed hypo-methylation when compared to non-BLBC, signifying subtype specific epigenetic regulation. ASO knockdown of candidate lncRNAs significantly increased apoptosis and decreased proliferation while CRISPR-on overexpression decreased cell sensitivity to doxorubicin treatment. Depletion of specific lncRNAs caused an increased proportion of cells in G2 phase of the cell cycle, indicating that overexpression of particular lncRNAs may contribute to aberrant cell cycle progression. Cis and trans-regulation of gene expression by lncRNAs is currently under investigation through RNA immunoprecipitation and genome-wide gene expression profiling. Conclusion: We propose that subtype specific lncRNAs function as onco-RNAs specific to BLBC, driving cellular proliferation and increasing resistance to DNA damaging chemotherapies through regulation of the cell cycle. Citation Format: Boatman SM, Han Y-J, Zhang J, Zheng Y, Yeh AC, Olopade OI. Molecular subtype-specific expression of long noncoding RNA regulates proliferation of basal-like breast cancer cells [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 P3-07-08.

SOX2 promotes lineage plasticity and antiandrogen resistance in TP53- and RB1-deficient prostate cancer.

Some cancers evade targeted therapies through a mechanism known as lineage plasticity, whereby tumor cells acquire phenotypic characteristics of a cell lineage whose survival no longer depends on the drug target. We use in vitro and in vivo human prostate cancer models to show that these tumors can develop resistance to the antiandrogen drug enzalutamide by a phenotypic shift from androgen receptor (AR)-dependent luminal epithelial cells to AR-independent basal-like cells. This lineage plasticity is enabled by the loss of TP53 and RB1 function, is mediated by increased expression of the reprogramming transcription factor SOX2, and can be reversed by restoring TP53 and RB1 function or by inhibiting SOX2 expression. Thus, mutations in tumor suppressor genes can create a state of increased cellular plasticity that, when challenged with antiandrogen therapy, promotes resistance through lineage switching.

Rb1 and Trp53 cooperate to suppress prostate cancer lineage plasticity, metastasis, and antiandrogen resistance.

Prostate cancer relapsing from antiandrogen therapies can exhibit variant histology with altered lineage marker expression, suggesting that lineage plasticity facilitates therapeutic resistance. The mechanisms underlying prostate cancer lineage plasticity are incompletely understood. Studying mouse models, we demonstrate that Rb1 loss facilitates lineage plasticity and metastasis of prostate adenocarcinoma initiated by Pten mutation. Additional loss of Trp53 causes resistance to antiandrogen therapy. Gene expression profiling indicates that mouse tumors resemble human prostate cancer neuroendocrine variants; both mouse and human tumors exhibit increased expression of epigenetic reprogramming factors such as Ezh2 and Sox2. Clinically relevant Ezh2 inhibitors restore androgen receptor expression and sensitivity to antiandrogen therapy. These findings uncover genetic mutations that enable prostate cancer progression; identify mouse models for studying prostate cancer lineage plasticity; and suggest an epigenetic approach for extending clinical responses to antiandrogen therapy.

Knockdown of CAVEOLIN-1 Sensitizes Human Basal-Like Triple-Negative Breast Cancer Cells to Radiation

Background/Aims: Triple-negative breast cancer (TNBC) is a high-risk breast cancer phenotype without specific targeted therapy options and is significantly associated with increased local recurrence in patients treated with radiotherapy. CAVEOLIN-1 (CAV-1)-mediated epidermal growth factor receptor (EGFR) nuclear translocation following irradiation promotes DNA repair and thus induces radiation resistance. In this study, we aimed to determine whether knockdown of CAV-1 enhances the radiosensitivity of basal-like TNBC cell lines and to explore the possible mechanisms. Methods: Western blotting was used to compare protein expression in a panel of breast cancer cell lines. Nuclear accumulation of EGFR as well as DNA repair and damage at multiple time points following irradiation with or without CAV-1 siRNA pretreatment were investigated using western blotting and confocal microscopy. The radiosensitizing effect of CAV-1 siRNA was evaluated using a clonogenic assay. Flowcytometry was performed to analyse cell apoptosis and cell cycle alteration. Results: We found that CAV-1 is over-expressed in basal-like TNBC cell lines and barely expressed in HER-2-positive cells; additionally, we observed that HER-2-positive cell lines are more sensitive to irradiation than basal-like TNBC cells. Our findings revealed that radiation-induced EGFR nuclear translocation was impaired by knockdown of CAV-1. In parallel, radiation-induced elevation of DNA repair proteins was also hampered by pretreatment with CAV-1 siRNA before irradiation. Silencing of CAV-1 also promoted DNA damage 24 h after irradiation. Colony formation assays verified that cells could be radiosensitized after knockdown of CAV-1. Furthermore, G₂/M cell cycle arrest and apoptosis enhancement may also contribute to the radiosensitizing effect of CAV-1 siRNA. Conclusion: Our results support the hypothesis that CAV-1 knockdown by siRNA causes increased radiosensitivity in basal-like TNBC cells. The mechanisms associated with this effect are reduced DNA repair through delayed CAV-1-associated EGFR nuclear accumulation and induction of G₂/M arrest and apoptosis through the combined effects of CAV-1 siRNA and radiation.

Targeting of PYK2 Synergizes with EGFR Antagonists in Basal-like TNBC and Circumvents HER3-Associated Resistance via the NEDD4-NDRG1 Axis.

Triple-negative breast cancer (TNBC) is a highly aggressive, heterogeneous disease with poor prognosis and no effective targeted therapies. EGFR is highly expressed in basal-like TNBC and is considered as a potential therapeutic target. However, EGFR targeting exerts only marginal clinical benefits, possibly due to activation of compensatory signaling pathways, which are frequently associated with HER3 upregulation. Here we show that concomitant targeting of EGFR and the nonreceptor tyrosine kinases PYK2/FAK synergistically inhibits the proliferation of basal-like TNBC cells in vitro and attenuates tumor growth in a mouse xenograft model. Dual targeting of EGFR and PYK2/FAK inhibited complementary key growth and survival pathways mediated by AKT, S6K, STAT3, and ERK1/2 activation. PYK2 inhibition also abrogated HER3 upregulation in response to EGFR antagonists, thereby circumventing HER3-associated drug resistance. Mechanistically, PYK2 inhibition facilitated the proteasomal degradation of HER3 while inducing upregulation of NDRG1 (N-myc downstream regulated 1 gene). NDRG1 enhanced the interaction of HER3 with the ubiquitin ligase NEDD4, while PYK2, which interacts with NEDD4 and HER3, interfered with NEDD4-HER3 binding, suggesting that the PYK2-NDRG1-NEDD4 circuit has a critical role in receptor degradation, drug response, and resistance mechanism. Our studies offer a preclinical proof of concept for a strategy of cotargeting the EGFR and PYK2/FAK kinases to improve TNBC therapy. Cancer Res; 77(1); 86-99. ©2016 AACR.

FBP1 modulates cell metabolism of breast cancer cells by inhibiting the expression of HIF-1α.

This study aimed to investigate the function of fructose-1, 6-bisphosphatase 1 (FBP1) in regulating cell growth and metabolism through hypoxia-inducible factor 1α (HIF-1α)-dependent hypoxic response in breast cancer cells. Two human breast carcinoma cell lines, including luminal-like cell line MCF-7 and basal-like cell line MDA-MB-468, were cultured under hypoxia condition, then the expressions of FBP1 and HIF-1α were detected by western blotting. In addition, up-regulated FPB1 in MDA-MB-468 cells were induced by lentivirus. Next, cell growth, migration and glucose metabolism were evaluated by MTT assay, Transwell and commercial kits, as well as the expressions of HIF-1α target genes, including pyruvate dehydrogenase kinase 1 (PDK1), lactate dehydrogenase A (LDHA), glucose transporter 1 (GLUT1) and vascular endothelial growth factor (VEGF) were detected by RT-qPCR. Furthermore, chromatin immunoprecipitation was used to estimate whether the hypoxia response elements (HREs) of PDK1, LDHA, GLUT1 and VEGF promoters were incorporated with FBP1. FBP1 was downregulated in MDA-MB-468 cells compared with MCF-7 cells. Overexpression of FBP1 in MDA-MB-468 cells reduced cell growth (p < 0.05) and migration (p < 0.05) as well as glycose consumption (p < 0.05) and lactate production (p < 0.05). In addition, overexpressed FBP1 inhibited HIF-1α protein expression and the mRNA levels of PDK1, LDHA, GLUT1 and VEGF (p < 0.05) under hypoxia condition. Also, FBP1 was revealed to have a concrete connection with PDK1. This study reveal that overexpressed FBP1 may repress tumor growth, migration and glycolysis via targeting HIF-1α in BLBC.

Syringocystadenoma papilliferum in the middle ear

Syringocystadenoma papilliferum is a rare adnexal tumor of controversial origin that often occurs as a solitary tumor derived from apocrine and eccrine glands. It is more common in children and adolescents and affects the head and neck regions, although occurrences on other anatomical locations have been described. Routine H&E-stained sections revealed cystic epidermal invaginations with papillary projections. The superficial portions of the cyst were lined by stratified keratinizing epithelium, whereas the deeper papillated portion exhibited a double layer of basal-like cells and luminal eosinophilic columnar cells with focal decapitation secretions. A female patient, aged 29 years, presented to us in the outpatient clinic suffering from left earache and hearing loss since the past 3 months, with gradual onset and a slowly progressive course. On examination, we found a left aural polyp, which was firm and grayish in color, but extremely tender on touch. Biopsy for histopathological examination was consistent with a diagnosis of syringocystadenoma papilliferum. The patient received regular follow-up and is doing well with regard to her earache with marked hearing improvement. To conclude, solitary lesions seen in unusual locations generate multiple differential diagnoses and should be confirmed by histopathology in order to exclude other pathologies and define the appropriate therapeutic approach.

BRD4 promotes p63 and GRHL3 expression downstream of FOXO in mammary epithelial cells.

Bromodomain-containing protein 4 (BRD4) is a member of the bromo- and extraterminal (BET) domain-containing family of epigenetic readers which is under intensive investigation as a target for anti-tumor therapy. BRD4 plays a central role in promoting the expression of select subsets of genes including many driven by oncogenic transcription factors and signaling pathways. However, the role of BRD4 and the effects of BET inhibitors in non-transformed cells remain mostly unclear. We demonstrate that BRD4 is required for the maintenance of a basal epithelial phenotype by regulating the expression of epithelial-specific genes including TP63 and Grainy Head-like transcription factor-3 (GRHL3) in non-transformed basal-like mammary epithelial cells. Moreover, BRD4 occupancy correlates with enhancer activity and enhancer RNA (eRNA) transcription. Motif analyses of cell context-specific BRD4-enriched regions predicted the involvement of FOXO transcription factors. Consistently, activation of FOXO1 function via inhibition of EGFR-AKT signaling promoted the expression of TP63 and GRHL3. Moreover, activation of Src kinase signaling and FOXO1 inhibition decreased the expression of FOXO/BRD4 target genes. Together, our findings support a function for BRD4 in promoting basal mammary cell epithelial differentiation, at least in part, by regulating FOXO factor function on enhancers to activate TP63 and GRHL3 expression.

Aging is associated with an expansion of CD49fhi mammary stem cells that show a decline in function and increased transformation potential.

Breast cancer incidence increases during aging, yet the mechanism of age-associated mammary tumorigenesis is unclear. Mammary stem cells are believed to play an important role in breast tumorigenesis, but how their function changes with age is unknown. We compared mammary epithelial cells isolated from young and old mammary glands of different cohorts of C57BL6/J and BALB/c mice, and our findings revealed that old mammary glands were characterized by increased basal cell pool comprised of mostly CD49fhi cells, altered luminal-to-basal cell ratio, and irregular ductal morphology. More interestingly, basal stem cells in old mice were increased in frequency, but showed a functional decline of differentiation and increased neoplastic transformation potential. Gene signature enrichment analysis revealed a significant enrichment of a luminal cell gene expression signature in the basal stem cell-enriched population from old mice, suggesting some luminal cells were expressing basal markers. Immunofluorescence staining confirmed the presence of luminal cells with high CD49f expression in hyperplastic lesions implicating these cells as undergoing luminal to basal phenotypic changes during aging. Whole transcriptome analysis showed elevated immune and inflammatory responses in old basal stem cells and stromal cells, which may be the underlying cause for increased CD49fhi basal-like cells in aged glands.

Genetic and Epigenetic Regulation of TOX3 Expression in Breast Cancer.

Genome wide association studies (GWAS) have identified low penetrance and high frequency single nucleotide polymorphisms (SNPs) that contribute to genetic susceptibility of breast cancer. The SNPs at 16q12, close to the TOX3 and CASC16 genes, represent one of the susceptibility loci identified by GWAS, showing strong evidence for breast cancer association across various populations. To examine molecular mechanisms of TOX3 regulation in breast cancer, we investigated both genetic and epigenetic factors using cell lines and datasets derived from primary breast tumors available through The Cancer Genome Atlas (TCGA). TOX3 expression is highly up-regulated in luminal subtype tumors compared to normal breast tissues or basal-like tumors. Expression quantitative trait loci (eQTL) analyses revealed significant associations of rs3803662 and rs4784227 genotypes with TOX3 expression in breast tumors. Bisulfite sequencing of four CpG islands in the TOX3 promoter showed a clear difference between luminal and basal-like cancer cell lines. 5-Aza-2’-deoxycytidine treatment of a basal-like cancer cell line increased expression of TOX3. TCGA dataset verified significantly lower levels of methylation of the promoter in luminal breast tumors with an inverse correlation between methylation and expression of TOX3. Methylation QTL (mQTL) analyses showed a weak or no correlation of rs3803662 or rs4784227 with TOX3 promoter methylation in breast tumors, indicating an independent relationship between the genetic and epigenetic events. These data suggest a complex system of TOX3 regulation in breast tumors, driven by germline variants and somatic epigenetic modifications in a subtype specific manner.

P53 Regulates Progenitor Cell Quiescence and Differentiation in the Airway

Mechanisms that regulate progenitor cell quiescence and differentiation in slowly replacing tissues are not fully understood. Here, we demonstrate that the tumor suppressor p53 regulates both proliferation and differentiation of progenitors in the airway epithelium. p53 loss decreased ciliated cell differentiation and increased the self-renewal and proliferative capacity of club progenitors, increasing epithelial cell density. p53-deficient progenitors generated a pseudostratified epithelium containing basal-like cells invitro and putative bronchioalveolar stem cells invivo. Conversely, an additional copy of p53 increased quiescence and ciliated cell differentiation, highlighting the importance of tight regulation of p53 levels. Using single-cell RNA sequencing, we found that loss of p53 altered the molecular phenotype of progenitors and differentially modulated cell-cycle regulatory genes. Together, these findings reveal that p53 is an essential regulator of progenitor cell behavior, which shapes our understanding of stem cell quiescence during homeostasis and in cancer development.

Abstract 3319: Characterization of the CD44+/CD49f+/CD24- subpopulation in basal-like DCIS cells

Abstract The molecular mechanisms responsible for Ductal Carcinoma In Situ (DCIS)-Invasive Ductal Carcinoma (IDC) transition have yet to be elucidated. Due to the lack of molecularly targeted therapies, basal-like DCIS has a high risk of recurrence and progression to invasive and metastatic cancers. In this study, using a human DCIS cell line model and a novel single-cell clonogenic approach, we have identified and characterized the aggressive clones derived from single cells with the CD49f+/CD44+/CD24- surface markers. We found that aggressive clones had enhanced self-renewal, migratory and invasive capacities. The aggressive clones had elevated ALDH activity, lower global DNA methylation and increased expression of stem cell related genes including OCT4 and SOX2. Further mechanistic studies showed that linc-ROR and miR-10b were overexpressed in aggressive clones and are key regulators of their self-renewal and invasive abilities. Finally, we confirmed our in vitro results in vivo, demonstrating that aggressive clones were capable of forming tumors in nude mice, whereas non-aggressive clones were not. We believe that characterization of aggressive clones within the heterogeneous CD49f+/CD44+/CD24- DCIS population and having linc-ROR and miR10b as additional markers to distinguish them from non-aggressive clones would provide the basis to develop new chemoprevention agents. Citation Format: Nadire Duru, Ramkishore Gernapudi, Pang-Kuo Lo, Yuan Yao, Benjamin Wolfson, Yongshu Zhang, Qun Zhou. Characterization of the CD44+/CD49f+/CD24- subpopulation in basal-like DCIS cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3319.

Abstract 2386: Microfluidic single cell exome-seq and RNA-seq analysis of tumor composition

Abstract Human breast tumors have been shown to exhibit extensive inter- and intra-tumor heterogeneity. While recent advances in genomic technologies have allowed us to deconvolute this heterogeneity, few studies have addressed the functional consequences of diversity within tumor populations. Here, we identified an index case for which we have derived a patient-derived xenograft (PDX) as a renewable tissue source to identify subpopulations and perform functional assays. On pathology, the tumor was an invasive ductal carcinoma which was hormone receptor-negative, HER2-positive (IHC 2+, FISH average HER2/CEP17 2.4), though the FISH signal was noted to be heterogeneous. On gene expression profiling of bulk samples, the primary tumor and PDX were classified as basal-like. We performed single cell RNA and exome sequencing of the PDX to identify population structure. Using a single sample predictor of breast cancer subtype, we have identified single basal-like, HER2-enriched and normal-like cells co-existing within the PDX tumor, a finding replicated in several independent experiments. Genes differentially expressed between these subpopulations are involved in proliferation and differentiation. One population was characterized by high MYC and the other high EGFR/KRT14. These findings were validated using immunohistochemistry on PDX and primary tumor material. Further functional experiments showed that that EGFR subpopulation has stem cell character istics and forms metastasis in the animal model. Microfluidic whole genome amplification followed by whole exome capture of 81 single cells, along with exome sequencing of the germline, primary and post treatment tumor and whole PDX showed that BRCA1 and TP53 are mutated in all single cells, as well as a number of sub-clonal mutations that are being investigated further. Loss of heterozygocity was observed in 16 TCGA cancer driver genes and novel mutations in 7 known cancer driver genes. Careful comparison of the exome sequencing data allowed the association of driver gene mutation prevalence with tumor progression. These findings are important in our understanding the functional consequences of intra-tumor heterogeneity with respect to clinically important phenotypes such as invasion, metastasis and drug-resistance. Citation Format: Ioannis Ragoussis, Paul Savage, Yu-Chang Wang, Timothee Revil, Dunarel Badescu, Sadiq Saleh, Ernesto Iacucci, Nicolas Bertos, Anie Monast, Attila Omeroglou, Dongmei Zuo, Morag Park. Microfluidic single cell exome-seq and RNA-seq analysis of tumor composition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2386.

Abstract 1982: BRD4-FOXO axis maintains differentiated mammary epithelial phenotype by regulating p63 and GRHL3 expression

Abstract Bromodomain-containing protein 4 (BRD4) is an important epigenetic reader which promotes gene transcription to modulate cell-specific functions and is under intensive investigation for its potential as an anti-tumor therapeutic target. However, the role of BRD4 in non-transformed cells remains unclear. Using RNA sequencing and chromatin immunoprecipitation-sequencing analyses in normal basal-like breast epithelial cells, we demonstrate that BRD4 is required for the expression of epithelial-specific genes and suppression of stem cell-like properties by binding to the distal regions of epithelial-related genes. Moreover, BRD4 occupancy correlates with enhancer activity and enhancer RNA (eRNA) transcription of these genes. Interestingly, we show that BRD4 perturbation regulates the expression of p63 and Grainy Head-like transcription factor-3, GRHL3, whose depletion partially mimics BRD4 inhibition and impairs a differentiated phenotype. By binding to the distal regions of p63 and GRHL3, BRD4 promotes RNA polymerase-II occupancy and thus affects eRNA transcription. Motif analyses on cell-specific BRD4 binding sites predict the involvement of FOXO transcription factors. Consistently, inhibition of EGFR-AKT signalling activates FOXO1 function and promotes the expression of p63 and GRHL3. Activation of Src kinase signaling decreases RNA polymerase-II occupancy on BRD4 target genes and enhancers. Altogether, these findings provide evidence that BRD4 promotes a differentiated epithelial phenotype in non-transformed mammary cells at least in part through the activation of p63 and GRHL3 expression by associating with FOXO factors. Citation Format: Sankari Nagarajan, Upasana Bedi, Anusha Budida, Wanhua Xie, Feda Hisham Moh’d Hamdan, Vivek Kumar Mishra, Zeynab Najafova, Malik Alawi, Daniela Indenbirken, Stephan Knapp, Cheng-Ming Chiang, Adam Grundhoff, Christina H. Scheel, Vijayalakshmi Kari, Florian Wegwitz, Steven A. Johnsen. BRD4-FOXO axis maintains differentiated mammary epithelial phenotype by regulating p63 and GRHL3 expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1982.

Abstract 3661: ALDH1A3-inducible RARRES1 is a tumor suppressor in triple-negative breast cancer and is methylated in claudin-low breast cancers

Abstract The retinoic acid (RA) signalling pathway plays an important role in breast cancer progression and has either a pro-tumorigenic or tumor-suppressive role depending upon the effector function of RA-inducible genes that are expressed or epigenetically silenced. To study this paradigm in breast cancer, we focused on a controversial RA-inducible gene, the retinoic acid receptor responder 1 (RARRES1) protein that is often hypermethylated in cancer and has been reported to have tumor-suppressive function in prostate and nasopharyngeal carcinomas. However, in a study focused on a rare subtype of breast cancer, inflammatory breast cancer, RARRES1 is pro-tumorigenic. This functional discrepancy requires further investigation to determine its role in breast cancer in general. First, analysis of patient data sets revealed that RARRES1 is predominantly expressed in triple-negative breast cancers (TNBCs). Knockdown of RARRES1 in claudin-low MDA-MB-231 and basal-like MDA-MB-468 and HCC1937 significantly increased tumor growth and cell proliferation, suggesting RARRES1 has a tumor suppressive function in (TNBC), regardless of position on the differentiation hierarchy. Expression analyses of 24 breast cancer cell lines (including 18 TNBC and 2 normal-like cell lines) revealed that RARRES1 is predominantly expressed in basal-like TNBC cells We found that RARRES1 expression is dependent on, and strongly correlates with, the cancer stem cell marker, and RA-producing, ALDH1A3 in fixed breast cancer patient samples. Immunohistochemistry of the same patient tumor samples revealed RARRES1 expression is localized to the endoplasmic reticulum. Importantly, however, the presence of ALDH1A3 or RA is not sufficient to induce RARRES1 expression. RARRES1 is hypermethylated in claudin-low breast cancer cell lines, and release of this silencing is required for full induction of RARRES1 expression. We have identified sites of regulation by methylation in RARRES1 using Illumina 450K methylation arrays and 5-methylcytosine ChIP. We conclude that RARRES1 is an ALDH1A3/RA-inducible tumor suppressor in TNBC with methylation and expression profiles distinct to the differentiation hierarchy observed in breast cancer. Citation Format: Krysta M. Coyle, Patrick Murphy, Dejan Vidovic, Cheryl A. Dean, Margaret L. Thomas, Derek Clements, Mohammad Sultan, Ahmad Vaghar-Kashani, Carman Giacomantonio, Lucy Helyer, Ian Weaver, Shashi Gujar, Patrick WK Lee, Paola Marcato. ALDH1A3-inducible RARRES1 is a tumor suppressor in triple-negative breast cancer and is methylated in claudin-low breast cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3661.

Characterization of the CD49f+/CD44+/CD24- single-cell derived stem cell population in basal-like DCIS cells.

The molecular mechanisms responsible for the Ductal Carcinoma in Situ (DCIS)-Invasive Ductal Carcinoma (IDC) transition have yet to be elucidated. Due to the lack of molecularly targeted therapies, basal-like DCIS has a high risk of recurrence and progression to invasive and metastatic cancers. In this study, by applying a novel single-cell clonogenic approach with the CD49f+/CD44+/CD24− surface markers, we characterized the aggressive clones that have enhanced self-renewal, migratory and invasive capacities derived from a human DCIS model cell line MCF10DCIS. The aggressive clones had elevated ALDH1 activity, lower global DNA methylation and increased expression of stem cell related genes, especially concurrent activation of SOX2/OCT4. In addition, we showed that the aggressive clones have increased expression of lincRNA-RoR and miR-10b compared to non-aggressive clones, which enhance their self-renewal and invasive abilities. Finally, we confirmed our in vitro results in vivo, demonstrating that aggressive clones were capable of forming tumors in nude mice, whereas non-aggressive clones were not. Our data suggest that lincRNA-RoR and miR10b could be used to distinguish aggressive clones from non-aggressive clones within the heterogeneous CD49f+/CD44+/CD24− DCIS population. Our findings also provide the foundation to develop new chemoprevention agents for DCIS-IDC transition.

The Ribonucleic Complex HuR-MALAT1 Represses CD133 Expression and Suppresses Epithelial-Mesenchymal Transition in Breast Cancer.

Epithelial-to-mesenchymal transition (EMT) is a core process underlying cell movement during embryonic development and morphogenesis. Cancer cells hijack this developmental program to execute a multi-step cascade, leading to tumorigenesis and metastasis. CD133 (PROM1), a marker of cancer stem cells, has been shown to facilitate EMT in various cancers, but the regulatory networks controlling CD133 gene expression and function in cancer remain incompletely delineated. In this study, we show that a ribonucleoprotein complex including the long noncoding RNA MALAT1 and the RNA-binding protein HuR (ELAVL1) binds the CD133 promoter region to regulate its expression. In luminal nonmetastatic MCF-7 breast cancer cells, HuR silencing was sufficient to upregulate N-cadherin (CDH2) and CD133 along with a migratory and mesenchymal-like phenotype. Furthermore, we found that in the basal-like metastatic cell line MDA-MB-231 and primary triple-negative breast cancer tumor cells, the repressor complex was absent from the CD133-regulatory region, but was present in the MCF-7 and primary ER+ tumor cells. The absence of the complex from basal-like cells was attributed to diminished expression of MALAT1, which, when overexpressed, dampened CD133 levels. In conclusion, our findings suggest that the failure of a repressive complex to form or stabilize in breast cancer promotes CD133 upregulation and an EMT-like program, providing new mechanistic insights underlying the control of prometastatic processes. Cancer Res; 76(9); 2626-36. ©2016 AACR.

Abstract P5-04-09: Copper chaperons as novel targets for therapy in triple-negative breast cancer (TNBC)

Abstract Background: Copper metabolism is frequently dysregulated in cancer and promotes tumorigenesis. Copper chelation was shown to delay tumor development, attenuate tumor growth, block angiogenesis and inhibit metastases in preclinical breast cancer models. Copper depletion with tetrathiomolybdate (TM) in on-going phase II study for breast cancer patients at high risk for relapse resulted in significant improvement in progression-free survival, especially in patients with TNBC. We hypothesized that targeting ATOX-1 and CCS, copper chaperons that are major regulators of copper trafficking, with novel selective inhibitor may disrupt cellular copper transport and suppress TNBC cell growth, block angiogenic activity, and enhance cytotoxicity of available chemotherapy. Methods:We measured ATOX-1 and CCS protein expression using western blot in a panel of breast cancer cell lines including TNBC cell lines with basal-like (BL) and claudin-low (CL) subtypes. We compared potency and efficacy of ATOX-1/CCS inhibitor to induce cytotoxicity in MDA-MB231, MDA-MB436, MDA-MB468 and primary normal mammary HMECs. We evaluated ability of the inhibitor to disrupt tubulogenesis of endothelial cells. To determine if blocking copper transport can enhance sensitivity of TNBC to chemotherapy we used novel ATOX-1/CCS inhibitor in combination with Cisplatin to treat TNBC in a schedule-dependent manner. Results: ATOX-1 protein expression was elevated in all tested TNBC cell lines compared to normal HMEC (1.7±0.2 and 2.1± 0.3 folds higher in BL and CL cells, respectively). Upregulated CCS protein expression was also observed in majority of tested cell lines compared to HMEC (2.8±0.6 and 1.2±0.1 times higher in BL and CL cells, respectively). Treatment of MDA-MB231, MDA-MB436, MDA-MB468 with the inhibitor resulted in reduced cell proliferation. IC50 doses for 72h treatment with single agent were: 0.23±0.02uM (MDA-MB468), 0.29±0.03uM (MDA-MB231) and 0.35±0.02uM (MDA-MB436). Additional cytotoxicity was observed in TNBC when ATOX-1/CCS inhibitor was applied in combination with Cisplatin. Interestingly, sequential treatment resulted in synergistic effect (CI&amp;lt; 1). Treatment with the inhibitor reduced growth of HMECs and HuVECs in vitro, and inhibited angiogenesis in tube formation assay with HuVECs. Conclusions: Targeting copper trafficking by selective inhibition of chaperons ATOX-1 and CCS is promising and could potentially serve as a therapeutic approach to overcome resistance to chemotherapy in TNBC. In vivo studies investigating efficacy and biological activity of the novel compound in a xenograft model are ongoing and will help to elucidate molecular mechanisms of action, and further estimate potential clinical relevance of this approach. Citation Format: Karginova O, Song A, Wang J, Luo C, Jiang H, He C, Olopade OI. Copper chaperons as novel targets for therapy in triple-negative breast cancer (TNBC). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-09.

Abstract P6-04-03: Transcriptome profiling reveals enrichment of luminal feature in aged basal cell population in murine mammary gland

Abstract Aging is the number one risk factor for breast cancer development. Increasing evidence suggests the potential of mammary stem cells (MaSCs) and their progenitors to generate certain types of breast cancers through neoplastic transformation. Our previous study has shown increased percentage of MaSC-enriched basal cell population (Lin-CD49fhighCD24med) and increased MaSC frequency during aging in murine models. On the other hand, a recent study from another group showed an increased frequency of CD49fhigh cells in the human luminal population (CD227+) during aging, indicating possible aberrant expression of CD49f in the aged luminal cells. However, how these age-related luminal cells with basal markers are generated and how they contribute to potential breast cancer development remains unknown. Here we apply bioinformatics analysis on Next Generation Whole Transcriptome Sequencing data of MaSC-enriched basal cell population (Lin-CD49fhighCD24med) and luminal progenitor-enriched cell population (Lin-CD49flowCD24high) of both young (4 to 6 months) and old (26 to 31 months) mouse mammary gland to test the hypothesis that age-associated increase of basal cell population and MaSCs may be due to the gain of basal cell markers and features by luminal cells. By Gene Set Enrichment Analysis (GSEA) we found a significant loss of basal cell and basal mammosphere signatures and a significant enrichment of luminal cell and luminal mammosphere signature in the old basal cell population and mammospheres in comparison with the young basal cell population and mammospheres. The core enrichment luminal genes from GSEA are able to cluster the old MaSC-enriched basal cell population as well as mammospheres closer to the cluster of luminal population than to the young basal population. These analyses indicate that aging may be associated with an expansion of aberrant MaSCs with both basal and luminal markers in mice, which may be the precursors of certain types of breast cancer. We are now studying the potential function of the basal-like luminal cells in the aged basal population. Citation Format: Gu X, Gao H, Wu A, Bandyopadhyay A, Dong Q, Sun L-Z. Transcriptome profiling reveals enrichment of luminal feature in aged basal cell population in murine mammary gland. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-04-03.